The influence of inorganic phosphate and ATP on the kinetics of bovine heart muscle pyruvate kinase

Summary The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/ mg (phosphate buffer, pH 7.6, 25 °C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.K_m(phosphoenolpyruvate) –0.04 mM, app.K_m(ADP) =0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the V_max without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in V_max was observed.The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app. K_m(P_i) = 20 mM and V_max = 167 units/ mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, P_i is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them.These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.Abbreviations PEP phosphoenolpyruvate - Pi inorganic phosphate - TEA triethanolamine - EDTA ethylenediaminetetraacetate     

Ribonucleoprotein complexes of pyruvate kinase from human skeletal muscle

Summary About 25% of total pyruvate kinase activity in muscle appears in a bound form which is insoluble in water or diluted salt solutions at pH 5.8. That activity is associated with the ribonuc-leoprotein complexes and is soluble at high ionic strength. A procedure is described for the purification and crystallization of this enzyme form herein called pyruvate kinase M_B and water soluble form M_A.Crystalline nucleoproteins are composed of active and inactive RNA-protein complexes with varying RNA content. By fractional crystallization and gel filtration a number of crystalline complexes were separated, two of them highly purified. One preparation was homogenous, contained 0.5% RNA and had a specific activity of 265 U/mg protein, the other one 10% RNA and 200 U/mg protein respectively.Forms M_A and M_B share the same protein as shown in immunodiffusion test with the anti-M_A sera. They differ in solubility and stability in diluted solutions. In Tris-HCl buffer, pH 7.6 form M_B is rapidly inactivated whereas form M_A is quite stable under the same conditions. Both forms have different K_m for phosphoenolpyruvate and ADP and V_max as well.Digestion of pyruvate kinase M_B with RNase was without marked effect on specific activity of the enzyme.The presence of numerous ribonucleoprotein complexes with a polynucleotide content in the range of 0.5 to 20% and specific activity of 160–220 U/mg protein suggests the control by RNA binding of pyruvate kinase activity from human skeletal muscle.This work was supported by a grant from the Biochemical and Biophysical Committee of the Polish Academy of Sciences.     

Isolation, purification and study of the properties of pyruvate kinase from the bovine adrenal cortex

Pyruvate kinase (ATP: pyruvate 2-0-phosphotransferase, EC 2. 7. 1. 40) from bovine adrenal cortex was purified 243 fold. The whole purification procedure included ammonium sulphate fractionation, heat treatment, Sephadex HW-55 chromatography and phosphocellulose chromatography. The specific activity of the preparation is 15.6 U/mg at 30 degrees C, the yield--36%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. The enzyme displayed a hyperbolic saturation curve with respect to P-enolpyruvate. The apparent Km for this substrate was 0.55 X 10(-4) M, pH optimum--6.8-7.0. K+ concentrations above 0.1 M inhibit the enzyme.     

The polysome as a terminal for the creatine phosphate energy shuttle

The role of the creatine phosphate shuttle in the energetics of muscle protein synthesis in isolated polysomes, from rat hindlimb muscle, was studied. Triton X-100-treated polysomes, following their centrifugation through a 1 M sucrose gradient, contained 38 mU/mg RNA of bound creatine kinase. In the presence of pH 5 enzyme (obtained from rat liver), 0.5 mM ATP, and 1 microM GTP, amino acid (leucine) incorporation by polysomes in the presence of 8 mM creatine phosphate was twice that in the presence of an exogenous ATP regenerating system of 10 mM phospho(enol)pyruvate and 10 U/ml pyruvate kinase. Since added creatine kinase had no effect on incorporation supported by creatine phosphate it is clear that endogenous creatine kinase allows sufficient regeneration of ATP. These data also suggest that nucleoside diphosphokinase must have been associated with the polysome for phosphate was transferred to GTP from [33P]creatine phosphate, and the specific activities of ATP and GTP increased at equal rates, reaching the specific activity of creatine phosphate at 8 min. We conclude that skeletal muscle polysomes have bound creatine kinase activity and they act as terminals for the creatine phosphate energy shuttle. Creatine phosphate regenerates GTP, probably through an intermediate reaction catalyzed by nucleoside diphosphokinase. This provided an added support for the hypothesis of compartmentation of enzymes and substrates and that the transport form of energy between the mitochondria and energy utilizing sites in muscle is creatine phosphate rather than ATP, which extends the general role of the creatine phosphate energy shuttle.     

Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.     

Purification and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.     

The control by RNA of pyruvate kinase activity in human skeletal muscle

Summary About 25% of total pyruvate kinase activity in human skeletal muscle is associated with the ribonucleoprotein complexes soluble in salt solutions of high ionic strength. These complexes, called form M_B, crystallize readily from 48% saturated ammonium sulfate at pH 5.6.Crystalline preparations represent a heterogenous population of ribonucleoprotein complexes displaying a graduated activity and a variable RNA content. Free protein was not detected in the preparations.Fractionation of crystalline complexes in salt solutions of varying ionic strength and pH, followed by gel filtration on Sephadex G-200 led to the separation of two nucleoprotein fractions with very high specific activity. Fractions containing 30% RNA and 85% RNA respectively revealed a specific activity of 660–670 U/mg protein at 25°C.Pyruvate kinase form M_A was extracted from muscle homogenate with distilled water, purified to homogeneity and crystallized. It contained less than 0.2% RNA and had a specific activity of 270 U/mg. Active ribonucleoprotein complexes gave in double immunodiffusion test the precipitation bands with the anti-M_A sera at the same protein concentration of both antigens, M_B and M_A.Pyruvate kinase M_B with high activity is sensitive to treatment with RNase. Digestion with RNase for 10 min at 25°C diminished the initial specific activity to about one third. Similar residual activity was found in crystalline ribonucleo protein complexes with low RNA content (3.5–20% RNA) which are resistant to further inactivation by RNase.These results implicate the enhancement and control of pyruvate kinase activity by RNA bound to the enzyme.This work was supported by a grant from the Biochemical and Biophysical Committee of Polish Academy of Sciences.     

Alteration in liver pyruvate kinase protein and catalytic activity upon starvation and refeeding

Liver pyruvate kinase (L-type isozyme) was purified from the livers of rats fed a high carbohydrate, low protein diet for 4 days. The protein was homogeneous as judged by polyacrylamide-gel electrophoresis with and without added sodium dodecyl sulfate and as judged by high speed sedimentation and low speed equilibrium centrifugation. The specific activity of the purified protein was 190–220 international units (IU)/mg. A precipitating antiserum directed specifically against liver pyruvate kinase was obtained from rabbits and was used to determine the amount of liver pyruvate kinase protein present in the 80,000g supernatant fraction of rat liver homogenates in response to the dietary status of the animal. Rats maintained on a high carbohydrate, low protein diet for 4 days prior to sacrifice have at least 20 mg of precipitable liver pyruvate kinase protein per liver. Starvation of the animal results in a marked reduction in liver pyruvate kinase so that by 3 days of starvation less than 7 mg of liver pyruvate kinase protein per liver remains. Refeeding the animal a high carbohydrate, low protein diet results in a return of the liver pyruvate kinase protein to the prestarvation level of 20 mg per liver. The liver pyruvate kinase activity per liver varies in the same direction as does the liver pyruvate kinase protein but does not parallel the change in protein. Animals fed a high carbohydrate, low protein diet for 4 days have 60–70 IU/mg of liver pyruvate kinase protein whereas animals starved for periods exceeding 30 h have greater than 100 IU/mg of liver pyruvate kinase protein. Refeeding starved animals with a high carbohydrate, low protein diet initially causes a large increase in activity per milligram of liver pyruvate kinase protein followed by a return of this value to the prestarvation level. The observed rise in the ratio of activity per milligram of liver pyruvate kinase protein during starvation suggests a modification in the enzyme protein resulting either in an increase in the specific activity of the enzyme or in a decrease in the affinity of the enzyme for the antibody.     

