Characterization of cytosolic phosphoglucoisomerase from immature wheat (Triticum aestivum L.) endosperm

Phosphoglucoisomerase from cytosol of immature wheat endosperm was purified 650-fold by ammonium sulphate fractionation, isopropyl alcohol precipitation, DEAE-cellulose chromatography and gel filtration through Sepharose CL-6B. The enzyme, with a molecular weight of about 130,000, exhibited maximum activity at pH 8.1. It showed typical hyperbolic kinetics with both fructose 6-P and glucose 6-P withK _m of 0.18 mM and 0.44mM respectively. On either side of the optimum pH, the enzyme had lower affinity for the substrates. Using glucose 6-P as the substrate, the equilibrium was reached at 27% fructose 6-P and 73% glucose 6-P with an equilibrium constant of 2.7. The ΔF calculated from the apparent equilibrium constant was +597 cal mol^-1. The activation energy calculated from the Arrhenius plot was 5500 cal mol^-1. The enzyme was completely inhibited by ribose 5-P, ribulose 5-P and 6-phosphogluconate, withK _i values of 0.17, 0.25 and 0.14 mM respectively. The probable role of the enzyme in starch biosynthesis is discussed.     

Purification and regulatory properties of pyruvate kinase from Veillonella parvula.

The nonglycolytic, anaerobic organism Veillonella parvula M4 has been shown to contain an active pyruvate kinase. The enzyme was purified 126-fold and was shown by disc-gel electrophoresis to contain only two faint contaminating bands. The purified enzyme had a pH optimum of 7.0 in the forward direction and exhibited sigmoidal kinetics at varying concentrations o-f phosphoenol pyruvate (PEP), adenosine 5'-monophosphate (AMP), and Mg-2+ ions with S0.5 values of 1.5, 2.0, and 2.4 mM, respectively. Substrate inhibition was observed above 4 m PEP. Hill plots gave slope values (n) of 4.4 (PEP), 2.8 (adenosine 5'-diphosphate), and 2.0 (Mg-2+), indicating a high degree of cooperativity. The enzyme was inhibited non-competitively by adenosine 5'-triphosphate (Ki = 3.4 mM), and this inhibition was only slightly affected by increasing concentration of Mg-2+ ions to 30 mM. Competitive inhibition was observed with 3-phosphoglycerate, malate, and 2,3-diphosphoglycerate but only at higher inhibitor concentrations. The enzyme was activated by glucose-6-phosphate (P), fructose-6-P, fructose-1,6-diphosphate (P2), dihydroxyacetone-P, and AMP; the Hill coefficients were 2.2, 1.8, 1.5, 2.1, and 2.0, respectively. The presence of each these metabolites caused substrate velocity curves to change from sigmoidal to hyperbolic curves, and each was accompanied by an increase in the maximum activity, e.g., AMP greater than fructose-1,6-P2 greater than dihydroxyacetone-P greater than glucose-6-P greater than fructose-6-P. The activation constants for fructose-1,6-P2, AMP, and glucose-6-P were 0.3, 1.1, and 5.3 mM, respectively. The effect of 5 mM fructose-1,6-P2 was significantly different from the other compounds in that this metabolite was inhibitory between 1.2 and 3 mM PEP. Above this concentration, fructose-1,6-P2 activated the enzyme and abolished substrate inhibition by PEP. The enzyme was not affected by glucose, glyceraldehyde-3-P, 2-phosphoglycerate, lactate, malate, fumerate, succinate, and cyclic AMP. The results suggest that the pyruvate kinase from V. parvula M4 plays a central role in the control of gluconeogenesis in this organism by regulating the concentration of PEP.     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch I. Its isolation and some enzymic properties

A method is described for isolating phosphomannose isomerasefrom young konjak corms. The enzyme is believed to catalyzethe mannose forming reaction in growing corm tissues. The purifiedenzyme preparation was free from phosphoglucose isomerase activity,and was stable at pH 6–9. Maximum enzyme activity wasobserved at pH 6.5–7.0. The molecular weight of the enzymewas estimated as 45,000 using Sephadex gel filtration. The followingkinetic parameters were obtained: Km (mannose-6-P), 0.73 mM,K_eq (fructose-6-P/mannose-6-P), 1.06 at pH 6.5, and activationenergy, 11,600 cal/mole. The enzyme was inhibited by the metalbinding agents EDTA, o-phenanthroline, and a,a'-bipyridyl. Theinhibitory effect of these agents was markedly influenced bythe pH level of the incubation mixture, being more pronouncedat pH 6 than at pH 8. ¹ This paper constitutes part 3 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Isomerase activity of the C-terminal fructose-6-phosphate binding domain of glucosamine-6-phosphate synthase from Escherichia coli

