---------~---------------
CALIFORNIA STATE UNIVERSITY, NORTHRIDGE
STUDIES OF ISOZYMES OF GLUCOSE-6-PHOSPHATE
\I
DEHYDROGENASE IN SEA URCHIN EGGS
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
David Martin Kolan
_..,-··
August, 1980
· - - - ------·--·--------------------·------------·------.
The Thesis of David Martin Kolan is approved:
llp€1
'
'
California State University, Northridge
i;
.
ACKNOWLEDGEMENTS
This work was completed through the endless patience
and assistance of Dr. Mary Lee Barber.
My warmest thanks
and appreciation go to her.
My wife, Susan, realized the importance of this work
to me and gave more gentle urging, understanding and
support than anyone could have a right to expect.
this work, with all my love, to her.
i i i
I give
[1
TABLE OF CONTENTS
Acknowledgements
iii
List of Figures
v
Abstract
vi
Introduction
1
Materials and Methods
4
Obtaining Gametes
4
Treatment Prior to Homogenization
4
Extraction for pH Studies
5
Extraction for Other Studies
6
Gel Electrophoresis and Staining
7
Results
9
Extraction at Various pH•s
9
OTT Added to Extracts Prior to Electrophoresis
9
Papain Added to Extracts Prior to Electrophoresis
li
Glucose-6-phosphate and/or NADP Added to Extracts
14
Tests of Cofactor and Substrate Specificity
14
Discussion
21
References
31
'
FIGURES
1
Extraction of Egg Pellet Fractions at Various pH•s
2
Extraction of Egg Gel Fractions at Various pH•s
3
OTT Added to Pellet and Gel Extracts Prior to
Electrophoresis
(3A-unfertilized; 38-fertilized)
4
Papain Added to Pellet and Gel Extracts Prior to
Electrophoresis
(4A-unferti1ized; 48-fertilized)
5
NADP and/or Glucose-6-phosphate Added to Extracts
Prior to Electrophoresis
(5A-contro1; 58-NADP; 5C-g1ucose-6-phosphate;
50-NADP and glucose-6-phosphate)
6
Cofactor and Substrate Specificity of Extract
Isozymes
(6A-NADP and Various Substrates
68-NAD and Various Substrates)
v
ABSTRACT
STUDIES OF ISOZYMES OF GLUCOSE-6~PHOSPHATE
DEHYDROGENASE IN SEA URCHIN EGGS
by
David Martin Kolan
Master of Science in Biology
In sea urchin eggs the hexose monophosphate shunt is
the main glycolytic pathway before and after fertilization,
and glucose-6-phosphate dehydrogenase (G6PD) is the main
regulatory enzyme of the pathway.
Activity of the enzyme
increases with fertilization, thought to occur as a result
of activation of NAD kinase and glycogen phosphorylase and
simultaneously increased titers of NADP and glucose-6phosphate (G6P).
Part of the enzyme activity is associated
with the egg surface membrane complex.
Experiments have
been done to determine the effects of extraction pH upon
isozymes in unfertilized eggs, and what effects dithiothreitol (OTT, a sulfhydryl reducer), papain (a well known
thiol protease), NADP, NAD, G6P, and other substrates have
when added exogenously, upon the formation of isozymes and
the mechanics of isozyme change as a part of the program
of fertilization.
Control extracts of pellet and gel fractions of unfertilized egg homogenates of the species Strongylocentrotus
purpuratus show three isozyme bands when extracted using
vi
O.OlM MgC1 2 ; these bands are designated 81, 82, and 83, the
slowest to fastest migrating respectively with electrophoresis.
Extraction with Lubrol WX yields an even slower mov-
ing band, 8L.
Fertilized eggs are shown to lack a B3 band.
Extraction pH has been found to have no effect upon formation of isozymes between pH 6.4 and 7.9, whether pellet or
gel extract.
Unfertilized and fertilized egg pH 7.9 ex-
tracts with OTT added show the apparent extinction of activity of all but the B2 isozyme band, thought to be due to
sulfhydryl inhibition of the binding of NAOP to cysteine
residues of the G6PO isozymes inactivated by OTT.
Papain
added to unfertilized egg extracts has no visible effect
upon the isozymes, while fertilized egg extracts take on
somewhat the appearance of unfertilized eggs, gaining the
B3 band and partially losing Bl, indicating a relationship
between Bl and B3.
Addition of NAOP and G6P together
causes the apparent disappearance of the B3 isozyme in unfertilized egg extracts and slightly increased mobility in
both fertilized and unfertilized cases.
With the experi-
mental results in mind, it is proposed that increased NAOP
titers cause a release of lower molecular weight isozymes
from membranes to soluble phases of the cell, and increased
concentrations of substrate and specific cofactor complete
formation of a larger G6PO isozyme, with the greater enzyme
activity available to supply the energy for the metabolism
of the newly formed sea urchin zygote.
vii
INTRODUCTION
The generalized program for metabolic activation of
the echinoderm egg at fertilization is said to take place
in two parts.
