scholarly journals Collagenolytic cathepsin activity in rabbit peritoneal polymorphonuclear leucocyte granules

1978 ◽  
Vol 172 (1) ◽  
pp. 83-89 ◽  
Author(s):  
W T Gibson ◽  
D W Milsom ◽  
F S Steven ◽  
J S Lowe
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polymorphonuclear Leucocyte ◽  
Polymorphonuclear Leucocytes ◽  
Differential Centrifugation ◽  
Ph Optimum ◽  
Cathepsin Activity ◽  
Granule Fraction ◽  
Alpha Beta

Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles.

scholarly journals The locations of cathepsin activity and β-glucuronidase in the Guerin T8 tumour

1970 ◽  
Vol 118 (3) ◽  
pp. 543-549 ◽  
Author(s):  
A. R. Poole
Keyword(s):  
Endoplasmic Reticulum ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Unit Membrane ◽  
Electron Microscopic ◽  
Cathepsin Activity ◽  
Density Gradient Sedimentation

Tumour homogenate fractions, isolated by differential centrifugation, were subfractionated by density-gradient centrifugation. Biochemical and electron microscopic analyses revealed that β-glucuronidase and cathepsin activity were associated with a class (possibly two) of lysosomal particles of density greater than those of mitochondria and the endoplasmic reticulum. Lysosomes sedimented by low g forces were vacuolar, electron-dense, delineated by a unit membrane and about 0.2μm in diameter. β-Glucuronidase was also apparently associated with ribosomes whereas cathepsin was bound in part to the endoplasmic reticulum. Catalase and glucose 6-phosphatase possessed slightly different density-gradient sedimentation profiles.

scholarly journals Intramitochondrial localization of δ-aminolaevulate synthetase and ferrochelatase in rat liver

1969 ◽  
Vol 114 (3) ◽  
pp. 455-461 ◽  
Author(s):  
Roxane McKay ◽  
R. Druyan ◽  
G. S. Getz ◽  
M. Rabinowitz
Keyword(s):  
Glutamate Dehydrogenase ◽  
Density Gradient ◽  
Malate Dehydrogenase ◽  
Rat Liver ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Inner Mitochondrial Membrane

Intramitochondrial loci for δ-aminolaevulate synthetase and ferrochelatase, the initial and final enzymes in haem synthesis, have been found in rat liver. Two different methods of fractionation were applied to mitochondria: (a) sonication and density-gradient centrifugation; (b) treatment with digitonin and differential centrifugation. Similar results were obtained with each technique. δ-Aminolaevulate synthetase is distributed similarly to two known matrix enzymes, malate dehydrogenase and glutamate dehydrogenase. Ferrochelatase is firmly bound to the the inner mitochondrial membrane. These results are considered in terms of the regulation of haem synthesis and in relation to mitochondrial biogenesis.

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 Some Properties of Rose Mosaic Virus from South Australia

1970 ◽  
Vol 23 (5) ◽  
pp. 1197 ◽  
Author(s):  
AA Basit ◽  
RIB Francki
Keyword(s):  
North America ◽  
Physical Properties ◽  
Mosaic Virus ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
South Australia ◽  
Sucrose Density Gradient ◽  
Differential Centrifugation ◽  
Sucrose Density Gradient Centrifugation

Isolates of rose mosaic virus (RMV) from South Australia were purified by differential centrifugation of cucumber extracts clarified by emulsification with ether, followed by sucrose density-gradient centrifugation. The virus was shown to be serologically similar to and to have many physical properties in common with RMV from North America. However, the Australian isolates studied appear to hlwe narrower host ranges.

