CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
NEUROSPORA CRASSA PHOSPHOSERHJE PHOSPHATASE
The effect of pH on the hydrolysis of
phospho sed ne by enzymes present in
Hi 1 d type and ser(JBN-5) extracts and
partial purification and characterization
of wild type phosphoserine phosphatase.
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Frances Wallace Johnston Beck
June, 1981
The Thesis of Frances Wallace Johnston Beck is
approved:
Kenneth
C.~ones,
Ph.D.
Dais~ A. Kuhn, Ph.D.
(Joyce B.
~1axwell,
Ph.D., Chairman
California State University, Northridge
ii
ACKNOWLEDGEt1ENT
I wish to gratefully acknowledge the assistance of my,graduate
studies
committee~
Dr. Joyce fvlaxt'iell, Chairman, for continuous
inspiration, guidance and encouragement; Dr. Ken Jones for advice
and editorial assistance; and Dr. Daisy Kuhn for excellent and
detailed critical editing of the manuscript.
I would also like to thank Drs. S.R.V. Raghavan and David
Knutson for assistance and cooperation in arranging work schedules
to permit expediting thesis work; and Drs. Sandra Jewett and Robert
Holcott for their interest in this study and assistance with
chemistry problems.
Grateful appreciation is also extended to Mrs. Evelyn Tackels
for her assistance in preparing the manuscript and to Ralph V.
Beck, my understanding and patient husband, who by now must know
the thesis by heart.
iii
TABLE OF CONTENTS
Page
LIST OF FIGURES
LIST OF TABLES
v
•
e
G
e
•
e
e
5
e
e
0
vii
e
ABSTRACT
INTRODUCTION
viti
.....
1
MATERIALS AND METHODS •
6
Chemical reagents
6
Strains used· •••
6
Preparation of crude extract •
7
Protein determinations •••
8
Phosphoserine phosphatase assay
9
Ammonium sulfate fractionation
12
Colu!lln chromatography
13
RESULTS
15
Effect of pH on the hydrolysis
of phosphoserine • • • • • • •
15
Effect of cations and inhibitors •
23
Thennostabil i ty
•
•
•
0
•
44
Ammonium sulfate fractionation
46
Column chromatography
46
DISCUSSION
64
REFEREt~CES
72
iv
LIST OF FIGURES
Figure
1.
Page
Effect of pH on phosphosP.rine hydrolysis
by enzymes present in wild type crude
extract
2.
• • • • • • • • • • • • • •
18
Effect of pH on phospt10serine hydrolysis
by enzymes present in ser(JBM-5)
extract
3.
4.
5.
6.
7.
8.
9.
10.
11.
• • •
o
•
•
•
•
•
•
•
•
•
20
•
Comparison of 20-minute pH rrotiles
obtained from wi 1 d type and ser( JBM-5) _
crude extracts • • • • • • • • • • •
22
Effect of Be++ on wild type enzymes
capable of phosphoserine hydrolysis
25
Effect ot· Be++ on ser(JBt'l-5) enzymes
capable of phosphoserine hydrolysis
27
Effect of pH and Be++ on the hydrolysis
of _B -glycerophosphate by enzymes present
in wild type and ser(JBt~-5) crude extracts
++
Effect of pH and Be
on the hydrolysis of pnitrophenyl phosphate by enzymes present in
wild tyoe and ser(J8~1-5) crude extracts
31
Effect of NaF on the hydrolysis of
phosphoserine by enzymes present in wild
type crude extract • • • • • • • •
35
Effect of NaF on the hydrolysis of
phosphoserine by enzymes present in
ser(JBM-5) crude extract • • • •
37
Effect of Mg++ on the hydrolysis of
phosphoserine by enzymes present in
wild type crude extract • • • •
40
Enzyrnatic activity of ammonium sulfate
fractions of wild type crude extract
o
12.
•
29
48
Q.A.E.-Sephadex column chromatography at
pH 7. 0 fo1·· wi 1 d type crude extract
• • • • 50
v
Page
Figure
13.
14.
15.
16.
1t.
18.
Q.A.E. - Sephadex column chromatography
at pH 7.0 for ser(JBM-5) crude extract
50
Q.A.E.-Sephadex column chromatography
at pH 8. 5 for \'/il d type crude extract
52
Q.A.E.-Sephadex column chromatography
at pH 8.5 for ser(JBM-5} crude extract
52
Hild type Q.A.E.-Sephadex cohnnn
chromatography \Ali th a 0 to 0. 3M NaCl
1 inear gradient at pH 7. 0 • • • • •
56
Ser(JBM-5) Q.A.E.-Sephadex column
chromatography with a 0 to 0. 3t~ NaCl
linear gradient at pH 7.0.
58
Effect of pH on the hydrolysis of
phosphoserine by enzymes present in
pooled colu~n fractions • • • • • •
61
v ·i
LIST OF TABLES
Table
2.
3.
4.
5.
Page
Effect of divalent cations on wild
type crude extract phosphoserine
and alkaline phosphatase activiti~s
42
Effect of selected compounds on
wild type crude extract phospho serine
and alkaline phosphatase activities
43
Effect of heat treatment on wild type
crude extract phosphoserine and
alkaline phosphatase activities
45
Relative rate of hydrolysis of wild
type pooled column fractions compared
to the crude extract preparations
62
Specific activity and purification
factor • • • • • • • • • • • • • •
63
vii
ABSTRACT
NEUROSPORA CRASSA PHOSPHOSERINE PHOSPHATASE
The effect of pH on the hydrolysis of
phosphoserine by enzymes present in wild
type and ser(JBM-5) extracts and partial
purifi cafi on and characterization of
wild type phosphoserine phosphatase
by
Frances Wallace Johnston Beck
Master of Science in Biology
The effect of pH on the hydrolysis of phospho serine by enzymes
extracted from Neurospora crassa was found to be different for wild
type than for the serine requiring mutant
ser(JB~1-5).
The pH profile
of activity in ser(JBH-5) extracts lacks a shoulder at pH 7.0 which
is present in the wild type extracts, while the activity from pH 7.5
to pH 9.5 has the characteristics of alkaline phosphatase and is
similar in both mutant and wild type extracts.
The presence of a specific phosphoserine phosphatase is
indicated by the fact that the hydrolytic
activity at pH 7. 0 is not
inhibited by Be++ or KCtJ, both of which al·e known inhibitors of
alkaline phosphatase.
Saturated ammonium sulfate fractionation was
consistent with two separate specificities.
Q. A. E. -Sephadex chromatography did not resolve the t\-Jo enzymatic
specificities but did increase the purity of both wild type and
mutant preparations.
As the purification increased, the specific
viii
/
activity of ser(JBM-5) phosphoserine phosphatase became more
convergent \'Jith the specific activity exhibited by the wild type.
This observation is consistent with the idea that the mutant produced
reduced amounts of a normal enzyme, rather than normal amounts of a
defective enzyme.
ix
INTRODUCTION
Tht·ee separate pathways have been proposed for the biosynthesis
of the. amino acid serine.
1) The non-phosphorylated path\'lay begins
with the oxidation of d-glyceric acid by an NAD+-linked d-glyceric
aci•d dehydrogenase to yield hydroxypyruvic acid.
The hydroxypyruvic
acid is then aminated by serine transaminase to yield serine
{Shallack, 1956).
2) The phosphorylated pathway proposed by Ichihara
and Greenberg (1957), involves the oxidation of 3-phosphoglyceric
acid to phosphohydroxypyruvic acid.
This acid is aminated by ·
phosphoserine transaminase to yield phosphoserine and in the final
biosynthetic step, phosphoserine phosphatase catalyses the hydrolysis
of phosphoset'ine to serine.
3) The glyoxylate pathway involves
sequentially the transamination of glyoxylate to glycine by
glyoxylate transaminase and then the conversion of glycine to serine
through the addition of a single carbon fragment from 5-10
methylenetetrahydrofo1ate.
The latter reaction is catalyzed by
serine hydroxymethyltransferase (DeBoiso and Stoppani, 1967).
The phosphorylated pathway is the major pathway or the only
pathway of serine biosynthesis for such bacteria as Escherichia coli
(Pizer, 1963), Desulfovibrio desulfuricans (Germano and Anderson,
1969), !-la£._mophilus inf1uenzae (Pizer, Ponce de Leon and t·1ichalka,
1969) and Rhodopseudor.10nas
caps_!:l_~ta
(Scl:midt and Sojka, 1973).
1
It
2
is also the major serine biosynthet"ic pathway for baker's yeast
Saccharomyces cerevisiae (Schramm, 1958).
Sojka and Garner (1967) found the mold Neurospora crassa to
possess the enzymes necessary for both the phosphoryl a ted and the
non-phosphoryl a ted pathways.
They suggest that the phosphoryl a ted
pathway is the predominant pathway activated when the organism is
grown on glucose medium.
Wright (1951), in contrast, studied a
serine-glycine mutant which exhibited optimal growth on media
supplemented with glyoxylic acid or glycine rather than on media
supplemented with serine.
She suggested that the biosynthesis of
serine in Neurospora crassa involved the conversion of glycolic acid
to glyoxylic acid, the amination of glyoxylic acid to glycine and
finally the conversion of glycine to serine.
Use of the glyoxylate
pathway may be unique to this mutant or may be an example of what is
now termed
"conditional auxotrophy" (Heptinstall and Quayle, 1970;
Ulane and Ogur, 1972) a condition in which an organism activates one
biosynthetic pathway when grown on media supplemented with a
particular carbon source but, activates a different pathway for
synthesizing the same end product when a different carbon source is
provided.
Conditional auxotrophy has been described with the
facultative methylofroph Pseudomonas
Harder and Quayle, 197lb).
A~11
(Harder and Quayle, 197la,
This mutant, when grown on methanol or
succinate is unable to produce serine by the phosphorylated pathway.
Although it was unable to gro\>J on succinate, it did grow slowly on
c1
compounds, indicating that it was capab1e of synthesizing serine
by an a_·! tern ate path\IJaY.
3
Sojka and Garner ( 1967) cultivated the Neurospora \'li 1 d type on
glucose or fructose medium, while Wright (1951} gre\'1 the mutant on
glycerol medium.
Hright's mutant is no longer available and no
presently known serine requiring mutant of Neurospora crassa grows
better on sucrose medium supplemented with giycine than on sucrose
medium supplemented \'Jith serine_
(r~ax\•!ell,
1970}.
The final step in the phosphorylated pathway is the
dephosphorylation of phosphoserine by phosphoserine phosphatase.
This phosphatase, also called phosphoserine phosphohydrolase
(Barman, 1969}, is specific for phosphoserine.
