Protein synthesis in Sarcophaga bullata

by Robert Roy Allen

Institution: Oregon State University
Department: Chemistry
Degree: PhD
Year: 1964
Keywords: Proteins
Record ID: 1514913
Full text PDF: http://hdl.handle.net/1957/48030


An investigation was made into certain aspects of protein synthesis at different developmental stages of the flesh fly Sarcophaga bullata. This included measurements of the incorporation of labeled amino acids into protein by intact cells from a single organ, the larval fat body, and a study of amino acid activating enzymes in the fat body and in the developing pupa. Fat bodies were dissected from Sarcophaga larvae and incubated for three hours with ¹⁴C-labeled amino acids. The incorporation of amino acids into protein was greater with fat bodies from two or three day larvae than with those from six day larvae. In vitro protein synthesis by this tissue seemed to be correlated with the overall growth rate of the larvae. Incorporation of radioactivity into the lipid fraction of larval fat bodies followed the same pattern as that described for protein. It was also evident from these experiments that Sarcophaga fat bodies were capable of extensive degradation of amino acids to carbon dioxide. The conversion of amino acids to carbon dioxide was most extensive with fat bodies from two day larvae, suggesting a greater energy requirement at this stage of rapid growth. Amino acid activating enzymes were studied by the pyrophosphate exchange method in which the exchange of radioactivity between ³²P-labeled inorganic pyrophosphate and adenosine triphosphate is a measure of the enzymatic activity. Larval fat bodies were homogenized, fractionated by differential centrifugation, and the different fractions assayed for the presence of amino acid activating enzymes. The supernatant from one hour's centrifugation at 110,000 g. contained enzymes exhibiting activity toward nineteen amino acids, although there was considerable variation in the activation observed with different amino acids. It was further observed with this soluble fraction that the endogenous activity was always significant, probably as a result of the high level of free amino acids found in insect tissue; that the relative rates of activation did not agree with the relative rates of incorporation into fat body protein for those amino acids studied; that the sum of the activities observed with single amino acids was greater than the activity in the presence of a mixture of these amino acids; that preparations from different batches of larvae differed in their content of activating enzymes; and that the activity in the presence of D-valine was only slightly above the endogenous, although the greatest activity with a single amino acid was observed with L-valine. Activating enzymes could be precipitated from the soluble fraction by adjusting the pH from 7.6 to 5.0 or lower. Although some separation of…