Department of Molecular & Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095, USA
* Author for correspondence (e-mail: cgundersen{at}mednet.ucla.edu )
Accepted 12 December 2001
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Summary |
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Key words: Secretion, Cortical reaction, protein kinase C
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Introduction |
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As an important step toward clarifying the mechanism by which activators of
PKC trigger cortical granule exocytosis in Xenopus oocytes, we sought
to identify the isoform(s) of PKC that participate in this regulated secretory
event. This endeavor was complicated by the fact that the family of PKC
enzymes includes more than 10 members
(Nishizuka, 1995;
Jaken, 1996
), many of which
are expressed in oocytes and eggs of Xenopus
(Chen et al., 1989
;
Sahara et al., 1992
;
Dominguez et al., 1992
;
Stith et al., 1997
;
Johnson and Capco, 1997
). For
instance, Johnson and Capco (Johnson and
Capco, 1997
) reported that Xenopus oocytes express six
isoforms of PKC, including: (i) all four members (
, ß1,
ß2 and
) of the conventional PKC (cPKC) sub-family
(whose members are activated by both Ca ions and diacylglycerol or phorbol
esters) (Nishizuka, 1995
);
(ii) one member of the novel (or new) PKC family (
, which is activated
by diacylglycerol or phorbol esters, but not by Ca)
(Nishizuka, 1995
) and (iii)
the zeta isoform of PKC which belongs to the atypical group of PKC isozymes
which are insensitive to both Ca and phorbol esters
(Nishizuka, 1995
). To identify
the PKC isoform (or isoforms) that regulates the cortical reaction in
Xenopus oocytes, we used a combination of pharmacological, immunoblot
and heterologous expression strategies. Our data indicate that the eta (
)
isoform of PKC is centrally involved in triggering cortical granule exocytosis
in these cells.
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Materials and Methods |
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Assay of secretion
Protein secreted from oocytes in response to PMA (Sigma) was assayed using
SDS-polyacrylamide gel electrophoresis
(Laemmli, 1970) by
A280 or by the Bio-Rad dye-binding assay. For gel analysis (unless
otherwise indicated), individual oocytes were incubated in 10-20 µl of
Barth's solution (or OR-2 solution, see below) with PMA at the concentration
specified. After 30-60 minutes, the solution was collected and mixed with
5x concentrated Laemmli (Laemmli,
1970
) sample buffer (with 10-20 mM DTT as indicated) and incubated
for 5 minutes at 70°C. Samples were resolved on 10-12.5% gels using
molecular weight markers from Amersham-Pharmacia and either stained with rapid
Coomassie stain (Diversified Biotech) or transferred to nitrocellulose for
immunoblot analysis of cortical granule lectin (cgl) as described
(Gundersen et al., 2001
). To
detect protein release using the Bio-Rad dye-binding assay, groups of 10-15
oocytes were incubated in 0.1-0.15 ml of OR-2 solution (83 mM NaCl, 2.5 mM
KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM
NaH2PO4, pH 7.4 with NaOH) with PMA at the
concentrations indicated. After 30-60 minutes, the fluid surrounding the
oocytes was diluted into water for the protein assay using the Bio-Rad dye
reagent and human IgG as a standard. Independent estimates of the amount of
protein released from oocytes was made by incubating groups of 50 oocytes in
0.5 ml of OR-2 with 1 µM PMA for 30 minutes and reading the absorbance at
280 nm. For this assay we assumed that one A280 unit corresponded
to 0.8 mg/ml protein. After the absorbance measurements, samples of this
solution were also submitted to the Bio-Rad dye-binding assay and to
electrophoretic detection of released protein. When PKC inhibitors
(staurosprine, Gö6976, Gö6983 from Calbiochem) were used, they were
prepared as 10 mM stocks in DMSO and used under low light conditions. Oocytes
were preincubated (10-15 minutes) with these drugs prior to exposure to PMA
(20 nM). IC50s for these drugs were determined by densitometric
analysis of Coomassie-stained cgl resolved on SDS-gels. Release of cgl from
individual inhibitor-treated oocytes was normalized to the mean value for
controls without inhibitor. Results are the mean±s.d. from at least 15
oocytes for each drug concentration.
