Search results for "Differential centrifugation"
showing 10 items of 20 documents
Cercospora beticola Toxin Inhibits Vanadate-Sensitive H+ Transport in Corn Root Membrane Vesicles
1988
The effect of Cercospora beticola toxin on the transport of protons by vanadate-sensitive ATPase was studied with corn (Zea mays) root microsomal vesicles prepared by differential centrifugation, sedimentation through a sucrose cushion, and washing with Triton X-100 plus KBr. In these preparations, addition of ATP induced intravesicular H(+)-accumulation as evidenced by a rapid quenching of the fluorescence of 9-amino-6-chloro-2-methoxy acridine. This quenching was relatively unaffected by inhibitors of mitochondrial and tonoplast-type ATPases, but was strongly reduced by inhibitors of plasma membrane H(+)-ATPase. C. beticola toxin markedly inhibited ATP dependent H(+)-transport, and this e…
2019
Recent studies on extracellular RNA raised awareness that extracellular vesicles (EVs) isolated from cultured cells may co-purify RNAs derived from media supplements such as fetal bovine serum (FBS) confounding EV-associated RNA. Defined culture media supplemented with a range of nutrient components provide an alternative to FBS addition and allow EV-collection under full medium conditions avoiding starvation and cell stress during the collection period. However, the potential contribution of serum-free media supplements to EV-RNA contamination has remained elusive and has never been assessed. Here, we report that RNA isolated from EVs harvested from cells under serum-replacement conditions…
Multi-virion infectious units arise from free viral particles in an enveloped virus
2017
Many animal viruses are enveloped in a lipid bilayer uptaken from cellular membranes. Since viral surface proteins bind to these membranes to initiate infection, we hypothesized that free virions may also be capable of interacting with the envelopes of other virions extracellularly. Here, we demonstrate this hypothesis in the vesicular stomatitis virus (VSV), a prototypic negative-strand RNA virus composed by an internal ribonucleocapsid, a matrix protein, and an external envelope1. Using microscopy, dynamic light scattering, differential centrifugation, and flow cytometry, we show that free viral particles can spontaneously aggregate into multi-virion infectious units. We also show that, f…
Sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of Neurospora crassa.
1989
Marked differences in the pattern of sedimentation of cellular structures were observed after isopycnic centrifugation of crude cell-free preparations from the Neurospora crassa wall-less 'slime' variant and mycelial wild-type strain. Kinetic studies of particle sedimentation showed that the various types of subcellular components, as revealed by turbidity, UV absorption, polypeptide patterns, and chitin synthetase activity determinations, sediment independently of one another. An important feature was the finding that chitin synthetase from 'slime' peaked at a median specific gravity of 1.1201 +/- 0.0036, whereas that from wild-type strain sedimented at a higher buoyant density (specific g…
Identification and isolation of the primary aggregation factor from the cell membrane of the sponge Geodia cydonium
1985
The primary aggregation factor (pAF) of sponge cells is a glycoprotein that is firmly associated with the cell membrane. Polyspecific antibodies (anti-GM) prepared from sera raised against membranes of cells from the siliceous sponge Geodia cydonium were found to inhibit initial aggregation of homologous cells. The inhibition of aggregation, caused by anti-GM was neutralized by pAF. The pAF had been successfully solubilized and enriched by affinity chromatography, gel filtration and density gradient centrifugation, if checked by polyacrylamide gel electrophoresis in the presence of urea. The Mr of the native pAF was approximately 40 000 as estimated by gel filtration; under denaturing condi…
Quantification of bacterial subgroups in soil : comparison of DNA extracted directly from soil or from cells previously released by density gradient …
2001
All molecular analyses of soil bacterial diversity are based on the extraction of a representative fraction of cellular DNA. Methods of DNA extraction for this purpose are divided into two categories: those in which cells are lysed within the soil (direct extraction) and those in which cells are first removed from soil (cell extraction) and then lysed. The purpose of this study was to compare a method of direct extraction with a method in which cells were first separated from the soil matrix by Nycodenz gradient centrifugation in order to evaluate the effect of these different approaches on the analysis of the spectrum of diversity in a microbial community. We used a method based on polymer…
Separation of deoxyribonucleases (DNases) of normal human stratum corneum and psoriatic scales by micro-disc-electrophoresis.
1975
Normal stratum corneum and psoriatic scales were homogenized and a differential centrifugation was performed. The DNase activity of the individual fractions was investigated by micro-disc-electrophoresis. At pH 5 only in the 600 × g pellet and 105.000 × g supernatant of normal keratin DNase activity could be observed. However, all psoriatic fractions showed distinct enzyme activities. At pH 7.4 little psoriatic DNase activity could only be demonstrated in the 105.000 × g supernatant. Except from the 15.000 × g pellet all fractions of normal stratum corneum displayed marked activities. In addition the 105.000 × g supernatant showed two different DNase bands.
A new method for the in situ determination of phospholipids after thin-layer separation
1973
Abstract A very sensitive method has been devised for the in situ determination of phospholipids after thin-layer chromatographic separation, which enabled us to investigate the phospholipid content of erythrocytes and ATPase preparations. The phospholipid compositions of the ATPase preparations and of the erythrocytes are different, and the relative phospholipid compositions of the Na,K-ATPase preparations are also different, which indicates that Na,K- and Ca-ATPase seem to be different with regard to their phospholipid composition. An increase in temperature during the preparation procedure yields a Ca-ATPase preparation (II), which exhibits different kinetic properties and a different ph…
Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes
2012
Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.