263 - Proteolytic Studies of Chain Cleavage and Proton Pump Activity of Bacteriorhodopsin in Purple Membranes

Abstract

Cleavage of bacteriorhodopsin in purple membranes of Halobacterium halobium by proteolytic digestion led to fragments, the molecular weights of which depended on the enzyme used. On SDS-polyacrylamide gels trypsin gave bands corresponding to 23,000 and 22,000 daltons, pronase — 22,000 and 17,400 daltons, and papain — 21,000 and 16,600 daltons, when the enzyme treatment was carried out in the dark. When the purple membranes were illuminated during the pronase digestion, an additional small fragment (M.W. ∼ 800), of primarily hydrophobic amino acid composition, was split off. The kinetics of the light-induced pH changes in suspensions of reconstituted proteoliposomes incorporating enzymatically modified bacteriorhodopsin could be fitted by two first-order processes, one being about twenty times faster than the other. After trypsin treatment this kinetics was not significantly changed. Pronase treatment drastically reduced the light-induced pH changes, acting mainly on the amplitude of the slower process. The apparent rate constants of both processes were markedly increased. Similar, but less drastic effects were found in papain-treated membranes. The increases in the rate constants of the slow phase could be accounted for by assuming that the proton leak through the proteoliposome membrane was increased by the proteolytic cleavage of the bacteriorhodopsin chain.