PMC:3248852 / 16960-27262 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3248852","sourcedb":"PMC","sourceid":"3248852","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3248852","text":"Discussion\nThe results presented here and elsewhere [2,3] indicate that progesterone binding to the catalytic subunit of the Na/K-ATPase at the oocyte plasma membrane initiates a sequence of changes in high energy phosphate compounds during the first meiotic division. In Rana pipiens oocytes, breakdown of the nuclear membrane occurs 8-10 hours after exposure to inducing levels of progesterone, followed by arrest at second meiotic metaphase at 15-16 hours (reviewed in [17]). An 80% increase in PCr precedes nuclear membrane breakdown accompanied by a marked decrease in pseudo first order rate constant (kf) for the PCr → ATP reaction (Figure 5). This increase in phosphoryl potential coincides with an increased phosphorylation of the phosphate-rich yolk protein, phosvitin, beginning at the onset of nuclear membrane breakdown (Figure 6).\n\nChanges in ATP utilization and plasma membrane surface area during the meiotic divisions\nThe decrease in kf shown in Figure 4 indicates a drop in cytosolic ADP concentration and an increase in the cytosolic phosphoryl potential of the oocyte prior to nuclear membrane breakdown (4 - 6 h). The decreased availability of ADP, normally generated in large part by hydrolysis of ATP during ion-transport by Na/K-ATPase, could arise from the observed internalization of the plasma membrane Na/K-ATPase (Na-pump) over the same time period [2]. This seems probable, based on our data showing a simultaneous 50 - 60% decrease in membrane capacitance (cell surface area), increased endocytosis [18], and the disappearance of more than 95% of the high affinity [3H]ouabain binding sites in progesterone-treated denuded Rana pipiens oocytes over the same time period [2].\n[3H]ouabain specifically binds to the α-subunit of the Na/K-ATPase (reviewed in [2,3]). By 6 h the [3H]ouabain is recovered bound to intracellular vesicles of progesterone-treated oocytes; it does not diffuse out of the progesterone-treated denuded oocytes [2]. Both scanning [18] and transmission (Figure 1) electron micrographs indicate that the prophase oocyte is covered with numerous microvilli. Estimates of the surface area in control oocytes reveal that the microvilli increase the oocyte surface area 10-12 times compared with that of a sphere of the same diameter [18]. Scanning electron micrographs reveal that the microvilli of progesterone-treated oocytes disappear coincident with the decrease in membrane capacitance, leaving only stumps on the oocyte surface [18].\n\nPhosphorylation of yolk platelet phosvitin\nAs seen in the 31P-NMR spectrum in Figure 2, phosvitin is the major phosphoprotein in the amphibian oocyte. Phosvitin is a glycosylated, serine-rich peptide with reported masses of 16-19 kDa, 25 kDa, or 31 kDa (reviewed in [19]). A single resonance (2.59 ppm) dominates the proton-decoupled 31P-NMR spectrum of Xenopus phosvitin [20]. Comparison of the phosvitin spectra with and without proton decoupling suggests a triplet splitting pattern for the major resonance, presumably due to coupling to methylene protons.\nRabinowitz and Lipmann [21] were the first to demonstrate reversible phosphate transfer between yolk phosphoprotein and ATP. Attempts were made to determine the equilibrium constant of the reaction between ATP and phosphoprotein. Figures varying from 20 to 50 were obtained for the forward reaction. However, their experiments indicated a non-homogenous phosphate population. The authors suggested that the \"thermodynamic potential of phosphoryl (groups) in phosvitin to be not far below that of ATP\". Mano and Lipmann [22] subsequently found that only more highly phosphorylated forms of phosvitin were good acceptors of phosphate from protein kinase and ATP. This suggests that a large fraction of the phosvitin serine phosphates do not turn over in situ. Our data indicate (Table 1) that only a small fraction of the serine phosphates in yolk phosvitin may be available for reversible phosphoryl exchange with ADP/ATP.