6-Phosphogluconate dehydrogenase in cell free extracts of Escherichia coli K-12

Summary Investigations into the properties of 6-PG dehydrogenase in cell free extracts of Escherichia coli revealed a pH optimum at pH 9.5 with a sharp decline on both sides of the optimum. The addition of 1.0×10^-2 m MgCl₂ produced maximal activity, whereas higher concentrations caused inhibition. The K _m values were 2.5×10^-4 m for 6-phosphogluconate and 2.5×10^-5 m for NADP⁺ as substrate. The enzyme was extremely stable for at least 5 hours if stored at 4°C in Tris–NaCl–MgCl₂ buffer at pH 7.5. 6-PG dehydrogenase activity was shown to be proportional to cell free extract concentration over the range 0–0.3 mg protein. An assay method based on the new optimal conditions has been established and has been shown to be 33% more sensitive than a number of commonly used methods.Meinem hochverehrten Lehrer Herrn Professor A. Rippel zum 80. Geburtstage.     

Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Phormidium sp.

the native enzyme was 104,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits with an identical molecular weight of 52,000. The optimum pH of the reaction was 8.0. The Km values for 6-phosphogluconate and NADP were 3.6×10^?5m and 1.3 × 10^?5m, respectively. The enzyme showed no Mg^2𠀫 requirement for the activity, but was activated by Mn^2𠀫 and Ca^2𠀫. The enzyme was inhibited by sulfhydryl reagents, indicating that a sulfhydryl group may be involved in the active site of the enzyme. The enzyme was also inhibited by NADPH₂, ATP, and the intermediates formed during photosynthesis. The substrate 6-phosphogluconate and cofactor NADP partially protected the enzyme from inactivation. The enzyme had enzymological and physicochemical properties similar to enzymes isolated from other sources.     

Purification and properties of the NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum

Summary NADP-dependent glutamate dehydrogenase from Dictyostelium discoideum was purified 9300 fold with a yield of 4.6%. The enzyme is a hexamer of apparent molecular weight 294 kDa on Sephacryl S400 and a subunit molecular weight of 52 kDa as determined by SDS gel electrophoresis. The apparent K_mS for -ketoglutarate, NADPH and NH _inf4 ^sup+ are 1.2 mM, 9.7 µM and 2.2 mM respectively, and the purified enzyme has a broad pH optimum with a peak at pH 7.75. GTP has a slight stimulatory effect (22% at 83 µM) as does ADP (11% at 1 mM), and AMP is slightly inhibitory (9% at 1 mM) whereas adenosine, ATP and cAMP have little or no effect. Neither the Zn²⁺ chelating compound 1,10-phenanthroline nor EDTA have any effect on the enzyme while p-hydroxymercuribenzoic acid inhibits enzyme activity (50% at 80 µM) yet N-ethylmaleimide does not.In addition, the NADP-GDH activity varies little during the various stages of morphogenesis.Abbreviations EDTA Ethylenediamine Tetraacetic Acid - Tris Tris(hydroxymethyl)aminomethane - Bis-tris bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane - TRITON X-100 iso-octylphenoxypoly-ethoxyethanol - pHMB p-Hydroxymercuribenzoic acid     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

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

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

Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His₆-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K _m and V _max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10⁻³ mM min⁻¹, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His₆-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.     

d-Ribulose-1,5-biphosphate carboxylase fromThiobacillus neapolitanus

d-Ribulose-1,5-bisphosphate carboxylase fromThiobacillus neapolitanus was isolated by differential centrifugation, sucrose density gradient centrifugation, and DEAE-Sephadex column chromatography. The specific activity of the purified enzyme was 2.8 μmol CO₂ fixed/min/mg protein. The enzyme's homogeneity was indicated by a single migrating band during polyacrylamide disc gel electrophoresis and as a single symmetrical schlieren peak that sedimented at a constant rate during ultracentrifugation. TheS _20,w was 18.2; the molecular weight, 500,000±20.000. Sodium dodecyl sulfate polyacrylamide disc gel electrophoresis resolved two polypeptide chains of 55,000 and 11,000 daltons. The pH optimum 0f 7.75 with 9 mM MgCl₂ shifted to 7.45 with 59 mM MgCl₂. Enzyme dialyzed free of Mg⁺⁺ was inactive and no other divalent cation substituted for Mg⁺⁺. TheK _m (Mg⁺⁺),K _m (CO₂), andK _m (RuBP) were 0.59 mM, 0.85 mM, and 0.092 mM, respectively. The inhibition by 6-phosphogluconate was competitive and no stimulation of activity could be demonstrated.     

d-Ribulose 1,5-diphosphate carboxylase from the blue-green alga Aphanocapsa 6308

d-Ribulose 1,5-diphosphate carboxylase from extracts of the unicellular blue-green alga Aphanocapsa 6308 has been purified by ammonium sulphate precipitation and linear sucrose density gradient centrifugation. The molecular weight was estimated to be 525 000 and the enzyme consisted of two types of sub-unit of molecular weights 51 000 and 15 000. The small sub-units were not detected after purification involving acid precipitation but were observed if the acid precipitation step was omitted. The Michaelis constants for Mg²⁺ and CO₂, when tested under air, were 0.35 mM and 0.071 mM respectively. Oxygen acted as a competitive inhibitor with respect to CO₂, suggesting that the enzyme also acts as an oxygenase. This was confirmed by measuring ribulose diphosphate-dependent O₂ uptake. A 1:1 stoichiometry between ribulose diphosphate utilization and O₂ consumption was observed. 6-Phosphogluconate inhibited carboxylase activity both at high (20 mM) and low (1 mM) bicarbonate concentrations. The data are compared with the properties of ribulose diphosphate carboxylase from other autotrophic prokaryotes and from chloroplasts.Abbreviations RuDP d-Ribulose 1,5-diphosphate - EDTA ethylene diamine tetraacetic acid - GSH reduced glutathione - SDS sodium dodecyl sulphate - 6PGluc 6-phosphogluconate - STB supplemented Tris buffer     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Partial purification and some properties of biopterin synthase and dihydropterin oxidase from Drosophila melanogaster

