Differentiation of fluoride-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase by calcium ions, ethyleneglycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid and triton X-100

Abstract

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84–88% inhibited by 5 · 10 −5 M ethyleneglycol-bis-( β-aminoethyl ether) N, N′-tetraacetic acid in the absence of F − but only 50–60% inhibited by 5 · 10 −5 M ethyleneglycol-bis-( β-aminoethyl ether) N, N′-tetraacetic acid in the presence of F −. In either case, further increase in EGTA concentration did not later the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 · 10 −5 M Ca 2+, (yielding a [free Ca 2+] of approximately 2 · 10 −5 M) and 5 · 10 −5 M Mn 2+ or Co 2+ and partially by 5 · 10 −5 M Sr 2+ but not by addition of 5 · 10 −5 M Ba 2+, Zn 2+, Ni 2+ or Fe 2+. A [free Ca 2+ or 7.2 · 10 −5 M markedly inhibited cyclase activity in the presence of F −. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 · 10 −5 M ethyleneglycol-bis-( β-aminoethyl ether) N, N′-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-( β-aminoethyl ether) N, N′-tetraacetic acid, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-) β-aminoethyl ether) N, N′-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concenrations of Ca 2+ and Mg 2+. A second component is totally Ca 2+ dependent and inhibited by high concentrations of F −. Mn 2+, Co 2+ and Sr 2+ appear to be in vitro Ca 2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca 2+ dependent cyclase component but a complete abolition of the NaF stimulated component.