Purification and properties of a 5'-nucleotidase from rat renal membranes.
5'-Nucleotidase activity was solubilized from a particulate fraction of rat renal homogenates by Sulphobetaine 14. An 11,430-fold purification was achieved by a two-step chromatographic procedure using concanavalin-A Sepharose and ADP-agarose. SDS-PAGE of the purified material revealed a single polypeptide band with a Mr of 69,000. The enyzme exhibited absolute specificity for 5'-mononucleotides. Among 7 tested substrates, adenosine monophosphate (AMP) showed the highest value of V/Km. The Km for 5'-AMP is 5.1 mumol/l and V is 632 mumol/min/mg. The plot of activity versus pH shows a broad plateau between pH 6.8 and 8.0. The hydrolysis of 5'-AMP was competitively inhibited by adenosine 5'-triphosphate (ATP; Ki = 1.2 mumol/l), adenosine 5'-diphosphate (ADP; Ki = 0.032 mumol/l) and alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP; Ki = 0.005 mumol/l). All of the 5 detergents tested activated the enzyme. Sulphobetaine 14 was the most potent and resulted in a 4-fold stimulation by increasing V without change of Km. Addition of exogenous divalent cations was not required for activity. However, the enzyme was inhibited by EDTA. This inhibition was overcome by the addition of Co2+, Mn2+ and to a lesser extent of Mg2+. Hg2+, Zn2+, Cu2+ and Pb2+ inhibited in the low micromolar range. The properties of this enzyme from the rat kidney are similar to those reported in the literature for ecto 5'-nucleotidases from other sources.
Le Hir
M
M
Department of Forschung, Medizinische Universitäts-Poliklinik, Kantonspital Basel, Schweiz.
Gandhi
R
R
Dubach
U C
UC
D015720:5'-Nucleotidase; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D003208:Concanavalin A / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D051381:Rats
D002413:Cations, Divalent; D003902:Detergents; C523965:alpha,beta-methyleneadenosine 5'-diphosphate; D003208:Concanavalin A; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; D015720:5'-Nucleotidase