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PubMed1982

Characterization of [L-3H]aspartate binding to chick retinal subcellular fractions.

Binding of [L-3H]aspartate to synaptic receptors was examined in membranes from whole chick retina and subcellular fractions enriched with photoreceptor terminals (P1) or terminals from the inner plexiform layer (P2), Na+-independent, stereospecific, high affinity binding was concentrated in the P1 fraction (Kb = 40 nM). P2 fraction also showed a high affinity binding system (KB = 11.8 nM) with lower capacity than in the P1 fraction. Comparative studies with [L-3H]-aspartate, [L-3H]-glutamate and [H3]-kainate showed that L-aspartate and L-glutamate are the most potent inhibitors of the binding of the three ligands. Aspartate and glutamate binding were effectively displaced by N-methyl-DL-aspartate and alpha-amino adipate, whereas only [3H]-glutamate binding was significantly inhibited by glutamate-diethyl-ester. Kainic acid exhibited negligible affinity for aspartate and glutamate binding sites. Results indicate the presence of different receptors for glutamate and aspartate in both plexiform layers of the retina.

Animals↗

PubMed1979

De novo fatty acid synthesis and fatty acid elongation catalyzed by subcellular fractions from hog and human aorta.

De novo synthesis and mitochondrial elongation of fatty acids have been demonstrated in subcellular fractions from hog and human aorta. Microsomal fatty acid elongation has been shown in hog aorta. The activity catalyzing the formation of fatty acids from acetyl and malonyl CoA was associated with a high molecular weight complex in the 6 x 10(6) g x min supernatant fraction. The principal product was palmitic acid. Some myristic and stearic acids were also formed. One elongation system was associated with protein which sedimented between 4500 g x min and 150,000 g x min. It used acetyl CoA but not malonyl CoA, and NADH was the preferred reducing agent. Radioactivity from acetyl CoA was incorporated into many fatty acids. In hog aorta a second elongation system was found associated with protein which sedimented at 6 x 10(6) g x min. It used malonyl CoA preferentially as substrate and either NADH or NADPH as reducing agent.

Acetyl Coenzyme A↗

PubMed1980

Cytotoxic and cytolytic action of the subcellular fraction (60-F) of live hemolytic streptococci against Ehrlich ascites carcinoma cells.

The cytotoxic effect of 60-F, a subcellular fraction obtained from 0.5 approximately 0.6 saturation of cell-free extract of live hemolytic streptococci (Su-strain) with ammonium sulfate on Ehrlich ascites carcinoma cells was studied by the assay of incorporation of nucleosides and amino acid, and by the in vitro-in vivo cytocidal experiment. Additionally, the cytolytic effect of 60-F was examined by the assay of RNA and DNA released from the tumor cells. It was found that the tumor cells treated with 60-F did not take up thymidine, uridine, or leucine, that 60-F eradicated the transplantability of tumor cells in mice, and that 60-F released RNA but not DNA from the tumor cells.

Animals↗

PubMed1989

Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions.

1. The effect of chronic ethanol treatment on the level of lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that after chronic ethanol treatment the level of Fe/ADP-ascorbate-induced lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. Dilution of the homogenates prevented depressive effect of ethanol on lipid peroxidation. 3. Chronic ethanol treatment did not affect the intensity of the Fe/ADP-ascorbate-induced process in rat liver mitochondria and microsomes. 4. Peroxidative alteration of the liver lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). It was shown that ethanol did not increase the level of u.v. absorption of lipids from mitochondria and microsomes. Chronic alcohol treatment did not influence the steady-state concentration of malonic dialdehyde in the whole liver homogenate. 5. The data obtained indicate that cytosol from the ethanol treated rat liver contains a factor(s) which prevents Fe/ADP-ascorbate-dependent lipid peroxidation in biological membranes.

Adenosine Diphosphate↗

PubMed1981

Ethanol- or acetone-pretreatment of mice strongly enhances the bacterial mutagenicity of dimethylnitrosamine in assays mediated by liver subcellular fraction, but not in host-mediated assays.

