Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.     

Mouse-liver glutathione reductase. Purification, kinetics, and regulation.

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.     

Methionyl-tRNA-Met-f deacylase. Purification, characterization, and effects on translational initiation complexes.

A methionyl-tRNA-Met-f deacylase was found in ribosomal salt wash from cultured human cells of the HeLa line. This enzyme was purified by the use of DEAE-cellulose, ammonium sulfate precipitation, gel filtration and isoelectric focusing, and appears to be a protein with a native molecular weight of 80,000, which consists of two 40,000-Mr subunits. The mechanism of the Met-tRNA-Met-f deacylase is shown to involve end-product inhibition by the deacylated form of Met-tRNA-Met-f. The methionyl-tRNA-Met-f deacylase is rather specific for Met-tRNA-Met-f as opposed to Met-tRNA-Met-m, has a KCl optimum of 85 mM, is inhibited by MgCl2 and is inhibited by GTP and NAD+ at physiological concentration. 40-S and 60-S subunits inhibit the enzyme, possibly by binding to it. The stability of translational initiation complexes, containing methionyl-tRNA-Met-f, was investigated in the presence of the enzyme. Purified ternary complex was slowly broken down by the enzyme, while the 40-S-subunit . Met-tRNA-Met-f complex was stable in the presence of enzyme. The 80-S complex formed with A-U-G trinucleotide as the message molecule was broken down, whereas the 80-S complex formed with globin mRNA was stable in the presence of the enzyme. The physiological role of this enzyme is unclear, but it might act to regulate initiation by deacylating Met-tRNA-Met-f.     

Simian adenovirus 20 genome. I. DNA mapping using restriction enconucleases BamHI, EcoRi, XbaI, HindIII

The effect of specific restriction endonuclease on the simian adenovirus SV20 DNA was studied. It was shown that endonucleases SalI, XbaI, EcoRI, BamHI, HindIII cleaved the viral DNA into 3, 4, 5, 5, 8 specific fragments respectively. The sequence of fragments (physical map) was determined and found to be B-C-A for enzyme SalI, C-D-B-A--for enzyme Xbal, E-A-C-D-B--for enzyme EcoRI, B-E-C-A-D--for enzyme BamHI and B-E-A-C-(GH)-D-F--for enzyme HindIII. The G-C content of specific fragments was studied. The "right"-"left" orientation of the physical map of the simian adenovirus 20 DNA based on the G-C content was made in respect with the nomenclature of human adenoviruses.     

Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver. Purification, properties, and immunochemical studies.

Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder. The purification was accomplished by a six-step procedure. The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts. Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis. The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein. Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies. The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively. Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...     

Isolation, purification, and characterization of a new enzyme from Pseudomonas sp. M-27, carboxypeptidase G3.

A new type of carboxypeptidase was found in a strain of Pseudomonas sp. M-27 isolated from soil. The cell-free extract, solubilized by colistin sulfate, was purified to homogeneity. This enzyme had a single peak with a molecular weight of 60,000 on a calibrated Superdex column and consisted of four subunits of identical molecular weights (M(r): 15,000). The enzyme hydrolyzed predominantly acidic peptides and N-acyl amino acids with Glu or Asp in the C-termini. This enzyme was not strongly affected by thiol enzyme inhibitors (PCMB, iodoacetic acid) or serine protease inhibitors (DFP, PMSF), but was inhibited by metal chelators. The enzyme resembles carboxypeptidase G1 or G2 in its glutamate-releasing activity. However, it acts not only on the L-form but also on the D-form of acidic amino acids and shows affinity for the long-chain fatty acyl group but not the benzoyl group. Thus, as this enzyme differs from carboxypeptidase G1 or G2, it was named carboxypeptidase G3.     

A novel type of family 19 chitinase from Aeromonas sp. No.10S-24. Cloning, sequence, expression, and the enzymatic properties.

