Biosynthesis of a proinsulin-like molecule in the pancreas of a lizard

The biosynthesis of insulin, in particular the occurrence of a proinsulin-like molecule (PILM), in the pancreas of the green anole, Anolis carolinensis, was investigated. The laboratory rat was used for comparison and validation of the procedures. Anolian pancreases were incubated in vitro with radiolabeled leucine or proline. Radioactivity incorporated into the acidic ethanol-soluble (AES) phase increased in an essentially linear manner with time. Selective incorporation of labeled amino acids into AES material was not enhanced by a high concentration of glucose (6 mg/ml), by the addition of glucagon, which increases cyclic AMP levels, or by the addition of cytochalasin B; yet all three conditions result in stimulated insulin secretion. Gel filtration of the AES material on columns of Bio-Gel P-30 revealed a major peak of radioactivity whose apex followed closely the apogee of porcine proinsulin. When this presumptive PILM was treated with trypsin and carboxypeptidase B, the radioactivity was shifted towards later elution volumes, but the peak of the converted anolian material was not coincidental with marker insulin. Immunopurification techniques and additional molecular filtrations resulted in the isolation of fractions with both insulin-like immunoreactivity (IRI) and radioactivity derived from tritium-labeled leucine. One peak of radiolabel was present in the region of the proinsulin standards. The level of radioactivity in the region of the insulin standards was relatively high after two days of organ culture of splenic pancreases. By polyacrylamide gel electrophoresis proteins that incorporated radiolabeled amino acids and had insulin-like immunoreactivity possessed migration patterns similar to those of mammalian proinsulin and insulin. The insulin-like component was less readily demonstrated than the proinsulin-like component and raises the possibility that anolian PILM may be a major storage form in this species. The results are consistent with the synthesis of a proinsulin-like precursor in the beta cells of the green anole. The results also provide evidence for a precursor-product relationship in the anolian beta cell.