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At least 19 recordsLinked to original sources
PubMed1988

Phosphatidylinositol kinase, phosphatidylinositol-4-phosphate kinase and diacylglycerol kinase activities in rat brain subcellular fractions.

Subcellular fractions isolated and purified from rat brain cerebral cortices were assayed for phosphatidylinositol (PI-), phosphatidylinositol-4-phosphate (PIP-), and diacylglycerol (DG-) kinase activities in the presence of endogenous or exogenously added lipid substrates and [gamma-32P]ATP. Measurable amounts of all three kinase activities were observed in each subcellular fraction, including the cytosol. However, their subcellular profiles were uniquely distinct. In the absence of exogenous lipid substrates, PI-kinase specific activity was greatest in the microsomal and non-synaptic plasma membrane fractions (150-200 pmol/min per mg protein), whereas PIP-kinase was predominantly active in the synaptosomal fraction (136 pmol/min per mg protein). Based on percentage of total protein, total recovered PI-kinase activity was most abundant in the cytosolic, synaptosomal, microsomal and mitochondrial fractions (4-11 nmol/min). With the exception of the microsomal fraction, a similar profile was observed for PIP-kinase activity when assayed in the presence of exogenous PIP (4 nmol/20 mg protein in a final assay volume of 0.1 ml). Exogenous PIP (4 nmol/20 mg protein) inhibited PI-kinase activity in most fractions by 40-70%, while enhancing PIP-kinase activity. PI- and PIP-kinase activities were observed in the cytosolic fraction when assayed in the presence of exogenously added PI or PIP, respectively, but not in heat-inactivated membranes containing these substrates. When subcellular fractions were assayed for DG-kinase activity using heat-inactivated DG-enriched membranes as substrate, DG-kinase specific activity was predominantly present in in the cytosol. However, incubation of subcellular fractions in the presence of deoxycholate resulted in a striking enhancement of DG-kinase activities in all membrane fractions. These findings demonstrate a bimodal distribution between particulate and soluble fractions of all three lipid kinases, with each exhibiting its own unique subcellular topography. The preferential expression of PIP-kinase specific activity in the synaptic membranes is suggestive of the involvement of PIP2 in synaptic function, while the expression of PI-kinase specific activity in the microsomal fraction suggests additional, yet unknown, functions for PIP in these membranes.

1-Phosphatidylinositol 4-Kinase↗
PubMed1974

Immunogenic properties of rat hepatoma subcellular fractions.

Subcellular fractions from an aminoazo dye induced rat hepatoma (D23) were examined for their ability to evoke rejection responses in syngeneic hosts to transplanted tumour cells and to induce the production of humoral antibody. Membrane fractions isolated by zonal centrifugation and displaying an increased activity of tumour specific antigen (Price and Baldwin, 1974), as well as crude membrane fractions and purified tumour cell ghosts, all elicited tumour specific antibody demonstrable by membrane immunofluorescence staining of viable hepatoma D23 cells. Tumour cell nuclei or soluble cytoplasmic protein were, however, lacking in this capacity. Resistance to tumour cell challenge was not observed in rats treated with any of the hepatoma D23 subcellular fractions administered by various routes either alone or in admixture with bacterial adjuvants. These findings are relevant to current views that tumour immunity may be more optimally achieved by inoculation of intact (viable or attenuated) tumour cells.

Animals↗
PubMed1975

P-Chloro-N-methylaniline demethylation by rat kidney subcellular fractions.

Subcellular fractions prepared from kidney homogenates catalyzed the demethylation of p-chloro-N-methylaniline (PCMA). The activity of the renal preparations were forty-one percent of the liver 9,000 xg supernatant fraction activity. A differential susceptibility to the addition of carbon monoxide, p-chloromercuribenzoate or the omission of NADPH and magnesium characterized the two preparations. Comparison of the Lineweaver-Burk plots of the PCMA demthylation activities indicated that the maximal velocities and the apparent Michaelis constants (Km) of the two preparations differed. The apparent Km of the renal enzyme was approximately two fold greater than that of the liver. The PCMA demethylation activity of renal preparations is distributed between the cytosol and microsomal subcellular fractions. The results suggest the presence of tissue-related differences between renal and hepatic enzymes which catalyze the demethylation of PCMA.

