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PubMed1981

Developmental changes in polypeptide composition of, and precursor incorporation into, cellular and subcellular fractions of rat cerebral cortex.

Neuronal-enriched and glial-enriched fractions from rat cerebral cortex at 2, 5, 9, 14 and 23 days postnatally, and subcellular fractions from 2, 14 and 46 day old rat were prepared. The polypeptide composition of all fractions was analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and quantified by densitometry. Fifty-nine polypeptides (mol. wts., 13,200-251,000) were resolved in the cell fractions of which the majority remained unchanged throughout postnatal development. Three polypeptides (mol. wts., 102,000, 56,000, 53,700) were found to increase in amount developmentally in both cellular fractions, the latter two showing a peak in relative amount on day 14 and a subsequent decline. Three polypeptides (mol. wts., 47,000, 28,200, 17,400) were found to be common to the glial-enriched fraction as well as the myelin fraction, and all showed a developmental increase. The neuronal-enriched fraction was found to be enriched in five polypeptides of which one (mol. wt., 51,900) showed a developmental increase after ten days postnatally, the others (mol. wts., 178,700, 142,000, 109,000, 24,000) showing a decrease. In vitro incorporation of [35S]-methionine into the glial-enriched fraction was carried out, and a developmental decline was observed in the labelling of a polypeptide of 42,000 mol. wt.

Aging↗
PubMed1990

Muscarinic cholinergic receptor mediated inhibitory transduction of adenylate cyclase activity in subcellular fractions from rat heart: improved detection in sodium phosphate buffer.

Cholinergic inhibition of myocardial adenylate cyclase activity in cell-free fractions has been known for many years, although the reported degrees of inhibition have been rather modest (20-30%), notably in rat heart fractions. The present study conducted with rat heart subcellular fractions document following major findings: (1) Myocardial adenylate cyclase activity and notably its cholinergic inhibition in cell-free fractions are notoriously labile to storage at 4 degrees C whereas its stimulation by beta adrenergic receptor agonists or forskolin are reasonably well preserved during storage. (2) Among four buffers (Tris, glycylglycine, imidazole and sodium phosphate) examined, sodium phosphate buffer afforded the best preservation of cholinergic inhibitory response of adenylate cyclase. (3) The commonly used biochemical buffers, notably imidazole, exerted deleterious effect on the cholinergic inhibition of myocardial adenylate cyclase such that it was considerably attenuated or barely detectable; this explains, in part, the reported poor inhibition of myocardial enzyme by others. (4) Imidazole buffer, on the other hand, augmented beta adrenergic and forskolin stimulated adenylate cyclase activity. The likely significance of these findings is discussed from consideration that the observed differential influence of buffers results from differential actions on the interactions between the components (receptor/coupling G proteins/catalyst) comprising autonomic receptor coupled adenylate cyclase system in rat heart.

Adenylyl Cyclases↗
PubMed1983

[3H]imipramine binding in subcellular fractions of rat cerebral cortex after chemical lesion of serotonergic neurons.

The specific high affinity binding of [3H]imipramine was investigated in subcellular fractions of rat cerebral cortex before and after chemical denervation of serotonergic neurons. In control animals the proportion of the total number of [3H]imipramine binding sites in the nuclear (N), heavy mitochondrial (M), light mitochondrial (L) and microsomal (P) fractions corresponded respectively to 3, 45, 16 and 36% of the total number of binding sites. After chemical lesion of serotonergic neurons with 5,7-dihydroxytryptamine (5,7-DHT) the density of [3H]imipramine binding sites in fractions M and L was decreased by 42 and 52% respectively. In these experiments the uptake of [3H]5-HT in fractions M and L decreased by approximately 80%. The Bmax of [3H]imipramine binding in fraction P was decreased by 80% after chemical denervation with 5,7-DHT. In the control group there was no detectable [3H]5-HT uptake while the endogenous serotonin levels in fraction P were rather low. Our results support the view that the high affinity binding of [3H]imipramine is partly located on serotonergic nerve terminals. The significance of the [3H]imipramine binding sites present in the microsomal (P) fraction remains to be clarified.

