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PubMed1989

Translocation of protein kinase C to subcellular fractions of human neutrophils.

The subcellular localization of protein kinase C in unstimulated human neutrophils and neutrophils stimulated by phorbol-myristate-acetate (PMA), 1-oleoyl-2-acetyl-rac-glycerol (OAG), and ionomycin was investigated in subcellular fractions obtained by nitrogen cavitation and Percoll density gradient centrifugation. Protein kinase C was found to be localized mainly in the cytosol in unstimulated cells, whereas significant translocation to fractions containing the plasma membrane was observed after stimulation by PMA, OAG, and ionomycin. At the same time, phospholipid-insensitive protein kinase activity appeared in the cytosol and the plasma membrane fractions. To determine whether binding of protein kinase C occurred to the plasma membrane or to intracellular membranes that had translocated to the plasma membrane, we investigated the ability of isolated azurophil, specific and secretory granules, and plasma membrane vesicles to bind protein kinase C in response to addition of PMA and OAG. Only fractions containing plasma membranes and secretory granules were able to bind protein kinase C. The observation explains the selective activation of plasma membrane structures by protein kinase C.

Binding Sites↗
PubMedDate not supplied

Insulin effect on lung subcellular fraction phospholipids and cholesterol.

Lung subcellular fraction (microsomes, mitochondria, 105 000 X g supernatant and plasma membranes) were isolated from normal and insulin treated rats. Phospholipid and cholesterol contents were determined, phosphatidylcholines and phosphatidyl-ethanolamines separated and their fatty acid profiles assayed. Teh investigations carried out indicated that insulin influenced the phospholipid content of all fractions. Cholesterol levels in the 105 000 X g supernatant and especially in the plasma membrane fraction were raised significantly. Insulin administration increased the contribution of the unsaturated long chain fatty acids in both phospholipids studied. The results are discussed in relation to stimulated lipid biosynthesis and to lipid fluidity, of which fatty acyl unsaturation and cholesterol level are main parameters.

Animals↗
PubMed1975

Distribution of LH-RH in subcellular fractions of the basomedial hypothalamus.

Subcellular fractionation of the mediobasal hypothalamus (MBH) and other brain structures was achieved by differential and sucrose gradient centrifugation. The fractions were monitored by measuring lactate dehydrogenase (LDH) activity (a marker for the soluble cytoplasmic fraction) and by electron microscopic examination. The luteinizing-hormone-releasing-hormone (LH-RH) content of the fractions was evaluated both by bioassay and radioimmunoassay. Significant amounts of LH-RH were found only in the MBH and in an anterobasal location corresponding to the organum vasculosum of the lamina terminalis. Within these areas, LH-RH activity was present in the first supernatant (homogenate with the exclusion of the nuclear pellet). Seventy percent of the LH-RH activity was recovered in the crude mitochondrial fraction. After further fractionation on a sucrose gradient, the distribution of LH-RH was parallel with that of LDH activity. Since LDH is predominantly located in the synaptosomal soluble fraction, it is concluded that the vast majority of LH-RH is contained within nerve endings. This finding is consistent with cyto-immunological data on the distribution of the neuropeptide in the rat hypothalamus.

Animals↗
PubMed1973

Phospholipids and acyl groups in subcellular fractions from human cerebral cortex.

Subcellular fractionation of human brain cortex obtained at autopsy yielded microsomal and synaptosome-rich fractions from the gray matter and microsomal and purified myelin fractions from the white matter. The phospholipids of myelin were high in plasmalogens, and the molar ratio of alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 4. The acyl groups of the myelin phosphoglycerides were enriched in monoenes (mainly 18:1 and 20:1) and a tetraene, 22:4(n - 6). The phospholipids in the synaptosome-rich fraction were high in diacyl sn-glycero-3-phosphorylcholine, and the molar ratio of the alkenyl acyl sn-glycero-3-phosphorylethanolamine to diacyl sn-glycero-3-phosphorylethanolamine was 0.88. The acyl groups of synaptosomal ethanolamine phosphoglycerides were rich in 22:6(n - 3) but contained a very low amount of 20:1. The lipid composition of microsomes from the gray matter was different from that of microsomes from the white matter but was nearly identical with that of the synaptosome-rich fraction. Except for a slightly lower proportion of alkenyl acyl sn-glycero-3-phosphorylethanolamine and sphingomyelin, the lipid composition of microsomes from the white matter was also similar to that of the myelin. There were also species-related differences between the brain lipid composition of human and subhuman primates and that of the rodents. Furthermore, the brain lipid composition in normal human subjects is rather constant and does not seem to be affected much by individual variations.