Subcellular fractionation and immunoblot analysis
We used a modified fractionation protocol that effectively sediments
cortical granules from triturated oocytes
(Gundersen et al., 2001)
(Fig. 2). Single oocytes were
disrupted by repeated passage (8-12 times) through the orifice of a yellow
pipet tip using 0.1 ml of HB (0.25 M sucrose, 10 mM Hepes, 1 mM EGTA, 2 mM
MgCl2, 1 mM phenylmethyl-sulfonylfluoride, 1 µg/ml pepstatin A,
1 µg/ml leupeptin, pH 7.4) followed by centrifugation for 1 minute at
14,000 g at 20-22°C. The supernatant was recovered and the
pellet was extracted with 0.1 ml of 10 mM Chaps (or 0.1% TritonX-100) in 0.1 M
NaCl, 50 mM Tris, 1 mM EDTA (pH 7.4) by trituration and centrifugation (1
minute at 14,000 g) to sediment yolk platelets and pigment
granules that did not completely solubilize. Protein was recovered
(Wessel and Flugge, 1984
)
separately from the supernatant and detergent extracts and subjected to
immunoblot analysis (Gundersen et al.,
2001
) using PKC isoform-specific antibodies from Santa Cruz
Biotechnology and ECL or ECL Plus (Amersham-Pharmacia) for detection. For
these analyses, the secondary antibody was preadsorbed using oocyte acetone
powder as described (Gundersen et al.,
2001
). As a positive control for the PKC antibodies, we used a
Xenopus brain P2 fraction prepared as in Gundersen et al.
(Gundersen et al., 2001
). As a
negative control, the PKC isoform-specific antibodies were pre-adsorbed
against the peptide immunogen following the supplier's protocol. Tests for
expression of PKC isoforms in stage II oocytes used extracts of 20-30 oocytes
homogenized directly in SDS sample buffer.
|
|
Autophosphorylation assay
Recombinant PKC (25 ng; from Calbiochem) was incubated in the buffer
in which it was provided with 5 µCi of 32P-ATP for 10 minutes at
20-22°C and resolved electrophoretically for autoradiography. PKC
antagonists were added 5 minutes before the ATP and the percentage inhibition
of the autophosphorylation reaction was quantitated by densitometry.
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Results |
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We sought independently to quantify protein secretion from oocytes exposed to PMA. As a first step in this direction, we were interested in the extent to which PMA induced cortical granule exocytosis in stage VI oocytes. The results of Fig. 2 show that while all of the detectable cgl immunoreactivity was retained in the supernatant and pellet fractions of a control oocyte, more than 90% of the cgl immunoreactivity was released from an oocyte exposed for 30 minutes to PMA (1 µM). In six separate trials, densitometry indicated that control oocytes secreted no detectable cgl, whereas PMA-treated oocytes released 80±10% of their total cgl immunoreactivity. Thus, PMA is an efficient trigger of cortical granule exocytosis in these cells. To quantify the amount of protein released, we used either the Bio-Rad protein assay or absorbance at 280 nm. In eight separate measurements using oocytes from three different frogs, A280 analyses indicated that PMA (1 µM for 30 minutes) evoked the secretion of 1.25±0.20 µg of protein per stage VI oocyte. Interestingly, when the same samples that had been submitted to A280 readings were subsequently analyzed using the Bio-Rad protein assay (with human IgG as the standard), protein release per stage VI oocyte was 0.70±0.06 µg. A possible explanation for the discrepancy in these estimates of protein secretion is addressed in the Discussion.
Pharmacological and ionic modulation of cortical granule
exocytosis
As a first step toward identifying which isoform(s) of PKC participate in
the PMA-triggered secretory process documented above (see also
Bement and Capco, 1989), we
conducted a series of experiments to investigate the impact of a variety of
ionic and pharmacological manipulations.
Prior work by Bement and Capco (Bement
and Capco, 1989; Bement and
Capco, 1990
) and Scheuner and Holz
(Scheuner and Holz, 1994
)
strongly suggested that PMA-triggered cortical granule exocytosis was
independent of Ca ions in stage V-VI oocytes. This is because the Ca ionophore
A-23187, which promotes cortical granule exocytosis in Xenopus eggs,
is ineffectual in oocytes (Bement and
Capco, 1989
). Similar results were obtained using ionomycin
(Scheuner and Holz, 1994
).
