Subcellular localization of hexose kinases in pea stems: mitochondrial hexokinase

The subcellular localization of hexose phosphorylating activity in extracts of pea stems has been studied by differential centrifugation and sucrose density gradient centrifugation. The hexokinase (EC 2.7.1.1) was associated with the mitochondria, whereas fructokinase (EC 2.7.1.4) was in the cytosolic fraction. Some properties of the mitochondrial hexokinase were studied. The enzyme had a high affinity for glucose (K_m 76 micromolar) and mannose (K_m 71 micromolar) and a relatively low affinity for fructose (K_m 15.7 millimolar). The K_m for MgATP was 180 micromolar. The addition of salts stimulated the activity of the hexokinase. Al³⁺ was a strong inhibitor at pH 7 but not at the optimum pH (8.2). The enzyme was not readily solubilized but, in experiments with intact mitochondria, was susceptible to proteolysis. A location on the outer mitochondrial membrane is suggested for the hexokinase of pea stems.     

A simple procedure to obtain yeast hexokinase free of glucosephosphate isomerase and mannosephosphate isomerase

A hexokinase preparation was obtained from aSaccharomyces cereviaiae mutant strain deficient in glucosephosphate isomerase (GPI) and mannosephosphate isomerase (MPI) by precipitation with ammonium sulfate. The supernatant fraction corresponding to 40 – 60 % saturation showed the lowest content in GPI and MPI activity. The fraction was used without further purification in the determination of glucose, either free or in a mixture with fructose and mannose. The results were similar to those obtained with pure commercial hexokinase.     

CHANGE IN THE ACTIVITY OF ARYLESTERASE IN SEA URCHIN EGGS FOLLOWING FERTILIZATION

The activity of arylcsterase in sea urchin eggs ( Anthocidaris craxsispina ), increases at 5 min after fertilization to about 1.5-fold that in unfertilized eggs, and decreases at 15 min to a lower level than that in unfertilized eggs. Then the activity of the enzyme increases again. The enzyme activity in unfertilized eggs is enhanced by either fructose 1, 6-diphosphate (FDP) at concentrations between 4 and 10 μM, or guanosine 3', 5'-cyclic monophosphalc (cGMP) at concentrations between 0.1 and 0.3 μM. The activity is detectable in the crude microsomal fraction and also in the supernatant fraction obtained from sea urchin egg homogenates by centrifugation at 105,000 × g for 2 hr.     

Characterization and compartmentation,in green leaves,of hexokinases with different specificities for glucose,fructose, and mannose and for nucleoside triphosphates

When green leaves of spinach (Spinacia oleracea L.) were surveyed for the presence of hexokinases which utilize glucose, fructose and-or mannose as a substrate, four kinases could be distinguished by their order of elution during chromatography on diethylaminoethyl (DEAE)-cellulose: (i) a hexokinase I with a specificity for fructose, glucose, and mannose, (ii) a fructokinase I with a specificity for fructose, (iii) a hexokinase II with a specificity for glucose, fructose and mannose, and (iv) a fructokinase II with a specificity for fructose. Hexokinases I and II had high apparent K_m values for fructose (8 and 15 mM, respectively) and medium or low apparent K_m values for glucose (150 and 18 μM, respectively) and mannose (18 and 15 μM, respectively). Maximal velocities were highest with fructose, medium with glucose and lowest with mannose. That hexokinases I and II used several sugars as substrate was concluded (i) from their identical elution profiles during enzyme separation and (ii) because their activities with two or three sugars at a time was always lower than the sum of activities with one substrate, indicating competition of the sugars for the reaction with the enzymes. Fructokinases I and II were very specific for fructose (85 and 140 μM, respectively) and had only little, if any, activity with glucose or mannose. All kinases showed varying degrees of activity with nucleoside triphosphates other than ATP. In the presence of all three sugars, hexokinases I and II were considerably more active with ATP than with uridine-, cytidine-, and guanosine 5'-triphosphate (UTP, CTP, GTP) except that, in the presence of glucose, hexokinase I was almost as active with UTP as with ATP. In the presence of fructose, fructokinase I exhibited highest activity with GTP and a gradually decreasing level of activity with CTP, UTP, and ATP. The activities in the presence of the other two sugars were highest with ATP. Fructokinase II was most active with ATP and fructose and progressively less active with GTP, UTP, and CTP. Cell fractionation by isopycnic density-gradient centrifugation or differential centrifugation indicated that fructokinase II was associated with chloroplasts, hexokinase II with mitochondria, and the other two kinases with the non-particulate cell fraction. In green leaves of pea (Pisum sativum L.), only a hexokinase (II) and fructokinase (II) were present. Corn (Zea mays L.) leaves exhibited only very low hexokinase activity. Dedicated to Prof. Dr. Hans Mohr on the occasion of his 60th birthday     