5,7-Dihydroxytryptamine↗
PubMed1978

Cyanide-insensitive NADH oxidation by subcellular fractions isolated from human polymorphonuclear blood cells.

The biochemical triad, NADH oxidation, oxygen (O2) uptake and hydrogen peroxide (H2O2) formation, by subcellular fractions of human blood polymorphonuclears (PMNs) was investigated. It was found that this biochemical triad (1) was under the control of the granule-rich fraction (GRF) only; (2) was not inhibited by cyanide; (3) occurred stoichiometrically for its three components, and (4) accounted quantitatively for the respiratory burst of the stimulated PMN. It was also shown that the above biochemical triad (1) involved an enzymatic step; (2) was enhanced by acidic pH (0.5) and Mg++; (3) was inhibited by Cu++ or low concentration of Mn++; (4) was dependent on H2O2, perhydroxyl radical (HO2) and hydroxyl radical (HO) since either catalase or superoxide dismutase or scavengers of HO2 or HO were inhibitor, and (5) involved multistep reactions. Evidence is provided that the sequence of the reactions is first a generation of H2O2, (spontaneously from NADH in our incubation medium), secondly the production of HO from H2O2, thirdly the oxidation of NADH with further production of HO2,O2 uptake and H2O2 formation, probably through a chain reaction. The identification of the enzyme(s) involved in these multistep reactions needs further studies.

Cyanides↗
PubMed2002

Inhibition of zaleplon metabolism by cimetidine in the human liver: in vitro studies with subcellular fractions and precision-cut liver slices.

1. The effect of cimetidine on the metabolism of zaleplon (ZAL) in human liver subcellular fractions and precision-cut liver slices was investigated. 2. ZAL was metabolized to a number of products including 5-oxo-ZAL (M2), which is known to be formed by aldehyde oxidase, N-desethyl-ZAL (DZAL), which is known to be formed by CYP3A forms, and N-desethyl-5-oxo-ZAL (M1). 3. Human liver microsomes catalysed the NADPH-dependent metabolism of ZAL to DZAL. Kinetic analysis of three microsomal preparations revealed mean (+/-SEM) S(50) and V(max) of 310 +/- 24 micro M and 920 +/- 274 pmol/min/mg protein, respectively. 4. Human liver cytosol preparations catalysed the metabolism of ZAL to M2. Kinetic analysis of three cytosol preparations revealed mean (+/-SEM), K(m) and V(max) of 124 +/- 14 micro M and 564 +/- 143 pmol/min/mg protein, respectively. 5. Cimetidine inhibited ZAL metabolism to DZAL in liver microsomes and to M2 in the liver cytosol. With a ZAL substrate concentration of 62 micro M, the calculated mean (+/-SEM, n = 3) IC50 were 596 +/- 103 and 231 +/- 23 micro M for DZAL and M2 formation, respectively. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver cytosol with a mean (+/-SEM, n = 3) K(i) of 155 +/- 16 micro M. 6. Freshly cut human liver slices metabolized ZAL to a number of products including 1, M2 and DZAL. 7. Cimetidine inhibited ZAL metabolism in liver slices to M1 and M2, but not to DZAL. Kinetic analysis revealed that cimetidine was a competitive inhibitor of M2 formation in liver slices with an average (n = 2 preparations) K(i) of 506 micro M. 8. The results demonstrate that cimetidine can inhibit both the CYP3A and aldehyde oxidase pathways of ZAL metabolism in the human liver. Cimetidine appears to be a more potent inhibitor of aldehyde oxidase than of CYP3A forms and hence in vivo is likely to have a more marked effect on ZAL metabolism to M2 than on DZAL formation. 9. The results also demonstrate that precision-cut liver slices may be a useful model system for in vitro drug-interaction studies.

Acetamides↗
PubMed1978

Analytical subcellular fractionation of neutrophils from patients with chronic granulomatous disease. Demonstration of the enzyme defect in four cases.

