Microbial metabolism of quinoline and related compounds. XII. Isolation and characterization of the quinoline oxidoreductase from Rhodococcus spec. B1 compared with the quinoline oxidoreductase from Pseudomonas putida 86.

Quinoline oxidoreductase from Rhodococcus spec. B1 was purified 39-fold to apparent homogeneity in a 5-step procedure with a recovery of 26%. The Mr of the native enzyme as determined by gel chromatography was 300,000. SDS polyacrylamide gel electrophoresis of the enzyme revealed 3 protein bands corresponding to Mr 82,000, 32,000, and 18,000. The enzyme contains 1.3 atoms of molybdenum, 8 atoms of iron, 8 atoms of acid-labile sulphur, 2 molecules of FAD and 2 molecules of molybdopterin cytosine dinucleotide. Cyanide, 4-hydroxymercuribenzoate and methanol were effective as inhibitors. The amino-terminal protein sequences of the 3 subunits of quinoline oxidoreductase from Rhodococcus B1 compared to those of quinoline oxidoreductase from Pseudomonas putida 86 revealed no difference among 71 amino acids examined.     

Microbial metabolism of quinoline and related compounds. VIII. Xanthine dehydrogenase from a quinoline utilizing Pseudomonas putida strain.

The xanthine dehydrogenase from Pseudomonas putida 86 was purified 68-fold to homogeneity with 47% recovery. SDS-polyacrylamide gel electrophoresis of the enzyme revealed two protein bands corresponding to an Mr of 87,000 and 52,000. The Mr of the native enzyme was calculated to 550,000 by gel chromatography. The enzyme contained 4 atoms of molybdenum, 16 atoms of iron, 16 atoms of acidlabile sulphur and 4 molecules of FAD. Due to the composition of the cofactors the xanthine dehydrogenase belongs to the class of molybdo-iron/sulphur-flavoproteins. Form A, an oxidation product of the molybdenum cofactor, was identified. Methanol and cyanide were effective inhibitors.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Purification and characterization of pyruvate:NADP+ oxidoreductase in Euglena gracilis

Pyruvate:NADP+ oxidoreductase was homogeneously purified from crude extract of Euglena gracilis. The Mr of the enzyme was estimated to be 309,000 by gel filtration. The enzyme migrated as a single protein band with Mr of 166,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the enzyme consists of two identical polypeptides. The absorption spectrum of the native enzyme exhibited maxima at 278, 380, and 430 nm, and a broad shoulder was observed around 480 nm; the maximum at 430 nm was eliminated by reduction of the enzyme with dithionite. Reduction of the enzyme with pyruvate and CoA and reoxidation with NADP+ were proved from changes of absorption spectra. The enzyme contained 2 molecules of FAD and 8 molecules of iron. It was also indicated that the enzyme was thiamine pyrophosphate-dependent. The enzyme was oxygen-sensitive, and the reaction was affected by the presence of oxygen. Pyruvate was the most active substrate, but the enzyme was slightly active for 2-oxobutyrate, 3-hydroxypyruvate, and oxalacetate, but not for glyoxylate and 2-oxoglutarate. The native electron acceptor was NADP+, whereas NAD+ was completely inactive. Methyl viologen, benzyl viologen, FAD, and FMN were utilized as artificial electron acceptors, whereas spinach and Clostridium ferredoxins were inactive. Pyruvate synthesis by reductive carboxylation of acetyl-CoA with NADPH as the electron donor occurred by the reverse reaction of the enzyme. The enzyme also catalyzed a pyruvate-CO2 exchange reaction and electron-transfer reaction from NADPH to other electron acceptors like methyl viologen. These results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruvate dehydrogenase multienzyme complex or pyruvate:ferredoxin oxidoreductase.     