Isolation of the 67Gallium Accumulating Fraction in Normal Rat Liver

1974 ◽  
Vol 13 (01) ◽  
pp. 72-84
Author(s):  
K. Hierholzer ◽  
K. zum Winkel ◽  
U. Haubold ◽  
E. Aulbert
Keyword(s):  
Electron Microscopy ◽  
Density Gradient ◽  
Intravenous Injection ◽  
Rat Liver ◽  
Soluble Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Normal Rat

SummarySubcellular 67Gallium distribution was investigated in normal rat liver after intravenous injection. By differential centrifugation and density gradient centrifugation 67Gallium accumulating bodies were isolated and identified as lysosomes by enzyme determination and electron microscopy. 67Gallium enrichment in this fraction was 23-fold. Using the isolated 67Gallium accumulating lysosomes the binding state of the isotope inside the lysosomes was studied. 67Gallium was found to be associated with the soluble fraction of lysosomes.

scholarly journals Purification and characterization of kynurenine-2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats

1975 ◽  
Vol 151 (2) ◽  
pp. 399-406 ◽  
Author(s):  
T Noguchi ◽  
Y Minatogawa ◽  
E Okuno ◽  
M Nakatani ◽  
M Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Small Intestine ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Wide Range

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.

scholarly journals The effects of pH and citrate on the activity of nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria

1969 ◽  
Vol 113 (5) ◽  
pp. 813-820 ◽  
Author(s):  
G. F. Cox ◽  
D. D. Davies
Keyword(s):  
Density Gradient ◽  
Isocitrate Dehydrogenase ◽  
Nicotinamide Adenine Dinucleotide ◽  
Substrate Concentration ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Nicotinamide Adenine ◽  
Ph Optimum ◽  
Effects Of Ph ◽  
Metabolic Significance

1. The effect of pH on the co-operative activation of the NAD-specific isocitrate dehydrogenase from pea mitochondria by isocitrate is shown. 2. The interlinked effects of pH on the affinity of the NAD-specific isocitrate dehydrogenase for isocitrate and the dependence of the pH optimum on the substrate concentration are presented. 3. A consideration of the conditions of pH and substrate concentration under which citrate activates the NAD-specific isocitrate dehydrogenase demonstrates similarities between the binding of isocitrate and citrate. 4. A comparison of the effects of citrate and pH on the gross structure of the enzyme is investigated by density-gradient centrifugation. 5. The kinetic interpretations of these results are briefly considered. 6. The metabolic significance of these studies is discussed.

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.

scholarly journals ISOLATION AND FRACTIONATION OF RAT BRAIN NUCLEI

1966 ◽  
Vol 30 (2) ◽  
pp. 405-415 ◽  
Author(s):  
H. Løvtrup-Rein ◽  
B. S. McEwen
Keyword(s):  
Rat Brain ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Brain Nuclei ◽  
Isolated Nuclei ◽  
Isolation Media

A method for isolating pure and unaltered nuclei from rat brain by means of differential centrifugation is described. The isolated nuclei are further separated into discrete fractions of neuronal, astrocytic, and glial nuclei, with a yield amounting to 20 to 25% of the DNA of the original homogenate. Both the morphology and size of the nuclei remained unchanged. Problems concerning the composition of the isolation media, the use of detergents, as well as those raised by density gradient centrifugation in sucrose, Ficoll, and Dextran are discussed. Some values for the density of each type of brain nuclei are suggested.

Uranyl Acetate and Rotary Shadowing to Increase the Contrast of Small Aggregated Virus Particles

1969 ◽  
Vol 27 ◽  
pp. 424-425
Author(s):  
Lee F. Ellis ◽  
Richard M. Van Frank ◽  
Walter J. Kleinschmidt
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Uranyl Acetate ◽  
Interferon Inducer ◽  
Virus Particles ◽  
Staining Methods ◽  
Rotary Shadowing

The extract from Penicillum stoliniferum, known as statolon, has been purified by density gradient centrifugation. These centrifuge fractions contained virus particles that are an interferon inducer in mice or in tissue culture. Highly purified preparations of these particles are difficult to enumerate by electron microscopy because of aggregation. Therefore a study of staining methods was undertaken.

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