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PubMed1994

Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications.

To identify amino acid residues of the Torpedo nicotinic acetylcholine receptor (AchR) interacting with membrane lipid, we have used the photoactivatable, hydrophobic probe 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine([125I]TID). The pattern of [125I]TID incorporation into the M3 and M4 hydrophobic segments of each subunit was the same both in the presence and absence of the agonist carbamoylcholine and in the presence of an excess of nonradioactive TID, consistent with nonspecific photoincorporation from the lipid-protein interface. [125I]TID reacted with five residues in alpha-M4 [Blanton, M.P., & Cohen, J. B. (1992) Biochemistry 31, 3738-3750] but with only two or three residues in M4 segments of beta-, gamma-, and delta-subunits. In delta-M3, [125I]TID reacted with Met-293, Ser-297, Gly-301, Val-304, and Asn-305 as well as with Ile-288 preceding M3. Residues at corresponding positions were labeled in beta-M3 (Met-285, Ile-289, Phe-293) and in gamma-M3 (Phe-292, Leu-296, Met-299, and Asn-300) as well as gamma-Ile-283. Within alpha-M3, Phe-284 and Ser-287 were labeled. The periodicity of labeled residues provides the first direct evidence that M3 as well as M4 segments of each subunit are organized as transmembrane alpha-helices each with substantial contact with lipid. In addition, in alpha-M1 [125I]TID reacted nonspecifically with Cys-222, Leu-223, Phe-227, and Leu-228, a pattern of incorporation inconsistent with the labeling pattern expected either for a "face" of an alpha-helix or a beta-sheet.

Amino Acid Sequence↗
PubMed2001

Site of resting state inhibition of the nicotinic acetylcholine receptor by a hydrophobic inhibitor.

The lipophilic photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine (TID) is a noncompetitive, resting-state inhibitor of the nicotinic acetylcholine receptor (nAChR) that requires tens of milliseconds of preincubation to inhibit agonist-induced cation efflux. At equilibrium, [(125)I]TID photoincorporates into both the ion channel and the lipid-protein interface of the Torpedo nAChR. To determine which of these regions is responsible for resting-state inhibition, we characterized the interactions between [(125)I]TID and nAChR-rich membranes milliseconds after mixing, by use of time-resolved photolabeling. Photolabeling was performed after preincubation times of 2 ms or 600 s (equilibrium), and the efficiencies of incorporation at specific residues were determined by amino-terminal sequence analysis of nAChR-subunit proteolytic fragments isolated by SDS-PAGE and/or reversed-phase HPLC. Equilibration of TID with lipid was complete within a millisecond as determined by both stopped-flow fluorescence quenching of diphenylhexatriene in lipid bilayers and photoincorporation into nAChR-rich membrane phospholipids. Equilibration with the lipid-protein interface (alphaM4) was slightly slower, reaching approximately 50% that at equilibrium after 2 ms preincubation. In contrast, equilibration with the channel region (alpha 2 and deltaM2) was much slower, reaching only 10% that at equilibrium after 2 ms preincubation. Within the ion channel, the ratio of [(125)I]TID incorporation between M2 residues 9', 13', and 16' was independent of preincubation time. We conclude that TID's access to the ion channel is more restricted than to the lipid-protein interface and that TID bound within the ion channel is responsible for flux inhibition upon activation of the nAChR.

Amino Acid Sequence↗
PubMed1994

A hydrophobic inhibitor of the nicotinic acetylcholine receptor acts on the resting state.

