Apparent Michaelis Constant Values Research Articles

Purification and characterization of phosphatidylethanolamine N-methyltransferase from Zymomonas mobilis.

Phosphatidylethanolamine N-methyltransferase, which catalyzes all the three-step methylation from phosphatidylethanolamine to phosphatidylcholine, was purified to homogeneity from the membrane fraction of Zymomonas mobilis. The purified enzyme exhibited a single band on SDS- polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42,000 on comparison with those of marker proteins. The three activities dependent on phosphatidylethanolamine, phosphatidyl-N monomethylethanolamine and phosphatidyl-N Af-dimethylethanolamine of the purified enzyme showed similar pH profiles with an optimum of pH 8.5, and were enhanced in the same manner by Triton X-100 and l-cysteine. The maximal velocities of the three reactions for S-adenosyl-l-methionine were 0.04, 1.36 and 0.69 nmol/mg protein/min with apparent Michaelis constant values of 3.6, 1.9 and 3.9 fiM, respectively, indicating that the first-step methylation is rate-limiting for the pathway in the organism.

Interaction of substrates with glutamine synthetase after limited proteolysis

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic lipase purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55000, 66000 and 2600 μmol fatty acid/mg per h with apparent Michaelis constant ( K m ) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotien C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.

Properties of uridine diphosphate glucose pyrophosphorylase from Golgi apparatus of liver

Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:α- d-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.

Susceptibilities of cardiac myofibrillar proteins to cathepsin D-catalyzed degradation.

The binding of various myofibrillar proteins to cathepsin D and consequent susceptibility to degradation was assessed by determination of apparent Michaelis constant (Km) values for the purified enzyme using myosin, alpha-actinin, actin, and tropomyosin as substrates. Cathepsin D, purified 1,000-fold to homogeneity from canine cardiac tissue, was incubated (0-100 min at 37 degrees C) with myofibrillar proteins isolated from homologous tissue and labeled radioactively with 14C by reductive alkylation. The reaction was terminated by addition of trichloroacetic acid (10% wt/vol), and radioactivity in the supernatant fraction was determined after centrifugation (5 min) at 100,000 g. Double reciprocal graphs (1/reaction velocity vs. 1/substrate concn) were constructed for each substrate from the linear portion of graphs of disintegrations. min-1 X ml-1 100,000-g supernatant vs. time (min). Apparent Km values (+/- SD) calculated for myosin, alpha-actinin, actin, and tropomyosin were found to be 2.7 +/- 0.3 X 10(-6) (n = 6), 10.0 +/- 2.3 X 10(-6) (n = 13), 13.0 +/- 1.3 X 10(-6) (n = 7), and 45.5 +/- 9.0 X 10(-6) (n = 6) mol/l, respectively. The results show that myofibrillar proteins differ in their binding and thus susceptibility to hydrolysis by cathepsin D in order of molecular weight (i.e., myosin greater than alpha-actinin greater than actin greater than tropomyosin). Because the relative turnover rates of myofibrillar proteins are known to be independent of molecular weight, our results suggest that the degradation of these proteins by cathepsin D is not rate limiting and that other factors including lysosomal or nonlysosomal enzymes must determine the rate-limiting steps of myofibrillar protein degradation in cardiac tissue.

Environmental adaptation of proteins: Strategies for the conservation of critical functional and structural traits