Phosphoserine
phosphatase has been fsolated from bacteria (Piser, 1963), yeast
(Schramm, 1958), chicken liver (Neuhaus and Byrne, 1959}, rat liver
(Borkenhagen and Kennedy, 1959}, bovine tissue (Paoli, Guiraud and
Brunel, 1974) and human tissue 0 11oro-Furlani, Turner and Hopkinson,
1980).
These enzyf!les are specific for phospho serine and ex hi bit
optimal activity within the neutral pH range.
Using crude extracts from the Neurospora
l
wi 1 d type and the ser-
mutant, Lynne Crosby (unpublished observation) found that at pH
5.5 or below phosphoserine phosphatase activity was non-existent in
the mutant, \•Jhile it was measurable in the wild type.
When the
phosphoserine phosphatase activity of the mutant was assayed at pH
7.0 (Dodson, 1980}, it was 1/3 to 1/2 of the activity exhibited by
the wild type.
The observation that there was no phosplloserine
phosphatase activity at pH 5 in the crude extract from the serine
mutant, suggests that a pH profile of this extract may be different
4
from that of the wild type crude extract.
Another serine requiring mutant,
ser(JBf~-5)
VJhi ch is allelic to
ser-3 n1axvJell, et al., 1978), was studied by Chuck -(1980) and
Dodson (1980).
Both of these mutants were found to exhibit
decreased phosphoserine phosphatase activity at neutral pH as
compared to their respective prototrophic strains.
This decrease in
enzymatic activity could be due to the production of normal levels
of' a .defective enzyme or to reduced production of a normal enzyme.
One way to resolve the question why there was residual phosphoserine
phosphatase activity in the crude extracts obtained fror.1 the two
serine requiring mutants, ser-3 and ser(JBt·1-5), would be to purify
both the
Vv'il
d type and mutant phospho serine phosphatases.
If the
wild type and mutant specific activities converge with increasing
purity, it \'loul d suggest that the mutants exhibit reduced production
of a normal enzyme.
If the specific activities remain divergent
upon increasing purity of the enzyme, it vmuld suggest that the
mutants possess an altered enzyme.
To more fully understand the nature of the serine requiring
mutant
ser(JB~1-5)
and the role of both wild type and ser(JBt,l-5).
phosphoserine phosphatases in the phosphoryl a ted· biosynthetic
pathway, the foll
1.
m<~i
ng objectives were set forth in this study:
To study the effect of pH on phosphoserine hydrolysis by
determining the pH profiles for the wild type strain and
the ser(JBM-5) strain.
2.
To evaluate methods by \'lhich the purity of the enzyme may
5
be increased.
3.
To compare specific activity of the wild type with that of
ser(JBH-5) as phospho serine phosphatase purity is
increased and determine whether the specific activities
converge or remain divergent.
4.
To determine the properties of the enzyme.
MATERIALS AND f· 1ETHODS
1
Chemical reagents
All chemicals used in this study were reagent grade. flglycerophosphate, Ortho-phospho-D,L-serine, L-serine, p-nitrophenyl
phosphate, Tris[Tris-hydroxymethyl(amino methane)] and Q.A.E.Sephadex ·(bead size 40-120 urn) were obtained from Sigma Chemical
Company (St. Louis, Missouri).
Double distilled de-ionized water
was used throughout these studies.
Glass columns (10 mm x 50 rrm)
used for chromatography with ·Q. A. E. -Sephadex were purchased from
Bio-Rad Laboratories (Richmond, California).
Pentex-fraction V, was purchased from
r~iles
Bovine serum albumin,
Laboratories, Inc.
(Elkhart, Indiana).
Strains used
Mary B. Mitchell, previously of the Division of Biology of the
California Institute of Technoiogy, provided the nutritionally wild
type Neurospora crassa strain C-102-15300-4-2A.
The serine
requiring mutant strain, C-1 02-15300-4-2/\ ser(JB1'·1-5) was provided
by Joyce B. Maxwell.
The mutant strain was isolated from the wild
type strain C-l02-l5300-4-2A by Joyce B. Maxwell and Paul West
{unpublished).
This mutant, hereafter referred to as ser(JBM-5),
is allelic to 47903 ser-3 (FGSC #1213) (Maxwell, et al., 1978).
6
7
Both strains were grown on agar slants containing Horowitz
complete medium (Horowitz, 1947).
After 10 days, 0.5 ml of a dense
suspension of conidia in water was transferred to 20 ml of Vogel •s
Medium (Vogel, 1956; Davis and DeSerres, 1970) supplemented with 2%
sucrose, 10 mM glycine and 10 mM sodium formate.
I
incubated for 100 hours at 25°C.
The cultures were
The mycelia were harvested at 4°-
60C on a Buchner funnel with suction and rinsed with chilled
distilled water •. Mycelial pads \tere hand pressed between paper
towels to remove as much water as possible before their wet weight
was determined on a Mettler balance, model H30.
These pads were
then stored at -22°C or were mixed with distilled water and
lyophilized or were extracted immediately.
Preparation of crude extract
Crude extract was prepared by grinding the mycelium (fresh,
defrosted or lyophilized) with reagent grade sea sand and a small
volume of O.lM Tris buffer, pH 7.0, in a chilled mortar with pestle
until a uniformly thick paste was obtained. This paste was diluted
with the
neces~ary
amount of Tri s buffer to ensure its complete
removal from the mortar.
The final volume to weight ratio was 2 to
5 ml per gram wet weight of mycelium or lyophilized material.
This
mixture was then centrifuged at 22,000 g for 30 minutes at 4°C in a
8
Sorvall centrifuge, model RC-5.
The resulting supernatant was
exhaustively dialyzed against 0.1 Tris buffer, pH 7.0, and was
stored at -22°C until assayed.
All operations were carried out at
4°-6°C with 0.1 t,1 Tris buffer at pH 7.0.
(fvlost crude extracts were
not treated further but \'Jere stored at -22°C following dialysis).
However, one extract was fractionated three times with sufficient
chilled saturated
am~onium
sulfate solution to give a final
concentration of 70% saturation.
This 70% saturated ammonium
sulfate fraction was suspended in buffer and exhaustively dialyzed
against 0.1 M Tris buffer pH 7.0, before being stored or assayed.
This procedure did not·significantly inerease the phosphoserine
phosphatase activity of the wild type and was, therefore, not
included as a standard procedure.
Protein determinations
Protein determinations of the crude extracts and dialyzed 70%
saturated ammonium sulfate fractions were performed either by the
biuret assay {Gornwall, Bardawill and David, 1949) or by the FolinCi ocal teau assay ( Lm'iry, et al • , 1951).
To reduce the l·evel of
possible interference by Tris buffer (Rej and Richards, 1974), bovine
serum albumin was prepared and diluted in O.H1 Tris buffer pH 7.0,
when it v1as used as the standard.
Protein content of column
fractions was determined by reading the optical density at 280 nm and
260 nm.
To <;>btain mg protein per ml solution, concentrations were
estimated from the following fonnula (Segal, 1976):
9
. ] mg/ml
[ Prote1n
= 1.55
lcm
Alcm
280 - 0.76 A260
The first and last fractions collected from the column, which
contained O.H! Tris buffer and
0.3~1
NaCl in O.lM Tris buffer
respectively and were free of protein, served as the negative or zero
blanks.
Phosphoserine phosphatase assay
.The enzymatic activities of the crude extracts, column fractions
and ammonium sulfate fractions were measured in a modified
phospho serine phosphatase assay (Schramm, 1963) containing the
following reagents in .a final volume of.l.O ml.
Reagent
Concentration
of reagent
Volume
added
Final concentration
in reaction mixture
Phospho-D
L-sedne
0.1 M
66.6 ul
MgC1 2
1. 0 f•1
16.6 ul
16.6 mM
Tri s buffer
0.1 M
835 ul
83.5 mM
Tris buffered
.. enzyme ..
10 - 120
ug protein
33.5 ul
8.35 mt~ Tris buffer
plus protein
6. 6
m~1
When inhibitors or additional cations were used, 10 ul of an
aqueous solution containing the compound were added to the mixture
before the addition of .the enzyme.
In addition to phosphoserine (P-ser) as a substrate, alkaline
phosphatase activity was assayed by using
and p-nitrophenyl phosphate (p-NP).
these substrates were 6.6
m~1
~-glycerophosphate
(B-GP)
The final concentrations of
and 10.5 mM respectively, except where
. 10
noted otherwise.
All substrates were prepared in distilled water and
neutralized with either 10 N NaOH or J N HCl.
A solution containing the substrate, Tris buffer and MgC1 2 was
equilibrated to 25°C for approximately 2 minutes before the reaction
was begun by the addition of the enzyme in Tris buffer.
Routine
incubation times were 20 minutes for the crude extracts and 30
minutes for samples from the column fractions.
I
.
The blank in all cases consisted of the reaction mixture and the
enzyme to· which had been added 0.1 ml 50% trichloroacetic acid (TCA)
before the incubation was begun.
The reaction was terminated by the addition of 0.1 ml 50% TCA to
the reaction mixture.
For the timed studies, 0.9 ml samples were
removed periodically and added to clean test tubes containing 0.1 ml
50% TCA.
For assays in which samples from the column fractions were
used, the reaction was terminated by the addition of the ascorbic
acid-molybdate solution directly to the reaction mixture.
a.~idity
The
of the molybdate reagent used to assay inorganic phosphates
was sufficient to precipitate the minute amounts of protein present
in these samples.
This small amount of protein did not interfere
with the inorganic phosphate assay.
In contrast, when crude extracts
were used, the amount of protein was so great it interfered with the
assay of the phosphates, so it was removed with TCA prior to the
addition of the molybdate reagent.
11
. The 1 .0 ml samples, with or Vli'thout TCA, were assayed for the
presence of inorganic phosphates by a method described by Ames
(1966), except that the samples were incubated at 37°C for 1 hour
before reading the optical density at 820 nm on a Gilford
spectrophotometer.
TCA-treated samples were centrifuged at 1500 g
for 5 minutes to remove any particulate matter and then
0.7~ml
of the
resulting supernatant was transferred to separate tubes for the
phosphate assay.
Sodium and potassium mono-basic phosphates \ltere used as
standards and prepared in O.lM Tris buffer at vari?us pH values.
Varying the pH of the standard solutions did not significantly affect
the optical density readings.
When the protein concentrations could be determined, the
specific activity was calculated and expressed as 10- 2 uMoles
inorganic phosphate (Pi) produ~ed per mg protein per minute (10- 2
ut1oles Pi/mg Pr./min).