3-(Trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) has recently been found to be a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAcChoR) by both photolabeling and flux assays (White et al., 1991). However, these experiments were done when TID was in equilibrium with the nAcChoR, and thus only its interactions with the resting and the desensitized states of the nAcChoR were studied. In this work we characterized the interaction between TID and nAcChoR in the open and resting states using a flux assay. When TID and acetylcholine were simultaneously mixed with the nAcChoR in native Torpedo vesicles, TID did not inhibit agonist-induced 86Rb+ flux. However, following prolonged preincubation (4 min) of TID with nAcChoR, complete inhibition was observed with a half-inhibition constant of 0.4 microM TID and a Hill coefficient of 0.9. This suggested that TID might act either on the resting or the desensitized state in preference to the open state of the nAcChoR. Preincubation of nAcChoR with TID, followed by a 7 ms agonist-induced flux assay, showed that the flux response declined exponentially with preincubation time. Assuming a pseudo-first-order process, analysis revealed the rate constant for the onset of inhibition of nAcChoR in the resting state to be in the range of (1.2-3.4) x 10(6) M-1 s-1. To test if fast desensitization was enhanced by TID under these conditions, we used a fluorescent analog of acetylcholine. Stopped-flow fluorimetry showed that the fraction of nAcChoR in the predesensitized state did not increase during preincubation with TID.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1982

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium.

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Hydrophobic labeling of a single leaflet of the human erythrocyte membrane.

The photoactivatable phospholipid 1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[9-3H] -8-oxadecanoyl]-sn-glycero-3-phosphocholine [( 3H]PTPC) was synthesized with high specific radioactivity. When a sonicated dispersion of [3H]PTPC was incubated with human erythrocyte membranes (ghosts), the radiolabel was inserted spontaneously into the erythrocyte membrane. Photo-cross-linking of [3H]PTPC to membrane components and subsequent analysis of the distribution of radiolabel among polypeptide fragments of glycophorin allowed conclusions concerning the transbilayer distribution of [3H]PTPC in the erythrocyte membrane. Thus, [3H]PTPC was inserted exclusively into the outer leaflet of resealed ghosts, whereas with unsealed (leaky) ghosts, the photosensitive lipid was incorporated into both halves of the membrane simultaneously. These results are incompatible with fusion of [3H]PTPC liposomes with the erythrocyte membrane being responsible for the lipid transfer observed and suggest instead that PTPC exchanges spontaneously between membranes. This property of PTPC could be related to the critical micelle concentration of this lipid [(6 +/- 2) X 10(-9) M at 23 degrees C].

Affinity Labels↗
PubMed1984

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a hydrophobic, photoreactive probe, labels calmodulin and calmodulin fragments in a Ca2+-dependent way.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means to characterize calmodulin sites that play a role in the interaction with targets.

Amino Acids↗
PubMed1984

Hydrophobic and electrostatic interactions between adrenocorticotropin-(1-24) -tetracosapeptide and lipid vesicles. Amphiphilic primary structures.

Hydrophobic photolabeling with 3-(trifluoromethyl) -3-(m-[125I]iodophenyl) diazirine ( [125I]TID [Brunner, J., & Semenza , G. (1981) Biochemistry 20, 7174-7182] ) and equilibrium dialysis were used to study hydrophobic and electrostatic interactions between three adrenocorticotropin fragments and liposomes prepared from mixtures of phosphatidylcholine with phosphatidic acid or phosphatidylserine. Corticotropin-(1-10)-decapeptide (ACTH1-10, net charge 0) formed hydrophobic clusters with [125I]TID in aqueous solutions at peptide concentrations above 1 microM but did not interact appreciably with neutral or anionic liposomes. Corticotropin -(11-24)-tetradecapeptide ( ACTH11 -24, net charge 6+) reacted electrostatically with anionic liposomes but showed no hydrophobic interactions. Corticotropin-(1-24)-tetracosapeptide (ACTH1-24, net charge 6+), a covalent combination of the two fragments, exhibited both hydrophobic and electrostatic interactions with lipid vesicles. Edman degradation and chymotryptic hydrolysis of labeled ACTH1-24 revealed that the hydrophobic interaction involved the N-terminal decapeptide "message" segment (corresponding to ACTH1-10) which entered the membrane and that the electrostatic interaction was caused by the C-terminal tetradecapeptide "address" segment (corresponding to ACTH11 -24) which remained on the aqueous membrane surface. This surface is in complete analogy to that reported for dynorphin- (1-13)-tridecapeptide by Gysin and Schwyzer [ Gysin , B., & Schwyer , R. (1983) FEBS Lett. 158, 12-16; Gysin , B., & Schwyzer , R. (1983) Arch. Biochem. Biophys. 225, 467-474]: in both cases, the specific, hydrophobic membrane interaction of the "message" critically depended on the presence of the hydrophilic "address". The results reported here were consistent with those obtained by infrared attenuated total reflection spectroscopy [ Gremlich , H.-U., Fringeli , U.-P., & Schwyzer , R. (1983) Biochemistry 22, 4257-4263] and were crucial for their interpretation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1984

