Thiosulfate Reductase of Desulfovibrio vulgaris

The thiosulfate reductase of Desulfovibrio vulgaris has been purified and some of its properties have been determined. Only one protein component was detected when the purified enzyme was subjected to polyacrylamide gel electrophoresis at pH values of 8.9, 8.0, and 7.6. In the presence of H(2), the enzyme, when coupled to hydrogenase and with methyl viologen as an electron carrier, catalyzed the reduction of thiosulfate to hydogen sulfide. The use of specifically labeled (35)S-thiosulfate revealed that the outer sulfur atom was reduced to sulfide and the inner sulfur atom was released as sulfite. Thus, the enzyme catalyzes the reductive dismutation of thiosulfate to sulfide and sulfite. The molecular weight of the enzyme was determined by sedimentation equilibrium (16,300) and amino acid analysis (15,500). The enzyme sedimented as a single, symmetrical component with a calculated sedimentation coefficient of 2.21S. Amino acid analysis revealed the presence of two half-cystine residues per mole of enzyme and a total of 128 amino acid residues. Carbohydrate and organic phosphorus analyses revealed the presence of 9.2 moles of carbohydrate and 4.8 moles of phosphate per mole of enzyme. The substrate specificity of the enzyme was studied.     

A Crystalline Aminopeptidase from Streptomyces peptidofaciens: Physico-chemical Properties and Characteristics as a Ca-Metalloprotease

A crystalline aminopeptidase obtained from the culture filtrate of Streptomyces peptidofaciens KY 2389 appeared to be homogeneous on ultracentrifugation and acrylamide gel electrophoresis. The sedimentation coefficient, s_{20, w}., was determined to be 2.6 S. The molecular weight was estimated to be approximately 19,000 by sedimentation equilibrium studies. The amino acid analyses indicated that the enzyme was composed of 147 amino acid residues and contained no sulfhydryl group. The isoelectric point was found to be around pH 7.4 by isoelectric focusing on ampholites.

The enzyme required Ca²⁺ for its maximal activity and was strongly inhibited by some metal-chelating agents such as ethylenediaminetetraacetic acid (EDTA) and o-phenanthroline. The EDTA-inactivated enzyme restored its activity almost completely by the addition of Ca²⁺ The crystalline preparation of aminopeptidase contained 1 g-atom of calcium and about 2 g-atoms of magnesium per mole of enzyme protein, and the calcium was essential for the activity of the enzyme.     

Purification and characterization of putative alkaline [Ni-Fe] hydrogenase from unicellular marine green alga, Tetraselmis kochinensis NCIM 1605

Hydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be approximately 89kDa by SDS-PAGE. This enzyme consists of two subunits with molecular masses of approximately 70 and approximately 19kDa. The hydrogenase was found to contain 10g atoms of Fe and 1g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50micromol H(2)/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value approximately 9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425nm indicating that the enzyme had iron-sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.     

Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

Purification and properties of maize polyamine oxidase: a flavoprotein

Polyamine oxidase was purified from maize shoots to homogeneityby the criteria of polyacrylamide gel electrophoresis and ultracentrifugation.The purified yellow enzyme showed absorption maxima at 278,380 and 460 nm. The molecular weight estimated by gel filtrationwas about 65,000 and the sedimentation coefficient was 5.95S. Sodium dodecylsulfate gel electrophoresis yielded a singleband at a molecular weight of 65,000. The enzyme contained 1mole of FAD per mole of enzyme. Amino acid composition and kineticproperties of the enzyme are presented. (Received April 30, 1980; )     

Pulmonary angiotensin-converting enzyme. Structural and catalytic properties.

