CHANGE IN THE ACTIVITY OF PYRUVATE DEHYDROGENASE COMPLEX IN SEA URCHIN EGGS FOLLOWING FERTILIZATION*

The activity of the pyruvate dehydrogenase complex in sea urchin eggs is localized in the crude mitochondrial fraction. The activity of the enzyme complex in the intact mitochondrial fraction of unfertilized eggs is too low to be estimated and is enhanced upon fertilization with a 5-min lag period. The activity of the enzyme complex in unfertilized eggs is enhanced by Ca²⁺at concentrations between 5 × 10^?5 M and 10^?3 M. The activity in fertilized eggs is blocked after incubation with 2 mM ATP, and the block of the activity is also released by Ca²⁺. The blockage of the enzyme complex activity is accompanied by phosphorylation of proteins, and release of the block by Ca²⁺ is concomitantly followed by the dephosphorylation of proteins in the mitochondrial fraction. The enzyme complex in unfertilized eggs will be assumed to be the one inhibited by phosphorylation. The enzyme complex will be activated upon fertilization as a consequence of the dephosphorylation, that is caused by the increase in intracellular concentration of Ca²⁺.     

Ovostatin,an endogenous trypsin inhibitor of sea urchin eggs: Purification and characterization of ovostatin from eggs of the sea urchin,Strongylocentrotus intermedius

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC₅₀ (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10^?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10^?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10^?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca²⁺ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.     

Factors which may affect removal of protamine from sperm DNA during fertilization in the rabbit

In order to derive information about possible mechanisms by which the sperm head is converted into the male pronucleus during fertilization in the rabbit, unfertilized egg homogenate was assayed for two enzyme activities. Protamine was extracted from rabbit sperm, purified, and labelled with [¹⁴C] in an in vitro reaction and used as a probe to assay for a protein kinase which could transfer [³²P]PO₄ from [γ-³²P]ATP onto the substrate. A kinase with a pH optimum of approximately 8.0 to 8.5 is described. Assays for the enzyme glutathione reductase were performed using homogenates from eggs or embryos at three early stages of development. Results suggest that oocytes can oxidize 2.58 × 10^?6 μmol NADPH per minute per oocyte, unfertilized eggs 5.16 × 10^?7 μmol NADPH per minute per ovum, and 20- to 24-hour postcoitus fertilized eggs 2.30 × 10^?6 μmol NADPH per minute per ovum. The relevance of these observations to male pronuclear formation is discussed.     

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10^?6 M and 10^?5 M). At higher concentrations of VLB (5×10^?5 M and 10^?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.     

The periodic change in adenosine 3′,5′-monophosphate concentration in sea-urchin eggs

The level of adenosine 3′,5′-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0·10^?7 M and 1.5·10^?6 M, respectively. The activity of adenylate cyclase in a 105 000 × g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl₂ increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5·10^?6 M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Phosphate transport in fertilized sea urchin eggs. II. Effects of metabolic inhibitors and studies on differentiation

