Chemical studies on the subunit structure of rabbit muscle phosphoglucose isomerase. Identification of N-acetylated NH 2 -terminal alanine and COOH-terminal glutamine.

Tryptic hydrolysates of native, denatured, or chemically modified phosphoglucose isomerase from rabbit skeletal muscle were subjected to standard peptide mapping techniques. Maps prepared from hydrolysates of either native, guanidine hydrochloride-denatured, or carboxymethylated enzyme were found to have only two-thirds to three-fourths of the number of ninhydrin-positive spots that would be expected (a) on the basis of the amino acid composition (82 lysyl and 42 arginyl residues per enzyme molecule of 132,000 mol. wt.) and (21) if it is assumed that the enzyme is composed of 2 identical subunits (physical studies have shown it to consist of 2 subunits of equal size). The lower-than-expected number of peptides obtained under these conditions was shown to be the result of incomplete hydrolysis, due to partial resistance of the enzyme to tryptic attack. However, tryptic peptide maps prepared from fully carbamylated phosphoglucose isomerase (all lysyl residues modified) yielded 20 to 22 unique peptides, the theoretical number for 2 identical subunits. The technique of peptide mapping after carbamylation of all of the lysyl residues proved to have numerous experimental advantages over mapping of the unmodified protein, suggesting this procedure as the method of choice for any protein composed of relatively large polypeptide chains. Standard methods of NHz-terminal analysis gave only negative results, leaving the possibility that the NH2 termini might be blocked. This was confirmed by gas chromatography which showed phosphoglucose isomerase to contain two acetyl groups per enzyme molecule. Applicatibn of gas chromatography and amino acid analysis after extensive proteolytic hydrolysis of the enzyme, followed by cation exchange chromatography of the products, yielded two equivalents of acetylalanine per enzyme molecule, i.e. one