Analytical subcellular fractionation studies were performed on neutrophils from five patients, including two females, with chronic granulomatous disease. The density distribution and marker enzyme activities of the principal subcellular organelles in unstimulated cells were similar to those in unstimulated neutrophils from control subjects. NADH dependent reduction of nitroblue tetrazolium was measured in four of the patients including one female. In homogenates of whole cells the specific activity of this enzyme expressed as milliUnits/mg protein was lower in the patients than in the controls, but the difference was not statistically significant. There was however a highly significant difference between the specific activities of this enzyme in the plasma membrane fractions isolated from neutrophils of the four patients and the three controls. These findings suggest that the primary microbicidal oxidase of neutrophils, defective function of which manifests as the syndrome of chronic granulomatous disease, is a plasma membrane NADH oxidoreductase.

Adolescent↗
PubMed2001

Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

Actins↗
PubMed1993

[3H]2-(4-phenylpiperidino)cyclohexanol (AH5183) binding to synaptosomes and subcellular fractions obtained from rat brain.

We investigated the binding of [3H]AH5183 (2-(4-phenylpiperidino)cyclohexanol) to rat brain synaptosomes and subcellular fractions. A high content of specific binding was observed in crude synaptosomes obtained from the striatum, cerebral cortex and hippocampus. The highest density of subcellular binding sites was observed in the synaptic vesicle-rich fraction. The affinities of AH5183 binding to crude synaptosomes and the synaptic vesicle-rich fraction were almost equivalent, but the density of binding sites was higher in the synaptic vesicle fraction. The present findings indicate that [3H]AH5183 binding to both synaptosomes and the synaptic vesicle-rich fraction is useful as a cholinergic marker, and that for quantitative studies, binding to the latter fraction is more preferable.

Animals↗
PubMed1981

The effects of administration of semicarbazide and aminooxyacetic acid on B6 vitamer levels in subcellular fractions of mouse brain.

The in vivo effects of semicarbazide (SC) and aminooxyacetic acid (AOAA) on the B6 vitamer concentrations in brain subcellular fractions, especially in extrasynaptosomal, synaptosomal plasma, extrasynaptosomal and synaptosomal mitochondria, were investigated. Treatment of mice with SC decreased levels of pyridoxal phosphate (PLP) in extrasynaptosomal and synaptosomal plasma, while it had no effect on those in extrasynaptosomal and synaptosomal mitochondria. On the contrary, treatment with AOAA more significantly decreased pyridoxamine phosphate (PMP) levels in extrasynaptosomal and synaptosomal mitochondria than those in extrasynaptosomal and synaptosomal plasma. According to these results, the difference between in vivo effects of SC and AOAA on vitamin B6-enzymes was discussed.

Acetates↗
PubMed1978

Biosynthesis of chenodeoxycholic acid: side-chain hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha-diol by subcellular fractions of guinea pig liver.

Side-chain hydroxylation of 5 beta-cholestane-3 alpha, 7 alpha diol was studied in subcellular fractions of guinea pig liver. The purity of the microsomal and the mitochrondrial fractions was determined with marker enzymes, and relatively little cross contamination between the particulate fractions was detected. Methods for the analysis of the incubation mixture by thin-layer chromatography and gas-chromatography-mass spectrometry were developed. Optimal assay conditons were established for the major hydroxylation reactions, namely the mitochondrial 26-hydroxylation and the microsomal 25-hydroxylation, It was found that the most active side-chain hydroxylation in the guinea pig was the microsomal 25-hydroxylation. The mitochondrial omega-hydroxylation was stereospecific, in that the rate of formation of (25R)-5 beta-cholestane-3 alpha, 7 alpha, 26-triol was 8 times greater than that of the 25S isomer. The microsomal "25" hydroxylation was not stereospecific under the conditions employed. It is concluded that the mitochondrial "26" hydroxylation (leading to the formation of (25R)-5 beta-cholestane-3 alpha, 7 alpha, 26-triol) plays an important role in the biosynthesis of chenodeoxycholic acid. The participation of microsomal 25-hydroxylation in the formation of chenodeoxycholic acids requires further investigation.

Animals↗
PubMed1995

Responses of antioxidant system to acute and trained exercise in rat heart subcellular fractions.