Metabolic inhibitors were applied after the transport system was fully developed in concentrations sufficient to block cleavage. 0.5–1.0 × 10^?4 M cyanide and anaerobiosis caused from negligible to moderate (40%) inhibition of phosphate uptake. The inhibition occurred late in the breeding season, and the inhibitory action of cyanide on uptake was associated with irreversible developmental effects. Azide (3 × 10^?3 M) did not inhibit uptake when the chamber method was used, but the aliquot and Hopkins' tube methods gave considerable inhibition. Purified preparations of 2,4-dinitrophenol (1 × 10^?4 M) did not inhibit uptake. Sodium iodoacetate (up to 0.05 M) and phlorizin (0.005 M) exerted no effect. Calculations of the minimal work requirement for the transport process reveal that this amounts to only a small fraction (0.24% at an external phosphate concentration of 2 μM) of the total available metabolic energy. Exposure of eggs at five minutes after insemination (lag phase) to cyanide (5 × 10^?5 M), anaerobic conditions, or azide (3 × 10^?3 M) blocked the expected increase of phosphate uptake. Removal of the inhibitors led to resumption of development and the appearance of the phosphate transport system in an essentially normal pattern. Exposure of eggs to 1.4–2.0 × 10^?4 M p-chloromercuribenzoate (p-CMB) during the accumulation phase severely depressed phosphate uptake, but cleavage was not inhibited nor delayed; recovery from the inhibition was accelerated by 1 × 10^?3 M cysteine. Exposure to p-CMB during the lag phase blocked the appearance of the transport system; cleavage proceeded normally. After the removal of p-CMB little reversal occurred until the addtion of 1 × 10^?3 M cysteine, when the phosphate transport system developed in an essentially normal manner. Trypsin (0.001–0.01%) neither activates the transport system in unfertilized eggs, nor inactivates it in denuded fertilized eggs by removal of surface proteins. The data are consistent with the conclusion that (1) the phosphate transport system is newly synthesized at fertilization in energy dependent reactions, and (2) phosphate transport is a carrier mediated process not directly dependent on metabolic energy.     

Fertilization-induced changes in concanavalin A binding to mouse eggs

The binding of concanavalin A (ConA) to zona-free unfertilized and fertilized mouse eggs has been investigated using tritiated ConA. At low lectin concentrations (1–5 μg ml^?1) the fertilized egg shows a higher affinity for [³H]ConA than does the unfertilized egg. In saturation conditions, however, unfertilized and fertilized eggs show the same binding capacity (1.55 × 10⁸ ConA molecules/egg). The results indicate that ConA-binding sites change qualitatively following fertilization; possible connections between this change and other fertilization-induced changes in the egg surface are discussed.     

Some features of the vinblastine-induced assembly of porcine tubulin.

Porcine tubulin precipitated by 10^?3, m vinblastine (VLB) contains approximately 0.50 molecule of VLB bound per 110,000-molecular-weight tubulin dimer. The amount of precipitate, followed by turbidity, is a linear function of the initial tubulin concentration. The rate of precipitation is roughly first order in protein concentration. Vindoline and velbanamine halves of VLB are ineffective separately or together in producing the tubular aggregates observed for VLB precipitates by electron microscopy. At 10^?3, m concentrations no turbidity is observed nor is there any competition with VLB-induced turbidity. Removal of GTP from tubulin by dialysis or incubation of tubulin in the absence of added GTP blocks VLB-induced assembly. Readdition of GTP at room temperature or above restores sensitivity to VLB precipitation. The β,γ methylene analog of GTP cannot substitute for GTP in this process. About 0.7 mol of added GTP is found bound per mole of tubulin dimer. During the course of VLB-induced assembly, roughly half of this GTP is displaced. These results show interesting similarities and differences in the VLB-induced assembly of tubulin and the normal in vitro assembly of microtubules. Further comparisons between both assembly processes should be useful.     

Pool sizes of the deoxynucleoside triphosphates in the sea urchin egg and developing embryo.

The four deoxynucleoside triphosphate pools in unfertilized eggs of L. pictus and S. purpuratus were measured and found to be very large, ranging from 10^?3 to 10^?2 pmoles per egg. The high levels of the individual dNTP pools are sufficient for one to eight rounds of DNA synthesis. During the first division cycle these pools fluctuate with the highest levels being attained prior to DNA synthesis. The pools then decrease just preceding or during the S period. There is a large reduction in the total cellular dNTP in later stages of development when DNA synthesis is reduced relative to the cleavage stages.     

