Decay accelerating factor of complement is anchored to cells by a C-terminal glycolipid

Membrane-associated decay accelerating factor (DAF) of human erythrocytes (Ehu) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the Ehu acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact Ehu with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated from urine. Nitrous acid deamination cleavage of Ehu DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) released the [125I]TID label in a parallel fashion as from [125I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [3H]ethanolamine resulted in rapid 3H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. Our findings indicate that DAF and AChE are anchored in Ehu by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi.     

Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the gamma-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist alpha-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effects on [125I]TID labeling of the AChR. The regions of the AChR alpha-subunit that incorporate [125I]TID were mapped by Staphylococcus aureus V8 protease digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the alpha-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology.     

Changes in conformation upon agonist binding, and nonequivalent labeling, of the membrane-spanning regions of the nicotinic acetylcholine receptor subunits

All four subunits of the acetylcholine receptor (AChR) are labeled by the lipid-soluble photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) with different stoichiometries and levels of saturable modification sites, dependent on the conformational state of the AChR. This probe is specific for hydrophobic targets such as the membrane-spanning regions of intrinsic proteins. In the resting state, the gamma subunit is labeled 4.5 times greater and the beta and delta subunits 1.65-1.69 greater than the alpha subunit. Carbamylcholine-induced desensitization of the AChR lowers the level and alters the stoichiometry of [125I]TID incorporation into each subunit. This effect is shown to be specific in two ways. First, it is eliminated by prior equilibration with excess alpha-bungarotoxin, which does not change the [125I]TID-labeling pattern of the AChR from that of the resting state. Second, bacteriorhodopsin is labeled by [125I]TID to the same extent both in the presence and absence of carbamylcholine. The noncompetitive blocker phencyclidine does not alter [125I]TID labeling of the AChR relative to the resting state. The 43-kDa protein, which is believed to cross-link the AChR to the cytoskeleton at the synapse, is not modified by [125I]TID, in agreement with earlier conclusions that the 43-kDa protein is not an intrinsic membrane protein.     

A unique hydrophobic domain of rat brain globular acetylcholinesterase for binding to cell membranes

Both salt-soluble and detergent-soluble rat brain globular acetylcholinesterases (SS- and DS- AChE EC 3.1.1.7) are amphiphiles, as shown by detergent dependency of enzymatic activity and binding to liposomes. Proteinase K and papain treatment transformed SS-AChE and DS-AChE into forms that, in absence of detergent, no longer aggregated nor bound to liposomes. In contrast, phosphatidylinositol-specific phospholipase C had no effect on these properties. Labeling DS-AChE with 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine ([¹²⁵I]TID) revealed, by polyacrylamide gel electrophoresis under reducing conditions, one single band of 69 kD apparent molecular mass. The same pattern was previously obtained with Bolton and Hunter reagent-labeled enzyme (1). Proteinase K treatment transformed the 11 S [¹²⁵I]TID labeled AChE into a 4 S form which no longer showed¹²⁵I-radioactivity and was unable to bind to liposomes. These results are compatible with the existence of a hydrophobic segment present both on salt-soluble and detergent-soluble 11 S AChE as well as on the minor forms 4 S and 7 S. This segment is not linked to the catalytic subunits by disulfide bounds in contrast to the 20 kD non-catalytic subunit described by Inestrosa et al. (2).Abbreviations used AChE acetylcholinesterase - SS-AChE salt-soluble AChE - DS-AChE detergent-soluble AChE - BSA bovine serum albumin - ChE serum (butyryl) cholinesterase - ConA-Sepharose concanavalin A-Sepharose 4B - DMAEBA-Sepharose dimethylaminoethylbenzoic acid-Sepharose 4B - PC-Chol-SA liposomes phosphatidylcholine-cholesterol-stearylamine liposomes - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - ¹²⁵I-TID 3-(trifluoromethyl)-3-(m-(¹²⁵I)-iodophenyl) diazirine     

The hydrophobic photoreagent 3-(trifluoromethyl)-3-m-([125I] iodophenyl) diazirine is a novel noncompetitive antagonist of the nicotinic acetylcholine receptor

