title: The amino acid sequence of yeast enolase. Preparation and characterization of peptides produced by chemical and enzymatic fragmentation.
abstract: Yeast enolase was subjected to chemical and enzymatic fragmentation, and the individual peptides produced were isolated by gel filtration and ion exchange chromatography. The chemical fragmentation was achieved by cleavage at the single cysteine residue with 2-nitro-5-thiocyanobenzoic acid, or at the 5 methionine residues with cyanogen bromide. The assignment of the two 2-nitro-5-thiocyanobenzoic acid fragments to the NH2-terminal or COOH-terminal regions (designated C1 and C2, respectively) of the enolase subunit could be done unequivocally on the basis of NH2-terminal and COOH-terminal analysis, and the same was the case for the NH2-terminal and COOH-terminal cyanogen bromide peptides (designated M1 and M6, respectively). From a comparison of the CNBr peptides from enolase with those from Fragment C1, the identity of methionine peptides M4, of which only the NH2-terminal half is present in C1, and M5, which along with M6 is missing in C1, could also be established. The major enzymatic fragmentation was achieved by tryptic cleavage at the 14 arginine residues after acetylation of the lysine residues. Based on overlaps with methionine peptides, most of the arginine peptides could be ordered in proper sequence during the early phases of the work. Because of the size of several of the primary fragments, secondary cleavages were required for optimal sequencing data. These secondary cleavages were accomplished by digestion with Staphylococcus aureus protease, or by tryptic cleavage at cysteine after aminoethylation.
mesh_terms: D000595:Amino Acid Sequence; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D010751:Phosphopyruvate Hydratase; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000201:enzymology; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D010447:Peptide Hydrolases; D014357:Trypsin; D010751:Phosphopyruvate Hydratase