Effects of Mn2+ on the exchange reaction of phosphoenolpyruvate carboxykinase in the presence of high concentrations of Mg2+

The mitochondrial phosphoenolpyruvate carboxykinase (GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32), purified from chick embryo liver, was synergistically activated by a combination of Mn2+ and Mg2+ in the oxaloacetate ---- H14CO-3 exchange reaction. Increases in the Mg2+ concentration caused decreases in the K0.5 value of Mn2+ in line with the earlier finding that the enzyme was markedly activated by low Mn2+ (microM) plus high Mg2+ (mM). In the presence of 2.5 mM Mg2+, increases in the Mn2+ level first enhanced the activity of phosphoenolpyruvate carboxykinase, and then suppressed it to the maximal velocity shown in the presence of Mn2+ alone. Kinetic studies showed that high Mn2+ inhibited the activity of Mg2+ noncompetitively, and those of GTP and oxaloacetate uncompetitively. The inhibition constant for oxaloacetate (K'i = 550 microM) was lower than that of Mg2+ (Ki = K'i = 860 microM) or GTP (K'i = 1.6 mM), and was nearly equal to the apparent half-maximal inhibition concentration of Mn2+. These results suggested that Mn2+ can play two roles, of activating and suppressing phosphoenolpyruvate carboxykinase activity in the presence of high Mg2+.     

Phosphoenolpyruvate carboxykinase assayed at physiological concentrations of metal ions has a high affinity for CO2.

The effect of Mn2+/Mg2+ concentration on the activity of intact, homogeneous phosphoenolpyruvate carboxykinase (PEPCK) from leaves of the C4 grass, Guinea grass (Panicum maximum), have been investigated. Assay conditions were optimized so that PEPCK activity could be measured at concentrations of Mn2+/Mg2+ similar to those found in the cytosol (low micromolar Mn2+ and millimolar Mg2+). PEPCK activity was totally dependent on Mn2+ and was activated at low micromolar concentrations of Mn2+ by millimolar concentrations of Mg2+. Therefore, at physiological concentrations of Mn2+, PEPCK has a requirement for Mg2+. Assay at physiological concentrations of Mn2+/Mg2+ led to a marked decrease in its affinity for ATP and a 13-fold increase in its affinity for CO2. The Km (CO2) was further decreased by assay at physiological ATP to ADP ratios, reaching values as low as 20 microM CO2, comparable with the Km (CO2) of ribulose 1,5-bisphosphate carboxylase-oxygenase. This means that PEPCK will catalyze a reversible reaction and that it could operate as a carboxylase in vivo, a feature that could be particularly important in algal CO2-concentrating systems.     

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.     

Purification and characterization of phosphoenolpyruvate carboxykinase from the parasitic helminth Ascaris suum

Phosphoenolpyruvate carboxykinase has been purified from homogenates of Ascaris suum muscle strips to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purification is a three-step procedure which yields pure enzyme in milligram quantities with good yield. The subunit molecular weight of the Ascaris enzyme is between 75,000 and 80,000. The native molecular weight is 83,000 as determined by gel filtration. The kinetic constants for substrates of the carboxylation reaction were determined and compared to those measured for the avian liver enzyme. From kinetic studies it appears likely that two separate roles for divalent metal ions exist in the catalytic process. Studies conducted with Mn2+ or with micromolar concentrations of Mn2+, in the presence of millimolar concentrations of Mg2+ suggest that Mn2+ but not Mg2+ binds directly to and activates the enzyme while either Mn2+ or Mg2+ may bind to the nucleotide resulting in the metal-nucleotide complex. The metal-nucleotide is the active form of the substrate for the reaction. In the presence of Mg2+, an increase in the Mn2+ concentration results in a decrease in the Km for P-enolpyruvate suggesting a direct role for Mn2+ stimulation and regulation of activity. The concentrations of Mn2+ and Mg2+ in Ascaris muscle strips were determined by atomic absorption spectroscopy and support the proposed hypothesis of a specific Mn2+ activation of the enzyme. The nucleotides ATP and ITP act as competitive inhibitors against GTP with KI values of 0.50 and 0.75 mM, respectively. ITP is a competitive inhibitor against both IDP and P-enolpyruvate, suggesting overlapping binding sites for the two substrates on the enzyme.     

