Abstract
The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.
Highlights
Halorhodopsin, in the membrawneas determined using HR was isolated according to themethod described in Ref. 8 from antibodies directed against a synthetic peptide which represents the C-terminal segmeonftthe protein
Partial inversion by soni- was synthesized by a modified Merrifield solid phase strategy using cation or lysis under low salt conditions exposedthis fluorenyl-methoxy-carbonyl-protecteadmino acids
The COOH terminus of themolecule is The amino acid composition was determined after hydrolysis in 6 located on the cytoplasmic surfaceof the membrane
Summary
From the $Max-Planck-Institut fur Bwchemie, 0-8033 Martinsried, Federal Republic of Germany. The orientation of the light-driven chloride pump, tested with the assay described in Ref. 9. Halorhodopsin, in the membrawneas determined using HR was isolated according to themethod described in Ref. 8 from antibodies directed against a synthetic peptide which represents the C-terminal segmeonftthe protein. Antibodies against this decapeptide did not bind to strain OD-2. Antibody binding was resins from Bachem) on a semi-automatic Labotec peptide syntheremoved by preincubationwith the decapeptide. The COOH terminus of themolecule is The amino acid composition was determined after hydrolysis in 6 located on the cytoplasmic surfaceof the membrane. M HCl on aKontronamino acid analyzer; its purity was further controlled by fast atom bombardment mass spectrometry on a Finnigan MAT 312 mass spectrometer
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