74 RAT LIVER PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE: EXISTENCE AS HETEROGENEOUS AGGREGATES AND IDENTIFICATION OF CATALYTIC SUBUNIT

Abstract

Phosphoribosylpyrophosphate (PRPP), an important substrate in the synthesis of purine, pyrimidine and pyridine nucleotides, is synthesized by the catalysis of PRPP synthetase. The enzyme from human erythrocytes exists as various associated forms and is subject to complex regulation. However, the in situ regulation of PRPP synthesis is poorly understood. As an approach to the problem, we purified rat liver PRPP synthetase and partially characterized the complex physical nature of the enzyme. It was purified to a specific activity of 7.28 μmol/min/mg, the highest value so far reported for a mammalian enzyme, but still contained several components of 34 kDa, 38 kDa and 40 kDa in relative amounts of that order. In various usual chromatographies, these components were coeluted, suggesting that the heterogeneous aggregate is the native form of the enzyme. To identify the catalytic subunit, the enzyme was subjected to gel filtration on TSK G2000SW in 1 M MgCl2, an agent to dissociate proteins. The enzyme appeared as a 68 kDa peak and the last quarter of the fractions contained only the 34 kDa species, with a definite catalytic activity. Other components in the aggregate appear to exert suppressive effects on the activity. Sequence analyses of N-terminal region and two tryptic peptides of the 34 kDa subunit, together with our cDNA cloning experiments (J.Biol.Chem., 262, 14867, 1987), revealed that the 34 kDa component consists of two homologous isoforms (PRS I and II).