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JBC, Vol. 253, Issue 15, 5286-5292, Aug, 1978

Soluble adenylate cyclase from thyroid membranes

R. F. Asbury, G. H. Cook and J. Wolff

Adenylate cyclase from purified beef thyroid membranes has been solubilized by the use of Triton N-101 after preactivation with guanosine 5'-(beta, gamma-imido)-triphosphate. The soluble activity passed a 0.22- micron filter, was not sedimented at 100,000 X g for 2 h, and behaved like aldolase in sucrose density gradients and on Sepharose 6B. From comparison of the sedimentation in D2O and H2O the partial specific volume was found to be like that of globular proteins (0.75 +/- 0.006), hence little detergent appeared to be bound to the enzyme. The sedimentation coefficient was 7.4 +/- 0.15, the Stokes radius 45 A, and the molecular weight 159,000. Prestimulation by thyrotropin did not survive solubilization. The stimulation produced by guanosine 5'-(beta, gamma-imido)triphosphate persisted as did the more active state resulting from pretreatment with both this nucleotide plus thyrotropin. Thyrotropin did not stimulate the solubilized enzyme. The Km for ATP, thermal stability, and inhibition by Ca2+ were identical for the membrane-bound and soluble enzyme, while the pH optimum was increased 0.5 unit in the latter. Polyanions and phenothiazines inhibited both preparations equally, whereas only membranes responded to stimulation by polylysine and ribonuclease.
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