Biomedical subjects

G M Carman

Publications and source records attributed to G M Carman.

At least 55 records · Page 3Linked to original sources

PubMed1995

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by CTP.

In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways. We examined the regulation of phosphatidylcholine synthesis by CTP. The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP. The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway. CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway. The apparent Km of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline. Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase. This was not due to a transcriptional repression mechanism. Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP. These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized. This regulation includes the supple of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗

PubMed1995

Phosphorylation of CTP synthetase from Saccharomyces cerevisiae by protein kinase C.

Phosphorylation of CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) from Saccharomyces cerevisiae protein kinase C was examined. Using pure CTP by synthetase as a substrate, protein kinase C activity was dose- and time-dependent and required calcium, diacylglycerol, and phosphatidylserine for full activation. Protein kinase C activity was also dependent on the concentration of CTP synthetase. Protein kinase C phosphorylated CTP synthetase on serine and threonine residues in vitro whereas the enzyme was primarily phosphorylated on serine residues in vivo. Phosphopeptide mapping analysis of CTP synthetase phosphorylated in vitro and in vivo indicated that the enzyme was phosphorylated on more than one site. Most of the phosphopeptides derived from CTP synthetase phosphorylated in vivo were the same as those derived from CTP synthetase phosphorylated by protein kinase C in vitro. The stoichiometry of the phosphorylation of native CTP synthetase was 0.4 mol of phosphate/mol of enzyme whereas the stoichiometry of the phosphorylation of alkaline phosphatase-treated CTP synthetase was 2.2 mol of phosphate/mol of enzyme. This indicated that CTP synthetase was purified in a phosphorylated state. Phosphorylation of CTP synthetase resulted in a 3-fold activation in enzyme activity whereas alkaline phosphatase treatment of CTP synthetase resulted in a 5-fold decrease in enzyme activity. Overall, the results reported here were consistent with the conclusion that CTP synthetase was regulated by protein kinase C phosphorylation.

Adenosine Triphosphate↗

PubMed1995

Regulation of lipid biosynthesis in Saccharomyces cerevisiae by fumonisin B1.

The regulation of lipid biosynthesis in the yeast Saccharomyces cerevisiae by fumonisin B1 was examined. Fumonisin B1 inhibited the growth of yeast cells. Cells supplemented with fumonisin B1 accumulated free sphinganine and phytosphingosine in a dose-dependent manner. The cellular concentration of ceramide was reduced in fumonisin B1-supplemented cells. Ceramide synthase activity was found in yeast cell membranes and was inhibited by fumonisin B1. Fumonisin B1 inhibited the synthesis of the inositol-containing sphingolipids inositol phosphorylceramide, mannosylinositol phosphorylceramide, and mannosyldiinositol phosphorylceramide. Fumonisin B1 also caused a decrease in the synthesis of the major phospholipids synthesized via the CDP-diacylglycerol-dependent pathway and the synthesis of neutral lipids. The effects of fumonisin B1 and sphingoid bases on the activities of enzymes in the pathways leading to the synthesis of sphingolipids, phospholipids, and neutral lipids were also examined. Other than ceramide synthase, fumonisin B1 did not affect the activities of any of the enzymes examined. However, sphinganine and phytosphingosine inhibited the activities of inositol phosphorylceramide synthase, phosphatidylserine synthase, and phosphatidate phosphatase. These are key enzymes responsible for the synthesis of lipids in yeast. The data reported here indicated that the biosynthesis of sphingolipids, phospholipids and neutral lipids was coordinately regulated by fumonisin B1 through the regulation of lipid biosynthetic enzymes by sphingoid bases.

Amidohydrolases↗

PubMed1994

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.

Binding Sites↗

PubMed1994

Purification and characterization of CTP synthetase, the product of the URA7 gene in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, CTP synthetase [EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] is the product of the URA7 gene. CTP synthetase was purified 503-fold to apparent homogeneity from cells bearing the URA7 gene on a multicopy plasmid that directed a 10-fold overproduction of the enzyme. The purification procedure included ammonium sulfate fractionation of the cytosolic fraction followed by chromatography with Sephacryl 300 HR, Q-Sepharose, Affi-Gel Blue, and Superose 6. The N-terminal amino acid sequence of purified CTP synthetase was identified and aligned perfectly with the deduced sequence of the URA7 gene. The minimum subunit molecular mass (68 kDa) of purified CTP synthetase was in good agreement with the size (64.7 kDa) of the URA7 gene product. Antibodies were raised against a maltose-binding protein-CTP synthetase fusion protein which immunoprecipitated CTP synthetase from wild-type cells. Immunoblot analysis was used to identify CTP synthetase in wild-type cells and cells bearing the URA7 gene on a multicopy plasmid. The results of gel filtration chromatography indicated that the size of native CTP synthetase was consistent with a dimeric structure for the enzyme. CTP synthetase oligomerized to a tetramer in the presence of its substrates UTP and ATP. Maximum CTP synthetase activity was dependent on magnesium ions (4 mM) and 2-mercaptoethanol at the pH optimum of 8.0. CTP synthetase exhibited positive cooperative kinetics with respect to UTP and ATP and negative cooperative kinetics with respect to glutamine and GTP. CTP synthetase was potently inhibited by the product CTP which also increased the positive cooperativity of the enzyme toward UTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1994

Regulation of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by CDP-diacylglycerol.

