Topography of the C terminus of cytochrome b5 tightly bound to dimyristoylphosphatidylcholine vesicles

• Published in: The Journal of biological chemistry Vol. 262; no. 32; pp. 15563 - 15567
• Main Authors: Arinç, E, Rzepecki, L M, Strittmatter, P
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15-11-1987; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cattle; Chymotrypsin - metabolism; Cytochrome b Group - analysis; Cytochromes b5; Dimyristoylphosphatidylcholine - metabolism; Fundamental and applied biological sciences. Psychology; Hemoproteins; Male; Metalloproteins; Microsomes, Liver - enzymology; Peptide Fragments - analysis; Proteins; Taurine - metabolism; C terminal-Sequence; Vesicle; Hemoprotein; Cytochrome b; Topography; Phosphatidylcholine; Molecular interaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47763-0

Cytochrome b5 holoenzyme was bound asymmetrically in the tightly bound form to small unilamellar dimyristoylphosphatidylcholine vesicles. [3H]Taurine, a membrane-impermeant nucleophile, was added to the external medium and was then cross-linked to cytochrome carboxyl residues by the addition of a water-soluble carbodiimide. Nonpolar peptide was isolated after trypsin digestion of taurine-labeled apocytochrome b5 and contained 1.7-1.9 residues of taurine. The C-terminal tetrapeptide containing residues Thr130-Asn133 was generated by chymotryptic hydrolysis of radiolabeled nonpolar peptide and was purified by gel filtration and ion exchange chromatography. Amino acid analysis of the C-terminal tetrapeptide showed that about 1.6 mol of taurine was cross-linked per mol of peptide. When the experiment was performed with taurine trapped inside the vesicles, no cross-linking was observed. The results suggest that when cytochrome b5 holoenzyme is bound to vesicles in the tight binding form, the C terminus is located on the external surface of the vesicles.