CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
A PHOSPHOSERIN3 PHOSPHATASE MUTANT OF
NEUROSPORA CRASSA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Laurence King Chuck
June, 1980
The Thesis of Laurence King Chuck is approved:
California State University, Northridge
ii
For my Parents
iii
ACK1JJI.JLEDGMEHT
The author is greatly indebted to Joyce B. Maxwell for her help,
suggestions, and encouragement.
The author is further indebted to
Sandra Jewett for her technical help, suggestions, and answers to our
technical and theoretical questions.
The author wishes to thank the members of
t~e
committee- Kenneth
C. Jones and Mary Lee Barber- for their help and suggestions in the
com~letion
of this thesis.
The author is indebted to Lynne Crosby and Peter Ringold whose
preliminary works this thesis was based on.
iv
TABLE OF CONTENTS
page
ACKNOi:JLEDffi1ENT. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • i v
LIST OF FIGUR.ES. • • • • • • • • • • • • • • • • . • . • • • . • • • • • • • . • . • • • • • • • • • . . • • • Vi
LIST OF TABLES. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • vii
ABSTRACT. • • • • • • • • • • • • • • . • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • viii
I.
II.
IN'rR.ODUCTIO~J .
••.••.•.•••••.•.•••..••.••.••••••••..•.••
1
ffi TE..'R.~S Al\ID
li~TIIODS • ...••...•.•.......••....••......
5
J'...
Strai..lls . ......•....•.......................•.•........
Chemicals • ••.....•............•.••.••••..•••....•..••.•
Maintenance and Growth of Neurospora Cultures •••••••••
Extraction of Soluble Enzymes ••.•...•.•••••
Calculation of Specific Activity ••
III.
RES T..JI.J TS • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •
15
Experilnent 1 • .............................•.•....•....•
Experiment 2 . ..................................•......
IV.
V.
DISCUSSION •••••••••••••••••••
........................ . 25
BIBLIOG.t-rtAPHY • •••••••••••••••••••••••••••••••••••••••• ~ 30
v
LIST OF
FI~URES
page
Figure
l.
2.
3.
h.
5.
6.
The three postulated pathways of serine bioS';nthesis
in lJeu.rosuora crassa.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . •
2
An example of an enzyme activity graph showing
specific activities of phosphoserine phosphatase
from the mutant and the prototrophic strain •••••••••••••
11
An exa'llple of a phosphate production graph shmving
phosphoserine phosphatase activity in a salted +(min)
extract. . . . . . . . . . . . . . . . . . . . . . . . . . . ............. -:-. . . . . . . .
13
Phosphoserine phosphatase activities of the mutant
and the prototrophic strain •••••••••••••••••••••••••••••
17
Phosphoserine phosphatase activities of (a) salted
extracts, (b) dialyzed salted extracts, and (c)
dialyzed crude extracts •••••...•••.•.••••.••..•..•....••
20
(a) Comparison of the phosphoserine phosphatase
activity between dialyzed crude ser(g+f), dialyzed
salted ~(g+f), and salted ~(g+f) of figure- Sa-c •••••
22
vi
LIST of TABLES
Table
1.
page
Phosphoserine phosphatase activity in experiment 1
and 2.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . •
,yti
16
ABSTRACT
A PHOSPHOSERINE PHOSPHATASE MUTANT OF
NEUROSPORA CRASSA
by
Laurence King Chuck
Master of Science in Biology
Three pathways of serine biosynthesis are postulated to exist in
Neurospora crassa.
The three pathways are the phosphorylated, the
nonphosphorylated, and the glyoxylate-serine pathway.
Evidence now
exists that the phosphorylated pathway may be the major pathway of
serine biosynthesis in Neurospora crassa.
ser(JBH
A serine requiring mutant,
5), was found to have a lower specific activity of extractable
phosphoserine phosphatase, one of the enzymes in the phosphorylated
pathway, than its isogenic wildtype.
requiring mutant, ser( JBr1
In this investigation, the serine
5), and its isogenic wild-type strain of
Neurospora crassa were tested for phosphoserine phosphatase activity.
The Ames' assay procedure and two modifications thereof were used to
assay the extracts for phosphatase activity.
The mutant had lower
phosphoserine phosphatase activity per gm extract than the wild-type,
suggesting that the phosphorylated pathway is the major pathway of
serine biosynthesis.
viii
INTRODUCTION
Three pathways of serine biosynthesis are postulated to.exist in
Neurosnora crassa.
The three pathways are the phosphorylated pathway,
the nonphosphorylated pathway and the glyoxylate-serine pathway.
The
phosphorylated and nonphosphorylated pathways v1ere studied by Sojka
and Garner (1967).
(19)1).
