Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction.
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ABSTRACT:
SUBMITTER: Dawson AP
PROVIDER: S-EPMC1154238 | biostudies-other | 1983 Feb
REPOSITORIES: biostudies-other
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Project description:1. By using Ca-EGTA buffers, the Km for Ca2+ uptake into rat liver heavy microsomes (microsomal fraction) was found to be 0.2 microM free Ca2+. 2. In the absence of oxalate, these vesicles accumulate about 20 nmol of Ca2+/mg of protein. Efflux of Ca2+ from the vesicles is much faster at pH 7.6 than at pH 6.8, but does not apparently show saturation kinetics or any stringent requirement for external ions. 3. The steady-state distribution of Ca2+ between the microsomes and the medium in the presence of ATP and the absence of oxalate is dependent on Ca2+ load. When the vesicles are loaded to 50% capacity, the external free Ca2+ concentration is 70 nM. 4. The affinity of heavy microsomes for Ca2+ is such that is seems likely that they has a dominant role in the determination of cytoplasmic free Ca2+ concentrations.
| S-EPMC1158551 | biostudies-other
Project description:GTP, when added to a rat liver microsomal fraction that had previously been allowed to accumulate Ca2+, causes a slow release of Ca2+, which is greatly enhanced by addition of inositol trisphosphate (IP3). The Ca2+ release caused by IP3 under these conditions is very much greater than that observed in the absence of GTP. The effect of GTP is dependent on the presence of polyethylene glycol in the incubation medium and is not due to inhibition of the Ca2+-accumulation system. The response to GTP is time-dependent, particularly at low (4 microM) GTP concentrations, and cannot be mimicked by ATP, ITP, CTP, UTP and GDP. Studies with [gamma-32P]GTP show that, during incubation with microsomal fractions, the terminal phosphate of GTP is transferred to two protein species, of Mr 38 000 and 17 000. These protein phosphorylations are still present when an excess of unlabelled ATP is included in the incubation mixture, but appear to be unaffected by the presence or absence of IP3 and polyethylene glycol. As a working hypothesis, it is suggested that a protein, phosphorylated by GTP, has to bind to the microsomal membranes before IP3 can stimulate Ca2+ release, and that, in vitro, the binding of this protein is favoured by the presence of polyethylene glycol.
| S-EPMC1146567 | biostudies-other
Project description:Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.
| S-EPMC1147047 | biostudies-other
Project description:Measurements of the initial rate of Ca2+ transport by rat liver microsomal preparations reveal the existence of two phases of transport activity. The first, a phase of rapid transport, is complete by 3-5 min, at which time the second (slower) phase begins; this remains linear for up to at least 40 min. The initial phase is minimal in the absence of MgATP. The initial rate of Ca2+ transport reaches values as high as 25 nmol/min per mg of protein; the Km for Ca2+total is 1-2 micrometer and that for MgATPtotal about 500 micrometer. Ruthenium Red (3-5 nmol/mg of protein) has little effect on the initial rate of transport, whereas tributylin (2 micrometer) inhibits equally in a KC1- or a KNO3-containing medium. Compunds that collapse components of the proton electrochemical gradient in mitochondria (valinomycin and carbonyl cyanide m-chlorophenylhydrazone) each inhibit by 70-80% the initial rate of microsomal Ca2+ transport.
