Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle.
1985; Elsevier BV; Volume: 260; Issue: 4 Linguagem: Inglês
10.1016/s0021-9258(18)89585-0
ISSN1083-351X
AutoresAiyang Cheng, Thomas J. FitzGerald, G M Carlson,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoEquilibrium binding andactivity studies indicate that adenosine 5"diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site.Equilibrium dialysis at pH 6.8 and 8.2, with and without M 8 + , and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per cy4947464 hexadecamer, with Kd values ranging from 0.26 to 17 p ~. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP.At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme.Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity.ADP seems to alter the conformation of the @ subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene.The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase.Phosphorylase kinase (ATP:phosphorylase 6 phosphotransferase, EC 2.7.1.38)from white skeletal muscle of the rabbit is a hexadecamer of four dissimilar subunits, ( ~~p ~y ~6 ~ (Cohen et al., 1978).Although the regulation of this complex enzyme by phosphorylation/dephosphorylation has been extensively studied in connection with the cascade activation of glycogenolysis (for a review see Carlson et al., 1979), only a few potential regulatory molecules have been described for this enzyme.Of these, probably the most well characterized are Ca2+ ions (Ozawa et al., 1967;Krebs et al., 1968;Brostrom et al., 1971;Grand et al., 1981) and plus Ca2+ ions (Kilimann and Heilmeyer, 1977;King and Carlson, 1981).Previous data from this laboratory obtained during protection studies against affinity labeling with analogs of 5'-ATP revealed the existence of a binding site on phosphorylase kinase with a high affinity for free 5'-ADP (King and Carlson, 1982;King et al., 1982).Such a binding site has also been inferred from protection by ADP against inactivation of the enzyme by phenylglyoxal (Soman and Graves, 1983).In these studies, ADP was more effective than ATP in protecting ~~~
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