scholarly journals Purification and properties of S-alkyl-L-cysteine lyase from seedlings of Acacia farnesiana Willd.

1975 ◽  
Vol 147 (3) ◽  
pp. 485-491 ◽  
Author(s):  
M Mazelis ◽  
R K Creveling
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkyl Substituent ◽  
Partial Specific Volume ◽  
Ph Optimum ◽  
Acacia Farnesiana ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Km Value

1. An S-alkyl-L-cysteine lyase (EC 4.4.1.6) was purified to apparent homogeneity from extracts of acetone-dried powders of the hypocotyls of etiolated 5-day-old seedlings of Acacia farnesiana Willd. 2. The enzyme catalyses a beta-elimination reaction and will utilize both the thioether and sulphoxide form of the substrate. 3. There is a braod specificity with regard to the alkyl substituent, but cystathionine is utilized very poorly. 4. The pH optimum is 7.8 and the Km value for the probable natural substrate L-djenkolate is 0.3 mM. 5. Both sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and ultracentirfugal analysis give a molecular weight of about 144000. 6. One mol of pyridoxal phosphate is bound/mol of enzyme. 7. The energy of activation with L-djenkolate as the substrate is 53.1 kJ/mol. 8. The enzyme has a partial specific volume of 0.56 and S20,w 7.26S.

scholarly journals Cathepsin D from pig myometrium. Characterization of the proteinase

1984 ◽  
Vol 219 (3) ◽  
pp. 899-904 ◽  
Author(s):  
R Barth ◽  
E G Afting
Keyword(s):  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Equilibrium Studies ◽  
Main Band ◽  
Km Value ◽  
Ph Range

The purification of cathepsin D from pig uterus by two-step affinity chromatography on concanavalin A- and pepstatin-Sepharose was described previously [Afting & Becker (1981) Biochem. J. 197, 519-522]. In this paper, chemical and physical properties of the proteinase are presented. The purified enzyme showed three bands on SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis, one main band corresponding to an Mr of 31 000 and two minor bands with Mr values of 43 000 and 15 000 respectively. Gel filtration on Bio-gel P-150 and sedimentation-diffusion equilibrium studies give an Mr for the main band of about 35 000. The pI of the enzyme was determined to be 7.2. Haemoglobin was the best substrate, with a Km value of 6.4 X 10(-6)M. It was hydrolysed with a pH optimum between 3.0 and 3.3 for a substrate concentration of 100 microM. The proteinase was stable over the pH range of 3.5-6.5. At pH 6 the enzyme showed stability up to a temperature of 50 degrees C; at pH 3 the activity was already decreased below 40 degrees C. Carbohydrate studies resulted in the staining of all three bands on an SDS/polyacrylamide gel by thymol/H2SO4. After treatment with endo-beta-N-acetylglucosaminidase H, all three bands were shifted to a region of lower Mr. Of various inhibitors tested, only pepstatin was strongly inhibiting, with a Ki of 2.1 X 10(-9)M.

scholarly journals Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids

1999 ◽  
Vol 181 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
Shunji Takahashi ◽  
Tomohisa Kuzuyama ◽  
Haruo Seto
Keyword(s):  
Amino Acid ◽  
Coenzyme A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Hmg Coa Reductase ◽  
Apparent Homogeneity ◽  
Key Enzyme ◽  
Coa Reductase

ABSTRACT The eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34 ) was purified 3,000-fold fromStreptomyces sp. strain CL190 to apparent homogeneity with an overall yield of 2.1%. The purification procedure consisted of (NH4)2SO4 precipitation, heat treatment and anion exchange, hydrophobic interaction, and affinity chromatographies. The molecular mass of the enzyme was estimated to be 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 100 to 105 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of around 7.2, with apparent Km values of 62 μM for NADPH and 7.7 μM for HMG-CoA. A gene from CL190 responsible for HMG-CoA reductase was cloned by the colony hybridization method with an oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The amino acid sequence of the CL190 HMG-CoA reductase revealed several limited motifs which were highly conserved and common to the eucaryotic and archaebacterial enzymes. These sequence conservations suggest a strong evolutionary pressure to maintain amino acid residues at specific positions, indicating that the conserved motifs might play important roles in the structural conformation and/or catalytic properties of the enzyme.

scholarly journals Characterization of an Inducible Chlorophenol O-Methyltransferase from Trichoderma longibrachiatum Involved in the Formation of Chloroanisoles and Determination of Its Role in Cork Taint of Wines