The glyoxylate-serine pathway was studied by Wright
In the phosphorylated pathway, serine is synthesized by a
pathv-ray containing three enzymes: the first, phosphoglyceric acid dehydrogenase, converts 3-phosphoglyceric acid into 3-phosphohydroxypyruvic acid; the second, phosphoserine transaminase,
transa~inates
3-phosphohydroxypyruvic acid to 3-phos?hoserine; and the third, phosphoserine phosphatase, dephosnhorylates phosphoserine to serine.
In
the nonphosphorylated pathway, serine is synthesized by a pathway containing two enzymes: the first, glyceric acid dehydrogenase, converts
glyceric acid to hydroxypyruvie acid; and the second, serine transaminase, transaminates hydroxypyruvic acid into serine.
In the glyoxy-
late-serine pathway, serine is synthesized by the enzyme serine hydroxymethyltransferase which converts glycine to serine by the addition of
a single carbon fragment (5-10 methylenetetrahydrofolate) to glycine.
Glycine is synthesized from glyoxylate.
The three pathways are dia-
grammed in figure 1.
The major pathway by which serine· is synthesized in Neurospora
crassa had not been elucidated before this investigation.
exists that in such diverse organisms as
1963),
£..
!·
Evidence
coli (Umbarger et al.,
ty:phirnuriurn (Umbarger et al., 1963), ~· subtilis (Ponce-De-
1
2
glucose
GLYCOLYSIS
l
-Pi
H2o3POCH 2CH-COOH - - - - - - - - - - - HOCH 2CH-COOH
OH
OH
3-phosphoglyceric acid
glyceric acid
PHOSPHOGLYCERIC ACID
DEHYDROGENASE
GLYCERIC ACID
DEHYDROGENASE
1
1
HOCH 2-C-COOH
H203POCH2C-COOH
0
0
hydroxypyruvic acid
3-phosphohydroxypyruvic acid
PHOSPHOSERINE
TRANSAMINASE
SERINE
TRANSAHINASE
!
H203POCH2CH-COOH
(2)
NH2
3-phosphoserine
PHOSPHOSERINE
PHOSPHATASE
HOCH 2CH-COOH
NH2
serine
SERINE
HYDROXYHETHYLTRANSFERASE
5-10 methylenetetrahydrofolate
r
glyoxylate
GLYOXYLA TE SHUNT
glycine
T
TRICARBOXYLIC ACID
CYCLE
Figure 1. The three postulated pathways of serine biosynthesis in
Neurospora crassa. (1) phosphorylated pathway (2) nonphosphorylated pathway . (3) glyoxylate-serine pathway
3
Leon and Pizer, 1972),
~·
~·
lysodeikticus (Nelson and Naylor, 1971),
desulfuricans (Germano and Anderson, 1969), ~· influenzae (Pizer
et al., 1969), and higher plants (Hanford and Davies, 1958) the phosphorylated pathway is the major pathway of serine biosynthesis.
The
nonphosphorylated pathway can be the major contributor of serine in
certain animal tissues (Ichihara and Greenberg, 1957; Walsh and Sallac~
1966).
Of the two pathways studied by Sojka and Garner, the phospho-
rylated pathway was judged to be the major pathway of serine biosynthesis in Neurosnora crassa because the enzymes in the phosphorylated
pathway had higher specific activities in cell extracts than the
enzymes in the nonphosphorylated pathway.
On the other hand, the glyoxylate-serine pathway can be the major
pathway of serine biosynthesis in some animal and plant tissues (1\Teissbach and Sprinson, 1953; Weinhouse and Friedmann, 1951; Tolbert and
Cohan, 1953).
In Saccharomyces cerevisiae, when grown on acetate, the
glyoxylate-serine pathway is the major pathvray of serine biosynthesis
(Gilvarg and Bloch, 1951; Ulane and Ogur, 1972).
Similarly, evidence
exists that under particular growth conditions, the
glyo~rlate-serine
pathway may be an alternative pathwav by which serine is synthesized
in Neurosnora.
Wright (1951) found that a serine-glycine mutant of
Neurospora growing on a medium with glycerol as the sole carbon source
grew better when supplemented with glyoxylic acid or glycine than when
fed serine.
Wright suggested that glyoxylic acid is converted to
glycine and then to serine.
The glyoxylate-serine pathway may not be
the major pathway by which serine is synthesized in Neurospora grown
on sucrose as a carbon source as no presently available serine requir-
4
ing mutant grows better on sucrose medium supplemented with glycine
than on medium supplemented with serine (Maxwell, 1970).
Wright's
mutant is no longer available for study.
To demonstrate that a biosynthetic pathway exists in an organism,
a series of auxotrophic mutants are examined to find the blocked steps
in the pathway.