An enzyme which has been named biopterin synthase has been discovered in Drosophila melanogaster. This enzyme, which has been purified 200-fold from extracts of Drosophila, catalyzes the conversion of sepiapterin to dihydrobiopterin, or oxidized sepiapterin to biopterin. The K _m values for the two substrates are 63 µm for sepiapterin and 10 µm for oxidized sepiapterin. NADPH is required in this enzymatic reaction. An analysis of enzyme activity during development in Drosophila indicates a correlation between enzyme activity and biopterin content at various development stages. Another enzyme, called dihydropterin oxidase, was also discovered and partially purified. This enzyme catalyzes the oxidation of dihydropterin compounds to the corresponding pterin compounds. For example, sepiapterin (a dihydropterin) is oxidized to oxidized sepiapterin in the presence of this enzyme. The only dihydropterin that has been tested that is not a substrate for this enzyme is dihydroneopterin triphosphate, the compound thought to be a precursor for all naturally occurring pterins and dihydropterins. Since the action of dihydropterin oxidase is reduced significantly when the concentration of oxygen is very low, it is likely that this enzyme uses molecular oxygen as the oxidizing agent during the oxidation of dihydropterins. Neither NAD⁺ or NADP⁺ is required. In the presence of the two enzymes dihydropterin oxidase and biopterin synthase, sepiapterin is converted to biopterin. However, in the presence of biopterin synthase alone, sepiapterin is converted to dihydrobiopterin.This work was supported by research grants from the National Institutes of Health (AMO3442) and the National Science Foundation (PCM75-19513 AO2).     

Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K _m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K _i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K _i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.Abbreviations PEG polyethylene glycol - PMT putrescine-N-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to C.R. Waspe, M.G. Hilton and P.D.G. Wilson for assistance with the provision of roots from fermenters. We thank W. Martin and S.D. Barr, Chemistry Department, University of Glasgow, and T.A. Smith, Long Ashton Research Station, Bristol, for the supply of compounds not commercially available, as indicated in the text. For helpful discussion and comment, we are grateful to A.J. Parr, W.R. McLauchlan and P. Bachmann. H.D.B, thanks the Science and Engineering Research Council for a research studentship and the Agricultural and Food Research Council Institute of Food Research for additional support.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Purification and characterization of two allelic forms of l-glycerol 3-phosphate dehydrogenase from inbred strains of mice

l-Glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) was purified from the muscle of BALB/cJ and C57BL/6J mice. The half-lives of the enzyme at 50 C were 6 and 33 min, respectively, for the BALB/cJ and C57BL/6J strains. Enzyme preparations from the two strains of mice were compared with respect to the following properties and found to be essentially indistinguishable: K _m values for dihydroxyacetone phosphate, NADH, l--glycerophosphate, and NAD⁺; maximum velocity; competitive inhibition by inorganic phosphate; pH optimum; energy of activation; electrophoretic mobility; molecular weight and subunit molecular weight. From these data, it is concluded that the kinetic properties of the purified enzyme are not the factors responsible for the differences in activity found in crude homogenates of mouse tissues.This work was supported by NIH Research Grant HD 06712 from the National Institute of Child Health and Human Development and by an allocation from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Purification and characterization of a new type of serine carboxypeptidase from<Emphasis Type="Italic"> Monascus purpureus</Emphasis>

Carboxypeptidase produced by Monascus purpureus IFO 4478 was purified to homogeneity. The purified enzyme is a heterodimer with a molecular mass of 132 kDa and consists of two subunits of 64 and 67 kDa. It is an acidic glycoprotein with an isoelectric point of 3.67 and 17.0% carbohydrate content. The optimum pH and temperature were 4.0 and 40 °C, respectively. The enzyme was stable between pH 2.0 and 8.0 at 37 °C for 1 h, and up to 50 °C at pH 5.0 for 15 min. The enzyme was strongly inhibited by piperastatin A, diisopropylfluoride phosphate (DFP), phenylmethylsulfonylfluoride (PMSF), and chymostatin, suggesting that it is a chymotrypsin-like serine carboxypeptidase. Monascus purpureus carboxypeptidase was also strongly inhibited by p-chloromercuribenzoic acid (PCMB) but not by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline, indicating that it requires cysteine residue but not metal ions for activity. Benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu), among the substrates tested, was the best substrate of the enzyme. The K_m, V_max, K_cat, and K_cat/K_m values of the enzyme for Z-Tyr-Glu at pH 4.0 and 37 °C were 0.86 mM, 0.917 mM min^–1, 291 s^–1, and 339 mM^–1 s^–1, respectively.     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[¹⁴C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K _m values of 2.92 mM and 63 M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K _i of 1.63 M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn²⁺, Zn²⁺ and Cu²⁺ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.Abbreviations DTT dithiothreitol - m6OMT myo-inositol 6-O-methyltransferase - SAH S-adenosyl-l-homocysteine - SAM S-adenosyl-l-methionine We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).     

Purification and characterization of a highly thermostable α-<Emphasis Type="SmallCaps">l</Emphasis>-Arabinofuranosidase from <Emphasis Type="Italic">Geobacillus caldoxylolyticus</Emphasis> TK4

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.