Aged↗
PubMed1982

An electrophoretic analysis of proteolipids from different rat brain subcellular fractions.

Proteolipid proteins were extracted from adult rat brain subcellular fractions and purified by chromatography on Sephadex LH-60. Polyacrylamide gel electrophoresis of the delipidized proteins, in the presence or absence of 8 M urea, was carried out with all fractions. The distribution of the various types of proteolipid proteins was studied and their molecular weight calculated by the Ferguson relationship. Several bands of proteolipid proteins were found in the five membrane fractions analyzed. Some of them, such as the 17.5 K and 37 K components were very prominent in mitochondria and synaptosomes. The 30 K component was found in myelin-derived membranes and in microsomes, while the 20 K and 25 K proteolipid proteins were present in all subcellular fractions. The 30 K component (proteolipid protein (PLP)), typical of the purified myelin membranes, showed a similar distribution to that of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (EC 3.1.4.37) activity, while the other major proteolipid protein present in all subcellular fractions (25 K) did not show such parallelism, indicating that it might not be an exclusive component of myelin. The electrophoretic pattern of microsomal proteolipid proteins did not show the high molecular weight components (aggregates of PLP) which are found in myelin. Furthermore, the 30 K component showed a smaller Y0 value than that of the 30 K found in myelin. Thus the presence of 30 K proteolipid protein in microsomes should not be considered as being due to myelin contamination.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1976

Inactivation of Oxytocin and its analogues by subcellular fractions of hen tissues.

The enzymic inactivation of oxytocin by liver, kidney, uterus and pancreas homogenate subcellular fractions of hens was studied. Oxytocin was most rapidly degraded by the soluble fraction of tissues examined. All the subcellular fractions of liver and kidney inactivated oxytocin, but only the microsomal and soluble fractions of uterus and pancreas showed the oxytocin-inactivating activity. The location of enzymes inactivating oxytocin in subcellular fractions of hen tissues was investigated with the aid of synthetic analogues of oxytocin (deamino-oxytocin and deamino-carba1-oxytocin). The carboxamidopeptidase activity, hydrolyzing the amide bonds in the linear portion of oxytocin was located in the soluble fraction of hen liver, kidney and uterus. No carboxamidopeptidase activity in the pancreatic soluble fraction was found. These results showed that aminopeptidase activity is bound to heavy subcellular particles in the hen tissue. An action of unknown endopeptidases was observed in the microsomal fraction of uterus and pancreas.

Animals↗
PubMed2005

New application of a subcellular fractionation method to kidney and testis for the determination of conjugated linoleic acid in selected cell organelles of healthy and cancerous human tissues.

