John P. Perkins

Abstract A protein kinase that catalyzes an adenosine 3',5'-monophosphate (cyclic AMP)-dependent phosphorylation of casein and protamine has been purified from rabbit skeletal muscle. The Km values of cyclic AMP for these reactions are 1 x 10-7 and 6 x 10-8 m, respectively. The protein kinase markedly increases the rate of the cyclic AMP-dependent activation and phosphorylation of phosphorylase kinase by ATP.

I-HYP, '251-hydroxybenzylpindolol; Gpp(NH)p, guanyl-5'-yl imido

Abstract Activation of skeletal muscle phosphorylase kinase, which occurs when the purified enzyme is incubated with ATP, Mg2+, and cyclic adenosine 3',5'-monophosphate (cyclic AMP), has been shown to involve two catalytic components. One of the catalysts is a cyclic AMP-dependent protein kinase and the other is phosphorylase kinase itself. The process was elucidated in part through a study of the effects of inhibitors on the activation process. One of the inhibitors that was used is a heat-stable protein from skeletal muscle which was shown to block the cyclic AMP-dependent protein kinase component of the reaction, and the other inhibitor was ethylene glycol bis(β-aminoethyl ether)-N, N'-tetraacetate which inhibits phosphorylase kinase. Clarification of the mechanisms operating at the phosphorylase kinase activation step makes it possible to describe more precisely how glycogenolysis is regulated by hormones, and for the first time to identify a specific site of action of cyclic AMP in a physiological process.

Incubation of 1321N1 human astrocytoma cells with 1 microM isoproterenol rapidly results in the conversion of a portion of the beta-adrenergic receptors to a membrane form that can be separated from markers for the plasma membrane by sucrose density gradient or differential centrifugation. This "light peak" form of the receptor reaches a maximal level within 10 min of incubation of cells with catecholamine. Two types of experiments suggest that the early phase of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase can be separated into at least two reactions. First, the agonist-induced loss of catecholamine-stimulated adenylate cyclase activity precedes the appearance of beta-adrenergic receptors in the light peak fraction by 1-2 min. Second, pretreatment of cells with concanavalin A prior to induction of desensitization blocks the formation of the light peak form of beta-adrenergic receptors without blocking the "uncoupling" reaction as measured by catecholamine-stimulated adenylate cyclase activity. Specificity for the reaction that converts beta-adrenergic receptors to the light peak form is indicated by the lack of a catecholamine-induced alteration in the sucrose density gradient distribution of muscarinic cholinergic receptors, adenylate cyclase or the guanine nucleotide-binding proteins, Ns and Ni. The light peak of beta-adrenergic receptors migrates at a density similar to that of at least a portion of the activity of galactosyltransferase, a marker for Golgi. Enzyme marker activities for lysosomes and endoplasmic reticulum are not associated with this population of beta-adrenergic receptors. Taken together, these and other data suggest that incubation of 1321N1 cells with isoproterenol results in a rapid uncoupling of beta-adrenergic receptors from adenylate cyclase which is followed by a change in the membrane form of the receptor. This latter step most likely represents internalization of receptors into a vesicular form which may then serve as the precursor state from which receptors are eventually lost from the cell.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSubunit structure of rabbit skeletal muscle phosphorylase kinaseTaro Hayakawa, John P. Perkins, and Edwin G. KrebsCite this: Biochemistry 1973, 12, 4, 574–580Publication Date (Print):February 1, 1973Publication History Published online1 May 2002Published inissue 1 February 1973https://pubs.acs.org/doi/10.1021/bi00728a002https://doi.org/10.1021/bi00728a002research-articleACS PublicationsRequest reuse permissionsArticle Views46Altmetric-Citations167LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts