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Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P
Author(s) -
Shaw C.,
Foy W. L.,
Johnston C. F.,
Buchanan K. D.
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.tb08551.x
Subject(s) - substance p , neurokinin a , chemistry , high performance liquid chromatography , chromatography , radioimmunoassay , neuropeptide , antiserum , gel permeation chromatography , ethanol , neurokinin b , biochemistry , biology , receptor , organic chemistry , antibody , immunology , polymer
Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse‐phase HPLC. Using an antiserum specific for the C‐terminal hexapeptide amide of substance P, levels of 3.43 ± 0.33 ng g −1 and 12.45 ± 0.76 ng g −1 (mean ± SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross‐reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (<0.1%) were 12.46 ± 0.47 ng g −1 and 7.20 ± 0.37 ng g −1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse‐phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co‐eluting with the respective sulphoxides was higher in acidified ethanol extracts, and sub stance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse‐phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide. Deamidation was ruled out on the basis of the unequivocal demonstration of both glutamine residues and a methionine amide residue on each metabolite. These data demonstrate the presence of the mammalian tachykinins substance P, neurokinin A, neurokinin B, and neuropeptide K in the bovine retina. The susceptibility of substance P to methionine oxidation has been confirmed by incubation with dispersed retinal cells, and this putative inactivation system may be of physiological relevance within the retina.