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Partial Purification and Characterization of the Defective Cyclic Adenosine 3':5'-Monophosphate Binding Protein Kinase from Adrenocortical Carcinoma¹

Department of Biochemistry, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163

Previous studies with isolated adrenocortical carcinoma 494 cells from this laboratory have indicated that the lack of cyclic adenosine 3':5'-monophosphate (cyclic AMP) control in steroidogenesis in the tumor may be due to the defective cyclic AMP-dependent protein kinase enzyme system. This paper describes the partial purification of such an enzyme.

Purification was achieved by precipitation of the tumor homogenate with 30 and 45% ammonium sulfate, adsorption on 3% calcium phosphate gel, and chromatography on DEAE-cellulose. Four major protein peaks were isolated. Peak 2 showed cyclic AMP-binding activity and was investigated further for its kinetic properties.

In contrast to the cyclic AMP-dependent protein kinase enzyme found in the normal adrenal gland, the enzyme specifically bound cyclic AMP but failed to phosphorylate exogenous histone. It is postulated that lack of the cyclic nucleotide-dependent kinase activity of the protein kinase enzyme may be responsible for the loss of cyclic AMP-regulated corticosterone synthesis in adrenocortical carcinoma cell. It is further shown that the tumor cyclic AMP-binding enzyme undergoes endogenous phosphorylation, which indicates that it has kinase activity but it is independent of cyclic AMP.

¹ This investigation was supported by Grant CA-16091 from the National Cancer Institute and Grant DRG-1237 from the Damon Runyon Memorial Fund for Cancer Research.

Received 1/ 7/77. Accepted 6/21/77.