The development of new classes of multisubstrate affinity chromatography media directed toward deoxycytidine kinase--and related purine-specific activities--now makes it possible to resolve and isolate these enzymes in a pure state. These activities are essential for activating important chemotherapeutic nucleosides. However, it has not been clear which protein, of several isoenzymes reported, is responsible for phosphorylating each drug, nor is it known whether such isoenzymes are distinct gene-products or merely proteolytic artifacts. The ability to obtain pure proteins provides the opportunity to obtain peptide maps and partial amino acid sequences to each, and raise polyclonal antibodies. Preliminary structural comparisons will be made, and immunological cross-reactivity determined, to assess the relatedness of putative isoenzymes. Each enzyme isolated will be characterized in detail as to its substrate specificities, and its physical and kinetic properties. The mechanism of deoxynucleoside triphosphate end-product inhibition of these enzymes has never been defined, but new evidence from this laboratory suggests that these nucleotides are acting as multisubstrate inhibitors. This model will be tested kinetically now, and reactive analogs will be employed later to map the deoxynucleotide triphosphate binding sites. The final objective during this period is to clone the DNAs complementary to the kinase mRNAs (cDNAs) and to determine their sequences; these will be related to amino acid sequence data and will permit more complete structural comparison of isoenzymes. Cloning of cDNAs will be accomplished with probes generated by the Polymerase Chain Reaction, using as primers oligonucleotides reflecting amino acid sequences. Availability of cDNAs will open the way to future isolation of genomic sequences, study of the regulation of gene expression and to structure-function studies based on site-directed mutagenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA047828-03
Application #
3191614
Study Section
Biochemistry Study Section (BIO)
Project Start
1989-05-01
Project End
1993-04-30
Budget Start
1991-05-01
Budget End
1993-04-30
Support Year
3
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Ohio State University
Department
Type
Schools of Arts and Sciences
DUNS #
098987217
City
Columbus
State
OH
Country
United States
Zip Code
43210