Kinetic and regulatory properties of pyruvate kinase isozymes from flight muscle and fat body of the cockroach,Periplaneta americana

Summary Pyruvate kinases from flight muscle and fat body of the cockroach,Periplaneta americana, were purified to homogeneity. The two tissues contained different forms of the enzyme which were separable by starch gel electrophoresis and isoelectric focusing (pI=5.75 for flight muscle and 6.15 for fat body). Both enzymes had molecular weights of 235,000±20,000.Flight muscle pyruvate kinase displayed Michaelis-Menten kinetics with respect to both ADP and P-enolpyruvate withK _m values of 0.27 and 0.04 mM, respectively.K _m for Mg²⁺ was 0.60 mM andK _a for K⁺ was 15 mM. The enzyme was weakly inhibitied by four compounds, ATP, arginine-P,l-alanine and citrate with apparentK _i values of 3.5, 15, 20 and 24 mM, respectively. Competitive inhibition by 3 mM ATP or 10 mM arginine-P raised theK _m for P-enolpyruvate to 0.067 or 0.057 mM. Fructose-1,6-P₂ did not activate the enzyme but reversed inhibitions by ATP and arginine-P.Fat body pyruvate kinase showed sigmoidal kinetics with respect to P-enolpyruvate with S_0.5=0.32 mM andn _H=1.43.K _m values for ADP and Mg²⁺ were 0.30 and 0.80 mM, respectively with aK _a for K⁺ of 10 mM. ATP andl-alanine were inhibitors of the enzyme; 2 mM ATP raised S_0.5 for P-enolpyruvate to 0.48 mM while 3 mMl-alanine increased S_0.5 to 0.84 mM. Neither citrate nor arginine-P inhibited the enzyme but citrate affected the enzyme by reversingl-alanine inhibition. Fat body pyruvate kinase was strongly activated by fructose-1,6-P₂ with an apparentK _a of 1.5 M. Fructose-1,6-P₂ at 0.1 mM reduced S_0.5 for P-enolpyruvate to 0.05 mM andn _H to 1.0.Flight muscle and fat body pyruvate kinases from the cockroach show properties analogous to those of the muscle and liver forms of mammalian pyruvate kinase. Fat body pyruvate kinase is suited for on-off function in a tissue with a gluconeogenic capacity. Strong allosteric control with a feed-forward activation by fructose-1,6-P₂ is key to coordinating enzyme function with glycolytic rate. The function of flight muscle pyruvate kinase in energy production during flight is aided by a lowK _m for P-enolpyruvate, weak inhibitor effects by high energy phosphates and deinhibition of these effects by fructose-1,6-P₂.     

Purification and characterization of two glutathione peroxidases from embryo of the camel tick <Emphasis Type="Italic">Hyalomma dromedarii</Emphasis>

Two glutathione peroxidase isoenzymes were purified from 24-day old embryos of the camel tick Hyalomma dromedarii and designated tick embryo glutathione peroxidase 1 and 2 (TEGPx1 and TEGPx2). The purification procedure involved ammonium sulfate precipitation, as well as ion exchange and gel filtration column chromatography. Glutathione peroxidase isoenzymes subunit molecular mass was determined by SDS-PAGE to be 36 ± 2 kDa and 59 ± 1.5 kDa for TEGPx1 and TEGPx2, respectively. TEGPx1 isoenzyme exhibited a dimeric structure with native molecular mass of 72 kDa while TEGPx2 was a monomeric protein. TEGPx1 and TEGPx2 displayed their pH optima at 7.6 and 8.2. Both isoenzymes cleaved preferentially H₂O₂ with K _m values of 24 and 49 μM. Iodoacetamide competitively inhibited TEGPx1 with K _i value of 0.45 mM and 1.10; phenanthroline competitively inhibited TEGPx2 with K _i value of 0.12 mM. These results indicate the presence of two different forms of glutathione peroxidase in the developing camel tick embryos. This finding enhances our knowledge and understanding of the physiology of these ectoparasites and will encourage the development of new and untraditional control methods.     

