title: Bovine heart pyruvate dehydrogenase kinase stimulation by monovalent ions.
abstract: The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase.
authors.affiliation: Department of Biochemistry, University of Texas Health Science Center San Antonio 78284.
authors.forename: J G
authors.initials: JG
authors.lastname: Robertson
authors.forename: L L
authors.initials: LL
authors.lastname: Barron
authors.forename: M S
authors.initials: MS
authors.lastname: Olson
mesh_terms: D000818:Animals; D002414:Cations, Monovalent; D002417:Cattle; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D066298:In Vitro Techniques; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000494:pharmacology
chemical_list: D002414:Cations, Monovalent; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D004228:Dithionitrobenzoic Acid; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D013835:Thiamine Pyrophosphate