Zinc inhibition of cAMP signaling.
Zn(2+) is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a variety of important biological processes. We report here that low micromolar concentrations of Zn(2+) inhibited hormone- or forskolin-stimulated cAMP production in N18TG2 neuroblastoma cells. Similarly, low concentrations inhibited hormone- and forskolin-stimulated adenylyl cyclase (AC) activity in membrane preparations and did so primarily by altering the V(max) of the enzyme. Zn(2+) also inhibited recombinant isoforms, indicating that this reflects a direct interaction with the enzyme. The IC(50) for Zn(2+) inhibition was approximately 1-2 microm with a Hill coefficient of 1.33. The dose-response curve for Zn(2+) inhibition was identical for AC1, AC5, and AC6 as well as for the C441R mutant of AC5 whose defect appears to be in one of the catalytic metal binding sites. However, AC2 displayed a distinct dose-response curve. These data in combination with the findings that Zn(2+) inhibition was not competitive with Mg(2+) or Mg(2+)/ATP suggest that the inhibitory Zn(2+) binding site is distinct from the metal binding sites involved in catalysis. The prestimulated enzyme was found to be less susceptible to Zn(2+) inhibition, suggesting that the ability of Zn(2+) to inhibit AC could be significantly influenced by the coincidence timing of the input signals to the enzyme.
Klein
Claudette
C
Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. kleinc@slu.edu
Sunahara
Roger K
RK
Hudson
Tracie Y
TY
Heyduk
Tomasz
T
Howlett
Allyn C
AC
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007313:Insecta; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000378:metabolism* / Q000494:pharmacology; D017967:Zinc Compounds / Q000494:pharmacology
D002712:Chlorides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D017967:Zinc Compounds; D005576:Colforsin; C016837:zinc chloride; D000242:Cyclic AMP; D008274:Magnesium; D015032:Zinc