N epsilonB29-(+)-biotinylinsulin and its complexes with avidin. Synthesis and biological activity.
Insulin was modified with d-biotin-N-hydroxysuccinimide ester in dimethylformamide. Mono-, di-, and triacylated insulins were separated by preparative isoelectric focusing. Monoacylated derivatives (isoelectric point 5.1) were fractionated twice on DEAE-cellulose to yield pure N epsilonB29-biotinylinsulin. The structure of the product was established by amino acid analysis before and after deamination. N epsilonB29-biotinylinsulin had biological activity indistinguishable from insulin on glucose oxidation and lipid synthesis assays using isolated rat epididymal fat cells. Complexes of N epsilonB29-biotinylinsulin with avidin, having essentially all but one binding site filled with biotin, were prepared in order to obtain a 1:1 insulin:avidin ration. The elicited identical maximal biological responses, but showed a potency decreased to 5% of that of insulin. Such complexes conjugated with ferritin will provide a useful tool in the development of electron microscopic stains of insulin receptors.
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001360:Avidin; D001710:Biotin / Q000031:analogs & derivatives*; D002848:Chromatography, DEAE-Cellulose; D004589:Electrophoresis, Disc; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D007525:Isoelectric Focusing; D008297:Male; D010047:Ovalbumin / Q000031:analogs & derivatives; D051381:Rats
D007328:Insulin; D001360:Avidin; D001710:Biotin; D010047:Ovalbumin; D005947:Glucose