Purification of bacteriorhodopsin and characterization of mature and partially processed forms.
Bacteriorhodopsin (BR) essentially free of native lipids has been prepared in a highly stable state. Purple membrane was solubilized in Triton X-100 and BR was purified by size exclusion chromatography using 3-[cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonic acid (CHAPSO) detergent at pH 5. Molar ratios of phospholipid/BR ranged from 0.4 to 0.05 corresponding to 94-98% phospholipid removal. Purified BR has an absorbance ratio (A280nm/A548nm) of 1.5-1.6 in the dark-adapted state which is the highest purified BR/protein ratio reported to date. The purified BR in CHAPSO shows maximum stability in the pH range 5.0-5.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of native purple membrane and solubilized BR from most Halobacterium halobium JW-3 cultures show 3 higher molecular weight bands in addition to BR. Immunological staining and amino acid sequencing indicates that these additional proteins are partially processed forms of the BR precursor protein. The BR preprotein contains 13 additional amino acids on the NH2 terminus which are removed by post-translational processing in at least four steps. Isoelectric focusing separated most delipidated and non-delipidated BR samples into 8 bands. Incomplete BR post-translational processing BR is thought to be largely responsible for the multiplicity of isoelectric BR species. The principal components have pI values of 5.20 and 5.24 and both have absorption maxima at 550 nm, characteristic of detergent-solubilized BR. BR in Triton X-100 or nonylglucoside, delipidated BR in CHAPSO, and BR in intact purple membrane all have a dark-adapted ratio of 13-cis to all-trans-retinal of 1.9:1.
Miercke
L J
LJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Ross
P E
PE
Stroud
R M
RM
Dratz
E A
EA
D001436:Bacteriorhodopsins / Q000302:isolation & purification*; D015153:Blotting, Western; D002793:Cholic Acids; D003902:Detergents; D006217:Halobacterium / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008823:Micelles; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D012172:Retinaldehyde / Q000378:metabolism; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D008823:Micelles; D001436:Bacteriorhodopsins; C048531:chapso; D012172:Retinaldehyde