Action of trypsin on the eggs of Dendraster excentricus

Crystalline trypsin in 3 × 10^?8 M concentration and higher, elicits fertilization membranes in the unfertilized eggs of Dendraster excentricus. These membranes are adequate in artificial parthenogenesis. If the action of trypsin on these eggs is continued for two or three hours the result is first, digestion of the membranes, followed later by reduction of the egg to amoeboid form. When fertilized, some of the partially digested eggs segment and form irregular cell masses, thus demonstrating that, in response to trypsin, there is first the cortical reaction giving rise to the fertilization membrane, and second, the progressive digestion and disintegration of the cytoplasm.Chymotrypsin causes rounding of the unfertilized eggs and, in rare instances, a few membranes, but the enzyme is not an adequate parthenogenetic agent.Fertilization of the egg renders the cytoplasm resistant to trypsin. The facts lead to the suggestion that fertilization liberates trypsin inhibitors in the cytoplasm.     

Double-sides sticking mechanism of vinblastine interacting with α,β-tubulin to get activity against cancer cells

Abstract

Vinblastine (VLB) and its derivatives have been used for clinical first-line drugs to treat various cancers. Due to the resistance and serious side effects from using VLB and its derivatives, there is a need to discover and develop novel VLB derivatives with high activity against cancer cells. In order to better discover and develop new VLB derivatives, we need to study the structural basis of VLB's anti-cancer cytotoxicity and the mechanism of its interaction with α,β-tubulins. Based on the crystal structure of α,β-microtubule complex protein, the molecular dynamics method including the sampling PMF method was used to study the variation of dissociation free energy (ΔG) of α,β-tubulins under different system conditions, and then from which to study the mechanism of the interaction between VLB and α,β-tubulins. The obtained results show that the dissociation of pure α,β-tubulins requires 197.8?kJ·mol^?1 for ΔG. When the VLB molecule exists between the interface of α,β-tubulins, the dissociation ΔG of α,β-tubulins reaches 220.5?kJ·mol^?1, which is greater than that of pure α,β-tubulin. The VLB molecule is formed by connecting a vindoline moiety (VM) molecule with a catharanthine moiety (CM) molecule through a carbon-carbon bond, which is a larger molecule. When the CM molecule exists in the middle of α,β-tubulin interface, the dissociation ΔG of α,β-tubulins is 46.2?kJ·mol^?1, during which the CM moves with β-tubulin. When the VM molecule exists between the middle of α,β-tubulin interface, the dissociation ΔG of α,β-tubulins is 86.7?kJ·mol^?1, during which it moves with α-tubulin. Therefore, the VLB molecule is like a double-sides tape to stick α-tubulin and β-tubulin together. The VLB molecule intervenes the dynamic equilibrium between dissociation and aggregation of α-tubulin and β-tubulin by a double-sides sticking mechanism to exert high activity with toxicity against cancer cell. Besides, our results demonstrate that VLB has its structural basis for anticancer cytotoxicity due to its two compositions composed of a CM molecule and a VM molecule although they have little toxicity against cancer cell alone.     