Structure of the membrane-embedded F0 part of F1F0 ATP synthase from Escherichia coli as inferred from labeling with 3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) is a photoactivatable carbene precursor designed to label selectively the hydrophobic core of membranes. We have used this reagent to obtain information on the topological organization of the membrane-embedded subunits of F1F0 ATP synthase from Escherichia coli. The study included [125I]TID labeling of F0 subunits in different structural (conformational) states and Edman degradations of the labeled polypeptides in order to assign the covalently bound radioactivity to individual amino acid residues. Released phenylthiohydantoin amino acids were analyzed by thin-layer chromatography, and the radioactive derivatives were visualized by autoradiography. The data suggest that labeling patterns can be correlated in a meaningful manner with reagent accessibility and hence with protein-lipid contact. Subunit b appears to be anchored to the membrane by a short N-terminal segment. As almost all of the amino acids of this part are accessible to the reagent, it is inferred that this segment has little interaction with the other subunits. In contrast, in the two segments of subunit c that were labeled with [125I]TID, only certain amino acids reacted with the label. The pattern of these labeled residues is compatible with that of tightly packed alpha-helices.

Adenosine Triphosphatases↗
PubMed1985

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

Animals↗
PubMed1986

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase.

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.

Acetylcholinesterase↗
PubMed1986

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)

Animals↗
PubMed1987

Interaction of blood coagulation factor Va with phospholipid vesicles examined by using lipophilic photoreagents.

Two different lipophilic photoreagents, [3H]adamantane diazirine and 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID), have been utilized to examine the interactions of blood coagulation factor Va with calcium, prothrombin, factor Xa, and, in particular, phospholipid vesicles. With each of these structurally dissimilar reagents, the extent of photolabeling of factor Va was greater when the protein was bound to a membrane surface than when it was free in solution. Specifically, the covalent photoreaction with Vl, the smaller subunit of factor Va, was 2-fold higher in the presence of phosphatidylcholine/phosphatidylserine (PC/PS, 3:1) vesicles, to which factor Va binds, than in the presence of 100% PC vesicles, to which the protein does not bind. However, the magnitude of the PC/PS-dependent photolabeling was much less than has been observed previously with integral membrane proteins. It therefore appears that the binding of factor Va to the membrane surface exposes Vl to the lipid core of the bilayer, but that only a small portion of the Vl polypeptide is exposed to, or embedded in, the bilayer core. Addition of either prothrombin or active-site-blocked factor Xa to PC/PS-bound factor Va had little effect on the photolabeling of Vl with TID, but reduced substantially the covalent labeling of Vh, the larger subunit of factor Va. This indicates that prothrombin and factor Xa each cover nonpolar surfaces on Vh when the macromolecules associate on the PC/PS surface. It therefore seems likely that the formation of the prothrombinase complex involves a direct interaction between Vh and factor Xa and between Vh and prothrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adamantane↗
PubMed1988

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.

Azirines↗
PubMed1988

Mischarging Escherichia coli tRNAPhe with L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine, a photoactivatable analogue of phenylalanine.