The activation of dimethylnitrosamine (DMN) to a bacterial mutagen in liver subcellular fraction and in intrasanguinous host-mediated assays was studied, in particular the effect of pretreatment of the animals with ethanol or acetone. Salmonella typhimurium TA 92 was much more sensitive to DMN mutagenicity than TA 100 and TA 1535 or Escherichia coli WP2uvrA and was used for the main part of the study. Noteworthy, in part already known, features of the in vitro activation are the relatively low pH optimum (pH 6-6.4), the non-linear dose-mutagenic response-relationship and the relatively high doses of DMN required for activation with control preparations. Pretreatment of mice with ethanol or acetone greatly reduced the minimal mutagenically effective concentration of DMN in the in vitro assay. Pretreatment with Aroclor 1254, an inducer frequently used in mutagenicity research, showed little effect when used alone, but reduced the potentiation by acetone. The results of the host-mediated assays substantially differed from those of the in vitro activation assays (a) in the relatively low dose of DMN required for mutagenicity to occur and (b) in the lack of potentiation by acetone-or ethanol-pretreatment. Acetone even led to a marginal decrease in mutagenicity. As a possible explantation for this apparent discrepancy were assume that with the in vitro system the activity of the dilute metabolizing system is limiting for the activation of DMN and induction therefore will increase the mutagenicity, whereas in vivo DMN is quantitatively metabolized in both induced and non-induced animals. The results show that caution has to be taken in the interpretation from in vitro results to the in vivo situation. In particular our in vivo experiments do not support the hypothesis that the induction by ethanol of an activating system with a low Km (which would strongly activate traces of DMN ingested with many foods) is one of the reasons for the increased risk of liver tumors in alcoholics.

Acetone↗

PubMed1989

A study of 5'-nucleotidase activity in subcellular fractions of rat cerebellum after the administration of the convulsant 3-mercaptopropionic acid.

The activity of 5'-nucleotidase in cerebellum subcellular fractions after the administration of the convulsant 3-mercaptopropionic acid was studied. This membrane enzyme presented an increase in its activity in certain fractions containing nerve endings and microsomes (Mic20, Mic100) in seizure and postseizure periods. 5'-nucleotidase activity in nuclear and myelin fractions did not show differences between the control and treated fractions. On the other hand, a decreased activity in the crude mitochondrial fraction and in a nuclear subfraction was found. It is suggested that the changes in the enzyme activity in some cerebellum fractions might be related to structural alterations previously observed in this laboratory and with the anticonvulsant actions of adenosine.

3-Mercaptopropionic Acid↗

PubMed1990

Haloperidol-sensitive (+)[3H]SKF-10,047 binding sites (sigma sites) exhibit a unique distribution in rat brain subcellular fractions.

The distribution of haloperidol-sensitive (+)[3H]N-allylnormetazocine ((+)[3H]SKF-10,047) binding sites (sigma sites) in subcellular fractions of rat brain homogenates was extensively characterized. In synaptosomal fractions, enriched in choline acetyltransferase activity, sigma sites were present in lower concentrations than in whole brain homogenates. On the other hand, microsomal and myelin fractions were found to be enriched in sigma sites. A similar pattern of enrichment was seen for 5'-nucleotidase activity, a general plasma membrane marker. However, subsequent experiments in which microsomes were subfractionated on linear sucrose gradients led to the recovery of sigma sites over a significantly lower density range than 5'-nucleotidase activity or ATP-stimulated [3H]ouabain binding, an additional plasma membrane marker. In addition, previously reported distributions of a number of other subcellular markers, including those for endoplasmic reticulum, were found to contrast with the observed distribution of sigma sites. It is concluded that rat brain sigma sites are not concentrated at synaptic regions of plasma membrane. However, the possibility that sigma sites are localized to specialized areas of nonsynaptic plasma membrane cannot be excluded.

5'-Nucleotidase↗

PubMed1994

One- and two-electron reduction of quinones by rat liver subcellular fractions.

