Studies on the different forms of material reacting with antiinsulin antibodies in the fetal and adult rat.
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 degrees 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 alpha I 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 alpha globulins and albumin (but not beef albumin) cross-react with the DA procedures; 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.
D000818:Animals; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D005033:Ethylmaleimide; D005260:Female; D005333:Fetus; D007328:Insulin / Q000276:immunology*; D007330:Insulin Antibodies; D007467:Iodoproteins; D046911:Macromolecular Substances; D008970:Molecular Weight; D011247:Pregnancy; D011485:Protein Binding; D051381:Rats; D014357:Trypsin; D014508:Urea
D007328:Insulin; D007330:Insulin Antibodies; D007467:Iodoproteins; D046911:Macromolecular Substances; D014508:Urea; D014357:Trypsin; D005033:Ethylmaleimide