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Michael Schweizer - One of the best experts on this subject based on the ideXlab platform.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology, 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1–PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Δprs1 and Δprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology (Reading England), 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing Phosphoribosyl Pyrophosphate in Saccharomyces cerevisiae.
The Journal of biological chemistry, 1999Co-Authors: Yolanda Hernando, Andrew T. Carter, Adrian Parr, Bjarne Hove-jensen, Michael SchweizerAbstract:Abstract The PRS gene family inSaccharomyces cerevisiae consists of five genes each capable of encoding a 5-Phosphoribosyl-1(α)-Pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 orPRS3; simultaneous deletion of PRS1 andPRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their Phosphoribosyl Pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions ofPRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast Phosphoribosyl Pyrophosphate synthetase exists in vivo as multimeric complex(es).
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PRS5, the Fifth Member of the Phosphoribosyl Pyrophosphate Synthetase Gene Family inSaccharomyces cerevisiae, Is Essential for Cell Viability in the Absence of either PRS1 or PRS3
Journal of bacteriology, 1998Co-Authors: Yolanda Hernando, Adrian J. Parr, Michael SchweizerAbstract:In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-Phosphoribosyl-1(alpha)-Pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a Phosphoribosyl Pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Deltaprs5 strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S. cerevisiae.
Yolanda Hernando - One of the best experts on this subject based on the ideXlab platform.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology, 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1–PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Δprs1 and Δprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology (Reading England), 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing Phosphoribosyl Pyrophosphate in Saccharomyces cerevisiae.
The Journal of biological chemistry, 1999Co-Authors: Yolanda Hernando, Andrew T. Carter, Adrian Parr, Bjarne Hove-jensen, Michael SchweizerAbstract:Abstract The PRS gene family inSaccharomyces cerevisiae consists of five genes each capable of encoding a 5-Phosphoribosyl-1(α)-Pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 orPRS3; simultaneous deletion of PRS1 andPRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their Phosphoribosyl Pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions ofPRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast Phosphoribosyl Pyrophosphate synthetase exists in vivo as multimeric complex(es).
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PRS5, the Fifth Member of the Phosphoribosyl Pyrophosphate Synthetase Gene Family inSaccharomyces cerevisiae, Is Essential for Cell Viability in the Absence of either PRS1 or PRS3
Journal of bacteriology, 1998Co-Authors: Yolanda Hernando, Adrian J. Parr, Michael SchweizerAbstract:In Saccharomyces cerevisiae, an open reading frame, YOL061w, encodes a polypeptide with sequence similarity to the four known 5-Phosphoribosyl-1(alpha)-Pyrophosphate synthetase (PRS) genes since it contains a divalent cation binding site and a Phosphoribosyl Pyrophosphate binding site. We regard YOL061w as the fifth member of the PRS gene family, PRS5. Loss of Prs5p has a significant impact on PRS enzyme activity, causing it to be reduced by 84%. On the other hand, Deltaprs5 strains are not affected in growth or in the size of their nucleotide pools. However, simultaneous deletion of PRS1 and PRS5 or PRS3 and PRS5 rendered the strains inviable, which implies that PRS5 plays an important role in the maintenance of PRS function in S. cerevisiae.
Michael N G James - One of the best experts on this subject based on the ideXlab platform.
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the structures of thermoplasma volcanium Phosphoribosyl Pyrophosphate synthetase bound to ribose 5 phosphate and atp analogs
Journal of Molecular Biology, 2011Co-Authors: Maia M Cherney, Leonid T Cherney, Craig R Garen, Michael N G JamesAbstract:Abstract Phosphoribosyl Pyrophosphate (PRPP) synthetase catalyzes the transfer of the Pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium ( Tv ) was expressed in Escherichia coli , crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P 2 1 . The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 A and 1.5 A resolutions, respectively. Crystals of the ternary complex with ADP–Mg 2+ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 A resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.
Patricia Mclean - One of the best experts on this subject based on the ideXlab platform.