The effects of acute and trained exercise on antioxidant enzymes (AOE), glutathione (GSH), and malondialdehyde (MDA) were compared in rat heart subcellular fractions and red blood cells. Fischer-344 rats were exercised acutely to 100% VO2 max and another group of Fischer-344 rats were given trained exercise for 10 weeks. The AOE and MDA were measured by spectrophotometry and GSH and oxidized GSH (GSSG) by high pressure liquid chromatography. Trained exercise significantly increased cytosol GSH to 131% of sedentary control (SC). Acute exercise significantly increased mitochondrial superoxide dismutase, catalase, and glutathione peroxidase by 167%, 358%, and 129% of SC, respectively, whereas enzyme activities following trained exercise were increased by 133%, 166%, and 128% of SC. The mitochondria/cytosolic ratio for superoxide dismutase, catalase, and glutathione peroxidase after acute exercise increased to 1.9, 2.7, and 1.7, respectively, whereas the respective ratios of these enzymes after trained exercise were 1.3, 1.6, and 1.3. Acute exercise contributed to oxidative stress more than trained exercise. Acute exercise resulted in a larger increase in enzyme activities than trained exercise, possibly as a compensatory mechanism to cope with the enhanced production of superoxides and oxyradicals during exhaustive exercise.

Animals↗
PubMed1975

Distribution of renin in subcellular fractions from the rabbit kidney.

Subcellular localization of renin in the rabbit kidney cortex was investigated using two centrifugation techniques. Renin was indirectly assayed on the basis of pressor activity and the reference enzymes such as succinic dehydrogenase, acid phosphatase and D-glucose-6-phosphatase for the other subcellular particulates were biochemically determined. Renin activity was found mainly in the mitochondrial fraction with very little in the microsomal fraction by a differential centrifugation. By a discontinuous sucrose density gradient centrifugation, renin granules were mainly recovered in the fraction corresponding to 1.5 M sucrose, while most of mitochondria, lysosomes and microsome equilibrated in the upper fractions. This renin granular fraction contained approximately 50% of total granular renin activity having a specific activity about six times that seen in the homogenate. After recentrifugation of the renin granular fraction, most of renin activity was recovered in the sediment. Repeated freezing and thawing of this fraction resulted in an increase of renin activity. On the basis of these experimental data it is assumed that renin located in the different subcellular particulates from mitochondria, lysosomes and microsomes in the rabbit kidney cortex.

Angiotensin II↗
PubMed1992

Effects of taurine on the phosphorylation of specific proteins in subcellular fractions of the rat retina.

The effects of 20 mM taurine on the phosphorylation of specific proteins in mitochondrial and rod outer segment subcellular fractions of the rat retina were measured. A band of protein with an apparent molecular weight of approximately 20K was consistently inhibited by taurine. Densitometry measurements performed on gel electrophoresis autoradiograms from the mitochondrial fraction demonstrated a 42.7 +/- 8.3% decrease due to taurine (20 mM) in the area corresponding to radioactivity from the approximately 20K phosphoprotein. However, only a 21.2 +/- 9.0% decrease was observed due to taurine in the rod outer segment preparation. These data suggest that taurine is exerting its primary effect on the phosphorylation of the approximately 20K molecular weight protein in the mitochondria of the retina. In addition, calmodulin and phorbol ester had no effect on the phosphorylation of the approximately 20K molecular weight protein.

Animals↗
PubMed1991

Subcellular fractionation of murine erythroleukemic cells: distribution of protein kinases.

A method for subcellular fractionation of murine erythroleukemic cells is described; a highly purified cytosol fraction and significantly enriched membrane, mitochondrial, and nuclear fractions were obtained. During development of the procedure, we demonstrated how the composition of the extraction buffers and the techniques used can affect activity and distribution of protein kinases. Protein kinases in the various fractions were separated by nondenaturing electrophoresis and detected by phosphorylation and autoradiography. Differences in the relative proportions of the kinases, which may be significant in relation to differentiation, were seen in all the fractions on hexamethylenebisacetamide treatment of the cells.

Animals↗
PubMed1992

Internalization of intact poliovirus by HeLa cells as shown by subcellular fractionation in isoosmotic Nycodenz gradients.

