Release and activation of a particulate bound acid phosphatase from Tetrahymena pyriformis.

A sedimentable form of acid phosphatase (EC 3.1.3.2) from Tetrahymena pyriformis was found to be solubilized by Triton X-100. The total enzyme activity in the insoluble cell fraction increased almost 200% upon solubilization with Triton X-100 or Nonidet P-40. Removal of membrane lipids and Triton X-100 from the particulate wash solution with a chloroform extraction resulted in non-specific enzyme-protein aggregation which was reversible upon addition of Triton X-100. The results indicate that this acid phosphatase is an integral membrane protein. The pH optima for this particulate bound acid phosphatase was 3.5 with o-carboxyphenyl phosphate and 4.0 with p-nitrophenyl phosphate as substrates. The Km values of each substrate were 3.1 and 0.031 mM, respectively.     

Phytase activity in Cryptococcus laurentii ABO 510

Ten Cryptococcus strains were screened for phytase activity, of which the Cryptococcus laurentii ABO 510 strain showed the highest level of activity. The cell wall-associated enzyme displayed temperature and pH optima of 62 degrees C and 5.0, respectively. The enzyme was thermostable at 70 degrees C, with a loss of 40% of its original activity after 3 h. The enzyme was active on a broad range of substrates, including ATP, D-glucose 6-phosphate, D-fructose 1,6-diphosphate and p-nitrophenyl phosphate (p-NPP), but its preferred substrate was phytic acid (K(m) of 21 microM). The enzyme activity was completely inhibited by 0.5 mM inorganic phosphate or 5 mM phytic acid, and moderately inhibited in the presence of Hg(2+), Zn(2+), Cd(2+) and Ca(2+). These characteristics suggest that the Cry. laurentii ABO 510 phytase may be considered for application as an animal feed additive to assist in the hydrolysis of phytate complexes to improve the bioavailability of phosphorus in plant feedstuff.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Screening for beta-poly(L-malate) binding proteins by affinity chromatography

Poly(beta-L-malic acid) is a cell type-specific polymer of myxomycetes (true slime molds) with the physiological role to organize mobility of certain proteins over the giant multinucleated plasmodia. We have developed an affinity chromatography employing 1,6-diamino-n-hexane-Sepharose-coupled poly(malic acid) to identify such proteins in cellular extracts of Physarum polycephalum. Molecular masses were measured by SDS-PAGE and non-denaturing PAGE after silver staining and/or Western blotting. Protein complexes/subunits were detected by 2-dimensional non-denaturing PAGE/SDS-PAGE. A simplified gel shift experiment displayed binding to fragmented calf thymus DNA. Nuclei were richest in poly(malate) binding proteins followed by cytoplasm and membranes. A protein of 370 kDa dissociated into 11 subunits of 11-29 kDa, indicative of a highly complex protein. This and other proteins displayed binding to nucleic acid in gel shift experiments. Poly(malate) is considered a structural and functional equivalent of long contiguous aspartate repeats in proteins of eukaryotes.     

Purification and characterization of carbonic anhydrase from bovine erythrocyte plasma membrane

Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membrane and characterized in this study. For this purpose, the blood taken from young animals was hemolysed, the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was removed from the membrane with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography.The CA obtained from erythrocyte membrane has an esterase activity as well as hydratase activity. The Vmax and Km of the enzyme for the substrate (p-nitrophenyl acetate) are 1.948x10(-3) mM/L x dak, and 3.596 mM, respectively. The purification degree of the enzyme was controlled by SDS-PAGE (3-10), which showed two distinct bands. It was determined that the enzyme had activity within the pH range of 4.5-9.5 and that the optimal pH was 7.5. The temperature at which it showed activity was 20-60 degrees C and optimal temperature was 37 degrees C. Molecular weight of CA was found to be 29844 and 61706 Dalton by gel filtration. On the other hand, sulfanilamide and acetazolamide affected the enzyme.     

Proctolin degradation by membrane peptidases from nervous tissues of the desert locust (Schistocerca gregaria).

