Evidence for the existence of isoenzymes of glycerol phosphate acyltransferase.

Subcellular-fractionation studies confirmed previous findings that rat liver glycerol phosphate acyltransferase was located in both mitochondria and the microsomal fraction. Studies of the two activities revealed several differences between them. The mitochondrial enzyme had a lower Km for sn-glycerol 3-phosphate and was more resistant to heat inactivation than was the microsomal enzyme. Some preparations of the mitochondrial enzyme were inhibited by high concentrations of glycerol phosphate. The mitochondrial enzyme was not inactivated by thiol-group reagents, whereas the microsomal enzyme was very rapidly inactivated by these compounds. However, the microsomal enzyme could be specifically protected against this inactivation by low concentrations of palmitoyl-CoA. The results indicate the existence of distinct isoenzymes of glycerol phosphate acyltransferase with different intracellular locations.     

Crystallization and properties of cathepsin B from rat liver.

Cathepsin B from rat liver was purified to apparent homogeneity by cell-fractionation, freezing and thawing, acetone treatment, gel filtration, DEAE-Sephadex and CM-Sephadex column chromatography, and was crystallized. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by disc gel electrophoresis in the presence of sodium dodecyl sulfate and by ultracentrifugal analysis. Its s20,w value was 2.8 S and its relative molecular mass was calculated to be 22,500 (+/- 900) by sedimentation equilibrium analysis. Crystalline cathepsin B was shown to consist of four isozymes with isoelectric points between pH 4.9 and 5.3, the main isozyme having an isoelectric point of pH 5.0. The enzyme was irreversibly inactivated by exposure to weak alkali. The pH optimum was 6.0 with alpha-N-benzoyl-DL-arginine-4-nitroanilide as substrate. Amino acid analysis showed that the enzyme contained hexosamine, glucosamine and galactosamine. Cathepsin B inactivated aldolase, glucokinase, apo-ornithine aminotransferase, and apo-cystathionase, but the rates of inactivation of glucokinase, apo-ornithine aminotransferase, and apocystathionase were lower than that of aldolase. Studies by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate showed that cathepsin B degraded apo-ornithine aminotransferase to two polypeptide chains differing in relative molecular mass and electrophoretic mobility.     

Irreversible inactivation of hypoxanthine phosphoribosyltransferase by periodate oxidized nucleotides.

Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) from rat brain or human erytherocytes can be irreversibly inactivated by incubation with periodate-oxidized analogues of the enzyme products GMP or IMP. This inhibition is specific and directed against the product binding site of the enzyme. Inactivation is not produced by periodate-oxidized AMP or other aldehydes, for example periodate-oxidized glycerol. The inactivation is concomitant with the binding of the inhibitor to the enzyme protein. The bound inhibitor cannot be removed from the protein by dialysis, Sephadex chromatography or polyacrylamide-gel electrophoresis. Adenine phosphoribosyltransferase (EC 2.4.2.7), on the other hand, is not influenced by any of the inhibitors mentioned above.     

Competitive inhibition of liver glucokinase by its regulatory protein

The regulatory protein of rat liver glucokinase (hexokinase IV or D) behaved as a fully competitive inhibitor of this enzyme when glucose was the variable substrate, i.e. it increased the half-saturating concentration of glucose as a linear function of its concentration without affecting V (velocity at infinite concentration of substrate). The inhibition by the regulatory protein and that by palmitoyl-CoA were synergistic with that by N-acetyl-glucosamine, indicating that the two former inhibitors bind to a site distinct from the catalytic site. In contrast, the effects of the regulatory protein and palmitoyl-CoA were competitive with each other, indicating that these two inhibitors bind to the same site. The regulatory protein exerted a non-competitive inhibition with respect to Mg-ATP at concentrations of this nucleotide less than 0.5 mM. At higher concentrations, the latter antagonized the inhibition by the regulatory protein partly by decreasing the apparent affinity for fructose 6-phosphate. The following anions inhibited glucokinase non-competitively with respect to glucose: Pi, sulfate, I-, Br-, No3-, Cl-, F- and acetate. Pi and sulfate, at concentrations in the millimolar range, decreased the inhibition by the regulatory protein by competing with fructose 6-phosphate. Monovalent anions also antagonized the inhibition by the regulatory protein with the following order of potency: I- greater than Br- greater than NO3- greater than Cl- greater than F- greater than acetate and their effect was non-competitive with respect to fructose 6-phosphate. Glucokinase from Buffo marinus and pig liver were, like the rat liver enzyme, inhibited by the regulatory protein, as well as by palmitoyl-CoA at micromolar concentrations. In contrast, neither compound inhibited hexokinases from rat brain, beef heart or yeast, or the low-Km specific glucokinase from Bacillus stearothermophilus.     

Opposing effects of two osmolytes--trehalose and glycerol--on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 degrees C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t0.5 for thermal inactivation of 56+/-5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 degrees C. Indeed, when phosphofructokinase was incubated for 45 min at 50 degrees C in the presence of lower concentrations of glycerol (7.5-25%, v/v), the remaining activity was 2-4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules.     