ON THE RATE OF OXYGEN CONSUMPTION BY FERTILIZED AND UNFERTILIZED EGGS : V. COMPARISONS AND INTERPRETATION

1. The rate of oxygen consumption by eggs may not merely undergo no change at fertilization, as in the case of the starfish, but it decreases to about half in Chaetopterus and in Cumingia. 2. The absolute rate of oxygen consumption in mm.³ O₂ per hour per 10 mm.³ eggs differs widely in several species of unfertilized eggs. It is very low in the sea urchin, intermediary in Nereis, and high in Chaetopterus and Cumingia. The range for these eggs is approximately 0.4 to 3.1 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C., in the ratio of about 1:8. 3. The absolute rates of oxygen consumption by the same fertilized eggs are much more nearly the same. They lie within the range 1.3 to 2.0 mm.³ O₂ per hour per 10 mm.³ eggs at 21°C., in the ratio of approximately 1:1.5. Within this same range lie the values obtained by a number of investigators using a variety of eggs of invertebrates from several phyla. Amoeba proteus and frog skin also are within this range (see Fig. 2). 4. The changes in rate of oxygen consumption at fertilization by the different species of eggs, differing both in direction and magnitude, appear to be such as to bring the rate, when development is initiated, to about the same rate, which is also the rate of other comparable normally growing cells. 5. The direction and magnitude of the change in rate at fertilization therefore appears in the cases cited to be primarily a function of the absolute rate of oxygen consumption by the unfertilized eggs, which are characterized in their peculiar inhibited condition, among other things, by a wide range of respiratory rates. 6. It is not to be supposed that this range of rates will apply at all universally to eggs, especially to eggs of extremes in proportional content of inert materials, such as large yolky eggs. Fish and amphibian eggs for example respire at a much lower rate per unit volume. The effect on surface: volume ratios attending extremes of cell size might also be expected to shift the absolute rate. 7. The absolute rate of oxygen consumption by the eggs of the alga Fucus vesiculosus is considerably higher than the rates of the animal eggs measured. It is of the same order of magnitude as the rates of several other small-celled algae, which respire at a greater rate per unit volume than most non-motile animal cells. 8. The comparatively high rates of oxygen consumption by the inhibited (unfertilized) eggs of Chaetopterus and Cumingia are not directly associated with nuclear or morphological activity of the cell since they continue at the high rate for hours after cessation of the brief initial nuclear activity, which takes place when the eggs are placed in sea water. 9. It is concluded that the rate of oxygen consumption is not necessarily and probably not generally the limiting factor which causes inhibition of the unfertilized egg. Increase in rate of oxygen consumption is not directly related to the initiation of development, in general, nor even necessarily concomitant. It is not improbable that the low rate of oxygen consumption is an immediate part of the cause of inhibition of the unfertilized sea urchin egg, but this is a special case. 10. This thesis, that the rate of oxygen consumption is not necessarily nor ordinarily the limiting factor in the inhibition of the unfertilized egg, and conversely that increase in the rate of oxygen consumption is not usually the essential feature of fertilization, is quite in agreement with the general relations between the rate of oxygen consumption on the one hand and anesthesia, growth, and development on the other in fertilized eggs and other organisms. 11. This conclusion is opposed to Loeb''s explanation of the essential feature of fertilization, as an increase in oxidation rate or more strictly to generalization of his hypothesis to include eggs other than those of the sea urchins (or of other similar special cases which may be discovered). It extends to fertilization (the initiation of development) his and Wasteney''s well established conclusion that "oxidation is not the independent variable in development." 12. It is suggested that the crux of the problem of fertilization lies in the nature of the inhibition of the unfertilized egg. Certain similarities between this condition, arrived at spontaneously in the case of the egg cell, and the condition of cells in narcosis or anesthesia are pointed out. 13. Although the rate of oxygen consumption by the unfertilized eggs of Chaetopterus and Cumingia cannot be regarded as the limiting factor which causes the inhibition of the eggs, in these and other cases with different absolute rates, it appears highly probable that the rate of oxygen consumption is in some way, at present obscure, tied up with or related to the condition of inhibition. This seems probable especially in view of the sharp change in rate which in most cases immediately attends cessation of the inhibition, but the relationship may be a non-causal one, as in narcosis. 14. It must be borne in mind that oxygen consumption is not necessarily a complete measure of oxidation, and that other measures such as of heat and metabolite production are necessary before the complete amount of oxidation is known. When these are completely worked out, if free energy relations are known, it is probable that more direct and inclusive relations may be found between oxidation, growth, development, and anesthesia. Generalization of Loeb''s hypothesis, using "oxidation" in the broad sense might then turn out to hold, with fertilization fitting into the general scheme, but there is no basis for it at the present time.     

