Values For Phosphoenolpyruvate Research Articles

Regulation of Maize (Zea mays L.) Phosphoenolpyruvate Carboxylase Isoenzymes by pH

Summary The activity of phosphoenolpyruvate carboxylase (PEPC) isoenzymes from maize leaves is regulated by pH. The S0.5 values for phosphoenolpyruvate (PEP) and Mg2+ ions decrease for both isoenzymes at pH below 7.5 and above 8.5. The Hill coefficient values for PEP are about 1.0 at pH above 8.0 for both isoenzymes, however, they increase to values higher than 1.0 with the decrease of pH below 7.5 (mainly with the PEPC I isoenzyme). The Hill coefficient values for Mg 2+ are pH-independent. The pK values of the amino acid groups present in the active site of the protein molecule of both PEPC isoenzymes are determined from the dependence of the enzyme's activity on the concentration of hydrogen ions. The pK values of about 7.3, 8.5 and 9.0 indicate that histidine, cysteine and lysine residues, respectively, are present in the active centre of both isoenzymes.

Purification and molecular and kinetic properties of phosphoenolpyruvate carboxylase from Amaranthus viridis L. leaves

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) was purified 43-fold from Amaranthus viridis leaves by using a combination of ammonium-sulphate fractionation, chromatography on O-(diethylaminoethyl)-cellulose and hydroxylapatite, and filtration through Sepharose 6B. The purified enzyme had a specific activity of 17.1 μmol·(mg protein)(-1)·min(-1) and migrated as a single band of relative molecular weight 100000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A homotetrameric structure was determined for the native enzyme. Phosphoenolpyruvate carboxylase from Zea mays L. and A. viridis showed partial identity in Ouchterlony two-dimensional diffusion. Isoelectric focusing showed a band at pI 6.2. Km values for phosphoenolpyruvate and bicarbonate were 0.29 and 0.17 mM, respectively, at pH 8.0. The activation constant (Ka) for Mg(2+) was 0.87 mM at the same pH. The carboxylase was activated by glucose-6-phosphate and inhibited by several organic acids of three to five carbon atoms. The kinetic and structural properties of phosphoenolpyruvate carboxylase from A. viridis leaves are similar to those of the enzyme from Zea mays leaves.

Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 m m glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogenous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with M r 49,000–55,800. K m values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 8 μ m, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate ( K i = 0.17 μ m). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.

Alfalfa Root Nodule Carbon Dioxide Fixation

A nonphotosynthetic phosphoenolpyruvate carboxylase (EC 4.1.1.31) was partially purified from the cytosol of root nodules of alfalfa. The enzyme was purified 86-fold by ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite chromatography, and reactive agarose with a final yield of 32%. The enzyme exhibited a pH optimum of 7.5 with apparent K(m) values for phosphoenolpyruvate and magnesium of 210 and 100 micromolar, respectively. Two isozymes were resolved by nondenaturing polyacrylamide disc gel electrophoresis. Subsequent electrophoresis of these isozymes in a second dimension by sodium dodecyl sulfate slab gel electrophoresis yielded identical protein patterns for the isozymes with one major protein band at molecular weight 97,000. Malate and AMP were slightly inhibitory (about 20%) to the partially purified enzyme. Phosphoenolpyruvate carboxylase comprised approximately 1 to 2% of the total soluble protein in actively N(2)-fixing alfalfa nodules.

Phosphoenolpyruvate Carboxylase of Escherichia coil. Inhibition by Various Analogs and Homologs of Phosphoenolpyruvate1

In an attempt to investigate the topography of the catalytic site of phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of Escherichia coli, the inhibitor constants (Ki) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme. The Ki values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme. The Ki value for L-phospholactate (0.30 mM) was almost equal to the Km value for PEP (0.25 mM), whereas that for D-phospholactate (0.89 mM) was about 3-fold larger than the Km value. It was presumed that PEP binds with the enzyme on its si-side. Among 6 PEP homologs, the Ki values for phosphoenol alpha-ketobutyrate (0.024 mM) and phosphoenol alpha-ketovalerate (0.034 mM) were about one-tenth the Km value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur. DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a Ki value of 2.20 mM.

Short-term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes.

Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271-278] was further investigated. The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively. Half-maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h. Dexamethasone acted both as a long-term and short-term modulator. Long-term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin-independent basal glycolytic rate. In short-term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half-maximal dose of dexamethasone was 10 nM. The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left-shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.