These parts are described by Epel (1975) as
the "early 11 events and
11
late" events of fertilization and
illustrate an intimate relationship between cell surface
changes and manifold alterations of the biochemical milieu
of the cytoplasm.
With binding of the sperm to the egg
plasma membrane the early events start, causing an immediate flooding of calcium ions from bound sites into the
egg cytoplasm.
This is postulated to cause a change in
sodium ion conductance, activation of NAD kinase (E.C.2.
7.1.23) and glycogen phosphorylase (E.C.2.4.1.1), and a
brief respritory surge (seen as increased oxygen consumption).
A change in intracellular pH of 0.3 to 0.4 units
fills the four-minute gap between early and late events.
Next,· increases in the synthesis of DNA and protein occur
along with increased membrane permeability and potassium
conductance, increased transport across the membrane, and
greater protease (E.C.3.4.24.4) and adenyl cyclase (E.C.4.
6.1.1) activity.
These early and late changes provide the
materials to permit the zygote to enter cell division.
Clycolysis is important in the program both before
and after fertilization for it provides metabolic energy
for cell processes.
In sea urchin eggs the hexose mono-
1
2
phosphate shunt (HMS) is the main glycolytic pathway before
and after fertilization (Epel, 1977; Isono and Yasumasu,
1968; Krahl, 1956), and glucose-6-phosphate dehydrogenase
(G6PD, E.C. 1. 1. 1.49) is the first enzyme of the pathway.
G6PD is viewed as the main regulatory enzyme of the HMS
(Bonsignore and De Flora, 1972; Scott and Mahoney, 1976),
catalyzing the conversion of glucose-6-phosphate(G6P) to
6-phosphogluconate(6PG) with nicotene adenine dinucleotide
phosphate (NADP) as the cofactor of the reaction.
Its
availability is the rate limiting component of the reaction
(Lehninger, 1970; White, Handler, and Smith, 1968).
G6PD has undergone much study, with its role in echinoderm egg fertilization being closely examined.
Under ex-
perimental conditions of Isono's work (1963) enzyme activity shifted from a particulate fraction to a soluble phase.
It was found that activity of the enzyme in sea urchin eggs
increases with fertilization (Isono and Yasumasu, 1968),
thought to occur as a result of activation of NAD kinase
(E.C.2.7. 1.23) and glycogen phosphorylase (E.C.2.4. 1.1) and
simultaneously increased titers of NADP and G6P (Epe1,
,1977).
Part of the enzyme was found to be associated with
the egg surface membrane complex (Barber and Foy, 1973).
Other work has been done that sheds more light on the
role of G6PD in fertilization.
Work by Barber and King
(1978) illustrates the presence of different soluble and
particle-bound isozymes before and after fertilization in
3
sea urchin eggs.
Ammonia activation of sea urchin eggs,
stimulating only the late events of fertilization (Epel
et al., 1974), has been found to induce a pattern of isozymes intermediate to those of fertilized or unfertilized
eggs (Barber and Shutt, 1978).
It is also noted that di-
thiothreitol (OTT) affects G6PO activity in fertilized
and unfertilized whole eggs and homogenates (Barber and
Edidin, 1974) in conjunction with its release from bound
to soluble form.
Bearing in mind some of the previous work, it was
decided to determine more fully the parameters of G6PO
activity in fertilized and unfertilized eggs with respect
to isozyme formation, structure, and interaction.
More
specifically, an examination was made of the effects of
extraction pH upon the formation of isozymes in unfertilized eggs, and what effects OTT (a sulfhydryl reducer),
papain (a well known thiol protease), NAOP, NAO, G6P, and
other substrates have, when added
exogenously~
upon the
formation of isozymes and the mechanics of isozyme change
as a part of the program of fertilization.
MATERIALS AND METHODS
Obtaining Gametes
The sea urchins used were of the species Strongylocentrotus purpuratus and were collected in the intertidal region of the Palos Verdes Peninsula, California.
Gametes
were obtained by intracoelomic injection of 0.55 M KCL.
Eggs were shed into milipore filtered sea water, pH 8.00
with 0.001 M Tris (tris-(hydroxymethyl) aminomethane)
(FSW).
Sperm were collected undiluted in a petri dish.
Collection of gametes and subsequent washes were done in
an ice bath.
Eggs were washed once in FSW and then treated
to remove the jelly coat by settling in pH 5.00 FSW for 3
min. (Sakai, 1960).
The pH was readjusted to 8.00 using
1.0 M Tris.
Treatment Prior to Homogenization
After removing jelly coats, all eggs were washed 3
times in FSW.
Eggs were then divided into two aliquots -
one to be fertilized.
Eggs were fertilized using a 1% v/v
solution of sperm in FSW, 0.1 ml per 10 ml of egg suspension.
Fertilization was considered complete if 95% of the
eggs had an elevated fertilization membrane.
Sperm cells
were removed after 15 min. by washing eggs twice in FSW.
