Heat-stable enterotoxin activation of immunopurified guanylyl cyclase C. Modulation by adenine nucleotides.

We studied the activation and inactivation of recombinant guanylyl cyclase (GC) C stably expressed in human kidney 293 cells. Activation of GC-C by heat-stable enterotoxin (STa), Cd2+, hemin, or the detergent Triton X-100 was followed by a rapid inactivation of the enzyme. Adenine nucleotides were found to prevent the inactivation process in native membranes, as well as following enzyme solubilization and immunopurification. Inactivation of GC-C was not associated with dephosphorylation. However, the phosphorylation of GC-C was promoted by phorbol esters, known activators of protein kinase C. The activation of purified GC-C by STa required the presence of a nonspecific factor as exemplified by bovine serum albumin. When immunopurified GC-C, stabilized by ATP and bovine serum albumin, was analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions, proteins with almost twice the molecular mass (220 and 245 kDa) of the monomer were observed. The mobility of these high M(r) GC-C forms was reduced by STa, suggesting that STa induces a conformation change in a preexisting GC-C dimer. These results indicate that ATP interacts directly with GC-C, stabilizing its active conformation and that the activation of GC-C may occur in the absence of other specific regulatory factors.