The isomerase activity of the C-terminal fructose-6P binding domain (residues 241-608) of glucosamine-6-phosphate synthase from Escherichia coli has been studied. The equilibrium constant of the C-terminal domain k(eq) ([glucose-6P]/[fructose-6-P]) = 5.0. A non-competitive product inhibition of the isomerase activity by the reaction product glucose-6-P has been detected. The existence of more than one binding and reaction sites for the substrate fructose-6P on the molecule of glucosamine-6-phosphate synthase can be expected. The fructose-6P binding domain possibly includes a regulatory site, different from the catalytic center of the enzyme.     

Activation by phosphate of yeast phosphofructokinase.

The activity of yeast phosphofructokinase assayed in vitro at physiological concentrations of known substrates and effectors is 100-fold lower than the glycolytic flux observed in vivo. Phosphate synergistically with AMP activates the enzyme to a level within the range of the physiological needs. The activation by phosphate is pH-dependent: the activation is 100-fold at pH 6.4 while no effect is observed at pH 7.5. The activation by AMP, phosphate, or both together is primarily due to changes in the affinity of the enzyme for fructose-6-P. Under conditions similar to those prevailing in glycolysing yeast (pH 6.4, 1 mM ATP, 10 mM NH4+) the apparent affinity constant for fructose-6-P (S0.5) decreases from 3 to 1.4 mM upon addition of 1 mM AMP or 10 mM phosphate; if both activators are present together, S0.5 is further decreased to 0.2 mM. In all cases the cooperativity toward fructose-6-P remains unchanged. These results are consistent with a model for phosphofructokinase where two conformations, with different affinities for fructose-6-P and ATP, will present the same affinity for AMP and phosphate. AMP would diminish the affinity for ATP at the regulatory site and phosphate would increase the affinity for fructose-6-P. The results obtained indicate that the activity of phosphofructokinase in the shift glycolysis-gluconeogenesis is mainly regulated by changes in the concentration of fructose-6-P.     

The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme-catalysed reactions.

The effects of pH and temperature on Michaelis constant (Km) and maximum velocity (Vmax.) and of NaCl on the activity of the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase EC 3.2.1.21) from cultures of Botryodiplodia theobromae Pat. have been studied. 2. Donor binding and inhibition of activity by glucose were dependent on the ionization of a group (pK 6.0) that appeared to be an imidazole group. 3. Catalytic activity and the stimulation of activity by glycerol were dependent on the ionization of two groups, which appeared to be a carboxy group and an imidazole group. 4. The Arrhenius activation energy (Ea) calculated from results obtained at pH 4.0 and 5.0 was about 45--46kJ.mol-1. 5. The enthalpies (delta H0) calculated from results obtained at pH 4.0 and 5.0 were similar (about -4kJ.mol-1), whereas at pH 6.5 the value was about -33kJ.mol-1. 6. The entropies (delta S0) calculated from these results at 37 degrees C were -21, -22 and -118J.K-1.mol-1 at pH 4.0, 5.0 and 6.5 respectively. A low concentration of NaCl (16.6 mM) stimulated enzymic activity and decreased the Km for the donor, whereas high concentrations (up to 500 mM) inhibited enzymic activity, increased the Km and had no effect on Vmax. 8. Plots of initial velocity data obtained in the presence of dioxan as 1/v against the ratio of the molar concentration of dioxan to that of water were linear. 9. The results are discussed in terms of the enzyme mechanism.     