After 30 min. both fertilized and unfertilized eggs were
pelleted with a hand centrifuge to remove the FSW, which
4
5
was replaced with 0.1 M MgC1 2 pH 6.50 (Tris/BES (N, N-bis(2-hydroxymethyl )-2-aminomethanesulfonic acid) buffer,
0.01 M) to stabilize the membranes.
Extraction for pH Studies
After a rinse in 0.1 M MgC1 2 , pH 6.50, unfertilized
eggs were divided into 9 aliquots, hand centrifuged to
pellet them, and then diluted with a 1:1 volume ratio with
0.01 M MgC1 2 , pH 6.50 (0.01 M Tris/BES buffer).
Each ali-
quot was homogenized for 30 sec. using a Sorvall OmniMixer with "micro" attachment at maximum speed (5 x 10 4
r.p.m.) on ice.
Fertilized eggs were homogenized for 45
sec. in MgC1 2 as above. After homogenization, all samples
were centrifuged at 2 x 10 4 r.p.m. (4.1 x 10 4 g) at oo C
for one hour.
The resulting pellet, gel (a gelatinous
mass neither supernatant nor pellet), and supernatant fractions of each sample were separated, the unextracted supernatants from unfertilized homogenates pooled and kept at
4° C.
Both gels and pellets of unfertilized egg homage-
nates were extracted for one hour at 4° C, 1:1 in 0.01
MgCl (0.01 M Tris/BES) as follows:
~1
UEl, unextracted super-
natant, pH 6.50; El, pH 6.40 extract; E2, pH 6.40 extract
with 1% w/v Lubrol WX; E3, pH 6.58 extract; E4, pH 6.85;
E5, pH 7.10; E6, pH 7.30; E7, pH 7.48, E8, pH 7.70; E9,
pH 7.90; FEl, pH 6.50 fertilized unextracted supernatant;
F2, pH 6.49 fertilized pellet extract.
Samples designated
6
Gl through G9 correspond to the extracted gel fractions of
unfertilized egg homogenates El through E9.
Sample FG was
the pH 6.40 extract of the fertilized egg gel fraction.
After the one hour extraction period, all gel and pellet
extracts were centrifuged for one hour at 2 x 10 4 r.p.m.
at
oo
C.
Pellet and gel fractions were discarded and
supernatants were recovered.
All samples were placed in
separate vials with a few crystals of sucrose and 0.05 ml
of Bromphenol Blue tracking dye per ml of supernatant before electrophoresis.
Extraction for Other Studies
After rinsing in 0.1 M MgC1 2 , pH 7.90 (0.01 M Tris/
BES), unfertilized eggs were hand centrifuged to pellet
and diluted to a 1:1 volume in 0.01 M MgC1 2 , pH 7.90 and
homogenized in two batches in the Sorvall Omni-Mixer, as
described above.
Unfertilized eggs were homogenized for
30 sec. and fertilized eggs were homogenized for 45 sec.,
each in two batches.
All four batches were centrifuged
for one hour at 2 x 10 4 r.p.m. (4.1 x 10 4 g). Unextracted
fertilized and unfertilized
hom~genate
supernatants were
decanted and placed in vials with sucrose and tracking dye
as described above, the samples designated UEl and FEl respectively.
Pellet and gel fractions from each batch were
separated and resuspended in 0.01 M MgC1 2 ~ pH 7.90, with
or without ~ubrol WX (1% w/v) to a 1:1 volume ratio and
7
allowed to extract at 4° C for one hour.
Suspensions were
then centrifuged for one hour as described above and the
extract supernatants recovered.
Unfertilized pellet ex-
tracts without or with 1% w/v Lubrol WX were designated El
and E2; unfertilized gel extracts, Gl and G2; fertilized
pellet extracts without or with Lubrol WX, Fl and F2; and
fertilized gel extracts, FGl and FG2.
Supernatants re-
covered were placed in vials with sucrose and tracking dye,
as above, before freezing.
Before electrophoresis, samples were thawed by allowing to come to room temperature (20° C) slowly.
gentle agitation to remix the samples, 50
removed and added to 50
~1
~1
After
aliquots were
of OTT, papain, NAOP (or NAO)
and/or glucose-6-phosphate (or glucose-1-phosphate, fructose-6-phosphate, 2-deoxyglucose-6-phosphate, or 6-phosphogluconate) in 0.01 M MgCl 2 , pH 7.90 (0.01 M Tris/BES),
such that the final concentration of the variables (OTT
etc.) was 1 mg/ml for electrophoresis.
Samples were kept
refrigerated (4° C) for one hour prior to loading.
Gel Electrophoresis and Staining
The polyacrylamide stacking gel was 3% w/v at pH 6.80
and the sample gel was pH 8.80 and 10% w/v.
buffer was 25 mM Tris-Glycine, pH 8.40.
gels for pH studies were 70
~1
The reservoir
Samples loaded on
per slot and for electro-
phoresis with OTT and other agents, 90
~1
of mixed sample
8
per slot was used.
