Biosynthesis of an insulin precursor by islet tissue of cod (Gadus callarias)

1. At 15°, slices of cod islet tissue incorporated [U−14C]proline into proteins soluble in acid–ethanol at a linear rate for 6hr. 2. Initially, all the radioactivity was associated with a polypeptide that had a molecular weight of about 10000 and was appreciably more basic than cod insulin. After 1hr. there was also a significant and progressive increase in the radioactivity of insulin and of fractions intermediate in molecular size and basicity between the polypeptide and insulin. 3. O-Ethyl O-p-nitrophenyl phenylpropylphosphonate markedly decreased the radioactivity both of the intermediate fractions and of insulin, but had no significant effect on the biosynthesis of the polypeptide. In contrast, puromycin inhibited the incorporation of radioactivity into all the fractions. 4. The polypeptide had an activity of less than 0·2 international unit/mg. in the epididymal-fat-pad bioassay. Treatment with low concentrations of trypsin caused a progressive increase in the formation of an insulin-like material, judged by bioassay and ion-exchange chromatography of the digest. 5. Gel filtration of the polypeptide after oxidative sulphitolysis indicated that it was a single polypeptide chain. 6. The results suggest that the polypeptide is an insulin precursor whose formation is inhibited by puromycin and that the steps involved in the conversion of precursor into product are sensitive to O-ethyl O-p-nitrophenyl phenylpropylphosphonate.

Characterization of the cleavage products of human serum immunoglobulin disulphide bonds. II. S-Cyano derivatives

The displacement by cyanide ions of S-sulpho substituents from sulphitolysed human serum immunoglobulins yielded S-cyano derivatives, the solubility and sedimentation characteristics of which were examined with respect to pH. Exclusion chromatography gave rise to an aggregated and an unaggregated fraction in proportions similar to those found after oxidative sulphitolysis. Evidence obtained from immunoelectrophoresis, N-terminal amino acid assay, and hexose and amino acid analysis showed that both S-cyano fractions contained polypeptide chains in the same ratios as in the native protein.

Oxidative sulphitolysis of the disulphide groups in insulin and cystine

All three disulphide bonds in insulin are broken by the combined action of sulphite and oxygen, the reaction proceeding at a reasonable rate at room temperature, pH 8-10. This " oxidative sulphitolysis " (RSSR'+RSSO< +RISSO<) is strongly catalysed by metal ions, particularly Cu(11). For a 1% solution of insulin at pH 10 containing 2x 10-4iu CU(II), reaction is complete in about 24 hr at 20 OC, and acidification then gives a precipitate of the S-sulpho B-chain, the S-sulpho A-chain remaining in solution. Catalysis of the air-oxidative sulphitolysis of cystine to S-sulphocysteine (Clarke 1932 ; Kolthoff and Stricks 1951a, 1951b) is confirmed, CU(II), M~(II), CO(IIL), and F~(III) being most effective in that order. In these reactions the binding of metal ions by intermediate thiols prevents or retards the usual metal-catalysed air oxidation of sulphite to sulphate, so that a large excess of sulphite is not required. When a mixture of insulin and sulphite is oxidized with CU(II) with exclusion of oxygen, two end-points are readily detected (cf. cystine ; Kolthoff and Stricks 1951a, 1951b), corresponding to the reactions In addition there is evidence for the formation, in the early stages, of cupric mercaptide according to the equation Cupric sulphitolysis can also be carried out in two stages by &st titrating the insulin with CU(I) to the end-point of the reaction and then with CU(II) to complete the reaction according to RSCU+SO;- +2Cu2++RSS0c +3Cu+.