The membrane-embedded segment of cytochrome b5 as studied by cross-linking with photoactivatable phospholipids.
Vesicles were prepared from a 9:1 (mole/mol) mixture of dipalmitoyl phosphatidylcholine and the radioactively labeled phospholipids, 1-palmitoyl-2-omega-(m-diazirinophenoxy)undecanoyl-sn-glycero-3-phosphocholine (PC-I) or 1-palmitoyl-2-omega-(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.
D000818:Animals; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000378:metabolism*; D003488:Cyanogen Bromide; D003573:Cytochrome b Group / Q000378:metabolism*; D015786:Cytochromes b5; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D008099:Liver / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010777:Photochemistry; D011247:Pregnancy; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D003432:Cross-Linking Reagents; D003573:Cytochrome b Group; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; C038804:1-palmitoyl-2-omega-(2-diazo-3,3,3-trifluoropropionyloxy)lauroyl-sn-glycero-3-phosphocholine; C026005:1-palmitoyl-2-(11-(3-diazirinophenoxy)undecanoyl)-sn-glycero-3-phosphocholine; D015786:Cytochromes b5; D010450:Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D003488:Cyanogen Bromide