Change in the fructose 1,6-bisphosphatase activity in sea urchin eggs following fertilization.

The activity of fructose 1,6-bisphosphatase [EC 3.1.3.11] in sea urchin eggs decreased following fertilization. During the first 30 min after fertilization, the activity was considerably lower than that in unfertilized eggs, but by 30 min the activity was similar to that in unfertilized eggs. The enzyme activity in fertilized eggs, estimated in the presence of EGTA, was similar to that in unfertilized eggs. The activity in unfertilized eggs was reduced by Ca2+ at concentrations between 1 X 10(-5) M and 5 X 10(-3) M. Immediately after fertilization, the enzyme was insensitive to concentrations of Ca2+ lower than 2 X 10(-4) M, but the Ca2+ sensitivity of the enzyme recovered 30 min after fertilization. In the presence of Ca2+ at concentrations higher than 2 X 10(-4) M, the enzyme activity in unfertilized eggs was similar to that in fertilized eggs. Mg2+ restored the Ca2+-induced inhibition of fructose 1,6-bisphosphatase. 3-Phosphoglycerate and citrate hardly affected the enzyme activity, and AMP at concentrations above 10 mM inhibited it.     

Respiration of Sugars in Spinach (Spinacia oleracea), Maize (Zea mays), and Chlamydomonas reinhardtii F-60 Chloroplasts with Emphasis on the Hexose Kinases

The role of hexokinase in carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of 14CO2 from darkened spinach (Spinacia oleracea), maize (Zea mays) mesophyll, and Chlamydomonas reinhardtii chloroplasts externally supplied with 14C-labeled fructose, glucose, mannose, galactose, maltose, and ribose. Glucose and ribose were the preferred substrates with the Chlamydomonas and maize chloroplasts, respectively. The rate of CO2 release from fructose was about twice that from glucose in the spinach chloroplast. Externally supplied ATP stimulated the rate of CO2 release. The pH optimum for CO2 release was 7.5 with ribose and fructose and 8.5 with glucose as substrates. Probing the outer membrane polypeptides of the intact spinach chloroplast with two proteases, trypsin and thermolysin, decreased 14CO2 release from glucose about 50% but had little effect when fructose was the substrate. Tryptic digestion decreased CO2 release from glucose in the Chlamydomonas chloroplast about 70%. 14CO2 evolution from [1-14C]-glucose-6-phosphate in both chloroplasts was unaffected by treatment with trypsin. Enzymic analysis of the supernatant (stroma) of the lysed spinach chloroplast indicated a hexokinase active primarily with fructose but with some affinity for glucose. The pellet (membranal fraction) contained a hexokinase utilizing both glucose and fructose but with considerably less total activity than the stromal enzyme. Treatment with trypsin and thermolysin eliminated more than 50% of the glucokinase activity but had little effect on fructokinase activity in the spinach chloroplast. Tryptic digestion of the Chlamydomonas chloroplast resulted in a loss of about 90% of glucokinase activity.     