To clarify the mechanism of the anticarcinogenic effect of conjugated linoleic acid (CLA), its intracellular distribution needs to be determined. Subcellular fractionation using centrifugation techniques is a method that is frequently used for isolation of cell organelles from different tissues. But as the size and density of the organelles differ, the method needs to be optimised for every type of tissue. The novelty of this study is the application of a subcellular fractionation method to human healthy and cancerous renal and testicular tissue. Separation of total tissue homogenate into nuclei, cytosol, and a mixture of mitochondria and plasma membranes was achieved by differential centrifugation. As mitochondria and plasma membranes seemed to be too similar in size and weight to be separated by differential centrifugation, discontinuous density-gradient centrifugation was carried out successfully. The purity of the subcellular fractions was checked by measuring the activity of marker enzymes. All fractions were highly enriched in their corresponding marker enzyme. However, the nuclear fractions of kidney and renal cell carcinoma were slightly contaminated with mitochondria and plasma membrane fractions of all tissues with lysosomes. The fraction designated the cytosolic fraction contained not only cytosol, but also microsomes and lysosomes. The CLA contents of the subcellular fractions were in the range 0.13-0.37% of total fatty acids and were lowest in the plasma membrane fractions of all types of tissue studied. C16:0, C18:0, C18:1 c9, C18:2 n-6, and C20:4 n-6 were found to be the major fatty acids in all the subcellular fractions studied. However, marked variations in fatty acid content between subcellular fractions and between types of tissue were detectable. Because of these differences between tissues, no general statement on characteristic fatty acid profiles of single subcellular fractions is possible.

Adipose Tissue↗
PubMed1993

Maternal dietary fish oil enriches docosahexaenoate levels in brain subcellular fractions of offspring.

Prompted by the speculated essentiality of docosahexaenoic acid (DHA) for neural development, this study was undertaken to investigate the incorporation of (n-3) fatty acids in the maternal diet into various phospholipids of infant rat brain subcellular fractions: microsomes (Ms), synaptosomes (Sy), myelin (My), and mitochondria (Mt). Two groups of infant rats were nourished by dams fed diets containing 20% of either corn oil (CO) or menhaden oil (MO) from 2 until 12 days of age. DHA but not eicosapentaenoic acid (EPA) was distributed to all subcellular fractions of infant rats in the CO group. The levels of DHA were higher in Ms and Mt than Sy and My, and higher in phosphatidylethanolamine (PE) and phosphatidylserine (PS) than phosphatidylcholine (PC) and phosphatidylinositol (PI). The MO feeding enriched DHA in PE of all subcellular fractions, PS of all subcellular fractions, except My, PC of Sy, My and Mt, and PI of My. EPA was enriched in phospholipids in all subcellular fractions, except mitochondrial PS of the MO group. In the MO group, the ratios of EPA/DHA, ranging from 0.01 to 0.85, in all subcellular phospholipids were markedly lower than that found in the mother's milk (i.e., 1.5), suggesting an ability to elongate and desaturate EPA to DHA and/or disproportional uptake of the fatty acids by the brain. In PE of all subcellular fractions, the increased levels of DHA and EPA, with a concomitant reduction of arachidonic and/or linoleic acid, yielded higher ratios of total (n-3)/(n-6) fatty acids in the MO than the CO group. The inclusion of preformed DHA and EPA in the maternal diet provides an effective means to enrich these fatty acids in developing brains.

Animals↗
PubMed2002

Preparation of crude subcellular fractions by differential centrifugation.

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. Buoyant density gradient purification of peroxisomes or lysosomes for example is almost invariably carried out on a light mitochondrial fraction so as to eliminate smaller particles that may have similar densities. Unless they are first removed, large rapidly sedimenting particles in homogenates may also disturb shallow gradients designed to fractionate small low-density microsomes.

Animals↗
PubMed1984

[Phospholipids of the subcellular fractions of brain and liver tissues in rats in total oxygen starvation of the body].

The rate of phospholipid turnover in brain subcellular fractions differ from that in the liver tissue. Hypoxia of medium severity (pressure chamber 240 mm Hg) influences neither the content nor the turnover rate of phospholipids in liver subcellular fractions. Meanwhile in brain subcellular fractions, it results in a decreased phospholipid turnover rate. Severe hypoxia (less than 200 mm Hg) leads only to the suppression of the phospholipid turnover rate in the liver, whereas in the brain, it results in the decline of the phospholipid turnover rate as well as in the reduction of phospholipid content. Thus, the well-known concept of the higher brain sensitivity to oxygen deficiency as compared to the liver is exemplified by phospholipid turnover in subcellular fractions.

