The biological potency of covalent insulin-receptor complexes. Dependence on site of cross-linkage

A radioactive photosensitive insulin analogue, 125I-N epsilon B29-(4-azido-2-nitrophenyl-acetyl)insulin, was covalently bound to the receptors of isolated rat adipocytes by irradiation with UV light. This caused a stimulation of lipogenesis. The relative potency of the covalent complexes to that of normal reversible complexes was calculated by comparing the amounts of radioactivity required to be covalently or reversibly bound by adipocytes to cause the same levels of stimulation. For several different occupancies , this relative potency was constant at 50 +/- 3%. Previous studies had shown that the relative potency of covalently bound 125I-N alpha B2-(4-azido-2- nitrophenylacetyl )des- PheB1 -insulin was only 25 +/- 4% under identical conditions. This demonstrates that the sites of crosslinking have a marked effect on the potency of the covalent hormone-receptor complex. It appears that attachment through the C-terminus of the B-chain leads to a better stabilization of the biologically active form than linking through the more flexible N-terminus.