Analysis of Site Occupancies in [32P] Phosphorylated Pyruvate Dehydrogenase Complexes by Aspartyl‐Prolyl Cleavage of Tryptic Phosphopeptides

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Abstract
Activity of mammalian pyruvate dehydrogenase complexes from all sources so far tested is regulated by phosphorylation involving 3 sites. To facilitate understanding of the precise biological roles of the individual phosphorylation sites, a method was developed, using pig heart [32P]phosphorylated complexes, which enables unambiguous measurement of their occupancies. Established methods of tryptic digestion give 2 peptides that contain the 3 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. Peptide TA may be specifically and quantitatively cleaved by formic acid at an Asp-Pro bond located between the 2 phosphorylation sites. Equal amounts of 2 new peptides each containing a different phosphorylation site (site 1 or site 2) are produced. The 32P-labeled 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 > 95% accuracy. This method was used to show that during phosphorylation and inactivation of pig heart complexes (inactivated to between 1.5-90%), > 98% o the observed inactivation was primarily attributable to phosphorylation of site 1; the contribution of site 2 was < 2% if at all. Relative initial rates of phosphorylation, site 1:site 2:site 3, were .apprx. 90:3:1.