scholarly journals Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1973 ◽  
Vol 133 (2) ◽  
pp. 335-347 ◽  
Author(s):  
J. C. Gray ◽  
R. G. O. Kekwick
Keyword(s):  
Phaseolus Vulgaris ◽  
Competitive Inhibition ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
French Bean ◽  
Exchange Chromatography ◽  
Mevalonate Kinase ◽  
Ph Optimum ◽  
Enzyme Preparations ◽  
Green Leaves

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a Km of 4.26×10−5m for RS-mevalonate and 1.54×10−3m for ATP. The preparation from green leaves had a Km of 4.55×10−5m for RS-mevalonate and 1.75×10−3m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

Purification and characterization of the extracellular α-amylase activity of the yeast Schwanniomyces alluvius

1987 ◽  
Vol 65 (10) ◽  
pp. 899-908 ◽  
Author(s):  
F. Moranelli ◽  
M. Yaguchi ◽  
G. B. Calleja ◽  
A. Nasim
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Proteinase K ◽  
Ph Optimum ◽  
Sds Page

The extracellular α-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS–PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0–7.0. Under the conditions tested, the activity is maximal between 45 and 50 °C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

scholarly journals Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

1978 ◽  
Vol 175 (2) ◽  
pp. 635-641 ◽  
Author(s):  
Ulla Christensen ◽  
Inge Clemmensen
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Active Site ◽  
Competitive Inhibition ◽  
Enzyme Inhibitor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Comparison Of The Results ◽  
Sepharose 4B

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH4)2SO4 precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen–CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor–plasminogen complex was determined to be approx. 3μm at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme–inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26μm) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor–plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.

scholarly journals Biochemical characterization of a leukemia-associated inhibitor (LAI) suppressing normal granulopoiesis in vitro

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 983-991 ◽  
Author(s):  
T Olofsson ◽  
I Olsson
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Charge Heterogeneity ◽  
Sucrose Density Gradient Centrifugation ◽  
Peripheral Cell

Low-density (less than 1.077 g/ml) marrow or blood cells from patients with acute or chronic leukemia release a high molecular weight substance called “leukemia-associated inhibitor” (LAI) that reduces the fraction of normal marrow CFU-c in S-phase as measured with the 3H-TdR suicide technique. LAI from conditioned media or 3M KCl extracts of subcellular fractions behaved homogeneously on gel chromatography, showing an apparent molecular weight greater than 500,000. However, ion- exchange chromatography and isoelectric focusing indicated considerable charge heterogeneity for LAI molecules. Results from SDS-polyacrylamide gel electrophoresis indicated that the biologic activity resides in a subunit of 150,000–170,000 daltons. The findings of marked affinity for Con-A-Sepharose, marked susceptibility to mild periodate treatment, partial susceptibility to protease digestion, and relative resistance to heating suggest that LAI is a glycoprotein. Data from radiolabeling of cell surface components and sucrose density gradient centrifugation are consistent with LAI being a peripheral cell membrane glycoprotein, which may suppress normal granulopoiesis in leukemia.

Isolation and characterization of cathepsin D from human gastric mucosa

1981 ◽  
Vol 46 (13) ◽  
pp. 3302-3313 ◽  
Author(s):  
Jan Pohl ◽  
Ladislav Bureš ◽  
Karel Slavík
Keyword(s):  
Gastric Mucosa ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Polyacrylamide Gel ◽  
Human Gastric Mucosa ◽  
Ph Optimum ◽  
Isolation And Characterization

The molecular weight of the enzyme, purified by ion-exchange chromatography and affinity chromatography, was determined by gel filtration on Sephadex G-100 as 49 000. After treatment with 2-mercaptoethanol, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate resolved the enzyme into two chains, of molecular weights 33 000 and 18 000. This shows that in the native state the enzyme is composed of one light and one heavy chain. Isoelectric focusing in polyacrylamide gel gave four bands, the isoelectric points being 5.5, 6.1, 6.5 and 7.1. The optimum protein substrate (pH optimum 3.2-3.6) was haemoglobin. The best synthetic substrate was methyl ester of pyroglutamyl-histidyl-phenylalanyl-phenylalanyl-alanyl-leucine. The protease was inhibited by the inhibitor of cathepsin D from the potato tubers. It is concluded that the enzyme is cathepsin D from gastric mucosa.