1.1. Comparative studies of lactate dehydrogenases (LDHs) and skeletal muscle actins from vertebrates adapted to widely different temperatures and hydrostatic pressures reveal major conservative trends in protein evolution and adaptation. For enzymes, ligand binding, as estimated by apparent Michaelis constant (Km) values, is strongly conserved at physiological temperatures, pressures, intracellular pH values and osmotic compositions of different organisms. The catalytic rate constants (kcatvalues) of enzyme homologues are highest for enzymes of low-body-temperature organisms, a trend that can be interpreted in terms of temperature compensation of metabolism. For skeletal muscle actins. the enthalpy and entropy changes accompanying the assembly of filamentous (F) actin from globular (G) actin are highest in high-body-temperature species and especially low in polar and deep-sea fishes. The thermal stability of G-actin is positively correlated with adaptation temperature, except in the case of actins of deep-sea fishes, which are also highly heat stable. Hydrophobic interactions between actin subunits may be of reduced importance in low-body-temperature animals and, especially, in deep-sea fishes. The differences in enthalpy and entropy changes during the G-to-F transformation favor a close conservation of the equilibrium constant for actin assembly under physiological conditions of temperature and pressure for different species. These adaptive patterns in enzymes and actin are likely to reflect changes in protein primary structure.2.2. The appropriate values for protein traits such as ligand binding abilities and catalytic rates are also shown to be established by the composition of the low molecular weight constituents of the cytosol. For example, the use of a combination of urea and methylamine solutes for osmoregulation by marine elasmobranchs is shown to be a mechanism which permits the conservation of key protein traits at high osmolarities. The methylamine solutes such as trimethylamine-N-oxide have effects on proteins opposite to those of urea, and at the approximately 2:1 concentration ratio of urea to methylamines, these counteracting effects are virtually complete.3.3. Regulation of hydrogen ion activity (pH) also is shown to play a major role in the conservation of critical protein traits. The importance of temperature-dependent pH in ectotherms is discussed in terms of stabilizing binding abilities and maintaining correct regulatory and structural sensitivities of proteins. The buffering capacity of tissues reflects the potential of the tissue for generating acidic end-products during anaerobic metabolism. Skeletal muscle, especially white locomotory muscle of fishes, is highly buffered relative to red locomotory muscle and heart muscle. Much of the difference in buffering may be due to different amounts of histidine-containing dipeptide buffers in the tissues. However, comparisons of muscle-type (M4) and heart-type (H4) LDHs show that the M4 isozyme has approximately twice the number of histidine residues per tetramer of the H4 isozyme. This difference between M4 and H4 isozymes which occur predominantly in anaerobically- and aerobically-poised tissues, respectively, suggests that selection for buffering capacity may be a heretofore unappreciated facet of protein evolution.

Metabolism of ketone bodies in pregnant sheep.

A combination of isotope-dilution and arteriovenous-difference techniques was used to determine the significance of ketones to energy homoeostasis in fasted pregnant ewes. 2. There was incomplete interconversion of D(-) 3-hydroxybutyrate (3HB) and acetoacetate (AcAc) and therefore neither entry rate nor oxidation of total ketone bodies could be estimated by assuming circulating ketone bodies represent a single metabolic compartment. Total ketone body metabolism was satisfactorily summarized using a three-compartment model. In fasted pregnant ewes the mean entry rate of total ketones was 1 mmol/h per kg body-weight and of the ketones entering the circulating 87% were promptly oxidized to carbon dioxide accounting for 30% of the total CO2 production. 3. Ketone bodies are readily utilized by hind-limb skeletal muscle such that if completely oxidized, 18 +/- 4 and 48 +/- 3% of the oxygen utilized could be accounted for in fed and fasted pregnant ewes respectively. For both 3HB and AcAc there was a hyperbolic relationship between utilization and arterial concentration. The apparent Michaelis constant (Km) values were 0.55 and 1.42 mM respectively and the maximum velocity (Vmax) 2.9 and 5.6 mmol/h per kg muscle. The arterial concentration of AcAc is always below the Km value and this limits the utilization rate. THe D(-) 3HB concentration however, may surpass that required for maximum utilization and ketoacidosis may be a consequence of this. 4. A two-compartment model was used to analyse ketone body metabolism by hind-limb skeletal muscle. The results suggested substantial interconversion and production of AcAc and 3HB. 5. The pregnant uterus utilized 3HB which if completely oxidized accounted for 12 +/- 2 (fed) and 25 +/- 4 (fasted) % of its O2 consumption. At least 64% of the net 3HB utilized was oxidized. AcAc was not utilized in significant quantitites.

Complexes of creatine kinase MM and immunoglobulin A.

Isoenzymes of creatine kinase (CK) with irregular electrophoretic mobility were found in sera from 5 patients. Enzyme immunoelectrophoresis and electroimmunofixation revealed that these unusual isoenzymes were complexes of CK and IgA. Light chain of CK linked IgA was lambda type.The native CK isoenzymes in the complexes were identified as CK-MM, since apparent Michaelis constant values of the complexes were similar to that of CK-MM. It is interesting that all donor patients were elder and had malignant tumors near the terminal stage.