We have shown previously that the lipophilic photoreagent 3-(trifluoromethyl)3-m-([125I]iodophenyl)-diazirine ([125I]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that greater than 70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B.H., and Cohen, J.B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of [125I]TID, (b) AChR-mediated ion transport, and (c) AChR binding of several cholinergic ligands. We find that TID inhibits [125I]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of [125I]TID photolabeling is half-maximal at 4 microM [125I]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 microM [125I]TID. Repeated labeling of membranes at a fixed [125I]TID concentration gave results consistent with a maximal incorporation of one [125I]TID molecule per AChR. Nonradioactive TID also noncompetitively inhibits agonist-stimulated 22Na+ efflux from Torpedo vesicles with an IC50 of 1 microM. Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [125I]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The [125I]TID site is presumably located within the central pore of the AChR.     

Interaction of water-soluble form of apoproteolipid of bovine brain myelin with phospholipid vesicles

The water-soluble form of apoproteolipid (APL) from bovine brain myelin was found to bind with phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (6:4) vesicles below pH 5. The protein bound to vesicles was photoactively labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I)TID) and was digested with trypsin. A [125I]TID-labeled fragment with an apparent molecular weight of approximately 2,500 was extracted. An APL fragment with an identical Mr value was also obtained from the tryptic digest of APL/vesicle complex without prior labeling with [125I]TID. Determination of amino acid composition and the identification of the N-terminal amino acid residue of this unlabeled fragment showed that this protected segment covers the amino acid residues from Met-205 to Lys-228. In another experiment, the [125I]TID-labeled APL obtained from the above experiment without the proteolysis step was extracted and reconstituted into PC vesicles. Subsequent tryptic digestion of the exposed segment and comparison of the elution profile of the extracted polypeptides on a Sephadex LH-60 column with the published profile of these polypeptides indicated that the membrane-inserted segment of the water-soluble form of APL when bound to vesicles is the C-terminal region of this apoprotein within the amino acid residues between Met-205 and Lys-268.     

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessing hydrophobic regions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae.

The hydrophobic, photoactivatable probe TID [3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine] was used to label the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The H(+)-ATPase accounted for 43% of the total label associated with plasma membrane protein and incorporated 0.3 mol of [125I]TID per mol of 100 kDa polypeptide. The H(+)-ATPase was purified by octyl glucoside extraction and glycerol gradient centrifugation, and was cleaved by either cyanogen bromide digestion or limited tryptic proteolysis to isolate labeled fragments. Cyanogen bromide digestion resulted in numerous labeled fragments of mass less than 21 kDa. Seven fragments suitable for microsequence analysis were obtained by electrotransfer to poly(vinylidene difluoride) membranes. Five different regions of amino-acid sequence were identified, including fragments predicted to encompass both membrane-spanning and cytoplasmic protein structure domains. Most of the labeling of the cytoplasmic domain was concentrated in a region comprising amino acids 347 to 529. This catalytic region contains the site of phosphorylation and was previously suggested to be hydrophobic in character (Goffeau, A. and De Meis, L. (1990) J. Biol. 265, 15503-15505). Complementary labeling information was obtained from an analysis of limited tryptic fragments enriched for hydrophobic character. Six principal labeled fragments, of 29.6, 20.6, 16, 13.1, 11.4 and 9.7 kDa, were obtained. These fragments were found to comprise most of the putative transmembrane region and a portion of the cytoplasmic region that overlapped with the highly labeled active site-containing cyanogen bromide fragment. Overall, the extensive labeling of protein structure domains known to lie outside the bilayer suggests that [125I]TID labeling patterns cannot be unambiguously interpreted for the purpose of discerning membrane-embedded protein structure domains. It is proposed that caution should be applied in the interpretation of [125I]TID labeling patterns of the yeast plasma membrane H(+)-ATPase and that new and diverse approaches should be developed to provide a more definitive topology model.     

