Specific inhibition of L-type pyruvate kinase by the triazine dye Procion Blue MX-R.
Incubation of the triazine dye Procion Blue MX-R with L- and M-type pyruvate kinase resulted in rapid time- and dye-concentration-dependent loss of activity. L-type pyruvate kinase was protected only by a low concentration of Mg2+; this was not the case with the M-type enzyme. Modification of the L-type form resulted in the incorporation of 1.54 +/- 0.057 mol of dye/mol of enzyme subunit in the absence of Mg2+, but only 0.73 +/- 0.024 mol of dye/mol of enzyme subunit in the presence of Mg2+. Tryptic peptide mapping of L-type pyruvate kinase modified in the presence and in the absence of Mg2+ further indicated that there were two sites modified in the enzyme, one of which was protected by Mg2+. The pKa of the nucleophile involved in the modification was calculated to be 7.1, implicating the possible involvement of a histidine residue. L-type enzyme was bound to Sepharose-immobilized Procion Blue MX-R specifically in the presence of Mg2+, whereas binding of the M-type enzyme was Mg2+-independent. The specific interaction of L-type pyruvate kinase with the dye was exploited in the large-scale purification of the enzyme and in the isolation of the phosphorylated enzyme.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity; D004396:Coloring Agents / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D008024:Ligands; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D051381:Rats; D014227:Triazines / Q000494:pharmacology*
D000345:Affinity Labels; D004396:Coloring Agents; D007527:Isoenzymes; D008024:Ligands; D010446:Peptide Fragments; D014227:Triazines; C036598:procion blue MX-R; D011770:Pyruvate Kinase