III. Negative Feedback Control of Cyclic Nucleotide 
Levels. 
The purified C subunit of cAK produced a two- 
fold activation of the low phosphodiesterase in 
crude microsomes (P-2 pellet) of rat adipocytes. 
This activation was C subunit-concentration de- 
pendent, ATP dependent, blocked by a specific pep- 
tide inhibitor, and lost if the C subunit was first 
-■heat-denatured. The concentration of ATP neces- 
sary for half-maximal activation of the low phos- 
phodiesterase was 4.5 X 1.1 |jlM, which was nearly 
the same as the known K of C subunit for ATP 
m 
(3.1 mM) using other substrates. The concentration 
of C subunit producing half-maximal activation of 
phosphodiesterase was 0.22 x 0.04 |jlM, slightly 
less than the measured concentration of total C 
subunit in adipocytes (0.45 |jM). The activation of 
the low phosphodiesterase by C subunit was 
specific, since on an equimolar basis, myosin light 
chain kinase, cGK, or Ca^^/calmodulin-dependent 
protein kinase II did not activate the enzyme. The 
percent stimulation of phosphodiesterase by C sub- 
unit was about the same as that produced by incu- 
PUBLICATIONS 
bation of adipocytes with a cAMP analogue, and the 
enzyme first activated in vivo with the analogue 
was not activated to the same extent (on a percent- 
age basis) by in vitro treatment with C subunit. 
Treatment of the crude microsomes with trypsin 
resulted in transfer of phosphodiesterase cata- 
lytic activity from the particulate to the superna- 
tant fraction, but the enzyme in the supernatant 
was minimally activated by C subunit, suggesting 
either loss or dislocation of the regulatory com- 
ponent. The C subunit-mediated activation of 
phosphodiesterase was preserved after transfer of 
phosphodiesterase activity to the supernatant 
fraction by non-ionic detergents or partial purifi- 
cation of the transferred enzyme. These findings 
are consistent with the suggestion that protein 
kinase regulates the concentration of cAMP 
through phosphodiesterase activation and pro- 
vide direct evidence that the mechanism of activa- 
tion involves phosphorylation. 
Dr. Corbin is Professor of Molecular Physiology 
and Biophysics at the Vanderbilt University School 
of Medicine. 
Articles 
Francis, S.H. , Noblett, B.D., Todd, B.W, Wells, J.N., and Corbin, J. D. 1988. Relaxation of vascular and tracheal 
smooth muscle by cyclic nucleotide analogs that preferentially activate cGMP-dependent protein kinase. 
Mol Pharmacol 34:506-517. 
Paveto, C, Passeron, S., Corbin, J. D., and Moreno, S. 1989. Two different intrachain cAMP sites in the cAMP- 
dependent protein kinase of the dimorphic fungus Mucor rouxii. EurJBiochem 179:429-434. 
Wagner, C, Decha-Umphai, W, and Corbin, J. 1989. Phosphorylation modulates the activity of glycine A^- 
methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate 
ligand. J Biol Chem 264:9638-9642. 
Weber, I.T, Shabb, J.B. , and Corbin, J.D. 1989. Predicted structures of the cGMP binding domains of the 
cGMP-dependent protein kinase: a key alanine/threonine difference in evolutionary divergence of cAMP 
and cGMP binding sites. Biochemistry 28:6122-6127. 
Wolfe, L., and Corbin, J.D. 1989. Cyclic nucleotides and disease. Curr Opinion Cell Biol 1:215-219. 
Wolfe, L., Corbin, J.D. , and Francis, S.H. 1989. Characterization of a novel isozyme of cGMP-dependent pro- 
tein kinase from bovine aorta. J Biol Chem 264:7734-7741. 
Wolfe, L., Francis, S.H., and Corbin, J.D. 1989. Properties of a cGMP-dependent monomeric protein kinase 
from bovine aorta. J Biol Chem 264:4157-4162. 
Woodford, T.A. , Correll, L.A., McKnight, G.S., and Corbin, J.D. 1988. Expression and characterization of mu- 
tant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP 
binding on holoenzyme activation. J Biol Chem 264:13321-13328. 
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