MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase.

Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.

Inhibitory effect of glucocorticoid on coronary artery endothelial function.

Acute and chronic stresses are implicated in cardiovascular diseases including coronary artery disease. The present study was designed to examine the direct effects of the stress hormone cortisol on nitric oxide (NO) release and endothelial NO synthase (eNOS) expression in cultured bovine coronary artery endothelial cells (BCAEC). Nitrate, nitrite, and NO (NO(x)) were measured by the chemiluminescence method. At 24 h after treatment, cortisol (1 nM-10 microM) produced a dose-dependent decrease in NO(x) release, which was attenuated in the presence of the 11beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone (3 microM). In accordance, eNOS protein levels were significantly decreased by cortisol in a dose-dependent manner. Cortisol pretreatment significantly increased the rate of eNOS protein degradation in the presence of cycloheximide. In addition, cortisol pretreatment decreased ATP-induced intracellular Ca(2+) elevation and NO(x) release in BCAEC. The presence of glucocorticoid receptors in BCAEC was demonstrated by Western blot. The results suggest that cortisol, through activation of glucocorticoid receptors, suppresses NO(x) release in BCAEC by downregulating eNOS proteins and inhibiting intracellular Ca(2+) mobilization. Decreased NO(x) is likely to result in an increase in contraction of coronary arteries, leading to a decrease in coronary blood flow.