硅藻土在青霉素G酰化酶提纯中的应用

国产硅藻土经氢氧化铵处理后可用于从发酰液中直接取青霉素Ｇ酰化酶,平均吸附量为９０Ｕ／ｇ。吸附的酶可用２２％硫酸铵－０．３ｍｏｌ／ＬＰＢＳ（ｐＨ８．０）溶液洗脱。平均比１８Ｕ／１８Ｕｍｇ蛋白（ＮＩＰＡＢ法）。硅藻土可反复使用。苯乙酰胺－Ｓｅｐｈａｒｏｓｅ４Ｂ树脂可对酶作进一步的纯化。     

Acute hormonal control of pyruvate kinase and lactate formation in the isolated rat hepatocyte.

The activity of pyruvate kinase from the isolated rat hepatocyte was studied under conditions which allow investigation into the hormonal regulation of the enzyme. Incubating hepatocytes from fed or fasted rats with 1 μm glucagon gives approximately 60% inhibition of the enzyme activity determined at 1.6 mm P-enolpyruvate. A good correlation between the regulation of pyruvate kinase and lactate formation from 10 mm dihydroxyacetone is observed in hepatocytes from fasted rats. When hepatocytes are incubated in a Krebs-Ringer phosphate buffer, the inhibition of the pyruvate kinase activity by 1 μm glucagon is not accompanied by a marked inhibition of lactate production from fructose. Half-maximal regulation is observed at 0.26 ± 0.02 nm glucagon and 0.37 ± 0.05 nm glucagon for the enzyme and lactate formation from dihydroxyacetone respectively. Incubating hepatocytes with 10 mm l-alanine enhances inhibition of pyruvate kinase by physiological concentrations of glucagon, lowering the half-maximally effective concentration of glucagon from 0.3 nm to approximately 0.1 nm. A small but consistent inhibition of pyruvate kinase by 10 μm epinephrine is also observed and this inhibition is enhanced by 0.5 mm theophylline and by 10 mm l-alanine. The inhibition of pyruvate kinase by epinephrine both in the absence and presence of theophylline is blocked by the α-adrenergic antagonist phenoxybenzamine. The β-adrenergic blocker propranolol has no influence on the inhibition of the enzyme by epinephrine. Adenosine 3′:5′-monophosphate, N⁶O²-dibutyryl adenosine 3′:5′-monophosphate, and guanosine 3′:5′-monophosphate also inhibit glycolysis from dihydroxyacetone and modulate pyruvate kinase activity in hepatocytes from fasted rats. Oleate, ethanol, and 3-hydroxybutyrate inhibit dihydroxyacetone glycolysis, but they do not influence the activity of pyruvate kinase. The divalent metal ionophore A23187 slightly stimulates lactate synthesis from dihydroxyacetone, but it has no influence on pyruvate kinase activity.     

Isolation and various properties of alpha-aminocaprolactam hydrolase from Klebsiella aerogenes]

L-alpha-aminocaprolactam hydrolase possessing the L lysine amidase activity was isolated from Klebsiella aerogenes and purified. The procedure of enzymes purification included cell destruction on USDN-I, fractionation by ammonium sulfate, gel chromatography on G-200. The preparation of the purified enzyme possessed specific activity of 50 mumol of lysin per 1 mg of protein per hour. Km was 2.6 mM in case of phosphate buffer (ph 7.2) for I-alpha-aminocaprolactam. Besides L-alpha-aminocaprolactam the enzyme hydrolyses lysine amide, leucine amide tryptophanamide. Magnesium ions are necessary for manifestation of catalytic activity of the enzyme.     