Concomitantly, buffering of cytosolic Ca ions (to low nM concentrations) with
BAPTA does not prevent cortical granule exocytosis in Xenopus oocytes
treated with PMA (Bement and Capco,
1990
). These results are significant because they imply that the
four conventional PKC isoforms (
, ß1,
ß2 and
), which are Ca-dependent
(Takai et al., 1979
; for a
review, see Nishizuka, 1995
),
are unlikely to participate in the activation of cortical granule exocytosis
in these cells. To extend these observations we found that injection of
oocytes with the Ca ion chelator EGTA (to a final concentration of 5-10 mM)
did not block PMA-induced secretion of cgl (data not shown). Independently,
injection of oocytes with enough Ca ions to achieve a calculated concentration
of 1-2mM (in the absence of buffering and assuming a cystolic volume of 0.5
µl) did not trigger cgl exocytosis nor was the Ca ionophore ionomycin (10
µM) an effective secretogogue (data not shown). Collectively, these data
indicate that changes of Ca ion activity are not important for the exocytotic
release of cgl. These results greatly diminish the likelihood that
Ca-dependent isoforms of PKC are involved in cgl secretion in stage VI
oocytes.
As a prelude to investigating the effects of more selective PKC inhibitors, we evaluated the threshold concentration for PMA to activate cgl secretion in Xenopus oocytes. (This proved to be important because pilot experiments using 1 µM PMA to evoke cgl secretion revealed no effect of inhibitors even at concentrations up to 20 µM.) As illustrated in the two examples of Fig. 3, low nM concentrations of PMA efficiently promoted the release of cgl. In eight experiments using oocytes from four different frogs, the EC50 (effective concentration for half-maximal secretion) was 6±2 nM PMA.
|
Over the last decade, two staurosporine derivatives (Gö6976 and
Gö6983) were found to exhibit specificity in their inhibition of
Ca-independent subtypes of PKC
(Martiny-Baron et al., 1993;
Gordge and Ryves, 1994
;
Gschwendt et al., 1995
;
Gschwendt et al., 1996
). Thus,
while Gö6976 inhibited PKCµ with an IC50 of 20 nM
(Gschwendt et al., 1996
), it
had a negligible effect (no inhibition at 10 µm) on the activity of PKC
or
(Martiny-Baron et al.,
1993
). By comparison, Gö6983 inhibited PKC
with an
IC50 of 100 nM (Gschwendt et
al., 1995
), but much higher concentrations (>10 µM) were
needed to inhibit PKCµ (Gschwendt et
al., 1996
). Because our pilot studies revealed that staurosporine
inhibited PMA-induced secretion of cgl with an IC50 of about 1
µM, we inferred that the Gö compounds could provide insight into the
PKC isoform(s) that initiate(s) this process. As indicated in
Fig. 4, Gö6976 and
Gö6983 inhibited PMA-evoked secretion of cgl. The IC50 for
these agents was 15±5 µM and 1±0.2 µM, respectively. Given
the pharmacological sensitivity of the PKC isoforms alluded to above, these
results provisionally excluded PKCs
,
and µ as triggers of
cgl secretion. As a final step in these pharmacological experiments, we
determined the IC50 for Gö6976 or Gö6983 to block
autophosphorylation of commercially available PKC
. For Gö6976 the
IC50 was 7.5±1 µM, whereas for Gö6983 it was
200±50 nM. As reviewed in the Discussion, the fact that these drugs
inhibit PKC
in vitro and antagonize PMA-induced secretion of cgl lend
support to the hypothesis that PKC
plays a role in the cortical
reaction.
|
Immunoblot investigations of PKC isoform distribution in
Xenopus oocytes
The rationale for these experiments was that we could exclude certain PKC
isoforms from being involved in the exocytotic release of cgl if these
isoforms were not detectably present in oocytes. To this end, we obtained
PKC-isoform-specific antibodies and first verified that these antibodies
recognized a protein of the appropriate mass in an extract from
Xenopus brain. Morever, since these were anti-peptide antibodies, we
verified that pre-adsorption of the antibody with the peptide immunogen
abolished the immunolabeling of the appropriate mass protein in
Xenopus brains (data not shown). These antibodies were then used to
probe soluble and particulate extracts from Xenopus oocytes that had
been prepared as in Fig. 2. As
shown previously (Gundersen et al.,
2001) and in Fig.
2, cortical granules (as reflected by the presence of cgl)
partition almost exclusively into the pellet in these experiments.
Interestingly, all five of the novel isoforms (
,
,
,
and µ) of PKC could be detected in these extracts
(Fig. 5A). With the exception
of PKC
, these PKC isoforms remained almost exclusively in the supernatant
after the brief centrifugation of the oocyte homogenate
(Fig. 5A). Since it is often
observed that lipid activators of PKCs induce translocation of these enzymes
to membranes (Nishizuka, 1995
;
Jaken, 1996
), we investigated
whether treatment of oocytes with PMA (1 µM for 30 minutes) would alter the
distribution of PKC isoforms shown in Fig.
5A. In no instance did we detect any significant re-distribution
of PKC isoform immunoreactivity in response to PMA
(Fig. 5A). Thus, whereas the
isoform of PKC is present in a fraction of oocytes that is enriched in
cortical granules, none of the other novel isoforms of PKC are significantly
distributed in this fraction, even after treatment with PMA
(Fig. 5A). The inference from
these studies is that PKC
is the only isoform in oocytes that is
associated with the structures that participate in the cortical reaction.
|
Results in Fig. 5B exemplify
the criteria that were used to identify PKC immunoreactive species in oocytes
(Fig. 5A). In this example
PKC antibodies identify a strongly immunoreactive species in
Xenopus brain at 76 kDa and a weaker signal at about 60 kDa. In the
oocyte pellet fraction, strong immunoreactivity is detected at 76 kDa, along
with weaker signals at about 60 kDa and 52 kDa
(Fig. 5B). Oocyte supernatant
reveals weakly immunoreactive species at about 47 kDa and 33 kDa
(Fig. 5B). Pre-adsorption of
the PKC
antibody with the peptide immunogen eliminates all of these
immunoreactive entities except for the signal at 52 kDa. Because the deduced
mass of PKC
in most organisms is close to 75 kDa
(Osada et al., 1990
;
Bacher et al., 1991
;
Nishizuka, 1995
), we conclude
that the prominent, 76 kDa species in frog brain and oocyte pellets in
PKC
. Concomitantly, the identity of the other immunoreactive species,
which could be due to cross-reactivity of these anti-peptide antibodies or to
degradation products of PKC
remains to be clarified.
Because stage II oocytes have the machinery to release cgl in response to
PMA (Fig. 1), we reasoned that
the PKC isoform(s) responsible for transducing the PMA effect must also be
expressed in these early stage oocytes. In this context, we note (data not
shown) that stage II oocytes (we used extracts from 20-30 oocytes) detectably
expressed immunoreactive PKC using the same criteria
(Fig. 5) for identification of
this protein in stage VI oocytes. By the same token, we could also detect
PKC
and PKC
in these early stage oocytes, so this strategy did
not enable us to exclude these PKC isoforms from playing a role in the
cortical reaction in these cells. Nevertheless, these results verify that
PKC
is present in oocytes at the stage when they first become competent
to secrete cgl.
Expression of wild-type and constitutively active PKC and
in oocytes and their effect on cgl secretion
As addressed in the Discussion, both the pharmacological data and the
immunoblot data were compatible with the hypothesis that the isoform of
PKC was important for initiating cgl secretion in this system. To obtain more
direct evidence for this conclusion, we investigated whether expression of
recombinant, constitutively active PKC
could trigger cgl secretion. As
controls, we also over-expressed wild type and constitutively active
PKC
, as well as wild-type PKC
. Results in
Fig. 6 show that oocytes
expressing constitutively active PKC
secrete cgl. However, oocytes
over-expressing (Fig. 6B)
wild-type PKC
or constitutively active PKC
did not release
detectable cgl (Fig. 6A).
Although the results in Fig. 6 are representative of data from a total of 15-20 oocytes expressing each PKC
isoform, it was interesting that in several oocytes over-expressing wild-type
PKC
, there was a low level of cgl secretion (less than half of that
obtained with the constitutively active isoform). These latter observations
indicate that under some conditions, over-expression of wild-type PKC
is
sufficient to trigger cortical granule exocytosis in these frog oocytes. Taken
together, these data support the hypothesis that PKC
is prominently
involved in triggering cortical granule discharge in these cells.