Two antigenically distinct forms of β-1,3-glucanase in sea urchin embryonic development

The enzyme β-1,3-glucanase is contained in the unfertilized eggs of most species of sea urchin. In some species, including Lytechinus variegatus, there is also substantial activity following gastrulation, and during remaining larval development. To determine if the same form of β-1,3-glucanase is present in both unfertilized eggs and after gut differentiation, an affinity purification procedure was utilized to isolate enzyme from unfertilized Lytechinus eggs. β-1,3-Glucanase is a 70,000-Da protein in this species, similar to the molecular weight of enzyme isolated from Strongylocentrotus purpuratus. Purified enzyme was used to generate an antibody that specifically recognized a 70,000-Da protein in unfertilized eggs by Western blot analysis, and stained the cortical granules of unfertilized eggs by immunofluorescence. The antibody also specifically immunoprecipitated β-1,3-glucanase activity from egg sonicates. The antibody was used to demonstrate that the form of β-1,3-glucanase present following gastrulation is antigenically distinct from the egg form. The 70,000-Da protein recognized by the antibody was no longer present by 24 hr, but embryos of this and later stages contained substantial amounts of activity, indicating the enzyme at these stages differs from the egg-specific form. In addition, the antibody was not capable of immunoprecipitating enzyme activity from pluteus sonicates. β-1,3-Glucanase has been partially purified from pluteus stage embryos, and appears to be a complex of approximately 200,000 Da. The enzyme is specific to endoderm and appears following differentiation of the gut, suggesting that it may function in larval digestion.     

12S and 9S RNA species. Localization and fate during development of Strongylocentrotus purpuratus eggs

Polyacrylamide gel electrophoresis of total RNA obtained from Strongylocentrotus purpuratus eggs and embryos have shown this material to contain a 12S and 9S RNA species, among others. These two species, found during early development of these cells, were not present in late prism or plutei stages. The RNA was extracted by cold phenol deproteinization in the presence of bentonite and precipitated by alcohol. RNAse, obtained from the 140 000 g egg-supernatant was tested against total RNA extracted from unfertilized eggs, blastulae, gastrulae and plutei embryos. The amount of nucleotides liberated was identical in all cases. Egg-RNAse, in the presence of plutei RNA, did not give rise to the 12S and 9S RNA species.     

Mechanism for Increase in Intracellular Concentration of Free Calcium in Fertilized Sea Urchin Egg : A Method for Estimating Intracellular Concentration of Free Calcium

Intracellular free calcium concentration in the sea urchin egg was calculated to increase from 0.1 mM in an unfertilized egg to 1 mM in a fertilized egg 10 min after fertilization, based on measurement of the dissociation constant between free calcium and sea urchin egg homogenate. The dissociation constant between free calcium (dialyzable calcium) and homogenate of sea urchin eggs was measured by means of dialysis equilibrium. The dissociation constant of the unfertilized egg was about 10^–4 M and that of the fertilized egg was about 10–3 M in three species of sea urchin, Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Pseudocentrotus depressus. An increase in the dissociation constant of the unfertilized egg homogenate was observed after the addition of calcium ion at a concentration above 0.3 mM, the dissociation constant becoming the same as that observed in the fertilized egg homogenate after the administration of CaCl₂ at a concentration above 1 mM. Sodium ion also caused a decrease in the calcium-binding ability of the unfertilized egg homogenate. Therefore, penetration of calcium ion or sodium ion upon fertilization might induce an increase in the dissociation constant and then intracellular concentration of free calcium would increase at fertilization. Almost all calcium-binding ability of the egg homogenate was found in the microsomal fraction, and the substance which bound calcium was thought to be protein in nature, since trypsin could decrease the level of calcium-binding substance in the homogenate of the eggs.     

γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

• 1.1. γ-Glutamyltranspeptidase is present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex.
• 2.2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min–6 hr) 37% increase in the activity of γ-glutamyltranspeptidase in homogenate fractions.
• 3.3. Relative to these homogenate levels, the specific activity of γ-glutamyltranspeptidase are ≈60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs.
• 4.4. The subcellular distribution of γ-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000 g particulate fractions, 22% in 40,000 g soluble fractions.
• 5.5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions.
• 6.6. In homogenates of Pisaster ochraceous larvae, γ-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Cloning, biochemical characterization and expression of a sunflower (Helianthus annuus L.) hexokinase associated with seed storage compounds accumulation

A full-length hexokinase cDNA, HaHXK1, was cloned and characterized from Helianthus annuus L. developing seeds. Based on its sequence and phylogenetic relationships, HaHXK1 is a membrane-associated (type-B) hexokinase. The predicted structural model resembles known hexokinase structures, folding into two domains of unequal size: a large and a small one separated by a deep cleft containing the residues involved in the enzyme active site. A truncated version, without the 24 N-terminal residues, was heterologously expressed in Escherichia coli, purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography and biochemically characterized. The purified enzyme behaved as a monomer on size exclusion chromatography and had a specific activity of 19.3 μmol/min/mg protein, the highest specific activity ever reported for a plant hexokinase. The enzyme had higher affinity for glucose and mannose relative to fructose, but the enzymatic efficiency was higher with glucose. Recombinant HaHXK1 was inhibited by ADP and was insensitive either to glucose-6-phosphate or to trehalose-6-phosphate. Its expression profile showed higher levels in heterotrophic tissues, developing seeds and roots, than in photosynthetic ones. A time course of HXK activity and expression in seeds showed that the highest HXK levels are found at the early stages of reserve compounds, lipids and proteins accumulation.     

Compartmentation of thymidine kinase in unfertilized sea urchin eggs and possible release of thymidine kinase from particulates in activated eggs

The thymidine kinase activity of homogenates of unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, in 1 M NaCl was always lower than that of homogenates of the unfertilized eggs in hypotonic media or homogenates of the fertilized or ammonia-activated eggs in 1 M NaCl by 30–50%. Sonication of the unfertilized egg homogenates in 1 M NaCl resulted in the elevation of thymidine kinase activity up to a level in the fertilized or ammonia-activated egg homogenates which is not affected by sonication. Differential centrifugation of unfertilized egg homogenates in 1 M NaCl revealed that the latent thymidine kinase is associated with the 1500g pellet or even with the 200g pellet. Exposure of the 1500g pellet to sonication, hypotonic media, 0.3% Triton X-100 in 1 M NaCl, and 2 M propyleneglycol resulted in the elevation of thymidine kinase, which was eventually shown to be no longer bound to the pellet fraction. Latent thymidine kinase was not detected in the 1500g pellet prepared from the fertilized egg homogenate in 1 M NaCl. These findings seem to suggest that thymidine kinase in unfertilized eggs may be sequestered, at least partly, in some large intracellular structures but may be released from them upon fertilization or ammonia activation, in accordance with our earlier observation on the apparent activation of thymidine kinase afer fertilization.     

INHIBITION OF DIHYDROFOLATE REDUCTASE BY PALMITOYL-CoA AND THE REVERSAL OF THE INHIBITION BY SPERMINE AND SPERMIDINE IN THE EGGS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Dihydrofolate reductase activity in fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, was almost the same as in unfertilized eggs. Aminopterin inhibited the enzyme competitively with dihydrofolate (FH₂). The apparent K_m value for FH₂ in the dihydrofolate reductase reaction was about 0.1 μM in the crude homogenate of both unfertilized and fertilized eggs. Dihydrofolate reductase in the eggs was also inhibited by palmitoyl-CoA. The inhibition was canceled by polyamines, especially by spermine, but putrescine failed to prevent the enzyme from the inhibition. The change in long-chain acyl-CoA and polyamine concentrations during fertilization are discussed as possible regulatory factors of the enzyme.     

Phosphatase active antigens in sea urchin eggs and embryos. II. A comparison between the activities in unfertilized eggs and plutei.

