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Construction of a Physiologically Active Photoaffinity Probe Based on the Structure of Bradykinin: Labelling of Angiotensin Converting Enzyme but Not Candidate Bradykinin Receptors on NG108–15 Cells
Author(s) -
Vries Johannes G.,
Phillips Elsa,
Snell Christopher R.,
Snell Penelope H.,
Webb Michael
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb09201.x
Subject(s) - bradykinin , chemistry , receptor , angiotensin ii , biochemistry , bradykinin receptor , angiotensin converting enzyme , agonist , ligand (biochemistry) , photoaffinity labeling , enzyme , biophysics , biology , endocrinology , blood pressure
The peptides bradykinin and kallidin are released in response to noxious stimuli and mediate various physiological effects, including a direct stimulation of nociceptive afferent neurones. The nature of the receptor molecules through which these ligands act is presently unknown. We synthesised an iodinatable photoaffinity probe, N ′‐4‐azido‐salicylylkallidin, and used it in an attempt to identify candidate bradykinin receptors on the NG108–15 neuroblastoma X glioma hybrid cell line. The ligand bound in subdued light to a particulate fraction of NG108–15 tumours and could be displaced by bradykinin with an IC 50 of 0.33 n M . In a physiological assay, it behaved as an agonist equipotent with bradykinin. Gel analysis of the labelled products after photolysis of the iodinated ligand in the presence of NG108–15 cells or tumour membranes revealed bradykinin‐blockable labelling of a glycoprotein with an M r of 166,000. The probe was also able to label purified commercial angiotensin converting enzyme. The band labelled in NG108–15 cells was immunoprecipitable with a polyclonal antiserum to angiotensin converting enzyme, an enzyme shown to be present in low amounts in these preparations by direct binding using the iodinatable specific ligand MK351A.