NAD(P)H-quinone (menadione, Trolox C quinone, and alpha-tocopherol quinone) reductase activity of rat liver subcellular fractions was observed optically at 340-400 nm, and oxygen radical generation was demonstrated using the ESR spin trap, 5,5'-dimethyl-1-pyrroline-1-oxide. NAD(P)H-menadione reductase activity of the fractions decreased in the order: cytosol > microsomes > plasma membranes. Although more than 65% of the activity of microsomes and plasma membranes was inhibited on the addition of dicoumarol, no change in the menadione-mediated formation of oxygen radicals by either fraction was observed. As judged from the intensity of ESR signals, the menadione-mediated oxygen radical formation by plasma membranes was only one-tenth as great as that by microsomes. No generation of oxygen radicals in the NAD(P)H-menadione reductase reaction by cytosol was found, and the activity was abolished in the presence of dicoumarol, an inhibitor of DT-diaphorase. It is concluded that plasma membranes reduce quinones by way of two-electron transfer and that the activity may prevent cellular quinone toxicity. NAD(P)H-alpha-tocopherol quinone reductase activity was confirmed in all cellular fractions [Hayashi et al. (1992) Biochem. Pharmacol. 44, 489-493] and this activity was also inhibited by dicoumarol, suggesting that it was due to DT-diaphorase.

Animals↗

PubMed1986

Ischemia modifies protein distribution in gerbil brain subcellular fractions.

This work deals with changes in sedimentation of proteins in subcellular fractions isolated from the ischemic brain of Mongolian gerbils. We analysed protein content and the activity of marker enzymes in P2, S2, M and S3 subfractions isolated after 10 min ischemia, ischemia combined with apnea or 1 h later, after recirculation. On the basis of protein balance, the distribution of marker enzymes and electrophoretic analysis of proteins in brain subfractions it was shown that in ischemia some fraction of proteins which are normally cytosolic and microsomal, cosediment with the crude mitochondrial pellet. This effect, aggravated in ischemia complicated by apnea, is reversible and after 1 h recirculation the protein distribution in the brain normalizes completely.

Animals↗

PubMed2003

Trichomonas vaginalis: identification of a phospholipase A-dependent hemolytic activity in a vesicular subcellular fraction.

Trichomonad total extracts (TTE), or vesicular (P30) and soluble (530) subcellular fractions from 3 pathogenic Trichomonas vaginalis strains (GT-3. GT-13. and GT-15), lysed both human and Sprague-Dawley rat erythrocytes in a time- and dose-dependent manner. The entire hemolytic activity of TTE was located in P30, showing 2 peaks of maximum activity, one at pH 6.0 and another at pH 8.0. in the presence of 1 mM Ca2+. Hemolytic activity on rat erythrocytes was greater at pH 6.0 16.71 +/- 0.33 hemolytic units IHU]/mg/hr to 11.60 +/- 0.24 HU/mg/hr) than at pH 8.0 (3.81 +/- 0.30 HU/mg/hr to 5.75 +/- 0.65 HU/mg/hr). and it was greater than that on human red blood cells at pH 6.0 (2.67 +/- 0.19 HU/mg/hr to 4.08 +/- 0.15 HU/mg/hr) or pH 8.0 (2.24 +/- 0.0 9 HU/mg/hr to 2.81 +/- 0.06 HU/mg/hr). The alkaline and acidic hemolytic activity diminished (60-93% at pH 6.0 and 78-93% at pH 8.0) by the effect of 80 microM Rosenthal's inhibitor, which also inhibited 27-45% and 29-54% trichomonad alkaline and acidic phospholipase A activities, respectively. Vesicles, vacuoles, and hydrogenosomes were rich in P30. Trichomonas vaginalis has a hemolytic PLA, which could be involved in its cytopathogenic mechanism.

Animals↗

PubMed1993

Distribution kinetics of inorganic mercury in the subcellular fractions of fish liver.