2003 ◽  
Vol 69 (9) ◽  
pp. 5089-5095 ◽  
Author(s):  
Juan-José R. Coque ◽  
María Luisa Álvarez-Rodríguez ◽  
Germán Larriba
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Ph Optimum ◽  
Trichoderma Longibrachiatum ◽  
Molecular Weights ◽  
Apparent Homogeneity ◽  
Halogenated Phenols ◽  
Cork Taint

ABSTRACT A novel S-adenosyl-l-methionine (SAM)-dependent methyltransferase catalyzing the O methylation of several chlorophenols and other halogenated phenols was purified 220-fold to apparent homogeneity from mycelia of Trichoderma longibrachiatum CECT 20431. The enzyme could be identified in partially purified protein preparations by direct photolabeling with [methyl-3H]SAM, and this reaction was prevented by previous incubation with S-adenosylhomocysteine. Gel filtration indicated that the M r was 112,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme was composed of two subunits with molecular weights of approximately 52,500. The enzyme had a pH optimum between 8.2 and 8.5 and an optimum temperature of 28°C, with a pI of 4.9. The Km values for 2,4,6-trichlorophenol and SAM were 135.9 ± 12.8 and 284.1 ± 35.1 μM, respectively. S-Adenosylhomocysteine acted as a competitive inhibitor, with a Ki of 378.9 ± 45.4 μM. The methyltransferase was also strongly inhibited by low concentrations of several metal ions, such as Cu2+, Hg2+, Zn2+, and Ag+, and to a lesser extent by p-chloromercuribenzoic acid, but it was not significantly affected by several thiols or other thiol reagents. The methyltransferase was specifically induced by several chlorophenols, especially if they contained three or more chlorine atoms in their structures. Substrate specificity studies showed that the activity was also specific for halogenated phenols containing fluoro, chloro, or bromo substituents, whereas other hydroxylated compounds, such as hydroxylated benzoic acids, hydroxybenzaldehydes, phenol, 2-metoxyphenol, and dihydroxybenzene, were not methylated.

scholarly journals Purification and properties of threonine aldolase from Clostridium pasterianum

1970 ◽  
Vol 117 (3) ◽  
pp. 585-592 ◽  
Author(s):  
R. H. Dainty
Keyword(s):  
Pyridoxal Phosphate ◽  
Anaerobic Bacteria ◽  
Clostridium Pasteurianum ◽  
Strictly Anaerobic ◽  
Prosthetic Group ◽  
Ph Optimum ◽  
Threonine Aldolase ◽  
Purification And Properties ◽  
Km Value ◽  
The Relationship

1. Threonine aldolase was purified about 200-fold in 10% yield from Clostridium pasteurianum and its properties were examined. The final preparation gave three bands after ionophoresis on polyacrylamide gel. 2. The purified enzyme was shown to produce glycine and acetaldehyde in stoicheiometric amounts from threonine. The reverse reaction was demonstrated qualitatively. 3. The enzyme has a broad pH optimum at 6.5–7.0. 4. The enzyme is highly specific for l-threonine. 5. The enzyme is completely inhibited by 1mm concentrations of hydroxylamine and semicarbazide. Activity is decreased to 20% of the original by treatment with cysteine plus mercaptoethanol; most of the loss is regained on incubation with pyridoxal phosphate. It is concluded that pyridoxal phosphate is a prosthetic group. 6. The relationship between velocity and substrate concentration is atypical but indicates a Km value of 0.42mm. 7. The enzyme was demonstrated in several other strictly anaerobic bacteria.

scholarly journals Microbial metabolism of amino alcohols. Purification and properties of coenzyme B12-dependent ethanolamine ammonia-lysae of Escherichia coli

1978 ◽  
Vol 175 (2) ◽  
pp. 555-563 ◽  
Author(s):  
Carol M. Blackwell ◽  
John M. Turner
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Coenzyme B12 ◽  
Apparent Homogeneity ◽  
Purification And Properties ◽  
Protein Components ◽  
Inhibited Enzyme