Purification and properties of mitochondrial phosphoenolpyruvate carboxykinase from liver of Squalus acanthias

Liver from Squalus acanthias (spiny dogfish), a representative elasmobranch, contains approximately 1.4 units (mumol/min) of phosphoenolpyruvate carboxykinase activity per gram and approximately 90% of the total units of activity are localized in the mitochondria. The mitochondrial phosphoenolpyruvate carboxykinase was isolated and characterized. The purified enzyme has properties generally similar to those found in mammalian and avian species. The enzyme has a molecular weight of approximately 70,000 and exists in a functional state as a monomer. The isolated enzyme displays a dual cation requirement (e.g., 6 mM Mg2+ and 10 microM Mn2+) for maximal activity; very little activity is observed when Mg2+ is present alone, and the maximal activity attained with Mn2+ alone (millimolar concentrations required) is significantly less than that observed under optimal conditions with both cations present. When assayed in the direction of oxalacetate formation there is a lag in product formation with time; the lag can be eliminated by the presence of 50 microM GTP (product). The Km for substrates is not affected by Mn2+ concentration, suggesting that the role of Mn2+ may not be related to substrate binding. The apparent Km for phosphoenolpyruvate (approximately 1 mM) is substantially higher than that reported for phosphoenolpyruvate carboxykinase from other species. The activity of phosphoenolpyruvate carboxykinase is increased 70% by physiological concentrations of urea. Maximal velocity of the reaction in the direction of oxalacetate formation is approximately half that of the reverse reaction.     

Purification and characterization of cytosol phosphoenolpyruvate carboxykinase from bullfrog (Rana catesbeiana) liver.

Cytosol PEP carboxykinase has been purified to electrophoretic homogeneity from bullfrog liver homogenate. The enzyme is a single polypeptide chain with a molecular weight of approximately 72,000-75,000. The purified enzyme catalyzed oxaloacetate decarboxylation (nucleoside triphosphate-supported), phosphoenolpyruvate carboxylation, and an exchange reaction between oxaloacetate and [14C]HCO3-in the presence of ITP or CTP. Manganese is absolutely required for the enzyme-catalyzed phosphoenolpyruvate carboxylation, whereas it can be replaced by Mg2+ for the oxaloacetate decarboxylation and the exchange reaction. The optimal pH of each reaction is dependent on the divalent metal ion used. The dependence of the enzyme activity on Mn2+ is markedly different in the phosphoenolpyuvate carboxylation and the oxaloacetate decarboxylation reactions.     

Phosphoenolpyruvate carboxykinase in mouse pancreatic islets. ATP-induced changes in sensitivity to Mn2+ activation

The presence of high phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity in mouse islet cytosol has been demonstrated. The enzyme was activated by Mn2+ with a Ka of 100 X 10(-6) mol/l. The mean total activity of the Mn2+-stimulated phosphoenolpyruvate carboxykinase in islet cytosol estimated at 22 degrees C with saturating concentrations of the substrates oxaloacetate and ITP was 146 pmol/min per micrograms DNA. Km was calculated to be 6 X 10(-6) mol/l for oxaloacetate and 140 X 10(-6) mol/l for ITP. The islet phosphoenolpyruvate carboxykinase activity was not increased after starvation of the animals for 48 h. Preincubation of the cytosol at 4 degrees C with Fe2+, quinolinate, ATP, Pi, glucose 6-phosphate, fructose 1,6-bisphosphate, NAD+, NADH, oxaloacetate, ITP, cyclic AMP and Ca2+ had no effect on the enzyme activity. However, preincubation of the cytosol at 37 degrees C with ATP-Mg inhibited the Mn2+-stimulated phosphoenolpyruvate carboxykinase activity progressively with time and in a concentration-dependent manner. A similar but weaker inhibitory effect was observed with p[NH]ppA, whereas p[CH2]ppA, ADP, AMP, adenosine and Pi had no effect. It is tentatively suggested that ATP and p[NH]ppA either by adenylation or otherwise affect the interaction between islet phosphoenolpyruvate carboxykinase and the recently discovered Mr = 29000 protein modulator of the enzyme in such a way - perhaps by causing a dissociation between them - that phosphoenolpyruvate carboxykinase loses its sensitivity to Mn2+ activation.     

Differential effects of Mg2+ ions on the individual kinetic steps of human cytosolic and mitochondrial aldehyde dehydrogenases

Although the structures of mammalian cytosolic and mitochondrial ALDH have been determined, several differences, mainly functional, between these two 70% identical isozymes remain unexplained. A major difference is the differential effect of Mg(2+) ions that inhibits the cytosolic and activates the mitochondrial isozyme. Here, we have investigated the effect of Mg(2+) ions on each individual kinetic step of ALDH1 and ALDH2. The metal ions were found not to affect either acylation or hydride transfer for either isozyme. The lack of a Mg(2+) ion effect on hydride transfer was further demonstrated with an E399Q mutant of ALDH1 whose rate-limiting step had been changed from NADH dissociation to hydride transfer. The other steps, however, were affected by Mg(2+) ions for both isozymes. The metal ions inhibited NADH dissociation, the rate-limiting step for ALDH1, and enhanced deacylation, the rate-limiting step for ALDH2. Our results indicated that, with both isozymes, Mg(2+) ions tightened the binding of NADH, and by binding to the coenzyme, they increased the nucleophilicity of the nucleophile Cys302. The inhibition of ALDH1 and activation of ALDH2 at pH 7.4 are due to their different rate-limiting steps. Mg(2+) ions affected similarly the NADH activation of the esterase reaction for both isozymes. In contrast, the metal ions affected only the NAD(+) activation of ALDH1. This latter finding and other features described here can be rationalized on the basis of the known three-dimensional structures of the isozymes.     