The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory kinetic properties of liver and muscle phosphofructokinases

The effect of natural "activation factor" and synthetic fructose-2,6-P2 on the allosteric kinetic properties of liver and muscle phosphofructokinases was investigated. Both synthetic and natural fructose-2,6-P2 show identical effects on the allosteric kinetic properties of both enzymes. Fructose-2,6-P2 counteracts inhibition by ATP and citrate and decreases the Km for fructose-6-P. This fructose ester also acts synergistically with AMP in releasing ATP inhibition. The Km values of liver and muscle phosphofructokinase for fructose-2,6-P2 in the presence of 1.25 mM ATP are 12 milliunits/ml (or 24 nM) and 5 milliunits/ml (or 10 nM), respectively. At near physiological concentrations of ATP (3 mM) and fructose-6-P (0.2 mM), however, the Km values for fructose-2,6-P2 are increased to 12 microM and 0.8 microM for liver and muscle enzymes, respectively. Thus, fructose-2,6-P2 is the most potent activator of the enzyme compared to other known activators such as fructose-1,6-P2. The rates of the reaction catalyzed by the enzymes under the above conditions are nonlinear: the rates decelerate in the absence or in the presence of lower concentrations of fructose-2,6-P2, but the rates become linear in the presence of higher concentrations of fructose-2,6-P2. Fructose-2,6-P2 also protects phosphofructokinase against inactivation by heat. Fructose-2,6-P2, therefore, may be the most important allosteric effector in regulation of phosphofructokinase in liver as well as in other tissues.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

Enzyme activities associated with maize kernel amyloplasts

Activities of the enzymes of gluconeogenesis and of starch metabolism were measured in extracts of amyloplasts isolated from protoplasts derived from 14-day-old maize (Zea mays L., cv Pioneer 3780) endosperm. The enzymes triosephosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, phosphohexose isomerase, phosphoglucomutase, ADPG pyrophosphorylase, UDPG pyrophosphorylase, soluble and bound starch synthases, and branching enzyme were found to be present in the amyloplasts. Of the above enzymes, ADPG pyrophosphorylase had the lowest activity per amyloplast. Invertase, sucrose synthase and hexokinase were not detected in similar amyloplast preparations. Only a trace of the cytoplasmic marker enzyme alcohol dehydrogenase could be detected in purified amyloplast fractions. In separate experiments, purified amyloplasts were lysed and then supplied with radioactively labeled glucose-6-phosphate, glucose-1-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, glucose, fructose, sucrose, and 3-0-methylglucose in the presence of adenosine triphosphate or uridine triphosphate. Of the above, only the phosphorylated substrates were incorporated into starch. Incorporation into starch was higher with added uridine triphosphate than with adenosine triphosphate. Dihydroxyacetone phosphate was the preferred substrate for uptake by intact amyloplasts and incorporation into starch. In preliminary experiments, it appeared that glucose-6-P and fructose-1,6-bisphosphate may also be taken up by intact amyloplasts. However, the rate of uptake and incorporation into starch was relatively low and variable. Additional study is needed to determine conclusively whether hexose phosphates will cross intact amyloplast membranes. From these data, we conclude that: (a) Triose phosphate is the preferred substrate for uptake by intact amyloplasts. (b) Amyloplasts contain all enzymes necessary to convert triose phosphates into starch. (c) Sucrose breakdown must occur in the cytosol prior to carbohydrate transfer into the amyloplasts. (d) Under the conditions of assay, amyloplasts are unable to convert glucose or fructose to starch. (e) Uridine triphosphate may be the preferred nucleotide for conversion of hexose phosphates to starch at this stage of kernel development.     

The mechanism of activation of heart fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase

Partially purified fructose-6-P,2-kinase:fructose-2,6-bisphosphatase from beef heart was phosphorylated by cAMP protein kinase. The phosphorylated fructose-6-P,2-kinase shows lower Km for Fru-6-P (43 versus 105 microM) and for ATP (0.55 versus 1.3 mM) but no change in the Vmax, compared to those for unphosphorylated enzyme. There was no detectable change in Km or Vmax of fructose-2,6-bisphosphatase activity by the phosphorylation. These changes in heart fructose-6-P,2-kinase were in direct contrast to previous results for the liver isozyme in which phosphorylation led to inhibition of the kinase activity and activation of the phosphatase activity.     

Effect of manganese(ous) and sulfate on activity of human placental glucose 6-phosphate-dependent form of glycogen synthase.

