The activation of adenylate cyclase in lysed pigeon erythrocytes requires, among several cofactors, a nucleotide which may be ATP, GTP, or many other triphosphates. However, after removal of endogenous nucleotides by gel filtration o r by adsorption onto charcoal the requirement can be met only by GTP, or an analog of GTP. The GTP is required during the activation of the cyclase by toxin even if GTP is also included during the subsequent adenylate cyclase assay, conducted without toxin. In the presence of GTP it is possible t o assay for the cytosolic protein that is also required for the action of cholera toxin. By gel filtration, its apparent molecular weight is 15,000-20,000.
Key words: cholera toxin, GTP, pigeon erythrocyte, adenylate cyclase, cytosolic factor, phosphodiesterase protein activator
Fragment A l of cholera enterotoxin catalyses the activation of adenylate cyclase (ATP pyrophosphate-lyase [cyclizing] E.C. 4.6.1 .I .) in broken cells 113 . Besides plasma membranes, as a source of the cyclase, there are several soluble factors, omission of any one of which reduces the ability of the system t o respond t o the toxin and thus increases the toxin concentration required for a given response. We and others have attempted t o identify the relevant factors and hitherto have recognized four necessary components of the cytosol, namely NAD, nucleoside triphosphate, an unidentified protein, and thiol compounds that can reduce the cystine in toxin's subunit A [2] . The NAD requirement is specific, for it acts as an ADP-ribosyl donor [3,4]. The nucleoside triphosphate requirement is less specific and can be met by ATP, CTP, UTP, GTP, or even by their imido or ATP, CTP, GTP, TTP, UTP: adenosine, cytosine, guanosine, thymidine, and uridine triphosphate;