Angiotensin-converting enzyme has been solubilized from a particulate fraction of rabbit lung and purified to apparent homogeneity in 11% yield by a procedure including fractionation with DEAE-cellulose and calcium phosphate gel, elution from Sephadex G-200, and lectin affinity chromatography. The molecular weight estimated by equilibrium sedimentation was approximately 129,000, either in the absence or presence of 6 M guanidine hydrochloride. A slightly higher value of 140,000 determined for the reduced, denatured protein by gel electrophoresis in the presence of sodium dodecyl sulfate and a much higher figure derived from gel filtration are probably due to the glycoprotein nature of the enzyme. Its oligosaccharide content accounted for 26% of the weight calculated from its amino acid and carbohydrate composition. The estimated content of sugar residues per mole was: galactose, 57; N-acetylglucosamine, 53; mannose, 43; N-acetylneuraminic acid, 19; and fucose, 4. Threonine and alanine were identified, respectively, as NH2-terminal and COOH-terminal residues by the dansylation procedure and by digestion with carboxypeptidase A. The enzyme was found to contain approximately 1 g atom of zinc per mol. Km values for hydrolysis of hippurylhistidylleucine and angiotensin I were 2.3 and 0.07 mM, and the corresponding turnover numbers were 15,430 and 792 mol/min/mol at 37 degrees. Bradykinin was also a substrate, and release of its COOH-terminal dipeptide, Phe-Arg, was catalyzed at a comparable rate to that of His-Leu from the COOH terminus of angiotensin I. Enzyme activity required the presence of chloride ions and was inhibited by EDTA and by low concentrations of Bothrops bradykinin-potentiating peptides. In addition, hydrolysis of hippurylhistidylleucine was inhibited competitively by other defined peptides, including di- and tripeptides, which were not substrates.     

A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.     

Rat muscle 5'-adenylic acid aminohydrolase. I. Purification and subunit structure.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) has been purified to apparent homogeneity from rat muscle. The preparation exhibits a single polypeptide band with a molecular weight of 60,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme has a sedimentation coefficient of 11.3 S. Analysis by sedimentation equilibrium techniques showed the nat-ive enzyme to have a molecular weight of 238,000, whereas the enzyme, when analyzed in 6 M guanidine hydrochloride and 10 mM 2-mercaptoethanol, had a molecular weight of only 59,500. The amino acid composition of the enzyme was determined and peptide mapping was performed on a tryptic digest of S-carboxymethylated enzyme. NH2-terminal analysis by both the dansylation and cyanate procedures failed to identify a free NH2 terminus. Treatment of the enzyme with carboxypeptidase A resulted in the release of approximately 0.5 mol each of valine and leucine per 60,000 g of enzyme. The data presented indicate that hte native enzyme has a tetrameric structure consisting of four polypeptide chains each having a molecular weight of 60,000. The COOH-terminal analysis can be interpreted either as an indication of subunit heterogeneity or as a result of incomplete digestion of a -X-Leu-Val sequence at the end of a single type of polypeptide chain. Tryptic peptide maps strongly support the latter interpretation and suggest that the subunits are essentially identical.     

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Subunits of the alkaline phosphatase of Bacillus licheniformis: chemical, physicochemical, and dissociation studies.

The alkaline phosphatase (orthophosphoric monoester phosphydrolase, EC 3.1.3.1) of Bacillus licheniformis MC14 was studied in an attempt to determine the number of subunits contained in the 120,000-molecular-weight native enzyme. Two moles of arginine was liberated per mole of native enzyme by carboxypeptidases A and B in the presence of sodium dodecyl sulfate. The effect on the native enzyme of progressively lowering the solvent buffer pH was monitored by determining the molecular weight by sedimentation equilibrium analysis, the sedimentation coefficient, the frictional coefficient, and the percent alpha-helix content of the enzyme. The alkaline phosphatase dissociates into two subunits around pH 4. At pH 2.8 a further decrease in S value, but no change in molecular weight, is observed, indicating a change in conformation. The frictional coefficients and percent alpha-helix content agree with this interpretation. A subunit molecular weight of 59,000 was calculated from sodium dodecyl sulfate gels.     

