Studies on an ACTH-activated adenyl cyclase from a mouse adrenal tumor

Abstract

Adenyl cyclase from an ACTH sensitive adrenal tumor has been reduced to a molecular weight of 3 to 7 million by treatment of a lyophilized preparation of the particulate enzyme in a French pressure cell, a Nossal shaker or by sonication in the presence of both a phospholipid and fluoride. Phosphatidyl ethanolamine, phosphatidyl serine, dipalmitoyl lecithin, and sphingomyelin were all effective. No “solubilization” was achieved if either phospholipid or fluoride was omitted. The enzyme was activated by ACTH after the fluoride was removed by dialysis. The “soluble” adenyl cyclase was examined with the electron microscope. Negative staining with potassium phosphotungstate revealed a population of vesicular profiles ranging in size from 300–800 Å, studded with granules of an average diameter of 90 Å. Many of the 90 Å granules were distributed singly unattached to the vesicles. A negative stained preparation of phospholipid alone revealed only the vesicular profiles. Following exposure of the particulate or the “soluble” enzyme to fluoride, persistent enzymatic activity was present despite removal of fluoride by dialysis. The addition of more fluoride stimulated the enzymatic activity to levels exceeding that of the undialyzed enzyme, suggesting removal of an inhibitor.