The hydrolysis of the insect neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) by enzyme preparations from the nervous tissue of the desert locust (Schistocerca gregaria) was investigated. Neural homogenate degraded proctolin (100 microM) at neutral pH by cleavage of the Arg-Tyr and Tyr-Leu bonds to yield Tyr-Leu-Pro-Thr, Arg-Tyr and free tyrosine. Arg-Tyr was detected as a major metabolite when the aminopeptidase inhibitors amastatin and bestatin were present to prevent Arg-Tyr breakdown. Around 50% of the proctolin-degrading activity was isolated in a 30,000 g membrane fraction and was shown to be almost entirely due to aminopeptidase activity. The aminopeptidase had an apparent Km of 23 microM, a pH optimum of 7.0 and was inhibited by 1 mM-EDTA and amastatin [IC50 = 0.3 microM], but was relatively insensitive to bestatin, actinonin and puromycin. Phenylmethanesulphonyl fluoride (1 mM) and p-chloromercuriphenylsulphonic acid (1 mM) had no effect on this enzyme activity. Although the bulk of the Tyr-Leu hydrolytic activity was located in the 30,000 g supernatant, some weak activity was detected in a washed membrane preparation. This peptidase displayed a high affinity for proctolin (Km = 0.35 microM) and optimal activity at around pH 7.0. Synaptosome- and mitochondria-rich fractions were prepared from crude neural membranes. The aminopeptidase activity was concentrated in the synaptic-membrane preparation, whereas activity giving rise to Arg-Tyr was predominantly localized in the mitochondrial fraction. The subcellular localization of the membrane aminopeptidase is consistent with a possible physiological role for this enzyme in the inactivation of synaptically released proctolin.     

Characterization of a retinylmonophosphatase in the plasma membrane of mouse brain.

Retinylmonophosphatase (RMPase) activity in mouse brain paralleled the subcellular distribution of the plasma-membrane marker Na+ + K+-dependent ATPase. The enzyme had a pH optimum between 5.5 and 7.0. The enzyme demonstrated linear kinetics with respect to time and both protein and substrate concentrations. RMPase was saturated by low retinyl monophosphate (RMP) concentrations and exhibited an apparent Km of 4.6 microM. The enzyme did not require MgCl2 for activity, and in fact assays were routinely run in the presence of 10 mM-Na2EDTA. In general, detergents inhibited the enzyme, with 0.05% Triton X-100 causing a 30% loss of activity. Phosphatidic acid was also inhibitory, but phosphatidylcholine and sphingomyelin stimulated phosphatase activity. RMPase was inhibited 35% by 5 mM concentrations of fluoride, phosphate or pyrophosphate. A series of other phosphorylated compounds, including glucose 6-phosphate, alpha-glycerophosphate, ATP, AMP, p-nitrophenyl phosphate and thiamin pyrophosphate, showed little or no inhibition. RMPase activity differed in several characteristics from that previously reported for dolichylmonophosphatase. It is concluded that RMP could play a distinct role in the plasma membrane.     

Extracellular alkaline phosphatase is a sensitive marker for cellular stimulation and exocytosis in heterotroph cell cultures of Chenopodium rubrum

We investigated the response of extracellular phosphatase to heat shock in heterotrophic Chenopodium rubrum L. cell cultures. Surprisingly, in contrast to the generally used acid phosphatase, an extracellular alkaline phosphatase showed the most sensitive response. This phosphatase was characterized as a marker for cellular stimulation by its high correlations with induced changes of extracellular pH: 10microM nigericin (correlation coefficient r=0.91), 100microM salicylic acid (r=0.84), heat shock 5min 37 degrees C (r=0.79), and heat shock after pre-treatment with 5microM fusicoccin (r=0.92) or 0.5% ethanol (r=0.90). Cellular stimulation was estimated with concentrations of acids and bases, yielding similar levels of pH change (0.5 pH) in cell-free supernatant: salicylic acid (200microM), benzoic acid (600microM), HCl (140microM), NaOH (100microM), and KOH (100microM). The Golgi apparatus inhibitor Brefeldin A (200microM) reduced the heat-shock-induced phosphatase (-33%). The pH optimum of heat-shock-induced phosphatase was 3; however, there the proportion of constitutive phosphatase was higher than at pH 8-9.5, indicating different pH dependence of constitutive and induced activity. Thus, heat-shock-induced phosphatase was characterized by alkaline activity with inhibitors (10microM molybdate: -52%, 2.5mM phosphate: -64%, 10microM ZnCl(2): -82%), substrates (2.5mM, tyrosine phosphate: 255pkat g(-1), p-nitrophenyl phosphate: 92pkat g(-1), serine phosphate: 0, threonine phosphate: 0), Hill coefficient (nH=1.4) indicating two binding sites, and the extent of heat-shock stimulation (p-nitrophenyl phosphate: +190%, tyrosine phosphate: +180%). SDS-PAGE showed a correlation of alkaline phosphatase with the heat-shock-induced release of highly N-glycosylated 53kDa protein, detected by peroxidase-labeled concanavalin A affinoblotting after endoglycosidase H treatment. The 53kDa protein showed no in-gel phosphatase activity after SDS-PAGE and regeneration treatment, in contrast to a putative dimer (105kDa).     