Respiratory functions involved in the induction of puffs in Drosophila salivary glands

Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various substances affecting oxidative phosphorylation. After an incubation time of 1–3 h changes in puff size and in cellular ATP level were registered. 10^?6 M trinactin, 10^?5 or 10^?4 M oligomycin both induce puff 63BC together with some other puffs and reduce the cellular ATP level by about 80–90%. The trinactin-dependent puff induction can be inhibited, if the medium is supplemented with 10^?3 M ATP or 10^?3 M ITP or 10^?6 M antimycin or 10^?2 M KCN. The effect of exogenous ATP is prevented by adding 10^?6 M oligomycin to the incubation mixture; 10^?6 M oligomycin alone, however, has no inductive effect on 63BC. The presence of exogenous ITP, furthermore, prevents the ATP level from being reduced by trinactin. 10^?4 M atractyloside lowers the ATP level by about 75 %, whereas a puff induction cannot be observed. The same is true for various concentrations of KCN. It is concluded that ATP itself is not involved directly in the regulation of puff activity but that it acts on a phosphorylating reaction that can be inhibited by oligomycin.     

Inhibition of ribosomal peptidyltransferase with cytidyl-3' leads to 5'-[2'(3')-O-L-phenylalanyl]-L-adenosine.

The title compound 1e, obtained by chemical synthesis, is an inhibitor of E. coli ribosomal peptidyltransferase. A 50% inhibition of peptidyltransferase-catalyzed N-Ac-Phe-puromycin formation at puromycin concentration 1 × 10^?4 M with 70 S ribosome-poly U-N-Ac[¹⁴C-Phe-tRNA complex occurred at 5 × 10^?4 M of 1e. In contrast, the parent compound 2′(3′)-O-L-phenylalanine-L-adenosine (1b) is a much weaker inhibitor causing only 5% inhibition at 1 × 10^?3 M. Alkaline hydrolysis of compound 1e to cytidylyl-3′→5′-L-adenosine (1c) results in a greatly diminished inhibition which, however, exceeds that of 1b by a factor of two. The inhibition of peptidyltransferase with 1e can be reversed by puromycin. The latter effect levels off at 40% inhibition.     

Mechanism of the involvement of monoamine oxidase in the regulation of (Na+ + K+)-ATPase in rat brain

Increasing concentrations of dopamine fail to give a biphasic response to (Na⁺ + K⁺)-ATPase activity in various subcellular fractions of rat brain preincubated with monoamine oxidase inhibitors, viz. 1·10^?4 M clorgyline and 1·10^?4 M deprenyl. The product of the monoamine-oxidase-catalysed reaction with dopamine as substrate is 3-methoxy-4-hydroxyphenylacetaldehyde. An analogue of this product is 3-methoxy-4-hydroxybenzaldehyde. This analogue, when incubated with the subcellular fractions which had been preincubated with monoamine oxidase inhibitors and dopamine, gave a more pronounced biphasic response to (Na⁺ + K⁺)-ATPase activity than that observed in the fractions incubated with dopamine alone.     

Changes in the organization of the mouse egg plasma membrane upon fertilization and first cleavage: Indications from the lateral diffusion rates of fluorescent lipid analogs

Using the technique of fluorescence photobleaching recovery, we have measured the lateral diffusion of the lipid analogs 3,3′-diacylindocarbocyanine iodides of acyl chain lengths, n = 14 to 22 in the plasma membranes of unfertilized and fertilized CD-1 mouse eggs and two-cell embryos. Mean diffusion coefficients ranged from (3.4 to 7.9) × 10^?9 cm²/sec and mean percentage diffusing ranged from 65 to 82%. Within 10% diffusing fractions are independent of chain length and unaffected by fertilization or first cleavage. In unfertilized and fertilized eggs the diffusion coefficients show relative maxima at n = 16 and 20. In two-cell embryos the relative maximum at n = 16 is suppressed. As was observed by D. E. Wolf, W. Kinsey, W. Lennarz, and M. Edidin (1981, Develop. Biol.81, 133) on sea urchin eggs, the effect of fertilization on diffusion coefficients is chain length dependent. Little change is observed for n = 14 and 18; a slight increase is observed for n = 16; and small decreases are observed for n = 20 and 22. This is interpreted in terms of a reordering of the lipid domains in the plasma membrane upon fertilization and first cleavage.     