In Neurosnora crassa there exists a series of serine
requiring mutants, none of which had been linked previously to a particular enzyme of any of the three serine biosynthetic pathways.
Neurospora, identification of a mutant with its particular
In
enz}~e
was
difficult because most nonisogenic strains of Neurosnora have a wide
range of activities for a particular enzyme (Kline, 1973), which meant
that a comparison of a nonisogenic wild-type strain and a mutant for a
particular enzyme could not be done as the wild-type strain from which
the mutant was derived may have had a different enzyme activity than
the wild-type being used for comparison.
Findings by other members of this research group suggested that
the phosphoserine phosphatase of the phosphorylated pathway might be
the enzyme that was blocked in the serine requiring mutant ser( JBH
(unpublished data).
hypothesis that an
5)
The work described in this thesis confirms the
~xtract
of ser(JBM
5) does have significantly lower
phosphoserine phosphatase specific activity than an extract of its
isogenic wild-type.
This result indicates that the serine requirement
of ser(JBM 5) is due to this deficiencv and hence the phosphorylated
pathway is probably the major pathway of serine biosynthesis in Neurospora crassa under the conditions investigated in this study.
MATERIALS AND 11ETHODS
Strains
Two isogenic strains of Neurospora crassa were used in these
studies.
Hary B. }iitchell, formerly of the Division of Biology of the
California Institute of Technology, provided the nutritionally wildtype strain, Cl02-15300-4-2A, hereafter to be referred to as wild-type.
Joyce B. Maxwell and Paul West isolated from this
wild-tJ~e
strain a
serine requiring mutant, Cl02-15300-4-2A ser(JBM 5), hereafter to be
referred to as ser(JBM 5).
Chemicals
Reagent grade materials were used throughout the studies with the
exception that
~ractical
grade Tris, 2-Amino-2-(hydroxymethyl) 1,3-pro-
panediol, was used to prepare certain buffer solutions.
The saturated
ammonium sulfate solution was made by dissolving at room temperature an
excess of ammonium sulfate crystals (more than ?Og/lOOml) in 0.1 M Tris
HCl pH 7.5 buffer.
Company.
was
L-phosphoserine was obtained from Sigma Chemical
Fresh phosphoserine solutions were made each time the assay
~erformed
and were neutralized with NaOH to a pH range of 6.8 to
7.0.
Maintenance and Growth of Neurospora Cultures
The wild-type was maintained on agar slants of Vogel's minimal
medium N (Vogel, 1956).
Ser(JBM 5) was maintained on minimal slants
supplemented with 0.4 mg/rnl serine.
Stock cultures were grown in the
daylight at room temperature for at least four days before use to allow
the Neurospora sufficient time to conidiate.
5
A dense conidial suspen-
6
sion in sterile distilled water was prepared from each stock culture.
For each set of cultures, 0.2 ml
sa~~les
of the suspension were used to
inoculate 20 ml of liquid Vogel's minimal medium N and 2% (w/v) sucrose
in each 125 ml Erlenmeyer flask.
C in an incubator.
Cultures were grow-m stationary at 25°
The medium for ser(JBM 5) and half the medium used
to grow the wild-type were supulemented with lOmH glycine and lOm.Til
sodium formate.
Possible feedback inhibition of phosphatase synthesis
by serine was eliminated by using serine hydroxymethyltransferase of
the glyoxylate-serine pathway to synthesize serine.
The various cul-
tures and growth conditions are coded as follows:
+(min)
~(g+f)
~(g+f)
wild-type grown on Vogel's minimal medium N
wild-type grown on Vogel's minimal medium N
supplemented with glycine and formate
ser(JBM 5) grown on Vogel's minimal medium N
supplemented with glycine and formate
Extraction of Soluble Enzymes
The mycelial pads were harvested on a Buchner funnel with suction.
The pads were rinsed briefly with distilled water and blotted dry be~reen
sheets of paper towels.
autolysis.
The ?ads were kept on ice to reduce
Afterward, the pads were weighed and ground with washed
sea sand in an ice cold mortar and pestle.
Phosphoserine phosphatase and other soluble proteins were extracted from ground mycelium with ten volumes of 0.1 M Tris-HCl pH 7.5 buffer (hereafter to be referred to as Tris buffer).
as 1 ml/ 1 g of mycelial pad.
A volume is defined
The crude extracts were centrifuged at
11000 g for twenty minutes in a Sorvall RC-5B refrigerated SS centrifuge using a SA-600 rotor.
The protein in the supernatant was precipi-
tated by adding seven parts 100% saturated ammonium sulfate solution to
7
three parts extract, giving a final solution that was seventy percent
saturated with salt.
The use of liquid ammonium sulfate was easier
and less time consuming than earlier procedures using solid ammonium
sulfate.
