33 EVIDENCE FOR |[ldquo]|LOU Km|[rdquo]| AND |[ldquo]|HIGH Km|[rdquo]| SOLUBLE 5|[lsquo]|- NUCLEOTIDASES IN HUMAN TISSUES AND RAT LIVER

摘要

Three distinct 5′-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T- and B-lymphoblasts and rat liver using AMP-sepharose 4B affinity chromatography and a sequence of specific eluting agents. We have defined these activities as “low Km” 5′-nucleotidase, “high Km” 5′-nucleotidase and nonspecific phosphatase. “High Km” 5′-nucleotidase was eluted with 0.5 M NaCl, “low Km” 5′nucleotidase was eluted with 10 mM ADP and nonspecific phosphatase was not retained on the column. The relative content of “high Km” and “low Km” activities in the tissues studied ranged from 5.5 to 264. The molecular weight of the “low Km” enzymes ranged from 72.5 to 209 kD, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 and for IMP from 10 to 26 uM, respectively. ATP and ADP were inhibitors of “low Km” enzymes with the apparent Ki values of 55 to 100 and 8 to 20 uM, for ATP and ADP, respectively. The molecular weight of the “high Km” 5′-nucleotidases ranged from 182 to 210 kD, pH optimum was at 6.5, and Km for IMP was 0.3 to 0.5 mM and for AMP 1.0 to 9.4 mM. “High Km” enzymes were activated by ATP with A0.5 values, measured at 20 uM IMP, of 1.7 to 2.3 mM. The ATP activation was substrate dependent. The data indicate that soluble “low Km” and “high Km” 5′-nucleotidase coexist in mammalian cells and fulfill different functions. These observations suggest a complex system for the regulation of AMP and IMP dephosphorylation.