Phosphatase activities in sea urchin eggs and plutei were investigated by means of histochemical staining of immunoprecipitates. Two protein fractions were obtained by extraction in a hypotonic medium and by detergent treatment of the residual pellet. Three distinctly different phosphatase activities were discerned, nucleoside diphosphatase (EC 3.6.1.6.), acid phosphatase (EC 3.1.3.2.) and alkaline phosphatase (EC 3.1.3.1.). The nucleoside diphosphatase activity, which was confined to one antigen, was present in both water soluble and detergent extracts and at roughly the same concentration in eggs and plutei. By means of a monospecific antiserum the immunological identify of this antigen was established in all instances. The acid phosphatase activity, which was displayed by ten detergent extracted antigens in eggs, was only found in five detergent extracted antigens in plutei. This decrease in number of enzyme active antigens was also reflected by a general decrease in number of enzyme active antigens was also reflected by a general decrease in activity as assessed by quantitative determinations. Furthermore, by means of absorbed antisera it was established that two or three of the acid phosphatase active antigens were "egg specific". Another acid phosphatase active antigen, which was common to both developmental stages, was investigated by a monospecific antiserum. While this antigen was found in both soluble fractions, it was only enzymatically active when extracted with detergent. Alkaline phosphatase active antigens were only found in the detergent extract of plutei. However, immunoprecipitates with this activity appeared both with antiserum against unfertilized eggs and with antiserum against plutei. This suggests that the egg contained the antigens in an enzymatically inactive form.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

Studies on pituitary follitropin. III. Functional role of the amino groups in subunit and receptor interactions of the ovine hormone.

The effectiveness of Glc, mannose, 2-deoxyglucose, fructose, galactose, arabinose, and N-acetylglucosamine at protecting rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) from inactivation by chymotrypsin, glutaraldehyde, heat, and Ellman's reagent have been compared. The relative effectiveness at protecting against these inactivating agents decreases in the order Glc > mannose > 2-deoxyglucose > fructose, galactose, and arabinose, the last three providing no significant protection at all. The nonphosphorylatable substrate analog, N-acetylglucosamine, provides substantial protection against heat inactivation, but is ineffective against inactivation by the other agents. Similar inactivation studies were conducted using several hexose 6-phosphates. Glc-6-P and 1,5-anhydroglucitol-6-P provided substantial protection while 2-deoxyglucose-6-P, fructose-6-P, mannose-6-P, and galactose-6-P were all relatively ineffective at protecting hexokinase activity. The protective effect of these ligands is taken as an indication of ligand-induced conformational changes in brain hexokinase. The results are interpreted in terms of, and considered to support, a recently proposed model (J. E. Wilson, 1978, Arch. Biochem. Biophys.185, 88–99) in which the suitability of a carbohydrate as a substrate depends directly on its ability to induce specific conformational changes prerequisite for subsequent catalytic events while the inhibitory effectiveness of a hexose 6-phosphate is likewise related to its ability to evoke appropriate conformational change in the enzyme. Synergistic interactions between hexose and hexose-6-P binding sites, first reported for Glc and Glc-6-P by Ellison et al. (1975, J. Biol. Chem.250, 1864–1871), have been confirmed. Thus, although fructose and galactose were themselves quite ineffective at providing protection against inactivation of hexokinase by chymotrypsin or glutaraldehyde, they greatly increased the protection afforded by suboptimal (with respect to degree of protection in the absence of added hexose) levels of Glc-6-P. Conversely, the 6-phosphates of fructose, galactose, mannose, and 2-deoxyglucose, which were themselves ineffective at protecting the enzyme activity, markedly enhanced the protection provided by suboptimal levels of Glc or mannose. Based on the relationship between this enhancement of Glc-dependent protection and the hexose-6-P concentration, the dissociation constants for the complexes of hexokinase with 2-deoxyglucose-6-P and mannose-6-P were estimated to be ?0.5 mm.     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

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.     

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⁺.