1. The distribution of creatine and the creatine-synthesizing enzymes in the animal kingdom has been investigated. Creatine was found in tissues of all vertebrates examined, and in various invertebrates from phyla Annelida, Echinodermata, Hemichordata and Chordata, subphylum Cephalochordata. The activities of the creatine-synthesizing enzymes, arginine–glycine transamidinase and guanidinoacetate methylpherase, were not detected in the hagfish or in any of the invertebrates, including those in which creatine was found, with the exception that transamidinase activities were detected in the amphioxus and salt water clam; however, these activities are considered to be artifacts for reasons mentioned in the text. Additional evidence that the hagfish and various creatine-containing invertebrates could not synthesize creatine was the observation that these animals did not convert one or the other of the likely precursors of creatine (arginine and glycine) into creatine, in vivo. Further, the inability of these animals to synthesize creatine is correlated with the observations that all animals tested were able to abstract creatine from their aqueous environment. 2. The activities of the creatine-synthesizing enzymes were detected in the sea lamprey and in all but a few of the other vertebrates examined. Neither activity could be detected in the sharks and rays (cartilaginous fish), buffalo fish (bony fish) or the snapping turtle. Transamidinase or guanidinoacetate methylpherase activity could not be found in the salamander or garter snake, respectively. 3. The results obtained with the lamprey are in direct contrast with those obtained with the hagfish (both subphylum Agnatha, class Cyclostomata). The lamprey had the ability to synthesize creatine and did not abstract creatine from lake water. The hagfish did not have any apparent ability to synthesize creatine and did abstract creatine from sea water. The present report thus supports the theory that the myxinoid (hagfish) and petromyzoid (lamprey) agnathans are only distantly related. 4. The lack of creatine-synthesizing enzyme activities in the cartilaginous fishes may have phylogenetic significance, but may also be explained by the availability of creatine in the diet of these animals. The lack of one or both enzyme activities in vertebrates other than the hagfish and the cartilaginous fish is suggested to be the result of creatine in the diet.

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