Electrophoresis was carried out at 15
rna per gel, at room temperature for 12 hours.
After elec-
trophoresis, the gels were incubated for 10 min. in buffer
consisting of 50 mM triethanolamine, 10 mM MgC1 2 , and 5 mM
EDTA, (ethylenediamine tetraacetic acid), at a final pH of
7.40.
The gel staining solution was the above buffer con-
taining 2.5 mM G6P, 0.2
~M
NADP, 70
~M
Phenazine metho-
sulfate, 10 mM KCN, and 0.3 mM Nitro-blue tetrazolium.
In-
cubation was on a reciprocating shaker, in the dark, for
a minimum of 2 hours.
Next, gels were rinsed in distilled
water for one hour, and then photographed or soaked in a
solution of l% glycerine, 10% methanol, for 4-6 hours followed by vacuum drying and storage.
RESULTS
Extraction at Various pH's
Figure 1 shows the isozyme patterns for unfertilized
egg pellet salt extracts at pH's 6.40 through 7.90, samples
El through E9.
In addition, the unextracted unfertilized
egg supernatant (UEl) and fertilized supernatant and extracted samples are shown.
All unfertilized egg pellet ex-
tracts exhibit the same three bands, 81, 82, and 83, with
83 being the fastest moving electrophoretic band.
In sam-
ple E2 (pH 6.40 with 1% Lubrol WX) a very slow moving band
is noted, designated 8L.
Finally, both fertilized egg sam-
ples show the typical fertilized egg isozyme pattern with
only the two slower moving bands, isozymes 81 and 82.
Extracts of gel fractions of unfertilized egg homagenates (Fig. 2), ranging from pH 6.40 to 7.90 show isozyme
banding patterns identical to those of the unfertilized
supernatant and egg pellet extracts; that is, the normal
81/82/83 pattern with the additional 8L band when treated
with Lubrol WX.
The fertilized egg jelly extracts have
the same 81/82 electrophoretic pattern as the pellet extracts to which they correspond.
OTT Added to Extracts Prior to Electrophoresis
OTT has noticeable and consistent effects as seen in
Figures 3A and 38.
In all unfertilized egg samples, bands
9
10
FIGURE 1
Extraction of unfertilized egg pellet fractions at various
pH's. Bands from top to bottom (and in all other figures)
are BL, Bl, 82, and 83 respectively. From right to left:
1 )--El--pH 6.40 unfertilized pellet extract
2)--E2--pH 6.40 unfertilized pellet extract (with
Lubrol \.JX)
3)--E3--pH 6.58 unfertilized pellet extract
4)--E4--pH 6.85 unfertilized pellet extract
5)--E5--pH 7.10 unfertilized pellet extract
6)--E6--pH 7.30 unfertilized pellet extract
7)--E7--pH 7.48 unfertilized pellet extract
8)--E8--pH 7.70 unfertilized pellet extract
9)--E9--pH 7.90 unfertilized pellet extract
10)-UEl--pH 6.50 unfertilized unextracted supernatant
(spnt)
11)-FEl--pH 6.50 fertilized unextracted spnt
12)--Fl--pH 6.40 fertilized extracted spnt
FIGURE 2
Extraction of unfertilized egg gel fractions at various
pH's. Bands are as described above. From right to left:
1)--Gl--pH 6.40 unfertilized gel extract
2)--G2--pH 6.40 unfertilized gel extract (with
Lubrol WX)
3)--G3--pH 6.58 unfertilized gel extract
4)--G4--pH 6.85 unfertilized gel extract
5)--G5--pH 7. 1 0 unfertilized gel extract
6)--G6--pH 7.30 unfertilized gel extract
7)--G7--pH 7.48 unfertilized gel extract
8)--G8--pH 7.70 unfertilized gel extract
9)--G9--pH 7.90 unfertilized gel extract
10)-FGl--pH 6.40 fertilized gel extract
"!
--.
J
'-
}1
l {;
.0
7
8
8
7
6
6
5
5
4
4
J
2
1
ll
Bl and B3 are virtually eliminated.
(See Figure 3A, sam-
ples numbered 2, 4, 6, 8, and 10, compared to the untreated
samples 1, 3, 5, 7, and 9).
Accompanying the disappearance
of bands Bl and B3 is an apparent increase in the intensity
of the B2 band.
In the E2 and G2 samples where the BL band
is normally seen, the BL isozyme appears much reduced (compare sample 5 with 6 and 9 with 10).
A similar pattern is seen in the electrophoretic bands
of fertilized egg homogenate pellet and gel extracts.
As
Figure 3B shows, the Bl isozyme band present in untreated
samples (1, 3, 5, 7, and 9) is absent in the OTT treated
preparations (2, 4, 6, 8, and 10) and the BL band, normally
present in Lubrol extracts, is much reduced in the OTT samples.
Papain Added to Extracts Prior to Electrophoresis
Figure 4A shows a comparison of untreated and treated
unfertilized egg homogenate pellet and gel extracts.