Properties of 5′-nucleotidase from nodules of pigeonpea (Cajanus cajan)

5′-Nucleotidase from pigeonpea nodules has been resolved into two forms, N-I and N-II, having M,s of 52 000 and 119 000, respectively. Both forms had pH optima in the acidic range (between pH 5.2 and 5.7) with either CMP, GMP, XMP, IMP or AMP as the substrate. Up to pH 6.6, both forms showed higher activity with CMP followed by GMP, XMP, IMP and AMP, respectively. However, the activity changed with pH in the alkaline range making the enzyme relatively more active with purine nucleotides. Neither of the forms had a requirement for any of the metal ions tested. Fe³⁺ inhibited the enzyme activity; the inhibition at 5, 10 and 15 mM concentrations being 11, 43 and 47%, respectively with N-I and 14,47 and 52%, respectively with N-II. K_m values for AMP, IMP, GMP, CMP and XMP were 0.10, 0.18, 0.40, 0.40 and 0.77 mM, respectively with N-I and 0.12, 0.20, 0.40, 0.40 and 0.99 mM, respectively with N-II. The enzyme was inhibited non-competitively by adenosine and inosine; K_i values being 1.78, 0.25 and 0.30; 3.50, 2.12 and 0.75 mM, respectively with AMP, IMP and XMP as the substrate.     

Characterization of α-phosphoglucomutase isozymes from Toxoplasma gondii

The Toxoplasma gondii genome project has revealed two putative isoforms (TgPGM-I and TgPGM-II) of α-phosphoglucomutase (EC 5.4.2.2). We obtained recombinant proteins of these isoforms from the Beverley strain of T. gondii and characterized their properties, particularly the kinetic properties of these isoforms. The specific activities of TgPGM-I and TgPGM-II for α-d-glucose 1-phosphate were 338 ± 9 and 84 ± 6 μmol/min/mg protein, respectively, at 37 °C under optimal conditions. The Kcat and Km values of TgPGM-I were 398 ± 11/s and 0.19 ± 0.03 mM and those for TgPGM-II were 93 ± 7/s and 3.53 ± 0.91 mM, respectively, for α-d-glucose 1-phosphate. Magnesium ions were the most effective divalent cations for both the enzyme activities. The maximum activities of both the enzymes were obtained in the presence of more than 0.2 mM α-d-glucose 1,6-bisphosphate. Although both enzymes were attached to the α-phosphohexomutase superfamily, amino acid sequence homology between TgPGM-I and TgPGM-II showed very low overall identity (25%). No α-phosphomannomutase (EC 5.4.2.8) activity was detected for either enzyme. The data indicated that TgPGM-I, but not TgPGM-II, may play an important role in α-d-glucose 6-phosphate production.     

Characteristics of α-glucan phosphorylase from Chlorella vulgaris

α-Glucan phosphorylase from Chlorella vulgaris has been partially purified. In the direction of glucan phosphorolysis the apparent K_m for Pi was ca 2.4 mM at pH 7.1. In the direction of glucan synthesis the K_m for G1P was ca 0.12 mM at pH 6.2. The enzymic activity was inhibited by physiological concentrations of ADP, ATP, ADPG and UDPG. In the direction of starch degradation in the presence of 2.4 mM Pi the I_0.5 values for ADP and ATP were ca 1.6 and 2.9 mM, respectively, while in the direction of synthesis in the presence of 0.12 mM G1P the values were ca 0.23 and 1.4 mM, respectively. The Hill plots for starch degradation showed n values of 2.2 for ADP and 2.2 for ATP and values of 1.5 and 1.2, respectively, for starch synthesis. Both ADPG and UDPG were linear competitive inhibitors either with respect to Pi or with respect to GIP. The K_i values for ADPG and UDPG in the direction of phosphorolysis were shown to be ca 0.11 and 0.51 mM, respectively, and those in the direction of synthesis 0.033 and 0.15 mM, respectively.     

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P₂) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P₂), with K_i for Fru-2,6-P₂ of approximately 0.5 μm. Binding of Fru-2,6-P₂ to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P₂ is enhanced in the presence of a noninhibitory concentration (5 μm) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P_2, unlike the substrate Fru-1,6-P₂, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.     

In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K_a) values were obtained for each bisphosphate were: for glucose 1-6-P₂, 0.5 to 1.8; fructose 2,6-P₂, 6 to 11.7; and fructose 1,6-P₂, 7 micromolar, respectively. Fructose 2,6-P₂ is known to occur in both plant and animal tissues at changing levels encompassing the K_a values found in this study; hence, these results implicate fructose 2,6-P₂ as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P₂ has not been found in plants, and the method for measuring glucose 1,6-P₂ by monitoring the activation of phosphoglucomutase is not specific.     