Regulation of the 45- and 55-kDa forms of Saccharomyces cerevisiae membrane-associated phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase) by phospholipids was examined using Triton X-100/phospholipid-mixed micelles. CDP-diacylglycerol and phosphatidylglycerol inhibited 45-kDa PI 4-kinase activity in a dose-dependent manner. Kinetic analyses of the 45-kDa PI 4-kinase showed that phosphatidylglycerol was a competitive inhibitor with respect to PI (Ki = 2 mol %), and CDP-diacylglycerol was a mixed type of inhibitor with respect to PI (Ki = 4 mol %) and MgATP (Ki = 5 mol %). 55-kDa PI 4-kinase activity was not significantly affected by phospholipids. The physiological relevance of CDP-diacylglycerol inhibition of 45-kDa PI 4-kinase activity was examined using plasma membranes from inositol auxotrophic (ino1) cells. Immunoblot analysis showed that 45-kDa PI 4-kinase expression in plasma membranes was not affected by inositol starvation of ino1 cells. However, both 45-kDa PI 4-kinase activity and its product PI 4-phosphate were reduced in plasma membranes from inositol-starved ino1 cells. The CDP-diacylglycerol concentration (9.6 mol %) in plasma membranes of inositol-starved ino1 cells was 12-fold higher than its concentration (0.8 mol %) in plasma membranes of inositol-supplemented cells. Plasma membranes of inositol-starved ino1 cells also had increased levels of phosphatidate, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. However, these phospholipids did not affect pure 45-kDa PI 4-kinase activity. The concentration of CDP-diacylglycerol in plasma membranes of inositol-starved ino1 cells was in the range of the inhibitor constants determined for CDP-diacylglycerol by kinetic analyses using pure 45-kDa PI 4-kinase. These results raised the suggestion that 45-kDa PI 4-kinase activity may be regulated in vivo by CDP-diacylglycerol.

1-Phosphatidylinositol 4-Kinase↗

PubMed1994

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae.

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5'-triphosphate (UTP) to cytidine 5'-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.

Amino Acid Sequence↗

PubMed1993

Photoaffinity labeling of the 45-kDa and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

The membrane-associated 45- and 55-kDa forms of phosphatidylinositol (PI) 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) from Saccharomyces cerevisiae are inhibited by ADP by a competitive mechanism with respect to ATP. We initiated studies toward defining the ATP and ADP sites on the PI 4-kinases using azidonucleotide photoaffinity labeling probes. The photoprobe 8-azido-ATP fulfilled the criteria of a specific photoaffinity label for the 45- and 55-kDa PI 4-kinases. 8-Azido-ATP was a substrate and a competitive inhibitor of the PI 4-kinases with Ki values similar to the Km for ATP. 8-Azido-ATP photoinactivated the enzymes and was photoincorporated into the enzymes in a dose-dependent manner at concentrations similar to the Ki values for the photoprobe. ATP, the true substrate, provided specific protection against photoinactivation and photoincorporation of the PI 4-kinases with 8-azido-ATP, whereas GTP, a nonspecific nucleotide, provided no protection against photoinactivation and photoincorporation. Photoaffinity labeling of the PI 4-kinases with 8-azido-ATP was specifically prevented with ADP. The photoprobe 8-azido-ADP also fulfilled the criteria needed to validate its use as a specific photoprobe for the PI 4-kinases. Photoinactivation of the PI 4-kinases with 8-azido-ADP was prevented specifically with ATP. Taken together, these data supported the conclusion that the ATP and ADP sites on the membrane-associated 45- and 55-kDa PI 4-kinases from S. cerevisiae were the same.

1-Phosphatidylinositol 4-Kinase↗

PubMed1993

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases.

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.

Amines↗

PubMed1993

Regulation of the 45- and 55-kDa forms of phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae by nucleotides.

The synthesis of phosphatidylinositol (PI) 4-phosphate and PI 4,5-bisphosphate in the yeast Saccharomyces cerevisiae is stimulated by glucose. PI 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) catalyzes the committed step in the synthesis of these phosphoinositides. Previous studies have suggested that the glucose effect on phosphoinositide synthesis is mediated by cellular levels of ATP and ADP and by the RAS/cAMP pathway. Using purified preparations of the membrane-associated 45- and 55-kDa forms of PI 4-kinase, we examined the regulation of these activities by nucleotides and cAMP-dependent protein kinase. MgADP was a potent inhibitor of both forms of the enzyme. Detailed kinetic analyses of the 45- and 55-kDa enzymes using Triton X-100/PI-mixed micelles showed that MgADP was a competitive inhibitor (Ki = 0.14 and 0.25 mM, respectively) with respect to MgATP and a noncompetitive inhibitor (Ki = 1.3 and 0.9 mM, respectively) with respect to PI. The Ki values for MgADP were about 2-fold lower than the Km values the enzymes have for their substrate MgATP and about 2-fold lower than the cellular concentration of ADP. The 45- and 55-kDa forms of PI 4-kinase activity were regulated differentially by CTP, an important nucleotide involved in phospholipid biosynthesis. Whereas the 55-kDa PI 4-kinase was inhibited by CTP, the 45-kDa enzyme was unaffected by CTP. CTP was a mixed type of inhibitor (Ki = 1.5 mM) with respect to MgATP and a noncompetitive inhibitor (Ki = 4 mM) with respect to PI. The Ki value for CTP was 4-fold higher than the Km value for MgATP and 7-fold higher than the cellular concentration of CTP. The 45- and 55-kDa PI 4-kinases were neither phosphorylated nor regulated by cAMP-dependent protein kinase. These results did not support the previous conclusion that PI 4-phosphate synthesis was mediated by the RAS/cAMP pathway. Our kinetic studies supported the conclusion that the glucose effect on the synthesis of PI 4-phosphate was mediated by cellular levels of ATP and ADP through the regulation of membrane-associated PI 4-kinase activity.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.

Alleles↗

PubMed1992

Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.

A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.

1-Phosphatidylinositol 4-Kinase↗

PubMed1992