One disadvantage in using solid ammonium sulfate is that
frothing of the extract frequently occurs when the salt is dissolved
by stirring into the extract.
ivit3r by denaturation.
Frothing may cause loss of enz}rme act-
The seventy per cent saturated solution was
left on ice for ten minutes and then centrifuged at 11000 g for twenty
minutes.
The supernatant was decanted and discarded.
The top of the
pellet was rinsed with one ml of cold Tris buffer and resuspended in
three volumes of Tris buffer.
The s-olution was then precipitated with
seven volumes of 100% saturated ammonium sulfate solution, left on ice,
centrifuged, rinsed, and resuspended in fifteen volumes of Tris buffer.
Extracts prepared in this way will be referred to as salted extracts.
Approximately three ml samples of extract of each culture,
~(g+f),
~(min),
and ser(g+f) were dialyzed in one fourth inch diameter, 12000
dalton pore size dialysis tubing against Tris buffer.
Dr. Sandra
Jewett supplied the dialysis tubing, which was prepared by boiling in
1% sodium carbonate and 1% EDTA and stored in So% ethanol before use.
The three samples in separate dialysis bags were placed in a ope liter
Erlenmeyer flask containing one liter of Tris buffer.
The buffer was
constantly stirred with a magnetic stirrer in a cold room. Two changes
of buffer were made over a period of 48 hours.
The first change of
buffer occurred after twenty four hours, and the second change five to
six hours later.
Extracts prepared in this way will be referred to
as dialyzed salted extracts.
8
In a later experiment, five volumes of Tris buffer instead of ten
volumes were used to extract the soluble proteins.
After the crude
extracts were centrifuged at 11000 g for twenty minutes, two fifths of
the supernatant was taken from the top half of the centrifuge tube.
this supernatant an equal volume of Tris buffer was added.
samples were taken for dialysis as outlined previously.
To
Three ml
Extracts pre-
pared in this way will be referred to as dialyzed crude extracts.
To
the remaining supernatant, 100% saturated ammonium sulfate solution
was added to give seventy per cent saturation.
The extracts were
treated thereafter as described previously for the salted and dialyzed
salted extracts with the exception that the final pellet was resuspended
in ten volumes of Tris buffer rather than in fifteen volumes.
Generally, the extracts were frozen after being
thawed just before use.
~de
and were
In an earlier experiment, it was found that
freezing did not affect the extracts appreciably-as the specific activity of the frozen extract compared to the same extract refrigerated
showed no significant difference.
A problem inherent in the use of
refrigerated extracts was that the phosphate background increased with
time, whereas the initial phosphate concentration in the frozen
extracts was essentially zero.
Enzyme Assay
Phosphoserine phosphatase was assayed by determining the amount of
inorganic phosphate formed from phosphoserine in the presence of enzyme
as described by Ames (1966).
of Tris-HCl pH
7.5
buffer,
SO
The reaction mixture contained 200 umoles
umoles of MgC1 , 20 umoles of DL-phospho2
serine and approximately 0.1 mg of protein in a final volume of three
9
ml.
The reaction mixture and the extract were incubated separately at
2)°C before the reaction was begun by the addition of the extract.
The
reaction was stopped by transferring 0.7 m1 of the reaction mixture to
2.3 ml phosphatase mix (the mix contains one part 10% ascorbic acid and
six parts 0.42 per cent ammonium molybdate.4H 2o in lN H2so4). Phosphate
from the phosphatase reaction produces a blue colored complex with
molybdate in the presence of ascorbic acid.
Color is developed by in-
cubation of the mixture for twenty minutes at 45°C and the absorbance
at 815
~,
is determined in a Perkin-Elmer Coleman 124 Double Beam
Spectrophotometer.
Two modifications were made of this assay.
First, 20 umoles of
1-phosphoserine were used in place of the D1-phosphoserine.
Both phos-
sphoserine solutions, 1 and D1, were neutralized with NaOH to pH 6.8
to 7.0.
The phosphoserine solution was neutralized in order for the
enzyme reaction to occur at a physiological pH.
In the second modif-
ication, the enzyme reaction was stopped by removing 0.9 m1 samples of
the reaction mixture and transferring the mixture to test tubes containing 0.1 m1 of fifteen per cent trichloroacetic acid.
This treat-
ment removes soluble proteins which can associate with the blue colored complex and precipitate from the solution.
The trichloroacetic acid
treated mixture was centrifuged in a clinical centrifuge for ten minutes to pellet the precipitated proteins.
A 0.7 ml sample of supernat-
ant from the trichloroacetic acid treated mixture was then added to 2.3
ml of phosphatase mix.
as before.
Color was developed and absorbance determined
Protein concentration was measured by the Biuret reaction
as described by Gornall (1949).
' .
