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Partial purification and characterization of the defective cyclic adenosine 3':5'-monophosphate binding protein
Abstract:
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.
Insights
Adrenocortical carcinoma cells show defective cyclic AMP-dependent protein kinase, impacting steroidogenesis. This study partially purified a tumor enzyme that binds cyclic AMP but lacks kinase activity, potentially explaining the loss of hormonal control.
Area of Science:
- Biochemistry
- Endocrinology
- Oncology
Background:
- Adrenocortical carcinoma cells exhibit impaired steroidogenesis.
- This dysfunction is potentially linked to a defective cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase enzyme system.
Purpose of the Study:
- To partially purify and characterize the cyclic AMP-dependent protein kinase enzyme from adrenocortical carcinoma cells.
- To investigate the kinetic properties of the isolated enzyme and compare it to the normal adrenal gland enzyme.
Main Methods:
- Partial purification of the enzyme using ammonium sulfate precipitation, calcium phosphate gel adsorption, and DEAE-cellulose chromatography.
- Isolation of four major protein peaks, with Peak 2 exhibiting cyclic AMP-binding activity.
- Kinetic analysis of the purified enzyme.
Main Results:
- A tumor enzyme was purified that specifically binds cyclic AMP but fails to phosphorylate exogenous histone.
- This enzyme differs from the normal adrenal gland enzyme, which possesses kinase activity.
- The tumor enzyme undergoes endogenous phosphorylation, indicating intrinsic kinase activity independent of cyclic AMP.
Conclusions:
- The defective cyclic AMP-dependent protein kinase activity, specifically the lack of cyclic nucleotide-dependent phosphorylation, may cause the loss of cyclic AMP-regulated corticosterone synthesis in adrenocortical carcinoma.
- The tumor enzyme's kinase activity is independent of cyclic AMP, suggesting a regulatory defect.