Purification and properties of pyruvate kinase from Mycobacterium smegmatis

Pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from Mycobacterium smegmatis has been purified to homogeneity through a seven-step procedure with a yield of 16% and specific activity of 220 units/mg protein. The purified enzyme had a molecular weight of 230,700 and was composed of four subunits with identical molecular weights of 57,540. Analysis of amino acid composition revealed a low content of aromatic amino acids. The enzyme exhibited sigmoidal kinetics of varying concentrations of phosphoenolpyruvate, the degree of cooperativity and S 0.5 v value for phosphoenolpyruvate being strongly dependent on the pH of the reaction mixture. Among the nucleoside diphosphates acting as substrate for pyruvate kinase, ADP was the best phosphate acceptor, as judged by its lowest K m value. The enzyme showed an absolute requirement for divalent cations (either Mg 2+ or Mn 2+), but monovalent cations were not necessary for activity. Other divalent cations inhibited the Mg 2+-activated enzyme to varying degrees (Ni 2+ > Zn 2+ > Cu 2+ > Ca 2+ > Ba 2+). The differences in the kinetic responses of the enzyme to Mg 2+ and Mn 2+ are discussed.

Purification and properties of pyruvate kinase from Halobacterium cutirubrum

Pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from Halobacterium cutirubrum has been purified by neutral salt precipitation, exclusion, hydrophobic and hydroxyapatite chromatography. The main band, as determined by SDS-polyacrylamide gel electrophoresis, migrates with a molecular weight of 73000. Sepharose CL-4B gel filtration yielded a peak of maximal activity corresponding to a tetramer of M r 300000. The enzyme is highly unstable at less than 3 M NaCl or KCl, and cannot be renatured. Optimum stability occurs at pH 6.5 and room temperature. Maximum velocities are influenced by both cations and anions, in the following order: K + >Rb + >Cs + ⋍Na + >NH 4 + >Li + and Cl − >Br − >NO 3 >SCN − = 0. At 3 M salt, the apparent K 0.5 values for phospho enolpyruvate are identical for KCl, RbCl, CsCl and NH 4Cl (2 mM), and greater in the case of NaCl and KBr (7 mM). When KCl and KBr concentrations are varied, the apparent K 0.5 values vary similarly, showing a maximum at 2 M and diminishing towards 3 M salt. This effect can be explained by the formation of inactive complexes of K + and mutual depletion of ions and substrates. The enzyme displays homotropic cooperativity in the presence of phospho enolpyruvate. It is activated by ribose 3-phosphate, fructose 6-phosphate and adenosine 5′-monophosphate, and inhibited by fructose 1,6-diphosphate, 6-phosphogluconate and inorganic phosphate.

Purification and properties of pyruvate kinase type M 2 from rat lung

(1) Pyruvate kinase type M 2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of M r = 224 000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M 2, excepts that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K 0.5 value for phospho enolpyruvate is 0.26 mM ( n H = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM ( n H = 1.06). 1 μM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phospho enolpyruvate half-maximally. The K m value for ADP at 1 mM phospho enolpyruvate is 0.4 mM. The K m value for other nucleoside diphosphates increases in the order ADP<GDP<IDP<UDP. (3) No evidence for an interconversion of pyruvate kinase type M 2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M 2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047–1055) we suggest that the interconvertible form of pyruvate kinase type M 2 may represent a separate form of the pyruvate kinase type M 2 family.

The role of cations in avian liver phosphoenolpyruvate carboxykinase catalysis. Activation and regulation.

The activation of the avian liver phosphoenolpyruvate carboxykinase catalyzed reversible decarboxylation of oxalacetate by Mn2+ has been studied. The Mn2+ facilitates the interaction of oxalacetate to the enzyme. At saturating ITP and oxalacetate concentrations, Mn2+ has a Km = 2 microM. The cation Mg2+ can substitute for Mn2+ with 50% Vmax in the oxalacetate decarboxylation and 2% Vmax in the oxalacetate formation. The Km for Mg2+ is 3 orders of magnitude greater than the Km of Mn2+, however. Of the other cations tested (Ca2+, Zn2+, Fe2+, and Cd2+), Co2+ was the only other cation found to activate the enzyme in both directions. In the presence of Mg2+, the enzyme is extremely sensitive to trace metal contaminants which can cause activation. At 1 mM Mg2+, 20 microM Mn2+ causes a 15-fold activation of activity. The apparent Km for Mn2+ (2 microM) at high concentrations of Mg2+ is the identical value calculated for free Mn2+. In a mixed metal (Mg2+ and Mn2+) assay, the Km values for phosphoenolpyruvate and for oxalacetate are independent of the concentration of Mg2+ but decrease upon an increase in Mn2+. The kinetic results demonstrate two roles for the divalent cations for activity. The cation forms a metal . nucleotide complex which serves as the substrate. The results indicate that MgITP is a better substrate than MnITP, but MnIDP is a better substrate than MgIDP. The cation also binds to the enzyme to form an enzyme . metal complex which is the active form of the enzyme. This cation functions to aid in the interaction of either oxalacetate or phosphoenolpyruvate to the enzyme. The activation by micromolar amounts of Mn2+ at millimolar concentrations of Mg2+ suggests that this enzyme, and thus the pathway of gluconeogenesis, can be modulated by changes in concentration of Mn2+ within mitochondria.

