title: Biochemical characterization of halorhodopsin in native membranes.
abstract: Procedures are described for selectively radiolabeling the protein moiety (haloopsin) or the chromophoric prosthestic group (retinal) of the light-driven chloride pump halorhodopsin in intact cells of Halobacterium halobium. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, two retinal-binding polypeptides are observed to band near the known molecular weight of the halorhodopsin chromophoric polypeptide (25,000). Synthesis of one of these polypeptides is controlled by retinal and is sufficient for generation of complete halorhodopsin function. The other is constitutively produced by the cells and differs chemically from the haloopsin protein as indicated by differences in their V8 protease digestion patterns. V8 protease cleavage of haloopsin in its native membrane is compared with that of the protein in denaturing and nondenaturing detergents. Protease cleavage sites available in the denatured haloopsin molecule are hidden in its native membrane-integrated conformation and in nondenaturing detergent micelles. Treatment with a variety of proteases indicates susceptibility of a short terminal region of the haloopsin chain in its native conformation.
mesh_terms: D001436:Bacteriorhodopsins / Q000032:analysis*; D002338:Carotenoids / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D006217:Halobacterium / Q000032:analysis*; D025602:Halorhodopsins; D008566:Membranes / Q000032:analysis; D008823:Micelles; D008970:Molecular Weight; D011487:Protein Conformation; D012697:Serine Endopeptidases
chemical_list: C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D003902:Detergents; D025602:Halorhodopsins; D008823:Micelles; D002338:Carotenoids; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase