PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN 5'-METHYLTHIOADENOSINE PHOSPHORYLASE - DEFINITE IDENTIFICATION OF CODING CDNA

5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16(INK4A). Ch romosomal deletions encompassing both the phosphorylase and p16(INK4A) genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic difference s between malignant and normal cells. Recently, the cloning of the pho sphorylase gene has been reported on the basis of indirect evidence. I n order to demonstrate definitely the identification of 5'-methylthioa denosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria. The recombinant phosphorylase has been purified to homog eneity and its physicochemical, immunological and kinetic features hav e been characterized. The results obtained allowed the conclusive demo nstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization. (C) 1996 Aca demic Press, Inc.