Doctoral Dissertations

Date of Award

6-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Robert E. Bryant, Jorge E. Churchich

Committee Members

John Koontz, Otto Schwarz

Abstract

A previous investigation of the clonal cell line KG62 had shown it to have significantly reduced intracellular PRPP levels when compared to the parental V-79A cell line. In an attempt to explain this phenotypic difference in the two cell lines, the physical and kinetic properties of the enzyme responsible for PRPP production, PRPP-synthetase, have been examined. Initial examination of the catalytic properties of the V-79A and KC62 PRPP-synthetase was conducted using 110,000 x G supernatant preparations of each enzyme. Determination of the kinetic parameters (Km and Vmax) for the V-79A and KC62 enzyme provided no evidence for a difference in their catalytic properties. Subsequent studies of the enzymatic activity in the presence of various nucleotides, however, demonstrated the KC62 enzyme to have an increased sensitivity to a variety of nucleotide inhibitors compared to its V-79A counterpart.

A scheme for the purification of V-79A and KC62 PRPP-synthetase was devised and the kinetic and physical properties of the purified enzymes were examined. Km values for the V-79A and KC62 PRPP-synthetase were found to be very similar. Inhibition of both enzymes by AMP, GMP and XMP was examined with respect to both substrates. For any given inhibitor and variable substrate, the resulting type of inhibition was found to be the same for the V-79A and KC62 enzyme. The apparent Ki values for the KC62 enzyme, however, were in all cases less than those of the V-79A enzyme. These results suggested that the KC62 enzyme possessed a structural alteration rendering it more sensitive to inhibition by nucleotide inhibitors. Examination of the physical properties of the two enzymes failed to provide evidence of a major structural difference between the two enzymes. KC62's subunit molecular weight, molecular weight, and pattern of thermal inactivation at 55°C was identical to that of the V-79A enzyme.

Finally, the consequences of KC62's reduced PRPP levels on its nucleotide pool sizes have been assessed. The IMP, GMP NADH and CDP pools of the KC62 cells were significantly less than those of V-79A cells. While it is reasonable to attribute KC62's reduced nucleotide pools to its reduced PRPP levels, the specific mechanisms involved have not been determined.

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