Studies on the Different Forms of Material Reacting with Antiinsulin Antibodies in the Fetal and Adult Rat[*]

Abstract

The nature of peak B (MW = 10-12000, proinsulin? ) and peak C (MW = 50-100000, "big big" insulin? ) materials detected by the double antibody (DA) procedure in elution profiles of rat sera after Sephadex G 50 or G 100 chromatography (cf. preceding companion paper) is further investigated. Peak B is converted by mild tryptic digestion in an immunoreactive material behaving in rechromatography exactly like insulin monomer. Peak C is less easily detected by the dextran coated charcoal (DCC) method; it resists 8 M urea 37°C for 1 hr, is not an artifact due to the complement system; its relative importance is very much reduced in pancreatic extracts or perifusates. Incubation of biologically active ¹²⁵I labelled insulin in rat sera results in appearance of labelled material behaving on chromatography like peak C natural material, having the electrophoretic mobility of rat α1 globulins and albumin, and resisting 8 M urea, acidic pHs and 0.5 M NaCl. Similar incubation in buffer supplemented with bovine albumin results in appearance of a labelled material having the electrophoretic mobility of beef albumin; N-ethyl-maleimide provides against this binding, which might result from (S-S)-(SH) interchanges. Rat a globulins and albumin (but not beef albumin) cross-react with the DA procedure; they do not react with the DCC method. Insulin bound to plasma proteins reacts with both methods. It is suggested that peak C material, as detected by the DA method in rat serum, consists both of insulin covalently bound to plasma proteins and of certain plasma proteins; the DCC method detects only bound insulin. In streptozotocin treated rats, peak C material persists after the complete disappearance of insulin and proinsulin, when detected by the (DA) procedure, but disappears when detected by the DCC procedure.

Key words

IRI - Circulating Insulin - ¹²⁵I Insulin - In Vitro Incubation

1 Supported by ATP no 429912 and by D.G.R.S.T. grant no 71-7-3162

1 Supported by ATP no 429912 and by D.G.R.S.T. grant no 71-7-3162