Some kinetic properties of pyruvate kinase from Trypanosoma brucei: Influence of pH and fructose-1,6-diphosphate

The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The K m for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a p K a of 6.5–6.7. The cooperative interaction of the multiple phospho enolpyruvate (PEP) binding sites is independent of pH in the range 5.7–7.8. Variation of the V max value with pH indicates the presence of a dissociated group (p K a 6.2–6.3) and of an undissociated group (p K a 7.5–7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S 0.5 value for this substrate, as is an undissociated enzyme group with a p K a in the range 6.7–7.0. It is shown that PEP and fructose-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme. FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S 0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed.

Purification and characterization of pyruvate kinase from Trypanosoma brucei

Pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been partially purified by carboxymethylcellulose chromatography, and gel filtration. The enzyme is unstable in aqueous solution and requires the presence of a thiol protecting reagent as well as glycerol for the maintenance of activity. Dithiothreitol activates as well as stabilizes the enzyme. Phosphoenolpyruvate allosterically activates trypanosome pyruvate kinase whereas hyperbolic kinetics are found when ADP is the variable substrate. Mg 2+ or Mn 2+ ions and a monovalent cation are essential for enzyme activity. Fructose 1,6-diphosphate acts as a heterotropic allosteric activator, markedly decreasing the S 0.5 value for phosphoenolpyruvate from 1.34 to 0.25 m m at 1 m m fructose 1,6-diphosphate and transforms the phosphoenolpyruvate saturation curve from a sigmoidal to a hyperbolic form. The enzyme has a pH optimum of 6.5–7.0 and a molecular weight of 270,000 ± 27,000 as estimated by gel chromatography. Purine nucleotides are the preferred coenzymes for the reaction, having much lower K m values than the pyrimidine nucleotides. The possible role of pyruvate kinase in the regulation of glycolysis in T. brucei is discussed.

Regulation of lactose catabolism in Streptococcus mutans: purification and regulatory properties of phospho-beta-galactosidase

Phospho-beta-galactosidase (P-beta-gal), the enzyme which catalyzes the first step in the metabolism of intracellular lactose phosphate, occurred at high specific activity in the cytoplasm in 12 of 13 strains of streptococcus mutans grown on lactose but not other carbon sources. The P-beta-gal from S. mutans SL1 was purified 13-fold using diethylaminoethyl-cellulose ion exchange and agarose A--0.5 M molecular exclusion column chromatography. The molecualr weight of the enzyme was estimated to be 40,000, and its pH optimum was 6.5 in three different buffer systems. P-beta-gal activity was inhibited by Co2+, Zn2+, and Cu2+, but other cations, ethylenediaminetetraacetic acid, orthophosphate, and fluoride had no effect upon enzyme activity. The kinetic response of P-beta-gal to a model substrate, o-nitrophenyl-beta-D-galactopyranoside-6-phosphate, obeyed Michaelis-Menten kinetics, and the Km for this substrate was 0.19 mM. In addition to being under genetic control, P-beta-gal activity was regulated by a number of biologically active metabolites. Enzyme activity was inhibited in a sigmoidal fashion by phosphoenolpyruvate. The M 0.5 V value for phosphoenolpyruvate was 2.8 mM, and the Hill coefficient (n) was 3. In addition, P-beta-gal exhibited strong inhibition by ATP, galactose-6-phosphate, and glucose-6-phosphate. In contrast to inhibition of P-beta-gal activity by phosphoenolpyruvate, the inhibition exerted by ATP, galactose-6-phosphate, and glucose-6-phosphate obeyed classical Michaelis-Menten kinetics; the Ki values for these inhibitors were 0.55, 1.6, and 4.0 mM, respectively.

