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.
Vaandrager
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Department of Biochemistry, Medical Faculty, Erasmus University Rotterdam, The Netherlands.
van der Wiel
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de Jonge
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000494:pharmacology*; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004768:Enterotoxins / Q000494:pharmacology*; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D006162:Guanylate Cyclase / Q000302:isolation & purification / Q000378:metabolism*; D006418:Heme / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013816:Thermodynamics; D014162:Transfection
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; C034151:heat stable toxin (E coli); D012710:Serum Albumin, Bovine; D006418:Heme; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D013755:Tetradecanoylphorbol Acetate; D004229:Dithiothreitol