Over a period of approximately 15 months during which these
studies were conducted, five separate growth cultures of both wild
type and ser(JBM-5) mutant \-Jere .used.
There was no observable
difference in the specific activity from one culture to the next of
either wild type or its mutant strain even though some were extracted
immediately, others were frozen for future use as mycelial pads or
lyophilized.
Several 100 ml quantities of 0.1
M Tris buffer were prepared
12
and were individually adjusted to pH's extending from pH 5.5 to 10.5.
In all cases, the Tris buffers were adjusted to the desired pH at
25°C.
These different buffers were used in place of Tri s-pH 7. 0
buffer for a pH profile to assay crude extracts (or in a few cases,
column fractions) with three different substrates:
serine,~-glycerophosphate
phospho-D,L-
and p-nitrophenyl phosphate.
Since the
dialyzed extracts, column fractions and ammonium sulfate fractions
were usually in Tris buffer at pH 7.0 and the substrates and
t~gC1
2 in
water adjusted to pH 7.0, the final pH of the reaction mixture was
not the same as the buffer added.
However, the results are recorded
as the final pH of the reaction mixture which contained all of the
various components.
In the initial experi8ents, Tris-maleate, MES (2-[N-morpholino]
ethanesul fonic acid) and HEPES (N-2-Hydroxyethyl piperazine-N '2'ethanesulfonic acid) were evaluated as buffers because of their
extended pH ranges.
None of these were used because Tris-maleate had
a high absorption at 280 nm and HEPES interfered with both the FolinCiocalteau assay and the phosphate reaction.
MES buffering range, pH
5.5 to 6.7, was considered too limiting for a pH profile.
Ammonium sulfate fractionations
Individual 0. 2 ml ali quots of wild type crude extract containing
1 mg protein were fractionated with 0.8 ml of several different
percentages of saturated ammoni urn sulfate in 0.1 MTri s buffer at pH
7.0.
The mixture was allo\ried to stand on ice for 1 hour and was then
centrifuged for 30 minutes at 3000 g in a Beckman J-6B centrifuge.
13
After centrifugation, the supernatant was transferred to another test
tube and the pellet resuspended in 1.0 ml Tris buffer at pH 7.0.
Both supernatant and resuspended pellet were then assayed with pnitrophenyl phosphate at pH 9.0 and phosphoserine in the presence of
5 x 10- 6M Be++ at pH 7.0. Protein content of both supernatant and
pellet solutions were determined by optical density at 280 nm and 260
nm.
Column chromatography
For each 10 x 50
fiTl1
column, 2.5 grams of Q.A.E.-Sephadex (a
fully quarteni zed, strongly basic ani on exchanger)· 1vere added slowly
to 100 ml of 0.1 fvl Tri s buffer pH 7. 0 or pH 8. 5.
The Sephadex was
allowed to swell overnight at room temperature and was then rinsed 8
times with Tris buffer to remove the fine particles.
The Sephadex
was cooled to 6°C before being_ added to a pre-cooled, clean column.
Each column was equilibrated with 0.1 M Tris buffer overnight at 4°6°C.
One rnl of crude extract containing 5 to 7 mg protein was
layered onto the column, \'Jhich was developed by adding a linear
gradient of 0 to 0.5 N NaCl in 0.1
t~
Tris buffer.
The total volume
of gradient added was 300 ml and 100-three ml fractions were
collected.
Five col urnn.s each of wi 1d type and
ser(JB~·l-5)
extracts
were eluted \'lith the gradient at pH 7.0 and two each with the
gradient at pH 8.5.
Crude extracts prepared initially in pH 7.0
buffer were dialyzed overnight against 0.1 M Tris buffer pH 8.5
before being applied to the columns equilibrated at pH 8.5.
Samples
14
of each fraction were assayed for phosphoserine phosphatase activity
at both pH 7.0 and pH 8.5.
Four additional column chromatograms were performed, two each
of wild type and mutant extracts.
Columns, to which were applied
1.2 ml of the crude extract containing 15 mg protein, were developed
with 120 ml of a linear gradient of 0 to 0.3 M NaCl in 0.1 M Tris
buffer at pH 7.0.
Again, three ml fractions were collected.
Separate samples from each of the 45 fractions were assayed for:
1.
alkaline phosphatase activity with p-nitrophenyl phosphate
at pH 9.0
2.
phosphoserine phosphatase activity with phosphoserine at
pH 7.0.
3.
phosphoserine phosphatase activity with phosphoserine in
the presence of lo- 6 M Be++ at pH 7.0.
Fractions which contained maximum activity with these
substrates \<Jere pooled for both the wild type and mutant extracts.
A pH profile with phosphoserine was performed on the two pooled
samples.
RESULTS
Effect of pH on the hydrolysis of phosphoserine
In order to determine whether the hydrolysis of phospho serine
and the attendant release of inorganic phosphate would alter the pH
of the reaction mixture during the course of the reaction, the pH
was monitered as a function of time.
The pH of the reaction mixture
did not vary by more than 0.01 pH unit during the 20 minutes of
incubation at 25°C.
Thus, the release of the inorganic phosphate
did not significantly affect the pH of the solution used under the
condition of the
assay~
The effect of pH on the hydrolysis of phosphoserine by enzymes
contained in the wild type extract is shown in Figure 1.
Hydrolysis
of phospho-D,L-serine occurred at all pH values included in this
study, with the greatest activity between pH 8.0 and 8. 5.
Ser(JBfvl-5) exhibits low activity for all time periods at pH 6.2
to 7.5 but increased activity from pH 7.5 to 9.0 as shown in Figure
2.
A comparison of the two profiles at 20 minutes is shown in
Figure 3. The specific activity, defined as 10- 2 uMoles of
inorganic phosphate produced per mg protein per minute, of the wild
type at pl-1 7.0 and pH 7.5 was 3.52 and 2.17 times greater,
respectively, than that of ser(JBM-5).
wild type
e~tract
The specific activity of the
at pH 8.5 is only 1.35 times greater than that of
the mutant and suggests normal activity in the mutant at this pH.
The pH profile of the wild type extract exhibits a 11 Shoulder 11
15 ..
16
between pH 6. 5 and pH 7. 5 and caul d be interpreted as a profile of
two or more enzymes capable of hydrolyzing phosphoserine.
In this
case, each enzyme would show optimal activity at a different pH.
Alternatively, one enzyme with two separate active sites with
different pH optima for their catalytic activities could produce the
same re sul ts.
·The pH profile of the mutant suggests that the hydrolytic
activity may be due to only one type of enzyme and is similar to the
pH profile of alkaline phosphatase isolated from Neurospora crassa
by Kuo and Blurnenthal.(l96lb) and Davis. and Lees (1973).
These
investigators assayed the alkaline phosphatase with pg1ycerophosphate_(Kuo and Blumenthal, 196lb) and p-nitrophenyl
phosphate. (Davis and Lees, 1973).
Kuo and Blumenthal (196lb)
reported no hydrolysis of phosphoserine by their alkaline
phosphatase at pH 8.9.
In contrast, Davis and Lees (1972) reported
alkaline phosphatase hydrolysis of phosphoserine to be 70% of that
of p-nitrophenyl
phosphate at pH 9.0.
17
Figure 1
EFFECT OF pH ON PHOSPHOSERINE
HYDROLYSIS BY ENZYMES
PRESENT IN WILD TYPE CRUDE EXTRACT
Samples which contained 6.0 ml/tube of the reaction mixture
(3.96 x l0- 2t--l phospho-D, L-serine, 9.96 x 10- 2M MgC1 2 , _5 x l0- 1fvl
Tris buffer adjusted to the required pH) were equilibrated to 25°C
before the addition of Tris buffered wild type crude extract to
begin the reaction.
For each time period, 0.9 ml of the reaction
mixture was removed from each of the ten samples and placed in a
test tube containing 0.1 ml of 50% trichloroacetic acid.
were removed immediately
(non
Samples
minute), after 2.5, 5.0, 10.0, 15.0
and 20.0 minutes of incubation at 25°C.
The TCA samples were
centrifuged and 0.7 ml of the supernatants were assayed for free
inorganic phosphate.
The profile represents the average of five
profiles determined over a period of three months.
Samples of
crude extract were obtained from two separate cultures and the
variation was insignificant.
18
8.0
.,._
.Q)
;::)
c:
7.0
E
1.."
Q)
0.
6.0
c:
Q)
+0
1..
0.
20 min·
5.0
0>
E
1..
Q)
0.
"0
4.0
Q)
0
:J
"0
0
1..
0.
3.0
0..
CJ)
Q)
-:::;:
0
N
I
2.0
::1.
...
0
1.0
0
5.0
55
6D
p H
19
Figure 2
EFFECT OF pH ON PHOSPHOSERINE HYDROLYSIS BY DJZYf"lES
PRESENT IN SER(JBM-5) CRUDE EXTRACT
Samples·which contained 6.0 ml/tube of the reaction mixture
(3.96 x 10- 2M phospho-D,L-serine, 9.96 ~ 10- 2M Mg 2, 5 x 10- 1M Tris
buffer adjusted to the required pH) \'lere equilibrated to 25°C before
the addition of Tris buffered·
reaction.
ser(JB~1-5)
crude· extract to begin the
For each time period, 0.9 ml of the reaction mixture was
removed from each of the· ten samples and placed in a test tube
containing 0.1 ml of 50% trichloroacetic acid.
Samples were removed
immediately ("0" minute), after 2.5, 5.0, 10.0, 15.0 and 20.0
minutes incubation at 25°C.
The trichloroacetic acid saMples were
centrifuged and 0. 7 ml of the supernatants Here assayed for free
inorganic phosphate.
The profile is the average of five profiles performed over a
period of three months.
Sampl_ es of crude extract were obtai ned from
two separate cultures and the variation was insignificant.
20
....GJ
8.0
:;)
c
E
~
GJ
7.0
0.
·-....c
GJ
0
~
6.0
0.
0>
E
~
GJ
0.
5.0
"0
GJ
u
:;)
"0
0
~
4.0
a.
n..
fJ)
GJ
3.0
0
:E
::L
N
-
l
0
2.0
1.0
0
5.0
5.5
6D
6.5
7D
p
H
7.5
8JJ
8.5
9D
9.5
21
Figure 3
COMPARISON OF
20-~1INUTE
pH PROFILES OBTAINED
FR0!4 WILD TYPE AND SER (JBM-5) CRUDE EXTRACTS
This figure represents the effect of pH on the hydrolysis of
phosphoserine by enzymes present in both wild type and crude
extracts after 20 minutes of incubation at 25°C.
Each point
represents the average of 8 separate assays, each with duplicate
samples.