L(+)-lactate dehydrogenase of Clostridium acetobutylicum is activated by fructose-1,6-bisphosphate

Cells of Clostridium acetobutylicum contained an NADH-dependent L(+)-lactate dehydrogenase which was activated specifically by fructose-1,6-bisphosphate (F-1,6-P₂), with calcium or magnesium ions as positive effectors. During the purification steps the enzyme was very unstable. The purified enzyme existed in a tetrameric structure (apparent M_r of about 159 kDa) and had its pH optimum at pH 5.8. Little activity was left at pH values below 5.0. The enzyme was unidirectional, catalysing only the reduction of pyruvate. The half maximal activation of the reaction velocity with F-1,6-P₂ depended on the pyruvate concentration.     

UDP-galactose 4′-epimerase from vicia faba seeds

UDP-Galactose 4′-epimerase was purified ca 800-fold through a multi-step procedure which included affinity chromatography using NAD⁺ -Agarose. Three forms of the enzyme were separated by gel-filtration but only the major form was purified. The pH optimum of the enzyme was 9.5. Exogenous NAD⁺ was not required for enzymic activity but its removal caused inactivation. The enzyme was unstable below pH 7.0 but stable at pH 8.0 in the presence of glycerol and at ?20° for two months. The equilibrium constant for the enzyme-catalysed reaction was 3.2 ± 0.15. The K_m for UDP-galactose and UDP-glucose were 0.12 mM and 0.25 mM, respectively. The inhibition by NADH was competitive, with a K_i of 5 μM. The MW of the enzyme was 78 000; the two minor forms showed the values of 158 000 and 39 000, respectively.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Purification and properties of dihydroxyacetone kinase from Gluconobacter suboxydans

Different carbon and nitrogen sources had little effect on the level of dihydroxyacetone kinase formed in the cells of Gluconobacter suboxydans. The enzyme was purified to homogeneity from cell-free extract of the organism by ammonium sulfate fractionation and chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200 (60-fold purification, 6% yield). Its molecular weight was 260,000; it was stabilized by addition of ATP, dithiothreitol, 2-mercaptoethanol or EDTA, and it reacted optimally at pH 6.5. d-Glyceraldehyde was equally as effective as DHA as a phosphate acceptor (K_m: 0.30 mM each). UTP showed 15% of the reactivity of ATP as a phosphate donor. K_m values for ATP were 0.33 mM in phosphorylation of dihydroxyacetone and 0.39 mM with d-glyceraldehyde. The enzyme activity was dependent on Mg²⁺ but not on Mn²⁺. The reaction with dihydroxyacetone as an acceptor was inhibited by d-glyceraldehyde. The inhibition was competitive with respect to dihydroxyacetone 3K_i=0.09 mM) and noncompetitive with respective to ATP (K_i=5.7 mM).     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

Transport kinetics of maltotriose in strains ofSaccharomyces

Summary Maltotriose transport was studied in two brewer's yeast strains, an ale strain 3001 and a lager strain 3021, using laboratory-synthesized¹⁴C-maltotriose. The maltotriose transport systems preferred a lower pH (pH 4.3) to a higher pH (pH 6.6). Two maltotriose transport affinity systems have been indentified. The high affinity system hasK _m values of 1.3 mM for strain 3021 and 1.4 mM for strain 3001. The low affinity competitively inhibited by maltose and glucose withK _i values of 58 mM and 177 mM. respectively, for strain 3021, and 55 mM and 147 mM, respectively, for strain 3001. Cells grown in maltotriose and maltose had higher maltotriose and maltose transport rates, and cells grown in glucose had lower maltortriose and maltose transport rates. Early-logarithmic phase cells transported glucose faster than either maltose or maltotriose. Cells harvested later in the growth phase had increased maltotriose and maltose transport activity. Neither strain exhibited significant differences with respect to maltose and maltotriose transport activity.     

2-carboxy-d-hexitol 1,6-bisphosphate: An inhibitor of d-ribulose 1,5-bisphosphate carboxylase/oxygenase