Aniline Compounds↗
PubMed1977

Studies on intestinal adenosine triphosphatases. II. Stabilitiies in different rat subcellular fractions.

Subcellular fraction (brush border, mitochondria, microsomes and plasma membranes) are isolated from the rat intestinal epithelial cells. A comparison was made between the effect of cold storage, freeze-thawing, heating and of some chemicals (DMSO, DTT, glycerol, sucrose) on the stability of Mg2+ and (Na+-K+) dependent ATPases in these fractions in order to determine possible difference linked to the localization in the enterocyte. Enzymatic activities were found more stable at -20 degrees C than at +4 degrees C. Microsomal (Na+-K+)-ATPase increased in activity until the 8th day, then declined. Brush border (Na+-K+)-ATPase was the least resistant of all fractions. For Mg2+-ATPase, that from mitochondria was that had lost much more activity (84%) in 15 days at +4 degrees C. With freeze-thawing there was a comparable decrease in all activities (20-35%). by heating between 35 and 60 degrees C, Mg2+-ATPase was shown to be more heat resistant than (Na+-K+)-ATPase. The addition of some stabilizing chemicals (DMSO, glycerol, sucrose) improved the heat stability of the two enzymes: better results were obtained with glycerol for Mg2+-ATPase and sucrose for (Na+-K+)-ATPase. These differences might be due to the compositon in membraine lipids or to the nature of the enzymes studied.

Adenosine Triphosphatases↗
PubMed1986

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes.

A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A+ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.

Aminopeptidases↗
PubMed1988

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: viability of subcellular fractions.

The metabolic and functional viability of synaptosomes was examined in 5 subcellular fractions obtained after centrifugation of an S1 fraction from rat cerebral cortex on a discontinuous Percoll gradient (Brain Research, this volume, 1987). Fraction 4 was the most enriched for viable synaptosomes since, although it accounted for only 11.8% of the total protein recovered from the gradient, this fraction contained 23.7% of the basal synapsin I phosphorylation activity, the greatest degree of depolarisation-stimulated increase in synapsin I phosphorylation, 36.1% of the total [3H]noradrenaline uptake capacity and 46.9% of the total [3H]noradrenaline release capacity. Noradrenaline release from fraction 4 was consistent with a neuronal mechanism as it was increased with increasing K+ concentrations and was dependent on calcium. Fractions 1 and 2 contained few viable synaptosomes as judged by their capacity for noradrenaline uptake and release, yet these fractions accounted for some 62.6% of the endogenous content of noradrenaline. In part their lack of viability was due to a low content of intrasynaptosomal mitochondria, while their high content of endogenous noradrenaline was due to the presence of synaptic vesicles released from damaged nerve terminals. The synaptosomes in fraction 3 were metabolically and functionally viable, but their capacity for uptake and release of noradrenaline was lower than for fraction 4. The synaptosomes in fraction 5 showed only a small depolarisation-stimulated release of noradrenaline, suggesting a lack of viability. Part of the capacity for uptake of [3H]noradrenaline into fraction 5 was attributed to the presence of extrasynaptosomal mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors. III. Stimulation of syngeneic antitumor responses by subcellular fractions and molecular species of Moloney virus-induced tumors in CBA and A mice.

YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation. Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector: target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells. Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells. The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10(8) intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.

Animals↗
PubMed1980

Separation of the gut hormone endocrine-cell storage granules of human jejunum using analytical subcellular fractionation.

Analytical subcellular fractionation techniques in combination with specific radioimmunoassays have been used to investigate the properties of the hormone-containing granules in human jejunal biopsy specimens. After gentle homogenisation, for all hormones except VIP, over 90% of the immunoreactivity was released in intact granules into the post-nuclear supernatant. Approximately 50% of the VIP immunoreactivity sedimented with the low speed pellet, probably reflecting the presence of this peptide in nerve fibres. The following hormone granules (equilibrium densities between parentheses) were separated by isopycnic centrifugation on the sucrose density gradients: VIP (1 x 17), motilin (1 x 20), and secretin (1 x 24). Gastrin and GIP granules were not resolved, both having the same modal density of 1 x 22 g/cm3. This combination of procedures thus provides a new quantitative technique for studying the properties of the gastrointestinal hormone-containing granules, and should therefore be of value in investigating pathophysiological problems in a variety of gastrointestinal diseases.

