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At least 19 recordsLinked to original sources
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↗
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↗
PubMed1975

Effects of freezing and storage on subcellular fractionation of guinea pig and human brain.

Subcellular fractionations were carried out on guinea pig and human brains. Distributions of protein marker enzymes, and galactolipids were examined with guinea pig cerebral cortex that was (Group I) homogenized immediately; (Group II) stored 3 to 5 days at -70 degrees C prior to homogenization; (Group III) stored 3 to 6 months; (Group IV) homogenized after 3 h at R.T. and 16 to 18 h at 4 degrees C and then stored at -70 degrees C for 7 to 9 months. Human frontal lobe obtained at autopsy was fractionated immediately (Group V) or stored at -70 degrees C for 5 to 8 months prior to fractionation (Group VI). Protein recoveries in myelin, microsomal, synaptosomal, and supernatant fractions were decreased in brains that were not frozen for several hours prior to storage (Groups IV-VI). SDH and MAO recoveries in the nuclear and free mitochondrial fractions were increased in these groups. AChE, a membrane marker, was also increased in the free mitochondrial fractions in Groups IV-VI, suggesting increased contamination of mitochondria by synaptosomal membrane fragments. Arylsulfatase, a lysosomal enzyme, was decreased in the free mitochondrial fraction with freezing, but the distributions in tissues not frozen for several hours showed only an increase in the nuclear fraction and a decrease in the microsomal fraction. Freezing brought about an increase in supernatant LDH and a decrease in this enzyme in the free mitochondrial fractions. Total galactolipid contents in synaptosomal and free mitochondrial fractions were increased by freezing and storage. Though some redistribution of enzymes takes place, meaningful subcellular fractions can be obtained after storage of fresh and postmortem brain tissues.

Acetylcholinesterase↗
PubMed1992

Prostaglandin profile and synthetic capacity of the colon: comparison of tissue sources and subcellular fractions.

Although there has been intense interest in the physiology and pathophysiology of prostaglandins (PGs) synthesized in the colon, little is known about the PG profile and synthetic capacity of different tissue sources and subcellular fractions as enzyme sources. Subcellular fractions prepared from the mucosa and muscle layer of rat colon were incubated with or without exogenous arachidonic acid ([3H]20:4n-6) for 30 min. In experiments with exogenous [3H]20:4n-6, the prostaglandin synthetic capacity of the colonic muscle layer was significantly higher than that of the mucosa. Among the subcellular fractions, microsomes had the highest PG synthetic capacity in both mucosa and muscle. The major PG product was PGI2 and PGD2 in the mucosal microsomes and PGI2 and PGE2 in the muscularis microsomes. However, production of PGI2 in the mucosa and PGE2 in the muscle was significantly reduced in the fractions containing both cytosol and microsome, resulting in an alteration of the PG profile. Substrate availability (exogenous vs endogenous supply) appears to influence the PG profile of the colon. In the colonic mucosa with exogenous [3H]20:4n-6, the production of PGI2 was 5 times higher than that of PGE2, whereas the production of PGE2 was twice higher than that of PGI2 in experiments with endogenous 20:4n-6. These observations indicate: 1) different PG profile and synthetic capacity of tissue sources and subcellular fractions; 2) alteration of PG profile due to the variation of 20:4n-6 availability. Thus, the outcome of experiments on the physiological role of PG in the colon may be determined, in part, by the tissue source and subcellular fraction selected for analysis. The present study also suggests that the variation of substrate availability in physiological and pathophysiological processes may affect the PG profile of the colon.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
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↗
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↗
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↗
PubMed1979

Isolation of synaptic junction-enriched fraction from the forebrain of day-old chickens. Preparation and characterization of chick forebrain subcellular fractions.

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighed with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chick-brain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (sds). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and -insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.

Animals↗
PubMedDate not supplied

[Distribution of gamma-hexachlorcyclohexane in subcellular fractions of the liver and brain].

The character of the gamma-HCCH distribution in the subcellular fractions of the liver and brain of albino rats with peroral threefold introduction of the compound in doses of 34 mg/kg (1/5 LD50) and in a dose of 1.7 mg/kg (1/100 LD50) for a space of 1, 3 and 6 months was investigated. It was found that gamma-HCCH penetrates the cells of the liver and brain and is non-uniformly distributed among subcellular fractions, viz. nuclear, mitochondrial and supernatant. Both in acute and chronic experiments the greatest amounts of the preparation were found in the nuclear fraction of the cell. The established features distinguishing the accumulation and distribution of the preparation in the subcellular fractions of the liver and brain explain the previously elicited biochemical and physiological shifts occurring in the organism after entrance of gamma-HCCH thereinto.

Animals↗
PubMed1992

Effect of different diurnal lighting conditions on serotonin content in the pig pineal gland. Subcellular fractions of the tissue.

Serotonin content in the pineal glands and in their subcellular fractions of the pig housed under natural and limited to circa 2.5 hours per day lighting conditions has been examined spectrofluorometrically. The shortened circadian light phase caused dramatically high drop of serotonin content in the evening (1900-2000). Correlation between presence of dense bodies in subcellular fractions and content of serotonin has been not observed.

Animals↗
PubMed1976

Proteolytic activity of subcellular fractions from Streptomyces griseus no. 45-H.

