AMP and IMP dephosphorylation by soluble high- and low-Km 5'-nucleotidases.
Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define 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. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.
Spychala
J
J
Department of Internal Medicine, Clinical Research Center, University Hospital, Ann Arbor, Michigan 48101-0108.
Madrid-Marina
V
V
Nowak
P J
PJ
Fox
I H
IH
D015720:5'-Nucleotidase; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D007291:Inosine Monophosphate / Q000378:metabolism*; D007292:Inosine Nucleotides / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009708:Nucleotidases / Q000302:isolation & purification / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D051381:Rats; D013379:Substrate Specificity; D013601:T-Lymphocytes / Q000201:enzymology
D007292:Inosine Nucleotides; D007527:Isoenzymes; D007291:Inosine Monophosphate; D000249:Adenosine Monophosphate; D009708:Nucleotidases; D015720:5'-Nucleotidase