Effet de petites proteines basiques sur les activites phosphohydrolase et phosphotransferase de la glucose-6-phosphatase microsomique des hepatocytes de singe

The effects of histone 2A and some polycations on microsomal carbamylphosphate: d-glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities ( d-glucose-6-phosphate phoshohydrolase, EC 3.1.3.9), have been investigated. 1. 1. Histone 2A and polycations activate the enzymic activities. At a constant cation concentration, this activation increases with the number of cationic groups per molecule. 2. 2. Activation by histone 2A is related to its fixation on microsomal membranes. This fixation varies with quantities of histones and pH. 3. 3. The nature of the interactions between histones and microsomal membranes is shown to be electrostatic, probably between the cationic groups of histones and the anionic group of membranous lipids. 4. 4. Kinetic analysis reveal that histone 2A increases the maximal reaction velocity but does not affect the apparent Michaelis constant values for the substrates. 5. 5. The role played by the cationic groups of histone 2A on the microsomal glucose 6-phosphatase, is discussed.

The interaction of heparin with human α-thrombin: Effect on the hydrolysis of anilide tripeptide substrates

The interaction of heparin with human α-thrombin was investigated in the present report. Hydrolysis of synthetic tripeptide anilide substrates by thrombin was enhanced in the presence of heparin. With both N-α-benzoyl- l-phenylalanyl- l-valyl- l-arginine- p-nitroanilide (BzPheValArgNaN) and N-α- p-tosyl- l-glycyl- l-prolyl- l-arginine- p-nitroanilide (TosGlyProArgNaN), saturating concentrations of heparin enhanced the binding of substrate two-to threefold as determined by a decrease in the apparent Michaelis constant value, while having a marginal inhibitory effect on V. Substrate inhibition was observed with BzPheValArgNaN, which was enhanced in the presence of heparin. The enhancing effect of heparin on the binding of TosGlyProArgNaN was used to determine a dissociation constant value of 1.7 × 10 −9 m for the heparin · thrombin complex. This value is nearly two orders of magnitude lower than the dissociation constant value determined for the heparin · antithrombin III complex (B. Nordenman and I. Bjork, 1978, Biochemistry 17, 3339–3344), suggesting strongly that heparin must bind to thrombin to account for the enhancing effect of heparin on the antithrombin III/thrombin reaction. Heparin also enhanced the rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino- l-2-hepatonone, but had little effect on the inactivation rate with phenylmethanesulfonyl fluoride.

Stimulation by polyamines of carbamylphosphate: Glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase

The effects of added polyamines on carbamylphosphate (carbamyl- P): glucose phosphotransferase and glucose-6-phosphate (Glc-6- P) phosphohydrolase activities of rat hepatic D-Glc -6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6- P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl- P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH 4 + (0.25–5.0 mM) produced at best but a minor activation (0–14%), while poly( L-lysine) ( M r = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H 2O 2 formation; and were eliminated in the presence of Ca 2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl- P and Glc-6- P, but had no effect on the K m for glucose. Poly( L-lysine) increased the V value for both Glc-6- P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl- P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.

Immobilization of urea cycle enzymes. I. Characterization of immobilized carbamoylphosphate synthetase and ornithine carbamoyltransferase

Carbamoylphosphate synthetase (EC 2.7.2.5) and ornithine carbamoyl-transferase (EC 2.1.3.3) extracted from frog liver were successfully immobilized on CNBr-activated Sepharose 4B. The immobilized preparation had a better stability towards heat. The apparent Michaelis constant values for N-acetylglutamate, ammonia, and ATP were not significantly changed by immobilization.

Ferredoxin–NADP+ Reductase from Tsuga canadensis. A Procedure for Isolation and Properties

AbstractActivity of ferredoxin‐NADP+ reductase in leaf extracts of eastern hemlock [Tsuga canadensis (L.) Carr.] was relatively low, but could be markedly increased by use of protective agents. The best method employed polyvinylpolypyrrolidone (PVP) in the extraction medium plus removal of phenolic compounds by filtering the extracts through an insoluble PVP (Polyclar AT) column. Further purification of the enzyme was achieved by means of DEAE cellulose chromatography and DEAE Sephadex chromatography. A 94‐fold purification of the enzyme with a total recovery of 43% was obtained.The eastern hemlock ferredoxin‐NADP+ reductase was characterized by its diaphorase activity, i.e. the transfer of electrons from NADPH to an electron acceptor. 2,6‐dichlorophenol indophenol. The pH optimum for the oxidation of NADPH is between 8.5 and 9.0. The enzyme is highly specific for its electron donor. NADPH, but shows low specificity for electron acceptors. The apparent Michaelis constant values of the enzyme for NADPH. NADH, and 2,6‐dichlorophenol indophenol are 2.4 × 10−5, 5.4 × 10−3, and 4.7 × 10−5M respectively. The molecular weight of the enzyme, as estimated by gel filtration, is about 45,000. The enzyme is inhibited by both organic and inorganic mercurials and certain cations. Comparison of properties of eastern hemlock ferredoxin‐NADP+ reductase and spinach ferredoxin‐NADP+ reductase shows that both enzymes are similar.