2-Carboxy-d-hexitol 1,6-bisphosphate (CHBP) has been prepared from d-fructose 1,6-bisphosphate and cyanide. DEAE-Sephadex chromatography separated the reaction products into two fractions which were identified as CHBP and CHBP-lactone. CHBP is presumably a mixture of two diastereomers, 2-carboxy-d-glucitol 1,6-bisphosphate and 2-carboxy-d-mannitol 1,6-bisphosphate, but an attempt to separate these compounds was not successful. The material in the CHBP-lactone peak had no effect on d-ribulose 1,5-bisphosphate (RuBP) carboxylase. However, CHBP was a potent reversible inhibitor of RuBP carboxylases. This compound displayed an inhibition constant (K_i at pH 8.0 and 30 °C) of 1–2 μm with the enzymes from spinach and barley, while the K_i was 60–70 μm with bacterial RuBP carboxylases from Pseudomonas oxalaticus and Rhodospirillum rubrum. The mode of inhibition was competitive with respect to RuBP for all the carboxylases, and noncompetitive with respect to CO₂ for the enzymes from spinach, P. oxalaticus and R. rubrum. The results indicate that, in the binding of certain organic phosphates by RuBP carboxylases, there may be a fundamental difference between the enzymes isolated from microbial and from higher plant sources. RuBP oxygenase activities from spinach and P. oxalaticus were also inhibited by CHBP, with K_i values which were similar to those obtained with the carboxylase activity of the same enzymes. The mode of inhibition of the oxygenase activities was also competitive with respect to RuBP. Thus, it seems that the binding of CHBP is similar for the carboxylase and oxygenase reactions of the same enzyme.     

The binding of products, metal ion, and a substrate analog to rabbit liver fructose bisphosphatase.

Binding of fructose-6-P and P_i to rabbit liver fructose bisphosphatase has been analyzed in terms of four negatively cooperative binding sites per enzyme tetramer. The association of fructose-6-P occurs in the absence of divalent metal ion, although the extent of binding is increased in the order Mg²⁺ < Zn²⁺ < Mn²⁺. The binding of P_i shows an absolute requirement for divalent metal ion with Mn²⁺ being more effective than Mg²⁺. The interaction of the enzyme with the substrate analog, (α + β) methyl-d-fructofuranoside-1,6-P₂ in the presence of Mn²⁺ closely resembles that found for fructose-1,6-P₂ in the absence of Mn²⁺, although the measured constants are on average an order of magnitude smaller. Combination experiments with the three ligands show that the binding follows an identical ordered sequence, i.e., the tighter sites are initially occupied regardless of the ligand's identity. The binding of P_i or fructose-6-P is not altered by the presence of the other. Comparison of binding constant with K_i values obtained from steady-state assays permits identification of the catalytic sites expressed in the latter. The association of Mn²⁺ at the catalytic site can be induced by fructose-6-P or the substrate analog suggesting that a 1-phosphoryl group enhances but is not necessary for Mn²⁺ binding at this site. The binding of AMP is decreased in the presence of substrate analog relative to fructose-1,6-P₂, suggesting that the 2-hydroxyl serves as a “molecular signal.” From the single and combined binding experiments, a calculation of the equilibrium constant for the overall hydrolysis reaction on the enzyme surface in the presence of Mn²⁺ has been carried out and an estimate made for the Mg²⁺ case.     

Some properties of glutamine synthetase and glutamate synthase from Derxia gummosa

The incorporation of ¹⁵N into washed cells of Derxia gummosa from labelled-(NH₄)₂SO₄ and -KNO₃ respectively was inhibited by both L-methionine-DL-sulphoximine and azaserine. Glutamine synthetase purified to homogeneity from this bacterium had a molecular weight of 708 000 and was composed of 12 similar subunits each of 59 000. The enzyme assayed by γ-glutamyltransferase method had K_m values for L-glutamine and hydroxylamine of 12.5 and 1.2 mM, respectively. Optimal pH values for adenylylated and deadenylylated forms were pH 7.0 and pH 8.0, respectively. The adenylylated enzyme was deadenylylated by treatment with snake venom phosphodiesterase. The inhibitions by both glutamate and ammonia were competitive. The activity was markedly inhibited by L-methionine-DL-sulphoximine, alanine, glycine and serine and to a lesser extent by aspartate, phenylalanine and lysine. Various tri-, di- and mono-phosphate nucleotides, organic acids (pyruvate, oxalate and oxaloacetate) were also inhibitory. Glutamate synthase purified 167-fold had specific requirements for NADH, L-glutamine and 2-ketoglutarate. The K_m values for NADH, glutamine and 2-ketoglutarate were 9.6, 270 and 24 μM respectively. Optimal pH range was 7.2–8.2. The enzyme was inhibited by azaserine, methionine, aspartate, AMP, ADP and ATP.     

