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PubMed1985

Acid and alkaline phosphatase activities in homogenates and subcellular fractions of human brain tumors.

This study is a quantitative analysis of acid and alkaline phosphatase activity in human brain tumor homogenates and subcellular fractions, in parallel with normal brain tissue. Glioblastoma multiforme, meningioma, astrocytoma and normal tissue samples were separated by ultracentrifugation into five subcellular fractions: nuclei (N), mitochondria (M), microsomes (P), ribosomes (R) and supernatant (S). These two phosphatases showed significant increase in astrocytoma and meningioma tissue homogenates, compared with normal brain tissue. Alkaline phosphatase levels were determined to increase significantly in glioblastoma multiforme tissue homogenates as compared with normals, while those of acid phosphatase were observed to decrease. The results of this investigation also indicate that the subcellular distributions of acid and alkaline phosphatase show differences in the different tumor types. This observation is evidence against metabolic uniformity in tumoral tissue.

Acid Phosphatase↗
PubMed1976

Preparation of subcellular fractions from rat liver: comparison of the Polytron with the Dounce homogenizer.

The PolytronR and Dounce homogenizers have been evaluated for preparation of homogenates of rat liver prior to isolation of subcellular fractions by differential centrifugation. Marker enzymes used to evaluate the subcellular fractions included cytochrome oxidase, monoamine oxidase, D-amino acid oxidase, acid phosphatase, glucose-6-phosphatase, ethyl morphine demethylase, and lactate dehydrogenase. No significant difference in the distribution of enzymes (percent recovery or specific activity) was observed between the two methods of homogenization. In addition, there were no significant differences in the ultrastructural appearances and respiratory control ratios of the mitochondrial fractions prepared by the two methods of homogenization.

Animals↗
PubMed1977

Comparative studies on the influence of some radioprotectors on the postirradiation hydrolytic function of subcellular fractions of pancreas.

Comparative studies on the influence of the two radioprotectors Fenchlorfos and cysteamine on the hydroproteolytic activity of pancreatic subcellular fractions of rats were carried out. The animals were irradiated with the single dose of 800 R of X-rays. Biochemical estimation of lipase, beta-glucuronidase, acid phosphatase and catheptic (caseinolytic) activity were done 24 hours after exposition. The two radioprotective substances have a lot of common in their action against hydrolytic activity. They induce the decrease of some enzymes of subcellular fractions we checked both in control as well as in irradiated groups. The ambivalent influence of Trichlorfon on the lipase activity in granules and supernatant fraction seems to be of interest.

Animals↗
PubMed1984

Distribution of tin in brain subcellular fractions following the administration of trimethyl tin and triethyl tin to the rat.

The time course of tin distribution in homogenates and subcellular fractions of rat brain was determined following the acute administration of trimethyl tin (TMT) and triethyl tin (TET) to the rat. Exposure to TMT resulted in lower concentrations but greater persistence of tin in subcellular fractions compared to exposure to TET. A delayed accumulation of tin in the mitochondrial fraction was observed following the administration of TMT but not TET. Analysis of total protein and mitochondrial markers did not reveal differences between the compositions of mitochondrial fractions prepared from control and TMT-treated subjects.

Animals↗
PubMed1979

Taurine effects on 45Ca2+ transport in retinal subcellular fractions.

The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1--5 min in a Krebs--bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.

Amino Acids↗
PubMed1985

Identification of artifactual cathepsin D activity in cardiac subcellular fractions related to formation of an iron-EDTA complex.

The conventional assay procedure for cathepsin D (E.C.3.4.23.5) activity in tissue homogenates and subcellular fractions requires incubation with hemoglobin as substrate. Cathepsin D (CD) activity is calculated by determining the increase in absorbance at 280 nm after precipitation of all proteins with trichloroacetic acid. This increase in absorbance (presumably due to the release of tyrosine residues from hemoglobin) is converted to arbitrary CD activity units. Homogenization and fractionation of cardiac tissue frequently requires that ethylenediamine tetraacetic acid (EDTA) be included in the homogenization medium. We have observed that subcellular fractions of cardiac tissue prepared in the presence of EDTA demonstrate residual CD activity despite either quantitative removal of all CD protein by immunoprecipitation or complete inhibition of CD by pepstatin. The present study demonstrates that this 'apparent' CD activity (residual increase in absorbance at 280 nm) is due to the formation of an Fe-EDTA complex which absorbs at 280 nm. Data are presented which demonstrates that the EDTA of the medium complexes with non-heme iron which contaminates commercially available hemoglobin. A method for preparing hemoglobin free of contaminant non-heme iron is described for use in studies of CD metabolism when EDTA is present in the homogenization buffer.

Animals↗
PubMed1976

Calcium accumulation and enzymatic activities of subcellular fractions from aortas and ventricles of genetically hypertensive rats.