Purification and properties of membrane-bound hydrogenase isoenzyme 1 from anaerobically grown Escherichia coli K12

Hydrogenase isoenzyme 1 from the membrane fraction of anaerobically grown Escherichia coli has been purified to near homogeneity. The preparation involved dispersion of the membrane fraction with deoxycholate followed by ammonium sulphate precipitation, ion-exchange, hydroxyapatite and gel filtration chromatography steps. The enzyme was assayed by quantification of the H2:benzyl viologen oxidoreductase activity immunoprecipitated by a non-inhibitory antiserum specific for the enzyme. The enzyme constituted about 8% of the hydrogenase activity found in the detergent-dispersed membranes, the remainder being attributable to hydrogenase isoenzyme 2. Isoenzyme 1 was purified 130-fold and the specific activity of the final preparation was 10.6 mumol benzyl viologen reduced min-1 (mg protein)-1 (H2:benzyl viologen oxidoreductase). The final preparation contained polypeptides of apparent Mr 64,000, 31,000 and 29,000. Antibodies were raised both to the final preparation and to immunoprecipitation arcs containing hydrogenase isoenzyme 1, excised from crossed immunoelectrophoresis plates. The former cross-reacted with all three polypeptides in the enzyme preparation but the latter recognised only the Mr-64,000 polypeptide. Immunological analysis revealed that the polypeptides of apparent Mr 31,000 and 29,000 are fragments of a single polypeptide of Mr 35,000 which is present in the detergent-dispersed membranes. The fragmentation of the Mr-35,000 polypeptide during the preparation correlates with a change in the electrophoretic mobility of the enzyme. A similar electrophoretic mobility change was observed, accompanied by cleavage of the Mr-35,000 polypeptide to one of 32,000 when the enzyme was analysed after exposure of detergent-dispersed membranes to trypsin. The enzyme in the detergent-dispersed membranes consists minimally of two subunits of Mr 64,000 and two subunits of Mr 35,000. It contained 12.2 mol Fe and 9.1 mol acid-labile S2-/200,000 g enzyme. The enzyme, purified from bacteria grown in the presence of 63Ni, was found to contain 0.64 (+/- 0.20) mol Ni/200,000 g enzyme. A constant ratio of 63Ni immunoprecipitated to hydrogenase isoenzyme 1 activity immunoprecipitated by antiserum specific for the enzyme was observed during the preparation, consistent with Ni being part of the enzyme. The enzyme has a low Km for H2 (2.0 microM) in the H2:benzyl viologen oxidoreductase assay. It catalyses H2 evolution employing reduced methyl viologen as electron donor. It is inhibited reversibly by CO and irreversibly by N-bromosuccinimide.     

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.     

8-Hydroxy-5-deazaflavin-reducing hydrogenase from Methanobacterium thermoautotrophicum: 1. Purification and characterization

The 8-hydroxy-5-deazaflavin (coenzyme F420) reducing hydrogenase from the obligate anaerobe Methanobacterium thermoautotrophicum delta H has been purified 41-fold to apparent homogeneity. The major active enzyme form is a high molecular weight aggregate of Mr ca. 800,000, composed of three subunits, alpha (Mr 47K), beta (Mr 31K), and gamma (Mr 26K). The hydrogenase is purified aerobically in reversibly inhibited form, and conditions for anaerobic reductive activation with H2, high salt, thiols, and electron acceptors have been defined. The minimal species transferring electrons from H2 to coenzyme F420 appears to be an alpha beta delta (Mr 115K) complex. The tightly associated redox cofactors per 115K species are 0.6-0.7 nickel atom, 0.8-0.9 flavin adenine dinucleotide (FAD), and 13-14 iron atoms in iron-sulfur centers. The subunits have been separated by denaturing gel electrophoresis, which has permitted determination of amino acid composition, subunit N-terminal sequencing, and preparation of subunit-directed antibodies. There is iron associated with the alpha-subunit, but placement of the nickel and FAD has not been established.     