The human placental glucose-6-P-dependent form of glycogen synthase, in the absence of glucose-6-P, can be activated by MnSO4. Separately, Mn2+ and SO4(2-) have no significant effect. In the presence of glucose-6-P, Mn2+ activates the enzyme, but SO4(2-) inhibits; MnSO4 synergetically increases the enzyme activity. Mn2+ reduces the Ka for glucose-6-P to one-tenth of the control value; SO4(2-) increases the Ka 5-fold; however, MnSO4 has no effect on Ka. MnSO4, like glucose-6-P, increases the Vmax of the enzyme in the presence of its substrate, UDP-glucose; it slightly increases the Km for UDP-glucose. In the presence of glucose-6-P, Mn2+ increases and SO4(2-) decreases the Vmax of the enzyme, but neither has an effect on the Km for UDP-glucose. At physiological concentrations of UDP-glucose and glucose-6-P, either Mn2+ or MnSO4 at concentrations less than 1 mM increases the enzyme activity as much as 8 mM glucose-6-P does. At physiological concentrations of UDP-glucose and glucose-6-P, Mn2+ or MnSO4 reverses the inhibition of the enzyme by ATP.     

Properties of 1-phosphofructokinase from Pseudomonas putida.

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis-Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 X 10(-4) M and 4.08 X 10(-4) M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3',5'-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.     

Partial Purification and Characterization of Hydroxycinnamoyl-Coenzyme A:Tyramine Hydroxycinnamoyltransferase from Cell Suspension Cultures of Solanum tuberosum

A pathogen elicitor-inducible soluble acyltransferase (tyramine hydroxycinnamoyltransferase [THT], EC 2.3.1), which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-coenzyme A (CoA) esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was partially purified with a 380-fold enrichment and a 6% recovery from cell-suspension cultures of potato (Solanum tuberosum L. cv Datura). The enzyme showed specific activities of 33 mkat (kg protein)-1 (formation of feruloyltyramine). The apparent native Mr was found to be approximately 49,000. Highest activity was at pH 6.8 in K-phosphate. The isoelectric point of the enzyme was approximately pH5.2. The apparent energy of activation was calculated to be 96 kJ mol-1. The enzyme activity was stimulated more than 5-fold by 10 mM Ca2+ or Mg2+. The apparent Km values were 36 [mu]M for feruloyl-CoA and 85 and 140 [mu]M for cinnamoyl- and 4-coumaroyl-CoA, respectively. The Km value for tyramine in the presence of feruloyl-CoA was 22 [mu]M. In the presence of 4-coumaroyl-CoA, however, the Km for tyramine increased to about 230 [mu]M. The mode of action was an iso-ordered bi bi mechanism in which A, B, P, and Q equal hydroxycinnamoyl-CoA, tyramine, N-hydroxycinnamoyltyramine, and CoA, respectively. Thus, the reaction occurred in a ternary complex of the enzyme and substrates. The equilibrium constant of the reaction was determined to be 1.3 x 104. This gave a [delta]G[deg][prime] eq value of -23.5 kJ mol-1.     

A novel pathway for phosphorylated oligosaccharide biosynthesis. Identification of an oligosaccharide-specific phosphate methyltransferase in dictyostelium discoideum.

The N-linked oligosaccharides on three lysosomal enzymes in Dictyostelium discoideum were found to contain mannose 6-phosphomethyl residues. We have identified and partially characterized a novel S-adenosylmethionine-dependent methyltransferase that is probably responsible for the synthesis of this unusual diester from Man-6-P. The enzyme selectively methylates the phosphate group of Man-6-P (Km 4.3 mM). Glucose-6-P and fructose-1-P are relatively poor acceptors; however, the enzyme is inactive against a broad array of other phosphorylated compounds. Using model di-, tri-, and pentasaccharide acceptors that include portions of the three different branches of high mannose-type oligosaccharides, we found that the enzyme prefers terminal alpha 1----2-linked Man-6-P residues (Km 0.15-1.25 mM) found on the known phosphorylated branches. The enzyme is membrane bound, has a neutral pH optimum and cofractionates on sucrose gradients with GlcNAc-1-P transferase, which resembles its mammalian counterpart, and is, presumably, the first enzyme in the phosphorylation pathway. Based on the substrate specificity and colocalization with GlcNAc-1-P transferase, the phosphate methyltransferase is likely to be responsible for the generation of mannose-6-phosphomethyldiester on Dictyostelium oligosaccharides.     

Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions.

