Determination of an obligatory role for diadenosine 5'5'''-p1,p4- tetraphosphate (Ap4A) in the cellular responses to oxidative stress in the yeast, Saccharomyces cerevisiae, is the goal of the proposed experimentation. This goal is based on the hypothesis that Ap4A is a regulatory nucleotide that signals the onset of oxidative stress and initiates adaptive responses. The basic strategy is to use molecular genetics to manipulate the intracellular Ap4A level and, subsequently to measure perturbations in the cellular sponses to oxidative stress. The gene (AFP) for Ap4A phosphorylase will be isolated from a yeast genomic library using an immunologic or oligonucleotide probe. The gene will be sequenced and its identity confirmed by expression in E. coli. Yeast will be transformed with a shuttle vector containing AFP with a regulatable promoter. Induced expression of AFP should yield increased enzymic activity and decreased endogenous Ap4A. Null mutants of AFP will be constructed by disruption of the chromosomal gene. Null mutants should exhibit negligible enzymic activity and have an increased level of Ap4A. The expected changes in Ap4A phosphorylase activity and endogenous Ap4A will be confirmed by enzymic assay and hplc analysis, respectively. Under conditions of genetic manipulation of endogenous Ap4A, transformed yeast will be subjected to oxidative stress induced by hyperoxia, hypoxia, and various compounds. Viability, synthesis of stress proteins, and activities of superoxide dismutase and catalase will be analyzed as indices of the cellular response to oxidative stress. If Ap4A is required for cellular responses to oxidative stress, then such responses should be decreased or absent in transformants induced for high enzyme level expression. Conversely, null mutants may be more resistant to oxidative stress. This experimental approach of using molecular genetics and biochemistry should unequivocally determine if Ap4A is required for responses to oxidative stress. The strength of this strategy is that it is not restricted to testing this particular hypothesis for the function of Ap4A. Establishment of Ap4A as a regulatory nucleotide for oxidative stress would elucidate a fundamental biochemical component in adaptive metabolic regulation. Reactive oxygen species participate in normal physiologic processes such as the microbiocidal action of neutro- phils, and they have also been implicated in pathologic conditions such as carcinogenesis, mutagenesis, cellular degeneration related to aging, and post-ischemic tissue damage. Thus, elucidation of Ap4A as a regulatory nucleotide for oxidative stress would be relevant to physiologic and pathophysiologic processes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Biochemistry Study Section (BIO)
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University of Texas Health Science Center San Antonio
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Robinson, A K; de la Pena, C E; Barnes, L D (1993) Isolation and characterization of diadenosine tetraphosphate (Ap4A) hydrolase from Schizosaccharomyces pombe. Biochim Biophys Acta 1161:139-48
Garrison, P N; Koob, M; Barnes, L D (1992) Physical mapping of the Saccharomyces cerevisiae Ap4A phosphorylase I-encoding gene by the Achilles' cleavage method. Gene 111:131-4
Avila, D M; Robinson, A K; Kaushal, V et al. (1991) A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae. J Bacteriol 173:7875-80
Robinson, A K; Barnes, L D (1991) Chemical modification of a functional arginine residue in diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) phosphorylase I from Saccharomyces cerevisiae. Biochem J 279 ( Pt 1):135-9
Avila, D M; Kaushal, V; Barnes, L D (1990) Immunoaffinity chromatography of diadenosine 5',5'''-P1,P4-tetraphosphate phosphorylase from Saccharomyces cerevisiae. Biotechnol Appl Biochem 12:276-83
Kaushal, V; Avila, D M; Hardies, S C et al. (1990) Sequencing and enhanced expression of the gene encoding diadenosine 5',5'''-P1, P4-tetraphosphate (Ap4A) phosphorylase in Saccharomyces cerevisiae. Gene 95:79-84
Garrison, P N; Mathis, S A; Barnes, L D (1989) Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations. J Bacteriol 171:1506-12
Kaushal, V; Barnes, L D (1986) Effect of zwitterionic buffers on measurement of small masses of protein with bicinchoninic acid. Anal Biochem 157:291-4
Garrison, P N; Mathis, S A; Barnes, L D (1986) In vivo levels of diadenosine tetraphosphate and adenosine tetraphospho-guanosine in Physarum polycephalum during the cell cycle and oxidative stress. Mol Cell Biol 6:1179-86
Robinson, A K; Barnes, L D (1986) Three diadenosine 5',5''-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase. Arch Biochem Biophys 248:502-15

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