Included in these assays are samples of crude extracts
prepared from three separately harvested cultures.
22
• Wi I d type- mean + standard deviation
•
8.0
o.•••••••••• -6 Ser(JBM-5>-mean +- standard deviation
cv
· ::1
c 7.0
......
E
L
cv
0.
c
-
6.0
cv
0
1..
0.
0)
E
5.0
,
••
•
•••
••
••
1..
QJ
0.
"'0
QJ
0
4.0
:J
"'0
••
•••
0
L
0.
0..
••
•••
••
3.0
•
I/)
J
QJ
~ 2.0
N=i.
I
••
0
~
1 .o
0
5D
5.5
6D
6.5
7[)
7.5
8D
8.5
9.0
9..5
pH
/
23
Effect of cations and inhibitors
Both Kuo and Blumenthal (196lb) and Davis and Lees (1969)
reported that alkaline phosphatase of Neurospora crassa is sensitive
to inhibition by Be++.
This inhibition provided a means by which an
estimation could be made of the amount of phosphoserine hydrolysis
attrib(ltable to alkaline phosphatase activity at pH 8.9- 9.0.
The pH profile in Figure 4 indicates a 58% decrease in the
I
enzymatic activity of the wi 1d type extract at pH 8. 5 and an
insignificant decrease in the activity at pH 7.0 when the extract was
assayed in the presence of 5 x 10- 6M BeC1 2 as compared to the
activity in the absence of the divalent cation. Even more pronounced
v1as the effect of Be++ on the enzymatic activity of the mutant
ser(JBM-5} at pH
~.5,
producing a 96% decrease (Figure 5).
Thus,
hydrolysis of phosphoserine by enzymes in this wild type crude
extract could be interpreted as due in part to the activity of
alkaline phosphatase at pH 8.5, while in the mutant, ser(JBM-5),
virtually all of the activity (96%) could be attributed to alkaline
phosphatase.
Alkaline phosphatase activity of both wild type and mutant
appeared to be comparable when assayed with
~-glycerophosphate
and p-
nitrophenyl phosphate in the presence o~ absence of Be++ (Figures 6
and 7).
Ser(JBM-5) may be more strongly inhibited by Be++ than is the wild
type.
24
Figure 4
EFFECT OF Be++ ON WILD TYPE ENZY~1ES CAPABLE
OF PHOSPHOSERINE HYDROLYSIS
For each sample assayed, 1.0 ml of the reaction mixture (6.6 x
10- 3M phosphoserine, 16.6 x 10- 3M MgC1 2, and 83.5 x 1o- 2M Tris buffer
adjusted to the desired pH) was equilibrated ·to 25°C before the
addition of the Tris buffered crude extract to begin the reaction.
The reaction mixture wa·s incubated for 20 minutes at 25°C.
For
experiments in which the enzymatic activity was measured in the
presence of Be++, 5 x 10- 6M Bec1 2 dissolved in \'later was added to
the reaction mixture in lOul amounts before the addition of the
enzyme to begin the reaction.
four samples.
Each point represents the average of
25
8.0
-
j(.......... ..
P-serine
P-serine plus Be++
(I)
::J
c
7.0
E
L
(I)
a.
c
-
6.0
QJ
0
L.
a.
0'>
5.0
E
L.
(I)
a.
"0
(I)
4.0
0
::J
"0
0
L.
a.
.Ji···
~
••
•••
II)
(I)
0
. N
I
~
~~
••
3.0
Q..
:E
....
••
••
•
••
••
••
>t
•••"•.t
2.0
::1.
••
••
••
••
••
••
0
.1.0
••
••
••
••
•
~·.t.
•••
0
5D
6!5
7D
75
pH
8.0
9D
9.5
26
Figure 5
EFFECT OF Be++ ON SER(JBM-5) ENZYMES
CAPABLE OF PHOSPHOSERINE HYDROLYSIS
For each sample assayed, 1 .Oml of the reaction mixture (6.6 x
10- 3M phosphoserine, 16.6 x 10- 3MMgC1 2 , and 83.5 x 10- 2M Tris
buffer adjusted to the desired pH) was equilibrated to 25°C before
the addition of the Tris buffered crude extract to begin the
reaction.
25°C.
The reaction mixture was incubated for 20 minutes at
For experiments in which the enzymatic activity was measured
in the presence of Be++, 5 x 10~ 6 M BeCl 2 dissolved in water was
added to the reaction mixture in 10 ul amounts before the addition
of the enzyme to begin the reaction.
Each point represents the
averag~
of four samples.
27
•... ., .....
a.o
.}(
P-serine
P-serine plus Be++
...
Q)
::)
c
EI
7.0
'Q)
0.
c
...
6.0.
Q)
0
'0..
0)
E
5.0
'Q)
0.
"'0
Q)
0
:J
4.0
"'0
0
'0.
ll.
3.0
II)
Q)
0
:E
N
I
::1.
2.0
0
~
1.0
........,.,..
............. .t,~.••
0
5.0
5.5
6D
6.5
• y
....._......···~..
7.0
pH
.
·~
7!5
•
8D
··.Y.:
7\........ .
8b
........
9.0
9.5
28
Figure 6
THE EFFECT OF pH AND Be++ ON THE HYDROLYSIS OF ~-GLYCEROPHOSPHATE
0
BY ENZYMES PRESENT IN WILD TYPE AND SER(JBM-5) CRUDE EXTRACTS
For each sample assayed, 1.0 ml of the reaction mixture (6.6 x
l0- 3 t~ ~-glycerophos-phate, 16.6 x 10- 3Mt~gC1 2 , and 83.5 x 10- 2M Tris
buffer adjusted to the desired pH) was equilibrated to 25°C before
the addition of the crude extract in Tris buffer to begin the
reaction.
For experiments in which the enzymatic .activity was
.
++
6
measured in the presence of Be , 5 x 10- M BeC1 2 dissolved in water
was added to the reaction mixture in 10 ul amounts before the
addition of the enzyme in Tris buffered crude extract.
The figure represents the average of two profiles, each with
duplicate samples.
29
-·-
•
x
• Wi I d type
++
x Wild type plus 8e
Q)
:J
c
8.0
E
b----·-6
Ser ( JBM-5)
~···········~
Ser ( JBM-5) pI
Q)
Q_
-·
++
\
L
c
us Be
7.0
A\
ClJ
A"
0
,~
L
Q_
0>6.0
E
~\
\
\
\
\
\
.t\
L
Q)
Q_
"'0
ClJ
5.0
u
:J
"'0
0
L
Q_
4.0
a_
If)
ClJ
0
:;;E
:::1
3.0
N
I
0
..-
2.0
1.0
~..• •~..•
·~"
v
0
5JJ
55
6IJ
65
.···~••
•••
7D
75
pH
9D
9.5
30
p •
Figure 7
THE EFFECT OF pH AND Be++ ON THE HYDROLYSIS OF p-NITROPHENYL PHOSPHATE
BY ENZYMES PRESENT IN WILD TYPE AND SER(JBM-5} CRUDE EXTRACTS
For each sample assayed; 1.0 ml of the reaction mixture (10.5 x
10- 3M p-nitrophenyl phosphate, 16.6 x 10- 3MMgC1 2 and 83.5 x 10- 2M
Tris buffer adjusted to·the desired pH} was equilibrated to 25°C
before the addition of the crude extract in Tris buffer to begin the
reaction.
For experiments in which the enzymatic activity was
measured in the presence of Be++, 5 x 10- 6M BeC1 2 dissolved in water
was added to the reaction mixture in 10 ul amounts before the
addition of the enzyme in Tris buffered crude extract.
The figure represents the average of two profiles, each with
duplicate samples.
31
~----•
Wild type
++
:----x Wi I d type pI us Be
(1)
+-
6.-----6.
8.0
Ser ( JBM-5)
~·········~ Ser ( JBM-5
:;)
c
·-E
>
++
pI us Be
\....
QJ
0..
7.0
c
cu
+-
0
\....
0..
6.0
0'>
E
\....
QJ
0..
5.0
"0
(1)
u
:;)
"0
0
\....
0..
4.0
0..
Ill
(1)
0
3.0
:E
C\1
:1.
I
0
..-
2.0
1.0
0
5.0
5.5
6JJ
6.5
7D
pH
7b
8D
85
9D
9!5
32
The phosphoserine hydrolysis at pH 7.0 to 7.5, present in the
wild type and decreased or missing in the mutant ser(JBM-5), is
evidence for the existence of a specific phosphatase, namely
phosphoserine phosphatase.
the experimental results of Chuck (1980) and Dodson
(1980),
left unresolved whether ser(JBM-5) had an altered enzyme or less of
I
the normal enzyme.
The results presented here suggest that the
enzyme phospho serine phosphatase is present in the mutant ser(JBH-5)
but is much 1ess active than in the wi 1d type.
Phosphoserine
phosphatase is not knmm to be inhibited by beryllium and the
enzymatic activity at pH 7.0 of the vlild type used here is
unaffected by the presence of the cation.
phosphatase activity of the mutant
The phosphoserine
ser(JB~1-5)
was not completely
inhibited in the presence of Be++ at pH 7.0.
Phosphoserine phosphatases isolated from bovine tissues (Paoli,
Guiraud and Brunel, 1974), from E. coli (Pizer, 1963) and yeast
(Schramm, 1958) are specific for phosphoserine, are dependent upon
Mg ++ for maximal activity and exhibit optimal hydrolysis betvJeen pH
5.5 and pH 8.5.
In addition they are sensitive to inhibition by 10
mM NaF and do not significantly hydrolyze
~-glycerophosphate
or p-
nitrophenyl phosphate.
Several experiments were performed in an effort to determine
whether the enzyme active in Neurospora crassa at pH 7.0 and 7.5
against phosphoserine had properties similar to these known
33
phosphoserine phosphatases.
Enzymes present in both wild type and mutant extracts displayed
considerably lower rates of hydrolysis of
~-glycerophosphate
and p-
nitrophenyl phosphate at neutral pH than at alkaline pH (Figures 6
and 7).
These data agree with those of Kuo and Blumenthal (l96lb)
who reported 25% alkaline phosphatase activity with
.a-
glycerophosphate at 7.0 and with Davis and Lees (1973), who reported
10% alkaline phosphatase activity at pH 7.0 \'lith p-nitrophenyl
phosphate.
The fact that alkaline phosphatase activity is significantly
reduced at neutral pH as compared to alkaline pH•s suggests that the
hydrolysis of phosphoserine at pH 8.5 is due to alkaline phosphatase
while the hydrolysis of this substrate from pH 7.0 to 7.5 is due to
phosphoserine phosphatase.
