THE DETERMINATION OF THE PATHWAYS OF RETINAL FORMATION DURING RHODOPSIN PHOTOLYSIS
Abstract
The formation of retinal during rhodopsin photolysis has been examined in both digitonin extracts of bovine rhodopsin and excised retinas (Bufo marinus, Raja erinacea, Raja oscellata). As determined for bovine rhodopsin the earliest photoproduct to undergo hydrolysis was Meta II(,380). Dual pathways in the thermal decay of Meta II(,380) were determined; Meta II(,380) was demonstrated to decay to Meta III(,465) and to hydrolyze to retinal(,387) plus opsin. For bovine rhodopsin at pH 7.0, 3(DEGREES)C, approximately 75% Meta II(,380) thermally decayed to Meta III(,465) while 25% was hydrolyzed; in excised retinas (pH 7.0, 3(DEGREES)C) 69% Meta II(,380) decayed to Meta III(,465) and 31% hydrolyzed to retinal(,387) plus opsin. The amount of retinal produced via Meta II(,380) hydrolysis for digitonin extracts of bovine rhodopsin is pH dependent, having a transition at pH 6.8; at pH's 8.0, 7.6 and 7.0, 22%, 24% and 25% retinal was produced for Meta II(,380) while at pH's 6.7 and 6.5 the percentages increased to 39% and 42% respectively. The kinetics of retinal formation from Meta II(,380) hydrolysis in digitonin extracts of bovine rhodopsin were examined. The observed rate of formation of retinal was separated into the rate of formation of Meta III(,465) and rate of hydrolysis of Meta II(,380) to retinal(,387) plus opsin. Both of these processes were found to be pH dependent: at pH 8.0 the half-lives for Meta III(,465) formation and Meta II(,380) hydrolysis were 0.6 (+OR-) .1 hr and 2.0 (+OR-) .4 hr, respectively, and at pH 6.5 increased to 4.4 (+OR-) .6 hr and 6.1 (+OR-) .8 hr, respectively. It was also suggested that approximately 10% of the Meta III(,465) formed (8% of total rhodopsin) could be transformed to a form that hydrolyzed to retinal plus opsin following a pH decrease. A decay scheme for the photolysis of rhodopsin is proposed. The pathway of rhodopsin photolysis in excised retinas (toad and skate) was determined using a variety of methods. The decay of Meta II(,380) was demonstrated to occur by two pathways: Meta II(,380) decay to Meta III(,465) and also hydrolysis to retinal plus opsin. The pathway of Meta III(,465) decay to retinal(,387) followed by retinal(,387) reduction to retinol(,330) was also directly demonstrated using a variety of techniques. It is also suggested that the Meta II(,380) hydrolysis pathway for toad retinas is identical to that proposed for bovine rhodopsin extracts.
Degree
Ph.D.
Subject Area
Biochemistry
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