There
appears to be no significant effect of papain upon the extracts with only slight variations in band intensities
noted.
No bands were lost.
Fertilized egg extracts (seen in Figure 4B) show significant differences between control and papain-added
ples.
sam~
In all cases those samples with papain added appear
to mimic the normal unfertilized pattern in that a distinct
11
B3
11
isozyme is seen.
Along with the appearance of the B3
12
FIGURE 3A
OTT added to unfertilized egg pellet and gel extracts before electrophoresis (final concentration 1 mg/ml).
1)--unfertilized unextracted spnt (UEl)
2)--UEl with OTT added
3)--unfertilized extracted pellet spnt (El)
4)--El with OTT added
5)--unfertilized extracted (with Lubrol WX) pellet spnt
(E2)
6)--E2 with OTT added
?)--unfertilized extracted gel spnt (Gl)
8)--Gl with OTT added
9)--unfertilized extracted (with Lubrol WX) gel spnt (G2)
10)--G2 with OTT added
FIGURE 38
OTT added to fertilized egg pellet and gel extracts before
electrophoresis (final concentration 1 mg/ml ).
1)--fertilized unextracted spnt (FEl)
2)--FEl with OTT added
3)--fertilized extracted pellet spnt (Fl)
4)--Fl with OTT added
5)--fertilized extracted (with Lubrol WX) pellet spnt (F2)
6)--F2 with OTT added
?)--fertilized extracted gel spnt (FGl)
8)--FGl with OTT added
9)--fertilized extracted (with Lubrol WX) gel spnt (FG2)
10)--FG2 with OTT added
4
I
(
'.J
...
,.
r
.,
• j
• I
13
FIGURE 4A
Papain added to unfertilized egg pellet and gel extracts
before electrophoresis (final concentration 1 mg/ml).
1 )--UEl
2)--UEl with papain added
3)--El
4)--El with papain added
5)--E2
6)--E2 with papain added
7)- -G 1
8)--Gl with papain added
9)--G2
10)--G2 with papain added
FIGURE 48
Papain added to fertilized egg pellet and gel extracts
before electrophoresis (final concentration 1 mg/ml ).
1 )--FEl
2)--FEl with papain added
3)--Fl
4)--Fl with papain added
5)--F2
6)-- F2 with papain added
7)--FGl
8)--FGl with papain added
9)--FG2
1 O)--FG2 with papain added
2
2
4
5
7
6
)
10
..,
I
H
10
band, the Bl band in each of the papain-added samples is
significantly less intense than in the corresponding untreated samples.
In Lubrol extracted samples the BL band
seems unaffected by papain treatment.
Glucose-6-phosphate and/or NAOP Added to Extracts
Control isozyme electrophoretic patterns are seen in
Figure 5A.
With addition of exogenous NADP (Fig. 5B) to a
final concentration of lmg/ml, all samples seem to be much
in~reased
in intensity, though bands are present the same
as in the control samples.
With addition of G6P (Fig. 5C)
no appreciable differences are noted in any samples.
Addi-
tion of NAOP and G6P (Fig. 50) to the extracts causes the
same intensity increase noted in Figure 5B, but in addition
appears to cause the disappearance of the B3 isozyme in all
unfertilized extracts (samples 1 through 5), such that they
have somewhat the appearance of fertilized egg extracts.
It should be noted that in all samples the B2 band has a
slightly faster mobility than control and other treated
cases and lies in the area between B2 and B3, based on Rf
values.
Tests of Cofactor and Substrate Specificity
In an experiment to see if the banding and intensity
manifestations were substrate- or cofactor-specific, Figures 6A and 6B show the electrophoretic patterns of G6PO
14
with NADP in conjunction with glucose-6-phosphate, fructose-6-phosphate, glucose-1-phosphate, deoxyglucose-6phosphate, and 6-phosphogluconate.
Using NADP the apparent
intensity phenomena is seen with the seeming loss of the
B3 isozyme.
In Figure 6B, with NAD substituted for NADP,
only the normal fertilized and unfertilized isozyme patterns are seen.
15
16
FIGURE 5
Unfertilized and fertilized egg pellet and gel extracts,
untreated or with NADP and/or glucose-6-phosphate (final
concentration 1 mg/ml) added prior to electrophoresis.
Samples 1 through 10 are described below. (see next 2
pages for gels.)
1)--UEl-unfertilized spnt
2)--El--unfertilized extracted pellet
3)--E2--unfertilized extracted (with Lubrol WX) pellet
4)--Gl--unfertilized extracted gel
5)--G2--unfertilized extracted (with Lubrol WX) gel
6)--FEl-fertilized spnt
7)--Fl--fertilized extracted pellet
8)--F2--fertilized extracted (with Lubrol WX) pellet
9)--FGl-fertilized extracted gel
10)--FG2-fertilized extracted (with Lubrol WX) gel
17
FIGURE 5A
Untreated unfertilized and fertilized pellet and gel
extracts, as described on previous page.
