title: Ecto-5'-nucleotidase of intact cultured C6 rat glioma cells.
abstract: The characteristics of 5'-nucleotidase in a clonal line (C6) of rat glioma cells has been examined in detail. The cells liberated 6.80 +/- 0.33 mumol of inorganic phosphate/mg of cell protein/hour, producing nearly equimolar amounts of adenosine and inorganic phosphate from AMP in the extracellular fluid. No 5'-nucleotidase was released by the cells into the medium. Most of the 5'-nucleotidase activity was found to be located in the outer surface of the plasma membrane of C6 cells and rapidly accessible to exogenous AMP, by experiments based upon differential labeling of extracellular and intracellular compartments with 32P and 33P. The ecto-enzyme was active in the absence of divalent cations. However, Mn2+ or Co2+ were somewhat stimulatory. Zn2+ suppressed activity very markedly. The relationship of enzymatic reaction velocity to pH was complex, with an optimum at pH 7.4 for all substrates tested. The ecto-5'-nucleotidase readily hydrolyzed 5'-AMP and 5'-UMP. Other 5'-nucleoside monophosphates, including 5'-deoxy-AMP, were also hydrolyzed, but more slowly; 2'- or 3'-nucleoside monophosphates were not attacked. The ecto-5'-nucleotidase in the intact cell obeyed Michaelis-Menten kinetics. Apparent Km for AMP was 0.22 mM; apparent Km values for other substrates were similar and ranged from 0.16 to 0.18 mM. ADP exerted a very powerful inhibitory effect, behaving as a competitive inhibitor, and 5'-UMP behaved as a strictly competitive substrate for 5'-AMP. ATP and ITP were inhibitory. Of these, ITP served to increase Km for AMP. ATP did likewise, but also greatly lowered Vmax. These findings indicate that the intact cell is capable of rapid hydrolysis of exogenous 5'-AMP, to produce adenosine at the cell surface at a rate which responds directly to extracellular AMP concentration but which can be suppressed by extracellular ADP or ATP.
mesh_terms: D001667:Binding, Competitive; D002413:Cations, Divalent; D002460:Cell Line; D003035:Cobalt / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009708:Nucleotidases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D002413:Cations, Divalent; D003035:Cobalt; D004492:Edetic Acid; D009708:Nucleotidases; D008274:Magnesium