Animals↗
PubMed1993

Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions.

The oxidized (UQox) and reduced (UQred) forms of ubiquinone (UQ) homologues in rat tissues and subcellular fractions were analyzed to elucidate their distribution and physiological role. UQ-9 and UQ-10 were detected in all tissues studied, and UQ-9 was the predominant homologue. The total amount of UQox-10 and UQred-10 was 20-50% that of UQox-9 and UQred-9. The levels of these homologues were highest in heart with lesser amounts occurring in kidney, liver and other organs. In liver and blood plasma, the UQred homologue amounted to 70-80% of the total UQ (UQox + UQred = t-UQ). UQred was less than 30% of t-UQ in other tissues and blood cells. t-UQ was much higher in leukocytes and platelets in blood than in erythrocytes. In erythrocytes, t-UQ was exclusively located in the cell membranes. UQox and UQred were also found in all subcellular fractions isolated from liver and kidney in about the same ratio as UQred/t-UQ was present in the whole organ. The levels of UQox and UQred per mg protein in subcellular fractions from liver were highest in mitochondria, with lesser amounts present in plasma membranes, lysosomes, Golgi complex, nuclei, microsomes and cytosol. In the mitochondria, the outer membranes were richer in t-UQ than the inner membranes. In the Golgi complex, the light and intermediate fractions were rich in t-UQ when compared to the heavy fraction. The possible physiological role of UQox and UQred in tissues and subcellular fractions is discussed.

Animals↗
PubMed1975

Phosphatidate phosphohydrolase and palmitoyl-coenzyme A hydrolase in cardiac subcellular fractions of hyperthyroid rabbits and cardiomyopathic hamsters.

Activities of phosphatidate phosphohydrolase and palmitoyl-CoA hydrolase were determined in cardiac subcellular fractions prepared from rabbits which has received tri-iodothyronine and from hamsters with hereditary cardiomyopathy (strain BIO 14.6). 1. Both mitochondrial and microsomal fractions of hyperthyroid rabbit hearts produced 4-5 times as much diacylglycerol 3-phosphate from glycerol 3-phosphate and palmitate as did those of euthyroid hearts. 2. Phosphatidate phosphohydrolase, measured with phosphatidate emulsion, was activated by 1mm-Mg(2+) in all but the mitochondrial fraction of euthyroid rabbit hearts. The activation was more pronounced in subcellular fractions isolated from hyperthyroid hearts, so that the measured activities were significantly increased above those of the controls. The highest activity was found in the microsomal and lysosomal fractions. 3. In the absence of Mg(2+) during incubation, the difference in phosphohydrolase activities between eu- and hyper-thyroid states was not significant. 4. The phosphohydrolase of subcellular fractions of control hamsters did not respond to addition of 0.5-8.0mm-Mg(2+). The enzyme from cardiomyopathic hearts was slightly inhibited by this bivalent cation and therefore significant increases in activity were observed only in the absence of Mg(2+) from the assay system. 5. The rate of reaction by soluble phosphatidate phosphohydrolase was similar regardless of the nature of the substrate. Both when microsomal-bound phosphatidate was used as the substrate and when phosphatidate suspension was used, the activity of soluble enzyme was lower than that of the microsomal and lysosomal enzymes measured with phosphatidate suspension; this was especially so when the assay was carried out in the absence of Mg(2+). Neither tri-iodothyronine nor cardiomyopathy influenced the soluble phosphohydrolase activity in the two species. 6. Neither tri-iodothyronine nor cardiomyopathy significantly changed palmitoyl-CoA hydrolase activities in subcellular fractions. 7. Microsomal diacylglycerol acyltransferase and myocardial triacylglycerol content were also unchanged in the hyperthyroid state.

Acid Phosphatase↗
PubMed1985

Protective immunity and pathology induced by inoculation of mice with different subcellular fractions of Trypanosoma cruzi.

