PT  - JOURNAL ARTICLE
AU  - NJANOOR NARAYANAN
AU  - PRAKASH V. SULAKHE
TI  - 5'-Guanylylimidodiphosphate-Activated Adenylate Cyclase of Cardiac Sarcolemma Displays Higher Affinity for Magnesium Ions
DP  - 1977 Nov 01
TA  - Molecular Pharmacology
PG  - 1033--1047
VI  - 13
IP  - 6
4099  - http://molpharm.aspetjournals.org/content/13/6/1033.short
4100  - http://molpharm.aspetjournals.org/content/13/6/1033.full
SO  - Mol Pharmacol1977 Nov 01; 13
AB  - Activation of heart sarcolemmal adenylate cyclase by 5'-guanylylimidodiphosphate [Gpp(NH)p] resulted in enhanced affinity of the enzyme for Mg2+. The nucleotide increased the apparent affinity of adenylate cyclase for Mg2+ 3-fold in the absence of isoproterenol, and 5-fold in its presence. At various Mg2+ concentrations, the nucleotide (with and without isoproterenol) did not alter the apparent Km (0.10 ± 0.02 mM) for ATP. Activation of adenylate cyclase by Gpp(NH)p showed temperature dependence. The nucleotide failed to activate the enzyme below 30° but enhanced enzyme activity 3-7-fold between 30° and 37°. The synergistic influence of isoprotenenol and nucleotide resulted in 7-12-fold higher enzyme activity, depending on the Mg2+ concentration. Previous incubation of sarcolemma with or without isoproterenol in the absence of Gpp(NH)p above 15° led to a rapid decline in enzyme activity, whereas prior incubation in the presence of Gpp(NH)p not only prevented the decay but also enhanced both basal and isoproterenol-sensitive enzyme activity; the degree of activation depended on the prior incubation temperature. Treatment of sarcolemmal membranes with phospholipase A markedly reduced basal enzyme activity and activity in the presence of NaF, Gpp(NH)p, and Gpp(NH)p plus isoproterenol. The impairment was more pronounced in the case of enzyme activity stimulated by NaF and by Gpp(NH)p plus isoproterenol. Adenylate cyclase stimulated by NaF or by Gpp(NH)p in the presence of isoproterenol was relatively more resistant to phospholipase C treatment. Exposure of sarcolemmal membranes to extremely low concentrations of trypsin (ratio of trypsin to sarcolemmal protein, 0.0008-0.001) significantly enhanced basal and Gpp(NH)p-stimulated enzyme activity, but treatment with higher concentrations of trypsin (ratio of trypsin to sarcolemmal protein, 0.008-0.04) resulted in complete loss of enzyme activity. These observations suggest that activation of adenylate cyclase by Gpp(NH)p involves a conformational change in the enzyme that results from the interaction with Gpp(NH)p, presumably at the nucleotide binding site. Isoproterenol facilitates the nucleotide-induced transition in the enzyme. The conformational change is temperature-dependent. An altered enzyme configuration displays increased affinity for Mg2+ at the metal binding site.