FIGURE 5B
NADP added to unfertilized and fertilized pellet and gel
extracts before electrophoresis (1 through 10 as described).
1
2
1
4
2
3
4
.,
1 ()
'
5
6
7
8
9
10
18
FIGURE 5C
Glucose-6-phosphate added to unfertilized and fertilized
pellet and gel extracts (as earlier described) before
electrophoresis.
FIGURE 50
NADP and glucose-6-phosphate added to unfertilized and
fertilized pellet and gel extracts before electrophoresis.
-
1
2
3
)
'.
4
5
-
.
-~
7
6
f. J
8
.,
10
9
'
.
19
FIGURE 6A
Unfertilized and fertilized pellet extracts (with and
without Lubrol WX) with NADP and various substrates added
prior to electrophoresis. Samples are shown below with
El, E2, Fl, and F2 as described in Figure 5.
1)--El--with
2)--E2--with
3)--Fl--with
4)--F2--with
glucose-6-phosphate
glucose-6-phosphate
glucose-6-phosphate
glucose-6-phosphate
5)--El--with
6)--E2--with
7)--Fl--with
8)--F2--with
glucose-1-phosphate
glucose-1-phosphate
g1ucose-1-phosphate
glucose-1-phosphate
9)--El--with
10)--E2--with
11 )--Fl--with
12)--F2--with
fructose-6-phosphate
fructose-6-phosphate
fructose-6-phosphate
fructose-6-phosphate
13)--El--with
14)--E2--with
15)--Fl--with
16)--F2--with
deoxyglucose-6-phosphate
deoxyglucose-6-phosphate
deoxyglucose-6-phosphate
deoxyg1ucose-6-phosphate
17)--El--with
18)--E2--with
19)--Fl--with
20)--F2--with
6-phosphog1uconate
6-phosphogluconate
6-phosphogluconate
6-phosphogluconate
2
11
3
12
4
13
-
7
·~
14
15
16
17
lO
9
8
18
1.
20
20
FIGURE 6B
Unfertilized and fertilized pellet extracts (with and
without Lubrol WX) with NAD and various substrates added
prior to electrophoresis. Samples 1 through 20 are with
substrates and extracts as described in Figure 6A.
,
1
2
3
4
5
11
14
15
6
...,
I
JI
I
1l
2
16
17
18
20
DISCUSSION
Dividing the early and late events of fertilization
in sea urchin eggs is a change in intracellular pH (Epel,
1975).
This change is usually over a range of 0.3 to 0.5
pH units (demonstrated by Lopo and Vacquier (1977)) with a
range of pH 6.34 to 6.76
with~
purpuratus homogenates.
Shen and Steinhardt (1978) detected a pH range of 6.84 to
7.26 using microelectrodes inserted in eggs of Lytechinus
pictus.
This transitory elevation of pH, which is a nor-
mal event, is also induced by ammonia activation of the
sea urchin egg (Shen and Steinhardt, 1978).
A possible
relationship between the fertilization change in G6PD isozymes and pH elevation is formed when three
experimental evidence are included.
mor~
pieces of
First, it is well
known that G6PD undergoes a change from the particulate
fraction to soluble phase upon fertilization (Isono, 1963).
(His
\vas the supernatant resulting from
centrifugation at 2 x 10 4 g for 15 min. in 0.75 M man11
Soluble phase
nitol.)
11
Next, it was discovered that ammonia activation
of the sea urchin egg causes a release of surface-bound
proteins (Johnson and Epel, 1975).
Finally, recent work
in our laboratory has shown that ammonia activation results
in intermediate G6PD isozyme patterns in
and Shutt, 1978).
~
pictus (Barber
One can, therefore, postulate a possible
relationship between the change in intracellular pH at fer-
21
22
tilization and the changed observed in the G6PD isozymes.
Other factors, however, make this relationship at best a
secondary one.
Work by Barber and King (1978), in which
extraction was done at pH 8.00 only, showed isozyme differences before and after fertilization and led them to
conclude that
11
elevated pH may not be the only trigger for
the observed changes, since all of these extractions were
done at the higher pH 11
•
The data presented here (Figs. 1
and 2) indicate that, for the pH range 6.40 to 7.90, there
is no change in the G6PD isozymes extracted from the pellet, as shown by electrophoresis.
This pH range encom-
passes the actual pH change detected by both Lopo and
Vacquier (1977) and Shen and Steinhardt (1978) and thus,
is considered a valid experimental parameter.
It is noted
that the apparent failure of different extraction pH's to
induce a change in pellet extract isozyme patterns is also
found for gel extracts.
This is not to say, however, that
pH is not an effector of isozyme banding patterns in other
systems.
For example, in the cyanobacteria Anabaena sp.
ATCC27893 a hypoactive monomeric form (120,000 daltons) is
favored at pH 7.00 or greater, while the dimer (240,000
daltons) is found in its most active state at pH 6.50
(Schaeffer and Stanier, 1978).