A family 19 chitinase gene from Aeromonas sp. No.10S-24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N-terminal chitin-binding domains, which are separated by two proline-threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced alpha-anomer by hydrolyzing beta-1,4-glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N-acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (-2)(-1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (-3)(-2)(-1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.     

Histamine N-methyltransferase from rat kidney. Cloning, nucleotide sequence, and expression in Escherichia coli cells.

Complementary DNA clones encoding rat kidney histamine N-methyltransferase have been isolated using synthetic oligonucleotide probes based on partial amino acid sequences of tryptic peptides of the purified enzyme. The 1.3-kilobase cDNA consisted of a 5'-noncoding region of 8 nucleotides, a coding region of 885 nucleotides, and a 3'-noncoding region of 369 nucleotides. The encoded protein of 295 amino acid residues had a calculated molecular weight of 33,940.2. After introduction of a prokaryotic expression vector containing the isolated cDNA, Escherichia coli cells expressed histamine N-methyltransferase activity. The enzyme expressed in these cells was isolated and purified as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whose mobility was identical to the natural enzyme purified from rat kidney. The recombinant enzyme had Vmax and Km values for both histamine and S-adenosylmethionine identical to those of the natural enzyme. All of the inhibitors of the natural enzyme tested showed similar Ki values on both recombinant and natural enzyme.     

Purification, crystallization, and properties of D-ribose isomerase from Mycobacterium smegmatis.

D-Ribose isomerase, which catalyzes the conversion of D-ribose to D-ribulose, was purified from extracts of Mycobacterium smegmatis grown on D-ribose. The purified enzyme crystalized as hexagonal plates from a 44% solution of ammonium sulfate. The enzyme was homogenous by disc gel electrophoresis and ultracentrifugal analysis. The molecular weight of the enzyme was between 145,000 and 174,000 by sedimentation equilibrium analysis. Its sedimentation constant of 8.7 S indicates it is globular. On the basis of sodium dodecyl sulfate gel electrophoresis in the presence of Mn2+, the enzyme is probably composed of 4 identical subunits of molecular weight about 42,000 to 44,000. The enzyme was specific for sugars having the same configuration as D-ribose at carbon atoms 1 to 3. Thus, the enzyme could also utilize L-lyxose, D-allose, and L-rhamnose as substrates. The Km for D-ribose was 4 mM and for L-lyxose it was 5.3 mM. The enzyme required a divalent cation for activity with optimum activity being shown with Mn2+. the Km for the various cations was as follows: Mn2+, 1 times 10(-7) M, Co2+, 4 times 10(-7) M, and Mg2+, 1.8 times 10(-5) M. The pH optimum for the enzyme was 7.5 to 8.5. Polyols did not inhibit the enzyme to any great extent. The product of the reaction was identified as D-ribulose by thin layer chromatography and by preparation of the O-nitrophenylhydrazone derivative.     

Beef kidney 3-hydroxyanthranilic acid oxygenase. Purification, characterization, and analysis of the assay.

Beef kidney 3-hydroxyanthranilic acid oxygenase has been purified to homogeneity. It is a single subunit protein of Mr = 34,000 +/- 2,000 with a frictional coefficient (f/f0) of about 1.1. The enzyme readily aggregates to form, apparently inactive, higher molecular weight oligomers. The very rapid loss of enzyme activity during the assay was analyzed extensively. It was found to be due to inactivation of the enzyme by the substrate, 3-hydroxyanthranilate, and unrelated to enzyme turnover or oxidation of bound iron. The loss of activity was shown to be a first order decay process, and methods are given for obtaining accurate initial reaction rates under all conditions. Evidence was presented that the enzyme assumes a catalytically inactive conformation at pH 3.4, which only relatively slowly rearranges to an active form at pH 6.5; the rearrangement can be blocked by the presence of substrate. We have found that Fe2+, which is required for enzymatic activity, can equilibrate freely, albeit slowly, with the enzyme during the course of the enzyme reaction even in the presence of saturating 3-hydroxanthranilate. Under assay conditons, the Fe2+ has an apparent dissociation constant of 0.04 mM. The kinetic properties of the enzyme were found to be dramatically different in beta,beta-dimethylglutarate buffer and collidine buffer; both the rate of loss of activity during the assay and the substrate Km and Vmax were affected.     

