Purification and properties of boticin P produced by Clostridium botulinum

1974 ◽  
Vol 20 (3) ◽  
pp. 385-390 ◽  
Author(s):  
A. H. S. Lau ◽  
R. Z. Hawirko ◽  
C. T. Chow
Keyword(s):  
Density Gradient ◽  
Clostridium Botulinum ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Electron Microscopic Examination ◽  
Equilibrium Density ◽  
Type B ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation

A bacteriocin produced spontaneously by a nontoxigenic strain of Clostridium botulinum, type E, PM-15, has been isolated and designated boticin P. It was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-100, sucrose density gradient centrifugation, and cesium chloride equilibrium density gradient centrifugation. Boticin P is composed mainly of proteins (98.8%) with a trace amount of carbohydrates (0.4%), and has an apparent molecular weight in excess of 4 × 106 daltons as estimated by gel filtration on Sepharose 2B. Electron microscopic examination of boticin P reveals a phage tail-like structure of 100 nm in length.Boticin P exerted a static effect on vegetative growth and spore outgrowth but not on the initial events of germination. The boticin was active on 10/12 toxigenic and 3/6 nontoxigenic type E and 2/2 nonproteolytic type B strains of C. botulinum. The activity spectrum on 27 strains supports the proposal that type E and the nonproteolytic type B strains belong to the same taxosubspecies.

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

1980 ◽  
Vol 189 (3) ◽  
pp. 581-590 ◽  
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.

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 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

1971 ◽  
Vol 121 (4) ◽  
pp. 635-641 ◽  
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.

INTERMICROSOMAL DISTRIBUTION OF AROMATIZING ENZYME SYSTEM IN EQUINE TESTICULAR TISSUE

1973 ◽  
Vol 72 (2) ◽  
pp. 366-375 ◽  
Author(s):  
Ran Oh ◽  
Bun-ichi Tamaoki
Keyword(s):  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Enzyme System ◽  
Gradient Centrifugation ◽  
Electron Microscopic Examination ◽  
Sucrose Density Gradient ◽  
Testicular Tissue ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytochrome P 450

ABSTRACT The microsomal fraction (10 000–105 000 × g precipitate) of equine testes was fractionated into the smooth- and the rough-surfaced microsomal subfractions by a sucrose density-gradient centrifugation in the presence of CsCl. The validity of this fractionating procedure was confirmed by electron microscopic examination and also by chemical analysis of the RNA contents in these subfractions. The aromatizing enzyme system (19-hydroxylase and aromatase) which was concentrated in the microsomal fractions among the organellae was found to be localized in the smoothsurfaced microsomal fraction. The cytochrome P-450 which was also involved in the process of enzymatic aromatization was detected exclusively in the smooth-surfaced microsomal fraction. The distribution of the aromatizing system between the two microsomal subfractions of equine testes was discussed in comparison with that in human full term placentae.

scholarly journals A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure

1981 ◽  
Vol 197 (1) ◽  
pp. 155-162 ◽  
Author(s):  
J P Pearson ◽  
A Allen ◽  
S Parry
Keyword(s):  
Density Gradient ◽  
Protein Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Gastric Mucus ◽  
Mucus Glycoprotein

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 × 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 × 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.

scholarly journals Organ distribution of rat histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 157 (3) ◽  
pp. 635-641 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Isoelectric Focusing ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Organ Distribution ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Amino Donor

The organ distribution of rat histidine-pyruvate aminotransferase isoenzymes 1 and 2 was examined by using an isoelectric-focusing technique. Isoenzyme 1 (pI8.0) is present only in the liver and its activity is increased by the injection of glucagon, whereas isoenzyme 2 (pI5.2) is distributed in all tissues (liver, kidney, brain and heart) tested, and is not affected by glucagon injection. Isoenzyme 2 of the liver, kidney, brain and heart was purified by the same procedure and characterized. Isoenzyme 2 preparations from these four tissues were nearly identical in physical and enzymic properties. These properties differed from those previously found for the highly purified isoenzyme 1 preparation of rat liver. Isoenzyme 2 was active with pyruvate but not with 2-oxoglutarate as amino acceptor. Amino donors were effective in the following order of activity: tyrosine greater than histidine greater than phenylalanine greater than kynurenine greater than tryptophan. Very little activity was found with 5-hydroxytryptophan. The apparent Km for histidine was about 0.45 mM. The Km for pyruvate was about 4.5 mM with histidine as amino donor. The amino-transferase activities of isoenzyme 2 towards phenylalanine and tyrosine were inhibited by histidine. The ratio of aminotransferase activities towards these three amino acids was constant through gel filtration, electrophoresis, isoelectric focusing and sucrose-density-gradient centrifugation of the purified isoenzyme 2 preparations. These results suggest that these three activities are properties of the same enzyme protein. Sephadex G-150 gel filtration and sucrose-density-gradient centrifugation yielded mol.wts. of approx. 95000 and 92000 respectively. The pH optimum was between 9.0 and 9.3.

Methods for the Separation of Platelets from Plasma

1974 ◽  
Vol 31 (01) ◽  
pp. 119-132 ◽  
Author(s):  
R. A Hutton ◽  
Margaret A Howard ◽  
Daniel Deykin ◽  
R. M Hardisty
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Morphological Changes ◽  
Adenine Nucleotides ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Combined Method ◽  
Electron Microscopic ◽  
Platelet Factor

SummaryPlatelets have been separated from plasma of a series of normal volunteers by three methods: albumin density-gradient centrifugation, gel filtration, and a combination of the two. The efficacy of these three methods has been assessed by determination of the percentage recovery of the platelets, their aggregability by ADP, collagen and thrombin, platelet factor-3 availability, content and release of platelet adenine nucleotides and electron microscopic appearances. Platelets prepared by all three methods showed some loss of sensitivity to aggregating agents, minor activation of platelet factor 3 and morphological changes, as compared with platelets in PRP. Function and morphology were best preserved in platelets prepared by the combined method and most disturbed in those prepared by repeated washing on an albnmin density gradient.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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