Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Sequential degradation of chondroitin sulfate in molluscs. Desulfation of chondroitin sulfate without prior depolymerization by a novel sulfatase from Anomalocardia brasiliana

A sulfatase acting upon chondroitin sulfate polymers, free of beta-glucuronidase and beta-N-acetylhexosaminidases, was isolated from extracts of the mollusc Anomalocardia brasiliana. The enzyme totally desulfates both chondroitin 4- and 6-sulfates without concomitant depolymerization of the compounds. It has no activity upon heparan sulfate, heparin, dermatan sulfate, and chondroitin sulfate disaccharides. It shows a pH of 5.0 and a temperature of 37 degrees C for optimum activity with a Km of 4 x 10(-5) M. The sulfatase is inhibited by sulfate and phosphate ions and HgCl2. The latter inhibition is reverted by sodium tetrathionate. Contrary to the sulfatases described so far the enzyme is activated by the lactone of D-saccharic acid when in the presence of beta-glucuronidase and beta-N-acetylgalactosaminidase. Several experiments indicate that the sulfatase is the first enzyme in the sequential degradation of chondroitin sulfate in the mollusc. This differs from the pathway of degradation of this compound in vertebrates and bacteria.     

Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller.

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8--4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.     

Preparation and characterization of kappa-carrageenan immobilized urease

Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity.     

Dirofilaria immitis: comparison of cytosolic and mitochondrial glutamate dehydrogenases

Two membrane-bound glutamate dehydrogenases were found in adult Dirofilaria immitis, an NAD-linked enzyme (EC 1.4.1.2) in the cytosol (C-GDH) and an enzyme equally reactive with NAD or NADP (EC 1.4.1.3) in the mitochondria (M-GDH). The cytosolic enzyme had a pH optimum of 7.8-8.0 and exhibited 30% more activity at 25 C than at 37 C (pH 8.0). The mitochondrial enzyme had a pH optimum at 8.4 and exhibited 27% more activity at 37 C than at 25 C (pH 8.4); it was also more sensitive to heat denaturation. Gel filtration of worm subfractions separated four peaks of C-GDH activity with molecular weights of approximately 610, 285, 180, and less than 100 thousand, and a single major peak of M-GDH activity with a molecular weight of about 335,000. When assayed at pH 8, 37 C, and 200 microM NADH, the Km for the substrate, alpha-ketoglutarate, was equivalent for the two enzymes, but the Km for ADP (activator) was five times greater for M-GDH. When the two enzymes were assayed at pH 8.0, 37 C, and 100 microM NADH, 1 mM ADP approximately doubled and 1 mM ATP halved the velocity observed for each enzyme with no effector present. Under these assay conditions AMP, IDP, GDP, and GTP had opposite effects on the reaction velocities for the two enzymes. When the assay conditions were changed, the effects of added purine nucleotides varied, even directionally. Addition of up to 5 mM glutamate (product) had no significant effect on C-GDH kinetics, nor on the substrate Km of M-GDH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some properties of the pyruvate carboxylase from Pseudomonas fluorescens.

The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold from cells grown on glucose at 20 degrees C. The activity of this purified enzyme was not affected by acetyl-coenzyme A or L-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0-08 and 0-21 mM, from the lower and the higher concentration ranges, respectively. The apparent Km for HCO3 at pH 6-9, in the presence of the manganese ATP ion (MnATP2-), was 3-1 mM. The enzyme reaction had an optimum pH value of 7-1 or 9-0 depending on the use of MnATP2- or MgATP2-, respectively, as substrate. Free Mg2+ was an activator at pH values below 9-0. The enzyme was strongly activated by monovalent cations; NH4+ and K+ were the better activators, with apparent Ka values of 0-7 and 1-6 mM, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 degrees C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after pre-incubation for 10 min at 46 degrees C. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.     

Thiamine pyrophosphatase and nucleoside diphosphatase in rat brain

Two types of nucleoside diphosphatase were found in rat brain. One (Type L) had similar properties to those of the liver microsomal enzyme with respect to its isoelectric point, substrate specificity, Km values, optimum pH, activation by ATP and molecular weight. The other (Type B), which separated into multiple forms on isoelectric focusing, had lower Km values and a smaller molecular weight than the Type L enzyme, and was inhibited by ATP. The Type B enzyme catalyzed the hydrolysis of thiamine pyrophosphate as well as those of various nucleoside diphosphates at physiological pH, while Type L showed only nucleoside diphosphatase activity at neutral pH. These findings suggest that the two enzymes play different physiological roles in the brain.     