The present study tries to find out the kinetics of distribution of mercury in the different subcellular fractions of the liver in a freshwater perch Anabas testudineus over a period of 48 h after a single i.m. injection of [203Hg]mercuric nitrate at a dose of 4 mg/kg body weight. The fish were killed at 15 min, 2 h, 6 h and 48 h post injection. In addition the interaction of this metal with different biomolecules, viz., protein, DNA and RNA, was also investigated. Cytosol was found to be the major site of mercury accumulation. Moderate amounts of accumulation occurred in the nuclear, mitochondrial and microsomal fractions, although varying with time, while the lysosomal fraction did not reveal any spectacular retention of mercury. A significant increase in the protein content of nuclear, mitochondrial, lysosomal and cytosolic fractions was also noticed at different time periods of mercury injection. In the nuclear, microsomal and cytosolic proteins, mercury binding increased more significantly over time than the mitochondrial and lysosomal proteins. A biphasic binding pattern of mercury was seen in nuclear and mitochondrial DNA and mitochondrial and cytosolic RNA.

Animals↗

PubMed1977

Cooperation of various subcellular fractions in protein degradation in vitro.

In order to test the possibilities in protein degradation between cell organelles comparatively, [3H]- and [14C]-leucine short-time labelled subcellular fractions from rat liver were incubated with each other at pH 6.9. All fractions tested were able to degrade short-lived proteins from foreign fractions, whereby the lysosomal supernatant fraction showed the highest proteolytic activity, which declines in the sequence: lysosomes--nuclei--mitochondria--cytosol--microsomes. Short-lived cytosolic proteins were especially suited as substrate for neutral proteases from all other fractions, but also microsomal, mitochondrial and nuclear proteins were well degraded by foreign fractions in comparison with the substrate autoproteolysis. Therefore in vivo manyfold cooperations between several organelles in protein catabolism seem to be possible.

Animals↗

PubMed1978

[Similar distribution of the activities of neutral alpha-glucosidase (gamma-amylase) and glucose-6-phosphatase in subcellular fractions from rat and trout livers].

alpha-glucosidases are cellular enzymes, able to split the polysaccharides into glucose. In subcellular fractions from rat and trout hepatocytes, the distribution patterns of neutral alpha-glucosidase and of glucose-6-phosphatase appear to be very similar, i.e., closely linked to the endoplasmic reticulum, and are somewhat related to the particular glycogen. The data suggest a probable role of neutral alpha-glucosidase in cell physiology and in carbohydrates metabolism.

Amylases↗

PubMed1986

The metabolism of zearalenone in subcellular fractions from rabbit and hen hepatocytes and its estrogenic activity in rabbits.

The in vitro reduction of zearalenone (ZEN) by subcellular fractions from hen and rabbit hepatocytes clearly shows species-specific differences in the cofactor requirements, rate of metabolism and production of metabolites. The presence of NADH as cofactor in the reaction mixtures enhanced only the reducing activity of the microsomal fraction from rabbit hepatocytes, while NADPH enhanced the reducing activities of the cytosolic fraction from rabbit and both the microsomal and cytosolic fractions from hen hepatocytes. Furthermore, we observed that hen hepatocytes metabolize faster and produce beta-zearalenol (ZEL) as the major metabolite, whereas rabbit hepatocytes metabolize ZEN slowly and mainly into alpha-ZEL, the more uterotrophic metabolite. These last findings are closely related to the higher sensitivity to ZEN estrogenic effects observed in rabbits during the toxicity test involving p.o. administration of the mycotoxin to the animals at 3 dosage levels (0.1, 1, 2 mg/kg body wt).

Animals↗

PubMed1988

Studies on protein phosphorylation using subcellular fractions from insulin-treated white adipose tissue of rats.

