PMID: 8974452Sep 1, 1995Paper

High-yield affinity alkylation of the atrial natriuretic factor receptor binding site

Bioconjugate Chemistry
X HeK S Misono

Abstract

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 7...Continue Reading

References

Jan 1, 1990·Medicinal Research Reviews·P R Bovy
Aug 15, 1985·Biochemical and Biophysical Research Communications·K S MisonoT Inagami
Dec 1, 1972·Proceedings of the National Academy of Sciences of the United States of America·J Houmard, G R Drapeau
Aug 30, 1984·Biochemical and Biophysical Research Communications·M SugiyamaT Inagami
Mar 15, 1984·Biochemical and Biophysical Research Communications·K S MisonoT Inagami
Sep 17, 1984·Biochemical and Biophysical Research Communications·K S MisonoT Inagami
Oct 31, 1983·Biochemical and Biophysical Research Communications·R T GrammerK S Misono
May 17, 1994·Biochemistry·G Dormán, G D Prestwich

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