Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments.
Pronase treatment of aqueous suspensions of purple membrane fragments from H.halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000--21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme. Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated. Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M412 and the regeneration of bR570. Simultaneously the yield of O660 is reduced. As with untreated purple membrane, the correlation between the rates of decay of M412 and regeneration of bR570 is greatest when the yield of O660 is lowest.
D001426:Bacterial Proteins / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000378:metabolism* / Q000528:radiation effects; D002338:Carotenoids / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006217:Halobacterium / Q000378:metabolism; D008027:Light; D010446:Peptide Fragments; D010782:Photolysis; D011402:Pronase / Q000378:metabolism; D013053:Spectrophotometry
D001426:Bacterial Proteins; D010446:Peptide Fragments; D002338:Carotenoids; D001436:Bacteriorhodopsins; D011402:Pronase