Subcellular fractions were prepared from Streptomyces griseus No. 45-H at different stages of life cycle, and their proteolytic activity was examined. The highest proteolytic activity was found in the 24- and 72- h-old vegetative hyphae, the lowest in the resting spores. Spores contained about 9--30% of the proteolytic activity of vegetative cells. At the age of 16 h about 80%, at 26 h 70%, at 72 h 40%, and in spores about 60% of the proteolytic activity was particulate. The greatest part of the proteolytic activity could be inhibited by EDTA, lower levels of serine and sulfhydryl protease activities were detected in the cell-free extracts of vegetative cells.

4-Chloromercuribenzenesulfonate↗
PubMed1975

Essential fatty acid deficiency: metabolism of 20:3(n-9) and 22:3(n-9) of major phosphoglycerides in subcellular fractions of developing and mature mouse brain.

Essential fatty acid deficiency was initiated in young and mature mice. The metabolism of 20:3(n-9) and 22:3(n-9) in brain subcellular fractions was followed after the mice were switched from the deficient diet to a corn oil supplemented diet. After switching to the supplemented diet, the proportions of (n-9) polyunsaturated fatty acids in brain in both groups of mice decreased with time. The rate of disappearance of (n-9) polyunsaturated fatty acids was faster in the young groups than in the mature group. In the developing mice, the half-linves of the (n-9) polyunsaturated fatty acids in the total ethanolamine phosphoglycerides of brain microsomal, synaptosomal, and myelin fractions were 3, 10, and 15 days respectively. In the mature group, the half-lives for 20:3(n-9) in diacyl-glycerophosphorylethanolamine of microsome, synaptosome, and myelin fractions were 8-10, 10, and 22 days, respectively; and the half-lives for 22:3(n-9) in alkenylacyl-glycerophosphorylethanolamine of the same subcellular fractions were 8-12, 28, and rate of disappearance of 20:3(n-9) in brain was faster in the diacyl-glycerophosphorylethanolamine than in the alkenylacyl-glycerophosphorylethanolamine. These results demonstrate that the metabolism of (n-9) polyunsaturated fatty acid in brain phosphoglycerides during recovery from essential fatty acid deficiency not only varies with age, but also depends upon individual phosphoglycerides present in each subcellular fraction.

Aging↗
PubMed1975

Insulin degradation by isolated fat cells and their subcellular fractions.

Isolated frt cells and purified subcellular fractions of fat cells have been shown to degrade insulin to biologically inactive trichloroacetic-acid-soluble fragments. Further study of this activity has revealed the following characteristics: 1 Most of the insulin-degrading enzymes are intracellular, inaccessible to insulin or trypsin when fat cells are intact. More that 90 per cent of the recovered activity is found in the high-speed supernatant (cytosol) when cell fractionation studies are performed. 2. The plasma membrane contains significant insulin-degradative capacity, as shown by tryptic digestion of intact cells and cell fractionation. 3. The pH optimum of the cell-membrane insulin-degrading site is more acid than that of the cytosol activity, but the tow enzyme systems are similar with regard to substrate specificity, response to metabolic inhibitors, and elution volume of degradation products on gel filtration. 4. The plasma-membrane-degrading activity differs from the specific insulin-binding site with regard to saturation kinetics, optimum temperature, substrate specificity, sensitivity to sulfhydryl-blocking agents, and trypsin snesitivity.

Adipose Tissue↗
PubMed1976

Ca-activated ATPase activity in subcellular fractions of mouse pancreatic islets.

Ca-stimulated ATPase activity has been demonstrated in homogenates of mouse pancreatic islets. On subcellular fractionation Ca-ATPase activity was found in secretory granules, mitochondria, and microsomes, but not in the postmicrosomal fractions. Highest specific activity was found in the granules. In all active subcellular fractions two Km(Ca) values for Ca-ATPase around 7.0 X 10(-6) and 1.8 X 10(-7) M were estimated. Assuming an ATP hydrolysis:Ca pumping ratio of 1:2, the highest capacity for active Ca transport was found in secretory granules and mitochondria. Concentrations of 40 mM or higher of Na and 10(-5) M cyclic AMP inhibited Ca-ATPase in all subfractions. Caffeine at a concentration of 10 mM inhibited Ca-ATPase significantly in secretory granules and microsomes. Also MG-ATPase activity was demonstrated in the various subfractions. This activity was compared with that of Ca-ATPase at identical concentrations of free metal ions and in the absence or presence of various inhibitors. It was concluded that high-affinity Ca-ATPase and Mg-ATPase are two different enzymic entities. Ca-ATPase may tentatively be assumed to participate in active transport of Ca between intracellular compartments and to constitute a Ca-accumulating system which returns the cytosolic free Ca concentration to the resting state after stimulation of the beta-cells by secretagogues. This enzyme may therefore play a significant role in regulation of insulin release.

4-Chloromercuribenzenesulfonate↗
PubMed1979

The subcellular fractionation of embryonic chick tendon and cartilage cells: a re-examination.

A re-examination of the subcellular fractions obtained from matrix-free chick tendon and cartilage cells has been made since the discovery that three out of four of the micrographs of chick tendon microsomal fractions published in an earlier paper from this laboratory were not authentic. The present studies demonstrate that by using the procedures previously reported it is possible to isolate microsomal and submicrosomal fractions from tendon and cartilage cells which exhibit typical morphology when examined by electron microscopy. These observations are consistent with our original biochemical characterization of subcellular fractions, which we know to be valid. Other publications from this laboratory in which these fractionation procedures have been applied to studies of collagen biosynthesis are in no way compromised, and indeed, most of our data have been confirmed by several other laboratories.

Animals↗