10 mM NaF decreased phosphoserine hydrolysis at all pH values
tested (Figures 8 and 9).
At pH 8.5 the hydrolytic activity of the
wild type extract was decreased by 56% and the mutant extract
activity was decreased by 53%.
The decrease in phosphoserine
hydrolysis at pH 7.0 was 74% in the wild type and 66% in the mutant.
Yeast phosphoserine phosphatase activity has been reported to be
100% inhibited in the presence of 10 mM NaF (Schramm, 1958).
Although the inhibition by NaF at pH 7.0 in the experiments
presented here was not 100%, phosphoserine hydrolysis was more
sensitive to the effect of the fluoride ion at pH 7.0 than at pH 8.5.
34
Figure 8
EFFECT OF NaF ON THE HYDROLYSIS OF PHOSPHOSERINE BY ENZYMES
PRESENT IN WILD TYPE CRUDE EXTRACT
Samples containing 1.0 ml of the reaction mixture {6.6 x 10- 3M
phosphoserine, 16.6 x 10~ 3 M MgC1 2 , and 83.5 x 10- 2M Tris buffer
adjusted to the desired pH) were equilibrated to 25°C before the
addition of wild type crude extract in Tris buffer to begin the
reaction.
For experiments in which the enzymatic activity was
measured in the presence of NaF, 10 mM NaF dissolved in water was
added to the reaction mixture in 10 ul amounts before the addition
of the enzyme in Tris buffered crude extract.
· The figure represents the average of three profiles, each with
duplicate samples.
35
• P-ser i ne
•
8.0
-
a·········o P-ser i ne pI us 1OmM NaF
CIJ
::>
c
7.0
E
i..
CIJ.
0-
6.0
c
CIJ
0
i..
0-
5.0
0>
E
i..
CIJ
0-
,
,u
4.0
CIJ
::>
...
0
R
i..
a. 3.0
•• ••
Q..
en
CIJ
0
:E
•• ••
••• •••
•
••
••
•••
•
:
2.0
•
t?•
••
~
:
..•
1 .o
f
·•.
•
.• •.
t:J:
N:i.
I
0
......
~
•....
•h
~
•..
~
....fl
...
••
0
5D
5.5
7D
pH
75
8D
85
9D
95
36
Figure 9
THE EFFECT OF NaF ON THE HYDROLYSIS OF PHOSPHOSERINE BY ENZY~1ES
PRESENT IN SER(JBM-5) CRUDE EXTRACT
Samples containing 1.0 ml of the reaction mixture (6.6 x 103M
phosphoserine, 16.6 x 10- 3 t~ ~1gC1 , and 83.5 x lo- 2H Tris. buffer
2
adjusted to the desired pH) were equilibrated to 25°C before the
addition of the ser(JB~1-5) crude extract in Tris bl!ffer to begin the
reaction.
For experiments in which the enzymatic activity was
measured in the presence of NaF, 10
m~1
NaF dissolved in \'later was
added to the reaction mixture in 10 ul amounts before the addition
of the enzyme in Tris buffered crude extract.
The figure represents the average of two profiles, each 'r'lith
duplicate samples.
37
•
• P-ser i ne
a .•••••••• .o P-ser i ne pI us 1OmM NaF
8.0
7.0
Q)
~
::l
c
E
6.0
L.
Q)
a.
c
Q)
~
5.0
0
L.
a.
0>
E
\.,
4.0
Q)
a.
"0
Q)
0
::>
"0
3.0
0
L.
a.
a..
II)
Q)
~
•• •••
•
•• •••
••
•
2.0
-:E.
0
N-:J_
I
0
.:
·"
•
1 .o
•
y-o
0
.--.o-••
•
••
•
••
••
pH
75
•
··~••••
••
••
•
tl
·"iJ·
7D
\!1
8085
9.D 95
38
Divalent cations and other selected compounds were added to the
reaction mixture used in the phosphatase assays to study their
effects on the enzymatic activity of the vlild type crude extracts.
The mixtures always contained 16.6 uMoles MgC1 2 •
To establish the necessity for the magnesium ion, a separate
experiment was performed in which the activity in the
of the
abse~ce
ion was compared with the activity in the presence of Hg++.
The
resul.ts shown in Figure 10 indicate that t~g++ is necessary for
optimal activity of this (these) enzyme(s) at both pH 7.0 and at
alkaline pH levels.
The requirement for ~1g++ is well documented for
alkaline phosphatase (pH 8.9 to pH 9.0) as well as for phosphoserine
phosphatase.
The enzymes present in this Neurospora crassD:_ wild
type appear to be no exception to this requirement, although at pH
9.2 there seems to be little stimulation by Mg++.
Table 1 summarizes the results of several experiments
evaluating the effect of cations or inhibitors on the enzymatic
activity obtained in the routine assay.
Since alkaline and
phosphoserine phosphatase activities are reduced in the absence of
++
Mg , MgC1 2 was not omitted from the reaction mixture. Compounds
containing the cation_or inhibitors were dissolved in water and
added to the incubation in 10 ul volumes to be sure that the same
concentration was added to each sample and dissolved in the same
sol uti on.
None of the compounds 1 i sted in Tables 1 and 2 altered
the pH of the reaction mixture by more than 0.2 pH unit.
39
Figure 10
THE EFFECT OF Mg++ ON THE HYDROLYSIS OF PHOSPHOSERINE
BY ENZYf'1ES PRESENT IN HILD TYPE CRUDE EXTRACT
Samples containing 1.0 ml of the reaction mixture (6.6 x 10- 3M
phosphoserine, 16.6 x 10-3M MgC1 2 and 83.5 x 10- 2M Tris buffer
adjusted to the desired pH) were equilibrated ·to 25°C before the
addition of wild type crude extract in Tris buffer to begin the
reaction.
Ten test tubes containing 1 .0 ml of the reaction mixture minus
the MgC1 2 were equilibrated to 25°C before the addition of the wild
type crude extract to begin the reaction. ~1gC1 2 was replaced by
16.6 ul O.lM Tris buffer •
. The crude extracts were dialyzed overnight against 0.1 M Tris
buffer pH 7.0 before being used in the assay.
Any f>1g++ which may
have been bound to the enzyme. was not removed with E. D. T.A.
figure represents the average of duplicate samples.
The
40
,.16 .6!J.MO I es Mg++
++
O··········DNO Mg
•
8.0
QJ
+-
7.0
::l
c
e.
L
QJ
6.0
0.
c
QJ
+0
L
5.0
0.
C)
E
L
QJ
0.
4.0
"C
QJ
u
::l
-c
0
L
3.0
~···0
••
•
rp•
•
•
0.
Cl..
(/)
QJ
•
••
•
••
2.0
0
9 •• ••'i$
•••••
:E
N:J.
I
...
0
...
""•
1 .o
..··n-·.
.{}'"'.......u..
G"
0
5D
55
6D
6.5
7D
pH
7!5
8D
8.5
9. 0
95
41
Beryllium ions (5 x 10- 6M) inhibited the alkaline phosphatase
assayed with p-nitrophenyl phosphate at pH 9.0, while phosphoserine
phosphatase activity assayed with phosphoserine at pH 7.0 was only
slightly decreased.
This result was expected, since alkaline
phosphatase is only marginally active at pH 7.0 (Kuo and Blumental,
196lb; David and Lees, 1973).
It was interesting to note (Table 1) the extreme sensitivity to
· th a t HgCl was bare 1y
Hg ++ by alkal1"ne phasp ha t ase, cons1"d er1ng
2
soluble in water (Solubility of HgC1 2 is 1 gram per 13.5 ml water at
25°C).
Phosphoseri ne phosphatase activity assayed with phosphoseri ne
at pH 7.0 was inhibited by 5 x 1o- 3M ca++, 5 x 1o- 3M zn++ and 2.5 x
10- 3M Cd++.
Enzymatic activity of purified yeast phosphoserine
phosphatase is inhibited 94 tp 100% by these cations (Schramm,
1958)
0
Zinc ions have been reported to be a requirement
for~
coli
alkaline phosphatase (Reid and Wilson, 1971) and for Neurospora
alkaline phosphatase (Davis and Lees, 1969). Hov1ever, alkaline
phosphatase activity in the pre.sence of 1a- 3M Zn ++ added to the ~1g ++
was approximately 60% of that activity in the presence of the ~1g ++
alone (Davis and Lees, 1969). Alkaline phosphatase activity in the
presence of 2.5 x 10- 3M zn++ and 16.6 x 10- 3M Mg++ when assayed in
this study was 69% of that activity exhibited in the presence of
Mg++ alone.
42
TABLE 1
EFFECT OF DIVALENT CATIONS ON WILD TYPE CRUDE EXTRACT
PHOSPHOSERINE AND ALKALINE PHOSPHATASE ACTIVITIES
Divalent
Cation
u~1ol
es
per ml
P-ser
pH 7.0
J3-GP
pH 8.5
p-NP
pH 9.0
~1g++
16.60
1.00
1.00
1 .00
None
0.00
0.36
N.D.*
o. 32·>
2. 50
0.65
0.00
0.00
0.005
0. 97
0.00
0.00
Ca++
5.00
0.40
0.62
0.62
Cd++
2. 50
0.28
0.56
0.57
Co++
5.00
2. 50
o. 57
0.89
0.83
N.D.
0.33
0.55
Hg++
**
0.87
0.12
0.15
~1n++
5.00
2.5o·
o. 52
0. 72
0.49
N.D.
N.D.
N.D.
Zn++
5.00
2.50
0.05
0.19
o. 61
0.95
0.55
0.69
0.90
0.43
N.D.
o. 77
r~g++/pl
Be++
us
*
N.D. - not done
** Only small amounts Here soluble in water. The
amount of inorganic pho~phate produced per minute by
the enzyme in the absence of additional cation is
designated as 1.00. Relative rates of hydrolysis by
enzymes in the presence of the additional cations are
listed as decimal fractions of 1 .00.
.
Each relative rate is the average of t\'1o or more
assays, each with duplicate samples.
P-ser = phosphoserine, B-GP = ~-glycerophosphate, pNP = p nitrophenyl phosphate.
43
TABLE 2
EFFECT OF SELECTED COMPOUNDS ON WILD TYPE CRUDE EXTRACT
PHOSPHOSERINE AND ALKALINE PHOSPHATASE ACTIVITIES
Compound
MgCll
I
uMoles
per ml
P-ser
pH 7.0
B-GP
pH 8.0
p-NP
pH 9.0
16.6
1.00
1.00
1.00
5.00
0.97
0.06
0.02
2.50
1.24
N.D.
