Abstract
Abstract Agents that raise cyclic adenosine 3':5'-monophosphate (cyclic AMP) levels in chicken embryonic fibroblasts increase cyclic AMP phosphodiesterase activity. Cyclic guanosine 3':5'-monophosphate (cyclic GMP) phosphodiesterase activity is not increased indicating that these enzyme activities are under separate regulation. The increase in cyclic AMP phosphodiesterase activity requires cellular protein and RNA synthesis, and the regulation of induction probably occurs at the transcriptional level. The induced cyclic AMP phosphodiesterase activity is very unstable. The enzyme activity decays from the induced level with a half-life of 70 to 80 min. Increased enzyme activity is found in both the particulate and soluble cell fractions. The particulate enzyme can be solubilized by sonic disruption. DEAE-cellulose chromatography of the solubilized enzyme yields two fractions that hydrolyze cyclic AMP. Induction causes an increase in the activity of the second fraction. Neither of the particulate enzymes hydrolyze cyclic GMP. DEAE-cellulose chromatography of the soluble enzyme also yields two fractions. The first peak hydrolyzes both cyclic AMP and cyclic GMP and the second only cyclic AMP. Induction increases the activity of the second fraction. Chicken embryonic fibroblasts thus contain mechanisms for effectively modulating cyclic AMP phosphodiesterase activity via protein synthesis and enzyme turnover. Cyclic GMP phosphodiesterase activity is not affected by this regulatory scheme.
Highlights
The particulate enzyme can be solubilized by sonic disruption
We find that (a) cyclic AMP phosphodiesterase activity is increased by treatment of chicken embryonic fibroblasts with a potent cyclic AMP phosphodiesterase inhibitor (SC-2964) or analogues of cyclic AMP, [5] the induction requires RNA and protein synthesis and regulation of induction probably occurs at the level of transcription, (c) the induced enzyme is unstable and decays with a half-life of 70 to 80 min, (d) the induced enzyme is located in both the soluble and membrane fractions and is readily separated from the form that hydrolyzes cyclic GMP
Analogues and agents that raise intracellular cyclic AMP concentrations in chicken embryonic fibroblasts and that the regulation of induction occurs at the transcriptional level
Summary
The secondary chicken embryonic fibroblasts Eagle’s modified essential medium supplemented were grown in with fetal bo-. Vine serum, sodium glutamate (2 mM), pyruvate (1 mM), dextrose (2 g per liter), tylosine (I%), penicillin (I%), streptomycin (I%), and tryptose phosphate broth (lOyo). The cells were plated in medium containing 10% fetal bovine serum and the medium was changed daily with 5yo serum. Secondary chicken embryonic fibroblasts (1.5 to 2 mg of protein per 50-cm* dish) were used throughout. The cells were rinsed with cold phosphatebuffered 0.9% NaCl solution four times. Cells were homogenized (0.8 ml of buffer per dish) with a Dounce homogenizer
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