In these studies the incorporation of 32P into proteins within subcellular fractions, obtained from rat white adipose tissue upon incubation in the presence of [gamma-32P]ATP, was investigated. A stable increase in the activity of protein serine(threonine) kinase in high-speed supernatant fractions was observed following treatment of intact tissue with insulin. Protein kinase activity associated with the plasma membrane fraction of cells was diminished in response to insulin, but the decrease was apparently insufficient to account for increases observed in corresponding supernatant fractions. A range of assay conditions was employed to characterize the insulin-stimulated protein serine(threonine) kinase in in supernatant fractions. The insulin-stimulated protein serine(threonine) kinase displays properties that indicate it is distinct from a number of well-characterized protein kinases, including those regulated by cAMP, calcium ions (in the presence or absence of calmodulin or mixtures of phosphatidylserine-diacylglycerol), polyamines, or heparin. There were no apparent effects of insulin on incorporation of 32P into added casein or histones II-S or III-S. The protein serine(threonine) kinase activity (or activities) described here displays properties that also appear to differ from the properties of previously described insulin-stimulated activities able to catalyze the phosphorylation of the ribosomal protein S6. The differences in properties may, in part, be explained by the use of different cell types, but may also indicate that treatment of cells with insulin leads to activation of more than one protein serine(threonine) kinase.

Acetyl-CoA Carboxylase↗

PubMed1993

Identification and localization of 23,000 and glycosylated rat prolactin in subcellular fractions of rat anterior pituitary and purified secretory granules.

Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.

Animals↗

PubMedDate not supplied

L-Thyroxine effects upon ATPase activities of several subcellular fractions of liver of the rat and the guinea pig.

The effect of thyroxine administration upon ATPase activity of several subcellular fractions of livers from rats and guinea pigs has been studied. To determine a patho-physiological dose of levo thyroxine [T4] for guinea pigs, a dose-response curve was examined of T4 effect upon oxidative phosphorylatin of guinea pig liver mitochondria. Maximum stimulation of mitochondrial respiration without uncoupling of oxidative phosphorylation was found with 15 microgram of T4 per 100 g body weight per day. This dose of T4 stimulated Mg++ activated ATPase of plasma membranes of guinea pigs and slightly stimulated Mg++ activated ATPase of guinea pig liver nuclear membranes. Rat liver nuclear membrane ATPase was not responsive to thyroxine at doses from 5 to 150 microgram per 100 g body weight. T4 significantly stimulated Ca++ or Mg++ ATPase of mitochondria and microsomes from both rat and guinea pig liver. Microsomes from both species were maximally activated by Mg++ and no significant additional stimulation with Ca++ was found. Mitochondrial ATPase from both species showed significantly greater Ca++ plus Mg++ ATPase activity than did Mg++ alone. Ca++ activated ATPase was approximately equal to dinitrophenol stimulated mitochondrial ATPase. Maximum activation of microsomal ATPase in both species was found with 1 mM calcium. We conclude that at physiological-intracellular concentrations of Ca++ and Mg++, thyroxine probably stimulates Mg++ activated microsomal ATPase and Ca++ activated mitochondrial ATPase. A potential role of Ca++ as a moderator of thyroxine stimulated activity in mitochondria and the relation of calcium to other metabolic reactions that are thyroxine sensitive is discussed.

Adenosine Triphosphatases↗

PubMed1982

The distribution of radioligand binding activity and 5'-nucleotidase activity in bovine caudate nucleus subcellular fractions.

The distribution of 5'-nucleotidase activity, dopaminergic [3H]spiperone binding sites, and [3H]quinuclidinyl benzilate (QNB) binding sites in different subcellular fractions of bovine caudate nucleus has been studied. Each activity was enriched in a microsomal (P3) preparation from that tissue. The microsomal preparation was further fractionated by different techniques. First, the P3 fraction, or a sonicated P3 fraction, was fractionated on a discontinuous sucrose density gradient. Second, the P3 fraction, or a digitonin pretreated P3 fraction, was fractionated on a continuous sucrose density gradient. The results obtained demonstrate that 5'-nucleotidase activity does not cofractionate with radioligand binding activity, although no difference between the distributions of [3H]spiperone binding and [3H]QNB binding were seen. It is concluded that the two radioligand binding activities are located on nonglial membranes.

5'-Nucleotidase↗