Location of 14C in protein from isolated rat diaphragm incubated in vitro with [14C]amino acids and with 14CO2

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Abstract
Isolated rat diaphragm was incubated with various C14 amino acids and metabolites, and protein from the diaphragm was hydrolyzed and separated by column chromatography into its component amino acids. When diaphragm was incubated with U-C14-glucose or with 1-C14-pyruvic acid, C14 was incorporated mainly into the alanine fraction and to a less extent into aspartic acid and glutamic acid. Incubation of diaphragm in a medium containing C14O2 led to the recovery of similar quantities of C14 in aspartic acid and glutamic acid. Diaphragm incubated with U-C14-alanine incorporate C14 mainly into the alanine fraction of the protein, though some C14 was also found in aspartic acid and glutamic acid. Incubation with U-C14-aspartic acid gave rise to C14 in the glutamic acid and alanine fractions as well as in aspartic acid, whereas incubation with U-C14-glutamic acid led to an appreciable recovery of C14 in aspartic acid and glutamic acid alone. C14 incorporated into protein of diaphragm from 1-C14-glycine was recovered almost entirely as C14-glycine, the remainder being found in the serine+threonine fraction. Conversely, protein of diaphragm incubated with U-C14-serine contained C14 mainly in the serine+threonine fraction but also to a small extent in the glycine fraction. Addition of insulin to the medium, although increasing the amount of C14 incorporated by 25-50%, did not change the distribution of C14 incorporated from C14.gluose, alanine and glycine. The results are largely explicable in terms of rapid transaminations and operation of the tricarboxylic acid cycle in isolated rat diaphragm, together with incorporation of CO2 by pyruvate, succinyl-coenzyme A or related substances.