The Boc-protected derivative of a photoactivatable, carbene-generating analogue of phenylalanine, L-4'-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenylalanine [(Tmd)Phe], was used to acylate 5'-O-phosphorylcytidylyl(3'-5')adenosine (pCpA). A diacyl species was isolated which upon successive treatments with trifluoroacetic acid and 0.01 M HCl yielded a 1:1 mixture of 2'(3')-O-(Tmd)phenylalanyl-pCpA and of its 2'-5'-phosphodiester isomeric form. Adapting a procedure introduced by Hecht's group [Heckler, T.G., Chang, L.H., Zama, Y., Naka, T., Chorghade, M.S., & Hecht, S.M. (1984) Biochemistry 23, 1468-1473], brief incubation of a 15 molar excess of this material with Escherichia coli tRNAPhe, missing at the acceptor stem the last two nucleotides (pCpA), in the presence of T4 RNA ligase and ATP afforded "chemically misaminoacylated" tRNAPhe in approximately 50% yield. Following chromatographic purification on DEAE-Sephadex A-25, benzoylated DEAE-cellulose, and Bio-Gel P-6, the misaminoacylated tRNAPhe was characterized by (i) urea-polyacrylamide gel electrophoresis, (ii) enzymatic reaminoacylation under homologous conditions following chemical deacylation, and (iii) its ability to stimulate protein synthesis in an in vitro translation system which, through the addition of the phenylalanyl-tRNA synthetase inhibitor phenylalaninyl-AMP, was unable to charge its endogenous tRNAPhe. The data demonstrate that we have prepared a biologically active misaminoacylated tRNAPhe.

Azirines↗
PubMed1988

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.

Amino Acid Sequence↗
PubMed1981

Selective labeling of the hydrophobic core of membranes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a carbene-generating reagent.

The synthesis of a new photoactivatable probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]-TID), with a high specific radioactivity (10 Ci mmol-1) is described. It was tested as a probe for the hydrophobic core of membranes. TID partitions strongly in favor of the lipid phase of membranes, and the photogenerated carbene labels intrinsic membrane proteins in a highly selective manner. This conclusion was reached from the distribution of radioactivity among the proteins of [125I]TID-labeled human erythrocyte membranes. By far the most heavily labeled protein is band 3 [nomenclature of Fairbanks, G., Steck, T. L., & Wallach, F. G. H. (1971) Biochemistry 10, 2606-2617] while the labeling of glycophorin is approximately 5 times less than that of band 3. There is little or no labeling of known extrinsic proteins.

Affinity Labels↗
PubMed1982

Modification of catalytic groups in lysozyme with ethylenimine.

The reaction of lysozyme with ethylenimine has been studied at neutral and acidic pH. At least four singly modified lysozyme derivatives have been isolated. From the product analyses, only carboxyl groups in the protein were found to react. One of the derivatives, 1, was very labile and reverted back to native lysozyme during the isolation procedure. Its formation was markedly inhibited by the presence of tri(N-acetyl-D-glucosamine), indicating that the modified carboxyl residue in 1 is at or close to the binding site of lysozyme for the trisaccharide. Two other derivatives were identified as 2-aminoethyl esters of Glu-35 (2) and Asp-52 (3). At pH 10 and room temperature, these derivatives rearranged to the corresponding 2-hydroxyethylamide derivatives (5 and 6). A fourth derivative (4) did not contain an ethanolamine moiety; nevertheless, its Glu-35 was found to be modified by sequence analysis. Lysozyme derivatives modified at Glu-35 and Asp-52 were inactive toward glycol chitin but retained high affinity for tri(N-acetyl-D-glucosamine). Thus, Glu-35 and Asp-52 are essential for enzyme activity. A mechanism for the selective modification of these carboxyl residues in lysozyme has been proposed and related to the metal binding ability of lysozyme.

Animals↗
PubMed1980

Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane.

1-[3H]Spiro[adamantane-4,4'-diazirine], a lipophilic, photoactivatable reagent designed to label those segments of intrinsic proteins that lie within the lipid bilayer of biological membranes, has been evaluated. The reagent labels the intrinsic proteins of human erythrocyte membranes far more heavily than it labels the extrinsic proteins. This result, together with a detailed analysis of the label distribution in several well-characterized membrane proteins [Goldman, D.W., Pober, J.S., White, J., & Bayley, H. (1979) Nature (London) 280, 841], demonstrates that labeling with adamantanediazirine is a convenient and rapid method both for distinguishing intrinsic from extrinsic membrane proteins and for locating within intrinsic proteins those amino acid residues that are in contact with the hydrocarbon core of the lipid bilayer.

Adamantane↗