1. The 120-fold purification of ethanolamine ammonia-lyase from Escherichia coli extracts, to apparent homogeneity, is described. Ethanolamine, dithiothreitol, glycerol and KCl protected the apoenzyme from inactivation. 2. At the optimum pH7.5, Km values for ethanolamine and coenzyme B12 were 44μm and 0.42μm respectively. The Km for ethanolamine was markedly affected by pH, transitions occurring at pH7.0 and 8.35. 3. The enzyme was specific for ethanolamine as substrate, none of the 18 analogues tested being active. l-2-Aminopropan-l-ol (Ki 0.86μm), dl-1-aminopropan-2-ol (Ki 2.2μm) and dl-1,3-diaminopropan-2-ol (Ki 88.0μm) inhibited competitively. 4. Enzyme activity was inhibited, irreversibly and non-competitively, by the coenzyme analogues methylcobalamin (Ki 1.4nm), hydroxocobalamin (Ki 2.1nm) and cyanocobalamin (Ki 4.8nm). 5. Iodoacetamide inhibited in the absence of ethanolamine, but only slightly in its presence. p-Hydroxymercuribenzoate inhibited markedly even in the presence of ethanolamine. Dithiothreitol and 2-mercaptoethanol (less effectively) restored activity to the enzyme dialysed against buffer containing ethanolamine. 6. Although K+ ions stabilized the enzyme during dialysis or storage, they were not necessary for activity. 7. Gel filtration showed the enzyme to be of high molecular weight, ultracentrifugal studies giving s20,w of 16.4 and an estimated mol.wt. 560400. The isoelectric point for the apoenzyme was approx. pH5.0. inhibited enzyme activity at concentrations above 1m (95% inhibition at 3m) and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated protein subunits of mol.wt. 61400. 8. Immunological studies showed that the E.coli enzyme was closely related to those of other enterobacteria, but only distantly to that of Clostridium sp. A double precipitin band suggested that the apoenzyme may be made up of two protein components.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida

1986 ◽  
Vol 64 (12) ◽  
pp. 1288-1293 ◽  
Author(s):  
Josefa M. Alonso ◽  
Amando Garrido-Pertierra
Keyword(s):  
Pseudomonas Putida ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Catabolic Pathway ◽  
Ph Optimum ◽  
Semialdehyde Dehydrogenase ◽  
Standard Assay ◽  
Assay Conditions

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.

scholarly journals Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

1978 ◽  
Vol 175 (2) ◽  
pp. 743-750 ◽  
Author(s):  
P Calvo ◽  
A Reglero ◽  
J A Cabezas
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mixed Substrates ◽  
Ph Optimum ◽  
Terrestrial Mollusc ◽  
Buffer Solutions ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Ph Range

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8–4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

Purification and characterization of ferro- and cobalto-chelatases

1988 ◽  
Vol 66 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Eduardo T. Cánepa ◽  
Elena B.C. Llambías
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Degree Of Unsaturation ◽  
Apparent Homogeneity ◽  
Optimum Ph ◽  
Single Protein ◽  
Metal Insertion

Pig liver ferrochelatase was purified 465-fold with about 30% yield, to apparent homogeneity, by a procedure involving solubilization from mitochondria, ammonium sulfate fractionation, and Sephacryl S-300 chromatography. The fraction of each purification step had cobaltochelatase as well as ferrochelatase activity. A purified protein of molecular weight 40 000 was found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. A molecular weight of approximately 240 000 was obtained by Sephacryl S-300 chromatography. Both activities of the purified fraction increased linearly with time until 2 h. but nonlinear plots were obtained with increasing concentrations of protein. Their optimum pH values were similar. Km values were, for ferrochelatase activity, 23.3 μM for the metal and 30.3 μM for mesoporphyrin. and for cobaltochelatase activity. 27 and 45.5 μM, respectively. Fe2+ and Co2+ each protected against inactivation by heat. Pb2+, Zn2+, Cu2+, or Hg2+ inhibited both activities, while Mn2+ slightly activated; Mg2+ had no effect, at the concentrations tested. There appeared to be an involvement of sulfhydryl groups in metal insertion. Lipids, in correlation with their degree of unsaturation, activated both purified activities; phospholipids also had activation effects. We conclude that a single protein catalyzes the insertion of Fe2+ or Co2+ into mesoporphyrin.

Novel Catalatic Proteins of Bakers' Yeast. I. An Atypical Catalase

1973 ◽  
Vol 51 (11) ◽  
pp. 1551-1555 ◽  
Author(s):  
Tony C. M. Seah ◽  
A. R. Bhatti ◽  
J. G. Kaplan
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Native Protein ◽  
Wild Type ◽  
Major Band ◽  
Subunit Molecular Weight ◽  
Purification And Properties

At any stage of growth of a wild-type bakers' yeast, some 20% of the catalatic activity of crude extracts is not precipitable by means of antibody prepared against the typical catalase (catalase T), whose purification and properties have been previously described. Some of this catalatic activity is due to the presence of an atypical catalase (catalase A), a heme protein, with a molecular weight estimated as 170 000 – 190 000, considerably lower than that of the usual catalases (225 000 – 250 000). Preparations of catalase A were found to be homogeneous in the analytical ultracentrifuge and in polyacrylamide gel electrophoresis. Its subunit molecular weight, determined from its iron content, was 46 500, virtually the same as that of the major band obtained in gel electrophoresis in the presence of sodium dodecyl sulfate, suggesting that the native protein is tetrameric. Its specific activity is in the range of those reported for other typical catalases.

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