Subcellular fractions were obtained from aortas and ventricles of 6-month-old spontaneously hypertensive and normotensive Wistar rats by the use of differential and sucrose density gradient centrifugation. These preparations were studied to determine what alterations in calcium accumulation and enzymatic activities might be associated with hypertension. The total amount of calcium accumulation (in the presence of ATP and 17 muM free calcium) by the plasma membrane-enriched fraction from hypertensive rat aortas significantly less than that from normotensive rats (11.3 +/- 0.4 vs 16.2 +/- 1.6 mumol of calcium/g of protein, n = 8). In contrast the specific activities of the plasma membrane marker enzymes, 5'-nucleotidase and phosphodiesterase I, were 80% and 40% greater, respectively, in the hypertensive than in the normotensive fractions. On the other hand, various fractions from ventricles of the two types of rats were generally similar in enzyme activities and calcium accumulation. The decreased rate of relaxation of aortas from spontaneously hypertensive rats may be caused by the decreased rate of calcium transport demonstrated in this study.

Animals↗
PubMed1975

Distribution and properties of cholinesterases in subcellular fractions from rat jejunum and heart tissue.

Homogenates of rat jejunum and heart tissue in 0.3 M sucrose have been separated into different subcellular fractions by centrifugation. The distribution of cholinesterases in these two tissues is different. Acetylthiocholine (Ac), propionylthiocholine (Pc), and butyrylthiocholine (Bc) iodides were used as substrates. Using 1 mmol/1 Ac in the presence of hexafluorenium, a local anaesthetic drug, inhibition percentages of these subcellular cholinesterases were found to be different. The differences in the effect of hexafluorenium were most marked at a concentration of 5 mumol/1. Four electrophoretically distinct subcomponents of cholinesterase have been identified in different subcellular fractions from both jejunum and heart tissues. The electrophoretic profile, as determined by polyacrylamide disc electrophoresis, of different subcellular cholinesterases in these two tissues also appears to be different.

Acetylthiocholine↗
PubMed1973

Qualitative and quantitative morphological changes in subcellular fractions from mouse cerebral cortex during postnatal development.

Discontinuous Ficoll-sucrose gradients were used to prepare subcellular fractions from mouse cerebral cortex at various stages of postnatal development. Representative samples of each subcellular fraction were obtained by sedimentation in an analytical ultracentrifuge and each fraction was examined quantitatively and qualitatively by electron microscopy. The amount of synaptosomal material was determined for each fraction on the basis of volume percentage, obtained from a series of contiguous micrographs, to circumvent any sampling error. This allowed an accurate appraisal of synaptosomal distribution during neural development and a direct comparison of the Ficoll-sucrose gradient fractions to the original crude mitochondrial preparations. The distribution of synaptosomal material was shown to be quantitatively altered during neural development, and maturation-dependent changes, at a qualitative level, were described. In addition, the relationship between neural maturation and the relative proportion and distribution of subcellular particles which contain processes tentatively identified as growth cones were characterized.

Animals↗
PubMed2006

The subcellular fractionation properties and function of insulin receptor substrate-1 (IRS-1) are independent of cytoskeletal integrity.

Efficient insulin action requires spatial and temporal coordination of signaling cascades. The prototypical insulin receptor substrate, IRS-1 plays a central role in insulin signaling. By subcellular fractionation IRS-1 is enriched in a particulate fraction, termed the high speed pellet (HSP), and its redistribution from this fraction is associated with signal attenuation and insulin resistance. Anecdotal evidence suggests the cytoskeleton may underpin the localization of IRS-1 to the HSP. In the present study we have taken a systematic approach to examine whether the cytoskeleton contributes to the subcellular fractionation properties and function of IRS-1. By standard microscopy or immunoprecipitation we were unable to detect evidence to support a specific interaction between IRS-1 and the major cytoskeletal components actin (microfilaments), vimentin (intermediate filaments), and tubulin (microtubules) in 3T3-L1 adipocytes or in CHO.IR.IRS-1 cells. Pharmacological disruption of microfilaments and microtubules, individually or in combination, was without effect on the subcellular distribution of IRS-1 or insulin-stimulated tyrosine phosphorylation in either cell type. Phosphorylation of Akt was modestly reduced (20-35%) in 3T3-L1 adipocytes but not in CHO.IR.IRS-1 cells. In cells lacking intermediate filaments (Vim(-/-)) IRS-1 expression, distribution and insulin-stimulated phosphorylation appeared normal. Even after depolymerisation of microfilaments and microtubules, insulin-stimulated phosphorylation of IRS-1 and Akt were maintained in Vim(-/-) cells. Taken together these data indicate that the characteristic subcellular fractionation properties and function of IRS-1 are unlikely to be mediated by cytoskeletal networks and that proximal insulin signaling does not require an intact cytoskeleton.