Remarkable effect of mobile phase buffer on the SEC-ICP-AES derived Cu, Fe and Zn-metalloproteome pattern of rabbit blood plasma

The development of an analytical method to quantify the major Cu, Fe and Zn-containing metalloproteins in mammalian plasma has been recently reported. This method is based on the separation of plasma proteins by size exclusion chromatography (SEC) followed by the on-line detection of the metalloproteins by an inductively coupled plasma atomic emission spectrometer (ICP-AES). To assess whether the mobile phase buffer can affect the SEC-ICP-AES-derived metalloproteome pattern, thawed rabbit plasma was analyzed using phosphate buffered saline (PBS)-buffer (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) or Mops-buffer (0.1 M, pH 7.4). In contrast to the Cu-specific chromatograms, the Fe and Zn-specific chromatograms that were obtained with Tris, Hepes and Mops-buffer were considerably different from those attained with PBS-buffer. The Tris, Hepes and Mops-buffer mediated redistribution of ~25% plasma Zn(2+) from <100 kDa to >100-600 kDa plasma proteins and to a smaller extent to a <10 kDa (Tris)(2)Zn(2+)-complex can be rationalized in terms of the abstraction of Zn(2+) from the weak binding site on albumin. In contrast, only Hepes and Mops-buffer redistributed ~20% of plasma Fe(3+) from the <100 kDa to the >600 kDa elution range. Based on these results and considering that the utilization of PBS-buffer has previously resulted in the detection of a number of Cu, Fe and Zn-containing metalloentities in rabbit plasma that was most consistent with literature data, this mobile phase buffer is recommended for metallomic studies regarding mammalian blood plasma.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Purification and general properties of δ-aminolaevulate dehydratase from Nicotiana tabacum L

1. delta-Aminolaevulate dehydratase (EC 4.2.1.24) was purified 80-fold from tobacco leaves and its properties were studied. 2. The enzyme had optimum pH7.4 in potassium phosphate buffer, K(m)6.25x10(-4)m at 37 degrees and pH7.4, optimum temperature 45 degrees and an activation energy of 11100 cal./mole. 3. The enzyme lost activity when prepared in the absence of cysteine, and this activity was only partly restored by the later addition of thiols. Reagents for thiol groups inactivated the enzyme. 4. Mg(2+) was essential for activity, and EDTA and Fe(2+) were inhibitory; Mn(2+) was an activator or an inhibitor depending on the concentration.     

Properties of pyruvate kinase and phosphoenolpyruvate carboxykinase in relation to the direction and regulation of phosphoenolpyruvate metabolism in muscles of the frog and marine invertebrates