10
Calculation of Specific Activity
The specific activity of phosphoserine phosphatase in an extract
was determined from twelve to fifteen values of the rate of phosphate
production for a particular extract.
Alternatively, the specific acti-
vity can be determined from the slope of the change in absorbance per
minute versus milligram of protein as seen in figure 2.
Values for
the change in absorbance are calculated from the slope of absorbance
(phosphate production) versus time of reaction after the addition of a
given protein concentration as seen in
fi~e
3.
Generally in this
study, the rate of phosphate production is determined for four to five
different protein concentrations of a given extract at three time intervals.
'!he four to five rates are then used to determine the
specific activity of a particular extract.
~
Figure 2.
An example of an enzyme activity graph showing the specific activities of salted
-+(min)
(•--•), salted +(g+f) (•--•), salted ser(g+f) ( • - • ) , dialyzed salted +(min)
(.o.
b), dialyzed salted ~(g+f) (o--o), and dialyzed salted ~(g+f) (o
-
slope of each line is the
.
-
s~ecific
activity of the extract.
-
o).
The
Each point represents the rate
of phosphate production at a particular protein concentration.
The reaction mixture contained
200 umoles of Tris-HCl pH 7.5, 50 umoles of MgC1 2, 20 umoles of neutralized phosphoserine
and approximately 0.05 to 0.9 mg of protein.
the extract.
The reaction 1-1as started by the addition of
0.7 ml aliquots of this mixture were removed and assayed for inorganic phosphate.
7
~(g+
dialyzed salted
6
~
C!
5
0
-.
><
c
4•-
-/
//
•
/
·-e
.......
~
0
c:
31·
•• 2
c
.s::.
"' 1
0
~ ~o
f//
dialyzed
salted ~(g+f)
-~
aJ
1
2
3
4
5
6
7
8
9
Mg(X0.1) of Protein
f-1
1'\)
I-'
w
Figure 3.
An example of a phosphate uroduction graph showing phosphoserine phosphatase
activity in a salted ~(min) extract.
pH
?.S, So
umoles of MgCl2, 20 umoles of neutralized phosphoserine and 0.12 to 0.62 mg
of protein in a final volume of 3 ml.
extract.
The reaction mixture contained 200 umoles of Tris-HCl
At
S,
The reaction was started by the addition of the
11, and 16 minutes 0.9 ml samples were removed and placed in test tubes
containing 0.1 m1 of fifteen per cent trichloroacetic acid.
mixture were removed and assayed for inorganic phosphate.
0.7 m1 aliquots of this
From the slopes of each line
the rate of phosphate production for a particular amount of protein is determined.
14
c
,...
-
'0
Q)
~
c:
E
'-'
Q)
E
1-
.,.
.
CD
RESULTS
Exoeri.ment 1
The mycelial
~ad
weight per flask for
~(min)
~(g+f)
growth was about equal to the pad weight of
growth and about twice the pad
growth.
weight of
after two days'
after three days'
~(g+f)
after three days'
These growth patterns were consistent with growth patterns
observed in earlier experiments- after three days of growth the
mycelial
~ad
per flask weighed more than the
~(g+f)
weight of the
~(g+f)
~(mj_n)
pad and uvice the
pad.
Growth on medium supplemented with glycine and formate is not as
good as growth on medium supplemented with serine (Gomes da Costa,
unpublished results).
Growth of wild-type strains on glycine and
formate supplemented medium is not as good as growth on minimal medium,
at least for the first two days of growth; after two days, growth is
the same on the two media.
The S?ecific activities of the dialyzed extracts were different
from the undialyzed extracts for all three cultures.
1 and figure
4,
upon dialysis the specific activity for
decreased by 26%; the specific activity for
and the specific
As seen in table
activit~r
for
~( g+f)
~(min)
~(g+f)
increased by 28%;
decreased by 7%.
Ser( g+f) in
both the dialyzed salted and salted extracts had the lowest specific
activity,
~(g+f)
in both extracts had the highest activity, and
-
~(min)
---
in both extracts had activity closer to +(g+f) than to ser(g+f) spec,
ific activity.
The specific activity of .:!:,(g+f) was about three to
four times the specific activity of ser(g+f) and one and one half to
15
16
Table 1.
Phosphoserine phosphatase activity in experiment 1 and 2.
The specific activity is expressed as the change in optical density
per mg of protein per minute.
~(min)
~(g+f)
~(g+f)
Experiment 1
salted
dialyzed salted
0.120 + 0.008
0.154 ~ 0.012
0.230 + 0.020
0.213 ~ 0.015
o.053
Experiment 2
salted
dialyzed salted
dialyzed crude
0.107 + 0.004
0.162 + 0.009
0.191 ~ 0.022
0.264 + 0.017
0.236 + 0.019
0.195 ~ 0.025
0.081 + 0.005
0.086 + O.Oll
0.102 ~ 0.022
o.072 + o.oo6
~ o.oo6
@
•
t--1
--J
Figure
4.
