ECTO-5'-NUCLEOTIDASE OF INTACT CULTURED C6 RAT GLIOMA CELLS

The cells [clonal line (C6) of rat glioma cells] liberated 6.80 .+-. 0.33 .mu.mol of inorganic phosphate/mg of cell protein/h, 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 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. 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. The intact cell is apparently 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.