Cell Fractionation↗
PubMed1984

Kilham rat virus DNA replication in subcellular fractions.

A subcellular system actively replicating Kilham rat virus DNA in vitro was developed. Cellular lysates and isolated nuclei from infected cells showed an amplification of replicative forms in vitro. Solubilized replicative complexes, either partly purified or in the form of a crude extract, were able to synthesize replicative forms and single-stranded DNA. DNA polymerase alpha played a major role in Kilham rat virus DNA synthesis in vivo and in vitro. Furthermore, a factor present in the cytosol of infected cells increased the polymerizing activity of viral replicative complexes.

Animals↗
PubMed2004

A novel protocol for the subcellular fractionation of C3A hepatoma cells using sucrose density gradient centrifugation.

In this paper, we describe a method to obtain a relatively pure mitochondrial and microsomal fractions by subcellular fractionation of human hepatoma cell line C3A using sucrose as the hypoosmotic medium. The cells were subjected to osmotic stress with sucrose and homogenized. Osmolarity was then restored to the cells and the organelles were separated by density gradient centrifugation. The protein profiles were examined by SDS-PAGE and the purity was analysed by marker enzymes and Western blotting. Our results indicate a good separation of mitochondrial and microsomal fractions from human hepatoma C3A cells.

Blotting, Western↗
PubMed1983

Immunoreactive prolactin in subcellular fractions from bovine mammary tissue.

Mammary tissue from lactating Holsteins at slaughter was homogenized and fractionated into subcellular compartments. Bovine prolactin was quantified in each subcellular fraction and in preslaughter milk by radioimmunoassay procedures validated for this purpose. Prolactin concentrations were ng prolactin/mg protein for all samples. Immunoreactive prolactin was in rough endoplasmic reticulum (24.8 ng/mg), Golgi apparatus (18.8 ng/mg), and secretory vesicle (10.2 ng/mg). All of these concentrations exceeded that in samples of milk (2.7 ng/mg). Nuclear fractions contained a material that reacted in the prolactin radioimmunoassay, but criteria for valid quantification could not be satisfied. These results confirm prolactin inside bovine mammary cells, and they are consistent with an intracellular pathway for transfer of prolactin from plasma into milk.

Animals↗
PubMed1983

Membrane lipids of hepatic tissue. I. Neutral lipids from subcellular fractions of liver and hepatoma 7288CTC.

Enriched subcellular fractions of nuclei, mitochondria, endoplasmic reticulum (ER), plasma membrane, and cytosol were prepared from liver and hepatoma 7288CTC taken from male inbred BUF rats. Purity was established by marker enzyme activities and distribution of DNA, RNA, sialic acids, total phospholipids, and cholesterol. The subcellular fractions of hepatoma differed from those of liver: 5'-Nucleotidase activity was elevated in ER and mitochondria, cytosol RNA was increased, and cholesterol was elevated in all hepatoma subcellular fractions. Neutral lipid classes of hepatoma subcellular fractions differed quantitatively from those of liver: Hepatoma nuclei and mitochondria contained elevated levels of free fatty acids (FFA) and triglycerides (TG). Generally, the fatty acid profiles of FFA, TG, and sterol esters from hepatoma subcellular fractions were more uniform and showed less organelle specificity than did liver. Hepatoma FFA and TG contained lower percentages of palmitate and higher percentages of stearate in all subcellular fractions than did liver. The sterol esters from most hepatoma subcellular fractions compared to those from liver were characterized by much higher levels of long-chain fatty acids of 20 carbon atoms or longer. The oleate-to-vaccenate ratio in FFA of liver subcellular fractions exhibited some specificity, but not that of hepatoma subcellular fractions. The oleate-to-vaccenate ratio in the acyl chains of liver and hepatoma TG did not reveal organelle specificity.

Animals↗
PubMed1975

Effects of thrombin on washed, human platelets: changes in the subcellular fractions.

Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecysulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 plus or minus 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control (mol. wt 147 000 plus or minus 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37 degrees C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 plus or minus 3500) had disappeared. The distribution of protein and beta-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (greater than 77%). The granule fractions, D and E of the control contained 3.0% plus or minus 0.8% and 6.4% plus or minus 1.3% of the total amount of beta-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% plus or minus 1.0% of total while fraction D was lacking.

Animals↗
PubMed1988

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions.

A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.

Animals↗