Insulin biosynthesis in the brown bullhead, Ictalurus nebulosus (Le Sueur), was studied by measuring the incorporation in vitro of [3H]leucine into proteins of the principal islet. The tissue was incubated for 6–15h in Krebs–Ringer bicarbonate buffer with [3H]leucine, supplemented with amino acids and glucose. Proteins, precipitated with trichloroacetic acid and extracted with acid ethanol, were separated by gel-filtration on Biogel P-30 in 3m-acetic acid. Three major components were found after incubation of the islets at 22°C. On the basis of the results of sulphitolysis, biological activity and the demonstrated precursor–product relationship, components I and II were identified as proinsulin and insulin respectively. The third component was not identified. At 12°C, [3H]leucine was incorporated only into proinsulin. No radioactivity was found in insulin or the unidentified component III at 12°C as was found after incubation at 22°C. When the temperature was lowered from 22° to 12°C after 3h of a 15h incubation, decreased conversion of proinsulin into insulin resulted at the lower temperature compared with the control tissue maintained at 22°C. When the temperature was raised from 12° to 22°C at 3h of a 15h incubation, conversion of proinsulin into insulin occurred. No conversion occurred in the control tissue with the temperature maintained at 12°C. No qualitative difference in the incorporation of [3H]leucine into proinsulin and its conversion into insulin at 12° and 22°C could be demonstrated between islet tissue from fish acclimated to less than 12°C or to 22°C. The results suggest that the enzyme(s) responsible for converting proinsulin into insulin in the bullhead may be temperature sensitive with low activity at 12°C.

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