Nitration of the tyrosine residues of porcine pancreatic colipase with tetranitromethane, and properties of the nitrated derivatives.
The nitration of the long form (N-terminal valine) of porcine pancreatic colipase with tetranitromethane was investigated under a variety of conditions. Fractionation of the nitrated monomers on DE-cellulose led to well-defined derivatives containing one, two and three nitrotyrosines per mol. Automated Edman degradation of the nitrated peptides, especially that of the staphylococcal proteinase peptide (49-64) showed that Tyr-54 was nitrated very fast under all conditions. This residue was the only one to be nitrated in water. Partial nitration of Tyr-59 was induced by bile salt micelles, while both Tyr-59 and Tyr-58 reacted extensively in the presence of lysophosphatidylcholine micelles (in which tetranitromethane is concentrated 150-fold compared to water) or of a liquid tetranitromethane-water interface. The strong negative Cotton effect at 410 nm which has already been observed using unfractionated preparations of nitrated colipase (Behnke W.D. (1982) Biochim. Biophys. Acta 708, 118-123) is linked with the nitration of Tyr-59 and it is markedly reduced by taurodeoxycholate micelles, suggesting a conformational change induced by the micelles in the tyrosine region. Moreover, the pKa of the nitrotyrosine residues in nitrated colipase is the same as that of free nitrotyrosine (pKa = 6.8) and it is shifted to 7.6 in the presence of taurodeoxycholate micelles. Micelles protected colipase against polymerization during nitration. These data suggest that Tyr-58 and Tyr-59 are part of the interface recognition site of colipase. The participation of Tyr-55 in binding is not excluded. The upwards nitrotyrosine pKa shift in the colipase micelle complex may explain why nitrated colipase can reactivate lipase in a triacylglycerol-taurodeoxycholate system at pH 7.5.
D000818:Animals; D003089:Colipases; D008049:Lipase / Q000378:metabolism; D008697:Methane / Q000031:analogs & derivatives; D008823:Micelles; D010179:Pancreas / Q000201:enzymology; D011506:Proteins; D013552:Swine; D013657:Taurodeoxycholic Acid; D013774:Tetranitromethane; D014443:Tyrosine
D003089:Colipases; D008823:Micelles; D011506:Proteins; D014443:Tyrosine; D013657:Taurodeoxycholic Acid; D008049:Lipase; D013774:Tetranitromethane; D008697:Methane