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PubMed1979

Studies in the rat on the hepatic subcellular distribution and billary excretion of lithocholic acid.

1. A compartmental model has been used to derive the in vivo subcellular distribution of lithocholic acid in rat liver. The model is based on the values of the partition coefficients for the distribution of lithocholic acid between subcellular fractions and buffer. It also permits calculation of the amount of lithocholic acid which is in free solution in cytosol. 2. The hypothesis that the rate of biliary excretion of a bile acid depends on the proportion in free solution was investigated by comparing the rates of biliary excretion of lithocholic acid and glycocholic acid. The rate for lithocholic acid was substantially less than for glycocholic acid while the percentages of each bile acid in free solution were 0.8% and 10%, respectively. 3. The validity of the model was supported by the observation that the amounts of lithocholic acid predicted to be present in the nuclear and cytosolic fractions were similar to the amounts found after intravenous injection of the bile acid.

Animals↗

PubMed1980

Subcellular distribution of ascorbate in bovine adrenal medulla. Evidence for accumulation in chromaffin granules against a concentration gradient.

The subcellular distribution of ascorbate and catecholamines has been studied in homogenates of bovine adrenal medulla and cortex. 1. The recovery of the vitamin was found to be 4.10 +/- 0.22 and 9.57 +/- 1.37 mumol/g wet weight for the medulla and cortex, respectively. A major fraction (34.4%) of the vitamin was recovered in the particulate fraction of the medulla as compared to about 8% in the corresponding fraction of the cortex. In comparison, 78.9% of the catecholamines were found in the particulate fraction of the medulla. 2. Analytical differential centrifugation of medulla homogenates revealed a sedimentation profile of ascorbate which was identical to that obtained for noradrenalin and adrenalin. The co-sedimentation of these compounds indicates that ascorbate is an essential component of the heavy as well as the light population of chromaffin granules. The stoichiometry of catecholamines to ascorbate was approx. 25:1 in both subpopulations. 3. Based on an estimated volume fraction of approximately 13% for the chromaffin granules, as determined morphometrically (Kryvi, H., Flatmark, T. and Terland, O. (1979) Eur. J. Cell Biol. 20, 76-82), a concentration gradient (chromaffin granules:cytosol) of approx. 4 was estimated for ascorbate in the cells of adrenal medulla. 4. No ascorbate 2-sulfate was detected in any of the subcellular fractions isolated, and the content of dehydroascorbate in isolated chromaffin granules was less than 1% of the total ascorbate value.

Adrenal Cortex↗

PubMed1981

Subcellular distribution of enzymes in the yeast saccharomycopsis lipolytica, grown on n-hexadecane, with special reference to the omega-hydroxylase.

The subcellular localization of the omega-hydroxylase of Saccharomycopsis lipolytica was assessed by the analytical fractionation technique, originally described by de Duve C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F., and hitherto little, if at all, applied to yeasts. Protoplasts were separated in six fractions by differential centrifugation. Some of these fractions were further fractionated by density gradient centrifugation. The distribution of omega-hydroxylase and 15 other constituents chosen as possible markers of its subcellular entities. (1) Mitochondria were characterized by particulate malate dehydrogenase, particulate Antimycin A-insensitive NADH-cytochrome c reductase, oligomycin-sensitive and K+-stimulated ATPase pH 9. (2) Most if not all of the catalase and urate oxidase is peroxisomal. (3) Free ribosomes account for most RNA. (4) Nucleoside diphosphatase is for the first time reported in a yeast and appears to belong to an homogeneous population of small membranes. (5) The soluble compartment contains magnesium pyrophosphatase, alkaline, 5'-nucleotidase and part of the NADH-cytochrome c reductase. Latent arylesterase and ATPase pH 7 have an unspecific distribution. Alkaline phosphodiesterase I has not been detected.

Alkanes↗

PubMed1981

Analytical subcellular fractionation of rat pituitary homogenates, with special reference to prolactin proteolysis by lysosomes.