(It is best to note that
here and in later discussion, a monomer will be defined as
an active enzyme complex of molecular weight 100,000 to
120,000 daltons made up of two inactive subunits of 50,000
Q
•
23
to 70,000 daltons.
This makes consistent various refer-
ences• terminology (e.g., Levy, 1979) where the monomer
was viewed as the inactive subunit of an active, twosubunit complex.)
OTT is a potent sulfhydryl-reducing agent.
When used
here, added prior to electrophoresis, both 81 and 83 bands
are eliminated or inactivated.
In other systems, OTT has
similar but not identical effects.
For example, in mam-
mary tissue homogenates and supernatants of lactating mice
exposed to OTT prior to electrophoresis a shift occurs from
a dimer (260,000 daltons) to a monomer (118,000 daltons) of
the G6PO enzyme.
In preneoplastic HAN tissue supernatants
however, a conversion from the monomer to a faster migrating form occurs, drawing the conclusion that a reducing
environment favors the active monomeric form over the dimer
(Hilf et al., 1975).
Similar interconversions were noted
by Schumulker (1970) with erythrocyte G6PO, while other
workers have reported reconversion from faster- to slowermigrating forms in mouse or rat liver, when treated with
s u l f hy dry 1 reagents (Hi z i and Ya g i 1 , 1 9 74 ; Taketa and
Watanabe, 1971 ); thus, there is no doubt that OTT sulfhydryl groups are involved in some monomer-dimer interactions.
The apparent extinction of both the 81 and 83 iso-
zymes, however, makes any interconversion questionable
(although the work with papain to be discussed indicates
an intimate relationship, 81 being the slower moving dimer
24
and B3 the faster moving monomer).
The OTT effects imply
a possible interaction between the sulfhydryl reagent and
cysteine on some other level; that is, the interaction is,
perhaps through sulfhydryl groups that may be involved in
monomer subunit interactions.
This too, is questionable
in light of the lack of evidence that G6PO isozymes have
disulfide bonds that could be upset by OTT (Levy, 1979);
therefore, one can conclude that some other mechanism may
be at work.
This mechanism is perhaps some form of sul-
fhydryl inhibition of the binding of NAOP to cysteine residues, as concluded by Kuby et al. (1974), based on chemical modification studies of G6PO in Saccharomyces Carlsbergensis.
Other work supports this contention (Yoshida,
1973; Hsu and Joshi, 1977; Criss and McKerns, 1969), although the cysteine residue is not considered essential in
some systems.
One example is Leuconostoc mesenteroides,
where cysteine is not even present (Ishaque et al. 1974).
An explanation of the increased intensity of the B2
band (an isozyme apparently significantly different from
Bl and B2) might be found in the work of Hilf et al.
(1975), where it was noted that OTT prevented oxidation
and loss of total G6PO activity; apparently, OTT stabilized the enzyme.
Another explanation might be that more
NAOP, substrate, and other components of the enzyme stain
were available to the remaining active sample bands with
the inhibition of the Bl and B3 isozymes, although there
is no other evidence to support this suggestion.
25
The effect of papain on fertilized egg pellet and gel
extracts is to cause the partial reappearance of the B3
isozyme with corresponding disappearance of Bl.
This sug-
gests very strongly that there is a dimer-monomer relationship involving Bl and B3, as postulated earlier.
The
change normally observed at fertilization, namely the disappearance of B3 is probably a completion of the conversion
to the dimer.
Apparently, this is a more active form, in
light of the observation of increased enzyme activity in
egg ghosts (Barber and Foy, 1973) and in whole eggs (Isono
and Yasumasu, 1968) after fertilization.
There are two
possible mechanisms for the action of papain described
above.
Unfortunately, there is little or no evidence to
support either.
The first is based on two facts.
It has
been noted that papain is most active on arginine and
lysine amino acid residues (Fasman, 1975; Neilands and
Stumpf, 1958).
It has also been observed that both argi-
nine and lysine residues are essential in the binding of
glucose-6-phosphate to the active site of G6PD subunits
(Milhausen and Levy, 1975; Levy et al. 1977), based on
work using various inhibitory chemicals.
Finally, it is
well documented that G6PD and NADP titers increase at fertilization (Epel, 1977) and that isozyme patterns change
as well (Barber and King, 1978; Barber and Shutt, 1978).
If one assumes that the change in isozyme patterns brought
about by fertilization is due at least in part to the
26
change in NADP and G6P concentrations (perhaps a reasonable assumption based upon the work shown in Figure 5 as
well as work to be mentioned later), then one may conclude
that papain causes the
obs~rved
change in fertilized egg
extract banding patterns by interferring with the ability
of essential lysine and/or arginine residues to bind G6P
and thus, allow some dimer disassembly.
Another possible reason for papain's demonstrated action could be the fact that the sulfhydryl group of cysteine no. 159 is part of the active site of papain (Ferst,
1977).
