hp://w w w .jb.org/ D ow nladed from D Evain and W B Anderson Chinese hamster ovary cell membranes activated in vitro by cholera toxin
- J. Flores, G.W.G. Sharp
- J. Clin. Inuest
The adenylate cyclase activity of crude membranes prepared from Chinese Hamster Ovary (CHO) cells in culture is enhanced about a-fold with 0.1 mM GTP and 5to lo-fold with 0.1 mu guanyl-V-y1 imidodiphosphate (Gpp(NH)p). Incubation of intact CHO cells with cholera toxin results in pronounced activation of adenylate cyclase, and this activated form of the enzyme is also stimulated by GTP and Gpp(NH)p. Incubation of isolated CHO membranes with activated cholera toxin in the presence of 5 mM ATP, 5 mM MgCl,, 1 mM NAD+, and 1 mM GTP produces at least a 20-fold stimulation of cyclase activity. Of significance is the finding that with cell-free activation by toxin the adenylate cyclase system is converted to a form which is sensitive to inhibition by GTP and Gpp(NH)p. To achieve this state of the cyclase which exhibits this inhibitory response, it is essential that activation bj the A, fragment of cholera toxin be carried out in the presence of ATP, NAD’, and GTP. The guanyl nucleotides inhibit in a concentration-dependent manner; Gpp(NH)p is consistently observed to be a more potent inhibitor than GTP. The inhibitory response is transitory, with maximal inhibition during the first minute of assay followed by a progressive decline in inhibition with increasing time of incubation. These results raise the possibility that the CHO adenylate cyclase complex may possess both GTP activatory and inhibitory sites and that other factors or events modulate how the cyclase will respond to GTP.