Identification and characterization of multiple tachykinin immunoreactivities in bovine retina: evidence for the presence of a putative oxidative inactivation system for substance P.
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 (less than 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 substance 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Shaw
C
C
Department of Medicine, Queen's University, Belfast, Northern Ireland.
Foy
W L
WL
Johnston
C F
CF
Buchanan
K D
KD
D000577:Amides / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010084:Oxidation-Reduction; D011863:Radioimmunoassay; D012160:Retina / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism*; D015320:Tachykinins / Q000032:analysis*
D000577:Amides; D000596:Amino Acids; D015320:Tachykinins; D013373:Substance P