One can imagine sulfhydryl interactions analogous
to the interconversion phenomena described earlier (Hilf
et al., 1975; Schumulker, 1970; and others), with papain
acting not so much as an enzyme but rather as another
source of sulfhydryl groups to modify the G6PD quarternary
structure.
It was discovered, as shown in Figure 5, that NADP
and G6P added together to unfertilized egg extracts causes
a change in the G6PD isozyme patterns to an appearance
similar to fertilized egg extracts, the implications of
which may say a great deal about the mechanism of isozyme
changes brought about by the program of fertilization of
the sea urchin egg.
Complicating the picture, however, is
the fact that the supposed 82 band is no longer in its normal position, but rather occupying the space between the
usual 82 and 83 positions.
In addition, it is not known
27
whether 83 has also undergone some change or simply disappeared.
NADP has been cited many times as a factor necessary
in maintaining G6PD quarternary structure (see Levy, 1979
for several examples) through hydrophobic stabilization;
however, NADP has also been noted as the cause of conversion from a dimeric form (240,000 daltons) to a mixed
dimeric-monomeric form, with a substantial decrease in
activity, as in Anabaena sp. ATCC27893 (Schaeffer and
Stanier, 1978).
A contrasting effect was seen in sweet
potato G6PD where increased NADP concentration caused a
conversion from monomer to dimer (Muto and Uritani, 1972).
There is little evidence for the effects of G6P alone
upon isozymes of G6PD.
In Anabaena, the addition of
glucose-6-phosphate to the enzyme prior to ultracentrifugation has rro affect upon molecular weight
distri~ution
a sucrose gradient (Schaeffer and Stanier, 1978).
been found in
~
in
It has
Carlsbergensis that G6P can inhibit dimer
formation by NADP (Kuby et al., 1974).
This work indicates that glucose-6-phosphate added
alone has no effects on sea urchin egg G6PD isozymes (Fig.
5C), but as mentioned above, NADP and G6P together cause
somewhat the appearance of the fertilized egg enzyme pattern in unfertilized egg extracts.
Changes in isozymes
opposite to the work in Figure 50 have been noted in sweet
potato, where NADP at constant concentration was used with
28
G6P of increasing concentrations, causing a progressive
decrease in molecular weight from dimer to monomer (208,000
daltons to 110,000 daltons) (Muto and Uritani, 1972).
There is little, if any, other evidence for G6P and NADP
together promoting what may be a change from monomeric to
dimeric isozyme forms; that is, a possible change from unfertilized to fertilized pattern.
It is to be noted that the NADP concentration used
in this work was 8.5 times that used by Muto and Uritani
(1972) and the G6P concentration was 5.4 times as great,
suggesting that the effects seen are probably different
from those of Muto and Uritani.
In the work done here,
the faster-moving band is found in the range between the
normal B2 and B3 Rf values (not included in this paper).
This band is perhaps a "new" B2 or B3, or both, and a possible explanation for the change in location is that the
high concentrations of NADP and G6P (or their reaction
products) altered the shape and/or frictional resistance
to the isozyme in electrophoresis, thus, increasing electrophoretic mobility.
Another explanation arises from ob-
servations of fertilized egg extracts, (with the reminder
that the change in Rf value is found in both the fertilized
and unfertilized extracts).
Since B3 was not present prior
to treatment, we can assume that the faster-moving band
must be B2.
Although the data presented entitle us to no
certain explanation, we can say that part of the B2 isozyme
29
has changed to a smaller molecule or one with an increased
positive charge.
This would apply, as well, to the anal-
ogous region of enzyme activity in the unfertilized supernatant cases.
Since it is not known exactly what occurred to result
in the modified 82 isozyme or if 83 is indeed even present
after treatment of unfertilized egg extracts, we can still
be allowed some thoughts about increased concentrations of
cofactor and substrate relative to the program of fertilization.
A plausible mechanism for the change in G6PD of sea
urchin eggs emerges, based on the results of the work described here.
As outlined by Epel (1975), a release of
intracellular calcium ions initiates the transient respiratory burst by activating NAD kinase and glycogen phosphorylase, which results in increased titers of NADP and
glucose-6-phosphate.
The increased NADP concentration may
cause a release of particle-bound G6PD to "soluble 11 cellular components (Isono, 1963) due to a greater affinity of
the cysteine sulfhydryl groups in G6PD monomers (83) for
NADP than for the sulfhydryl groups of membranes or other
particulate cell fractions.
With the release of the mono-
mer molecules to the soluble fraction, the NADP, G6P, and
G6PD, all together in the intracellular milieu, complete
the formation of the dimer, with greater enzyme activity
(an observed change, with the assumed
disappearanc~
of the
30 .
B3 isozyme) started prior to fertilization.
The presence
of all three components of the first step of the pentose
phosphate pathway in the soluble phase of the cell permits
an increased chance of the molecular collision required
for the reaction to take place, providing the respiratory
energy necessary for the late events of fertilization and
later metabolism to support protein synthesis and cleavage
of the sea urchin egg.
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