Purification and Characterization of a Phosphoenolpyruvate Phosphatase from Brassica nigra Suspension Cells

Phosphoenolpyruvate phosphatase from Brassica nigra leaf petiole suspension cells has been purified 1700-fold to apparent homogeneity and a final specific activity of 380 micromole pyruvate produced per minute per milligram protein. Purification steps included: ammonium sulfate fractionation, S-Sepharose, chelating Sepharose, concanavalin A Sepharose, and Superose 12 chromatography. The native protein was monomeric with a molecular mass of 56 kilodaltons as estimated by analytical gel filtration. The enzyme displayed a broad pH optimum of about pH 5.6 and was relatively heat stable. Western blots of microgram quantities of the final preparation showed no cross-reactivity when probed with rabbit polyclonal antibodies prepared against either castor bean endosperm cytosolic pyruvate kinase, or sorghum leaf phosphoenolpyruvate carboxylase. The final preparation exhibited a broad substrate selectivity, showing high activity toward p-nitrophenyl phosphate, adenosine diphosphate, adenosine triphosphate, gluconate 6-phosphate, and phosphoenolpyruvate, and moderate activity toward several other organic phosphates. Phosphoenolpyruvate phosphatase possessed at least a fivefold and sixfold greater affinity and specificity constant, respectively, for phosphoenolpyruvate (apparent Michaelis constant = 50 micromolar) than for any other nonartificial substrate. The enzyme was activated 1.7-fold by 4 millimolar magnesium, but was strongly inhibited by molybdate, fluoride, zinc, copper, iron, and lead ions, as well as by orthophosphate, ascorbate, glutamate, aspartate, and various organic phosphate compounds. It is postulated that phosphoenolpyruvate phosphatase functions to bypass the adenosine diphosphate dependent pyruvate kinase reaction during extended periods of orthophosphate starvation.     

Purification and biochemical characterization of a pyruvate-specific class II aldolase, HpaI

HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.     

Purification and immobilization of acetate kinase from Desulfovibrio vulgaris

Summary The enzyme acetate kinase (EC 2.7.2.1) was purified fromDesulfovibrio vulgaris by a combination of ammonium sulfate precipitation, hydroxyl-apatite and dye-affinity chromatography. An overall-purification factor of 15 was obtained resulting in a specific activity of 24 U/mg protein. The purified enzyme was immobilized on differently derivatized controlled pore glass beads.     

Diaminopimelate decarboxylase of sporulating bacteria

The meso-diaminopimelate (DAP) decarboxylase of Bacillus licheniformis, a pyridoxal phosphate-requiring enzyme, was stabilized in vitro by 0.15 m sodium phosphate buffer (pH 7.0) containing 1 mm 2,3-dimercaptopropan-1-ol, 100 mug of pyridoxal phosphate per ml, and 3 mm DAP. When the meso-DAP concentration was varied, the enzyme in cell-free extracts of B. licheniformis exhibited Michaelis-Menten kinetics. Pyridoxal phosphate was the only pyridoxine derivative which acted as a cofactor. The enzyme was subject to both inhibition and repression by l-lysine. The inhibitory effect of lysine was on the K(m) (meso-DAP). A maximum repression of about 20% was obtained. No significant inhibition or activation was produced by cadaverine, dipicolinic acid, phenylalanine, pyruvate, ethylenediamine-tetraacetate, adenosine triphosphate, adenosine diphosphate, or adenosine monophosphate. When B. licheniformis was grown in an ammonium lactate-glucose-salts medium, an increase in DAP decarboxylase specific activity occurred during cellular growth with a maximal specific activity at the end of the exponential phase. As soon as growth ceased, the specific activity of the enzyme decreased to approximately one-half of the maximal specific activity and remained at this level thereafter. When B. cereus was grown in complex media, there was an increase in DAP decarboxylase specific activity up to the end of the exponential phase. Thereafter, the specific activity decreased to a nondetectable level in 4 hr. Dipicolinic acid synthesis was first detected 15 min later and was essentially complete after an additional 2.5 hr. The significance of the disappearance of DAP decarboxylase in B. cereus was discussed with regard to control of dipicolinic acid and spore mucopeptide biosynthesis.     

Induction of glycolytic enzyme synthesis in proliferating fibroblasts. Study of phosphofructokinase, glucose phosphate isomerase and pyruvate kinase.

