Measurement of Glycinamide Ribonucleotide Synthetase
Activity in Intact Human Fibroblasts and Chinese Hamster
Ovary Cells

Hards, Robert G.; Patterson, David

doi:10.1159/000469333

Enzyme

1986

DOI: 10.1159/000469333

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Measurement of Glycinamide Ribonucleotide Synthetase Activity in Intact Human Fibroblasts and Chinese Hamster Ovary Cells

Robert G. Hards¹,

David Patterson²

Abstract: An intact cell assay system based on Tween 80 permeabilization was used to investigate glycinamide ribonucleotide (GAR) synthetase activity in human fibroblasts and Chinese hamster ovary cells. Optimal conditions for the assay of the enzyme were determined with regards to ATP, MgCl(2), NH(4)C1 and ribose-5'-phosphate concentrations as well as pH. Using the optimal assay conditions, the V(max) values as determined by Lineweaver-Burke double reciprocal plots were found to be 5.19 nmol GAR formed/5 X 105 cells/30… Show more

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1986

1991

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Cited by 7 publications

References 11 publications

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Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

et al. 1989

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New mutant alleles of the adenosine2 locus (ade2; 2-17.7) have been isolated using the eye-color phenotype exhibited by the prototype auxotrophic allele ade2 as the screening criterion. The new mutants form a single complementation group, suggesting that they all exhibit purine auxotrophy and defective formylglycineamide ribotide amidotransferase enzyme, like ade2. Tests carried out on particular new alleles confirm these suggestions. The new mutants all exhibit more extreme physical defects than the prototype. They have wing abnormalities like mutants defective in pyrmidine biosynthesis and reduced bristles like those defective in protein synthesis; thus they exhibit the combined visible phenotype of rudimentary wings, rosy eyes, and bobbed bristles. Cytogenetic analysis places the locus in the interband proximal to 26B1-2.

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Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

et al. 1989

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Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade?A mutant deficient in this activity

Barton

Bleskan

Patterson

1991

Somat Cell Mol Genet

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We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

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Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities

Chang

Barnes

Schild

et al. 1991

Somat Cell Mol Genet

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The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

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Measurement of Glycinamide Ribonucleotide Synthetase Activity in Intact Human Fibroblasts and Chinese Hamster Ovary Cells

Cited by 7 publications

References 11 publications

Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade?A mutant deficient in this activity

Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities

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