螺旋藻氢酶的纯化与生化特性

本研究用DE－52、SephadexG－75、SephadexG－100柱层析从螺旋藻分离纯化得到比活性提高200倍的氢酶,回收率为14％。凝胶柱层析和SDS－PAGE显示一条带,其分子量为56kd。氨基酸分析结果表明酸性氨基酸比例较大,等电聚焦测定结果证明其等电点为pH4．2。吸收光谱结果显示氢酶是铁硫蛋白。甲基紫晶（MV）是氢酶催化放氢的最佳电子供体,其Km（MV）为0．31mmol／L,最适pH值为7．5－8．0。     

Purification and properties of a low-molecular-weight, high-alkaline pectate lyase from an alkaliphilic strain of Bacillus

A low-molecular-weight, high-alkaline pectate lyase (pectate transeliminase, EC 4.2.2.2) was found in an alkaline culture of Bacillus sp. strain KSM-P15, purified to homogeneity, and crystallized. The enzyme had a relative molecular weight of approximately 20,300 as measured by sedimentation equilibrium, with a sedimentation coefficient (s20,w0) of 1.73 S. It was a basic protein with an isoelectric point of pH 10.3, and the alpha-helical content was only 6.6%. In the presence of Ca2+ ions, the enzyme degraded polygalacturonic acid in a random manner to yield 4,5-unsaturated oligo-galacturonides and had its optimal activity around pH 10.5 and 50-55 degrees C. It also had a protopectinase-like activity on cotton fibers. The N-terminal amino acid sequences of the intact protein (28 amino acids) and its two lysyl endopeptidase-cleaved peptide fragments (8 and 12 amino acids) had very low sequence similarity with pectate lyases reported to date. These results strongly suggest that the pectate lyase of Bacillus sp. strain KSM-P15 may be a novel enzyme and belongs in a new family.     

Subunit structure of biodegradative threonine deaminase.

The molecule weight of the biodegradative threonine deaminase from Escherichia coli was determined to be approximately 147,000 by sedimentation equilibrium ultracentrifugation. Similar experiments using 5 M guanidinium chloride gave a value of 39,000 for the molecular weight of the enzyme subunit. On sodium dodecyl sulfate-gel electrophoresis the enzyme also dissociated into a single subunit with an estimated molecular weight of 38,000. The NH2 terminus of the enzyme was determined to be methionine by the dinitrophenylation procedure. Quantitative analysis revealed that 3.6 mol of methionine were detected per 147,000 g of enzyme. The selective tritium labeling method established alanine as the COOH-terminal residue. The sequence of residues at the NH2 terminus, determined using an automated sequence analyzer, was: (formula: see text). The fact that a single amino acid was released at each degradation step in the above experiment strongly suggests that the subunits in the enzyme contain the same amino acid sequence. Therefore, the native enzyme with a molecular weight of 147,000 appears to be composed of four identical polypeptide subunits.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

Studies of the structure of fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55 000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate that fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.     

Characterization of Human K-Casein Purified by FPLC

ABSTRACT

Because previous purification procedures for human K-casein may have caused the loss of some carbohydrate, relatively gentle methods were used. The protein was isolated by a four-step procedure which included isoelectric precipitation of whole casein, gel chromatography on Sephadex G-200 in the presence of SDS, removal of the SDS with Extracti-Gel D, and FPLC chromatography on Mono Q with buffers containing 6 M urea. The purified protein was nearly identical in amino acid composition to that found earlier by amino acid analysis and peptide sequencing and a molar extinction coefficient of 11.2 + 0.1 was determined on the basis of amino acid analysis with a norleucine internal standard. Hydrolysis, acylation, and methylsilylation of the carbohydrate, followed by gas chromatographic analysis on a fused silica column, yielded approximately 5% fucose, 17% galactose, 18% N-acetylglucosamine, 8% N-acetylgalactosamine and 7% sialic acid, totaling almost 55% by weight. The percentages from two different donors were almost the same. About 1 mole phosphorus per mole of K-casein was also detected. Using low-speed sedimentation equilibrium methods, a molecular weight of only 33,400 was obtained for human K- casein, suggesting carbohydrate lability. Human p-casein with four phosphoryls was stabilized against precipitation by 10 mM Ca ions at a level greater than 95% when the molar ratio of K/p exceeded 0.15.     

Purification and properties of proline reductase from Clostridium sticklandii.

Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.