Some regulative properties of glutamine synthetase in cucumber leaves

Abstract Glutamine synthetase (L-glutamate: ammonia ligase (ADP) EC 6. 3. t 2) was prepared from cucumber leaves grown on ammonium medium and some regulative properties were investigated. The apparent Michaelis constant (Km) values of the various substrates and cofactors were determined. Lineweaver-Burk plots gave a Km of 4.5mM with L-glutamate, 0.74mM with ATP and 3.1 mM with NH2OH. Metals, including Cu2+, Hg2+, Zn2+, Ni2+, Fe2+, and Fe3+, strongly inhibited the enzyme activity, Cd2+ did not inhibit the enzyme activity markedly, in contrast with its effect on the rice enzyme. Ca2+ was quite inhibitive to enzyme reaction and more than 50% of the activity was lost at 3 mM. The amino acids tested generally had no effect on the enzyme activity except alanine, which showed little but clear inhibition. Isocitrate and α-ketoglutarate were slightly promotive to enzyme activity while pyruvate and glyoxylate (24 mM) significantly inhibited the enzyme activity. Glucose-l- or -6-phosphate and fructose-6-phosphate we...

Metabolism of Uronic Acids in Plant Tissues

A new enzyme, named uronic acid oxidase, was extracted and purified 67-fold by (NH(4))(2)SO(4) fractionation and CM-Sephadex column chromatography from ethylene-treated Shamouti orange (Citrus sinensis L. Osbeck) leaves. The enzyme catalyzes the oxidation of d-galacturonic acid and d-glucuronic acid to the corresponding hexaric acids in the presence of molecular oxygen with the production of H(2)O(2). The pH optimum for the oxidation of d-galacturonic acid and d-glucuronic acid is between 7 and 8. The enzyme is highly specific for d-galacturonic acid and d-glucuronic acid. It also oxidizes polygalacturonic acid. The apparent Michaelis constant values of the enzyme for d-galacturonic acid and d-glucuronic acid are 0.13 and 0.5 mm, respectively. The molecular weight of the enzyme, as determined by gel filtration, is about 98,000. The enzyme is inhibited by sodium hydrosulfite and other sulfites, indicating that it contains a flavin prosthetic group.

Immunological and Biochemical Studies on Isozymes of Malate Dehydrogenase and Citrate Synthetase in Castor Bean Glyoxysomes

Rabbit gamma-globulin obtained after the injection of solubilized proteins of castor bean (Ricinus communis L var. Hale) glyoxysomes contains antibodies against some of the glyoxysomal enzymes. The gamma-globulin was shown to inhibit by 50% and 80%, respectively, the in vitro activities of the castor bean glyoxysomal citrate synthetase and malate dehydrogenase. The conditions required for the inactivation are described.The glyoxysomal and mitochondrial citrate synthetase from castor bean endosperm show no significant difference in the above immunological test, in their apparent Michaelis constant values for acetyl coenzyme A and oxaloacetate, or in their sensitivity toward ATP inhibition. In the immunological test, glyoxysomal malate dehydrogenase from castor bean endosperm appeared to be related more closely to the leaf peroxisomal malate dehydrogenase from spinach (Spinacia oleracea L.) and to the glyoxysomal malate dehydrogenase from a variety of other fatty seedlings than to the castor bean mitochondrial or soluble malate dehydrogenase. Unlike the mitochondrial or soluble malate dehydrogenase, the castor bean glyoxysomal malate dehydrogenase is easily inactivated by gentle heat treatment, as was reported previously by other workers for the leaf peroxisomal malate dehydrogenase. Thus, the malate dehydrogenases from various plant microbodies share some common characteristics which distinguish them from other isozymes of malate dehydrogenase.

