title: Analysis of site occupancies in [32P]phosphorylated pyruvate dehydrogenase complexes by aspartyl-prolyl cleavage of tryptic phosphopeptides.
abstract: Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving three sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites a method has now been developed, using pig heart [32P]phosphorylated complexes, which enables unambiguous measurement of their occupancies. Established methods of tryptic digestion give two peptides that contain the three phosphorylation sites: TA contains sites 1 and 2; TB contains site 3. Thus, while occupancy of site 3 may be determined unequivocally by tryptic digestion, occupancies of sites 1 and 2 cannot. The present paper shows that peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the two phosphorylation sites. Equal amounts of two new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labelled peptides may be completely separated and quantified by high-voltage paper electrophoresis at pH 2. A combination of tryptic digestion (determination of 32P in site 3) and formic acid cleavage of peptide TA (determination of 32P in sites 1 and 2) thus enables unequivocal assignment of occupancies of individual phosphorylation sites to greater than 95% accuracy. This method has been used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5 % and 90%) greater than 98% of the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was less than 2% if at all. Relative initial rates of phosphorylation site 1 X site 2 X site 3 were approximately 90:3:1.
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D005561:Formates / Q000494:pharmacology*; D009206:Myocardium / Q000201:enzymology; D010748:Phosphopeptides; D010766:Phosphorylation; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors*; D013552:Swine; D014357:Trypsin
chemical_list: D005561:Formates; D010748:Phosphopeptides; D011768:Pyruvate Dehydrogenase Complex; C030544:formic acid; D014357:Trypsin