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The absence of 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAase) activity in malignant cells, and the putative localization of its gene, suggest that this enzyme deficiency might be due to a genomic alteration also involving a tumour-suppressor gene. We studied the possible occurrence of inactive forms of the protein in two MTAase-negative cell lines, namely K562 and Jurkat, by immunochemical methods. Two highly specific antisera, directed against different epitopes of the phosphorylase [Della Ragione, Oliva, Gragnaniello, Russo, Palumbo & Zappia (1990) J. Biol. Chem. 265, 6241-6246], were used to carry out immunotitration and immunoblotting analyses, as well as to investigate the biosynthesis of the enzyme. No MTAase protein was detected by Western-blotting technique performed under conditions where all the phosphorylase-positive samples gave a clear band at the MTAase subunit molecular mass. No cross-reacting material was observed by a sensitive immunotitration method which permitted the detection of as low as 0.5 ng of protein. Moreover, the results obtained by [35S]methionine-labelling experiments ruled out phosphorylase biosynthesis in the negative cell lines. Altogether, these data suggest that an alteration at the gene level hampering the specific mRNA biosynthesis or resulting in an untranslatable mRNA is the cause of the enzyme deficiency in the MTAase-negative cell lines studied.

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAase) was purified to homogeneity (10,000-fold) from bovine liver with a recovery of 12%. The pure protein shows a molecular weight of about 98,000 +/- 3,000 and is composed of three apparently identical subunits. Several physicochemical features have been investigated including hydrodynamic properties, amino acid composition, and secondary structure. In particular, the CD spectrum of the protein indicates a very low alpha-helical content and a large percent of beta-structure and random coil. The pure protein was used to raise specific rabbit antisera but, because of the scarce antigenic properties of the native enzyme, different chemically modified forms were prepared and employed as immunogens. Among the antibodies obtained, those to keyhole limpet hemocyanin-MTAase recognize both the native and the denatured enzyme and are also active against the human protein. Therefore, they were employed as a tool to investigate the occurrence of inactive forms of MTAase in two human malignant cell lines lacking this enzymatic activity. The results obtained with K562 and Jurkat cells indicate that the protein is absent in these phosphorylase-deficient cell lines.

5'-Methylthioadenosine phosphorylase has been purified to homogeneity (30,000-fold) from human full-term placenta by a procedure involving covalent chromatography on organomercurial-agarose as the major step. The specific activity of the homogeneous enzyme is 10.2 mumol of 5'-methylthioadenosine cleaved per min per mg of protein, and the overall yield is about 20%. The enzyme has a molecular weight of 98,000, as determined by gel filtration on Sephacryl S-200 and Superose 6B, and is composed by three apparently identical subunits with a molecular weight of 32,500. The isoelectric point is 5.5, and the optimal pH ranges from 7.2 to 7.6. The resistance of the enzyme to thermal inactivation is increased remarkably by the addition of 5'-methylthioadenosine or phosphate. The homogeneous enzyme shows an absolute requirement for -SH-reducing agents and is specifically and rapidly inactivated by thiol-blocking compounds. The reaction catalyzed by the enzyme is fully reversible with a Keq of 1.39 X 10(-2) (in the direction of phosphorolysis) at 37 degrees C and pH 7.4. The Km values for 5'-methylthioadenosine, phosphate, adenine, and 5-methylthioribose 1-phosphate are 5, 320, 23, and 8 microM, respectively.