~
•), salted
Phosphoserine phosphatase activities of salted
~(g+f)
(•-----•), dialyzed salted
~(min)
:_(g+f) (o--u), and dialyzed salted ser(g+f) (o---o).
specific activity of the extract.
o.oS
7.5, SO
(•----•), salted
~(g+f)
(6-----A), dialyzed salted
The slope of each line is the
Each point represents the rate of phosphate production
at a particular protein concentration.
Tris-HCl pH
~(min)
The reaction mixture contained 200 umoles of
umoles of MgC12 , 20 umoles of neutralized phosphoserine and approximately
to 0.8 mg of protein in a final volume of 3 ml.
addition of the extract.
The
react~on
was started by the
At three time intervals 0.9 ml samples were
test tubes containing 0.1 ml of fifteen per cent trichloroacetic acid.
of this mixture were removed and assayed for inorganic phosphate.
removed and placed in
0.1 ml aliquots
salted +(min)
..
salted ser(g+f)
-.
-.
T"",
0
0
-
dialyzed salted
!(
5
•
>(
c
'
dialyzed
salted ~(g+f)
4
·-E
......
. 3
Q
d
·-c:
•cc:n
as
.s:
0
1
1
2
3
4
5
6
7
8
9
Mg(X0.1) of Protein
I-'
(X)
19
two times the specific activity- of -+(min).
Dialysis does not appear to
affect +(g+f) as much as +(min) or ser(g+f); specific activities are
-
-
nearly the same for
tracts.
For
~(g+f) in
~(min),
--
.
both the salted and dialyzed salted ex-
dialysis decreases the specific activity.
Experi.ment _g
The specific activities of the dialyzed extracts and the salted
extracts were similar except where noted otherwise.
As seen in table
1 and figures 5a-c and 6a-c, the specific activity is essentially the
same for dialyzed salted
~(g+f).
~(g+f)
~(g+f),
salted
~(g+f),
and dialyzed crude
On the other hand, different treatments of the
extracts yielded varying results.
dialyzed crude
~(min)
~(min)
Dialyzed salted ~(min) and
have roughly the same values.
Both of these
values are one and a half to two times the value for salted
Dialyzed salted
~(g+f)
and salted
~(g+f)
and
~(min).
have roughly the same value,
and both of these values were higher than dialyzed crude
~(g+f)
by
about 20-30%.
Ser(g+f) in each extract condition- dialyzed salted, salted, and
dialyzed crude-still had the lowest specific activity when compared to
=(min) or =(g+f).
Dialyzed salted ~(g+f) had about one third the
specific activity of dialyzed salted
~(g+f)
specific activity of dialyzed salted
~(min).
one third the specific activity of salted
fourtrnthe specific activity of salted
and about one half. the
Salted
~(g+f)
~(min).
~(g+f)
had about
and about three
Dialyzed crude
~(g+f)
had about one half the specific activity of dialyzed crude ~(g+f) and
about one half the specific activity of dialyzed crude ~(min).
Unlike the earlier experiment, dialysis does not appear to affect
20
Figure
5.
Phosphoserine phosphatase activities of (a) salted extracts,
(b) dialyzed salted extracts, and (c) dialyzed crude extracts.
slope of each line is the specific activitv of the extract.
The
Each point
represents the rate of phosphate production at a particular protein
concentration.
The reaction mixture contained 200 umoles of Tris-HCl
pH 7.5, 50 umoles of MgC1 2 , 20 umoles of neutralized phosphoserine and
approximately 0.05 to
o.S
mg of protein in a final volume of 3 ml.
The reaction was started by the addition of the extract.
At three
time intervals 0.9 rnl samples were removed and placed in test tubes
containing 0.1 ml of fifteen per cent trichloroacetic acid.
aliquots of this
phate.
~ixture
0.7
~~
were removed and assayed for inorganic phos-
21
7
a
c
e....
ci
0
.!:
•...
.;:
"
.s::.
u
2
3
4
5
li
•
7
Mg (X o.tl of Prol•ln
7
6 ·
dialy•od oalted _!(g•r)
dialy&Od"salt.d
b
.!hun)
~
0
0
)(
c
....E
ci
0
=
•...
c:
•
.s::.
u
•
Mg (X 0.11 of Prot• In
dialyud
7
6
~
C!
-
cruel• .!( o:•tl
c
-
5
dialyred
crude nr(g•tl
0
)(
. ,:
4
E
ci 3
0
.!: 2
•...
c:
•u
1:
2
4
s
Mg(XO.I) of Protein
•
7
•
22
Figure 6.