\nPhosvitin also contains firmly bound, non-heme iron [23]. Grant and Taborsky [24] suggested that at alkaline pH, autoxidation of iron converts phosvitin-bound serine phosphate to the corresponding enol phosphate, an energy-rich structure. However, subsequent studies by Rosenstein and Taborsky [25] failed to find evidence for the production of a stable phosphoenol product and a demonstration of the stability of the C-H bond at the α-carbon of the oxidized residue further ruled it out. Their finding that phosphate release occurs by P-O bond cleavage is consistent with a mechanism by which an oxidatively generated carbonium ion derivative of phosphoserine is converted into a stable product by the direct formation of the free aldehyde and a monomeric metaphosphate ion, the latter reacting with water to yield inorganic orthophosphate. Rosenstein and Taborsky [25] proposed that yolk phosvitin would provide the developing embryo with a potential phosphorylating agent (HPO42-) which becomes activated by oxidation.\n\nProgesterone-induced protein phosphorylation\nPhosvitin phosphate turnover is minimal in control oocytes (Table 1) and the 15-fold increase in 32PO4 incorporation in progesterone-treated oocytes reflects the increased phosphate turnover in one phosphate per block of 5 serine phosphates in hormone stimulated oocytes, indicating that specific serine residues of highly phosphorylated species of yolk phosvitin are further phosphorylated in response to progesterone. This is consistent with the proposal by Williams and Sanger [26] that structures containing serine-phosphoserine blocks could serve as active sites in cellular metabolism.\nThe failure to observe a progesterone-induced increase in 32P-labelling of phosvitin in Xenopus laevis ovarian follicles by Maller et al. [27] may be due, in part, to dephosphorylation by endogenous ferrous/ferric ions during protein isolation. Additionally, Xenopus laevis ovaries contain only a small subpopulation of \"banded\" oocytes that are selectively released by gonadotropin [4]. Thus, increased phosvitin phosphorylation may only occur in a small subpopulation of Xenopus laevis oocytes that are exposed to progesterone. In comparison, 100% of the large Rana pipiens oocytes respond to, and are released from, the follicles by gonadotropin. The Xenopus phosphorylation studies should be repeated using the \"banded\" oocyte population.\nTaken in toto, our findings indicate that progesterone initiates a selective internalization of plasma membrane rich in Na/K-ATPase (Na-pump sites). This decrease in Na/K-pump activity coincides with plasma membrane depolarization and significantly reduces the ATP utilized for ion transport. Increased phosphorylation of the yolk protein phosvitin coincides with membrane depolarization and occurs just prior to the breakdown of the nuclear membrane. Our previous studies showed [6] that intracellular pH rises from 7.37 ± 0.01 (N = 6) in the prophase-arrested oocyte to 7.82 ± 0.03 (N = 5) during the first 3 h after exposure to progesterone, consistent with utilization of H+ during ATP formation via the creatine kinase reaction.