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 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 Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

1970 ◽  
Vol 117 (1) ◽  
pp. 161-167 ◽  
Author(s):  
Keitaro Kato ◽  
Hiroyuki Ide ◽  
Tsuranobu Shirahama ◽  
William H. Fishman
Keyword(s):  
Endoplasmic Reticulum ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Mouse Kidney ◽  
Differential Centrifugation ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Glucuronidase Activity

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-14C]glucosamine or l-[U-14C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.

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 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.

scholarly journals Studies on the subcellular localization of human neutrophil alkaline phosphatase

1979 ◽  
Vol 36 (1) ◽  
pp. 401-412
Author(s):  
G.J. Rustin ◽  
P.D. Wilson ◽  
T.J. Peters
Keyword(s):  
Alkaline Phosphatase ◽  
Endoplasmic Reticulum ◽  
Density Gradient ◽  
Human Neutrophil ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Human Neutrophils ◽  
Gamma Glutamyl Transferase ◽  
Gamma Glutamyl ◽  
Glutamyl Transferase

The intracellular localization of alkaline phosphatase has been determined in human neutrophils with analytical subcellular fractionation by density gradient centrifugation and EM cytochemistry. Centrifugation on sucrose gradients containing 1 mM DETA and 5 units/ml of heparin showed that alkaline phosphatase was associated with a membranous component distinct from plasma membrane, mitochondria, specific granules and azurophil granules. There was no resolution from the endoplasmic reticulum. Density gradient centrifugation on a sucrose-imidazole-heparin gradient showed a clear resolution of the alkaline phosphatase-containing membranes from the Golgi and endoplasmic reticulum. Density gradient centrifugation of neutrophils that had been disrupted in the presenceof 0.12 mmol/l. digitonin clearly separated alkaline phosphatase-containing membranes from the endoplasmic reticulum. Part of the gamma-glutamyl transferase has a similar localization to that of alkaline phosphatase. EM cytochemistry of neutrophils, neutrophil homogenates and of the density gradient fractions identified alkaline phosphatase-containing granules as irregular-shaped, often tubular, structures. It is suggested that alkaline phosphatase and part of the gamma-glutamyl transferase activity are localized to a unique organelle in the human neutrophil.

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.

Changes in Hepatic Enzymes and Organelles in Alcoholic Liver Disease

1978 ◽  
Vol 55 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Carol A. Seymour ◽  
T. J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Liver Disease ◽  
Fatty Liver ◽  
Density Gradient ◽  
Alcoholic Liver Disease ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Marker Enzyme

1. Liver biopsy specimens obtained from patients with alcoholic liver disease of varying severity were assayed for lysosomal and microsomal enzyme activities, the results being compared with values previously obtained in control subjects. 2. Analytical subcellular fractionation by sucrose-density-gradient centrifugation was performed on extracts of the biopsies and the properties of the lysosomes, plasma membrane, biliary canaliculi and endoplasmic reticulum membranes were determined. Increased activities of plasma membrane marker enzymes, particularly γ-glutamyl transpeptidase believed to be localized to the biliary canalicular membrane, were demonstrated. These findings were most marked in alcoholic cirrhosis. The centrifugation studies revealed no abnormalities in the properties of these membranes. 3. Although the total activities of the endoplasmic reticulum marker enzyme neutral α-glucosidase were unaltered in alcoholic liver disease, centrifugation studies showed a decrease in the density distribution of the membrane-bound enzyme in cirrhosis indicating an increase in the proportion of smooth endoplasmic reticulum membranes. 4. Apart from a small decrease in activity of certain acid hydrolases in fatty liver and in cirrhosis the activities of the lysosomal enzymes were unaffected by alcoholic liver disease. 5. Measurements of lysosomal integrity and density-gradient-centrifugation studies revealed no significant abnormalities in the various patient groups apart from increased stability and reduced equilibrium density of certain lysosomes in fatty liver. It is concluded that lysosomal disruption is not implicated in the pathogenesis of alcoholic liver disease.

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 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.

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