1. The properties of pyruvate kinase and, if present, phosphoenolpyruvate carboxykinase from the muscles of the sea anemone, scallop, oyster, crab, lobster and frog were investigated. 2. In general, the properties of pyruvate kinase from all muscles were similar, except for those of the enzyme from the oyster (adductor muscle); the pH optima were between 7.1 and 7.4, whereas that for oyster was 8.2; fructose bisphosphate lowered the optimum pH of the oyster enzyme from 8.2 to 7.1, but it had no effect on the enzymes from other muscles. Hill coefficients for the effect of the concentration of phosphoenolpyruvate were close to unity in the absence of added alanine for the enzymes from all muscles except oyster adductor muscle; it was 1.5 for this enzyme. Alanine inhibited the enzyme from all muscles except the frog; this inhibition was relieved by fructose bisphosphate. Low concentrations of alanine were very effective with the enzyme from the oyster (50% inhibition was observed at 0.4mm). Fructose bisphosphate activated the enzyme from all muscles, but extremely low concentrations were effective with the oyster enzyme (0.13mum produced 50% activation). 3. In general, the properties of phosphoenolpyruvate carboxykinase from the sea anemone and oyster muscles are similar: the K(m) values for phosphoenolpyruvate are low (0.10 and 0.13mm); the enzymes require Mn(2+) in addition to Mg(2+) for activity; and ITP inhibits the enzymes and the inhibition is relieved by alanine. These latter compounds had no effect on enzymes from other muscles. 4. It is suggested that changes in concentrations of fructose bisphosphate, alanine and ITP produce a coordinated mechanism of control of the activities of pyruvate kinase and phosphoenolpyruvate carboxykinase in the sea anemone and oyster muscles, which ensures that phosphoenolpyruvate is converted into oxaloacetate and then into succinate in these muscles under anaerobic conditions. 5. It is suggested that in the muscles of the crab, lobster and frog, phosphoenolpyruvate carboxykinase catalyses the conversion of oxaloacetate into phosphoenolpyruvate. This may be part of a pathway for the oxidation of some amino acids in these muscles.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Lysine 213 and histidine 233 participate in Mn(II) binding and catalysis in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyses the reversible metal-dependent formation of oxaloacetate and ATP from PEP, ADP, and CO2 and plays a key role in gluconeogenesis. This enzyme also has oxaloacetate decarboxylase and pyruvate kinase-like activities. Mutations of PEP carboxykinase have been constructed where the residues Lys213 and His233, two residues of the putative Mn2+ binding site of the enzyme, were altered. Replacement of these residues by Arg and by Gln, respectively, generated enzymes with 1.9 and 2.8 kcal/mol lower Mn2+ binding affinity. Lower PEP binding affinity was inferred for the mutated enzymes from the protection effect of PEP against urea denaturation. Kinetic studies of the altered enzymes show at least a 5000-fold reduction in V(max) for the primary reaction relative to that for the wild-type enzyme. V(max) values for the oxaloacetate decarboxylase and pyruvate kinase-like activities of PEP carboxykinase were affected to a much lesser extent in the mutated enzymes. The mutated enzymes show a decreased steady-state affinity for Mn2+ and PEP. The results are consistent with Lys213 and His233 being at the Mn2+ binding site of S. cerevisiae PEP carboxykinase and the Mn2+ affecting the PEP interaction. The different effects of mutations in V(max) for the main reaction and the secondary activities suggest different rate-limiting steps for these reactions.     

Acid phosphatases of the mosquito Culex tarsalis coquillett

1. Spectrophotometric and isoelectric focusing (IEF) electrophoretic characterization of the acid phosphatases (ACP) of the mosquito, Culex tarsalis, are presented. 2. ACP hydrolysis of P-nitrophenylphosphate (Pnp) was optimal at 37 degrees C, pH 5.25 in the presence of 15 mM MgCl2 and 0.1% (w/v) polyvinylpyrollidone (PVP). Vmax and Km values varied significantly between the various mosquito strains examined. 3. Several divalent cations (i.e. Mn2+, Ca2+, Ba2+ and Co2+), either the chloride or sulphate salts, were stimulatory for ACP. Both Cu2+ and Fe2+ (15 mM) were inhibitory. 4. Slight inhibition (i.e. 10%) of ACP activity was observed with dithiothreitol (100 mM) and 50% inhibition by cysteine (100 mM). 5. ACP activity was cyclic during the 15-day post-adult emergence period of the study. No significant differences were noted between the ACP specific activities of males and females nor between geographic strains. 6. IEF electrophoresis revealed three alpha-naphthyl phosphate hydrolytic ACP isozymes within the pH 4.5-5.5 range (i.e. ACP4.8, ACP5.3 and ACP5.5). 7. IEF ACP isozymes were stimulated by PVP, Mg2+, Zn2+ and inhibited by cysteine, EDTA (except ACP5.3) and NaFl. 8. IEF detection of ACP with Pnp revealed an ACP isozyme (ACP4.3) distinct from those ACP isozymes capable of alpha-naphthyl phosphate hydrolysis.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Effects of divalent cations and pH on amiloride-sensitive Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule.

The effect of Ca2+, Cd2+, Ba2+, Mg2+ and pH on the renal epithelial Na(+)-channel was investigated by measuring the amiloride-sensitive 22Na+ fluxes into luminal membrane vesicles from pars recta of rabbit proximal tubule. It was found that intravesicular Ca2+ as well as extravesicular Ca2+ substantially lowered the channel-mediated flux. Amiloride sensitive Na+ uptake was nearly completely blocked by 10 microM Ca2+ at pH 7.4. The inhibitory effect of Ca2+ was dependent on pH. Thus, 10 microM Ca2+ produced 90% inhibition of 22Na+ uptake at pH 7.4, and only 40% inhibition at pH 7.0. The tracer fluxes measured in the absence of Ca2+ were pH independent over the range from 7.0 to 7.4. All the cations Ca2+, Cd2+, Ba2+ except Mg2+ inhibited the 22Na+ influx drastically when added extravesicularly in millimolar concentrations. The cations Cd2+, Ba2+ and Mg2+ in the same concentrations intravesicularly inhibited the 22Na+ influx only slightly. A millimolar concentration of Ca2+ intravesicularly blocked the amiloride-sensitive 22Na+ flux completely. The data indicate that Ca2+ inhibits Na+ influx specifically by binding to sites composed of one or several deprotonated groups on the channel proteins.     

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization.

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.