Purification of thymidine phosphorylase from human amniochorion

Abstract

Thymidine phosphorylase (thymidine : orthophosphate deoxyribosyltransferase, EC 2.4.2.4) has been purified 1500-fold from extracts of human amniochorion. The purified enzyme catalyzes the phosphorolysis of deoxythymidine and to a lesser extent deoxyuridine but not deoxycytidine nor uridine. Discontinuous gel electrophoresis of the freshly purified enzyme shows a band containing 95% of the stainable protein. Gradient gel electrophoresis resolves the preparations into an active fraction with an apparent molecular weight of about 120 000 and a heavier less active or inactive fraction of about 180 000. Storage of the enzyme results in a decrease of the 120 000 dalton component, a loss in activity, and an apparent increase in the high molecular weight component. Sodium dodecyl sulfate gel electrophoresis shows only a single subunit of about 58 000 daltons which does not change on storage. These data are consistent with an active enzyme dimeric in structure which is capable of being converted to a less active form larger in molecular weight and possibly trimeric or tetrameric in structure.