Guanine nucleotide inhibition of cyc- S49 mouse lymphoma cell membrane adenylyl cyclase

Abstract

Cyc- S49 mouse lymphoma cell membranes contain an adenylyl cyclase system which is deficient in the regulatory properties characteristic of a stimulatory guanine nucleotide-binding regulatory component (Ns), such as enhancement of activity in the presence of GTP, GTP analogues, and NaF. Detailed kinetic analysis of cycl- adenylyl cyclase showed, however, that it is not unresponsive to these agents, for their addition to assays elicited inhibitory effects. Inhibition of cyc- activity was seen in 11 different membrane preparations obtained from two different strains and was observable both in the absence and presence of the strongly stimulatory diterpene forskolin. The GTP analogues GTP gamma S and guanyl-5'-yl imidodiphosphate caused a maximum of 60% inhibition with IC50 values of 2 and 12 nM, respectively. GTP itself was less potent than its analogues, with an IC50 of 100 mM, and elicited less inhibition as well (a maximum of 25%). Cholera toxin treatment of cyc- S49 cell membranes, under conditions which appeared to maximally activate the adenylyl cyclase system of wild type S49 cell membranes, had no effect on inhibition of catalytic activity. Our results indicate the presence in cyc- S49 cell membranes of a guanine nucleotide-binding component which is inhibitory to adenylyl cyclase activity (Ni). This suggests (a) that these membranes cannot be considered as completely deficient of adenylyl cyclase guanine nucleotide-binding regulatory components, and (b) that these membranes offer a unique opportunity to study Ni-mediated effects of guanine nucleotides and fluoride in the functional absence of Ns-mediated effects of these agents.