Difference in the Effect of Glucagon and Starvation upon l‐Type Pyruvate Kinase from Rat Liver

The mechanism of the glucagon action upon pyruvate kinase in isolated hepatocytes from fed or starved rats has been investigated. Glucagon incubation of hepatocytes increases the reaction responsible for pyruvate kinase inactivation. This inactivation reaction is completely dependent upon the presence of adenosine 3′: 5′‐monophosphate (cAMP), (half maximal activation at 0.1 μM cAMP) with cells incubated without glucagon, while with cells incubated with glucagon cAMP addition only has a marginal effect. It is concluded that glucagon acts by activation of the protein kinase responsible for pyruvate kinase inactivation. Glucagon addition to isolated hepatocytes from fed and starved rats causes a similar decrease in pyruvate kinase activity of 40.7 ± 2.5% and 36.6 ± 3.6%, respectively, when measured at suboptimal phosphoenolpyruvate concentrations. Although optimal concentrations. of glucagon (1 μM) are used during the cell incubations, the pyruvate kinase activity can be inactivated further in vitro by incubation under conditions for optimal protein kinase activity. From the kinetic curves for the substrate phosphoenolpyruvate, obtained before and after further incubation in vitro, it can be concluded that pyruvate kinase by its presence inside the cell is protected against a complete inactivation by glucagon. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from glucagonincubated cells versus cells incubated without glucagon disappears after further incubation of the supernatants of these cells in vitro under optimal conditions for protein kinase activity (plus 10 μM cAMP) and an enzyme with a low affinity for phosphoenolpyruvate is obtained (K0.5= 4.4 mM). These results indicate that the glucagon‐induced change in kinetic properties of pyruvate kinase can be adequately explained by an increased phosphorylation state of pyruvate kinase. The difference in apparent affinity of pyruvate kinase for phosphoenolpyruvate from fed liver versus starved liver (P &lt; 0.001) still persists after further incubation of the liver supernatants under optimal conditions for protein kinase activity in vitro (P &lt; 0.001). For the completely inactivated enzyme from livers of fed and starved rats K0.5 values for phosphoenolpyruvate of respectively 2.8 and 4.4 mM are obtained. These results indicate that the nature of the effect of starvation differs from that of glucagon. It is suggested that the effect of starvation might be caused by partial proteolysis of the enzyme or oxidation of essential thiol groups.

Phosphoenolpyruvatcarboxykinase aus Phycomyces blakesleeanus Bgff. / Phosphoenolpyruvate Carboxykinase in Phycomyces blakesleeanus Bgff.

Phosphoenolpyruvate carboxykinase (PEPCK) from Phycomyces blakesleeanus was partially purified by protamine sulfate precipitation, ammoniumsulfate precipitation, and diethylamino ethyl cellulose (DEAE) treatment. This preparation was employed for the characterization of the enzyme. The Km values for phosphoenolpyruvate (PEP) and ADP were determined as 1.6 and 0.42 mM. The nucleotid specificity was demonstrated for ADP exclusively. The use of sulfuryl reagents showed the presence of thiol groups sensitive against p-hydroxymercuribenzoate but not effected by N-ethylmaleimide.

Kinetic studies with l-type pyruvate kinase from rats either fed a high carbohydrate, low protein diet or starved

Rat liver pyruvate kinase (ATP : pyruvate 2- O-phosphotransferase, EC 2.7.1.40) was purified to homogeneity from animals fed a high carbohydrate, low protein diet prior to killing and from animals starved 72 h prior to killing. The liver pyruvate kinase purified from the fed rats always has a specific activity around 220 International Units per mg of protein, whereas the maximum specific activity observed with enzyme purified from the starved rats was 80 units/mg. On polyacrylamide gel isoelectric focusing carried out in the absence of fructose-1,6-bisphosphate, the protein from fed rats resolved into one major band at pH 5.8–6.0 and into an apparent doublet at pH 5.2–5.3. The majority of the protein from starved rats focused into a broad band at pH 5.8–6.0. When isoelectric focusing was carried out in the presence of 5μM fructose-1,6-bisphosphate, both proteins focused at pH 5.2–5.3. The enzymes isolated from both fed and starved rats differed in three kinetic parameters. Firstly, in the difference in specific activities. Secondly, inthe absence of fructose-1,6-bisphosphate the K 0.5 value for phospho enolpyruvate was 0.96 mM for the enzyme from fed rats and was 1.6 mM for the enzyme from starved rats. In the presence of 1 mM fruxtose-1,6-bisphosphate the K 0.5 was 0.15 mM phospho enolpyruvate for both enzymes. Thirdly, at a phospho enolpyruvate concentration of 0.1 mM the K 0.5 values for fructose-1,6-bisphosphate were 0.05 μM for the enzyme from fed rats and 0.13 μM for the enzyme from starved rats. The enzyme from fed rats and from starved rats yielded the same kinetic values for ADP ( K m = 0.4 mM), for phospho enolpyruvate ( K m = 0.15 mM) in the presence of 1 mM fructose-1,6-bisphosphate, for fructose-1,6-bisphosphate ( K m = 1 μM) in the presence of 10 mM l-alanine, and for l-alanine ( K m = 10 mM), when assayed with 1 μM fructose-1,6-bisphosphate and 0.1 mM phospho enolpyruvate.