Prolactin proteolysis by rat pituitary homogenates was assayed by measuring the release of trichloroacetic acid-soluble peptides from 125I-labelled rat prolactin. There was a distinct optimum at pH 4.3, with only trace amounts of activity at neutral and alkaline pH. Rat pituitary homogenates were subjected to analytical subcellular fractionation by sucrose density gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their respective marker enzymes, including: cytosol (lactate dehydrogenase); plasma membrane (5'-nucleotidase); lysosomes (N-acetyl-beta-glucosaminidase, beta-glucuronidase); mitochondria (particulate malate dehydrogenase); endoplasmic reticulum (neutral alpha-glucosidase); prolactin granules (radioimmunoassayable prolactin). Acid prolactin protease had a similar distribution to the lysosomal marker enzymes. A localisation of the activity to lysosomes was confirmed by subcellular fractionation experiments in which the lysosomes were selectively disrupted with low concentrations of the membrane perturbant, digitonin. Experiments with specific inhibitors of the lysosomal cathepsins indicate that both cathepsins B and D are implicated in pituitary prolactin proteolysis.

Animals↗

PubMed1983

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture.

The properties and subcellular localization of the elastase-like activities of smooth muscle cells cultured from pig aortas have been investigated. Homogenates of the cells hydrolysed N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, a synthetic substrate for elastases, with a distinct pH optimum of 8.2 and hydrolysed insoluble elastin with a distinct pH optimum of 8.5. Both enzyme activities were directly proportional to the concentration of homogenate in the assay mixture. The activities toward both substrates were inhibited by phenylmethylsulphonyl fluoride and were therefore probably due to a serine peptidase(s). The activities were also inhibited by EDTA and, in a dose-related manner, by alpha 1-antiprotease. Pepstatin, which inhibits cathepsin D, and leupeptin, which inhibits cathepsin B, did not significantly inhibit the elastase-like activities in these cells. The cells were homogenized and a post-nuclear supernatant subjected to sucrose density gradient centrifugation. The distribution of elastase-like activity toward both substrates was similar to that of the plasma membrane marker 5'-nucleotidase, and distinct from those of marker enzymes for the other organelles. Cells were also homogenized with digitonin, which selectively increases the equilibrium density of the plasma membrane. The equilibrium densities of both 5'-nucleotidase and of the elastase-like activities were increased considerably, confirming the plasma membrane localization of the elastase-like activities. The subcellular localization of the elastase-like activities of arterial smooth muscle cells is therefore consistent with a role for them in the degradation of elastin in the normal arterial wall and in atherosclerotic lesions.

Animals↗

PubMed1984

Subcellular localization of prostaglandin E2 binding sites in bovine adrenal medulla.

Prostaglandins E1 and E2 are specifically bound by particulate fractions from bovine adrenal medulla. The subcellular localization of these binding sites has been investigated by comparing their distribution in subcellular fractions obtained by differential and gradient centrifugation to those of marker enzymes for various organelles. Prostaglandin E2 binding sites were purified about 16-fold with respect to the homogenate in a fraction which was highly enriched in plasma membranes on the basis of the activities of the marker enzymes acetylcholinesterase and calcium-dependent ATPase, which were both purified by about 12-fold in this fraction. The plasma membrane fraction contained relatively low activities of marker enzymes for mitochondria (monoamine oxidase), lysosomes (acid phosphatase), endoplasmic reticulum (glucose-6-phosphatase), Golgi (galactosyl transferase) and chromaffin granule membranes (dopamine beta-hydroxylase). The only other fractions enriched in prostaglandin E2 binding sites were those for the endoplasmic reticulum and the Golgi, in which the binding sites were purified about 4-fold and 7-fold, respectively. This is probably due mainly to contamination with plasma membranes, since calcium-dependent ATPase and acetylcholinesterase were each purified to a similar extent in these two fractions. These data suggest that the high-affinity prostaglandin E2 binding sites of the adrenal medulla are localized primarily on the plasma membranes of the medullary cells.

Adrenal Medulla↗

PubMed1985

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts.

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.

Cell Line↗

PubMed1986

Subcellular distribution and selectivity of the protein-binding component of the recognition system for sex-hormone-binding protein-estradiol complex in human decidual endometrium.

