3-((4-azidophenyl)dithio)propionic-n-hydroxysuccinimide and methyl-4-azidobenzimidate

Binding studies were performed with bovine adrenal cortex membranes, human 125I-labelled high-density lipoprotein (HDL) and modified photoactivable derivatives of 125I-labelled HDL, namely 125I-labelled HDL-amidinophenylazide and 125I-labelled HDL-amidopropionyldithiophenylazide. The purity of the apolipoprotein composition of the 125I-labelled HDL and photoactivable 125I-labelled HDL used in the binding studies was determined by Coomassie blue and silver staining, and by measuring 125I-labelled cpm after SDS-polyacrylamide gel electrophoresis. About 45% of the 125I-labelled HDL binding to the membranes occurred in the presence of excess EDTA and only unlabelled HDL competed for the binding site. The 125I-labelled interaction with this binding site on the membranes did not require calcium. In addition, 40% of the 125I-labelled HDL binding was to an EDTA-sensitive site, and unlabelled HDL and low-density lipoprotein (LDL) competed for the binding site. Consequently, adrenal cortex membranes have binding sites which show cross reactivity for both HDL and LDL. Modification of 58% of the apolipoprotein lysine residues of 125I-labelled HDL with methylazidophenylimidate, a reagent which maintains the positive charge at lysine residues, had little affect on binding to EDTA-sensitive and insensitive sites. In contrast, modification of 35% of apolipoprotein lysine residues of 125I-labelled HDL with N-succinimidyl(4-azidophenyldithio)propionate, a reagent which converts charged amino lysines to amide bonds, showed binding properties which were almost totally inhibited by EDTA.

Topics: Adrenal Cortex; Animals; Azides; Binding, Competitive; Calcium; Carrier Proteins; Cattle; Cross-Linking Reagents; Edetic Acid; Humans; Imidoesters; Lipoproteins, HDL; Lipoproteins, LDL; Lysine; Membranes; Photolysis; Receptors, Cell Surface; Receptors, Lipoprotein; RNA-Binding Proteins