| S-EPMC1183864 | biostudies-other
Project description:A membrane fraction enriched in endoplasmic reticulum was prepared from rat parotid glands by using sucrose-gradient centrifugation. The fraction showed a 10-fold increase in specific activity of NADPH: cytochrome c reductase activity over that of tissue homogenates and minimal contamination with plasma membranes or mitochondria. The endoplasmic reticulum fraction possessed both Mg2+ -stimulated ATPase as well as Ca2+, Mg2+-ATPase [( Ca2+ + Mg2+)-stimulated ATPase]activity. The Ca2+, Mg2+-ATPase required 2-5 mM-Mg2+ for optimal activity and was stimulated by submicromolar concentrations of free Ca2+. The Km for free Ca2+ was 0.55 microM and the average Vmax. was 60 nmol/min per mg of protein. The Km for ATP was 0.11 mM. Other nucleotides, such as GTP, CTP or ADP, could not substitute for ATP in supporting the Ca2+-activated nucleotidase activity. Increasing the K+ concentration from 0 to 100 mM caused a 2-fold activation of the Ca2+, Mg2+-ATPase. Trifluoperazine, W7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide] and vanadate inhibited the enzyme. The concentration of trifluoperazine and vanadate required for 50% inhibition of the ATPase were 52 microM and 28 microM respectively. Calmodulin, cyclic AMP, cyclic AMP-dependent protein kinase and inositol 1,4,5-trisphosphate had no effect on the ATPase. The properties of the Ca2+, Mg2+ -ATPase were distinct from those of the Mg2+-ATPase, but comparable with those reported for the parotid endoplasmic-reticulum Ca2+-transport system [Kanagasuntheram & Teo (1982) Biochem. J. 208, 789-794]. The results suggest that the Ca2+, Mg2+-ATPase is responsible for driving the ATP-dependent Ca2+ accumulation by this membrane.
| S-EPMC1146712 | biostudies-other
Project description:Hepatic metabolic stability is a key pharmacokinetic parameter in drug discovery. Metabolic stability is usually assessed in microsomal fractions and only the best compounds progress in the drug discovery process. A high-throughput single time point substrate depletion assay in rat liver microsomes (RLM) is employed at the National Center for Advancing Translational Sciences. Between 2012 and 2020, RLM stability data was generated for ~ 24,000 compounds from more than 250 projects that cover a wide range of pharmacological targets and cellular pathways. Although a crucial endpoint, little or no data exists in the public domain. In this study, computational models were developed for predicting RLM stability using different machine learning methods. In addition, a retrospective time-split validation was performed, and local models were built for projects that performed poorly with global models. Further analysis revealed inherent medicinal chemistry knowledge potentially useful to chemists in the pursuit of synthesizing metabolically stable compounds. In addition, we deposited experimental data for ~ 2500 compounds in the PubChem bioassay database (AID: 1508591). The global prediction models are made publicly accessible ( https://opendata.ncats.nih.gov/adme ). This is to the best of our knowledge, the first publicly available RLM prediction model built using high-quality data generated at a single laboratory.
| S-EPMC7693334 | biostudies-literature
Project description:The structural properties of purified palmitoyl-CoA synthetase from rat liver microsomal material were investigated. The active enzyme has a molecular weight of 168000 and contains about 8.2mol of phospholipid bound/mol of enzyme protein, as well as bound fatty acids. Association of the active enzyme was shown to occur, without impairment of catalytic activity. The lowest-molecular-weight species obtained under denaturing conditions was 27000 daltons.
| S-EPMC1177492 | biostudies-other
Project description:1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.
| S-EPMC1164521 | biostudies-other
Project description:Conditions were investigated for demonstrating the synthesis in vitro of the complete molecule of cytochrome c by isolated liver microsomal systems from partially hepatectomized rats. It was first found that in vivo the early labelled cytochrome c associated with the microsomal fraction required, by comparison with the mitochondrial pool, more drastic conditions of extraction and its binding was less affected by freezing and thawing of the subcellular particles. The procedure of extraction and purification of cytochrome c had to be modified accordingly, to assure the recovery of the recently synthesized molecule. Several subcellular fractions were isolated from regenerating liver with a homogenization medium containing either 5 or 10mm-Mg(2+) and most of them were active in the synthesis of the cytochrome c apoprotein. The microsomal fraction, in the presence of either cell sap or pH5.0 fraction, was also able to incorporate [(59)Fe]haemin, delta-amino[(3)H]laevulic acid and (55)Fe into the prosthetic group of cytochrome c. These experiments confirm firmly the conclusions of our previous results obtained in vivo showing that both the apoprotein and the haem moieties are made and linked together on cytoplasmic ribosomes and only then is the complete molecule transferred to the mitochondria.
| S-EPMC1177245 | biostudies-other
Project description:The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.
| S-EPMC1152211 | biostudies-other