In order to assess the subcellular distribution of the protein-binding component of the recognition system for sex-hormone-binding protein-estradiol complex (Strel'chyonok, O.A., Avvakumov, G.V. and Survilo, L.I. (1984) Biochim. Biophys. Acta 802, 459-466), plasma membranes and other subcellular fractions were prepared from homogenate of human decidual endometrium (8-12 weeks of pregnancy). Specific binding of 125I-labeled sex-hormone-binding protein complexed with estradiol was found preferentially in the plasma membrane fraction. The protein-binding component of the recognition system was found to specifically bind sex-hormone-binding protein complexed with estrogens (estradiol, estriol, estrone, with close affinities) but not with androgens (testosterone and 5 alpha-dihydrotestosterone). Specific membrane binding of 125I-labeled sex-hormone-binding protein complexed with 17 alpha-pregna-2,4-dien-20-yn-[2,3-d]isoxazol-17-ol (danazol) was also detected. This allowed us to suggest a putative mechanism for the danazol pharmacological action on endometrium which involves the interaction of the steroid-protein complex with the plasma membrane of endometrial cells. It is proposed that sex-hormone-binding protein takes part in the guided transport of steroids into endometrial cells.

Cell Fractionation↗

PubMed1986

Subcellular and organ distribution of cholesterol epoxide hydrolase in the rat.

The subcellular and organ distributions of microsomal epoxide hydrolases measured with cis-stilbene oxide and cholesterol 5,6 alpha-epoxide as substrates have been investigated. These two enzyme activities were found to have essentially the same subcellular distribution, with the highest total and specific activities localized in rough and smooth endoplasmic reticulum. Among the tissues studied (i.e., liver, kidney, lung, testis, spleen, brain and intestinal epithelium), the highest specific activities were recovered in liver microsomes, where the activities were at least 5-fold greater than in any of the other microsomal preparations.

Animals↗

PubMed1988

Subcellular localization of non-specific carboxylesterases, acylcarnitine hydrolase, monoacylglycerol lipase and palmitoyl-CoA hydrolase in rat liver.

The subcellular distribution and sidedness on the membranes of four chemically and genetically distinct esterases (esterases ES-3, ES-4, ES-8, ES-15) in rat liver was investigated using selective substrates. (1) Rat liver homogenate was divided into nine subcellular fractions by differential centrifugation techniques. The cell fractions were assayed for the enzymatic hydrolysis of acetanilide (ES-3), propanidid, palmitoyl-CoA and monopalmitoylglycerol (ES-4), methyl butyrate and octanoylglycerol (ES-8), and decanoylcarnitine (ES-15). With all substrates, the highest specific activities were found in the rough and smooth endoplasmic reticulum fractions. This localization of the esterases was confirmed by labelling the cell fractions with the specific, covalently binding inhibitor bis(4-nitro[14C]phenyl) phosphate. The enzymatic hydrolysis of the palmitoyl esters in differing cell fractions did not completely parallel that of propanidid. This confirms the well-known existence of palmitoyl-CoA hydrolases other than esterase ES-4. (2) Density gradient fractionations with crude mitochondria indicated that a low amount of at least one of these carboxylesterases was an integral part of these organelles too. (3) Proteinase treatment reduced the non-specific esterase activities as well as lipase activities versus dioctanoylglycerol, acylcarnitines and palmitoyl-CoA only in detergent-disrupted microsomal vesicles. This might indicate a lumenal orientation of these enzymes. However, of the charged substrates palmitoylcarnitine and palmitoyl-CoA only the latter one showed the typical latency to be expected for a hydrolysis in the lumen of the endoplasmic reticulum.

Animals↗

PubMed1990

Subcellular distribution of selenoproteins in the liver of the rat.

After in vivo labeling with [75Se]selenite, the intracellular distribution of selenoproteins in the liver was investigated in selenium-adequate and selenium-deficient rats. In the subcellular fractions, which were obtained by differential centrifugation, the proteins were separated by means of SDS-PAGE and the selenium compounds were identified via their 75Se activity. In this way twelve selenium-containing proteins or protein subunits with molecular weights between 12,100 and 75,400 were found. Glutathione peroxidase was concentrated in the cytosol and in the mitochondria. With the newly detected selenoproteins, some were enriched in the cytosol, one was mainly found in the nuclear fraction and some, which were present mainly in the mitochondrial and microsomal fractions, are most probably membrane-bound. In the liver of selenium-depleted rats the selenium administered was used predominantly to restore the levels of some of the newly found selenoproteins, while in the liver of selenium-adequate animals most of the selenium retained was incorporated into the glutathione peroxidase. The differences in the distribution among the subcellular fractions and the specific incorporation of the element in selenium deficiency into certain compounds suggest that there are several metabolic pathways for selenium and that the selenoproteins are involved in several different processes of intracellular metabolism.