Identifying the lipid-protein interface and transmembrane structural transitions of the Torpedo Na,K-ATPase using hydrophobic photoreactive probes

To identify regions of the Torpedo Na,K-ATPase alpha-subunit that interact with membrane lipid and to characterize conformationally dependent structural changes in the transmembrane domain, we have proteolytically mapped the sites of photoincorporation of the hydrophobic compounds 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) and the phosphatidylcholine analogue [(125)I]TIDPC/16. The principal sites of [(125)I]TIDPC/16 labeling were identified by amino-terminal sequence analysis of proteolytic fragments of the Na,K-ATPase alpha-subunit and are localized to hydrophobic segments M1, M3, M9, and M10. These membrane-spanning segments have the greatest levels of exposure to the lipid bilayer and constitute the bulk of the lipid-protein interface of the Na,K-ATPase alpha-subunit. The extent of [(125)I]TID and [(125)I]TIDPC/16 photoincorporation into these transmembrane segments was the same in the E(1) and E(2) conformations, indicating that lipid-exposed segments located at the periphery of the transmembrane complex do not undergo large-scale movements during the cation transport cycle. In contrast, for [(125)I]TID but not for [(125)I]TIDPC/16, there was enhanced photoincorporation in the E(2) conformation, and this component of labeling mapped to transmembrane segments M5 and M6. Conformationally sensitive [(125)I]TID photoincorporation into the M5 and M6 segments does not reflect a change in the levels of exposure of these segments to the lipid bilayer as evidenced by the lack of [(125)I]TIDPC/16 labeling of these two segments in either conformation. These results suggest that [(125)I]TID promises to be a useful tool for structural characterization of the cation translocation pathway and for conformationally dependent changes in the pathway. A model of the spatial organization of the transmembrane segments of the Na,K-ATPase alpha- and beta-subunits is presented.     

Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage.

Human decay-accelerating factor (DAF) proteins expressed on E and nucleated cells differ in their susceptibility to phosphatidylinositol (PI)-specific phospholipase C (PLC) cleavage/release. To investigate the mechanism of this difference, the glycoinositol-phospholipid anchoring moieties of E DAF, and of HeLa cell, polymorphonuclear cell, and lymphocyte DAF were structurally compared. Labeling of PI-PLC-resistant E DAF with 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)-diazirine ([125I]TID) and TLC analysis of nitrous acid deamination anchor fragments showed a predominant phospholipid species with less polar migration than the 125I-TID-labeled PI. Gas chromatographic analyses of methanolyzed E protein revealed 2.20 +/- 0.16 mol of fatty acids [16:0, 18:0, 18:1, 20:4, 22:4, and 22:5 (0.76, 0.36, 0.25, 0.15, 0.40, 0.28 mol, respectively)] and 0.86 +/- 0.05 mol of inositol per mol of N-terminal Asp. Gas chromatography-mass spectroscopy demonstrated principally myo-inositol but also variable amounts of the chiro-isomer. Nondenaturing polyacrylamide gel electrophoresis of 14C-radiomethylated E protein revealed that pretreatment with hydroxylamine, a reagent which removes ester-linked lipids, rendered it PI-PLC susceptible. In contrast, parallel analyses of 35S-cys-labeled PI-PLC-sensitive HeLa DAF protein revealed only minor amounts of the hydroxylamine-sensitive phospholipid species. Similar results were obtained with 125I-surface-labeled DAF from polymorphonuclear cells, as well as from unstimulated peripheral blood and anti-CD3-activated lymphocytes. These findings demonstrate that, rather than PI, the E DAF anchor contains an inositol alkylacylglycerol-phospholipid which is heterogeneous with respect to acyl groups and inositol isomers, that an ester-linked substitution in this inositolphospholipid underlies the resistance of E DAF protein to PI-PLC cleavage/release, and that this structural modification is cell-specific.     

Study of a hydrophobic site on bovine alpha-lactalbumin by labeling with [125I]-TID

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) labels apo-bovine alpha-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml-1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease the labeling of apo-alpha-lactalbumin with [125I]TID.     