The observed equilibrium constants (Kobs) of the creatine kinase (EC 2.7.3.2), myokinase (EC 2.7.4.3), glucose-6-phosphatase (EC 3.1.3.9), and fructose-1,6-diphosphatase (EC 3.1.3.11) reactions have been determined at 38 degrees C, pH 7.0, ionic strength 0.25, and varying free magnesium concentrations. The equilibrium constant (KCK) for the creatine kinase reaction defined as: KCK = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] [H+]) was measured at 0.25 ionic strength and 38 degrees C and was shown to vary with free [Mg2+]. The value was found to be 3.78 x 10(8) M-1 at free [Mg2+] = 0 and 1.66 x 10(9) M-1 at free [Mg2+] = 10(-3) M. Therefore, at pH 7.0, the value of Kobs, defined as Kobs = KCK[H+] = [sigma ATP] [sigma creatine] divided by ([sigma ADP] [sigma creatine-P] was 37.8 at free [Mg2+] = 0 and 166 at free [Mg2+] = 10(-3) M. The Kobs value for the myokinase reaction, 2 sigma ADP equilibrium sigma AMP + sigma ATP, was found to vary with free [Mg2+], being 0.391 at free [Mg2+] = 0 and 1.05 at free [Mg2+] = 10(-3) M. Taking the standard state of water to have activity equal to 1, the Kobs of glucose-6-P hydrolysis, sigma glucose-6-P + H2O equilibrium sigma glucose + sigma Pi, was found not to vary with free [Mg2+], being 110 M at both free [Mg2+] = 0 and free [Mg2+] = 10(-3) M. The Kobs of fructose-1,6-P2 hydrolysis, sigma fructose-1,6-P2 equilibrium sigma fructose-6-P + sigma Pi, was found to vary with free [Mg2+], being 272 M at free [Mg2+] = 0 and 174 M at free [Mg2+] = 0.89 x 10(-3) M.     

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.     

The Interactive Effects of pH,L-Malate,and Glucose-6-Phosphate on Guard-Cell Phosphoenolpyruvate Carboxylase

The interactive effects of pH, L-malate, and glucose-6-phosphate (Glc-6-P) on the Vmax and Km of guard-cell (GC) phosphoenolpyruvate (PEP) carboxylase (PEPC) of Vicia faba L. were determined. Leaves of three different physiological states (closed stomata, opening stomata, open stomata) were rapidly frozen and freeze dried. GC pairs dissected from the leaves were individually extracted and individually assayed for the kinetic properties of PEPC. Vmax was 6 to 9 pmol GC pair-1 h-1 and was apparently unaffected to a biologically significant extent by the investigated physiological states of the leaf, pH (7.0 or 8.5), L-malate (0, 5, or 15 mM), and Glc-6-P (0, 0.1, 0.5, 0.7, or 5 mM). As reported earlier, the Km(PEP.Mg) was about 0.2 mM (pH 8.5) or 0.7 mM (pH 7.0), which can be compared with a GC [PEP] of 0.27 mM. In the study reported here, we determined that the in situ GC [Glc-6-P] equals approximately 0.6 to 1.2 mM. When 0.5 mM Glc-6-P was included in the GC PEPC assay mixture, the Km(PEP.Mg) decreased to about 0.1 mM (pH 8.5) or 0.2 mM (pH 7.0). Thus, Glc-6-P at endogenous concentrations would seem both to activate the enzyme and to diminish the dramatic effect of pH on Km(PEP.Mg). Under assay conditions, L-malate is an inhibitor of GC PEPC. In planta, cytoplasmic [L-malate] is approximately 8 mM. Inclusion of 5 mM L-malate increased the Km(PEP.Mg) to about 3.6 mM (pH 7.0) or 0.4 mM (pH 8.5). Glc-6-P (0.5 mM) was sufficient to relieve L-malate inhibition completely at pH 8.5. In contrast, approximately 5 mM Glc-6-P was required to relieve L-malate inhibition at pH 7.0. No biologically significant effect of physiological state of the tissue on GC PEPC Km(PEP.Mg) (regardless of the presence of effectors) was observed. Together, these results are consistent with a model that GC PEPC is regulated by its cytosolic chemical environment and not by posttranslational modification that is detectable at physiological levels of effectors. It is important to note, however, that we did not determine the phosphorylation status of GC PEPC directly or indirectly (by comparison of the concentration of L-malate that causes a 50% inhibition of GC PEPC).     

