||A comparative chemical study of several ATP-creatine transphosphorylases from various species was undertaken in order to ascertain those similarities and differences which might ultimately serve to elucidate the mechanism of catalysis, as well as the relation of structure to enzyme function. The muscle-type enzymes from rabbit (Noltman, E, A., Maho-wald, T., and Kuby, S. A. (I962) J. Biol. Chem. 237, 11^6), calf, and human possess very similar amino acid compositions. Likewise, the brain-type enzyme from rabbit and calf are also very similar in composition. However, significant differences exist between the compositions of the muscle-and brain-type isoenzymes isolated from the same species; for example, in the calf there are 8 cysteinyl residues/mole in the muscle isoenzyme vs 10 in the brain, there are 11 fewer lysine residues/mole in the brain-type enzyme, and there are significant increases in a number of aliphatic residues (alanine, proline, leucine). Theoretical titration curves were constructed from the amino acid composition of the calf isoenzymes and the Linderstrom-Lang equation (Mahowald, T. A,, Noltman, E, A,, and Kuby, S. A. (I962) J. Biol.Chem. 237, 1535), assuming that the electrostatic factor is equal to 0. The theoretical curves gave isoionic points for the muscle-, hybrid-, and brain-type enzymes which agreed with the measured isoelectric point (pIo) within 0,5 units (Yue, R, H,, Okabe, K,, Keutel, H., and Kuby. S. A. (1968) Biochem. 7, 4291). In addition, theoretical titration curves were constructed for the brain-type enzyme at 0.02 and 0.1 ionic strength using an iterative procedure to determine the charge on a spherical molecule and assuming an electrostatic factor equal to one-half the theoretical factor. After correction for ion binding, these curves paralleled the measured electrophoretic mobility curves. The amino acid composition and primary structure of the normal and dystrophic human muscle enzymes were investigated in order to determine whether structural change in the dystrophic muscle-type enzyme had taken place. An abnormal presence of three isoenzymes in the dystrophic muscle tissue had been noted earlier (Jacobs, H., Okabe, K., Yue, R., Keutel, H,, Ziter, F., Palmieri, R., Tyler, F., and Kuby, S. A. (1969) Fed, Proc. 28, 346). The amino acid compositions of the two muscle-type enzymes (normal and dystrophic) were identical with the exception of glycine and alanine, which differed at most, by 0.9 residues. Two of the 8 sulfhydryl groups per mole which occur in the normal and dystropliic human muscle-type enzymes are exposed, and can be measured by reaction with DTNB (Jacobs et al. (1969) ibid.). Reaction between iodoacetic acid, iodoacet-amide, DTNB, and the 2 normally reactive -SH groups per mole leads to essentially complete loss of enzymatic activity. This permitted a selective labeling as the S-carboxymethylated derivatives of the 2 reactive sulfhydryl groups per mole with 2-14C-iodoacetate. After thin layer peptide mapping of the tryptic digest of the 2-14C-S-carboxymethylated normal and dystrophic human muscle enzymes, no significant differences could be detected between these two enzymes. Likewise, peptide mapping on Dowex 50-X2 cation exchange column gave essentially identical elution profiles for both enzymes as well as a single radioactive peak which was eluted in approximately 85% and 96% respectively for the normal and dystrophic enzymes. A final comparison was made between the single reactive sulfhydryl group containing tryptic peptides isolated from both enzymes. The amino acid sequence v/as determined to be: Val-Leu-Thr-S-C:.1C-Pro-Ser-Asn-Leu-Gly-Thr-Gly-Leu-Arg after purification by anion exchange for the normal enzyme. Although the sequence of residues 11 and 12 (Gly, Leu) could not be definitely assigned for the dystrophic enzyme, the tryptic peptides appeared to be essentially identical, A similar sequence surrounding the reactive sulfhydryl group has been reported for the rabbit muscle, ox muscle, and brain enzymes by Thomson and coworkers (Nature 203, 267,1964) (Biochem. J. 120,589,1970.