Lipoxygenase in trout gill tissue acting on arachidonic, eicosapentaenoic and docosahexaenoic acids

Lipoxygenase activity was characterized in the gill tissue of fresh-water trout. Incubation of arachidonic acid with gill preparations yielded 12-hydroxyeicosatetraenoic acid as the major product, suggesting a 12-lipoxygenase. Eicosapentaenoic acid was similarly converted to the 12-hydroxyeicosapentaenoic acid. Both arachidonic acid and docosahexaenoic acid were converted with equal apparent velocities and affinities into single monohydroxy derivatives. Analyses of the hydroxy product of docosahexaenoic acid were consistent with 14-hydroxydocosahexaenoic acid. This enzyme activity was localized to the cytosolic fraction and displayed a broad pH optimum around pH 7. The enzyme was insensitive to the cyclooxygenase inhibitors indomethacin and aspirin but activity was strongly inhibited in the presence of the lipoxygenase inhibitors, SnCl2 (5 mM), esculetin (10 microM) and eicosatetraynoic acid (100 microM).     

Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles

The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.     

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Properties of p-nitrophenyl phosphatase activity from porcine neutrophils

p-nitrophenyl phosphatase activity is high in porcine neutrophils and was found in plasma membrane and granule fractions isolated from sucrose density gradients after nitrogen cavitation to disrupt the cells. Very little activity was found in the cytosol. The enzyme has optimum activity at alkaline pHs with a pH optimum of 10.3. The pH profile was fairly broad with activity still remaining at physiological pH. Orthovanadate was shown to be a potent competitive inhibitor of the enzyme with a Ki of 14 microM. Phosphate also inhibited but at millimolar concentrations and the two inhibitors bind in a mutually exclusive fashion. Evidence from experiments using divalent ion chelators and zinc ions suggested that the phosphatase is a zinc metalloenzyme. Beryllium was found to be a very potent, non-competitive inhibitor of the neutrophil enzyme (Ki = 1.1 microM). Levamisole and theophylline were both shown to be uncompetitive inhibitors of the porcine phosphatase (Ki = 0.2 mM and 1.2 mM respectively). The neutrophil phosphatase was inhibited by L-homoarginine but unaffected by L-phenylalanine and L-glutamate.     

Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase

A particulate form of protein-phosphotyrosine phosphatase was solubilized and purified over 2,000-fold from the particulate fraction of rat spleen. Phosphorylated poly(Glu, Tyr), a random copolymer of glutamic acid and tyrosine, was used as substrate for measuring protein-phosphotyrosine phosphatase activity. Nonionic detergents like Triton X-100 increased the protein-phosphotyrosine phosphatase activity of the particulate fraction (but not of the soluble fraction) by 4-8-fold. Chromatography of the Triton extract of the particulate fraction on DEAE-Sephacel gave three peaks of protein-phosphotyrosine phosphatase activity. The major peak of activity was further purified on Bio-Gel HTP, Sephadex G-75, and phosphocellulose columns. On polyacrylamide gel electrophoresis in the presence of Na-dodecyl-SO4 the purified enzyme showed a major protein band of Mr 36,000 which comigrated with enzyme activity on the phosphocellulose column. The apparent Vmax and Km for phosphorylated poly(Glu,Tyr) were 6,150 nmol min-1 mg-1 and 1.6 microM, respectively. This enzyme was strongly inhibited by microM concentrations of orthovanadate and zinc acetate. Fluoride (50 mM) inhibited this enzyme only by 30-40%. Divalent metal ions Ca2+, Mg2+, and Mn2+ were inhibitory at 1-10 mM concentration. EDTA had no effect on the activity of the purified enzyme. This phosphatase could dephosphorylate and inactivate the phosphorylated form of a tyrosine-specific protein kinase (TK-I) previously purified from rat spleen. Dephosphorylation and inactivation of TK-I by purified phosphatase were inhibited by orthovanadate. After dephosphorylation and inactivation by phosphatase, TK-I could be rephosphorylated and reactivated on incubation with ATP. These results suggest that this protein-phosphotyrosine phosphatase may be involved in the regulation of the kinase activity of TK-I.     