HeLa cells were infected with radiolabelled poliovirus at different temperatures, and the intracellular distribution of input radioactivity was studied. To this end, homogenates were fractionated by rate zonal centrifugation in linear isoosmotic (2 to 30%) Nycodenz gradients. Further purification of subcellular fractions was achieved by recentrifugation to equilibrium in 10 to 30% Nycodenz. Temperatures were kept below 30 degrees C to prevent virus capsid modification. Under these conditions, the cell-associated virions remained fully infectious. Below 18 degrees C, most of the viral label was recovered from a bottom region (BR) of the rate zonal gradients. Marker enzyme analysis and antibody accessibility showed that the BR consisted of virions bound to the plasma membrane. Between 18 degrees C and 26 degrees C, viral label also accumulated in a top region (TR) of the rate zonal gradients. According to the criterion of antibody accessibility, the virions associated with the TR were present within intracellular structures, probably lipid membranes. Electron microscopy confirmed the presence of vesicles and tubules in this region of the gradient. No correlation was found between the TR and endosomal, lysosomal or plasma membrane markers. The TR equilibrated at low density (1.10 g/ml) in Nycodenz (free virus, 1.31 g/ml). The results confirm that intact poliovirions can enter the cell and do so via lipid-bound vesicles.

Biomarkers↗
PubMed1997

Regulation of phosphatidylethanolamine degradation by enzyme(s) of subcellular fractions from cerebral cortex.

Hydrolysis of 1-acyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine was studied in cerebral cortex homogenate and subcellular fractions. The enzyme(s) confined to the synaptic plasma membrane (SPM) hydrolyze(s) [14C-arachidonoyl]phosphatidylethanolamine (PE) in the presence of EGTA to [14C-arachidonoyl]diacylglycerol (DAG) and a small amount of [14C]arachidonic acid (AA). Degradation of PE is time-, protein- and substrate-dependent with a pH optimum of 7.8. The highest activity of PE degradation was observed in the presence of 10 mM EGTA. Under this condition GTP gamma S has no effect on PE hydrolysis. In the presence of Ca2+ ions degradation of PE was significantly lower as compared to the conditions with EGTA. However, the percentage distribution of free AA in the sum of both products of PE hydrolysis (AA + DAG) increases from 16 and 20% observed in the presence of EGTA 2 mM and 10 mM to 34% and 43% in the presence of 0.5 mM CaCl2 alone and together with GTP gamma S, respectively. Cytosolic enzymes also degrade PE in the presence of 2 mM EGTA with the formation of DAG and AA. Radioactivity in the AA represents about 80% of the total radioactivity of the products of PE degradation. The hydrolysis of PE by cytosolic enzymes is almost completely inhibited by neomycin but the hydrolysis by the SPM-bound enzyme(s) is inhibited only 70%. Other studies with quinacrine indicated that only a small pool of PE is degraded by SPM-bound Ca(2+)-independent phospholipase A2 (PLA2). All of these data suggest that PE in cerebral cortex is mainly degraded by cytosolic and SPM-bound Ca(2+)-independent phospholipase C. Further studies towards a better understanding of the mechanisms of cerebral degradation and the physiological significance of Ca(2+)-independent pathways of PE hydrolysis are necessary.

Animals↗
PubMed1982

Multiple sclerosis-lymphocyte transformation with multiple sclerosis and normal brain myelin basic protein and subcellular fractions.

Possible sensitisation of peripheral blood mononuclear cells from multiple sclerosis (MS) patients was studied in the presence of both normal and MS brain subcellular fractions. Myelin, synaptosomal and microsomal fractions and myelin basic protein were prepared by sucrose density gradient centrifugation from normal and MS brain. These preparations were added to mononuclear cell microcultures from the peripheral blood of nine acute and six chronic MS patients, 10 patients with other neurological diseases and 10 normal subjects. There was no significant increment in the peripheral blood mononuclear cell transformation of any of the acute MS patients with any of the fractions. Therefore there is no evidence that immunity to antigens in any of the fractions of MS or normal brain is of primary pathogenetic importance. A small increment in peripheral blood mononuclear cell transformation in the presence of several fractions was observed in two of six chronic MS patients, which might indicate the development of a secondary immune response after prolonged disease.

Acute Disease↗