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Effects of long-term experimental diabetes on adrenal gland growth and Phosphoribosyl Pyrophosphate formation in growth hormone-deficient dwarf rats
International journal of experimental pathology, 2012Co-Authors: Sirilaksana Kunjara, Patricia Mclean, A. Leslie Greenbaum, Henning Grønbæk, Allan FlyvbjergAbstract:The availability of growth hormone (GH)-deficient dwarf rats with otherwise normal pituitary function provides a powerful tool to examine the relative role of hyperglycaemia and the reordering of hormonal factors in the hypertrophy-hyperfunction of the adrenal gland that is seen in experimental diabetes. Here, we examine the effects of long-term (6 months) experimental diabetes on the growth of the adrenal glands; their content of Phosphoribosyl Pyrophosphate (PRPP); and the activity of the PRPP synthetase, G6P dehydrogenase and 6PG dehydrogenase enzymes in GH-deficient dwarf rats compared to heterozygous controls. These parameters were selected in view of the known role of PRPP in both de novo and salvage pathways of purine and pyrimidine synthesis and in the formation of NAD, and in view of the role of the oxidative enzymes of the pentose phosphate pathway in both R5P formation and the generation of the NADPH that is required in reductive synthetic reactions. This study shows that GH deficiency prevents the increase in adrenal gland weight, PRPP synthetase, PRPP content and G6P dehydrogenase and 6PG dehydrogenase. This contrasts sharply with the heterozygous group that showed the expected increase in these parameters. The blood glucose levels of the groups of long-term diabetic rats, both GH-deficient and heterozygous, remained at an elevated level throughout the experiment. These results are fully in accord with earlier evidence from studies with somatostatin analogues which showed that the GH-insulin-like growth factor I (IGF-I)-axis plays a key role in the adrenal diabetic hypertrophy-hyperfunction syndrome.
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Effects of long-acting somatostatin analogues on adrenal growth and Phosphoribosyl Pyrophosphate formation in experimental diabetes.
International journal of experimental pathology, 2012Co-Authors: Sirilaksana Kunjara, Milena Sochor, A. Leslie Greenbaum, Murad Ali, Allan Flyvbjerg, Henning Grønbæk, Patricia McleanAbstract:It is well established that, following the induction of diabetes, there is an early increase in adrenal size, with hypertrophy of the zona fasciculata and an increase of steroid production (De Nicola et al. 1976, 1977; Rhees et al. 1983; Penhoat et al. 1988). The most marked changes in adrenal weight and plasma corticosteroid levels were observed 5 days after the induction of diabetes in rats with streptozotocin (STZ), with a less marked, but persistent, adrenal hyperactivity occurring up to 6 weeks after the onset of diabetes (De Nicola et al. 1977). Kunjara et al. (1992) have shown that not only does the rat adrenal gland contain a notably high concentration of Phosphoribosyl Pyrophosphate (PRPP), some 20-fold greater than a range of normal tissues such as the liver, kidney and heart, but also that this nucleotide precursor is increased markedly within 3 days of STZ induction of diabetes. Phosphoribosyl Pyrophosphate is known to play a central role in nucleotide synthesis. It serves as a substrate for the de novo and salvage pathways of purine and pyrimidine synthesis, and as an activator of the first steps in both de novo routes (Becker et al. 1979; Becker 2001). In synthesis of nicotinamide mononucleotide (NMN) via Nampt, PRPP is the rate-limiting step in NAD synthesis (see Garten et al. 2009; Imai 2009a). Recent studies have emphasized the multiple roles of NAD in addition to its established function in redox systems, in glycolysis and energy production. These include a number of signalling pathways: poly ADP ribosylation in DNA repair (Menissier de Murcia et al. 2003), formation of cyclic ADP-ribose involved in calcium signalling (Lee 2001), and of Sir2, an NAD-dependent histone deacetylase and mono-ADP-ribosyl transferases that regulates a wide array of proteins involved in metabolism and cell survival (Imai et al. 2000; Landry et al. 2000; Revollo et al. 2004; Michan & Sinclair 2007; Imai 2009b). The linkages between PRPP formation and the multiple sites of cellular regulation are summarized in Scheme 1. These interrelated functions have highlighted the potential significance of the regulation of PRPP in growth processes such as those seen in the diabetic adrenal and prompted an investigation of PRPP- and PRPP-associated factors involved in the early stages of the adrenal response in experimental diabetes. Scheme 1 Pathways linking Phosphoribosyl Pyrophosphate (PRPP) formation with multiple sites of cellular regulation and growth. The present study demonstrated the increase in PRPP