0.48
fvlgC1 2 p1 us
KCN
---------------------------ComEound
mMo1es
eer m1
P-ser
pH 7.0
· B-GP
.2!! 8. 5
p-·NP
pH 9.0
MgC1 2plus
NaF
10
0.39
0.74
0.54
L-serine
10
0.80
0.81
0.75
The amount of inorganic phosphate produced per
·minute by the enzyme in the absence of the added
compounds is designated as l .00. Relative rates of
hydrolysis by the enzyme in the presence of the added
compounds are listed as decimal fractions of this 1 .00.
Each relative rate is the average of two or more
assays, each with duplicate samples.
P-ser = phosphoserine, B-GP = p-glycerophosphate, p-NP =
p-nitrophenyl phosphate.
44
Alkaline phosphatase activity was inhibited by KCN (Table 2).
This is in agreement with Kuo and Blumenthal (196lb).
Ba++ and sodium tartrate had no effect on the hydrolytic
activity of either alkaline phosphatase or phosphoserine
phosphatase.
The,rmostabil ity
The effect of heat treatment was evaluated in an attempt to
separate phosphoserine phosphatase activity from alkaline
phosphatase activity.
Davis and Lees (1969) had been able to use
heat treatment as a method to remove non-specific proteins from the
crude extracts in their isolation of Neurospora crassa alkaline
phosphatase since the activity of this enzyme is little affected by
heat treatment.
The heat stability of Neurospora crassa
phosphoserine phosphatase has not been determined previously.
1.0 ml samples of the wild type crude extract were heated to
56°C for 30 minutes in the presence and absence of 16.6 uMoles
1•1gC1 2 • The samples \'/er:e centrifuged to remove the precipitate and
the supernatant was assayed with phosphoserine at pH 7.0 and with pnitrophenyl phosphate at pH 9.0.
Although heat treatment may provide a means by which the
purity of the enzyme may be increased, it did not prove to be a
single method by which one could separate the phosphoserine
phosphatase from the alkaline phosphatase activity because
TABLE 3
EFFECT OF HEAT TREATMENT ON WILD TYPE CRUDE EXTRACT PHOSPHOSERINE AND ALKALINE
PHOSPHATASE ACTIVITIES
Relative rate of hydrolysis
30 min pre-incubation
16.6 uMo1 es
t~gC1
2
Heated to
56°
P-ser
pH 7.0
+
+
+
p-NP
pH 9.0
o. 94 -+ 0.16
1. 00
o. 20
0.71 + 0.19
0.86 + 0.09
0.98 + 0.09
0.94 + 0.05
1. 04 + 0. 07
0.63 + 0.17
0.74+0.16
0.95 + 0.07
1.00
+
P-ser + Be++
pH 7.0
o. 56
+
Each figure represents the average of eight or more assays, each with duplicate samples.
Included in these samples are samples of crude extracts from three separately prepared
cultures.
P-ser = phosphoserine, p-NP = p-nitrophenylphosphate
0
·
.;:.
tn
...
phosphoserine phosphatase activity, although somewhat reduced, was
still present in the supernatant in significant amounts (Table 3).
Ammonium sulfate fractionation
Ammonium sulfate fractionations were carried out in an effort
to separate phosphoserine phosphatase from the alkaline phosphatase
activity.
0.1 ml samples from the supernatants and resuspended
ammonium sulfate pelleted material were assayed for phosphoserine
phosphatase activity with phosphoserine in the presence of 5 x 10- 6M
Be ++ at pH 7. 0.
Separate samples from the same supernatants and
resuspended material were also assayed with p-nitrophenyl phosphate
at pH 9.0 for alkaline phosphatase activity.
Phosphoserine
phosphatase activity was enriched in the pellet at 40% saturated ammonium sulfate, whereas most of the alkaline phosphatase activity
remained in the supernatant (Figure 11).
These data suggest that
phosphoserine phosphatase activity can be separated from alkaline
phosphatase activity by this method.
This study is being pursued
by another student at this time.
Column chromatography
The results of a typical 100-fraction-Q.A.E. Sephadex column
eluted with 0 to 0.5 M NaCl linear gradient at pH 7.0 are shown in
Figures 12 and 13.
Figures 14 and 15 represent columns eluted with
the same gradient at pH 8.5.
Separate undialyzed samples from each
fraction were assayed, with phosphoserine at both pH 7.0 and pH
8.5.
In no case
\'JaS
there a distinct and separate peak which
47
Figure 11
ENZYME ACTIVITY OF AMMONIUM SULFATE FRACTIONS OF WILD TYPE
CRUDE EXTRACT
Individual 0.2 ml aliquots of wild type crude extract
containing 1 .0 mg protein were fractionated with sufficient
saturated ammonium sulfate in 0.1 MTris buffer to give ·a final
solution of 20, 30, 40, 50, 60, and 70% saturated ammonium sulfate.
The solutions were allowed to stand on ice for one.hour and then
centrifuged.
The supernatant was transferred to a clean tube and
the pellet resuspended in Tris pH 7.D.
Undialyzed samples of the
supernatant and resuspended pellet were assayed with phosphoserine
at pH 7.0 and p-nitrophenyl phosphate at pH 9.0.
Protein content
of the whole crude extract, the supernatant and the resuspended
pellet were determined by the following formula:
O.D. 260 x 0.76 = mg protein/ml.
O.D. 280 x 1.55-
The figures represeht the average
of duplicate samples assayed with 100 ug protein.
p-NP, p-nitrophenyl phosphate, P-ser = phosphoserine
--
-------
-·--
-----~
-------
-----·-
-----------------
48
-Precipitate
0
.
.......
]Supernatant
1.2
:::c
0.
Q.l
;:)
c
E
....10
t
CD
\1)
0.
.4
Q.l
0.
!I..
.2
Q.l
c
II)
Q.l
a..
-
.6
;:)
c
·-
.8
Q.l
!I..
Q.l
1.0
I
0
0
0
!I..
0.
en
E
1.8
!I..
Q.l
c..
1.6
"0
Q.l
()
1 •4
;:)
"0
0
1.2
~
c..
c..
II)
Q.l
0
:E
N:i.
I
0
..-
0
• 1.0
0'1
:::c
.8
....10
.6
0.
a..
z
I
c..
.4
.2
0
0
20
30
40
50
60
% SATURATED AMMONIUM SULFATE
70
49
Figures 12 and 13
Q.A.E.-SEPHADEX COLUMN CHROMATOGRAPHY AT pH 7.0
Q.A.E.-Sephadex columns were developed with a linear gradient
of 0 to 0.5 M NaCl in 0.1 M Tris at pH 7.0.
Separate undialyzed
samples from each of the 100 fractions were assayed with
phosphoserine at pH 7.0 or with phosphoserine at pH 8.5.
Figure 12 represents the five wild type column profiles, each
layered with 5 to 7 mg protein.
Figure 13 represents the five ser(JBM-5) column profiles each
layered with 5 to 7 mg protein.
50
.....cv
Figure 12
:l
c
E
pH
12.5
............
l.
cv
Q.
"0
cv
..'
10.0
\
~
7.5
f ~
.1 i-•".
l.
Q.
Q..
...
'.
:t
;
:l
0
pH 8.5
•
0
"0
7~0
:
•
5.0
..
~
ll)
cv
0
:£
2.5
N-:j_
I
0
....
0
20
40
....:lcv
60
80
Fraction number
Figure 13
c
E
l.
cv
pH 7.0
12.5
Q.
"0
cv
10.0
.•
1 •
7.5
'
'
Q.
Q..
ll)
pH 8.5
•" I)'I
:l
"0
0
........ ' ....
-~
1:
••
0
l.
100
'
•,
"•
•
•'
cv
..
pt
I
,'
5.0
~
•~
0
:E
N-:j_
2.5
I
....0
0
20
40
60
Fraction number
80
100
51
Figures 14 and 15
Q.A.E.-SEPHADEX COLUHN CHROMATOGRAPHY AT pH 8.5
Q. A.E. -Sephadex columns were developed \'lith a 1inear gradient
of 0 to 0.5 M NaCl in 0.1
r~
Tris at pH 8.5.
Separate undialyzed
samples from each of the 100 fractions collected were assayed with
phosphoserine at pH 7.0 or with phosphoserine at pH 8.5.
Figure 14 represents two wild type column profiles.
Figure 15 represents two ser(JBM-5) column profiles.
The amount of protein used in each column was 5 to 7 mg.
52
Figure 14
..••''
15.0
E
.••''.
•••
•• •••••
' ..
• .
•
•
'
Q)
10.0
0
::>
0
t
~
~
..
.
7.5
0.
a..
•
•
.:. .
.•• ••.,
•
Q)
~
\
\
••
5.0
••
(f)
Q)
.··l r··,,
I
••r.
~
N
I
,\•
),
.,. .J.
::i.
• /\
'r!
0
.-
pH 8.5
.•
I
\.
0.
"0
..........
~~
12.5
~
"0
pH 7.0
,'•.
: ; •t
I
•
c•I
•'
0
60
40
20
t
'~..............~., .. ,.
100
80
Fraction number
Figure 15
Q)
+-
::>
c:
15.0
••.,
12.5
:1
• •
1 o.o
. ...
•• ••:
0
:;:)
7.5
.'
•
5.0
.
-o
0
~
0.
••
•
~
a..
(f)
Q)
~
N
I
::i.
0
.....
pH 8.5
,• •
0.
Q)
,
.
Q)
"0
...... ....
..........••...
f!
E
~
pH 7.0
":t
~
2.5
••
.,
:•
•
"1
-- ......,. ...,..,.. ....
•
;-.
••
••
...,.
It
·~
0
.
•
.. J
20
~
"
40
60
80
Fraction number
100
53
contained enzyme activity at pH 7.0 but not at pH 8.5 or vice
ve1·sa.
Whether the columns were eluted at pH 7. 0 or at pH 8. 5,
hydrolysis of phosphoserine was always lower when assayed at pH 7.0
than when assayed at pH 8.5.
The activity at pH 7.0 of the peak
fract1ons isolated from wild type was always greater than that
obtained with the ser(JBH-5) mutant.
Both of these phenomena were
I
observed when crude extracts of the two strains were assayed as
seen in the original pH profiles and timed study experiments
(Figures 1 , 2 and 3).
Of the total 5 to 7 mg protein layered onto the columns only
10 to 15% was recovered, while 50 to 76% of the total activity of
both phosphoserfne phosphatase and alkaline phosphatase was
recovered from the columns.
Figures 16 and 17 represent the results obtained from the
columns eluted with 0 to 0.3 M NaCl linear gradient at pH 7.0.
