Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase.
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The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C. (+info)
Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein.
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Substantial purification of rat liver phosphatidylinositol (PtdIns) synthase has been achieved by a combination of Hecameg extraction, heat treatment, affinity chromatography and chromatography on PBE-94. The activity chromatographs as a single peak which has an apparent molecular mass between 150 and 200 kDa on Sepharose 4B. When analysed by SDS/PAGE, two major bands are seen. The enzyme activity is correlated with a protein band of 21 kDa. A second band, at 51 kDa, is eluted from a PBE-94 column slightly ahead of the activity. Manganese is an absolute requirement for stabilization of activity in the presence of detergent. The effect of manganese is optimal at 0.5 mM; magnesium at a concentration of 10 mM is only minimally effective. Substrate Kms are 1.3 mM and 9.5 microM for inositol and CDP-diacylglycerol respectively. The activity eluting from the PBE-94 column is purified 5000-fold over the post-mitochondrial supernatant. (+info)
Purification and characterization of phosphatidylinositol synthase from human placenta.
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Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C. (+info)
A somatic cell mutant defective in phosphatidylglycerophosphate synthase, with impaired phosphatidylglycerol and cardiolipin biosynthesis.
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Phosphatidylglycerophosphate (PGP) synthase catalyzes a reaction involved in the synthesis of phosphatidylglycerol (PG), which serves as a metabolic precursor for cardiolipin (CL), found primarily in the mitochondrial membranes of eukaryotic cells. We isolated a Chinese hamster ovary cell mutant (designated PGS-S) with a specific lesion in PGP synthase by using an in situ enzymatic assay for the enzyme. This mutant was obtained by introducing a second mutation into mutant PGS-P that had been generated by first-step mutagenesis. The PGP synthase activities in cell extracts of mutant PGS-S grown at 33 and 40 degrees C were 14 and 1% of those in the wild type cells, respectively; in addition, PGP synthase in cell extracts of mutant PGS-S exhibited higher sensitivity to heat than that of the wild type. Mutant PGS-S also showed a temperature-dependent defect in the synthesis of PG and CL in vivo, together with temperature sensitivity for cell growth. A temperature-resistant revertant of mutant PGS-S simultaneously restored PGP synthase activity and the ability to synthesize PG and CL in vivo to nearly the same levels as those of mutant PGS-P. These results constitute genetic evidence that PGP synthase is responsible for PG synthesis and is essential for cell growth. (+info)
Hydrolysis of short acyl chain inositol lipids by phospholipase C-delta 1.
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We investigated the relationship between substrate aggregation and activation of phosphoinositide-specific phospholipase C-delta 1 (PLC-delta 1), isolated from bovine brain cytosol. The inositol lipids 1,2-dibutyryl-sn-glycero-3-phosphoinositol (di-C4-PI), 1,2-dihexanoyl-sn-glycero-3-phosphoinositol (di-C6-PI), and 1,2-dioctanoyl-sn-glycero-3-phosphoinositol (di-C8-PI) were prepared from synthetic cytidine diphosphate diglyceride analogs in a reaction with myo-inositol catalyzed by yeast phosphatidylinositol synthase. All three lipids served as substrates for PLC-delta 1 at concentrations significantly below their critical micelle concentration (cmc). Under these conditions, steps that might limit the reaction rate, such as membrane adsorption or penetration into the phospholipid surface, were eliminated. Below the cmc, the concentration of lipid substrate required to produce hydrolysis followed the order: di-C8-PI < di-C6-PI << di-C4-PI. Calcium was essential for hydrolysis of the short chain substrates at all lipid concentrations tested. The dependence of the reaction on calcium suggests that this ion activates PLC-delta 1 at a step other than adsorption to or penetration of the membrane surface. As the concentration of di-C8-PI was raised above the cmc, the reaction velocity increased 2-3-fold. These results are consistent with the idea that micellar or bilayer aggregates of phosphoinositol are not required for PLC-catalyzed hydrolysis, although the reaction rate is enhanced by micelle formation. (+info)
Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes.
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Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system. (+info)
Characterization of reactions catalysed by yeast phosphatidylinositol synthase.
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The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E. coli has been investigated. The single enzyme is shown to carry both CDP-diacylglycerol-dependent incorporation of inositol into phosphatidylinositol (Km for inositol of 0.090 mM) and a CDP-diacylglycerol-independent exchange reaction between phosphatidylinositol and inositol (Km for inositol of 0.066 mM). The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates. These results suggest that CMP-stimulated exchange and CMP-dependent reverse reactions are distinct processes catalysed by the same enzyme, phosphatidylinositol synthase. (+info)
Direct labelling of hormone-sensitive phosphoinositides by a plasma-membrane-associated PtdIns synthase in turkey erythrocytes.
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We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids. (+info)