Isolation and characterisation of a soluble active fragment of hydrogenase isoenzyme 2 from the membranes of anaerobically grown Escherichia coli

An active tryptic fragment of membrane-bound hydrogenase isoenzyme 2 from anaerobically grown Escherichia coli has been purified. The soluble enzyme derivative was released from the membrane fraction by trypsin cleavage. The purification procedure involved ion-exchange, hydroxyapatite and gel permeation chromatography. The enzyme derivative was purified 100-fold from the membrane fraction and the specific activity of the final preparation was 320 mumol benzyl viologen reduced min-1 mg protein-1 (H2:benzyl viologen oxidoreductase). The native enzyme derivative had an Mr of 180,000 and was composed of equimolar amounts of polypeptides of Mr 61,000 and 30,000. It possessed 12.5 mol Fe, 12.8 mol acid-labile S2- and 3.1 mol Ni/180,000 g enzyme. Antibodies were raised to the purified preparation which cross-reacted with hydrogenase isoenzyme 2 but not with isoenzyme 1 in detergent-dispersed preparations. Western immunoblot analysis revealed that isoenzyme 2 which had not been exposed to trypsin contained cross-reacting polypeptides of Mr 61,000 and 35,000. Trypsin treatment of the membrane-bound enzyme to form the soluble derivative of isoenzyme 2, therefore, cleaves a polypeptide of Mr 35,000 to produce the 30,000-Mr fragment. Trypsin treatment of the detergent-dispersed isoenzyme 2 produces the same fragmentation of the enzyme. Neither of the subunits of the enzyme revealed any immunological identity with those of hydrogenase isoenzyme 1.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

A selenium-containing hydrogenase from Methanococcus vannielii. Identification of the selenium moiety as a selenocysteine residue

A 75Se-labeled hydrogenase was purified to near homogeneity from extracts of Methanococcus vannielii cells grown in the presence of [75Se]selenite. The molecular weight of the enzyme was estimated as 340,000 by gel filtration. The enzyme tends to aggregate and occurs also as a larger protein species (Mr = 1.3 x 10(6)). The same phenomenon was observed on native gel electrophoretic analysis. Hydrogenase activity exhibited by these two protein bands was proportional to protein and 75Se content. Both molecular species reduce the natural cofactor, 8-hydroxy-5-deazaflavin, and tetrazolium dyes with molecular hydrogen. Sodium dodecyl sulfate-gel electrophoresis of 75Se-labeled enzyme showed that 75Se is present exclusively in an Mr = 42,000 subunit. A value of 3.8 g atoms of selenium/mol of enzyme (Mr = 340,000) was determined by atomic absorption analysis. The chemical form of selenium in the enzyme was shown to be selenocysteine. This was identified as the [75Se]carboxymethyl and [75Se]carboxyethyl derivatives in acid hydrolysates of alkylated 75Se-labeled protein. The hydrogenase is extremely oxygen-sensitive but can be reactivated by incubation with molecular hydrogen and dithiothreitol.     

Purification and properties of a dissimilatory nitrate reductase from Haloferax denitrificans.

A membrane-bound nitrate reductase (nitrite:(acceptor) oxidoreductase, EC 1.7.99.4) from the extremely halophilic bacterium Haloferax denitrificans was solubilized by incubating membranes in buffer lacking NaCl and purified by DEAE, hydroxylapatite, and Sepharose 6B gel filtration chromatography. The purified nitrate reductase reduced chlorate and was inhibited by azide and cyanide. Preincubating the enzyme with cyanide increased the extent of inhibition which in turn was intensified when dithionite was present. Although cyanide was a noncompetitive inhibitor with respect to nitrate, nitrate protected against inhibition. The enzyme, as isolated, was composed of two subunits (Mr 116,000 and 60,000) and behaved as a dimer during gel filtration (Mr 380,000). Unlike other halobacterial enzymes, this nitrate reductase was most active, as well as stable, in the absence of salt.     

Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis.

The pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis is an extrinsic protein bound to the hydrogenosomal membrane. It has been solubilized and purified to homogeneity, principally by salting-out chromatography on Sepharose 4B. Low recoveries of active enzyme were caused by inactivation by O2 and the irreversible loss of thiamin pyrophosphate. It is a dimeric enzyme of overall Mr 240,000 and subunit Mr 120,000. The enzyme contains, per mol of dimer, 7.3 +/- 0.3 mol of iron and 5.9 +/- 0.9 mol of acid-labile sulphur, suggesting the presence of two [4Fe-4S] centres, and 0.47 mol of thiamin pyrophosphate. The absorption spectrum of the enzyme is characteristic of a non-haem iron protein. The pyruvate: ferredoxin oxidoreductase from T. vaginalis is therefore broadly similar to the 2-oxo acid: ferredoxin (flavodoxin) oxidoreductases purified from bacterial sources, except that it is membrane-bound.     