5
mM MgC1 2 was added to all Tri s buffer used for these columns
because it had been reported that f'ilg++ stabilizes the enzyme.
Separate samples from each of the 45 fractions collected were
assayed with phosphoserine at pH 7.0 in the absence and in the
presence of 5 x 10- 6M Be++ and with p-nitrophenyl phosphate at pH
9. 0.
The amount of p-ni trophenyl phosphate used here \'las one half
of that used in the routine assay or 5.25 uMoles/ml.
As in the
case of the previous columns assayed, both alkaline phosphatase and
phosphoserine phosphatase co-eluted.
Mutant enzymatic activity
54
with phosphoserine as a substrate at pH 7.0 was, as before, less
than that of the wild type.
Peak fractions from the columns used
to separate \'li 1d type extracts were pooled as were the peak
fractions obtained from the columns used to separate the mutant
ser(JBM-5) crude extract.
Again, only about 15% of the total
protein was recovered but the alkaline and phosphoserine
phosphatase activities recovered was 76 to 97% of the total
activity applied to the column.
~-glycerophosphate
was also used
~sa
substrate to measure
hydrolytic activity of each fraction at pH 6.0.
This was done to
evaluate the possible contribution of acid phosphatase to the total
phosphatase activity.
The profile of peak activity followed exactly the profile· for
p-nitrophenyl phosphate and phosphoserine (data not included).
These results suggest that an acid phosphatase was eluted in the
same peak fractions as those which contained the alkaline and
phosphoserine phosphatase, or, that one or both of the enzymes is
active at pH 6.0.
A pH profile was obtained with phosphoserine using the pooled,
undialyzed column fractions.
These fractions contained
approximately 0.2M NaCl in 0.1 M Tris buffer.
both wild type and
ser(JB~1-5)
The profiles for
activities \'/ere similar to the
profiles obtained with the crude extracts.
However, when the
55
Figure 16
WILD TYPE
Q.A.E.-SEPHADEX COLUMN CHROMATOGRAPHY WITH A 0.0 TO 0.3 M
NaCl LINEAR GRADIENT AT pH 7.0.
Q.A.E.-Sephadex columns were developed with a 0.0 to 0.3 M
linear gradient in 0.1 M Tris buffer at pH
7.p.
added to the Tris buffer used in these columns.
5 mM MgC1 2 was
Separate
undialyzed samples from_ each of the 45 fractions collected were
assayed with 6.6·x 10- 3 M phosphoserine at pH 7.0 or with 5.25 x
10- 3 M p-nitrophenyl phosphate at pH 8.5.
Protein content of each fraction was determined by reading
the optical density at 280 nm and 260 nm.
56
25
,
.....
QJ
::J
.
4
'
: I
r
I I
I I
E
I
t
:
QJ
0.
I
\
I
I
'
'
''
15
I
I
I
0
::J
"0
\
I
I
I
I
I
I
I
0
)....
I
I
0.
I
\
500
'
l=
'
(Q
'
.,
"0
I
I
I
I
400
'
300
0
N
I
...
0
200
I
I
I
:1.
I
I
5
' '
,,
t
,' I
I
I
I
I
0
I
I
I
I
'
\
t,' t•
.·!\~ ...I
I
,
100
I
I
0
10
20
30
.,CD
3
QJ
:;::
CD
"0
I
10
0
::J
\
I
Q_
IJ)
'
J
l
I I
I I
I I
)....
QJ
J
J
II
c
"0
I
I
II
20
t500
40
Fraction number
Phosphoserine-pH 7.0
p-Nitrophenyl phosphate-pH 9.0--Protein--·------------~-------------~
Figure 17
SER{JBH-5)
Q.A.E.-SEPHADEX COLUMN CHROMATOGRAPHY \<liTH A 0 TO
0.3~1
NaCl LINEAR GRADIENT AT pH 7.0
Q.A.E. -Sephadex columns\'lere developed with a 0 to 0.3t4 NaCl
linear gradient in O.lM Tris at pH 7.0.
5 mM MgC1 2 was added to the
Tris buffer used in these columns. Separate undialyzed samples from
each of the 45 fractions collected were assayed with 6.6 x 10- 3 M
phosphoserine at pH 7.0 or with 5.25 x 10- 3M p-nitrophenyl phosphate
at pH 8.5.
Protein content of each fraction \'las determined by reading the
optical density at 280 nm and 260 nm.
The figure represents both ser{JBM-5) columns profiles.
58
25
r--...
I
I
(1)
' ''
I '
~
''
'
I'
'\
,,~.
(1)
a.
••••
I•
15
o I
'
I I
••
::l
'0
I
I
'I
0
!...
.I
a.
0...
Ill
•
.
I
I
•
i'
(1)
I
:1.
.....
1=
<.0
400
'\
.
I
'. I
5
'•
I
I
I
I
I
I
I
I
I
300
\
''
200
\
\
\
100
0
0
20
30
40
Fraction number
Phosphoserine-pH
p-Ni trophenyl
-o
tl)
\
3
'
I
I
0
-to
::l
'
.
'II
"0
\
tl)
\
\
0
0
I
I
'
::
C'l
I
I
I
I
10
500
r
r
'...•
!...
(1)
(.)
600
J
I
20
c
E
'0
I
7.0---------------
phosphate-pH 9 . 0 - - -
Protein-····------------·--------·-
59
samples which were assayed at several selected intervening pH values
were included in the profile, two separate peaks for phosphoserine
hydrolysis appeared for wild type (Figure 18).
Phosphoserine
phosphatase in the partially purified wild type extract exhibits
optimal activity at pH 7.4 and is distinct from the alkaline
phosphatase activity with phosphoserine as substrate at pH 8.0.
The
pH profi 1e for ser(JBtvl-5), however, remained very simi 1ar to that
profile obtained with the crude extract, and indicates decreased
phosphoserine phosphatase activity at pH 7.0 to 7.5 in the mutant
compared to the wi 1 d type.
Small samples of pooled wild type fractions were assayed with
phosphoserine at pH 7.0 and p-nitrophenyl phosphate at pH 9.0 in the
presence of selected compounds (Tables 1 and·2).
Most of these
compounds affected the enzymatic activity of the column fractions and
the crude extract similarly, with the notable exception of those
listed in Table 4.
Data in Table 5 briefly summarize the increased specific
activity obtained vlith each purification procedure of both
and
ser(JB~1-5)
mycelia 1 extracts.
\'Iii
d type
Because enzymes re sponsi b1e for
both phosphoserine hydrolysis at·pH 7.0 and alkaline phosphatase
activity co-elute from the Sephadex columns, it is not yet known
exactly how much of the phosphoserine hydrolysis is due to alkaline
phosphatase at pH 7.0 in the mutant strain.
Clearly phosphoserine
phosphatase must be further purified before further characterization
of this enzyme can be accomplished.
60
Figure 18
EFFECT OF pH ON THE HYDROLYSIS OF PHOSPHOSERINE
BY
ENZY~1ES
PRESENT IN POOLED COLUtvlN FRACTIONS
Fourteen test tubes, each containing the reaction mixture (6.6
x 10- 3 M phosphoserine, 16.6 x 10- 3 M MgC1 2 , and 83.5 x 10- 2 M Tris
buffer adjusted to the desired pH), were equilibrated to 25°C before
the addition of samples from pooled column fractions.
The reaction
was terminated after 30 minutes by the addition of the ascorbicmolybdate solution used to assay for the presence of inorganic
phosphates.
Each point represents the average of duplicate samples.
61
•------• Wild type
b.-········~ S er
( J BM- 5 l
30
....:::>
<1.1
c
E'
"<1.1
0.
b.
•
••
••
••
• ••
••
••
••
•e
••
•
25
c
....0<1.1
"0.
/
20
0>
"<1.1
0.
"0
<1.1
u
15
:::>
"0
0
"0.
\f
••
••
••
••
••
••
••
••
••
••
••
•
••
•
.A
••
~
••
••
•••
•
•••
••
•••
•
•••
••
•••
~:s.··b.
1Q-
0
:E
::1
('.1
I
0
...-
.
•
••
••
•
•••
••
•••
••
•
a..
1/)
(1)
••
••
••
•
I
E
5
'\•
A
••
~
••
0
A
6.5
7.0
7.5
pH
8.0
8.5
9.0
62
TABLE 4
RELATIVE RATE OF HYDROLYSIS OF WILD TYPE POOLED COLUMN
FRACTIONS COMPARED TO CRUDE EXTRACTS
A.
Phosphoserine as the substrate at pH 7.0
MgC1 2pl us
CdC1 2
HgC1 2
NaF
L-serine
B.
Pooled
Fractions
16.6 uMoles
1.00
1.00
2.5 uMoles
0.28
0.87
0.39·
0.80
0.11
0.57
0.17
0.46
*
10.0 mMoles
10.0 mMoles
p-Nitrophenyl phosphate as the substrate at pH 8.5-9.0
MgC1 2
t4gC1 2 plus
BeC1 2
ZnS04
*
Crude
Extracts
Concentration
Compounds
16.6 uMoles
0.005 uMoles
0.50 uMol es
1.00
1.00
0.00
0.90
0.40
0.60
Slightly insoluble in water.
Relative rates are decimal fractions of 1.00
63
TABLE 5
SPECIFIC ACTIVITY* AND (PURIFICATION FACTOR)
Wild Type, C-102, 15300-4-2A
Substrate and pH assayed
P-ser + Be++
p-NP
Crude Extract
3.18
(1 .00}
6.70
(l
70% (NH 4 )2so4 :j:
Peak Column
Fraction
5.14
{1. 60)
8.98
( 1 •30)
34.5
(10.9)
80.0
(11 .9)
Mutant,
.00)
C-102, 15300-4-2A-ser(JBM-5)
Crude Extract
0.47
(1
.00)
5.64
( 1 •00)
70% (NH 4}2so 4
Peak Column
Fraction
1.74
(3.20)
9.14
(1 • 60)
6.25
(13. 3}
52.8
{9. 40)
* Specific activity is expressed as 10- 2 ut,1oles of inorganic
3
phosphate produced upon the hydroly~is of either 6.6 X 10-M
phosphoserine (P-ser} or 10.5 X 10- M p-nitrophenyl phosphate
(p-NP) by one milligram of protein per minute of incubation
at 25°C.
t Precipitation, resulting from fractionation in 70% saturated
ammonium sulfate, resuspended in 0.1 Tris buffer and dialyzed
overnight against 0.1 Tris buffer at pH 7.0 before being used.