The inhibition of glucokinase and glycerokinase from Bacillus stearothermophilus by the triazine dye Procion Blue MX-3G.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.     

A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline.

Glycerophosphate acyltransferase present in an extract of rat adipocytes is strongly inhibited by excess palmitoyl-CoA. This inhibition is released by serum albumin but an excess of serum albumin is inhibitory, particularly at low palmitoyl-CoA concentrations. An optimal activity is reached when the ratio palmitoyl-CoA/albumin is in the range of 3-6. In the absence of albumin, oleic acid inhibits the activity at all palmitoyl-CoA concentrations. This inhibition is released by albumin and, inversely, oleic acid releases the inhibition by high concentrations of albumin. Another effect of fatty acids is to favour the inactivation of the glycerophosphate acyltransferase in extracts of adipocytes kept at 0 degree C. This inactivation is time-dependent and cannot be reversed by the addition of albumin to the assay mixture. Treatment of adipocytes with noradrenaline had no effect on the activity of the enzyme as long as the cells had been separated from fatty acids and albumin. With extracts of unwashed cells, the effect of noradrenaline on both the activity and stability of glycerophosphate acyltransferase could be explained by the presence of fatty acids in the extract.     

Factors affecting the activity and stability of the palmitoyl-coenzyme A hydrolase of rat brain

Palmitoyl-CoA hydrolase (EC 3.1.2.2) catalyses the irreversible hydrolysis of long-chain acyl-CoA thioesters. This enzyme is found primarily in the postmicrosomal supernatant fraction prepared from homogenates of rat brain. Either of two forms of the hydrolase, a lower-molecular-weight species of approx. 70000 or a higher-molecular-weight species of approx. 130000 can be isolated by gel filtration. The higher-molecular-weight form is obtained from columns of Sephadex G-200 eluted with buffer containing 10mum-palmitoyl-CoA or 20% (v/v) glycerol, whereas the lower-molecular-weight form is obtained when the eluting buffer does not contain palmitoyl-CoA or glycerol. The two forms of the hydrolase have the same pH optimum of 7.5, are equally sensitive to the thiol-blocking reagents p-hydroxymercuribenzoate, HgCl(2), and 5,5'-dithiobis-(2-nitrobenzoic acid), and exhibit the same K(m) (1.8mum) with palmitoyl-CoA as substrate. The two forms differ in the availability or reactivity of certain external thiol groups, as determined by covalent chromatography with activated thiol Sepharose. Dilute solutions of the lower-molecular-weight form of the hydrolase rapidly lose activity (50% in 60min at 0 degrees C), but there is no change in the K(m) with palmitoyl-CoA as substrate during this progressive inactivation. Dilutions of the hydrolase in buffer containing 10mum-palmitoyl-CoA retain full activity. However, addition of palmitoyl-CoA to solutions of the lower-molecular-weight form will not restore previously lost hydrolase activity. The evidence supports the conclusion that the substrate palmitoyl-CoA promotes the formation of a relatively stable dimer from two unstable subunits. This process may not be reversible, since the removal of palmitoyl-CoA or glycerol from solutions of the higher-molecular-weight form does not result in the appearance of the lower-molecular-weight form of the hydrolase.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Chemical Modification of Specific Active Site Amino Acid Residues of Enterobacter aerogenes Glycerol Dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (GlDH EC 1.1.1.6), a tetrameric NAD + specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5′-phosphate (PLP) and o -phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced GlDH indicated the specific modification of ? -amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD + and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of GlDH by the bifunctional reagent OPA, which reacts specifically with proximal ? -NH 2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD + was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of GlDH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase

Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.     

Effect of glycerol on glucokinase activity: loss of cooperative behavior with respect to glucose

Glucose phosphorylation catalyzed by rat liver glucokinase measured at saturating concentrations of MgATP2- shows a cooperative response with respect to glucose in the concentration range 0.25-5 mM with a Hill coefficient of 1.6. In this range of glucose concentrations, the degree of cooperativity was dependent on the presence of glycerol in the assay mixture, and it decreased progressively and disappeared completely as the glycerol concentration reached about 20% (v/v) glycerol. If attention was confined to concentrations above 5 mM, no cooperativity could be detected either in the absence or in the presence of glycerol. The limiting velocity of the glucokinase reaction (measured at saturating concentrations of glucose and MgATP2-), and the half-saturation concentration for glucose and MgATP2- were all decreased by about 50-60% as the glycerol concentration was raised from zero to 30% (v/v). The presence of glycerol had no effect on the qualitative inhibition patterns of MgADP2-, glucose 6-phosphate, or N-acetylglucosamine, and only slight effects on the quantitative half-saturation values and inhibition constants. All of these effects caused by glycerol were fully reversible by decreasing the concentration of glycerol by dilution. Simulation studies based on the "mnemonical" model of glucokinase action proposed earlier [A. C. Storer and A. Cornish-Bowden (1977) Biochem. J. 165, 61-69] show that the effects of glycerol on glucokinase-catalyzed glucose phosphorylation can simply be explained assuming the glycerol favors the existence of the conformation of the enzyme with a higher affinity for glucose and thus supports the model.     

