The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids

Takagaki, Y. ; Radhakrishnan, R. ; Gupta, C. M. ; Khorana, H. G. (1983) The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids Journal of Biological Chemistry, 258 (15). pp. 9128-9135. ISSN 0021-9258

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Official URL: http://www.jbc.org/content/258/15/9128.abstract

Abstract

Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-ω -(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-ω -(2-diazo-3,3,3-trifluropropionyloxy)lauroyl-sn- glycero-3-phosphocholine (PC-II). Rabbit liver cytochrome b5 was inserted into these vesicles spontaneously and the resulting vesicles containing the cytochrome b5 in the transferable form were photolyzed. Cytochrome b5 containing covalently cross-linked phospholipids was isolated by Sephadex LH-60 column chromatography using ethanol/formic acid as the solvent. Of the total radioactivity, 4.6% (PC-I) or 11.3% (PC-II) was linked to the protein; of the former, up to 51% was base-labile, while in the latter, 22% was base-labile. The sites of cross-linking of PC-I to the protein were investigated by fragmentation with trypsin, Staphylococcus aureas V8 protease, CNBr, and o-iodosobenzoic acid followed by Sephadex LH-60 chromatography and Edman sequencing (solid phase) of the appropriate fragments. The distribution of cross-linking was broad (Ser-104 to Met-130), showing a bell-shaped pattern with a significant peak at Ser-118. The labeling pattern is consistent with the previously proposed loop-back model for the membranous segment in the transferable form of cytochrome b5.

Item Type:Article
Source:Copyright of this article belongs to American Society for Biochemistry and Molecular Biology.
ID Code:15936
Deposited On:16 Nov 2010 13:42
Last Modified:17 May 2016 00:46

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