Studies on Galactosyltransferase: KINETIC EFFECTS OF α-LACTALBUMIN WITH N-ACETYLGLUCOSAMINE AND GLUCOSE AS GALACTOSYL GROUP ACCEPTORS

Abstract Investigations have been made of the kinetic effects of α-lactalbumin on the reactions catalyzed by galactosyltransferase using both N-acetylglucosamine and glucose as galactosyl group acceptors. The results indicate that α-lactalbumin can cause (a) the reactions to occur via alternative pathways, (b) reductions in the apparent Michaelis constant values for both substrates, (c) a decrease in the apparent maximum velocity of the reaction with N-acetylglucosamine, and (d) an increase in the apparent maximum velocity of the reaction with glucose. Further, it has been shown that α-lactalbumin enhances the substrate inhibition by N-acetylglucosamine and glucose, as well as the dead-end inhibition by l-arabinose and N-acetylgalactosamine. The general conclusion has been reached that α-lactalbumin is a special type of modifier which combines with the enzyme only after the addition of a carbohydrate reactant.

Effects of cetyltrimethylammonium bromide on catalytic properties of kidney microsomal glucose-6-phosphatase, inorganic pyrophosphate-glucose phosphotransferase and inorganic pyrophosphatase activities

The modifying effects of the cationic detergent cetyltrimethylammonium bromide (CTAB) on catalytic properties of kidney microsomal d- glucose-6-P phosphohydrolase (EC 3.1.3.9) and associated inorganic pyrophosphatase and PP 1-glucose phosphotransferase activities have been studied. Initial reaction velocities, apparent Michaelis constant values and apparent maximal reaction velocity values were determined at various H + concentrations with microsomal preparations which had been supplemented with the detergent to a final concentration (w/v), of 0.00, 0.05, 0.10, 0.20 or 0.30% 15 min prior to assay. Measurements of initial reaction velocities at various assay pH values indicated that CTAB treatment produced a shift in pH optimum of glucose-6-P phosphohydrolase activity towards acid pH values and in that of inorganic pyrophosphatase and phosphotransferase activities in the direction of neutrality. Both on the basis of initial reaction velocity values and apparent maximal reaction velocity values extrapolated to infinite substrate concentrations, lower concentrations of CTAB (0.05 and 0.10%, w/v) were found to elevate all activities, while higher concentrations (0.20 and 0.30%, w/v) were less effective in this respect or actually inhibited. In contrast with these polyphasic effects of the detergent on velocity values, apparent Michaelis constant values for all phosphate substrates, assayed at pH 4.5, 5.5, and 6.5, all decreased progressively as CTAB concentrations were increased from 0.05% to 0.30% (w/v). Plots (see J. S. Friedenwald and G. D. Meangwyn-Davies, in W. D. McElroy and B. Glass, The Mechanism of Enzyme Action, Johns Hopkins Press, Baltimore 1954, p. 180), of reciprocals of apparent Michaelis constant values for these compounds versus CTAB concentrations were linear, supporting the concept that CTAB acts as a “coupling” activator at lower concentrations and as a noncompetitive inhibitor at higher concentrations. Apparent Michaelis constants for PP i—0.3–0.4 mM with 0.30% (w/v) CTAB (pH 5.5)—are the lowest such values yet reported. The apparent Michaelis constant value for glucose in the phosphotransferase reaction, in contrast, was only modestly changed by CTAB treatment of microsomes. Several alternative mechanistic interpretations are considered briefly.

Malarial parasite metabolism: The lactic acid dehydrogenase of Plasmodium berghei

The electrophoretic mobility, substrate stereospecificity, and apparent Michaelis constant ( K m ) values for lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized and reduced forms) were examined for the lactic dehydrogenase (LDH) of cell-free extracts of normal mouse erythrocytes and host cell-free P. berghei. The parasite enzyme was electrophoretically distinct from the LDH of the host cell. Both LDH's were stereospecific for l(+) lactic acid. No significant differences existed between the host cell and the parasitic LDH's in the K m values for lactic acid and nicotinamide adenine dinucleotide (oxidized and reduced forms). However, the value for pyruvate with the parasite LDH was 1 36 th that of the host cell enzyme showing an increased affinity of the former system for pyruvate. The parasitic LDH had higher specific acitivities with both lactic acid and pyruvic acid than did the host cell enzyme.