Actin Cytoskeleton↗
PubMed1989

Proteins of muscle subcellular fractions in Duchenne progressive muscular dystrophy stained with "stains-all" cationic carbocyanine dye and with Coomassie Blue.

The protein compositions of subcellular fractions of muscle obtained from 17 Duchenne dystrophy patients, 15 disease controls (10 different primary myopathies, 5 spinal muscular atrophy patients), and 10 normals were examined by polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue and with "Stains-all," which stains calcium-binding proteins, sialic acid-rich glycoproteins, and phosphoproteins. In muscle membrane fractions of Duchenne dystrophy patients there was a marked reduction in the concentrations of calsequestrin and a 39 kDa protein that stained blue with "Stains-all." There were changes in the proteins of all subcellular fractions of Duchenne's patients; some of these changes appear to be specific for Duchenne dystrophy (DD). There was no apparent correlation between the protein changes observed on acrylamide gels and the age of the patients, the duration of the disease, the degree of disability, or activity of creatine kinase. A decreased level of calsequestrin in DD sarcoplasmic reticulum may contribute to an increased level of free calcium seen in muscle from these patients.

Calcium-Binding Proteins↗
PubMed1982

Labeling of poly(A) associated RNA in synaptosomes and the other subcellular fractions of rat cerebral cortex in basal conditions and during training.

The labeling of total and poly(A) associated RNA from the subcellular fractions of cerebral cortex was studied after a 1-hour 3H-uridine pulse in control and trained rats. In the control animals, the fraction of newly synthesized brain cortex RNA which is found in synaptosomes is the minimal one. However, in synaptosomes the proportion of poly(A)+ RNA within newly synthesized RNA is maximal. Training of the rats does not influence total incorporation of label into RNA in the brain cortex and the distribution of newly synthesized RNA within the subcellular fractions. The proportions of poly(A)+ RNA within total labeled RNA in the various fractions are also unaffected.

Animals↗
PubMed1987

Very long chain fatty acid beta-oxidation by subcellular fractions of normal and Zellweger syndrome skin fibroblasts.

Very long chain fatty acid (VLCFA) beta-oxidation was compared in homogenates and subcellular fractions of cultured skin fibroblasts from normal individuals and from Zellweger patients who show greatly reduced numbers of peroxisomes in their tissues. beta-Oxidation of lignoceric (C24:0) acid was greatly reduced compared to controls in the homogenates and the subcellular fractions of Zellweger fibroblasts. The specific activity of C24:0 acid beta-oxidation was highest in the crude peroxisomal pellets of control fibroblasts. Fractionation of the crude mitochondrial and the crude peroxisomal pellets on Percoll density gradients revealed that the C24:0 acid oxidation was carried out entirely by peroxisomes, and the peroxisomal beta-oxidation activity was missing in Zellweger fibroblasts. In contrast to the beta-oxidation of C24:0 acid, the beta-oxidation of C24:0 CoA was observed in both mitochondria and peroxisomes. We postulate that a very long chain fatty acyl CoA (VLCFA CoA) synthetase, which is different from long chain fatty acyl CoA synthetase, is required for the effective conversion of C24:0 acid to C24:0 CoA. The VLCFA CoA synthetase appears to be absent from the mitochondrial membrane but present in the peroxisomal membrane.

Abnormalities, Multiple↗
PubMed1976

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro.

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.

Adenosine Triphosphate↗
PubMed1989

Dihydroxyacetone phosphate acyltransferase and alkyldihydroxyacetone phosphate synthase activities in rat liver subcellular fractions and human skin fibroblasts.

Dihydroxyacetone phosphate acyltransferase (DHAP-AT) and alkyldihydroxyacetone phosphate synthase (DHAP-synthase) activities were examined in subcellular fractions of rat liver. The results indicate that at least 80% of DHAP-AT (assays carried out at pH 5.4) activity in rat liver is in peroxisomes, and the remaining activity is mitochondrial. In contrast to DHAP-AT, DHAP-synthase was detected in all subcellular fractions analyzed but the activity in peroxisomes was 208-fold and 42-fold greater compared to mitochondria and microsomes, respectively. We estimate that at least 70% of the DHAP-synthase activity in rat liver is in peroxisomes. DHAP-AT and DHAP-synthase activities were also examined in homogenates of skin fibroblasts from patients with inherited defects in peroxisomal structure and/or function. Both the enzyme activities were deficient in Zellweger syndrome whereas the activities were only partially deficient in infantile Refsum's disease. Greater reduction in DHAP-synthase activity, but only a partial reduction in DHAP-AT activity was observed in rhizomelic chondrodysplasia punctata. However, both DHAP-AT and DHAP-synthase activities were either normal or near normal in Refsum's disease or X-linked adrenoleukodystrophy. The results reported suggest that various peroxisomal disease states can be identified based on DHAP-AT and DHAP-synthase activities in skin fibroblasts of patients.