Phytoalexin synthesis in soybean: purification and characterization of NADPH:2'-hydroxydaidzein oxidoreductase from elicitor-challenged soybean cell cultures

An NADPH:2'-hydroxydaidzein oxidoreductase (HDR) from elicitor-challenged soybean cell cultures was purified to apparent homogeneity by a five-step procedure. The purification procedure included affinity adsorption on Blue Sepharose and elution of the enzyme with NADP+. It was shown by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis that HDR consists of only one polypeptide, which has a Mr about 34,700. The pH optimum of the reaction was 7.0. Apparent Michaelis constants determined for 2'-hydroxydaidzein, 2'-hydroxyformononetin, and NADPH were, respectively, 50, 60, and 56 microM. A low conversion of 2'-hydroxygenistein to the corresponding isoflavanone was also observed but isoflavones lacking a 2'-hydroxyl group and various other flavonoids did not serve as substrates. Enzymatically derived 2'-hydroxydihydrodaidzein gave a positive CD spectrum at 328 nm, which shows its 3R stereochemistry. Antibodies against HDR were raised in rats.     

Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous.     

Purification and some characteristics of a cytochrome c-containing nitrous oxide reductase from Wolinella succinogenes

Nitrous oxide reductase from Wolinella succinogenes was purified very nearly to homogeneity. The enzyme was found to be dimeric, with Mr = 162,000 and subunit Mr = 88,000, and to contain three copper atoms and one iron atom (as cytochrome c) per subunit. The oxidized enzyme exhibited absorption bands at 410 and 528 nm, and the dithionite-reduced enzyme, at 416, 520, and 550 nm. The isoelectric point was 8.6; specific activity was at 25 degrees C and pH 7.1, 160 mumol x min-1 x mg-1; and Km was 7.5 microM N2O under the same conditions. alpha-Chymotrypsin cleaved the enzyme into cytochrome c-depleted dimers with an average Mr = 134,000 and cytochrome c-enriched fragments with an average Mr = 13,000. The enzyme was stable at 4 degrees C for at least 100 h under air and 3 h in the presence of 5 mM EDTA. It exhibited a dithionite-N2O oxidoreductase as well as a BV+-N2O oxidoreductase activity. During turnover with BV+ at 25 mM N2O, the enzyme was observed to undergo an initial activation and a subsequent inactivation. The kinetics of inactivation were approximately first-order in remaining activity, and the first-order rate constant was essentially independent of the initial enzyme concentration. These characteristics are consistent with the occurrence of turnover-dependent inactivation. Acetylene was a relatively weak inhibitor, but cyanide and azide were rather strong inhibitors. The nitrous oxide reductase of W. succinogenes is quite different from that of denitrifying bacteria. The amount of activity in cell extracts and the absence of O2-labile nitrous oxide reductase suggested that the cytochrome c containing enzyme may be the only one produced by W. succinogenes.     

The zinc-containing high Km cyclic nucleotide phosphodiesterase of bakers' yeast

The high Km cyclic nucleotide phosphodiesterase of Saccharomyces cerevisiae was purified by an improved procedure. Its amino acid composition is reported. Its pI is 5.85 +/- 0.1. Sedimentation equilibrium analysis of the native enzyme gave Mr = 88,000 +/- 6,000, whilst gel electrophoresis in the presence of dodecyl sulfate gave a molecular weight of 43,000, indicating that the enzyme is a dimer. Preparations of 94 +/- 4% purity contained about 2.4 atoms of zinc/43,000 daltons. Inactivation of the enzyme by 8-hydroxyquinoline was accompanied by removal of about 2 zinc atoms per monomer. Partially inactivated enzyme regained activity during dialysis against zinc, or, with less effect, cobalt salts. 8-Hydroxyquinoline (Ki = 1.1 mM) and 1,10-phenanthroline (Ki = 0.6 mM) were competitive inhibitors. The enzyme was also inhibited by the nonchelating 1,7-and 4,7-phenanthrolines and by thiols and KCN, but not by NaN3. These inhibitors probably act by binding to, but not chelating, enzyme-bound zinc.     