Purification and Properties of Phosphoenolpyruvate and Ribulose Diphosphate Carboxylases from C4 and C3 Plants

Summary Ribulose diphosphate (RuDP) and phosphoenolpyruvate (PEP) carboxylases were partially purified from leaves of three C4 plants: Setaria, Pennisetum and Amaranthus and two C3 plants: Oryza and Rumex. RuDP carboxylase from C4 plants had a lower Km for RuDP and HCO3− than that of the enzyme from C3 type. Their reaction velocities were similar in all cases, reflecting the potential of RuDP carboxylase in C4 plants as well as in C3 plants. PEP carboxylase from C4 plants possessed higher Km values for PEP and Mg++ than that from C3 plants, but the Km for HCO3− was similar in both the categories. The maximum reaction velocities of PEP carboxylase from C4 plants were nearly 15–20 times higher than those from C3 plants, suggesting the basically different nature of PEP carboxylases from C3 and C4 plants. It is envisaged that in C4 plants PEP carboxylase helps in rapid fixation of CO2 into C4 acids at a faster rate under atmospheric CO2 concentration and these C4 acids move into bundle sheath and are decarboxylated creating a much higher concentration of CO2 than outside. At thus elevated levels of CO2 in bundle sheath of C4 plants RuDP carboxylase operates effectively.

Physical Properties of Phosphoenolpyruvate Carboxylase and Malic Enzyme in Grape Berries

Phosphoenolpyruvate (PEP) carboxylase and malic enzyme were extracted from 2-to-3-week-old 9Carignane9 grape berries. The K_m values for PEP and L-malate as substrates for PEP carboxylase and malic enzyme activities were respectively 0.09 and 0.45 m<i>M</i>. Fifty percent inhibition of PEP carboxylase activity occurred with the addition of 35 m<i>M</i> malate.

Physical Properties of Phosphoenolpyruvate Carboxylase and Malic Enzyme in Grape Berries

Phosphoenolpyruvate (PEP) carboxylase and malic enzyme were extracted from 2-to-3-week-old 9Carignane9 grape berries. The K_m values for PEP and L-malate as substrates for PEP carboxylase and malic enzyme activities were respectively 0.09 and 0.45 m<i>M</i>. Fifty percent inhibition of PEP carboxylase activity occurred with the addition of 35 m<i>M</i> malate.

Biochemical Studies on the Shell Gland of Japanese Quail, Coturnix coturnix japonica: 2. Allosteric two Components and their Catalytic Properties of Pyruvate Kinase in the Shell Gland1

Pyruvate kinase (EC 2.7.1.40) in Japanese quail shell gland was examined for the multiple components and enzymic properties. Two components were obtained by isoelectric focusing. Isoelectric points of the two components were pH 4.5 (designated SGPK-I) and pH 6.1 (designated SGPK-II), respectively. In the shell gland mucosa, the existence of only SGPK-II was detected. SGPK-I and SGPK-II were eluted from DEAE-cellulose by 0.02M and 0.12M KC1, respectively at pH 7.4 (20mM Tris-HCl). In the absence of fructose-1,6-diphosphate (FDP), SGPK-I and SGPK-II, showed a sigmoidal response with respect to phosphoenolpyruvate (PEP) concentration, while in the presence of FDP, they both exhibited the Michaelis-Menten type response. Kinetical responses of SGPK-I and SGPK-II were markedly influenced by pH, K+ and Mg2+ concentration even in the presence of FDP. No significant difference in apparent Km value for PEP of the two components was obtained. The inhibition of the two components by ATP was pH-dependent and was reversed by the presence of FDP. The two components from the shell gland were inhibited by Ca2+. The inhibition by Ca2+ was dependent on pH, K+, and Mg2+ concentration. FDP altered the Km value for PEP several folds smaller, of the two components inhibited by Ca2+, but did not change the Vmax. During the egg formation process, the ratio of the summed activity of SGPK-I to that of SGPK-II varied from 0.5 (minimum) to 2.0 (maximum), depending on the position of a developing egg in the oviduct. Physiological significance of the two components in the shell gland is discussed.