Identifying the lipid-protein interface of the alpha4beta2 neuronal nicotinic acetylcholine receptor: hydrophobic photolabeling studies with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

Using an acetylcholine-derivatized affinity column, we have purified human alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChRs) from a stably transfected HEK-293 cell line. Both the quantity and the quality of the purified receptor are suitable for applying biochemical methods to directly study the structure of the alpha4beta2 nAChR. In this first study, the lipid-protein interface of purified and lipid-reconstituted alpha4beta2 nAChRs was directly examined using photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID photoincorporated into both alpha4 and beta2 subunits, and for each subunit the labeling was initially mapped to fragments containing the M4 and M1-M3 transmembrane segments. For both the alpha4 and beta2 subunits, approximately 60% of the total labeling was localized within fragments that contain the M4 segment, which suggests that the M4 segment has the greatest exposure to lipid. Within M4 segments, [125I]TID labeled homologous amino acids alpha4-Cys582/beta2-Cys445, which are also homologous to the [125I]TID-labeled residues alpha1-Cys418 and beta1-Cys447 in the lipid-exposed face of Torpedo nAChR alpha1M4 and beta1M4, respectively. Within the alpha4M1 segment, [125I]TID labeled residues Cys226 and Cys231, which correspond to the [125I]TID-labeled residues Cys222 and Phe227 at the lipid-exposed face of the Torpedo alpha1M1 segment. In beta2M1, [125I]TID labeled beta2-Cys220, which is homologous to alpha4-Cys226. We conclude from these studies that the alpha4beta2 nAChR can be purified from stably transfected HEK-293 cells in sufficient quantity and purity for structural studies and that the lipid-protein interfaces of the neuronal alpha4beta2 nAChR and the Torpedo nAChR display a high degree of structural homology.     

Isolation and characterization of acetylcholinesterase from Drosophila

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.     

GSH-dependent photolabeling of multidrug resistance protein MRP1 (ABCC1) by [125I]LY475776. Evidence of a major binding site in the COOH-proximal membrane spanning domain

Substrates transported by the 190-kDa multidrug resistance protein 1 (MRP1) (ABCC1) include endogenous organic anions such as the cysteinyl leukotriene C(4). In addition, MRP1 confers resistance against various anticancer drugs by reducing intracellular accumulation by co-export of drug with reduced GSH. We have examined the properties of LY475776, an intrinsically photoactivable MRP1-specific tricyclic isoxazole modulator that inhibits leukotriene C(4) transport by this protein in a GSH-dependent manner. We show that [125I]LY475776 photolabeling of MRP1 requires GSH but is also supported by several non-reducing GSH derivatives and peptide analogs. Limited proteolysis revealed that [(125)I]LY475776 labeling was confined to the 75-kDa COOH-proximal half of MRP1. More extensive proteolysis generated two major 125I-labeled fragments of approximately 56 and approximately 41 kDa, and immunoblotting with regionally directed antibodies showed that these fragments correspond to amino acids approximately 1045-1531 and approximately 1150-1531, respectively. However, an approximately 33-kDa COOH-terminal immunoreactive fragment was not labeled, inferring that the major [125I]LY475776-labeling site resides approximately between amino acids 1150-1250. This region encompasses transmembrane (TM) segments 16 and 17 at the COOH-proximal end of the third membrane spanning domain of the protein. [125I]LY475776 labeling of mutant MRP1 molecules with substitutions of Trp(1246) in TM17 were reduced >80% compared with wild-type MRP1, confirming that TM17 is important for LY475776 binding. Finally, vanadate-induced trapping of ADP inhibited [125I]LY475776 labeling, suggesting that ATP hydrolysis causes a conformational change in MRP1 that reduces the affinity of the protein for this inhibitor.     

