Studies on N alpha-acylated proteins: the N-terminal sequences of two muscle enolases.
A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.
D000215:Acylation; D000595:Amino Acid Sequence; D000818:Animals; D007527:Isoenzymes; D009132:Muscles / Q000201:enzymology*; D009928:Organ Specificity; D010446:Peptide Fragments / Q000302:isolation & purification; D010751:Phosphopyruvate Hydratase; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012474:Salmon; D013045:Species Specificity
D007527:Isoenzymes; D010446:Peptide Fragments; D010751:Phosphopyruvate Hydratase