Purification and some properties of sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans.

A sulfur:ferric ion oxidoreductase that utilizes ferric ion (Fe3+) as an electron acceptor of elemental sulfur was purified from iron-grown Thiobacillus ferrooxidans to an electrophoretically homogeneous state. Under anaerobic conditions in the presence of Fe3+, the enzyme reduced 4 mol of Fe3+ with 1 mol of elemental sulfur to give 4 mol of Fe2+ and 1 mol of sulfite, indicating that it corresponds to a ferric ion-reducing system (T. Sugio, C. Domatsu, O. Munakata, T. Tano, and K. Imai, Appl. Environ. Microbiol. 49:1401-1406, 1985). Under aerobic conditions, sulfite, but not Fe2+, was produced during the oxidation of elemental sulfur by this enzyme because the Fe2+ produced was rapidly reoxidized chemically by molecular oxygen. The possibility that Fe3+ serves as an electron acceptor under aerobic conditions was ascertained by adding o-phenanthroline, which chelates Fe2+, to the reaction mixture. Sulfur:ferric ion oxidoreductase had an apparent molecular weight of 46,000, and it is composed of two identical subunits (Mr = 23,000) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sulfur oxidation by this enzyme was absolutely dependent on the presence of reduced glutathione. The enzyme had an isoelectric point and a pH optimum at pH 4.6 and 6.5, respectively. Almost all the activity of sulfur:ferric ion oxidoreductase was observed in the osmotic shock fluid of the cells, suggesting that it was localized in the periplasmic space of the cells.     

A secreted beta-glucan-branching enzyme from Candida albicans.

A Mr 34,000 wall protein was isolated as a by-product of the purification of an endo-(1-3)-beta-glucanase from the culture filtrate of Candida albicans. The purified fraction contained no exo- or endo-beta-glucanase activity, and analysis by SDS poly-acrylamide gel electrophoresis (SDS-PAGE) showed one protein band at Mr 34,000. Analysis by gel filtration high performance liquid chromatography (HPLC) of reaction products from incubations of the protein fraction with laminarioligosaccharides of five glucosyl units or greater revealed a unique glucanosyl transferase activity. The enzyme specifically cleaved laminaribiaose (G2) from the reducing-end of a linear beta-(1-3)-glucan and transferred the remainder to another laminarioligosaccharide. The reaction with laminaripentaose (G5) produced G2 and a product eluting at the position of G8. Analysis of the latter transferase product by 13C- and 1H-nuclear magnetic resonance (NMR) spectroscopy shows it to be a branched molecule containing a beta-(1-3)-beta-(1-6)-branchpoint. It is suggested that the Mr 34,000 wall protein is a glucan branching enzyme, perhaps the key enzyme responsible for the transformation of the initial linear beta-(1-3)-glucan into the branched beta-(1-3)-beta-1-6)-glucan as found in the cell wall of C. albicans.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000.     

Purification, crystallization and preliminary X-ray investigation of quinoprotein methylamine dehydrogenase from Thiobacillus versutus

The enzyme methylamine dehydrogenase or primary-amine:(acceptor) oxidoreductase (deaminating) (EC 1.4.99.3) was purified from the bacterium Thiobacillus versutus to homogeneity, as judged by polyacrylamide gel electrophoresis. The native enzyme has a Mr of 123 500 and contains four subunits arranged in a alpha 2 beta 2 configuration, the light and heavy subunits having a Mr of 12900 and 47500 respectively. The isoelectric point is 3.9. The purified enzyme was crystallized from 37--42% saturated ammonium sulphate in 0.1 M sodium acetate buffer, pH 5.0. The space group is P3(1)21 or P3(2)21, with one alpha 2 beta 2 molecule in the asymmetric unit. The cell dimensions are: a = b = 13.01 nm; c = 10.40 nm. The X-ray diffraction pattern extends to at least 0.25-nm resolution.