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Discussion |
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As outlined in the Introduction, the identified isoforms of PKC have been
segregated into three categories that reflect the sensitivity of these enzymes
to activation by Ca ions and lipid modulators, such diacylglycerol and phorbol
esters (Stabel and Parker,
1991; Gordge and Ryves,
1994
; Nishizuka,
1995
; Jaken,
1996
). Thus, the rate of substrate phosphorylation by conventional
isoforms of PKC is regulated both by Ca ions and agents like PMA or
diacylglycerol. Novel isoforms of PKC are insensitive to changes of Ca ion
activity but show enhanced substrate phosphorylation in the presence of
appropriate lipid activators. And, atypical PKCs are unaffected by either Ca
ions or lipid activators. These criteria led us to focus on novel PKCs as the
likely mediators of PMA-induced cortical granule discharge in Xenopus
oocytes. This is because secretion of cgl, the major protein stored in
cortical granules (Greve and Hedrick,
1978
; Nishihara et al.,
1986
; Chamow and Hedrick,
1986
) is elicited by low nM concentrations of PMA, but it is
unaffected by treatments that alter Ca ion activity. For instance, use of
BAPTA (Johnson and Capco,
1997
) or EGTA to clamp cytosolic ionized Ca below the resting
level did not interfere with PMA-evoked secretion of cgl. Moreover, neither
injection of Ca ions nor use of the Ca ionophores A-23187
(Johnson and Capco, 1997
) or
ionomycin elicited cgl release. This lack of sensitivity to changes of Ca ion
activity implies that the Ca-dependent family of PKCs is not centrally
involved in secretory triggering in oocytes. Concomitantly, the fact that cgl
secretion is initiated by PMA (or diacyglycerol)
(Bement and Capco, 1989
)
excludes the atypical PKC isoforms, which are insensitive to these lipid
mediators. By this process of elimination, the novel PKCs emerged as the most
plausible targets of PMA action.
The efforts to identify pharmacological agents that selectively target
specific PKC isoforms has culminated in the selection of several drugs that
inhibit a subset of PKCs (Gordge and
Ryves, 1994). Thus, derivatives of staurosporine (a broad spectrum
protein kinase inhibitor, which blocked cgl secretion in the low micromolar
range), like Gö6976 and Gö6983, appear to be selective antagonists
of certain PKC isoforms (Martiny-Baron et
al., 1993
; Gordge and Ryves,
1994
; Gschwendt et al.,
1995
; Gschwendt et al.,
1996
). In our experiments, Gö6976 significantly inhibited cgl
secretion with an IC50 of 15 µM. Since in vitro kinase assays of
PKC
and
revealed that these enzymes were unaffected by
Gö6976 up to 10 µm ((Martiny-Baron
et al., 1993
); see also
(Gschwendt et al., 1995
) for
conflicting results concerning PKC
), these observations suggested that
neither
nor
was involved in secretory triggering in oocytes.
Similarly, Gö6983 was more potent than Gö6976 as an inhibitor of
PMA-evoked cgl secretion in our experiments. Since in vitro phosphorylation
assays revealed that Gö6976 was three orders of magnitude more potent
than Gö6983 as an inhibitor of PKCµ
(Gschwendt et al., 1996
), our
findings mitigated against a role for PKCµ in oocyte cgl secretion. Thus,
by a process of elimination, the pharmacological data favored PKC
or
as secretory triggers in oocytes. We independently verified that
Gö6976 and Gö6983 blocked autophosphorylation activity of
recombinant PKC
in a range compatible with the inhibitory effects of
these drugs on cgl secretion. However, similar experiments were not performed
for PKC
(which is not available commercially), and this precluded us
from drawing firm conclusions about the identity of the isoform of PKC that
evokes secretion based on the pharmacology alone.
In our next set of experiments, we sought to determine which of the novel
PKC isoforms were expressed in oocytes. For these experiments, we first
ensured that the isoform-specific antibodies detected a protein of the
appropriate mass in extracts from Xenopus brain and that this
antibody detection was occluded by an excess of the immunogen. With these
criteria of antibody specificity, it was interesting that we detected
immunoreactivity corresponding to all five of the novel PKC isoforms in
oocytes. In a previous study, PKC was detected in oocyte extracts
whereas PKC
and
were not detectable
(Johnson and Capco, 1997
). The
apparent discrepancy between our observations and this earlier report
(Johnson and Capco, 1997
) can
almost certainly be attributed to the different antibodies used in the current
experiments. Of greater interest was the fact that there was a distinctive
subcellular distribution of the novel PKC isoforms. Using a simple
sedimentation scheme, we found that almost all of the immunoreactivity
corresponding to PKCs
,
, µ and
remained in the
supernatant. Under identical conditions, almost all of the PKC
immunoreactivity was found in the pellet. (As discussed in the review by Jaken
(Jaken, 1996
), it will be
important to determine the basis for this differential subcellular
distribution of PKC isoforms.) Interestingly, the pellet fraction that was
obtained in these experiments was highly enriched in cortical granules (as
judged by the presence of cgl) (Fig.