P-ser = Phosphoserine,
p-NP = p nitrophenyl phosphate
DISCUSS ION
The final step in the phosphorylated biosynthetic pathway of
serine is the dephosphorylation of phosphoserine.
In many
organisms, this is accomplished by a specific phosphoserine
phosphatase (Ichihara and Greenberg, 1957).
However, in vitro, the
dephosphorylation of phospho serine can also be catalyzed by nonspecific phosphomonoesterases of which there are several in
Neurospora (Hochberg and Sargent, 1973).
Acid phosphatases from Neurospora exhibit optimal activity at
acidic pH's (pH ?-6) and demonstrate no requirement ·for the cation
Mg++ nor are they inhibited by zn++, Be++, Co++ or Cu++ (Kuo and
Blumenthal, 1961 a).
Neurospora acid phosphatases are simi1 ar to
acid phosphatases isolated from other organisms in these respects
(Hollander, 1971).
Alkaline phosphatases, in contrast, exhibit optimal activity
at alkaline pH values (pH 8.0 to 10).
These enzymes require t1g++
for optimal activity, are extremely sensitive to Be++ inhibition
and will, in most cases, hydrolyze phosphoserine,
~­
glycerophosphate and p-nitrophenyl phosphate (Kuo and Blumenthal,
196lb; Davis and Lees, 1972).
The Neurospora alkaline phosphatases are also similar to
alkaline phosphatases found in other organisms (Fernley, 1971; Reid
94
65
and Wi 1son, 1971) •
The extracts obtained from both wild type and ser(JBf·-1-5)
mycelia contain at least one alkaline phosphatase.
This enzyme,
isolated from both strains, dephosphorylates phosphoserine,
glyce~ophosphate
~-
and p-nitrophenyl phosphate at alkaline pH's, it
is ~1g++ dependent and it is sensitive to inhibition by Be++, co++,
++ '
++
Hg, , KCN and Zn • Thus the enzyme conforms quite well to the
alkaline phosphatase isolated from Neurospora by Kuo and Blumenthal
(196lb) and Davis and Lees (1969, 1972, 1973).
Unlike alkaline phosphatases isolated from other organisms
(Fernley, 1971; Reid and
l~ilson,
phosphatase is not activated
by
1971),
Neurospol~a
alkaline
the addition of zn++ to the
reaction mixture (Davis and Lees, 1969) and this investigator did
not observe any zn++ dependence in the enzymes present in either
wild type or mutant extracts.
The possibility remains that the
enzymes are zn++ dependent, but that sufficient zn++ is bound
tightly to the enzyme to permit optimal activity.
Chelating
experiments were not performed.
Since there is evidence that alkaline phosphatase obtained
from Neurospora crassa· will hydrolyze phosphoserine at alkaline
pH's (Davis and Lees, 1969) and is sensitive to inhibition by Be++
(Kuo and Blumenthal, l96lb; Davis and Lees, 1969), the maximal
activity at pH 8.5 from both wild type and mutant extracts is
thought to be due to alkaline phosphatase activity.
66
The presence of alkaline phosphatase in these extracts may
explain the peaks obtained at pH 8.5 to 9.0 in the pH profiles.
The
peak or shoulder at pH 7.0 to 7.5 in the \'lild type profile (which
is significantly decreased or absent in the profile from the mutant
ser(JB~1-5))appears
activity.
to be due to specific phosphoserine phosphatase
The results presented here indicate that the activity at
neutral pH may represent activity of an enzyme different from
alkaline phosphatase, but the enzyme could not be separated
completely from the alkaline phosphatase by the methods attempted
here.
The data from the ammonium sulfate experiments, in which
phosphoserine phosphatase activity was enhanced in the pellet while
most of the alkaline phosphatase activity remained in the
supernatant when 40% saturated ammonium sulfate was used, shows
promise as a method of separating the two enzymes.
Partial
purification was achieved with column chromatography on Q.A.E.Sephadex.
Phosphoserine phosphatase from other organisms is specific for
phosphoserine and rarely hydrolyzes other substrates.
Its optimal
pH is in the neutral range, pH 6.0 to 7.5, and it is dependent upon
Mg++ for maximal activity (Barman, 1969).
Data presented here show
that without the addition of Mg++, the activity exhibited by
Neurospora wild type enzyme at pH 7.0 is 32% of the activity in the
presence of 16.6 uMoles MgCl 0L •
The production of phosphate was greatly reduced at pH 7.0 when
67
~-glycerophosphate
and p-nitrophenyl phosphate were the substrates.
These data are in agreement with data reported by both Kuo and
Blumental (196lb) and Davis and Lees (1973) with respect to the
reduced alkaline phosphatase activity. in Neurospora at neutral pH.
Beryllium, a kno\'m inhibitor of alkaline phosphatase, reduces
.,
both wild type and
ser{JBt~-5)
enzymatic activity at pH 8.0 to 9.0
when assayed with phosphoserine, £-glycerophosphate and pnitrophenyl phosphate.
Dephosphorylation of phosphoserine is
reduced by an insignificant 3% and 9% when extracts from the wild
type and ser{JBf-,1-5), respectively, were assayed at pH 7. 0 in the
presence Of Be ++.
Beryllium is not known to inhibit phosphoserine
phosphatase activ.i ty and pro vi des a method to di sti ngui sh between
the two enzymatic activities.
Because Be++ does inhibit alkaline
phosphatase selectively, 97% of the dephosphorylation of
phosphoserine in the wild type and 90% in the mutant extract at pH
7.0 is considered to be the result of phosphoserine phosphatase.
However, at pH 8.5, the effect of Be++ on the hydrolysis of
phosphoserine is quite different.
Here, 42% of the phosphoserine
hydrolysis is considered to be due to phosphoserine phosphatase in
the wild type while 4% is thought to be due to that enzyme present
in the mutant.
42% and 4% represent the activity which remained
when phosphoserine vJas assayed at pH 8.5 in the presence of Be++.
Results obtai ned from the experiments performed 'r'Ji th
inhibitors indicate that the enzyme active at pH 7.0 is sensitive
68
to inhibition by Ca++, Cd++, Co++, Mn++ and Zn++.
Phosphoserine
phosphatase isolated from yeast is also inhibited by the presence of
++
these ions (Schramm, 1958). In contrast, KCN and Be , known
inhibitors of Neurospora alkaline phosphatase (Kuo and Blumental,
196lb; Davis and Lees, 1969) had little effect on the enzymatic
activity at pH 7.0. Inactivation of the alkaline phosphatase by KCN
and Hg ++ suggests the presence of thiol groups or disulfide bonds
within the complex of the enzyme ( Lehni nger, 1978), yet these two
compounds exhibited no effect on the enzymatic activity at pH 7.0
with phosphoserine.
L-serine has been used as an inhibitor of phosphoserine by many
investigators.
In wild type crude extract, phosphoserine
phosphatase activity was inhibited to the same extent by L-serine as
the alkaline phosphatase activity, approximately 20%.
However, when
the assay was performed on the· pooled column fractions from wild
type, L-serine inhibition of alkaline phosphatase remained the same,
while the inhibition of phosphoserine phosphatase was increased to
54%.
These figures represent the relative rate of hydrolysis as
compared to hydrolysis of the substrates in the absence of the
inhibitor.
Throughout this study, attempts have been made to separate the
alkaline phosphatase from the phosphoserine phosphatase.
saturated ammonium sulfate effort looks most promising.
The purity of both enzymes was increased by column
The
69
chromatography with the removal of 70% to 80% of non-specific
protein from the crude extracts.
The purification factors for both
wild type and ser(JBM-5) alkaline phosphatase were similar, 11.9 and
9.4 respectively.
The purification factor for phosphoserine
phosphatase in the wild type was 10.9 and was similar to the 11.9
for its alkaline phosphatase.
However, the purification factor for
the ser(JBM-5) phosphoserine phosphatase was 13.3 and was greater
than ·that for its alkaline phosphatase as well as the phosphoserine
phosphatase in the wild type.
Evidence has· been presented here to suggest that normal
phosphoserine phosphatase activity is present in the wild type
strain.
Since ser(JBM-5) possesses reduced phosphoserine
phosphatase activity, the question arises, is the genetic lesion of
ser(JBM-5) a defect in a regulator gene or in the structural gene
for phosphoserine phosphatase? A defective regulator gene would
reduce the amount of normal phosphoserine phosphatase produced but
as the purification of the enzyme increased, one would observe a
convergence of the wild type and mutant specific activities.
Ser(JBM-5) is allelic to ser-3 studied by Dodson (1980) and she
reported that the specific activity of ser-3 phosphoserine
phosphatase was increased sequentially from one experiment to
another.
She postulated that this \'/as due to increased purity of
the enzyme as her techniques improved.
As the purity of the enzyme
in the crude extracts was increased, the specific activity of the
70
mutant ser(JBM-5) phosphoserine phosphatase became more like that of
the wild type or the specific activities of the two enzymes became
more convergent.
This observation is consistent with the idea that
the enzyme present in the mutant is normal but is produced in
reduced quantity.
Another possibility is that the defect in
structural gene for phosphoserine phosphatase.
ser(JBt~-5)
is in the
If this were true,
one should observe no convergence of the specific activities of the
wild type and mutant strains as the purity of the preparations was
increased.
The specific activity of an altered enzyme (defective in
its hydrolytic capability) would be less than that of the normal or
wild type enzyme even if the two enzymes were used in their pure
form.
Therefore, the specific activities would not converge but
would remain divergent as the purity of the enzyme increased.
Conclusions based on the work presented here are tentative and
greater purification of the phosphoserine phosphatase must be
achieved before the answer to the true nature of the genetic lesion
can be found.
In summary, the effect of pH on the hydrolysis of phosphoserine
by enzymes in the .wild type is different than the effect on the
enzymes found in the mutant ser(JBM-5).
.§l lacks
The pH profile of
ser(JB~1-
a shoulder at pH 7.0 which is present in the \'/ild type,
while the ·activity from pH 7.5 to pH 9.0 has the characteristics of
71
alkaline phosphatase and is similar in both mutant and wild type.
Hydrolytic activity at pH 7.0 is not inhibited by Be++ or KCN,
but is inhibited by NaF and L-serine which argues that this
activity is due to a specific phosphoserine phosphatase.
Saturated ammonium sulfate fractionation is consistent with
two separate specificities.
·Q.A.E.-Sephadex chromatography increased the purity of the
preparations but did not resolve the two enzymatic specificities.
As the purification increased, the specific activity of
ser(JBM-5) phosphoserine phosphatase became more convergent with
the specific activity exhibited the wild type, suggesting that the
mutant may produce less of a normal form of the enzyme rather than
normal amounts of a defective enzyme.
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