Photoaffinity labeling of insulin receptor proteins of liver plasma membrane preparations

Abstract

The photoreactive insulin derivatives N.epsilon.B29-(azidobenzoyl)insulin (MAB-insulin) and N.alpha.A1,N.epsilon.B29-di(azidobenzoyl)insulin (DAB-insulin) were synthesized by reacting bovine insulin with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. These derivatives were purified by ion-exchange chromatography on SP-Sephadex, and their identities were established by polyacrylamide gel electrophoresis, amino acid analysis and end-group determination. Their biological activities were measured by receptor binding assay and fat cell assay. The photoreactivity of these 2 derivatives was demonstrated by spectral changes and by the formation of covalent polymers of high MW when exposed to light. Radioactive MAB-insulin and DAB-insulin were prepared by iodination with 125I. These radioactive derivatives were characterized for their photoreactivity, immunoreactivity and receptor binding to liver plasma membrane. Liver plasma membrane preparations of rat, mouse and guinea pig were incubated with these radioactive insulin derivatives and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization and reduction showed that 2 proteins were specifically labeled. The MW of the 2 radioactive bands were estimated to be about 130,000 and 90,000 in all 3 species of animals.