The phosphorylation of dadenosine and dguanosine is a critical step in the accumulation of toxic levels of intracellular deoxynucleoside triphosphates. In addition, these reactions are essential for the activation of antiviral and anticancer nucleoside analogs. This proposal will focus upon the enzymes which phosphorylate dadenosine and dguanosine. We will test the hypothesis that the regulation of dadenosine and dguanosine phosphorylation is mainly dependent upon the concentration of dadenosine and dguanosine. The approach to this problem will include: a) Purification and characterization of dadenosine and dguanosine phosphorylating activities from human placental cytoplasm, and b) performance of careful kinetic studies of the purified activities. The methods will include radiochemical assays of nucleoside kinase enzymes, affinity chromatography, ion exchange chromatography, other enzyme fractionation techniques, initial velocity studies and product inhibition studies. This approach will lead to an understanding of the regulation of the enzymes which phosphorylate dadenosine and dguanosine. It is hoped that new insights will be obtained into the metabolic basis for the accumulation of toxic deoxynucleotides in human cells and the development of noval antiviral and anticancer nucleoside analogs will be facilitated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032837-03
Application #
3281990
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-07-01
Project End
1988-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
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Spychala, J; Fox, I H (1989) The application of affinity chromatography for the separation of ""high Km"" and ""low Km"" 5'-nucleotidase and other AMP metabolizing enzymes. Adv Exp Med Biol 253B:119-27
Datta, N S; Shewach, D S; Mitchell, B S et al. (1989) Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase. J Biol Chem 264:9359-64
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Spychala, J; Madrid-Marina, V; Fox, I H (1989) Evidence for ""low Km"" and ""high Km"" soluble 5'-nucleotidases in human tissues and rat liver. Adv Exp Med Biol 253B:129-34
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Hurley, M C; Fox, I H (1985) Human placental nucleoside kinase activities. Ann N Y Acad Sci 451:42-53
Hurley, M C; Lin, B; Fox, I H (1985) Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase. J Biol Chem 260:15675-81