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.
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 epsilon-4-azidosalicylylkallidin, 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 IC50 of 0.33 nM. 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 Mr 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.
de Vries
J G
JG
Sandoz Institute for Medical Research, London, England.
Phillips
E
E
Snell
C R
CR
Snell
P H
PH
Webb
M
M
D000345:Affinity Labels; D000806:Angiotensin-Converting Enzyme Inhibitors; D001386:Azides / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005910:Glioma / Q000378:metabolism; D006822:Hybrid Cells; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D007609:Kallidin / Q000031:analogs & derivatives* / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D009447:Neuroblastoma / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D010782:Photolysis; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D000806:Angiotensin-Converting Enzyme Inhibitors; D001386:Azides; D004151:Dipeptides; D007457:Iodine Radioisotopes; D018002:Receptors, Bradykinin; D017981:Receptors, Neurotransmitter; C042037:L-proline, N2-((1S)-1-carboxy-3-phenylpropyl)-N6-((4-hydroxyphenyl)iminomethyl)-L-lysyl-; C059367:N(6)-4-azidosalicylylkallidin; D007609:Kallidin; D010447:Peptide Hydrolases; D007703:Peptidyl-Dipeptidase A; D001920:Bradykinin; D002118:Calcium