Animals↗

PubMed1991

Antioxidant role and subcellular location of hypotaurine and taurine in human neutrophils.

The subcellular location of taurine, and its precursor, hypotaurine, within human neutrophils has been examined by nitrogen cavitation, Percoll-gradient centrifugation and HPLC analysis. Hypotaurine and taurine were found to reside within the cytosolic compartment of the cell. The ratio of taurine to hypotaurine is approx 50:1. The cytosolic concentration of taurine is approx. 50 mM. The concentration of hypotaurine decreased by 80% when resting neutrophils were converted into actively respiring cells by exposure to opsonized zymosan. These results prompted in vitro studies on the antioxidant properties of hypotaurine. We demonstrate by EPR spectroscopy that hypotaurine competes with 5,5'-dimethyl-1-pyrroline N-oxide) (DMPO) for hydroxyl radicals, and that it is the sulfinyl group which confers hydroxyl radical scavenging activity to it. Following its exposure to hydroxyl radicals, two oxidation products were isolated by HPLC, one of which has been identified as taurine. The biological roles of hypotaurine and taurine in the neutrophil are discussed with respect to their antioxidant properties and subcellular location within the cell.

Antioxidants↗

PubMed1982

The influence of age, sex and genotype on the subcellular distribution of hepatic aldehyde dehydrogenase activity in the mouse.

1. The influence of age on hepatic aldehyde dehydrogenase (AlDH) was studied in both sexes of two selectively bred lines of mice (Mus musculus). 2. Whole liver AlDH activity increased from the 60-day-old level linearly up to 200 days of age. The enzyme activity of the females remained at the 200-day-old level, but in males. AlDH continued to increase up to 400 days of age. Males had higher AlDH activity than females. 3. The AlDH activities of 3 subcellular fractions: cytosol, mitochondria and microsomes also increased with age, but were more dependent on sex and genotype than was the whole liver activity. 4. It was concluded that AlDH activity increases with age in mouse liver but it was influenced greatly by sex, genotype and subcellular fraction studied.

Age Factors↗

PubMed1983

Tissue and subcellular distributions of cholecalciferol 25-hydroxylase: cytochrome P-450D25-linked monooxygenase system.

Cholecalciferol (vitamin D3) 25-hydroxylase activity and its tissue and subcellular distributions were studied using rabbit, rat, bovine, chick, duck, guinea-pig and yellowtail. Vitamin D3 25-hydroxylase was found not only rabbit and bovine livers, kidneys and intestines, but also in lungs, pituitary glands, adrenocortices, ovaries, adipose tissues, blood vessels, brains and testes. Subcellular fractions of rabbit livers were examined for the presence of this activity and it was detected in the nuclear membranes, smooth and rough endoplasmic reticula, Golgi membranes and mitochondrial membranes. The activity in microsomes was induced by sodium phenobarbital, but not by beta-naphthoflavone and 3-methylcholanthrene, and was inhibited by isocyanide and metyrapone as inhibitor of cytochrome P-450.

Animals↗

PubMed1984

The lipid composition of ovine placental tissue homogenate and its subcellular fractions.

Homogenates of the placental tissue of near term sheep were separated by differential centrifugation into mitochondrial, microsomal and cytosolic fractions. The relative proportions of the major neutral lipids and phospholipids, together with their fatty acid compositions, were determined in the homogenates and in each subcellular fraction. The cytosolic fraction contained the highest proportion of cholesteryl esters (CEs) and these possessed a fatty acid composition markedly different from the total CEs extracted from the homogenate. Both the mitochondrial and microsomal fractions contained significant proportions of solvent front phospholipid (SFP) and whereas the mitochondrial SFP displayed the relatively unsaturated fatty acid composition characteristic of diphosphatidylglycerol (cardiolipin), the fatty acids of the microsomal SFP were distinctly more saturated. These results are compared with those obtained from other mammalian tissues, both ruminant and non-ruminant, and discussed in terms of the function of the components of the subcellular fractions.