Photoaffinity probes for the alpha 1-adrenergic receptor and the calcium channel bind to a common domain in P-glycoprotein

P-glycoprotein is a 130-180-kDa integral membrane protein that is overproduced in multidrug-resistant cells. The protein appears to act as an energy-dependent drug efflux pump that has broad specificity for structurally diverse hydrophobic antitumor drugs. Many agents, such as the calcium channel blocker verapamil, reverse multidrug resistance and also interact with P-glycoprotein. The goal of this work was to determine if a common binding site participates in the transport of antitumor drugs and/or the reversal of drug resistance. This was done by comparing the peptide maps of P-glycoprotein (encoded by mdr1b) after it was labeled with a photoactive calcium channel blocker, [3H]azidopine, and a newly identified photoaffinity analog for P-glycoprotein 2-[4-(4-azido-3-[125I]iodobenzoyl) piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline [( 125I]iodoaryl azidoprazosin). [125I] Iodoaryl azidoprazosin, which classically has been used to identify the alpha 1-adrenergic receptor, bound to P-glycoprotein and was preferentially competed by vinblastine greater than actinomycin D greater than doxorubicin greater than colchicine. Peptide maps derived from P-glycoprotein labeled with [3H]azidopine or [125I]iodoaryl azidoprazosin were identical. After maximal digestion under conditions for Cleveland mapping, a single major 6-kDa fragment was obtained after digestion with V8 protease, whereas two major fragments, 6.5 and 5.5 kDa, were detected after digestion with chymotrypsin. The 6.0-kDa V8 fragment and the 6.5-kDa chymotrypsin fragment were both found when P-glycoprotein encoded by mdr1a and mdr1b was compared. Despite its specific interaction with P-glycoprotein, neither iodoaryl azidoprazosin nor prazosin markedly reversed resistance compared with verapamil or azidopine. Further, multidrug-resistant cells were 900-fold resistant to vinblastine but only 5-fold resistant to prazosin. These data demonstrate that structurally diverse reversal and/or antitumor agents are likely to have differential affinity for a small common domain of P-glycoprotein.     

Effects of lipids and detergents on the conformation of the nicotinic acetylcholine receptor from Torpedo californica.

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to characterize the effects of lipids and detergents on acetylcholine receptor (AChR) conformation. Affinity purified AChR reconstituted into dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidic acid (DOPA), and cholesterol showed the same pattern of [125I]TID-labeling and demonstrated the same reduction in labeling of all four subunits upon desensitization by the agonist carbamylcholine, as partially purified AChR in native lipids. On the basis of the patterns of [125I]TID incorporation, reconstitution into DOPC/DOPA also appeared to stabilize the resting (functional) conformation of the AChR, while reconstitution in DOPC/cholesterol or DOPC alone largely desensitized the AChR. The effects of lipids on the functional state of the AChR was determined independently by measuring the ability of AChR reconstituted into different lipid combinations to undergo the change in affinity for agonist diagnostic of desensitization. The dramatic reduction in the apparent levels of [125I]TID associated with the subunits of the AChR observed upon agonist-induced desensitization was shown not to be due to a change in affinity for tightly bound lipid. Solubilization of affinity purified AChR reconstituted into DOPC/DOPA/cholesterol by the non-ionic detergents octyl glucoside, Triton X-100, and Tween 20 (final detergent concentration = 1%) was shown to produce the same pattern of [125I]TID-labeling as desensitization by agonist, while solubilization in 1% sodium cholate appeared to stabilize a conformation of the AChR more similar to the resting state.     

Examining the noncompetitive antagonist-binding site in the ion channel of the nicotinic acetylcholine receptor in the resting state

