Difference in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases

W. L. Roberts, B. H. Kim, T. L. Rosenberry

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Abstract

Acetylcholinesterases (AcChoEases; EC 3.1.1.7) from bovine (E(bo)) and human (E(hu)) erythrocytes were purified to apparent homogeneity by affinity chromatography. The hydrophobic portion of the glycolipid membrane anchor of each enzyme was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Several cleavage procedures demonstrated that this radiolabel was highly selective for the fatty acid portion of the anchor in both enzymes. The labeled enzymes were digested with phosphatidylinositol (PtdIns)-specific phospholipase C (EC 3.1.4.10), and label release was assessed by polyacrylamide gel electrophoresis. About 85% of the radiolabel was cleaved from E(bo) AcChoEase, whereas only 5% was released from E(hu) AcChoEase. This finding agrees with a report that E(bo) AcChoEase was quantitatively released from intact erythrocytes by PtdIns-specific phospholipase C but E(hu) AcChoEase was not [Low, M.G. & Finean, J.B. (1977) FEBS Lett. 82, 143-146]. The two AcChoEases contained comparable amounts of the anchor components ethanolamine, glucosamine, and myo-inositol, but qualitative and quantitative differences were found in the fatty acids. Thin-layer chromatography of radiolabeled fragments generated from E(bo) and E(hu) AcChoEases by nitrous acid deamination revealed a major difference in the membrane anchors of the two enzymes. The fragment released from E(bo) AcChoEase by this procedure comigrated with PtdIns, whereas the corresponding fragment from E(hu) AcChoEase had a mobility much greater than that of PtdIns even though it contained myo-inositol and fatty acids. These studies show that 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine is useful for analysis of lipid-containing compounds and indicate that, whereas E(bo) AcChoEase contains PtdIns in its glycolipid anchor, E(hu) AcChoEase has a different anchor structure, which is resistant to PtdIns-specific phospholipase C. This observation suggests the existence of a class of glycolipid-anchored membrane proteins resistant to this phospholipase.

Original languageEnglish (US)
Pages (from-to)7817-7821
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number22
DOIs
StatePublished - Jan 1 1987

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