The objective of this research is to delineate biochemical and genetic mechanisms regulating purine nucleotide synthesis in man. Studies of inherited enzyme defects underlying excessive purine nucleotide and uric acid production in some patients with gout have contributed to concepts of the control of rates of purine synthesis de novo. Superactivity of phosphoribosylpyrophosphate (PRPP) synthetase (PRS), which catalyzes synthesis of the regulatory substrate PRPP, is one such defect. The proposed investigations focus on defining genetic and protein structural determinants of the activity of normal human PRS and on identifying genetic and protein structural aberrations underlying inherited PRS superactivity. Molecular cloning, protein structural, and enzyme kinetic methods will be employed to pursue this Specific Aim. Two cDNAs encoding proteins highly homologous to authentic PRSs have been cloned from human cDNA libraries. Despite striking sequence identity in protein coding regions, 3' noncoding regions of these hPRS cDNAs are divergent, suggesting separate hPRS genes coding for each. Full-length hPRS cDNAs complementary to hPRS mRNAs identified in human tissues will be isolated, characterized, and utilized as probes in several projects. 1) Kinetic characteristics and deduced amino acid sequences of normal and mutant hPRSs and PRSs characterized from bacterial and mammalian sources will be compared in order to define the structural basis of PRS activity. 2) Patterns of hPRS gene restriction and transcription and translation of hPRS genes will be examined to evaluate normal hPRS gene structure and expression and to identify provisional sites of derangement in genetic control of enzyme activity in cells with PRS superactivity. 3) Utilizing PCR and cDNA cloning methods, hPRS cDNAs will be cloned and characterized from cells of affected patients in order to define precise genetic defects underlying PRS superactivity. In these studies and in studies of the structure and function of normal hPRS gene products, expression of hPRS cDNAs will be attempted using an expression system in an E coli strain engineered to lack bacterial PRS. 4) Mapping of hPRS 1 and hPRS 2 genetic loci and identification of additional hPRS (pseudo)genes will be undertaken utilizing in situ chromosomal hybridization, pulsed field gel electrophoresis mapping and genomic DNA cloning. 5) Finally, genomic DNA libraries will be probed with hPRS cDNAs in order to clone and characterize normal and variant hPRS genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK028554-13
Application #
3228893
Study Section
Biochemistry Study Section (BIO)
Project Start
1980-09-01
Project End
1993-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
13
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Chicago
Department
Type
Schools of Medicine
DUNS #
225410919
City
Chicago
State
IL
Country
United States
Zip Code
60637
Garcia-Pavia, Pablo; Torres, Rosa J; Rivero, Manuel et al. (2003) Phosphoribosylpyrophosphate synthetase overactivity as a cause of uric acid overproduction in a young woman. Arthritis Rheum 48:2036-41
Becker, M A (2001) Phosphoribosylpyrophosphate synthetase and the regulation of phosphoribosylpyrophosphate production in human cells. Prog Nucleic Acid Res Mol Biol 69:115-48
Becker, M A; Ahmed, M (2000) Cell type-specific differential expression of human PRPP synthetase (PRPS) genes. Adv Exp Med Biol 486:10-May
Ahmed, M; Taylor, W; Smith, P R et al. (1999) Accelerated transcription of PRPS1 in X-linked overactivity of normal human phosphoribosylpyrophosphate synthetase. J Biol Chem 274:7482-8
Becker, M A; Taylor, W; Smith, P R et al. (1998) Regulation of human PRS isoform expression. Adv Exp Med Biol 431:215-20
Becker, M A; Taylor, W; Smith, P R et al. (1996) Overexpression of the normal phosphoribosylpyrophosphate synthetase 1 isoform underlies catalytic superactivity of human phosphoribosylpyrophosphate synthetase. J Biol Chem 271:19894-9
Becker, M A; Smith, P R; Taylor, W et al. (1995) The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity. J Clin Invest 96:2133-41
Fry, D W; Becker, M A; Switzer, R L (1995) Inhibition of human 5-phosphoribosyl-1-pyrophosphate synthetase by 4-amino-8-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine-5'- monophosphate: evidence for interaction at the ADP allosteric site. Mol Pharmacol 47:810-5
Becker, M A; Nosal, J M; Switzer, R L et al. (1994) Point mutations in PRPS1, the gene encoding the PRPP synthetase (PRS) 1 isoform, underlie X-linked PRS superactivity associated with purine nucleotide inhibitor-resistance. Adv Exp Med Biol 370:707-10
Roessler, B J; Nosal, J M; Smith, P R et al. (1993) Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene. J Biol Chem 268:26476-81

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