Specific activity of phosphofructokinase is 7-8-fold higher in exponentially growing human fibroblasts than in quiescent cells, but the difference is considerably less pronounced for two other glycolytic enzymes, glucose phosphate isomerase and pyruvate kinase. The ratio of the F-type to L-type phosphofructokinase subunits is essentially the same in growing and resting cells, 4:1. F-type-phosphofructokinase-related antigen concentration is decreased in resting cells as compared with proliferating fibroblasts, but relatively less than the enzyme activity; the ratio of the enzyme activity to the antigen concentration (immunological specific activity) is therefore lower in resting than in growing fibroblasts. Synthesis of phosphofructokinase, as a percentage of the total protein synthesis, is about 30-fold greater during the proliferative phase than in quiescent cells, but this difference is only 3-4-fold for glucose phosphate isomerase and pyruvate kinase. Modulation of the synthesis of phosphofructokinase therefore seems to be responsible for the changes of its specific activity in function of cell proliferation. The appearance of some inactive cross-reacting material in quiescent cells is probably due to post-translational alteration of the pre-synthesized molecules. Compared with other glycolytic enzymes, such as glucose phosphate isomerase and pyruvate kinase, phosphofructokinase seems to be the (or one of the) preferential target of glycolytic induction in proliferating cells.     

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.     

Demonstration of a heterogeneous distribution of glycolytic enzymes and of pyruvate kinase isoenzymes types M1 and M2 in unfertilized hen eggs

The intracellular distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and the pyruvate kinase isoenzymes type M1 and type M2 within unfertilized hen eggs was studied. Most of glycolytic enzyme activities were found in the yolk fraction; 8-24% of total glycolytic enzyme activities were found in the vitelline membrane fraction. However, the specific activities of these enzymes in the vitelline membrane fraction are 19-72-fold higher (U/mg protein) and 45-178-fold more concentrated (U/g wet weight) than in the yolk fraction. The study of intracellular localization of pyruvate kinase isoenzymes shows that the blastodisc, latebra and vitelline membrane contain only pyruvate kinase type M2, whereas pyruvate kinase types M1 and M2 are found in the egg yolk. The exclusive occurrence of pyruvate kinase type M2 in the blastodisc is consistent with the concept that this isoenzyme is involved in the cell proliferation. The heterogeneous distribution of the glycolytic enzymes hexokinase, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase, and the heterogeneous localization of the pyruvate kinase isoenzymes types M1 and M2 indicate that glycolysis is distributed heterogeneously within the unfertilized hen egg cell.     

Control of pyruvate kinase activity during glycolysis and gluconeogenesis in Propionibacterium shermanii

The concentrations of glycolytic intermediates and ATP and the activities of certain glycolytic and gluconeogenic enzymes were determined in Propionibacterium shermanii cultures grown on a fully defined medium with glucose, glycerol or lactate as energy source. On all three energy sources, enzyme activities were similar and pyruvate kinase was considerably more active than the gluconeogenic enzyme pyruvate, orthophosphate dikinase, indicating the need for regulation of pyruvate kinase activity. The intracellular concentration of glucose 6-phosphate, a specific activator of pyruvate kinase in this organism, changed markedly according to both the nature and the concentration of the growth substrate: the concentration (7-10 mM) during growth with excess glucose or glycerol was higher than that (1-2 mM) during growth with lactate or at growth-limiting concentrations of glycerol or glucose. Other glycolytic intermediates, apart from pyruvate, were present at concentrations below 2 mM. Glucose 6-phosphate overcame inhibition of pyruvate kinase activity by ATP and inorganic phosphate. With 1 mM-ATP and more than 10 mM inorganic phosphate, a change in glucose 6-phosphate concentration from 1-2 mM was sufficient to switch pyruvate kinase from a strongly inhibited to a fully active state. The results provide a plausible mechanism for the regulation of glycolysis and gluconeogenesis in P. shermanii.