(a) Comparison of the phosphoserine phosphatase activity
between dialyzed crude
~(g+f)
~(g+f),
dialyzed salted
~(g+f),
and salted
of figure 5a-c.
(b) Comparison of the phosphoserine phosphatase activity
be~~een
dialyzed crude ~(g+f), dialyzed salted ~(g+f), and salted ~(g+f) of
figure 5a-c.
(c) Comoarison of the phosphoserine phosphatase activity between
dialyzed crude ~(min), dialyzed salted ~(min), and salted ~(min) of
figure .5a-c.
23
7
6
;;
-
a
5
0
~
c:sa11s'>'~ ....1 t.e·l
m:t•r>
4
.:
...E
ci 3
d
.•
E 2
c:
•
u
I!
1
2
3
4
Mg(X0.1)
5
•
7
6
of Protein
7
b
;;
0
X
c
...E
0 3
d
.•.
E
c:
I!
u
2
7
Nlt.ed
3
·~·~
!<c+t)
4
5
Mf!(XO.I) ol
Proieln
Nltad
7
6
I
!<c•tl
dialJ'Mil cn<~e
!(c•tl
c
6
-....
5
0
~
c
...'E
4
ci 3 d
E
•ao
2 -
c:
•
II!
u
2
3
4
I
Mg(Xo.tl ol Protein
I
7
•
24
.s.ar.(g+.f) as the s-pecific activity for dialyzed salted
salted ser(g+f) were nearly identical.
~(g+f)
On the other hand, results
of the second exoeriment were similar to the first with
~(g+f)
and
~(min)-
~(min)
and had no effect on salted
and
res~ect
to
dialysis increased the soecific activity of salted
~(g+f).
DISCUSSION
This study demonstrates that in rreurospora crassa, the mutant
ser(JBM S) has a decreased specific activity of the extracted enzyme
phosphoserine phosphatase compared to its isogenic
wild~type
progenitor.
This demonstration supports the hypothesis that the phosphorylated
pathway is the major pathway of serine biosynthesis in Neurospora
crassa. The identification of a serine requiring mutant with its biochemical defect is the first such demonstration in Neurospora crassa
for isogenic strains of Neurospora. Preliminary studies by Sojka (1967)
suggested that ser-2 had a defective phosphoserine transaminase.
The
significance of this finding is difficult to determine as the wild-type
and the mutant used in the study had different genetic backgrounds.
Kline (1973) observed that the transaminase activity is quite variable
a"!long the various wild-type strains of Neurospora. Sojka (1967) tried
to reduce this heterogeneity by repeated backcrosses of the serine
mutants to a single wild-type; whether this attempt was successful is
not known.
Ha.xt..rell compared the mutant strain ser-4 with its immediate
progenitor, wild-type ST 74A, and found no differences in the specific
activity of any of the biosynthetic enzymes in either the phosphorylated or non?hosphorylated pathways.
Kline (1973) similarly compared
ser(JBM 4-13) with its parent strain Cl02-lS300-4-2A and found no
enzymic differences that would account for the serine requirement.
Gomes da Costa (manuscript in preparation) repeated Sojka's experiments
and found no difference in transaminase activity between the wild-type
STA 4 and ser-3.
25
26
Evidence exists that suggests that serine may be synthesized by
different pathways in the same organism growing on different carbon
sources.
Wright (1951) found a serine requiring mutant that grew
better on glycine or glyoxylic acid than on serine when grawn on glycerol medium.
Labeling studies by Ma.xtiell ( 1970) and Abelson and Vogel
(1955) have shown that the label of cl4 glycine is incorporated into
serine.
Maxwell (1970) has shown that glyoxylate can be used to satisfy
the serine requirement only i f glycerol is substituted for sucrose as
the carbon source.
Wild-type is very restricted in its growth under
these conditions.
Ulane and Ogur (1972) have shown that in Sacchar-
omyces, the phosphorylated pathway appeared to be the principal biosynthetic pathway to serine during growth on glucose media, while the
glyoxylate-serine pathway was the major pathway
tate.
durin~
growth on ace-
Ulane and Ogur suggested that the glvoxylate-serine uathway was
repressed by growth on glucose.
re"'Jressed enzyme.
Isocitrate lYase is thought to be the
In Neurospora this same enz:vme can be repressed by
growth on sucrose (Sjogren and Romano, 1967).
At the time this study was begun, no serine dependent mutant had
been linked to a particular enzyme defect in either of the two pathways
studied by Sojka, the phosphorylated and nonphosphorylated pathway, or
the pathway studied by Wright, the glyoxylate-serine pathway.
Ser(JBM 5) is a
bradytroph or "leaky" mutant; i.e. the mutant
growth on minimal medium is poor, but detectable.
