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At least 19 recordsLinked to original sources
PubMed1975

Lysosomes of cardiac and skeletal muscle: resolution by zonal centrifugation.

The application of zonal centrifugation to the analysis of homogenates of cardiac and skeletal muscle permits selection of fractions that are enriched in markers for lysosomes, sarcolemma, sarcoplasmic reticulum, and mitochondria. The method of disruption of normal and pathological tissue alters significantly the distribution of total protein and peaks of enzymatic activity on the gradient. Total activities of cathepsin, N-acetyl-beta-glucosaminidase, beta-glucuronidase, and para-nitrophenylphosphatase are distributed at different concentrations of sucrose on the gradient. Beta-Glucuronidase appears to "mark" the sarcoplasmic reticulum, as well as lysosomes, of skeletal muscle, para-Nitrophenylphosphatase, a common marker of acid phosphatase of lysosomes, is enriched in those fractions of cardiac muscle containing the highest specific activity of ouabain-inhibited Na-K-ATPase. Thus, these two enzymes appear to have a localization in at least two separate organelles. On the other hand, these results may indicate the isolation of several "populations" of lysosomes that are associated constantly with distribution peaks of other organelles. In any event, attempts to correlate changes in structure of organelles of normal and pathological specimens of tissue with functional impairment, e.g., Ca2+ uptake, activity of Na-K-ATPase, etc., must include consideration of dual localization of enzymatic markers or cross contamination by populations of other organelles.

4-Nitrophenylphosphatase↗
PubMed1975

Immunological properties of Rickettsia rickettsii purified by zonal centrifugation.

The properties of Rickettsia rickettsii purified from infected chicken yolk sacs or mouse L cell cultures by sucrose density gradient centrifugation in a zonal rotor were examined in various ways. Rickettsiae derived from both L cells and yolk sacs had similar compositions: about 12% nitrogen, 1.5% phosphorus, 5% carbohydrate, and 30% fatty acids. On a dry-weight basis, purified rickettsiae were at least 2,000 times as effective as a commercial spotted fever vaccine in protecting guinea pigs against infection with spotted fever rickettsiae and mice against death from toxin of R. rickettsii. Gradient-purified rickettsiae (0.6 mug) induced a serological response in guinea pigs of the same magnitude as that stimulated by 1,600 mug of the commercial vaccine. Gradient-purified rickettsiae had little group reactivity in complement fixation tests but became anti-complementary upon storage. Microagglutination and hemagglutination tests with the purified antigen gave promise of usefulness in diagnosis of spotted fever. These results suggest that zonal centrifugation may be a valuable procedure for the preparation of R. rickettsii vaccine and diagnostic reagent.

Animals↗
PubMed1979

Distribution of PNS myelin proteins and membrane enzymes in fractions isolated by continuous gradient zonal centrifugation.

Myelin was purified from adult rabbit sciatic nerve by two procedures: discontinuous gradient centrifugation and continuous gradient zonal centrifugation. Two fractions were obtained from the discontinuous gradient. The fraction floating on 0.32 M sucrose and the fraction recovered from the 0.32/0.85 M sucrose interface showed typical myelin membranes by electron microscopy and typical myelin proteins by gel electrophoresis. The specific activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased from the top to the bottom of the discontinuous gradient. The myelin separated by zonal centrifugation on a continuous sucrose gradient showed three distinct peaks (on monitoring optical density) at 0.10, 0.30 and 0.57 M sucrose. The latter peak yielded 92% of the material applied. The two minor peaks of low density exhibited high CNP and acetylcholinesterase (AChE) activities but the specific activity of both enzymes increased markedly at the heavy end of the gradient. The zonal fractions showed typical myelin proteins in all fractions by polyacrylamide gel electrophoresis but with important quantitative differences. These results indicate that PNS myelin shows significant heterogeneity.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1979

High-resolution flow-zonal centrifuge system.

A modified CF-32 Beckman flow centrifuge rotor has been developed that provides a long sedimentation path length with high gravitational force at the gradient sample interface. The modified rotor exhibits excellent separative capability and extraction efficiency when applied to purification of human influenza B and herpes simplex viruses.

Centrifugation, Zonal↗
PubMedDate not supplied

Zonal-centrifuged human diploid cell rabies vaccine.

In order to reduce the risk of allergic reactions, a purified human diploid cell culture rabies vaccine was developed. Pitman-Moore strain of rabies was propagated in WI-38 or MRC-5 cells. Both cell strains were found to be equally suitable for pro agation of rabies virus. The virus was purified and concentrated by continuous-flow sucrose density gradient ultracentrifugation. Virus peak fractions were diluted with a stabilized and then inactivated with BPL and freeze-dried. According to the standard NIH test for potency, the vaccine proved to be very potent and stable. The results of initial clinical applications of the vaccine will be presented.

