Depalmitylation with hydroxylamine alters the functional properties of rhodopsin.
Rhodopsin, the photosensitive protein found in rod photoreceptors, has two covalently attached palmitates that are thought to anchor a portion of the C terminus to the disc membrane, forming a fourth cytoplasmic loop. Using hydroxylamine (NH2OH) to cleave the thioester linkage, we have characterized the effect of depalmitylation on certain functional properties of rhodopsin. Treatment of rod outer segment membranes (prepared from rat retinas previously labeled in vivo with [3H]palmitate) with 1 M NH2OH typically removed greater than or equal to 75% of the [3H]palmitate initially bound to rhodopsin. Spectrophotometry of rod outer segment membranes that had been treated with 1 M NH2OH indicated preservation of 85% of the native rhodopsin and no effect on the shape of the absorbance spectrum of rhodopsin. In vivo labeled rhodopsin that had been treated with 1 M NH2OH did not reincorporate free endogenous [3H] palmitate over a 2-h incubation period. Both NH2OH-treated and untreated rhodopsin incorporated [14C]palmitate from exogenously added [14C]palmitoyl-CoA. This incorporation was substantially greater in the NH2OH-treated sample. The removal of palmitate by NH2OH inhibited rhodopsin regeneration by 44% and increased the ability of rhodopsin to activate transducin's light-dependent GTPase activity by 61%. However, the removal of palmitate from rhodopsin did not affect the light-dependent binding of transducin (T alpha and T beta gamma).
Department of Ophthalmology and Visual Sciences, Lions of Illinois Eye Research Institute, University of Illinois College of Medicine, Chicago 60612.
D F
DF
Morrison
P J
PJ
O'Brien
D R
DR
Pepperberg
D000818:Animals; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D015293:Transducin / Q000378:metabolism
D006898:Hydroxylamines; D010168:Palmitates; D019811:Hydroxylamine; D012243:Rhodopsin; D020558:GTP Phosphohydrolases; D015293:Transducin