A NOTE ON THE RESPIRATION OF ARBACIA EGGS

Methods used in preparing Arbacia eggs for respiration studies, in carrying through the manometric determinations, and in estimating egg quantities have been reexamined. Discrepancies in previous results are almost entirely due to a steady error in measuring egg volume by centrifuging. Volumes so obtained averaged 80 per cent too high. The respiration of unfertilized eggs of Arbacia punctulata at 21°C. is 0.9 c.mm. O₂ per hour per 10 c.mm. of eggs.     

Repair of a genetically-caused defect in oogenesis in Drosophila melanogaster by transplantation of cytoplasm from wild-type eggs and by injection of pyrimidine nucleosides

Eggs produced by homozygous mutant rudimentary (r⁹, 154.5) females of Drosophila melanogaster die during embryogenesis, apparently because the mutant female fails to incorporate certain substances into the egg during oogenesis. These eggs can be rescued by injecting them at the preblastoderm stage with wild-type nuclei and cytoplasm or wild-type cytoplasm alone from unfertilized eggs. Some preblastoderm eggs injected with 1.5% of egg volume of cytoplasm from unfertilized wild-type eggs were able to complete both embryonic and postembryonic development and emerged as adults, whereas not a single uninjected control egg was able to complete embryonic development. The eggs of rudimentary mothers can also be rescued by injecting each egg at the blastoderm stage with 0.01 μg of pyrimidine nucleosides. The results demonstrate that a pyrimidine deficiency is the cause of abortive embryogenesis, and confirm the previous finding that the rudimentary mutation leads to pyrimidine auxotrophy.     

Inhibition of protein synthesis on homologous and heterologous desensitization in the rat adipocyte

Desensitization of lipolysis was induced in isolated rat adipocytes by incubation with isoproterenol 10^?5M or ACTH 250 mU/ml for two and three hours, respectively. Those cells desensitized with isoproterenol were restimulated with either isoproterenol 10^?7M or ACTH 6 mU/ml and those cells desensitized with ACTH were restimulated with isoproterenol 10^?7M. Lipolysis was quantitated by the release of cyclic AMP and glycerol. No effect on either homologous or heterologous desensitization was observed when either cycloheximide 2 μg/ml or puromycin 10^?4M was included in the incubation media during the induction of desensitization. These findings support the conclusion that protein synthesis plays no role in the desensitization of lipolysis in the isolated rat adipocyte.     

Sodium-potassium exchange in sea urchin egg. I. Kinetic and biochemical characterization at fertilization

Biochemical and kinetic characteristics of the Na⁺-K⁺ exchange were studied in Paracentrotus lividus eggs. Measurement of the ⁸⁶Rb uptake shows that ouabain-sensitive ⁸⁶Rb uptake is dramatically stimulated within the first minute following fertilization. The Na⁺-K⁺ pump-mediated K⁺ entry presents a maximal rate at 8 min postfertilization and then decreases to reach a plateau within 30 min. We assess that the steep rise in cell K⁺ occurring at fertilization (J.P. Girard, P. Payan, C. Sardet, Exp. Cell. Res. 142:215–221, 1982) does not originate from a net entry of external K⁺. Measured 30 min postfertilization, the half-maximal activation by K⁺ of the ouabain-sensitive Na⁺-K⁺ exchange is 5–6 mM and the ouabain lC₅₀ is 5.10^?5 M. Egg cortices from unfertilized and fertilized eggs show comparable Na⁺-K⁺ ATPase activity with a 50% ouabain-sensitive fraction. V_m and K_m for Na⁺ and K⁺ of the enzyme are of the same order of magnitude in cortices of unfertilized and fertilized eggs. Cortical Na⁺-K⁺ ATPase from unfertilized eggs shows a ten fold increase of activity between pH 6.7 and pH 7.7. The results strongly suggest that the plasma membrane of unfertilized eggs contains a preexisting Na⁺-K⁺ transporting system which is obligatorily stimulated at fertilization.