Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

Abstract

AbstractA general feature of membrane‐bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane‐bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane‐bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane‐bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by Pi or PPi, and (4) and apparent loss of the Me2+‐reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane‐bound form.