Animals↗

PubMed1976

Accumulation of chlorporomazine by subcellular fractions of rat brain.

1. The study of the in vitro accumulation of [3H] chlorpromazine that a state of rapid equilibrium was reached for subcellular particles from cortex, midbrain and hindbrain of rats. 2. This accumulation was not saturable by increasing the concentration of chlorpromazine, and the relationship between accumulation and concentration was not modified by temperature, preincubation with high concentration of unlabelled chlorpromazine or by lowering the fraction concentration. 3. The highest accumulation of radioactivity was seen in the midbrain region and the lowest in the hindbrain. 4. In the cortex and midbrain the membrane fraction showed the highest accumulation. 5. The effect of pH on the accumulation indicated that a significnat rise was seen between pH 8 and 5. 6. This factor could also be explained in terms of solubility of the drug in membranes. 7. Studies on the metabolic fate of the drug during incubation indicated that there was a fairly rapid breakdown during incubation, the highest being seen with mitochondrail fractions and the least with membrane procedures. 9. It is concluded that accumulation of chlorpromazine by subcellular fractions in vitro may be associated with solubility in membranes and that the picture may be complicated by the presence of breakdown products.

Animals↗

PubMed1984

Subcellular distribution of pinazepam and its metabolite N-desmethyldiazepam in rat liver.

The distribution in subcellular fractions of pinazepam (Pz) and its metabolite N-desmethyldiazepam (N-Dz) was studied after a single oral administration of Pz (20 mg/kg body wt) to rats. Animals were killed 2 min and 6 hr after dosing. The amounts of the parent drug and its metabolite retrieved in the microsomal fraction were small, but significantly higher than those in the nuclear, mitochondrial and soluble fractions. The cytosol contained the lowest percentages of both drugs. No substantial difference was found in the subcellular distribution pattern of both Pz and N-Dz 2 min and 6 hr after administration.

Animals↗

PubMed1989

Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport.

The importance of subcellular storage, nerve impulse rate and pattern, and feedback regulation, as well as resupply by axonal transport for the release of noradrenaline and neuropeptide Y-like immunoreactivity, was studied in the blood perfused pig spleen in vivo. Vasoconstrictor responses were recorded as perfusion pressure changes. Subcellular fractionation experiments using sucrose density gradients showed a bimodal distribution of noradrenaline (peak concentrations at 0.8 and 1.1 M sucrose) while only one main peak of neuropeptide Y was present (at 1.1 M sucrose). Overflow suggesting release of noradrenaline and neuropeptide Y-like immunoreactivity could be detected after 10 s stimulation at 10 Hz. The ratio for the output of noradrenaline and neuropeptide Y upon continuous nerve stimulation in control animals decreased with frequency. After inhibition of noradrenaline reuptake by desipramine the vasoconstrictor response and noradrenaline output were enhanced while the corresponding overflow of neuropeptide Y was reduced by 50% at 0.5 Hz. Stimulation with the irregular or regular bursting patterns at high frequencies caused larger perfusion pressure increase and relative enhancement of neuropeptide Y output compared to noradrenaline than a continuous stimulation both before and after desipramine treatment. A similar fractional release per nerve impulse was calculated both for [3H]noradrenaline (5.6 +/- 1.0 x 10(-5) and neuropeptide Y (7.3 +/- 0.3 x 10(-5). After reserpine treatment combined with preganglionic denervation the vasoconstrictor responses were more long-lasting, neuropeptide Y release was enhanced while noradrenaline content and release were reduced by 99%. The difference in neuropeptide Y overflow between continuous and bursting types of stimulation was smaller after reserpine treatment. After prolonged intermittent stimulation with regular bursts (20 Hz) for 1 h the splenic content of neuropeptide Y was reduced by 58%, while no change was observed for noradrenaline. The maximal perfusion pressure increase upon prolonged nerve stimulation after reserpine was similar in control and reserpine-treated animals, but after reserpine the vasoconstrictor response and neuropeptide Y release were subjected to fatigue. Ligation experiments of the splenic nerves revealed the splenic neuropeptide Y content was resupplied by axonal transport with a calculated total tissue turnover time of 11 days. In contrast, axonal transport contributed only to a marginal extent for the resupply of noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