Catalytic properties of phosphoglucomutase from pea chloroplasts.

Electrophoretically homogeneous phosphoglucomutase (PGM) with specific activity of 3.6 units/mg protein was isolated from pea (Pisum sativum L.) chloroplasts. The molecular mass of this PGM determined by gel-filtration is 125 +/- 4 kD. According to SDS-PAGE, the molecular mass of subunits is 65 +/- 3 kD. The Km for glucose-1-phosphate is 18.0 +/- 0.5 microM, and for glucose-1, 6-diphosphate it is 33 +/- 0.7 microM. At glucose-1-phosphate and glucose-1,6-diphosphate concentrations above 0.5 and 0.2 mM, respectively, substrate inhibition is observed. The enzyme has optimum activity at pH 7.9 and 35 degrees C. Mg2+ activates the PGM. Mn2+ activates the enzyme at concentrations below 0.2 mM, while higher concentrations have an inhibitory effect. The activity of the PGM is affected by 6-phosphogluconate, fructose-6-phosphate, NAD+, ATP, ADP, citrate, and isocitrate.     

The kinetics of intact microsome glucose-6-phosphatase are sigmoid at physiologic glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were studied with intact and detergent-disrupted microsomes from normal and diabetic rats. Glucose-6-P concentrations employed (12 microM to 1.0 mM) spanned the physiologic range. With the enzyme of intact microsomes from both groups, plots of v versus [glucose-6-P] were sigmoid. Hanes plots (i.e. [glucose-6-P]/v versus [glucose-6-P]) were biphasic (concave upwards). A Hill coefficient of 1.45 was determined with substrate concentrations between 12 and 133 microM. Disruption of microsomal integrity abolished these departures from classic kinetic behavior, indicating that sigmoidicity may result from cooperative interaction of glucose-6-P with the glucose-6-phosphatase system at the substrate translocase specific for glucose-6-P. With the enzyme from normal rats the [glucose-6-P] at which the enzyme was maximally sensitive to variations in [glucose-6-P] (which we term "Smax"), determined from plots of dv/d [glucose-6-P] versus [glucose-6-P], was in the physiologic range. The Smax of 0.13 mM corresponded well with the normal steady-state hepatic [glucose-6-P] of 0.16 mM, consistent with glucose-6-phosphatase's function as a regulatory enzyme. With the diabetic enzyme, in contrast, values were 0.30 and 0.07 mM for the Smax and steady-state level, respectively. We suggest that the decreasing sensitivity of glucose-6-phosphatase activity to progressively diminishing glucose-6-P concentration, inherent in its sigmoid kinetics, constitutes a mechanism for the preservation of a residual pool of glucose-6-P for other hepatic metabolic functions in the presence of elevated concentrations of glucose-6-phosphatase such as in diabetes.     

Purification and characterization of myocardial fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase

Fructose-6-P,2-kinase:fructose-2,6-bisphosphatase has been purified to homogeneity from beef heart. The enzyme was bifunctional and the specific activities of the kinase and the phosphatase of the pure enzyme were 60 and 30 milliunits/mg, respectively. The molecular weight of the enzyme was 118,000, consisting of two subunits of 58,000. In some preparations of the enzyme a minor protein with a subunit Mr of 54,000 was present. This minor protein (54,000) was also bifunctional and showed the same immunoreactivity as the major protein. The specific activity of fructose-6-P,2-kinase of the minor component was three times higher than that of the major enzyme (58,000), but fructose-2,6-bisphosphatase activity was the same. These two forms have been separated by phosphocellulose chromatography. The tryptic peptide maps of these enzymes were very similar. The 58,000 enzyme was phosphorylated by cAMP-dependent protein kinase but the 54,000 enzyme was not. These results indicated that the minor 54,000 protein might be a proteolytically digested form of the 58,000 enzyme. The Km of the kinase for fructose-6-P and ATP was 70 microM and 260 microM, respectively for both the 58,000 and the 54,000 enzymes. Km for fructose-2,6-P2 and Ki for fructose-6-P of the phosphatase was approximately 40 and 11 microM, respectively. The enzyme was phosphorylated by fructose-2,6-P2 but the stoichiometry of the phosphate incorporation was 0.05 mol/mol subunit, while 0.4 mol/mol was incorporated in rat liver enzyme under the same conditions.