Studies on the soluble and membrane-bound amino acid 2-naphthylamidases in pig and human epidermis.

1. Membrane-bound (particulate) and soluble amino acid 2-naphthylamidases (EC 3.5.1.-) were present in subcellular fractions of epidermis from pig and human. 2. The particulate enzymes exhibited Michaelis-Menten kinetics, with Km 5.1x10(-5) (pig) and Km 7.3x10(-5)M (human) for the substrate L-leucine 2-naphthylamide. They were inhibited by puromycin and partially inhibited by EDTA. They did not require heavy metals and were not inhibited by thiol-group-blocking agents. Their pH optima were 7.0 (human) and 6.6 (pig). The particulate enzyme from pig epidermis retained 50% activity after 30 min at 70 degrees C. 3. The soluble amino acid 2-naphthylamidases gave sigmoidal curves for reaction velocity versus substrate concentration, and the kinetic data suggested that there was positive co-operativity between binding sites. This co-operativity was lost after treatment with 0.1mM-p-hydroxymercuribenzoate and the enzymes showed first-order kinetics at low substrate concentrations. The soluble enzymes were inhibited by puromycin and by thiol-group-blocking agents and activated by dithiothreitol. They were inactivated above 60 degrees C and lost activity on storage, but this could be restored with dithiothreitol. 4. The amino acid 2-naphthylamidases of human epidermis were much more active (2.5 times) towards L-alanine 2-naphthylamide than towards the commonly used substrate L-leucine 2-naphthylamide. 5. The kinetics of both the solube and particulate enzymes from epidermis of some elderly patients with either diabetes or ischaemia showed some differences from the kinetics of enzymes from healthy epidermis from younger individuals.     

Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

Purification and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath).

Crude soluble extracts of Methylococcus capsulatus strain Bath, grown on methane, were found to contain NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70 degrees C for 12 min). The non-dialysable, heat-sensitive component was isolated and purified, and has a molecular weight of about 115000. Sodium dodecyl sulphate gel electrophoresis of the protein suggested there were two equal subunits with molecular weights of 57000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low molecular weight protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized three other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45 degrees C for at least 10 min and had temperature and pH optima of 45 degrees C and pH 7.2 respectively. A number of metal-binding agents and substrate analogues were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.     

Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide supplemented with Streptomyces griseus amino-peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25 degrees C) with a catalytic efficiency (kcat = 1.2 x 10(2) s-1, Km = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (kcat = 1.2 x 10(3) s-1) but the overall efficiency is diminished by a higher Km (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidases and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.     

A specific L-asparaginase from Thermus aquaticus

The L-asparaginase from an extreme thermophile, Thermus aquaticus strain T351, was highly substrate- and stereospecific, with no activity against glutamine or D-asparagine. It had a high Km of 8.6 mM. In these aspects it closely resembled the corresponding enzymes from thermophilic bacteria. The enzyme had a molecular weight of 80,000, an isoelectric point of 4.6, and a pH optimum of 9.5. It showed some substrate inhibition above 20 mM asparagine and was also inhibited by L-aspartic acid, D- and L-lysine (Ki of 5.2 and 1.25 mM, respectively), and D- and L-serine. The half-life of the enzyme at 85 degrees C was 40 min. The Arrhenius plot showed a change in slope at 55 degrees C.     

Enzymatic properties of fish muscle aldolase

1. Aldolases were isolated from the ordinary muscle of red sea bream Pagrus major, Pacific mackerel Scomber japonicus, and carp Cyprinus carpio by ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and CM-Sepharose CL-6B columns, and examined for enzymatic properties. 2. The aldolases showed the highest activity in a pH range from 6.8-7.8 Km values for fructose-1,6-bisphosphate ranged from 0.025-0.10 mM. 3. Irrespective of fish species, aldolase activity was inhibited by ATP, ADP, and AMP. ATP showed the strongest inhibition and was competitive with fructose-1,6-bisphosphate. 4. The aldolases did not require divalent metal ions for activation and were completely inhibited at 0.1 mM Cu2+. 5. Thermal inactivation of the enzymes was of the first-order reaction. Red sea bream, Pacific mackerel and carp enzymes lost the activity by 50% when incubated at 50 degrees C for 8, 14 and 23 min, respectively.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Preparation and properties of immobilized papain and lipase.

Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.     

beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6.

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively.