Creatine amidinohydrolase of Pseudomonas putida: crystallization and some properties.

Creatine amidinohydrolase (EC 3.5.3.3, creatinase) of Pseudomonas putida var. naraensis C-83 was purified by column chromatography on sarcosine-hexamethylenediamine-Sepharose and Sephadex G-200 and then crystallized in the presence of ammonium sulfate. The purified preparation appeared homogeneous on disc gel electrophoresis and ultracentrifugal analysis. It was most active at pH 8 and showed a K_m value of 1.33 mm for creatine. Estimation of the molecular weight by the meniscus depletion method yielded a value of 94,000. A value of 47,000 was obtained, however, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the enzyme is composed of two subunits. Inhibition experiments suggested that a sulfhydryl group is closely related to the creatinase activity.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Purification and properties of invertase from a glutamate-producing bacterium

Invertase was purified from the cell extracts of the glutamic acid bacterium (Brevibacterium divaricatum) by ammonium sulfate fractionation, batch theatment with DEAE-cellulose, and column chromatographies on DEAE-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was proved to be almost homogeneous by polyacrylamide gel electrophoresis.The molecular weight of the enzyme was estimated to be 92,000 by both gel filtration and SDS-polyacrylamide gel electrophoresis methods. The optimum pH and temperature for the activity were 6.8 and 40°C. The enzyme was highly specific to sucrose as substrate, having only 10% as much activity toward raffinose as that toward sucrose, and being inert toward other disaccharides: maltose, trehalose, lactose, melibiose and cellobiose. The K_m value for sucrose was 0.19 M. The enzyme required phosphate or arsenate ions for activity. Monovalent or divalent Cu ions and sulfhydryl reagents inhibited the enzyme.     

Creatinine hydrolase and creatine amidinohydrolase: I. Presence in cell-free extracts of arthrobacter ureafaciens

Summary WhenA. ureafaciens are grown in a medium containing either creatinine or creatine as the sole source of carbon, an enzyme system capable of catabolizing creatine and creatinine is induced. This enzyme system has been isolated in a cell-free extract and is composed of two separate enzymes. The first, creatinine hydrolase, interconverts creatinine and creatine to form an equilibrium mixture of the two. The second enzyme, creatine amidinohydrolase, splits creatine into equimolar amounts of sarcosine and urea. The former enzyme is heat stable at 55°C for 30 min while the latter enzyme is completely destroyed at this temperature. The two enzymes have different solubilities in ammonium sulfate solutions.Predoctoral Fellow supported by NIH Training Grant GM00052.Submitted in partial fulfillment of the M.S. degree.     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Purification and properties of a fibrinolytic enzyme from Bacillus subtilis

A fibrinolytic enzyme obtained from B. subtilis was purified, using DEAE-cellulose column chromatography, and gel filtration on Sephadex G-100. The preparation was homogeneous as tested by gel filtration on Sephadex G-200, and disc electrophoresis. The molecular weight of this enzyme was 29.400 estimated by gel filtration on Sephadex G-100. The optimum pH for enzyme activity was 7.2 Copper ions significantly increased enzyme activity, while Zn++ and Mn++ caused marked inhibition.     

Acyl-CoA-ACP-transacylases of Mycobacterium smegmatis. An analytical study

Homogenates of Mycobacterium smegmatis have been prepared by sonication of bacteria in 0,1 M phosphate buffer, pH 7, containing 0,001 M EDTA and 0,01 M 2-mercaptoethanol. The 105 000 g supernatant of these homogenates has been fractionated by ammonium sulfate precipitation. The fraction precipitating between 25 and 65 per cent saturation in ammonium sulfate shows palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase activities. These activities are not associated, essentially, to a complexe of high molecular weight as they are retained on the column for 80 per cent or more upon gel filtration.By chromatographic procedures involving Sephadex G-150 gel filtration, and chromatography on DEAE-Sephadex A-50 the malonyl-CoA-ACP-transacylase activity is separated from the other two activities. The palmityl-CoA-ACP-transacylase activity and the acetyl-CoA-ACP-transacylase activity show two close but distinct major peaks either upon gel filtration on Sephadex G-150 or upon DEAE-Sephadex A-50 chromatography indicating thus that these two activities are attribuable to two distinct molecular entities. Besides the major peaks for these two activities, a supplementary minor peak for each activity is observed.An estimate of the approximate range of the apparent molecular weights for palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase has been made in using the curve of the K_av values of known proteins plotted against the logarithms of their molecular weights. The molecular weights thus estimated for these three enzymes are 100 000, 32 000 and 85 000 respectively.The void volume fractions of the chromatography of the homogenates on Sephadex G-150 present 20 per cent or less of the enzyme activity of the homogenates. Chromatography of the mixture of the void volume fractions on DEAE-Sephadex A-50 shows that the malonyl-CoA-ACP-transacylase activity associated to these fractions behaves differently from that of the fractions retained upon gel filtration; this may be taken as an indication for the presence of a complexe of the fatty acid synthetase present as a minor component of the homogenates.     

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

Purification and characterization of mucopolysaccharidase from an oral strain of Bacteroides sp.

A mucopolysaccharidase in the cell extract of an oral strain of Bacteroides sp. was purified to homogeneity by ammonium sulfate precipitation, DEAE-cellulose column chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. Specific activity increased 110-fold and recovery was 2%. The molecular weight was determined to be 89,000 by gel filtration, and the isoelectric point was 7.0. The optimum pH for the activity was 6.5. The enzyme was inactivated by heating at 60 degrees C for 5 min. The purified mucopolysaccharidase degraded hyaluronic acid more rapidly than chondroitin and chondroitin sulfate A and C. However, it had no activity against chondroitin sulfate B, heparin, and heparan sulfate. Since unsaturated disaccharides were derived from the enzyme substrate, this enzyme was considered to be a mucopolysaccharide lyase.     

Purification and characterization of mucopolysaccharidase from an oral strain of Bacteroides sp

A mucopolysaccharidase in the cell extract of an oral strain of Bacteroides sp. was purified to homogeneity by ammonium sulfate precipitation, DEAE-cellulose column chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. Specific activity increased 110-fold and recovery was 2%. The molecular weight was determined to be 89,000 by gel filtration, and the isoelectric point was 7.0. The optimum pH for the activity was 6.5. The enzyme was inactivated by heating at 60 degrees C for 5 min. The purified mucopolysaccharidase degraded hyaluronic acid more rapidly than chondroitin and chondroitin sulfate A and C. However, it had no activity against chondroitin sulfate B, heparin, and heparan sulfate. Since unsaturated disaccharides were derived from the enzyme substrate, this enzyme was considered to be a mucopolysaccharide lyase.     

Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase

Cholesterol ester hydrolase (sterol-ester acylhydrolase, EC 3.1.1.13) was purified from human pancreatic tissue by column chromatography and acetone precipitation, leading to a 400-fold enrichment. Isoelectric focusing of this product reveals a double-band at pH 4.5 and 4.6. The molecular weight was estimated at 320 kDa by means of Sephadex filtration on calibrated columns. Obviously these large molecules represent a tetrameric form of the monomeric subunit (molecular mass 76-80 kDa), which is also enzymatically active. It was found together with the dimeric form in pancreatic juice, where the tetrameric enzyme is responsible for the major part of the hydrolytic activity, splitting cholesterol ester as well as synthetic substrates, such as fluorescein or p-nitrophenyl esters. Attempts to split the tetrameric cholesterol ester hydrolase, isolated from pancreatic tissue, into active subunits found additionally in pancreatic juice by the influence of bile acids and proteolytic enzymes failed. The spectral shift method using Rhodamine fluorescence was employed in order to prove that fluorescein dilaurate forms micellar solutions and mixed micelles when bile salts are present.     

Purification and properties of pig liver kynureninase.

Kynureninase [L-kynurenine hydrolase, EC 3.7.1.3] was purified from pig liver by a procedure including DEAE-cellulose chromatography, hydroxyapatite chromatography, ammonium sulfate fractionation, DEAE-Bio Gel chromatography, Sephacryl S-200 gel filtration, kynurenine-Sepharose affinity chromatography, and Sephadex G-200 gel filtration. The enzyme was found to be homogeneous by the criterion of disc-gel electrophoresis. The enzyme has a molecular weight of about 100,000 and exhibits absorption maxima at 280 and 420 nm. The optimum pH and the isoelectric point of the enzyme are 8.5 and 5.0, respectively. The Michaelis constants were determined to be as follows: L-kynurenine, 7.7 X 10(-4) M; L-3-hydroxykynurenine, 1.3 X 10(-5) M; and pyridoxal 5'-phosphate, 1.8 X 10(-6) M. L-3-Hydroxykynurenine is hydrolyzed more rapidly than L-kynurenine; the liver enzyme can be regarded as a 3-hydroxy-kynureninase.     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

Purification and properties of the urea amidolyase from Candida utilis.

Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.     

Purification of an Apoplastic β-N-Acetyl-D-Hexosaminidase from Tomato Leaves Infected with Tobacco Mosaic Virus

Systematic infection of tomato (Lycopersicon esculenturn) leaves by tobacco mosaic virus (TMV) increased the levels of β-N-acetyl-D-hexosaminidase activity. The enzyme was purified from intercellular fluid by --20℃ acetone precipitation, CM-Sephadex C-25 ion exchange chromatography, Polybuffer Exchanger 94 chromatofocusing and Sephadex G-150 gel filtration column to homogeneity. The molecular weight obtained by SDS-PAGE and Sephadex G-150 gel filtration was 75 kD and 145 kD respectively. The enzyme hydrolysed p-nitrophenyl-N-acetyl-β-glucosaminide and p-nitrophenyl-N-acetyl-β-galaetosaminide, it was a glycoprotein. Most of the enzyme activity in the TMV-infected tomato leaves was found in the intercellular spaces.     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Purification and Some Properties of Maleylpyruvate Hydrolase and Fumarylpyruvate Hydrolase from Pseudomonas alcaligenes

Hydrolysis of the gentisate ring-cleavage product, maleylpyruvate (cis-2,4-diketohept-5-enedioic acid), was shown to be catalyzed by an enzyme, maleylpyruvate hydrolase 11, in Pseudomonas alcaligenes (P25X1) after growth with 3-hydroxybenzoate. This activity was separated from fumarylpyruvate hydrolase activity during the course of its purification which accomplished an approximately 50-fold increase in specific activity. An apparent molecular weight of 77,000 was assigned on the basis of Sephadex G-200 chromatography. Despite the presence of up to three similarly migrating bands of protein on polyacrylamide-gel electrophoresis of the purified enzyme, at least two of these bands possessed maleylpyruvate hydrolase activity. Electrophoresis on sodium dodecyl sulfate-polyacrylamide before and after reduction with mercaptoethanol gave a principal band of molecular weight of 33,000 (and a minor band of molecular weight 50,000). A number of substituted maleylpyruvates also served as substrates for maleylpyruvate hydrolase 11, but maleylacetoacetate and fumarylpyruvate were not attacked. Fumarylpyruvate hydrolase was purified approximately 40-fold to give a single band on polyacrylamide gels and with an apparent molecular weight of 73,000 by Sephadex G-200 chromatography. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis before or after reduction with mercaptoethanol, a subunit molecular weight of 25,000 was obtained. Neither maleylpyruvate nor fumarylacetoacetate served as substrates for fumarylpyruvate hydrolase. The activities of both maleyl- and fumarylpyruvate hydrolases were stimulated by Mn²⁺ ions. Reasons are discussed for the presence of both enzyme activities, one of which appears to be redundant.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.