Subcellular distribution of glycosylphosphatidylinositol-specific phospholipase D in rat liver.
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ABSTRACT:
SUBMITTER: Hari T
PROVIDER: S-EPMC1217933 | biostudies-other | 1996 Nov
REPOSITORIES: biostudies-other
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Project description:1. The thymidine kinase activity of rat liver is greatly enhanced on addition of phospholipase C to the assay mixture. 2. Most of the thymidine kinase activity of the liver is recovered in the mitochondrial and in the impure ;nuclear' fractions. No activity was detected in purified nuclei prepared in high-density sucrose. 3. A substantial thymidine kinase activity could be detected, with the aid of phospholipase C, in all rat tissues examined.
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Project description:Phospholipase D (PLD) is regulated by many factors including the small G-proteins, RhoA and ADP-ribosylation factor (ARF). The present study examined the distribution of RhoA- and ARF-responsive PLD in membranes, microsomes and cytosol of rat tissues and in rat liver subcellular fractions. PLD was present in all tissue fractions examined and was stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]), with the highes: specific activities being in lung, kidney and spleen. When myristoylated recombinant ARF (mARF) was added with GTP[S], the PLD activity was stimulated further, but the addition of RhoA was without effect. However, in extracts from crude membranes both mARF and RhoA enhanced the stimulation by GTP[S], with high specific activities of PLD being observed in all tissues except muscle. The response to mARF was usually greater than to RhoA, and the responses were additive, except for liver, which showed synergism. When the PLD activity of subcellular fractions of liver was examined, GTP[S] caused increases in all fractions except microsomes and mitochondria, which exhibited low activity. All fractions except mitochondria showed responses to RhoA and mARF, with the response to RhoA being greater in plasma membranes and that to mARF being greater in Golgi and nuclei. Western blotting showed that RhoA was located mainly in the cytosol and plasma membranes, whereas ARF was principally in the cytosol. These findings demonstrate the widespread occurrence of significant activity of both Rho- and ARF-responsive forms of PLD in membranes from all tissues except muscle, and the presence of both forms in liver subcellular fractions except mitochondria. The large variations in the relative responses of PLD to Rho and ARF observed in different tissues and fractions support the existence of different isoforms of the enzyme.
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Project description:1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a 'total-particles' fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.
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Project description:The distribution of pyruvate (glyoxylate) aminotransferases in the particulate fraction of rat liver homogenates was examined by centrifugation in a sucrose density graident. Aminotransferase activities towards serine, phenylalanine and histidine with pyruvate and those towards phenylalanine and histidine with glyoxylate were nearly identically distributed. Some 50-55% of the particulate activity was localized in the peroxisomes and the remainder in the mitochondria. Most of alanine-glyoxylate aminotransferase activity was localized in the mitochondria, with some activity in the peroxisomes. Glucagon injection resulted in increases of these enzyme activities in the mitochondria, but not in the peroxisomes.
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Project description:We investigated the subcellular distribution of dopamine D(1), D(2) and D(5) receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D(1) receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D(2) receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D(5) receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D(5) receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D(1) receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D(5) and D(2) receptor subtypes were not significantly altered by cocaine treatment. These data imply that D(5) receptors are predominantly cytoplasmic, D(2) receptors are diffusely distributed within the cell, whereas D(1) receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.
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Project description:Kynurenine transaminase activity in rat liver was found in both the mitochondrial and supernatant fractions. The mitochondrial and supernatant fractions contained (a) kynurenine-pyruvate transaminase, which showed a preference for pyruvate as amino acceptor and a pH optimum between 8.0 and 8.5, and (b) kynurenine-alpha-oxoglutarate transaminase, with a preference for alpha-oxoglutarate and a pH optimum between 6.0 and 6.5. Possible roles of these enzymes in tryptophan metabolism in the liver are discussed.
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Project description:1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.
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Project description:The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.
| S-EPMC1135559 | biostudies-other
Project description:This study investigated the enantioselective metabolism of benoxacor, an ingredient of herbicide formulations, in microsomes or cytosol prepared from female or male rat livers. Benoxacor was incubated for ≤30 min with microsomes or cytosol, and its enantioselective depletion was measured using gas chromatographic methods. Benoxacor was depleted in incubations with active microsomes in the presence and absence of NADPH, suggesting its metabolism by hepatic cytochrome P450 enzymes (CYPs) and microsomal carboxylesterases (CESs). Benoxacor was depleted in cytosolic incubations in the presence of glutathione, consistent with its metabolism by glutathione S-transferases (GSTs). The depletion of benoxacor was faster in incubations with cytosol from male than female rats, whereas no statistically significant sex differences were observed in microsomal incubations. The consumption of benoxacor was inhibited by the CYP inhibitor 1-aminobenzotriazole, the CES inhibitor benzil, and the GST inhibitor ethacrynic acid. Estimates of the intrinsic clearance of benoxacor suggest that CYPs are the primary metabolic enzyme responsible for benoxacor metabolism in rats. Microsomal incubations showed an enrichment of the first eluting benoxacor enantiomer (E1-benoxacor). A greater enrichment occurred in incubations with microsomes from female (EF = 0.67 ± 0.01) than male rats (EF = 0.60 ± 0.01). Cytosolic incubations from female rats resulted in enrichment of E1-benoxacor (EF = 0.54 ± 0.01), while cytosolic incubations from male rats displayed enrichment of the second eluting enantiomer (E2-benoxacor; EF = 0.43 ± 0.01). Sex-dependent differences in the metabolism of benoxacor in rats could significantly impact ecological risks and mammalian toxicity. Moreover, changes in the enantiomeric enrichment of benoxacor may be a powerful tool for environmental fate and transport studies.
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Project description:1. A simple new assay for glycerylphosphorylcholine phosphodiesterase is described, in which radioactive glycerylphosphorylcholine is used as substrate and the reaction products are separated by adsorption on an anion-exchange resin. 2. Rat liver subcellular fractions contained both particulate (58%) and soluble (42%) glycerylphosphorylcholine phosphodiesterase. Both activities released free choline from glycerylphosphorylcholine. 3. The particulate glycerylphosphorylcholine phosphodiesterase was recovered mainly in the nuclear and microsomal fractions and showed a distribution similar to those of 5'-nucleotidase and alkaline phosphodiesterase I, both of which are constituents of the liver plasma membrane. 4. During purification of plasma membranes glycerylphosphorylcholine phosphodiesterase, 5'-nucleotidase and alkaline phosphodiesterase I showed largely similar behaviour, indicating that glycerylphosphorylcholine phosphodiesterase is also localized in liver plasma membranes. Slight differences in the distributions of these three enzymes in density-gradient separations are discussed in relation to the possibility that they are unevenly distributed on different areas of the cell surface. 5. The differences between glycerylphosphorylcholine phosphodiesterase and alkaline phosphodiesterase I indicate that these two activities are not functions of a single enzyme. 6. The glycerylphosphorylcholine phosphodiesterase of liver plasma membranes has a pH optimum of 8.5 and a K(m) for glycerylphosphorylcholine of 0.95mm. It is inhibited by EDTA and fully reactivated by a variety of bivalent cations (and Fe(3+)).
| S-EPMC1178596 | biostudies-other