MOLECULAR-WEIGHT DETERMINATION OF THE HEPATIC VASOPRESSIN RECEPTOR WITH A HIGH-AFFINITY PHOTOPROBE

We report here a study of photoaffinity labeling of the V-1a-vasopress in receptor with high-affinity, V-1-specific radioiodinated antagonist ligands: one containing. an azidophenylalanine residue ([beta,beta-di methly-beta-mercaptopropionyl(1), p-azido-Phe(2), Val(4), Lys(8), D-Ty r(9)] vasopressin), two others containing nitrophenyl-alanine, and one , highly similar but without a photosensitive function, as control. Al l analogues competed in the dark for the same binding site with vasopr essin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specif ically labeled protein band at 53 kDa in SDS-PAGE. Identical experimen ts with the nitrophenylalanyl peptides produced only non-specific labe ling and control experiments vvith the non-photosensitive analogue pro duced no labeling at all, Chemical crosslinking of H-3-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deg lycosylation of the labeled receptor with endoglycosidase F reduced th e observed molecular weight of 53 kDa to 43 kDa. The molecular paramet ers reported herein of the presumed hepatic vasopressin receptor confi rm the values deduced from the molecular cloning of the rat V-1a recep tor. (C) Munksgaard 1996.