3-Trifluoromethyl-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) has been shown to be a potent noncompetitive antagonist (NCA) of the nicotinic acetylcholine receptor (AChR). Amino acids that contribute to the binding site for [(125)I]TID in the ion channel have been identified in both the resting and desensitized state of the AChR (White, B.H., and Cohen, J.B. (1992) J. Biol. Chem. 267, 15770-15783). To characterize further the structure of the NCA-binding site in the resting state channel, we have employed structural analogs of TID. The TID analogs were assessed by the following: 1) their ability to inhibit [(125)I]TID photoincorporation into the resting state channel; 2) the pattern, agonist sensitivity, and NCA inhibition of [(125)I]TID analog photoincorporation into AChR subunits. The addition of a primary alcohol group to TID has no demonstrable effect on the interaction of the compound with the resting state channel. However, conversion of the alcohol function to acetate, isobutyl acetate (TIDBIBA), or to trimethyl acetate leads to rightward shifts in the concentration-response curves for inhibition of [(125)I]TID photoincorporation into the AChR channel and a progressive reduction in the agonist sensitivity of [(125)I]TID analog photoincorporation into AChR subunits. Inhibition of [(125)I]TID analog photoincorporation by NCAs (e.g. tetracaine) as well as identification of the sites of [(125)I]TIDBIBA photoincorporation in the deltaM2 segment indicate a common binding locus for each TID analog. We conclude that relatively small additions to TID progressively reduce its ability to interact with the NCA site in the resting state channel. A model of the NCA site and resting state channel is presented.     

Hydrophobic photolabeling identifies BHA2 as the subunit mediating the interaction of bromelain-solubilized influenza virus hemagglutinin with liposomes at low pH

To investigate the molecular basis of the low-pH-mediated interaction of the bromelain-solubilized ectodomain of influenza virus hemagglutinin (BHA) with membranes, we have photolabeled BHA in the presence of liposomes with the two carbene-generating, membrane-directed reagents 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) and a new analogue of a phospholipid, 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl)diazirinyl]phenyl][2-3H] undecanoyl]-sn-glycero-3-phosphocholine ([3H]-PTPC/11). With the latter reagent, BHA was labeled in a strictly pH-dependent manner, i.e., at pH 5 only, whereas with [125I]TID, labeling was seen also at pH 7. In all experiments, the label was selectively incorporated into the BHA2 polypeptide, demonstrating that the interaction of BHA with membranes is mediated through this subunit, possibly via its hydrophobic N-terminal segment. Similar experiments with a number of other water-soluble proteins (ovalbumin, carbonic anhydrase, alpha-lactalbumin, trypsin, and soybean trypsin inhibitor) indicate that the ability to interact with liposomes at low pH is not a property specific for BHA but is observed with other, perhaps most, proteins.     

Domain mapping of the photoaffinity drug-binding sites in P-glycoprotein encoded by mouse mdr1b.

P-glycoprotein is an energy-dependent drug efflux pump with broad specificity for hydrophobic antitumor agents such as vinblastine, doxorubicin, and taxol. We have previously shown that [3H]azidopine and [125I] iodoaryl azidoprazosin, which are photoaffinity probes for the alpha 1-subunit of the L-type calcium channel and alpha 1-adrenergic receptor, respectively, specifically interact with P-glycoprotein, partially reverse multidrug resistance, and bind to a 6-kDa common domain in the 140-kDa P-glycoprotein molecule (Greenberger, L., Yang, C.-P. H., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401). An immunological approach was used to identify the position of photoaffinity drug-binding domains in P-glycoprotein. Analysis was done with a series of site-specific rabbit polyclonal antibodies to peptides that mimic domains in the mouse mdr1b gene product. The antibodies were made against amino acid residues 269-284, 356-373, 665-682, 740-750, 907-924, and 1203-1222. Upon trypsin digestion, cleavage products of 95 and 55 kDa were obtained, which after further digestion migrated at 60 and 40 kDa, respectively. The 40-kDa fragment was recognized by the antibodies to residues 1203-1222 and 919-1276, while the 55-kDa fragment was recognized by these antibodies plus antibodies to residues 740-750 and 907-924. In contrast, the 95- and 60-kDa trypsin fragments were recognized only by the antibody to residues 269-284. The 55- and 40-kDa fragments, as well as the 95- and 60-kDa fragments, were major photolabeled species after digestion of P-glycoprotein. The previously identified 6-kDa photo-labeled P-glycoprotein fragment was within the 40-kDa trypsin fragment. These data suggest that there are two photoaffinity drug-binding domains in P-glycoprotein encoded by mouse mdr1b. The C-terminal site most likely resides within or in close proximity to putative transmembrane domains 11-12.     

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.