\n\nPossible mechanism of progesterone-mediated increases in high energy phosphate compounds\nThe surface of the prophase amphibian oocyte [28], as well as that of most other cells [29], is studded with microscopic, flask-shaped invaginations called caveolae (~70 nm average outer diameter) that can either open for receiving and/or releasing material or close for processing and/or delivery to intracellular sites. Coinciding with the internalization of ouabain bound to the α-subunit of Na/K-ATPase, there is a net internalization of Rana oocyte plasma membrane and a disappearance of ouabain-sensitive K+-current [18]. Dersch et al. [30] report a similar progesterone-induced increase in cortical membrane trafficking in Xenopus laevis oocytes. By completion of the first meiotic division the cytoplasm of the progesterone-stimulated prophase oocyte becomes isopotential with the extracellular environment [31]. Xie and Askari [32] concluded from studies with cardiac cells that there are two pools of Na/K-ATPase with distinct but coupled functions. One is the classical pool in which the enzyme acts as an energy transducing ion pump and is localized in non-caveolar membranes. The other is the steroid -modulated, signal-transducing pool of the enzyme, which, through helix-helix interaction with membrane proteins called caveolins [33], is localized within the lipid rafts associated with the caveolae (reviewed in [34]). Our results suggest that progesterone acts to shift α-subunits from non-caveolar plasma membrane regions to the lipid rafts associated with caveolar membranes, followed by increased endocytosis of the caveolar vesicles. This is consistent with evidence indicating that the membrane regions containing \u003e95% of the Na/K-ATPase are selectively internalized over a 2-3 h period prior to nuclear membrane breakdown [2].\nThe α-subunit of Na/K-ATPase may thus cycle between the non-caveolar regions of the plasma membrane and the caveolar membranes. Our experiments indicate that decrease in the rate constant kf for the PCr → ATP reaction (Figure 6) coincides with internalization of the Na-pump. The decrease in rate constant must arise from reduced ADP availability and would result in an increased cytosolic phosphoryl potential. This increased phosphoryl potential would, in turn, contribute to enhanced phosphorylation of yolk phosvitin (Figure 7, Table 1).\n\nCation binding to yolk platelet phosvitin\nIn addition to non-heme iron [23], yolk phosvitin also contains Ca2+, Mg2+, Na+ and K+ [11]. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ [35]. A large fraction of the Rana oocyte Na+ was NMR-invisible and could be recovered in the yolk platelets [35]. During the first meiotic division there is a net increase in NMR-visible Na+; by completion of the second meiotic division (following fertilization), about 70% of the total Na+ becomes NMR-visible. Thus, phosvitin not only serves as a site for energy storage, but also as a storage site for iron and other ions essential for embryonic development in ponds and streams that contain little dissolved salts and minerals.\n","divisions":[{"label":"Title","span":{"begin":0,"end":10}},{"label":"Section","span":{"begin":846,"end":2486}},{"label":"Title","span":{"begin":846,"end":934}},{"label":"Section","span":{"begin":2488,"end":4991}},{"label":"Title","span":{"begin":2488,"end":2530}},{"label":"Section","span":{"begin":4993,"end":7106}},{"label":"Title","span":{"begin":4993,"end":5037}},{"label":"Section","span":{"begin":7108,"end":9488}},{"label":"Title","span":{"begin":7108,"end":7196}},{"label":"Section","span":{"begin":9490,"end":10301}},{"label":"Title","span":{"begin":9490,"end":9531}}],"tracks":[{"project":"2_test","denotations":[{"id":"22054214-16165176-9450421","span":{"begin":53,"end":54},"obj":"16165176"},{"id":"22054214-17936318-9450422","span":{"begin":55,"end":56},"obj":"17936318"},{"id":"22054214-16165176-9450423","span":{"begin":1379,"end":1380},"obj":"16165176"},{"id":"22054214-22080920-9450424","span":{"begin":1531,"end":1533},"obj":"22080920"},{"id":"22054214-16165176-9450425","span":{"begin":1702,"end":1703},"obj":"16165176"},{"id":"22054214-16165176-9450426","span":{"begin":1787,"end":1788},"obj":"16165176"},{"id":"22054214-17936318-9450427","span":{"begin":1789,"end":1790},"obj":"17936318"},{"id":"22054214-16165176-9450428","span":{"begin":1964,"end":1965},"obj":"16165176"},{"id":"22054214-22080920-9450429","span":{"begin":1983,"end":1985},"obj":"22080920"},{"id":"22054214-22080920-9450430","span":{"begin":2281,"end":2283},"obj":"22080920"},{"id":"22054214-22080920-9450431","span":{"begin":2482,"end":2484},"obj":"22080920"},{"id":"22054214-17314313-9450432","span":{"begin":2755,"end":2757},"obj":"17314313"},{"id":"22054214-2225770-9450433","span":{"begin":2861,"end":2863},"obj":"2225770"},{"id":"22054214-14435647-9450434","span":{"begin":3072,"end":3074},"obj":"14435647"},{"id":"22054214-5950403-9450435","span":{"begin":3568,"end":3570},"obj":"5950403"},{"id":"22054214-14220729-9450436","span":{"begin":4023,"end":4025},"obj":"14220729"},{"id":"22054214-5940939-9450437","span":{"begin":4048,"end":4050},"obj":"5940939"},{"id":"22054214-5461218-9450438","span":{"begin":4265,"end":4267},"obj":"5461218"},{"id":"22054214-5461218-9450439","span":{"begin":4837,"end":4839},"obj":"5461218"},{"id":"22054214-13651231-9450440","span":{"begin":5519,"end":5521},"obj":"13651231"},{"id":"22054214-885290-9450441","span":{"begin":5769,"end":5771},"obj":"885290"},{"id":"22054214-4109871-9450442","span":{"begin":6014,"end":6015},"obj":"4109871"},{"id":"22054214-6609721-9450443","span":{"begin":6854,"end":6855},"obj":"6609721"},{"id":"22054214-22080920-9450444","span":{"begin":7720,"end":7722},"obj":"22080920"},{"id":"22054214-12027880-9450445","span":{"begin":8034,"end":8036},"obj":"12027880"},{"id":"22054214-12606314-9450446","span":{"begin":8526,"end":8528},"obj":"12606314"},{"id":"22054214-16165176-9450447","span":{"begin":8943,"end":8944},"obj":"16165176"},{"id":"22054214-14220729-9450448","span":{"begin":9562,"end":9564},"obj":"14220729"},{"id":"22054214-3874869-9450449","span":{"begin":9770,"end":9772},"obj":"3874869"},{"id":"22054214-3874869-9450450","span":{"begin":9879,"end":9881},"obj":"3874869"}],"attributes":[{"subj":"22054214-16165176-9450421","pred":"source","obj":"2_test"},{"subj":"22054214-17936318-9450422","pred":"source","obj":"2_test"},{"subj":"22054214-16165176-9450423","pred":"source","obj":"2_test"},{"subj":"22054214-22080920-9450424","pred":"source","obj":"2_test"},{"subj":"22054214-16165176-9450425","pred":"source","obj":"2_test"},{"subj":"22054214-16165176-9450426","pred":"source","obj":"2_test"},{"subj":"22054214-17936318-9450427","pred":"source","obj":"2_test"},{"subj":"22054214-16165176-9450428","pred":"source","obj":"2_test"},{"subj":"22054214-22080920-9450429","pred":"source","obj":"2_test"},{"subj":"22054214-22080920-9450430","pred":"source","obj":"2_test"},{"subj":"22054214-22080920-9450431","pred":"source","obj":"2_test"},{"subj":"22054214-17314313-9450432","pred":"source","obj":"2_test"},{"subj":"22054214-2225770-9450433","pred":"source","obj":"2_test"},{"subj":"22054214-14435647-9450434","pred":"source","obj":"2_test"},{"subj":"22054214-5950403-9450435","pred":"source","obj":"2_test"},{"subj":"22054214-14220729-9450436","pred":"source","obj":"2_test"},{"subj":"22054214-5940939-9450437","pred":"source","obj":"2_test"},{"subj":"22054214-5461218-9450438","pred":"source","obj":"2_test"},{"subj":"22054214-5461218-9450439","pred":"source","obj":"2_test"},{"subj":"22054214-13651231-9450440","pred":"source","obj":"2_test"},{"subj":"22054214-885290-9450441","pred":"source","obj":"2_test"},{"subj":"22054214-4109871-9450442","pred":"source","obj":"2_test"},{"subj":"22054214-6609721-9450443","pred":"source","obj":"2_test"},{"subj":"22054214-22080920-9450444","pred":"source","obj":"2_test"},{"subj":"22054214-12027880-9450445","pred":"source","obj":"2_test"},{"subj":"22054214-12606314-9450446","pred":"source","obj":"2_test"},{"subj":"22054214-16165176-9450447","pred":"source","obj":"2_test"},{"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