Aspartokinase of Streptococcus mutans: purification, properties, and regulation.

Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine.     

Psychrophilic valine dehydrogenase of the antarctic psychrophile, Cytophaga sp. KUC-1: purification, molecular characterization and expression.

We found the occurrence of valine dehydrogenase in the cell extract of a psychrophilic bacterium, Cytophaga sp. KUC-1, isolated from Antarctic seawater and purified the enzyme to homogeneity. The molecular mass of the enzyme was determined to be approximately 154 kDa by gel filtration and that of the subunit was 43 kDa by SDS/PAGE: the enzyme was a homotetramer. The enzyme required NAD+ as a coenzyme, and catalyzed the oxidative deamination of L-valine, L-isoleucine, L-leucine and the reductive amination of alpha-ketoisovalerate, alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketocaproate. The reaction proceeds through an iso-ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment and the half-life at 45 degrees C was estimated to be 2.4 min. The kcat/Km (micro(-1).s(-1)) values for L-valine and NAD+ at 20 degrees C were 27.48 and 421.6, respectively. The enzyme showed pro-S stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of coenzyme. The gene encoding valine dehydrogenase was cloned into Escherichia coli (Novablue), and the primary structure of the enzyme was deduced on the basis of the nucleotide sequence of the gene encoding the enzyme. The enzyme contains 370 amino-acid residues, and is highly homologous with S. coelicolor ValDH (identity, 46.7%) and S. fradiae ValDH (43.1%). Cytophaga sp. KUC-1 ValDH contains much lower numbers of proline and arginine residues than those of other ValDHs. The changes probably lead to an increase in conformational flexibility of the Cytophaga enzyme molecule to enhance the catalytic activity at low temperatures.     

Histidine ammonia-lyase from rat liver. Purification, properties, and inhibition by substrate analogues.

Histidine ammonia-lyase (EC 4.3.1.3) from rat liver was purified more than 250-fold to near homogeneity. Electrophoretic determinations indicated a native molecular weight of approximately 200,000. The enzyme has a pH optimum of approximately pH 8.5. The minimum Km for L-histidine was 0.5 mM at pH 9.0. The Michaelis constant in the physiological pH range was, however, more than 2.0 mM. D-alpha-hydrazinoimidazolylpropionic acid was found to be a potent competitive inhibitor of liver histidine ammonia-lyase (Kis=75 muM); the L enantiomer of this compound was less effective in this regard. The enzyme was also inhibited competitively by L-histidine hydroxamate (Kis=0.4 mM), and to a lesser extent by L-histidinol, D-histidine, and glycine. Failure of a wide variety of other histidine analogues to inhibit the enzyme substantially indicates high specificity of the active site for L-histidine. No alternate substrates were identified for the enzyme. DL-alpha-Hydrazinophenylpropionic acid, the alpha-hydrzino analogue of phenylalanine, was similarly shown to be a very potent competitive inhibitor of a mechanistically similar L-phenylalanine ammonia-lyase purified from Rhodotorula glutinis. The properties of histidine ammonia-lyase from rat liver differ significantly from those of the enzyme from Pseudomonas fluorescens which has been studied most extensively to date.     

Insulin-sensitive phosphodiesterase. Its localization, hormonal stimulation, and oxidative stabilization.

