PREPARATION OF ZYMOGEN GRANULES WITH GEL FILTRATION: DISTRIBUTION OF AMYLASE AND CARBONIC ANHYDRASE ACTIVITY FOLLOWING “LINEAR-DISCRETE” DENSITY GRADIENT CENTRIFUGATION

Abstract This study was prompted by the observation that fresh platelet suspensions--prepared by gel filtration or albumin density gradient centrifugation--possessed only minimal factor V activity, whereas frozen-and-thawed platelet suspensions possessed striking factor V activity. Results of experiments with fresh suspensions suggested that unaltered platelets did not bind plasma factor V. The factor V activity of frozen-and-thawed platelet suspensions was markedly diminished after exposure to a factor V antibody, was not activated by thrombin, and was not associated with an increase in factor V antigen over that found in fresh platelet suspensions. Consequently, disruption by freezing and thawing must have resulted in the appearance of small amounts of an activated factor V molecule in platelet suspensions. Disrupted platelets were shown to activate native factor V, but an interaction between a platelet activator and traces of native factor V in fresh suspensions could not be demonstrated to account for the full activity of frozen-and-thawed suspensions. Apparently, therefore, platelets also contained an activated factor V molecule. Adding collagen, but not adenosine 5′-diphosphate to fresh platelet suspensions increased their factor V activity. Release of an activated platelet factor V molecule after exposure to collagen could represent a physiologically significant early step in hemostasis.

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Molecular parameters of 6-methylsalicylic acid synthetase from gel filtration and sucrose density gradient centrifugation

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(68)90668-1 ◽

1968 ◽

Vol 125 (1) ◽

pp. 326-333 ◽

Cited By ~ 17

Author(s):

Robley J. Light ◽

Lowell P. Hager

Keyword(s):

Density Gradient ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Molecular Parameters

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Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

Biochemical Journal ◽

10.1042/bj1890581 ◽

1980 ◽

Vol 189 (3) ◽

pp. 581-590 ◽

Cited By ~ 36

Author(s):

Etsuo Okuno ◽

Yohsuke Minatogawa ◽

Masayuki Nakamura ◽

Naoki Kamoda ◽

Junko Nakanishi ◽

...

Keyword(s):

Amino Acids ◽

Density Gradient ◽

Human Liver ◽

Analytical Ultracentrifugation ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Sucrose Density Gradient ◽

Sucrose Density Gradient Centrifugation ◽

Glyoxylate Aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.

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

Biochemical Journal ◽

10.1042/bj1550107 ◽

1976 ◽

Vol 155 (1) ◽

pp. 107-115 ◽

Cited By ~ 23

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.

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DETERMINATION OF SERUM PROTEIN BINDING OF 3H-ACTH USING DEXTRAN GEL FILTRATION AND DENSITY GRADIENT CENTRIFUGATION

Acta Endocrinologica ◽

10.1530/acta.0.056s182 ◽

1967 ◽

Vol 56 (1_Suppl) ◽

pp. S182 ◽

Cited By ~ 1

Author(s):

K. v. Werder ◽

F. Kluge ◽

K. Schwarz ◽

P. C. Scriba

Keyword(s):

Protein Binding ◽

Density Gradient ◽

Serum Protein ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Serum Protein Binding

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Proteoglycan Complexes from Bovine Heart Valve Fractionation by Density-gradient Centrifugation and Gel Filtration under Dissociative Conditions1

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a131044 ◽

1976 ◽

Vol 79 (1) ◽

pp. 17-25 ◽

Cited By ~ 1

Author(s):

Atsushi HONDA ◽

Yusuke KANKE ◽

Yo MORI

Keyword(s):

Density Gradient ◽

Heart Valve ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Gel Filtration ◽

Bovine Heart

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Nucleic acid enzymology of extremely halophilic bacteria. Gel-filtration and density-gradient-centrifugation studies of the molecular weights of Halobacterium cutirubrum polynucleotide phosphorylase and deoxyribonucleic acid- and ribonucleic acid-dependent ribonucleic acid polymerases

Biochemical Journal ◽

10.1042/bj1210635 ◽

1971 ◽

Vol 121 (4) ◽

pp. 635-641 ◽

Cited By ~ 8

Author(s):

B. Gregory Louis ◽

Pearl I. Peterkin ◽

P. S. Fitt

Keyword(s):

Rna Polymerase ◽

Density Gradient ◽

Ribonucleic Acid ◽

Density Gradient Centrifugation ◽

Gradient Centrifugation ◽

Halophilic Bacteria ◽

Gel Filtration ◽

Polynucleotide Phosphorylase ◽

Sucrose Density Gradient Centrifugation ◽

Molecular Weights

1. Conditions have been established for the estimation of molecular weights of proteins by analytical gel filtration and sucrose-density-gradient centrifugation in 2.5m-potassium chloride–1m-sodium chloride; Halobacterium cutirubrum polynucleotide phosphorylase, DNA-dependent RNA polymerase and RNA-dependent RNA polymerase have been studied by these methods. 2. The RNA-dependent polymerase has also been studied by density-gradient centrifugation in the absence of salt. 3. All three proteins are of unusually low molecular weight compared with similar enzymes from non-halophilic bacteria.

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