Properties of a Leu-Phe-Cleaving Endopeptidase Activity Putatively Involved in β-Endorphin Metabolism in Rat Brain

Incubation of beta-endorphin with cytosolic and particulate fractions of rat brain resulted in the formation of several peptides, including gamma-endorphin [beta-endorphin-(1-17)] and beta-endorphin-(18-31), indicating the presence of enzyme activity cleaving the Leu17-Phe18 bond of beta-endorphin. An assay for this Leu-Phe cleaving activity, based on the cleavage of the 14C-labeled substrate acetyl-Val-Thr-Leu-Phe-[epsilon-([14C]CH3)2]Lys-NHCH3, was used to examine the properties of this enzyme activity. beta-Endorphin-(1-31) competitively inhibited the Leu-Phe-cleaving enzyme activity on the pentapeptide substrate. Over 90% of activity was recovered in the cytosolic fraction. Leu-Phe-cleaving activity behaved like a thiol endopeptidase because it was inhibited by low concentrations of N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzoyl sulfate, and low concentrations of Hg2+. Low concentrations of sulfhydryl compounds stimulated Leu-Phe-cleaving activity. The activity was optimal between pH 8.5 and 9.0. The Km of Leu-Phe-cleaving activity in the cytosolic fraction was 35 microM and in the particulate fraction 88 microM with Vmax values of 193 and 15 nmol mg protein-1 h-1, respectively. The apparent molecular mass of the Leu-Phe-cleaving enzyme was estimated by gel filtration to be approximately 200 kilodaltons. These properties of Leu-Phe-cleaving activity indicate that the Leu-Phe-cleaving enzyme is distinct from any known brain endopeptidase.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

Spectrophotometric and cytochemical analyses of phosphatase activity in Beta vulgaris L

Spectrophotometric and cytochemical methods were used to investigate the localization and/or the sensitivity of phosphatase activities in aldehyde-fixed beet leaves and membrane fractions. The nonspecific acid phosphatase substrates, p-nitrophenyl phosphate and beta-glycerol phosphate, each exhibited unique spectrophotometric patterns of hydrolysis as a function of pH. Additionally, beta-glycerol phosphatase activity was primarily present on the tonoplast, whereas p-nitrophenyl phosphatase was present on the plasma membrane. Because of the unique pH response of each enzyme and their different localization, we conclude that they cannot be entirely "nonspecific." The spectrophotometric pattern of ATP hydrolysis differed from that of p-nitrophenol phosphate in that it decreased at pH 5.0-5.5 and was greatly inhibited by 10 mM sodium fluoride; however, both activities were on the plasma membrane. Therefore, we conclude that these activities represent either two enzymes or only one enzyme that differs in its ability to hydrolyze these two substrates. Generally, enzymatically produced lead deposits on the plasma membrane of non-vascular cells were as frequent and large as those on phloem cells; frequently, deposits on sieve element plasma membranes were relatively small. We therefore conclude that there is no evidence for the presence of relatively intense ATPase activity on the plasma membrane of phloem cells in beet leaf, in contrast to other species. Studies with membrane fractions indicated that formaldehyde could completely inhibit the inhibitor-sensitive phosphatase activities in mitochondrial and vacuolar fractions while preserving significant activity in the plasma membrane fraction.     

Purification and properties of cytosolic malic enzyme from human skeletal muscle

1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.