Radioiodinated, photoactivatable phosphatidylcholine and phosphatidylserine: transfer properties and differential photoreactive interaction with human erythrocyte membrane proteins

Abstract

An isotopically labeled cross-linking reagent, succinimido 3-(3-[125I]iodo-4-azidophenyl)propionate, has been synthesized and coupled to 1-acyl-2-(aminocaproyl)phosphatidylcholine according to previously described procedures [Schroit, A. J., & Madsen, J. (1983) Biochemistry 22, 3617-3623]. 125I- and N3-labeled phosphatidylserine (125I-N3-PS) was produced from the phosphatidylcholine (PC) analogue by phospholipase D catalyzed base exchange in the presence of L-serine. These phospholipid analogues are photoactivatable, are labeled with 125I at high specific activity, completely incorporate into synthetic vesicles, and spontaneously transfer between membranes. When an excess of acceptor vesicles or red blood cells (RBC) was mixed with a population of donor vesicles containing the 125I-N3-phospholipids, approximately 40% of the analogues transferred to the acceptor population. After transfer in the dark to RBC, all of the 125I-N3-PC incorporated into the cells could be removed by washing with serum, whereas the 125I-N3-PS could not. After photolabeling of intact RBC, approximately 50% of the PC and 20% of the PS cross-linked to membrane proteins as determined by their insolubility in CHCl3/MeOH. Analysis of probe distribution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 125I-N3-PS preferentially labeled a Mr 30,000 peptide which contained approximately 30% of the protein-bound label.

Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling.

“…The experiments in the present study demonstrate the use of a hydrophobic photoaffinity probe for covalent labeling of (27). These agents inhibit labeling of the proteins by probe diffusion through the aqueous phase.…”

Partial purification and characterization of the human erythrocyte Mg2+ ‐ATPase A candidate aminophospholipid translocase

“…A striking common feature is the PS requirement for a full activity expressed by the erythrocyte (this work), the coated vesicle [18] or the chromaffin granule Mg-ATPase (G.M., unpublished data). Labeling by ATP- [7)SS] confirmed that the apparent molecular weight of the erythrocyte Mg-ATPase, determined by electrophoresis under denaturating and reducing conditions, is in the 115-120 kDa range [11]. The labeling of that band is abolished under conditions where the ATPase is inactive, i.e.…”

Regulation of transbilayer plasma membrane phospholipid asymmetry

“…However, the enzyme displays an absolute requirement for the stereochemistry of the glycerol backbone; the sn -2,3-glycerol analog of the naturally-occurring sn -1,2-glycero-lipid is not a substrate for transport (72,80). In contrast to the polar headgroup specificity, the flippase is capable of accepting PS molecules containing fatty acids of various lengths and composition, including spin, fluorescent, and photoactivatable groups (25,55,58,59,63,(89)(90)(91), but prefers reporter groups attached to longer acyl chains (58, 59). Using an endocytosis mutant, Nichols recently demonstrated a similar ATP-dependent transport activity in yeast, although NBD-PC was transported in addition to NBD-PE (92,93).…”