Purification and Characterization of Recombinant Rat Phosphoribosylpyrophosphate Synthetase Subunit I and Subunit II

Abstract

Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the formation of PRPP from ATP and ribose 5-phosphate. The enzyme has been purified from bacteria [1, 2] and mammalian tissues [3–5]. The rat liver enzyme exists as complex aggregates of 34-, 38-, and 40-kDa components, the 34-kDa species being the catalytic subunit [5]. The 34-kDa component is actually a mixture of two isoforms, designated as PRS I and PRS II [5, 6]. The two isoforms are composed of 317 amino acid residues, and the sequences are highly conserved, differing only by 13 residues [6]. Furthermore, the amino acid sequences of human [7, 8] and rat [6] PRS II differ only by 3 residues and those of human [8, 9] and rat [6] PRS I are completely conserved. The PRS I and PRS II are encoded by two distinct genes located on X-chromosome [10, 11]. The two genes are expressed in almost all tissues of rats but the mRNA levels differ with the tissues [12]. These observations suggest functional differences between catalytic and/or regulatory properties of PRS I and PRS II. However, separation of the two proteins from the native enzyme was impossible. Therefore, the respective rat cDNAs were expressed in Escherichia coli. The expression vector was designed to produce the unfused proteins. The recombinant isoforms (named rPRS I and rPRS II) were isolated and characterized.