Mice were immunized with subcellular fractions obtained by differential centrifugation from epimastigotes of Trypanosoma cruzi (Tulahuén strain). In a chronic model of Chagas' disease, they were challenged each with 25 bloodstream trypomastigotes. Non-immunized, non-challenged and non-immunized challenged animals were kept as controls. Among the challenged mice, those immunized with 105,000 g pellet (Mc) and 105,000 g supernatant (Cs) fractions presented positive xenodiagnosis, myocarditis and myositis similar to those shown by non-immunized challenged controls. The fractions enriched in flagella, the 5000 g pellet (P5) and the flagellar fraction (F) resulted in fewer animals with positive xenodiagnosis and in hosts partially protected from the development of myocarditis. In the absence of infection, Mc and Cs induced an intense myocarditis while F induced mild lesions similar to those found in the controls. P5 caused a myocarditis intermediate between that elicited by Mc and that in the controls. 50% of the animals immunized with Cs presented pathological electrocardiograms in the absence of challenge. The animals immunized with F and P5 and challenged were protected against the development of pathological electrocardiograms, whereas those immunized with Mc and Cs behaved like the non-immunized controls. The immunized, non-challenged animals presented anti-T. cruzi IgG antibodies, with titres which were lower than those shown by the immunized and challenged mice.--The results show the possibility of obtaining tissue lesions with antigenic preparations of T. cruzi in the absence of infection, and suggest that the mechanisms involved in the generation of myocarditis and electrocardiographic alterations are probably different, since these pathologies can be elicited by different subcellular fractions. Among the antigenic components of the parasite, the flagellar fraction gave the best immunoprotective properties, with little or no immunoaggressive effects.

Animals↗
PubMed1983

[Effect of the linolenic acid content of the mother's diet on the polyunsaturated fatty acid composition of subcellular fractions in brain development in the rat].

In order to determine precisely the respective roles of linolenic acid and linoleic acid in the maternal diet on rat brain subcellular fractions during development, we used two diets with different percentages of linolenic acid (18:3 n-3). The animals were fed peanut oil (group A) or soybean oil (group B) during pregnancy and throughout lactation. Nature and amount of essential fatty acids had no incidence on saturated and monounsaturated fatty acid distributions in myelin, synaptosomal, mitochondrial and microsomal fractions. In adult rats, all subcellular fractions are marked by an increase of n-3 fatty acid and a decrease of n-6 fatty acid levels in group B compared to group A. In 15-day-old animals, on the contrary, only the synaptosomal fractions are significantly affected by the diet. Independent of diet, brain development is marked by a decrease of n-6 fatty acids in all subcellular fractions; on the other hand, the n-3 fatty acid level is increased in the synaptosomal and mitochondrial fractions, and decrease in the myelin and microsomal fractions. The sum of (n-3 + n-6) fatty acids remains constant in group B and in group A in all subcellular fractions. Finally, under our experimental conditions, we found no marked effect of diet composition upon linoleic acid conversion to arachidonic acid; only the delta 4-7-10-13-16-docosapentaenoic acid (22:5 n-6) level decreased in group B. delta 7-10-13-16-19-Docosapentaenoic acid (22:5 n-3) seemed to be a better substrate for delta 4 desaturase than delta 7-10-13-16-docosatetraenoic acid (22:4 n-6).

Animals↗
PubMed1985

The synthesis of reduced metabolites of aldosterone by subcellular fractions of rat kidney: effects of antimineralocorticoids.