concentration and PRPP synthetase activity in rat adrenal glands following induction ... The temporal parallelism between the growth response of the adrenal and the kidney in experimental diabetes (Kunjara et al. 1986a, 1992; Flyvbjerg et al. 1988) suggested the hypothesis that common hormonal signals might be involved in the two organs. Using adrenal cortical cell cultures in serum-free defined medium, a number of factors have been shown to be implicated. This includes both insulin and insulin-like growth factor I (IGF-I), which stimulate growth of bovine fasciculata cells (Penhoat et al. 1988). The characteristics of the IGF-I and insulin receptors, and the role of these hormones on adrenal cell function and steroidogenic response, were investigated, and at physiological concentrations IGF-I was shown to be the more potent factor. In fact, there has already been considerable interest in changes in IGF-I during early diabetic renal hypertrophy (Mendley & Toback 1988; Hammerman 1989; Fine et al. 1992). Increasing evidence supports the concept that IGF-I stimulates the initial renal hypertrophy (Flyvbjerg et al. 1988, 1990, 1991; Gronbaek et al. 2002; Flyvbjerg 2004). Flyvbjerg et al. (1991) infused IGF-1 into diabetic rats 5 days after treatment with STZ, that is at the time when the initial rapid growth phase had slowed, and growth acceleration was demonstrated. Administration of a somatostatin analogue prevented both the increase of kidney IGF-I and the initial renal growth in diabetes (Flyvbjerg et al. 1989; Gronbaek et al. 2002). Adrenal levels of IGF-1 were not documented in these studies. The similarities between kidney and adrenal gland responses in STZ diabetes have been related previously to a general and widely held concept of ‘glucose over-utilization’ in those tissues not requiring insulin for glucose uptake. These tissues include the ocular lens and peripheral nerve, and glycosylation of proteins such as haemoglobin A1c and lens α crystalline (Brownlee & Cerami 1981; Sochor et al. 1985) as well as the adrenal and kidney. This contrasts sharply with those tissues dependent upon insulin for glucose uptake and showing aspects of ‘glucose under-utilization’, such as muscle, adipose tissue and lactating mammary gland (Kunjara et al. 1986a,b, 1992) (Table 1). Table 11 Effect of diabetes on the activity of the pentose phosphate pathway in rat adrenal gland, kidney and lactating mammary gland: glucose under- and over-utilization To explore these questions, two different somatostatin analogues, Angiopeptin (AGP, Lantreotide), and Sandostatin (SMS, Octreotide), have been used. These analogues have been shown to have different effects on tissue growth (Alderton et al. 1998). Their effects on the early growth of the adrenal gland, on the tissue concentration of PRPP, on PRPP synthetase activity and on the enzymes of the pentose phosphate pathway (PPP) (which are involved in the provision of R5P, the immediate precursor of PRPP) were compared. In addition, they were also compared with the effect of insulin on the same parameters.
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Aspects of the regulation of hepatic Phosphoribosyl Pyrophosphate formation in the obese (ob/ob) mouse: relationship to the pentose phosphate pathway.
Biochemical medicine and metabolic biology, 1993Co-Authors: Sirilaksana Kunjara, Milena Sochor, A. L. Greenbaum, Patricia McleanAbstract:The content of Phosphoribosyl Pyrophosphate (PPRibP) and of intermediates involved in its synthesis has been measured in the livers of obese (ob/ob) mice 2 months and 3-4 months of age, a period of dynamic growth and marked hepatic hypertrophy and hyperplasia, and comparison made with the values found in the lean age-matched control groups. The total hepatic PPRibP content and PPRibP/mg DNA was significantly increased in the 3- to 4-month-old obese mice relative to both the lean control groups and the 2-month-old obese mice, illustrating the significant changes occurring in the obese mouse liver in this transition period. The change in hepatic PPRibP/mg DNA in the obese mice is positively correlated with age (up to 4 months, body weight 60 g) and with parameters linked to the activity of the pentose phosphate pathway. There is no apparent correlation between PPRibP concentration and inorganic phosphate, the energy status of the cell, or the hepatic PPRibP synthetase (EC 2.7.6.1.) activity. The increase in the bioavailability of PPRibP, which is both a substrate and activator of the de novo and salvage pathways of purine and pyrimidine synthesis, is considered in relation to the increased nucleotide requirement associated with the rise in total hepatic RNA, DNA, and adenine nucleotide in the obese mouse.
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Pyrimidine nucleotide synthesis in the rat mammary gland: changes in the lactation cycle and effects of diabetes.