Studies of rat adipose-tissue microsomal glycerol phosphate acyltransferase.

Incubation of rat adipose-tissue microsomal fractions with iodoacetate caused an inactivation of glycerol phosphate acyltransferase that could be prevented by the presence of palmitoyl-CoA. A microsomal protein of subunit Mr 54 000 was found to react with radioactively labelled iodoacetate in the absence, but not in the presence, of palmitoyl-CoA. It is suggested that this protein is a component of glycerol phosphate acyltransferase. Incubation of rat adipose-tissue microsomal fractions with the catalytic subunit of cyclic AMP-dependent protein kinase, ATP and Mg2+ caused an inactivation of glycerol phosphate acyltransferase whose magnitude depended on the conditions used for assay of the acyltransferase. Rat adipose tissue microsomal proteins were phosphorylated by using protein kinase and [gamma-32P]ATP. One of the phosphorylated proteins was very similar, but not identical, in mobility to the Mr-54 000 protein labelled by iodoacetate. In contrast with a previous report [Sooranna & Saggerson (1976) FEBS Lett. 64, 36-39], no changes could be detected in the activity of glycerol phosphate acyltransferase in adipocytes treated with adrenaline. Adipocytes were labelled with [32P]Pi and treated with adrenaline, but no 32P was incorporated into the Mr-54000 protein labelled by iodoacetate. The results suggest that the activity of adipose-tissue microsomal glycerol phosphate acyltransferase is not directly controlled by phosphorylation.     

Cobalt(III) labeling of methionyl-tRNA synthetase from Escherichia coli.

Native and trypsin-modified methionyl-tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP-Co(II). It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP-Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site. The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2-mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.     

Palmitate activation and esterification in microsomal fractions of rat liver.

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.     

In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography

Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.     

Opposing effects of two osmolytes – trehalose and glycerol – on thermal inactivation of rabbit muscle 6-phosphofructo-1-kinase

Trehalose and glycerol are known as good stabilizers of function and structure of several macromolecules against stress conditions. We previously reported that they have comparable effectiveness on protecting two yeast cytosolic enzymes against thermal inactivation. However, enzyme protection has always been associated to a decrease in catalytic activity at the stabilizing conditions i.e., the presence of the protective molecule. In the present study we tested trehalose and glycerol on thermal protection of the mammalian cytosolic enzyme phosphofructokinase. Here we found that trehalose was able to protect phosphofructokinase against thermal inactivation as well as to promote an activation of its catalytic activity. The enzyme incubated in the presence of 1 M trehalose did not present any significant inactivation within 2 h of incubation at 50 C, contrasting to control experiments where the enzyme was fully inactivated during the same period exhibiting a t_0.5 for thermal inactivation of 56± 5 min. On the other hand, enzyme incubated in the presence of 37.5% (v/v) glycerol was not protected against incubation at 50 C. Indeed, when phosphofructokinase was incubated for 45 min at 50 C in the presence of lower concentrations of glycerol (7.5–25%, v/v), the remaining activity was 2–4 times lower than control. These data show that the compatibility of effects previously shown for trehalose and glycerol with some yeast cytosolic enzymes can not be extended to all globular enzyme system. In the case of phosphofructokinase, we believe that its property of shifting between several different complex oligomers configurations can be influenced by the physicochemical properties of the stabilizing molecules. (Mol Cell Biochem 269: 203)     

Inactivation of glycerol dehydrogenase of Klebsiella pneumoniae and the role of divalent cations.

Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of O2, Fe2+, and ascorbate or dihydroxyfumarate. It appears that O2 and the reducing agent formed H2O2 and that H2O2 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD (but not glycerol) and could no longer be activated by Mn2+ which increased the Vmax of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.     

Purification of a new high activity form of glucose-6-phosphate dehydrogenase from rat liver and the effect of enzyme inactivation on its immunochemical reactivity.

A new form of cytoplasmic glucose-6-phosphate dehydrogenase (E.C.1.1.1.49) was purified from rat liver by protamine sulfate precipitation, ammonium sulfate fractionation, ion exchange chromatography with diethylaminoethyl cellulose, and affinity chromatography with Cibacron blue agarose and NADP agarose. This form of the enzyme has a specific activity of over 600 units/mg of protein and gives essentially a single band by polyacrylamide gel electrophoresis. The form of the enzyme isolated by this purification method is 3 times more active than the form purified from liver by previously reported procedures. The relative mass of this pure glucose-6-phosphate dehydrogenase enzyme was determined by disc gel electrophoresis to be 269,000. This high activity glucose-6-phosphate dehydrogenase enzyme, after inactivation by reaction with palmityl-CoA, was no longer precipitated by specific rabbit and goat antisera to this purified enzyme. Thus, the possibility still exists that starved fat-refed animals contain glucose-6-phosphate dehydrogenase (G6PD) enzyme protein in an inactivated form no longer detectable by either enzyme activity or immunoprecipitation.     

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.