Rat brain aryl acylamidase: multiple forms and inhibition effects of LSD, serotonin and related compounds

At least two forms of aryl acylamidase (E.C.3.5.1.13, AAA) were separated from rat brain extracts by ammonium sulfate precipitation (33–60% saturation) and subsequent Bio-Gel column chromatography. Fraction AAA-1 showed pH optimum at 7.5 whereas AAA-2 showed a pH optimum at 5.5 AAA-1 activity was markedly inhibited at pH 7.5 by d-LSD and 2-Br-LSD, moderately inhibited by 5-HT and slightly inhibited by tryptamine but it was not affected by 1-LSD, at 0.1 mM concentration. AAA-2 was only moderately inhibited at pH 5.5 by d-LSD and 2-Br-LSD but not affected by 1-LSD, 5-HT or tryptamine at the same concentrations. Catecholamines and their structurally related drugs had no significant effects on either enzyme activity. Kinetic studies with AAA-1 indicated competitive inhibition by d-LSD with a K_i value of 4.90 ± 0.61 μM.     

6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Kinetic properties of pig pyruvate kinases type A from kidney and type M from muscle.

Kinetic properties of homogeneous preparations of pig kidney and pig muscle pyruvate kinases (EC 2.7.1.40) were studied. Both isozymes showed a hyperbolic relationship to ADP with an apparent K_m of 0.3 mm. K⁺ and Mg²⁺ were necessary for the activity of both isozymes, and their dependences on these cations were similar. The muscle isozyme expressed Michaelis-Menten type of kinetics with respect to phosphoenolpyruvate, and the apparent K_m was the same (0.03 mm) from pH 5.5 to pH 8.0. In contrast, the dependence on phosphoenolpyruvate changed with pH for the kidney isozyme. It showed similar properties to the muscle isozyme at pH 5.5–7.0 (apparent K_m of 0.08 mm), while two apparent K_m values for this substrate were present at pH 7.5–8.0, one low (0.1 mm) and one high (0.3–0.6 mm). At pH 7.5, fructose 1,6-bisphosphate converted the kidney isozyme to a kinetical form where only the lower apparent K_m for phosphoenolpyruvate was detected. On the other hand, in the presence of alanine or phenylalanine the kidney pyruvate kinase showed only the higher K_m for this substrate. At low phosphoenolpyruvate levels both isozymes were inhibited by phenylalanine, and half-maximal inhibition was found at 0.3 and 2.2 mm for the kidney and muscle isozymes, respectively. At a 5 mm concentration of the substrate only the kidney isozyme was inhibited, the apparent K_i being the same. Alanine inhibited the kidney isozyme (apparent K_i at 0.3 mm, irrespective of substrate concentration). No effect was seen on the muscle isozyme. Fructose 1,6-bisphosphate was an activator of the kidney isozyme at phosphoenolpyruvate concentrations below 1.0 mm It also counteracted the inhibition by alanine or phenylalanine of this isozyme. ATP inhibited both isozymes, and this inhibition was not counteracted by fructose 1,6-bisphosphate. The kidney isozyme showed both a high and a low apparent K_m for phosphoenolpyruvate in the presence of ATP. The influence of the effectors on the activity of both isozymes varied markedly with pH, except for the action of ATP. At low substrate concentrations, however, the inhibitor action of ATP on the muscle enzyme was diminished around pH 7.5, in contrast to higher or lower pH values. Alanine or phenylalanine were more effective as inhibitors at higher pH values, and fructose 1,6-bisphosphate stimulated the kidney isozyme only at pH levels above pH 6.5. The influence of activators and inhibitors on the regulation of the kidney and muscle pyruvate kinases is discussed.     

Separation and characterization of four hexose kinases from developing maize kernels

Four forms of hexose kinase activity from developing maize (Zea mays L.) kernels have been separated by ammonium sulfate precipitation, gel filtration chromatography, blue-agarose chromatography, and ion exchange chromatography. Two of these hexose kinases utilized d-glucose most effectively and are classified as glucokinases (EC 2.7.1.2). The other two hexose kinases utilized only d-fructose and are classified as fructokinases (EC 2.7.1.4). All hexose kinases analyzed had broad pH optima between 7.5 and 9.5 with optimal activity at pH 8.5. The two glucokinases differed in substrate affinities. One form had low K_m values [K_m(glucose) = 117 micromolar, K_m(ATP) = 66 micromolar] whereas the other form had much higher K_m values [K_m(glucose) = 750 micromolar, K_m(ATP) = 182 micromolar]. Both fructokinases had similar substrate saturation responses. The K_m(fructose) was about 130 micromolar and the K_m(ATP) was about 700 micromolar. Both exhibited uncompetitive substrate inhibition by fructose [K_i(fructose) = 1.40 to 2.00 millimolar]. ADP inhibited all four hexose kinase activities, whereas sugar phosphates had little effect on their activities. The data suggest that substrate concentrations are an important factor controlling hexose kinase activity in situ.