Analysis of the cAMP-dependent protein kinase system using molecular genetic approaches.
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Amino Acid Sequence↗
Biomedical subjects
G G Cadd
Publications and source records attributed to G G Cadd.
Genetic characterization of a brain-specific form of the type I regulatory subunit of cAMP-dependent protein kinase.
An isoform (RI beta) of the regulatory type I subunit gene of cAMP-dependent protein kinase (EC 2.7.1.37) has been characterized in mouse. The open reading frame of the RI beta cDNA is 72% identical in nucleotide sequence with the previously cloned RI gene, now referred to as RI alpha. Both genes code for a protein of 380 amino acids and their proteins are 82% identical in amino acid sequence. Sequence similarity is highest in the regions that form the pseudosubstrate-binding site of the catalytic subunit and the two cAMP binding domains. The amino-terminal portion shows the greatest dissimilarity, suggesting that the isoforms may differ in their dimerization properties or interaction with other proteins. In contrast to RI alpha, which is constitutively expressed in all tissues, RI beta is expressed in a highly tissue-specific manner. Brain and spinal cord contained significant levels of RI beta mRNA, testis RNA gave a detectable signal, and all other tissues tested were negative. Expression of a RI beta cDNA in NIH 3T3 cells resulted in the appearance of a RI subunit protein that migrated more slowly than RI alpha after NaDodSO4/PAGE. The native form of RI beta in brain could also be distinguished from RI alpha by its abnormal migration on NaDodSO4/PAGE. RI beta protein produced in 3T3 cells was shown to be functional by its ability to form a cAMP-dependent holoenzyme with the catalytic subunit.
Amino Acid Sequence↗
Expression of wild-type and mutant subunits of the cAMP-dependent protein kinase.
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Amino Acid Sequence↗
Ketamine- and morphine-induced analgesia and catalepsy. I. Tolerance, cross-tolerance, potentiation, residual morphine levels and naloxone action in the rat.
The effects of ketamine and morphine on pain perception and catalepsy were compared in rats. Analgesia, as measured by the latency to withdrawal of the tail from a 55 degrees C water bath (tail-flick latency difference, TFLD), was produced by both ketamine and morphine, but at widely different doses, and in each case the effect was reversed by naloxone. Catalepsy, measured by the duration of loss of righting reflex (DLRR) in catatonic animals, was induced by larger doses of both ketamine and morphine and in each case was reduced by a larger dose of naloxone. DLRR and TFLD tolerance developed rapidly and with a similar time course after daily doses of ketamine or morphine. Rats tolerant to the DLRR effect of ketamine showed cross-tolerance to morphine. Rats tolerant to the DLRR effect of morphine did not show cross-tolerance to ketamine when administered the following day; instead, these rats showed potentiation of the ketamine-induced DLRR. The degree of potentiation noted 24 hr after a single or multiple daily doses of 45 mg/kg of morphine is the same as that seen when 2 mg/kg of morphine is given simultaneously with ketamine. The residual brain level of morphine 24 hr after 45 mg/kg is similar to the level 1 hr after a 2-mg/kg dose. The augmented ketamine response in morphine-tolerant rats is postulated to be a result of residual morphine still present in the brain 24 hr after the last DLRR-inducing dose of morphine.(ABSTRACT TRUNCATED AT 250 WORDS)
Analgesia↗
Inhibition of intracellular cAMP-dependent protein kinase using mutant genes of the regulatory type I subunit.
Expression vectors were constructed that code for mutated forms of the regulatory type 1 subunit (RI) of cyclic AMP-dependent protein kinase. These mutations alter a specific amino acid which is present in each of two homologous cAMP-binding domains of the RI protein. When these expression vectors were introduced into NIH 3T3 and Y1 adrenocortical tumor cells a mutant RI protein was produced that acted in a dominant fashion to cause a 20-400-fold inhibition of cAMP-dependent protein kinase activation. In addition, processes controlled by cAMP in adrenal cells were blocked; cells became resistant to the growth-inhibitory effects of cAMP and defective in steroid synthesis. Expression of mutant RI genes in cells provides a specific means to explore the role of cAMP and protein phosphorylation in the process of intracellular signalling.
Amino Acid Sequence↗
Isolation and characterization of bovine cardiac muscle cGMP-inhibited phosphodiesterase: a receptor for new cardiotonic drugs.
We have identified and highly purified a "low Km" cAMP phosphodiesterase from bovine cardiac muscle. This phosphodiesterase was inhibited by low concentrations of cGMP and has, therefore, been temporarily designated as cGMP-inhibited phosphodiesterase. After a 16,000-fold increase in specific activity, the highly purified enzyme had a specific activity of 6 mumol/min-mg and contained three major polypeptides. Initial data indicated that all of these polypeptides were derived from a single common precursor by proteolysis. We used this enzyme preparation to generate polyclonal antisera and monoclonal antibodies directed against the "low Km" phosphodiesterase. Immunoadsorption and immunoblot analysis allowed us to identify and isolate several molecular weight species of phosphodiesterase, including a larger form than previously reported for any purified low Km phosphodiesterase. This large form of the enzyme had a subunit molecular weight of approximately 110,000 and was the only one seen in fresh extracts of cardiac muscle. Full catalytic activity was recovered in the phosphodiesterase-antibody complex and enzyme prepared by immunoprecipitation exhibited Michaelis-Menten kinetics for cAMP hydrolysis and for inhibition by cGMP. The Km for cAMP hydrolysis was 0.15 microM and the Ki for cGMP inhibition of cAMP hydrolysis was 0.06 microM. This immunoprecipitation approach also allowed us to determine that the enzyme was phosphorylated on serine residues by cAMP-dependent protein kinase, and that the low Km, cGMP-inhibited phosphodiesterase was selectively inhibited by several new cardiotonic agents. Milrinone, amrinone, and fenoximone were highly selective inhibitors of this isozyme, and the relative affinities of these inhibitors were consistent with their order of potency as positive inotropic agents. These studies suggest that the cGMP-inhibited phosphodiesterase is a receptor for several new cardiotonic drugs.
3',5'-Cyclic-AMP Phosphodiesterases↗