2) (Gundersen et al.,
2001
), and plasma membrane (on the basis of the presence of Na-K
ATPase immunoreactivity; C.G., unpublished). Thus, PKC
colocalized with
elements (cortical granules and plasma membrane) that are likely to harbor
substrates of potential importance for triggering of cortical granule
exocytosis. Moreover, we obtained no evidence that any of the other PKC
isoforms redistributed into this fraction in a PMA-dependent fashion. Taken
together, the fact that PKC
is found in a subcellular fraction enriched
in cortical granules and plasma membrane and that none of the other novel PKCs
are significantly present in this fraction supports the conclusion that
PKC
participates in initiating cortical granule exocytosis in these
cells.
The strongest support for the hypothesis that PKC initiates cortical
granule exocytosis is the observation that constitutely active PKC
triggers cgl secretion and that, even in some cases, over-expression of
wild-type PKC
leads to detectable secretion of cgl. In contrast,
high-level expression of wild-type or constitutively active PKC
did not
induce secretion of cgl. These latter results imply that there is sufficient
substrate specificity between PKC
and PKC
such that PKC
does not initiate secretion in this system. Alternatively, it may be that
PKC
does not have access to the appropriate protein substrates, whereas
PKC
does have access. In either case, these results indicate that
activity of PKC
is sufficient to elicit the regulated secretion of cgl in
these frog oocytes.
Relative to the seminal reports of PMA-induced cortical granule exocytosis
in Xenopus oocytes (Bement and
Capco, 1989; Bement and Capco,
1990
) there are two issues worth noting. First, in our experience,
this secretory event can be initiated using low nanomolar concentrations of
PMA. In contrast, Bement and Capco (Bement
and Capco 1989
; Bement and
Capco, 1990
) used 0.1-3 µM PMA, which clearly triggers this
exocytotic event, as well as other changes in these cells, such as cortical
contraction and cleavage furrow formation. It remains to be determined whether
PKC
also contributes to these other physiological events. A second issue
is that we observed a considerable disparity between the levels of protein
secretion from oocytes measured using absorbance at 280 nm relative to the
Bio-Rad assay. Indeed, our A280 results were similar to those of
Bement and Capco (Bement and Capco,
1989
), who reported that single oocytes secreted about 1.5 µg
of protein in response to PMA. (Here, it is noteworthy that for a cell as
large as these oocytes, one can calculate that if cgl was packed in a hollow
sphere 1 µm thick that was located immediately below the plasma membrane,
this sphere could encapsulate approximately 5 µg of protein (this is
obtained by subtracting the volume of a sphere of 1.298 mm from a sphere of
1.300mm and assuming that cgl has a density of 1.2 gm per cm3); the
fact that one detects secretion of 25-30% of this amount of protein, which
ignores the glycosylation of cgl, indicates that these granules are very
abundant in these cells.) A plausible explanation for our
A280-Bio-Rad disparity can be seen in the results in
Fig. 1, where the overall
intensity of Coomassie staining of secreted protein is higher in samples
treated with DTT. Since the Bio-Rad dye-binding assay is incompatible with
reducing agents, the lower apparent level of protein secretion is probably due
to diminished binding of dye to un-reduced protein samples. Nevertheless, our
electrophoretic data show that one can readily detect protein secreted from
single stage VI oocytes (whereas Bement and Capco pooled exudates from 80
oocytes) and that even stage II oocytes are competent to secrete protein, the
bulk of which is cgl.
A final comment concerns prior observations about PKC, which was first
identified as a novel PKC isoform whose mRNA was prominently expressed in
lung, skin and heart (Osada et al.,
1990
; Bacher et al.,
1991
). Subsequent work with PKC
-specific antibodies indicated
a high level of expression of this protein in cytosolic fractions of brain
(Zang et al., 1994
).
Curiously, a later investigation revealed that PKC
was almost exclusively
associated with membranes in brain and other tissues of mice
(Frevet and Kahn, 1996
). While
this apparent discrepancy in the subcellular distribution of PKC
in the
brain needs to be resolved, work using a mast cell line showed that
over-expression of PKC
enhanced secretory responses of these cells
(Chang et al., 1997
). Thus, it
will be interesting to assess further the role of PKC
in regulated
secretory events in oocytes, mast cells and elsewhere.
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Acknowledgments |
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