Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by "P"-site agonists.
A detergent-dispersed adenylate cyclase from rat brain was used to study the effects of ammonium salts and polyamines on the proteolytic activation of the enzyme by a sperm protease and on the sensitivity of adenylate cyclase to inhibition via its "P"-site. A purified preparation of a trypsin-like, serine protease from bovine sperm was used to activate solubilized adenylate cyclase in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S). The proteolytically activated form of adenylate cyclase was found to be particularly sensitive to further activation by ammonium bicarbonate. The activation by NH4HCO3 was found to be due to the NH+4 cation and was characterized by an increased Vmax and by a decreased sensitivity of adenylate cyclase to inactivation by elevated concentrations of the sperm protease or by trypsin. NH4Cl and (NH4)2SO4 also caused biphasic effects on adenylate cyclase, which mimicked but were less effective than those caused by NH4HCO3. Consistent with observations of others, adenylate cyclase activity was enhanced by ammonium ions whether in the presence of reversible (Mn2+) or irreversible (GTP gamma S) activators. Mn2+- and GTP gamma S-stimulated activities were similarly optimally enhanced by 30 mM (NH4)2SO4 and by 30 to 150 mM NH4Cl or NH4HCO3. Ammonium ions did not increase the activity of the purified catalytic unit. Moreover, the effect of ammonium ions was not accompanied by an increased rate of activation by GTP gamma S, suggesting that the activation of Gs (guanine nucleotide-dependent stimulatory component) may not be the primary cause of stimulation by ammonium salts. Several polyamines at millimolar concentrations blocked the stimulatory effect of NH+4. This was observed when adenylate cyclase was activated by Mn2+, but not when it was activated by GTP gamma S or by the sperm protease + GTP gamma S. The inhibitory effect of polyamines was not due to the formation of a complex with ATP. Both the increase in Vmax of the Mn2+-stimulated enzyme by NH+4 and the decrease in Vmax caused by spermine were accompanied by an increase in the enzyme's Km MnATP app. Spermine increased the IC50 for inhibition of Mn2+-activated adenylate cyclase by 2',5'-dideoxyadenosine (2',5'-ddAdo) from 0.75 to 4.6 microM, consistent with the idea that increased sensitivity of P-site-mediated inhibition is associated with increased enzyme activity. In contrast, activation of Mn2+-stimulated adenylate cyclase by 30 mM (NH4)2SO4 also reduced sensitivity to inhibition by 2',5'-ddAdo(IC50 1.1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
Yeung
S M
SM
Department of Physiology and Biophysics, School of Medicine, State University of New York, Stony Brook 11794-8661.
Shoshani
I
I
Stübner
D
D
Johnson
R A
RA
D000176:Acrosin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008345:Manganese / Q000494:pharmacology; D011073:Polyamines / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology*; D051381:Rats; D012697:Serine Endopeptidases / Q000494:pharmacology*; D013094:Spermatozoa / Q000201:enzymology; D013096:Spermine / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
D001639:Bicarbonates; D011073:Polyamines; D000644:Quaternary Ammonium Compounds; D013873:Thionucleotides; D000643:Ammonium Chloride; D013096:Spermine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; C027043:ammonium bicarbonate; D006160:Guanosine Triphosphate; D012697:Serine Endopeptidases; C045523:ninhibin; D000176:Acrosin; D000262:Adenylyl Cyclases; D000645:Ammonium Sulfate