Antibody Formation↗
PubMed1976

Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae.

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, beta-fructofuranosidase, alpha-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.

Acid Phosphatase↗
PubMed1978

Isolation of enzymatically homogeneous populations of human lymphocytes, monocytes, and granulocytes by zonal centrifugation.

Human monocytes, lymphocytes, granulocytes, red cells, and platelets were completely separated from each other by zonal centrifugation on linear sucrose density gradient. The monocytes contained only one tenth the amount of myeloperoxidase, one half the amount of lysozyme, one half the amount of acid ,hosphatase, and one half the amount of beta-glucuronidase found in granulocytes; the monocytes contained no alkaline phosphatase or neutral protease. The lymphocyte fraction contained only acid phosphatase and beta-glucuronidase in amounts one half as much as in the monocytes. Fluctuations in enzyme levels of monocytes and granulocytes were noted following infection. In vitro, the isolated monocytes transformed into macrophages. The results suggest that lymphocytes, monocytes, and granulocytes may be linked biochemically in a differentiation sequence through sets of commonly shared enzymes as well as by groups of enzymes specific for each divergent cell line.

Acid Phosphatase↗
PubMed1979

A new gradient former and a simplified procedure for zonal centrifugation of immune complexes.

A new gradient former for production of optimized sucrose gradients for B-XIV zonal rotors is described. Optimized gradients are gradients with an initial shelf to provide capacity and a shape that ensures isokinetic sedimentation conditions. By exhaustive computer calculations it has been found that optimized gradients can be produced by a very simple two-compartment gradient former. The design is given. Moreover, graphs for choice of gradient strength and run duration for preparative and analytical runs of samples containing soluble immune complexes are calculated. The new gradient former allows the use of a very simple procedure for zonal centrifugation.

Antigen-Antibody Complex↗
PubMed1975

The large-scale isolation of renin-containing granules from rabbit renal cortex by zonal centrifugation.

A new method for the isolation of renin-containing granules from rabbit renal cortex by sequential two-dimensional centrifugation is presented. A mitochondrial fraction produced by preparative differential centrifugation was subjected to isopycnic centrifugation on combined discontinuous and linear density gradients in the same SZ-14 reorienting gradient zonal centrifuge rotor. Renin activity, as assayed by radioimmunoassay for angiotensin I, was localized in a region of the density gradient with a corresponding density of 1.196 Gm. per cubic centimeter or 1.53 M sucrose. The renin-containing granules were enriched 5.0-fold over whole homogenate levels, while peroxisomes were enriched 17.4-fold, lysosomes 23.4-fold, and mitochondria 4.3-fold. Electron microscopic examination of the fraction with highest renin activity showed a granular fraction almost free of contamination from other subcellular material. The data supports the view that renin-containing granules are a distinct subcellular particle. This new method for the large-scale isolation of renin-containing granules makes it possible to obtain the quantity of material necessary to study the release renin at the subcellular level and permit the further biochemical purification and characterization of the enzyme.

Acid Phosphatase↗
PubMed1978

Zonal centrifuge purification of human rabies vaccine obtained on bovine fetal kidney cells. Biological results.

An inactivated human rabies vaccine prepared on bovine fetal kidney cells is concentrated and purified by zonal centrifugation. The peak of rabies particles is monitored by hemagglutination. Immunogenicity of the purified particles was evaluated by titration of specific antibodies from vaccinated animals. Protective activity of the vaccine was assayed on guinea pigs challenged with street rabies. Biological results were compared with those obtained with other tissue culture vaccines.

Animals↗
PubMed1976

Fractionation by differential and zonal centrifugation of spheroplasts prepared from a glucose-repressed fission yeast Schizosaccharomyces pombe 972h-.

A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a3) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgCl2 or 0-4 mM-EDTA. Mitochondria were apparently smaller and of lower buoyant density in gradients containing EDTA. The bulk of the total units of malate dehydrogenase and NADH; cytochrome c oxidoreductase sedimented with mitochondria, whereas NADPH: cytochrome c oxidoreductase was located in fractions containing no mitochondria. The distributions of mitochondrial enzymes were heterogeneous in populations of mitochondria separated on the basis of size or density. The possible origins of mitochondrial heterogeneity in extracts of S. pombe are discussed with special reference to changes in the enzyme activities of cells during the cell cycle.

4-Nitrophenylphosphatase↗