Purification, characterization and cellular localization of 5'-nucleotidase from Torpedo electric organ.
5'-Nucleotidase was isolated from the electric organ of the electric ray Torpedo marmorata after solubilization in Triton X-100 and deoxycholate by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose. The purified enzyme has a Km for AMP of 38 microM, with a maximal velocity of 31 units/mg of protein. Of the purine and pyrimidine mononucleotides, AMP is hydrolysed most effectively. beta-Glycerophosphate, phosphoenolpyruvate and p-nitrophenyl phosphate are not substrates for the enzyme. Adenosine 5'-[alpha, beta-methylene]diphosphate, ADP and ATP are competitive inhibitors in this order of potency. Concanavalin A inhibits enzyme activity in a non-competitive manner. Whereas Mg2+, Ca2+ and Sr2+ activate enzyme activity in the millimolar range, Hg2+, and in particular Pb2+ and Zn2+, inhibit enzyme activity. On SDS/polyacrylamide-gel electrophoresis the enzyme has an apparent Mr of 62000, whereas that of the native deoxycholate-enzyme complex is 131000. An antiserum raised against the native enzyme inhibits enzyme activity. Inhibition studies suggest the presence of tissue-specific variants of the enzyme. By immunohistochemical analysis the enzyme can be localized to the ramifications of nerve terminals in the electric organ.
Grondal
E J
EJ
AK Neurochemie, Zoologisches Institut der J.W. Goethe-Universitt, Frankfurt am Main, Federal Republic of Germany.
Zimmermann
H
H
D015720:5'-Nucleotidase; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D004557:Electric Organ / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry; D007106:Immune Sera / Q000494:pharmacology; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014018:Tissue Distribution; D014101:Torpedo / Q000378:metabolism*
D002413:Cations, Divalent; D007106:Immune Sera; D009708:Nucleotidases; D015720:5'-Nucleotidase