Biochemical medicine and metabolic biology, 1992Co-Authors: Sirilaksana Kunjara, Milena Sochor, A. Leslie Greenbaum, Michael Bennett, Patricia McleanAbstract:Measurements have been made of the activities of the enzymes of the de novo and salvage pathways of pyrimidine synthesis (carbamoyl phosphate synthetase II (glutamine) (EC 6.3.5.5); dihydroorotate dehydrogenase (EC 1.3.99.11); the overall activity of Complex II (orotate Phosphoribosyl Pyrophosphate transferase (EC 2.4.2.10) and orotidine 5-phosphate decarboxylase (EC 4.1.1.23); uracil Phosphoribosyltransferase (EC 2.4.2.9)) in the mammary gland of rats at different stages of the lactation cycle and the effects of diabetes on the activity of these enzymes in lactation have been studied. From a consideration of the changes in enzyme activities and the changes in the tissue concentration of Phosphoribosyl Pyrophosphate, an activator of the de novo pathway and substrate for both the de novo and salvage routes, it is concluded that the de novo pathway is the major route of pyrimidine synthesis in mammary tissue. Diabetes decreases the activity of the enzymes of the de novo pathway; the effects are particularly marked for Complex II. The present results on pyrimidine synthesis are compared to the pattern for purine synthesis previously published.
Andrew T. Carter - One of the best experts on this subject based on the ideXlab platform.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology, 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1–PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Δprs1 and Δprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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The importance of the five Phosphoribosyl-Pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p.
Microbiology (Reading England), 2000Co-Authors: Roger Schneiter, Andrew T. Carter, Yolanda Hernando, Günther Zellnig, Lilian Mary Schweizer, Michael SchweizerAbstract:Phosphoribosyl-Pyrophosphate synthetase (Prs) catalyses the synthesis of Phosphoribosyl Pyrophosphate (PRPP), an intermediate in nucleotide metabolism and the biosynthesis of the amino acids histidine and tryptophan. The Saccharomyces cerevisiae genome contains a family of five PRS genes, PRS1-PRS5. Using anti-peptide antisera directed against two different epitopes of Prs1p it was shown that Prs1p localizes to granular cytoplasmic structures. This localization was confirmed by living cell microscopy of strains expressing a functional green fluorescent protein (GFP)-tagged Prs1p. Analysis of Prs1p distribution in conditional secretory-deficient (sec) mutants suggested that the observed distribution of Prs1p is independent of the secretory pathway. Electron microscopy revealed that plasma membrane invaginations and accumulation of cytoplasmic vesicles were more frequent in strains which lack some of the PRS genes than in the wild-type. The fact that Deltaprs1 and Deltaprs3 are hypersensitive to caffeine and unable to recover from exposure to it as judged by the release of alkaline phosphatase points to a possible link between Prs and the maintenance of cell integrity.
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Genetic analysis and enzyme activity suggest the existence of more than one minimal functional unit capable of synthesizing Phosphoribosyl Pyrophosphate in Saccharomyces cerevisiae.
The Journal of biological chemistry, 1999Co-Authors: Yolanda Hernando, Andrew T. Carter, Adrian Parr, Bjarne Hove-jensen, Michael SchweizerAbstract:Abstract The PRS gene family inSaccharomyces cerevisiae consists of five genes each capable of encoding a 5-Phosphoribosyl-1(α)-Pyrophosphate synthetase polypeptide. To gain insight into the functional organization of this gene family we have constructed a collection of strains containing all possible combinations of disruptions in the five PRS genes. Phenotypically these deletant strains can be classified into three groups: (i) a lethal phenotype that corresponds to strains containing a double disruption in PRS2 and PRS4 in combination with a disruption in either PRS1 orPRS3; simultaneous deletion of PRS1 andPRS5 or PRS3 and PRS5 are also lethal combinations; (ii) a second phenotype that is encountered in strains containing disruptions in PRS1 and PRS3together or in combination with any of the other PRS genes manifests itself as a reduction in growth rate, enzyme activity, and nucleotide content; (iii) a third phenotype that corresponds to strains that, although affected in their Phosphoribosyl Pyrophosphate-synthesizing ability, are unimpaired for growth and have nucleotide profiles virtually the same as the wild type. Deletions ofPRS2, PRS4, and PRS5 or combinations thereof cause this phenotype. These results suggest that the polypeptides encoded by the members of the PRS gene family may be organized into two functional entities. Evidence that these polypeptides interact with each other in vivo was obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3 polypeptides interact strongly with each other, and there are significant interactions between the PRS5 polypeptide and either the PRS2 or PRS4 polypeptides. These data suggest that yeast Phosphoribosyl Pyrophosphate synthetase exists in vivo as multimeric complex(es).