This study has shown
that the phosphoserine phosphatase specific activity in ser(JBM 5) is
not as great as the enzyme's activity in the wild-type.
Apparently,
sufficient serine is produced by the phosphatase for the mutant to
27
barely survive.
The nature of the mutation could be explained by either of
~10
alternative possibilities: either the nutant produces less of the wildtype form of the enzyme or it produces a normal level of defective
enzyme.
In the first possibility, a mutation may have occurred in the
regulatory gene controlling the biosynthesis of the phosphatase.
In
the second possibility, a mutation may have occurred in the structural
gene coding for the phosphatase.
Preliminary results in earlier ex-
periments suggest that the mutant phosphatase is more sensitive to acidic conditions than the wild-type phosphatase. Initial results by
Fran Beck (unpublished results) testing the possibility of an acid sensitive phosphatase suggest that the mutant phosphatase has a different
pH optimum than the wild-type enzyme.
This finding would also be ex-
pected i f the phosphatase used by the mutant was not the phosphoserine
phosphatase at all, but another non-specific phosphatase.
Neurospora
is known to possess the following phosphatases: repressible acid phosphatase, constitutive acid phosphatase, repressible alkaline phosphatase, and constitutive alkaline phosphatase.
However, Knox (1969)
reported that in certain rat tissues the substrate phosphoserine is
used principally by phosphoserine phosphatase and to a smaller extent
by other phosphatases.
On the other hand, the differences in pH optimum may mean that
the enzyme structure is altered in some way to account for this pH
difference.
If the enzyme structure were altered, the defect in the
enzyme may lie either in the active site or elsewhere in the structure
such that the tertiary structure of the enzyme is altered.
Such an
28
alteration in the tertiary structure may not affect the function of the
enzyme, but rather some other properties such as protein aggregation.
For example, Wagner (1967) has observed in Neurospora that a group of
isoleucine-valine requiring mutants did not show any apparent enzyme
dificiencies in the isoleucine-valine biosynthetic pathway based on
activities measured in extracts.
The enzymes of the mutants had higher
activity than the wild-type when extracted from the mycelium, yet in
vivo the enzymes were inactive.
Wagner suggested that in vivo the cell
requires a particular arrangement of the isoleucine-valine enzymes.
This arrangement is thought to be an multienzyme aggregate.
These
mutants are said to be organizational mutants-mutants unable to form
the multienzyme complex, although possessing functioning enzymes.
Dialysis and the precipitation of proteins by salting affected
the three extracts differently.
the least.
salted
Dialyzed crude
~(g+f)
Of the extracts, ser(g+f) was affected
~(g+f),
salted dialyzed
~(g+f),
all had roughly the same specific activity.
and
An earlier
eX?eriment (experiment 1) showed this not to be the case; salted dialyzed
~( g+f)
activity was lower than sal ted
~(
g+f).
Salting had different effects on the phosphoserine phosphatase
when the wild-type culture was grown under two different conditions.
Salting appears to significantly lower the specific activity of
~(min)
as the dialysis of a salted extract gave a higher specific activity.
On the other hand, salting appears to slightly raise the specific activity of
salted
~(g+f).
~(g+f)
Generally, differences in specific activity between
and dialyzed salted
~(g+f)
were small.
One of the
functions of salting was to purify the extract; however, little if
29
any purification is indicated by the results. Schramm (1958) in his
work on yeast phosphatase found phosphatase activity in the 70-100%
saturated salt solutions, and no activity in the 1-69% saturated salt
solutions.
In this study the activity was found in the 1-69% saturated
salt solutions.
The 70-100% fraction was tested for phosphatase act-
ivity by Snowdy Dodson (unpublished results) and found to be without
phosphatase activity.
In agreement with her finding, the 1-69%
fraction is likely to possess most of the phosphoserine phosphatase
as the activity of this fraction for
~(g+f)
and
~(min)
is comparable
to the dialyzed crude fractions which are assumed to possess all
soluble enzymes.
The variations in specific activities of the wild-type extracts
may be due to their impure nature.
Variations in the time of extract-
ion, precipitation or other factors are likely to give rise to variations in specific activity.
Two other serine mutants that have been tested for phosphoserine
phosphatase are ser-1 and ser-3.
Ser-1, genetically separate from
ser(JBH 5), was found to have normal levels of phosphoserine phosphatase activity compared to its isogenetic wild-type.
Ser-3, an allele
of ser( JB;1 5) (Maxwell et al., 1978), had lower levels of phosphoserine phosphatase activity compared to its isogenic wild-type (Snowdy
Dodson, unpublished results).
These two results give strong support
to the hypothesis that ser(JBH 5) has a defective phosphoserine
phosphatase and that the phosphorylated pathway is the major pathway
of serine biosynthesis.
30
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