Studies on the structure of ATP-creatine transphosphorylase from calf muscle

Identical amino acid composition and identical peptide map locus are sufficient criteria to establish the fact that two peptides are identical. Comparison of homologous proteins can be made on the basis of composition analysis and peptide mapping of peptides prepared under identical conditions. As a first step in the study of the homology of the isoenzymes of ATP-creatine transphosphorylase, the enzyme from the calf muscle was isolated and crystallized. Peptides from a tryptic digestion were separated initially by AG 50W-X2 cation exchange chromatography and then purified as individual peptides from two dimensional paper electrophoresis and chromatography (peptide mapping). Comparison of the peptide map on the original peptide mixture with that of the composite map indicated that not all peptides of the total tryptic digest can be resolved by peptide mapping; therefore, an initial separation of the peptides on AG 50W-X2 cation exchange resin allowed for purification of individual peptides from column fractions. Composition analysis of each pure peptide was performed on 20 hour acid hydrolysates on an amino acid analyzer utilizing high sensitivity techniques. The total amino acid composition of the enzyme was established, within experimental error, from the sum of the compositions of the individual peptides. This composition was compared with the composition obtained from total amino acid analysis of acid hydrolysates and gave good correlation within the limits of experimental error. Peptides containing S-carboxymethylcysteine, tryptophan and methionine were identified by amino acid composition and peptide map locus. These three residues are invariant throughout the series of creatine kinase isoenzymes and the 15 peptides in which these residues are found contain 38% of the total amino acids. The peptides containing S-carboxymethylcysteine are particularly important since enzymatic activity is associated with this residue. Titration with sulfhydryl seeking reagents equivalent to two moles/mole of enzyme resulted in complete loss of enzymatic activity. Comparison of the four (per subunit) S-carboxymethylcysteine containing peptides established, unequivocally, that the reactive sulfhydryl residue was contained in a 26 residue peptide, the sequence of at least a portion of this peptide is the same as similar peptides containing the reactive sulfhydryl residue in the rabbit and calf muscle enzymes and in the ox brain enzyme. The techniques of purification and identification of the individual tryptic peptides from the calf muscle enzyme that were established in this study can be utilized to compare the similarities and differences of other creatine kinase isoenzymes to the calf muscle enzyme with as little as one micromole of crystalline enzyme.