Acyltransferases↗
PubMedDate not supplied

[Ratio and composition of the plasmalogen and diacylated forms of phospholipids in subcellular fractions of the avian brain].

Studies have been made on the specific content of plasmalogen and diacylated forms of phosphatidylethanolamine and phosphatidylcholine in subcellular fractions (myelin, nuclei, microsomes, mitochondria, synaptosomes) from the brain of pigeons, as well as in the myelin fraction from the brain of the crow Corvus cornix and the hawk Accipiter gentelis. Fatty acid composition and fatty aldehyde composition of these two main phospholipids of the brain were studied in the subcellular fractions obtained. It was shown that plasmalogen forms of phospholipids are localized in birds mainly in the myelin fraction which exhibits the highest plasmalogen concentration as compared to the same fraction of all the vertebrates investigated. With respect to fatty acid and fatty aldehyde composition, as well as to the degree of their unsaturation, myelin plasmalogens from birds are similar to those from other cold-blooded and warm-blooded animals. This fact indicates that high relative content of plasmalogens together with their high unsaturation account for normal functional activity of myelin membranes in all vertebrates.

Aldehydes↗
PubMed1986

Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof.

In the present study we investigated the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. The O-methylation of 2-hydroxyoestradiol (2OHE2) and 2-hydroxyoestriol (2OHE3) displayed saturation kinetics in the intact tissue. The apparent Km and Vmax values for the O-methylation of 2OHE2 were determined to be 0.91 mumol/l and 104 pmol g-1 min-1, respectively, when 2OHE2 was used as substrate; and 1.14 mumol/l and 188 pmol g-1 min-1 when 2OHE3 was used as substrate. The inhibitors of the extraneuronal uptake process (viz; phenoxybenzamine 33 mumol/l; normetanephrine, 46 mumol/l; and deoxycorticosterone acetate 27 mumol/l) failed to inhibit the O-methylation of either 2OHE2 (3.4 mumol/l) or 2OHE3 (3.4 mumol/l) in intact segments of the rabbit thoracic aorta. (-)-Isoprenaline (40 mumol/l) abolished the O-methylation of 2OHE2 (3.4 mumol/l) and markedly reduced that of 2OHE3 (3.4 mumol/l). Pretreatment of tissues with phenoxybenzamine (33 mumol/l) partially restored the O-methylation of 2OHE2 and 2OHE3 in the presence of (-)-isoprenaline (40 mumol/l). The O-methylation of 2OHE2 (5 mumol/l) was significantly reduced in segments of aorta in which the endothelium was removed. The latter reduction could not be attributed to damage to components of the vessel media. The O-methylation of 2OHE2 and (-)-isoprenaline by subcellular fractions of the rabbit aorta also was examined. Both the microsomal and cytosolic fractions were shown to O-methylate 2OHE2 and (-)-isoprenaline, providing evidence for the existence of membrane-bound and soluble forms of COMT in the rabbit aorta. The O-methylation of 2OHE2 by cytosolic and microsomal fractions of the aorta was determined and compared to that of (-)-isoprenaline. The kinetic constants for the O-methylation of 2OHE2 by cytosolic (Km: 0.27 mumol/l; V max: 112 pmol g-1 min-1) and microsomal (Km: 0.15 mumol/l; Vmax: 161 pmol g-1 min-1) fractions were similar. In contrast, the kinetic constants for the O-methylation of isoprenaline by cytosolic (Km: 121 mumol/l; Vmax: 174 pmol g-1 min-1) and membranal (Km: 0.91 mumol/l; Vmax: 105 pmol g-1 min-1) fractions were very different. It is concluded that catechol oestrogens are excellent substrates for catechol-O-methyltransferase (COMT) in the rabbit aorta. Their O-methylation can occur in endothelial structures as well as in the smooth muscle-containing medial sections of the vessel.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

[Protein biosynthesis in rat liver subcellular fractions with quantitative and qualitative changes in the composition of the carbohydrate component of the diet].

The biosynthesis rate of total proteins of liver subcellular fractions was examined in rats kept for 40 days on a high-carbohydrate diet with the use of starch, and sucrose from 14C-lysine incorporation. It was shown that the majority of subcellular fractions with the exception of the microsomal experienced an increase in the rate of protein biosynthesis. Estimation of the protein content in the fractions allows the conclusion that enhancement of biosynthesis in membrane fractions of external, internal membranes of mitochondria and lysosomes is linked with the increased rate of renewability, while in the fraction of cytoplasm soluble proteins with induction of numerous enzymes because of the increased energy load.

Animals↗