As it was shown previoulsy by others, the membrane-bound phosphodiesterase (cyclic adenosine 3':5'-monophosphate phosphodiesterase) of rat epididymal fat cells was stimulated when intact cells were exposed to insulin. The levels of stimulation observed in the present study in the cell homogenate and microsomal fraction were approximately 2.0- to 2.5-fold and 2.5- to 3.0-fold, respectively, when the initial substrate level was 100 nM and insulin concentration was 1 to 3 nM. When the microsomal fraction was subjected to a sucrose density gradient centrifugation, most of the insulin-sensitive phosphodiesterase activity was fractionated into the "light" microsomal fraction which was rich in NADH2:potassium ferricyanide:oxidoreductase) and low in 5'-AMPase, adenylate cyclase, and insulin-binding activities. The latter three activities were mostly fractionated into the "heavy" microsomal fraction. Both basal and insulin-stimulated phosphodiesterase activities were low when cells were homogenized in the presence of N-ethylmaleimide or p-chloromercuribenzoate. The insulin-stimulated enzyme activity was also low when cells were homogenized in the presence of --SH compounds (e.g. dithiothreitol) or certain metal-chelating agents (e.g. ethylene glycol bis(beta-aminoethyl ehter)-N,N'-tetraacetate (EGTA)), or in a nitrogen atmosphere. The effect of EGTA was prevented by the addition of certain heavy metal ions but not by the addition of Ca2+ or Ca2+ plus Mg2+ ions. When cells were homogenized in the presence of certain oxidants (e.g. diamide, sodium tetrathionate, or air), a high plus-insulin activity was observed; this activity was not lowered by subsequent treatment of the enzyme with N-ethylmaleimede, EGTA, or fresh cell homogenate that was prepared in the presence of EGTA. However, the activity of an apparently oxidized enzyme could still be lowered by treatment woth dithiothreitol. A partially purified enzyme in the enzyme in the microsomal fraction was fairly stable both in basal and insulin-stimulated states (fully active after 35 days when kept at -20degrees). EGTA added to the homogenization buffer lowered the basal phosphodiesterase activity, but this effect was reversed by the addition of Ca2+ ions. EGTA also decreased the enzyme activity that was stimulated by norepinephrine. However, neither EGTA nor dithiothreitol had any effect on the activities of 5'-AMPase, NADH-dehydrogenase, and malate dehydrogenase of fat cells. The above data indicate that most of the insulin-sensitive phosphodiesterase and the so-called "cell membrane markers" are associated with different subcellular particles in the cell homogenate. In addition, the data seem to indicate that the insulin-stimulated phosphodiesterase has certain --SH groups and that the activity of the enzyme is stabilized when the --SH groups are oxidized by certain oxidants including molecular oxygen. It is suggested that the air oxidation of the enzyme is catalyzed by a trace of certain heavy metal ions and, therefore, can be blocked by a metal-chelating agent.     

gamma-Glutamylcysteine synthetase. Further purification, "half of the sites" reactivity, subunits, and specificity.

gamma-Glutamylcysteine synthetase was purified from rat liver by an improved method involving chromatography on Sepharose-aminohexyl-ATP to a specific activity of about 1600 units/mg, or approximately twice that previously obtained; it is thus the most active preparation of this enzyme thus far isolated. The earlier preparation, which is homogeneous on polyacrylamide gel electrophoresis, exhibits "half of the sites" reactivity in that it binds a maximum of 0.5 mol of the inhibitor L-methionine-S-sulfoximine phosphate per mol of enzyme. In contrast, the present enzyme preparation binds 1 mol of methionine sulfoximine phosphate per mol of enzyme; it also differs from the enzyme obtained earlier in exhibiting much less ATPase activity and less activity in catalyzing ATP-dependent cyclization of glutamate. gamma-Glutamylcysteine synthetase dissociates in sodium dodecyl sulfate into two nonidentical subunits of apparent molecular weights 74,000 and 24,000; after cross-linking with dimethyl-suberimidate, a species having a molecular weight of about 100,000 was found on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. New information has been obtained about the interaction of the enzyme with glutamate analogs; thus, the enzyme is active with such glutamate analogs as beta-glutamate, N-methyl-L-glutamate, and threo-beta-hydroxy-L-glutanate, and it is effectively inhibited by cis-1-amino-1,3-dicarboxycyclonexane, 2-amino-4-phosphonobutyrate, and gamma-methylglutamate.     

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.     

Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity

The enzyme N-succinyl-L-diaminopimelic acid desuccinylase from Escherichia coli has been purified 7,100-fold to apparent homogeneity. The enzyme is part of the diaminopimelic acid-lysine pathway in bacteria and catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to produce L-diaminopimelic acid and succinate. The enzyme exists as a mixture of dimeric and tetrameric species of identical subunits of molecular weight approximately 40,000. Activity was completely abolished following dialysis of the enzyme against metal chelators. Cobalt(II) and zinc were effective in restoring the activity. The apparent affinities of the apoenzyme for cobalt and zinc were similar (Kd values near 1 microM) and the cobalt enzyme was 2.2-fold more active than the zinc enzyme. The Km and turnover number for the hydrolysis of the natural substrate, N-succinyl-L-diaminopimelic acid, were 0.4 mM and 16,000 min-1, respectively. The substrate specificity of the enzyme was defined by preparing a number of substrate analogues that systematically lack the various functional groups present in the molecule. These studies show that the enzyme is highly specific for the natural substrate. These properties of N-succinyl-L-diaminopimelic acid desuccinylase and the fact that the enzyme is essential for bacterial growth make it an ideal target for the development of inhibitors with potential antibacterial activity.     

Studies on nitrate reductase of Clostridium perfringens. IV. Identification of metals, molybdenum cofactor, and iron-sulfur cluster

Nitrate reductase of Clostridium perfringens was purified by an improved method using immuno-affinity chromatography. The purified preparation contained Mo, Fe, and acid-labile sulfide; the Mo content was 1 mol per mol and the Fe 3.7 mol per mol of the enzyme. The inactive enzyme obtained from cells grown in the presence of tungstate did not hold Mo but contained 1 mol of W. The content of Fe was not increased. The presence of molybdenum cofactor in the nitrate reductase was indicated by the formation of molybdopterin form A in the oxidation of the enzyme by iodine and by the complementation of NADPH-nitrate reductase with the heart-treated enzyme in the extract of Neurospora crassa nit-1. The Clostridium nitrate reductase had an absorption maximum at 279 nm and shoulders at 320, 380, 430, and 520 nm. This enzyme seems to contain an iron sulfur cluster since the reduced enzyme showed decreased absorption in visible region. The CD spectrum of the enzyme has a positive peak at 425 nm and negative ones at 310, 360, and 595 nm. It was compared with the CD spectrum of ferredoxin (2Fe-2S or 4Fe-4S cluster) and the nitrate reductase of Plectonema boryanum.     

Protein-glutaminase from Chryseobacterium proteolyticum, an enzyme that deamidates glutaminyl residues in proteins. Purification, characterization and gene cloning.

A novel protein-deamidating enzyme was purified to homogeneity from Chryseobacterium proteolyticum and the gene encoding it was cloned. The enzyme is a monomer with a pI of 10.0, a measured M(r) of approximately 20,000 and a calculated M(r) of 19,860. Extensive comparison with Streptoverticillium transglutaminase showed that the protein-deamidating enzyme lacked transglutaminase activity in terms of hydroxamate-formation between benzyloxycarbonyl-Gln-Gly and hydroxylamine, or monodansylcadaverine incorporation into casein. The enzyme deamidated the two glutaminyl residues in the oxidized insulin A chain and deamidated both casein and the oxidized insulin B chain with higher catalytic efficiencies (k(cat)/K(m)) than with short peptides. The enzyme was active against several proteins, including insoluble wheat gluten, but did not deamidate asparaginyl residues in peptides, free glutamine or other amides. The enzyme was therefore named protein-glutaminase (EC 3.5.1). The gene encoding the protein was cloned and, when expressed in Escherichia coli, the protein product had protein-glutaminase activity and cross-reacted with antiserum raised against the purified enzyme. The protein-glutaminase was shown to be expressed as a prepro-protein with a putative signal peptide of 21 amino acids and a pro-sequence of 114 amino acids. The amino-acid sequence had no obvious homology to any published sequence and is therefore a novel protein-glutaminase.