Subcellular fractionation of male rat kidney revealed that the nuclear and plasma membrane fractions isolated from the 1,000 g pellet retained a significant proportion of the aldosterone ring-A reducing activity. Improved HPLC solvent systems separated all six possible ring-A reduced metabolites of aldosterone and revealed that 80-90% of the reduced metabolites synthesized by purified nuclei and plasma membranes were 5 alpha-reduced compounds consisting of 5 alpha-DHA and 3 alpha,5 alpha-THA in ratios of 1:2 (nuclei) and 1:1 (membranes). The 105,000 g cytosol also synthesized significant quantities of reduced, hydroxylated, and conjugated metabolites of aldosterone. In contrast, the majority of the reduced metabolites of aldosterone synthesized by kidney cytosol were 5 beta-products, consisting principally of 5 beta-DHA and smaller quantities of 3 alpha,5 beta-THA and 3 beta,5 beta-THA. The synthesis of reduced aldosterone metabolites in the cytosol, nuclear, and plasma membrane fraction was inhibited by both 5 and 50 microM concentrations of the antimineralocorticoids, progesterone, K+-canrenoate, and corticosterone. Progesterone was the strongest inhibitor of the synthesis of 5 alpha-DHA and 3 alpha,5 alpha-THA in both nuclei and plasma membranes. The overall order of inhibition of the synthesis of ring-A reduced metabolites in the kidney subcellular fractions was progesterone greater than K+-canrenoate greater than corticosterone; both progesterone and K+-canrenoate inhibited 5 alpha-reduction more than 5 beta-reduction.

Aldosterone↗
PubMed1987

Preparation and characterization of subcellular fractions from the liver of C57B1/6 mice, with special emphasis on their suitability for use in studies of epoxide hydrolase activities.

The present study was designed to prepare and characterize subcellular fractions from the liver of male C57B1/6 mice, with special emphasis on their suitability for use in studies of epoxide hydrolase isozymes. The effects of different washing and pelleting procedures on the mitochondrial, microsomal and cytosolic fractions were studied. It was found that 133,000 gav for 60 min (i.e. more extensive force than the usual 105,000 gav for 60 min) was necessary to obtain a membrane-free cytosolic fraction, while one wash for microsomes and two washes for mitochondria yielded reasonably pure fractions. The purity of the different fractions obtained by differential centrifugation was then determined using established enzyme markers and morphological examination with the electron microscope. Several enzymes involved in drug metabolism were also measured in these fractions. The subcellular distributions obtained here for marker enzymes closely resemble those reported for rat liver. Starvation had no significant effect on the epoxide hydrolase activities nor did the addition of mouse bile or rat liver cytosol, which might contain inhibitors. The change in epoxide hydrolase activities with time after preparation of the subcellular fractions was studied, as well as the effect of freeze-thawing. The subfractions prepared here are suitable for the further characterization of the different forms of epoxide hydrolase present in mouse liver, as well as for other studies requiring well-characterized subfractions.

Animals↗
PubMed1980

Subcellular fractionation of epiphyseal cartilage: isolation of matrix vesicles and profiles of enzymes, phospholipids, calcium and phosphate.

Epiphyseal cartilage was fractionated into subcellular components by nonenzymatic methods, and analyzed for activity of marker enzymes, for phospholipids, and for calcium and inorganic phosphate. Alkaline phosphatase, a marker enzyme for matrix vesicles and plasma membranes, was concentrated in the 100 000 X g (microsomal) pellet and, upon subsequent fractionation, in the low-density fractions from the sucrose gradient. Mitochondrial and endoplasmic reticular enzymes were localized primarily in the 20 000 X g pellet, lysosomal enzymes predominantly in the supernate from the microsomal pellet. Two phospholipids characteristic of matrix vesicles, sphingomyelin and phosphatidylserine, were enriched in the low-density sucrose fractions; however, unlike matrix vesicles, there was no depletion in phosphatidylcholine or increase in lysophospolipids. Ca and inorganic P were concentrated in the higher-density fractions, the amounts in the lower-density fractions being somewhat lower than those seen in matrix vesicles. The alkaline phosphatase-rich, low-density fractions were thus not identical to matrix vesicles isolated by collagenase digestion, but rather appear to be composed primarily of plasma membranes. Enzyme profiles indicate they were relatively free of mitochondrial, endoplasmic reticular and lysosomal contaminants. the data further indicate that significant modification of the phospholipid, electrolyte, and possibly enzyme content of chondrocyte plasma membranes, must occur during blebbing and matrix vesicle formation.

Alkaline Phosphatase↗