To facilitate characterization of the atrial natriuretic factor (ANF) receptor, we have developed an affinity labeling procedure, stepwise affinity labeling, which allows specific labeling of ANF binding sites in adrenal plasma membranes at high yields. An iodoacetyl (IAc-), bromoacetyl (BrAc-), or maleimidobenzoyl group was attached to the amino-terminal alpha-amino group of the ANF(4-28) peptide, and the peptide derivatives were radioiodinated at Tyr-28 to obtain affinity reagents, N4alpha-IAc-[125I]ANF(4-28), N4alpha-BrAc-[125I]ANF(4-28), and N4alpha-(maleimidobenzoyl)-[125I]ANF(4-28). Receptor labeling was carried out in a stepwise fashion as follows: (1) Membranes were treated with p-chloromercuriobenzenesulfonic acid (PCMBS) or N-ethylmaleimide to block sulfhydryl groups; (2) the affinity reagent was allowed to bind to the receptor at 0 degrees C for 1 h; and (3) the membranes were washed to remove unbound reagent and were incubated at room temperature to effect alkylation reaction. Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by autoradiography revealed specific labeling of a 130-kDa ANF receptor. On the basis of 125I-radioactivity incorporated, the labeling yields were estimated to be 70%, 52%, and 21% for the reactions with IAc-[125I]ANF(4-28), BrAc-[125I]ANF(4-28), and (maleimidobenzoyl)-[125I]ANF(4-28), respectively. The efficiency of receptor labeling by the stepwise procedure using IAc-[125I]ANF(4-28) was 27-fold greater than that obtained by photoaffinity labeling using N3Bz-[125I]ANF(4-28) and 63-fold greater than that by direct cross-linking using disuccinimidylsuberate and [125I]ANF(4-28) under comparable conditions. Digestion of the membrane protein labeled with IAc-[125I]ANF(4-28) by BrCN, endoproteinase Glu-C, and endoproteinase Lys-C gave single radiolabeled bands with apparent masses of 40, 18, and 29 kDa, respectively. Reversed-phase HPLC separation of the digests also gave single major peaks. The confinement of the affinity label to one major fragment in each digest suggests that the cross-linking occurred at a single or a limited number of sites. The stepwise affinity labeling with the high cross-linking yield and specificity may be useful for analyzing the ANF receptor binding site structure.
Adrenal Cortex Affinity Labels