Purine catabolism in man: characterization of placental microsomal 5′-nucleotidase

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Abstract
Human placental microsomal 5''-nucleotidase (EC 3.1.3.5) was prepared free of alkaline phosphatase by isoelectric focusing. A total of 7 electrophoretic variants were isolated during the preparation of 6 placentas. Only 3-6 variants were found in a single placenta. The isoelectric pH''s were 6.70, 6.44, 6.23, 6.02, 5.76, 5.63 and 5.44. These were found to be composed of variable quantities of a large, medium and low molecular weight form. The apparent molecular weights of the medium and light form of the enzyme were 86,500 and 43,500, respectively, as estimated from Stokes radius and sedimentation velocity determinations. The electrophoretic variants were not distinguishable with respect to specific activity and Km for AMP, GMP or CMP or inhibition by ATP, CTP or adenosine. These electrophoretic variants appeared to be pseudoisozymes based upon different states of aggregation of a common primary sequence. There was a wide range of substrate specificity among nucleoside 5''-monophosphates which included 2-deoxyribose compounds. With AMP as 100, substrate activity was: CMP, 122; NMN, 47; GMP, 68; IMP, 63; XMP, 28 and UDP-glucose, 68. The Km for AMP, GMP and CMP ranged from 12-18 .mu.M, from 33-67 .mu.M and from 170-250 .mu.M, respectively. Although 5''-nucleotidase was active in the absence of divalent cation, 5 mM MgCl2 stimulated the enzyme activity to 234% of control and shifted the pH optimum of 9.8 to a plateau from pH 7.4-9.8.