Template Specificity and Subunits of RNA Polymerase from Asporogenous Mutants of Bacillus subtilis

1974 ◽  
Vol 52 (11) ◽  
pp. 966-973 ◽  
Author(s):  
H. Nishimoto ◽  
I. Takahashi
Keyword(s):  
Rna Polymerase ◽  
Morphological Changes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Sedimentation Coefficient ◽  
Enzyme Preparations ◽  
Template Specificity ◽  
Α Subunits

In order to investigate relations between template specificity of RNA polymerase and sporulation, RNA polymerase activities in partially purified preparations from various asporogenous mutants were measured with poly[d(A-T)] or DNA from phage PBS 15 as template. Results obtained suggest that morphological changes occurring during sporulation may not be tightly linked temporally to transcriptional events.Subunits of RNA polymerase from these mutants were analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis after purification by (NH4)2SO4 precipitation, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Phenylmethylsulfonyl fluoride was present throughout the purification procedure to prevent proteolytic degradation. It was found that β and β′ subunits were present in 1:1 ratio in all preparations. In addition to β, β′, and α subunits, a protein having a molecular weight of 95 000 was found in enzyme preparations from a wild-type strain and stage II mutants harvested at t5–t9. This protein was absent in stage 0 mutants and in all strains harvested in log phase. The enzyme containing this protein was eluted from phosphocellulose column with 0.6 M KCl rather than 0.35 M KCl, which eluted the enzyme without the 95 000 dalton protein. Furthermore the enzyme with this protein showed a sedimentation coefficient higher than that of the enzyme without the 95 000 dalton protein.

scholarly journals Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds

1979 ◽  
Vol 177 (2) ◽  
pp. 509-520 ◽  
Author(s):  
R Casey
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Immunoaffinity Chromatography ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Salting Out ◽  
Wide Range ◽  
Gel Isoelectric Focusing

The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.

The Purification and Properties of an Amidohydrolase from Soybean

1974 ◽  
Vol 52 (10) ◽  
pp. 903-910 ◽  
Author(s):  
Robert E. Hoagland ◽  
George Graf
Keyword(s):  
Arrhenius Plot ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Hydrolysis Rate ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Hydrolysis Of

An amidohydrolase (EC 3.5.1.13) was isolated from the roots of soybean (Glycine max Merril, var. Hawkeye) seedlings and purified 130-fold over the crude extract with 30% recovery. The purification steps entailed ammonium sulfate precipitation, gel filtration, cellulose ion-exchange chromatography, and polyacrylamide gel electrophoresis. The specific activity of the purified enzyme for the hydrolysis of Nα-benzoyl-DL-arginine p-nitroanilide (BAPA) was 810 mU/mg. The Km of the enzyme for this substrate was 5.78 × 10−6 M. The enzyme possessed a broad substrate specificity and catalyzed the hydrolysis of BAPA, glycine p-nitroanilide, L-leucine p-nitroanilide, and L-lysine p-nitroanilide. Specificity studies with a series of aminoacyl β-naphthylamides revealed a high hydrolysis rate on Nα-benzoyl-L-arginine β-naphthylamide, and lower hydrolysis rates on several other aminoacyl-substituted β-naphthylamides. The enzyme also displayed dipeptide hydrolase activity on several dipeptide substrates. The enzyme had a pH optimum of 8.0 in 0.05 M phosphate buffer with Nα-benzoyl-DL-arginine p-nitroanilide as substrate. The temperature optimum was 50 °C. The apparent activation energy determined from an Arrhenius plot was 6.3 kcal/mol (26 400 J/mol). The molecular weight estimated by gel filtration was approximately 63 000. Mercury (II) ion, silver (I) ion, p-benzoquinone, p-chloromercuribenzoate, and N-ethylmaleimide were effective inhibitors of the enzyme.

scholarly journals Biochemical characterization of a leukemia-associated inhibitor (LAI) suppressing normal granulopoiesis in vitro

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 983-991 ◽  
Author(s):  
T Olofsson ◽  
I Olsson
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Charge Heterogeneity ◽  
Sucrose Density Gradient Centrifugation ◽  
Peripheral Cell

Abstract Low-density (less than 1.077 g/ml) marrow or blood cells from patients with acute or chronic leukemia release a high molecular weight substance called “leukemia-associated inhibitor” (LAI) that reduces the fraction of normal marrow CFU-c in S-phase as measured with the 3H-TdR suicide technique. LAI from conditioned media or 3M KCl extracts of subcellular fractions behaved homogeneously on gel chromatography, showing an apparent molecular weight greater than 500,000. However, ion- exchange chromatography and isoelectric focusing indicated considerable charge heterogeneity for LAI molecules. Results from SDS-polyacrylamide gel electrophoresis indicated that the biologic activity resides in a subunit of 150,000–170,000 daltons. The findings of marked affinity for Con-A-Sepharose, marked susceptibility to mild periodate treatment, partial susceptibility to protease digestion, and relative resistance to heating suggest that LAI is a glycoprotein. Data from radiolabeling of cell surface components and sucrose density gradient centrifugation are consistent with LAI being a peripheral cell membrane glycoprotein, which may suppress normal granulopoiesis in leukemia.

Sign in / Sign up

Export Citation Format

Share Document