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At least 199 records · Page 11Linked to original sources
PubMed1990

Photoaffinity labelling of the beta-methoxyacrylate binding site in bovine heart mitochondrial cytochrome bc1 complex.

A carbene-generating 14C-labelled beta-methoxyacrylate derivative ((E)-methyl-3-methoxy-2-[4-(3-trifluoromethyl-3-diazirinyl) benzoyloxyphenyl] propanoate, uniformly labelled with 14C in the benzene ring of the benzoyl group) has been used to locate the proteins involved in binding this class of inhibitors to bovine heart mitochondrial ubihydroquinone:cytochrome c oxidoreductase. The beta-methoxyacrylate photoaffinity label was shown to be a competent inhibitor of electron transport through the protein complex. Under illumination through a narrow bandpass filter, allowing specific photolysis of the diazirine group, the compound bound to cytochrome b and weakly to an 8 kDa polypeptide. Apart from some binding to a cytochrome b aggregate, other proteins were left unlabelled. The binding could be prevented in the presence of excess amounts of unlabelled beta-methoxyacrylate, myxothiazol or stigmatellin but not by antimycin A or HQNO. At high concentrations DBMIB partially competed for the binding site. The binding site for this class of inhibitors is therefore the 'o-site'. Our results indicate that this site is comprised of residues from cytochrome b and possibly the 8 kDa polypeptide and that the site may be close to the Reiske iron-sulphur protein.

Acrylates↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1983

Labeling of intramembrane segments of the alpha-subunit and beta-subunit of pure membrane-bound (Na+ + K+)-ATPase with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine.

The photoactivatable carbene precursor 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ( [125I]TID) was tested as a probe for labeling lipid-embedded segments of the proteins of pure membrane bound (Na+ + K+)-ATPase. The probe labeled the alpha-subunit (100 kDa), its major tryptic and chymotryptic fragments of 78 kDa, 58 kDa, and 46 kDa, and the beta-subunit (38 kDa) from within the lipid bilayer to nearly the same specific activity. The labeling was resistant to extensive proteolysis and the distribution of label among the proteolytic fragments and the two subunits was independent of a 47-fold variation in concentration of [125I]TID. The data show that several transmembrane segments are distributed along the sequence of the alpha-subunit and that also the beta-subunit traverses the bilayer. [125I]TID provides a more uniform labeling of the transmembrane segments of the alpha-subunit and beta-subunit than that obtained with other hydrophobic reagents. This will facilitate further studies of the primary structure and folding pattern of the Na+,K+-pump proteins in the membrane.

Animals↗
PubMed1983

A carbene-generating photoaffinity probe for beta-adrenergic receptors.

A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.

Affinity Labels↗
PubMed1986

The intramembranous domains of lipophilin in phosphatidylcholine vesicles are similar to those in the myelin membrane.

A membrane-permeable photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (125I-TID) has been used to label lipophilin in normal human myelin and after incorporation of purified lipophilin into phosphatidylcholine (PC) vesicles. The labelled protein was isolated and the specific activities for lipophilin from myelin and from PC vesicles was found to be 1.2 X 10(11) and 1.5 X 10(11) cpm/mol, respectively. The chromatographic profiles of tryptic peptides were similar in both cases and the specific activities of the C-terminal intramembranous fragments (residues 205-268) the same. We concluded that the organization of lipophilin in PC vesicles was similar to its organization in myelin and that the PC-vesicle system represents a good system in which to study the orientation and interaction of lipophilin with lipids.

Azirines↗
PubMed1988

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine.

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.

Animals↗
PubMed1989

Interaction of alpha-lactalbumin with phospholipid vesicles as studied by photoactivated hydrophobic labeling.

The alpha-lactalbumin segment which penetrates into phosphatidylserine/phosphatidylethanolamine vesicle bilayer under acidic condition was photoactively labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which had been partitioned into the hydrophobic interior of the bilayer. The hydrophobically labeled amino acid residues were identified by trypsin digestion of the alpha-lactalbumin/vesicle complex, extraction and Edman degradation of the membrane embedded fragment. The results are consistent with a notion that the segment exists in the membrane as an alpha-helix and that only one surface of this alpha-helix is exposed to the hydrophobic interior of the bilayer. Possible models are: (a) a loop of tightly held alpha-helix penetrating deep into the bilayer and (b) the helix being located on the interface between bilayer and the aqueous solution. The time-dependent [125I]TID labeling process revealed that the middle part of this segment goes into the bilayer first and is then followed by both ends. The penetration rate is comparable to that of the fusion of the lipid vesicles of the same composition by alpha-lactalbumin at the same pH, which further supports that the penetration is the cause of fusion.

Amino Acid Sequence↗
PubMed1991

Planar bilayer membranes from photoactivable phospholipids.

Planar bilayer membranes formed from photoactivable phospholipids have been characterized by low frequency voltametry. Cyclic voltametric measurements were applied for simultaneous registration of planar membrane conductivity and capacitance. The procedure has been utilized to characterize the formation and stability of planar bilayer membranes. Bilayer membranes were formed from N'-(1,2-dimyristoyl-sn-glycero-3-phosphoethyl)-N-((m-3- trifluoromethyldiazirine)phenyl)thiourea (C14-PED), a head-group photosensitive phospholipid. In situ photoactivation of C14-PED at wavelengths greater than or equal to 320 nm altered neither the mean conductivity nor the capacitance of the bilayer. Ionophore (valinomycin) and ion channel (gramicidin) activities were not impaired upon photoactivation. In contrast, bilayer membranes formed from 1,2-bis(hexadeca-2,4-dienoyl)-sn- glycero-3-phosphocholine (C16-DENPC) revealed short life times. In situ photopolymerization of the diene fatty acids significantly increased the membrane conductivity or led to membrane rupture.

Azirines↗
PubMed1994

The effects of drugs on the incorporation of a conformationally-sensitive, hydrophobic probe into the ion channel of the nicotinic acetylcholine receptor.

The pattern of incorporation of the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) into the nicotinic acetylcholine receptor (AChR) is a sensitive measure of AChR conformation (resting state or desensitized). We determined the ability of tetracaine, dibucaine, procaine, lidocaine, chlorpromazine or phencyclidine to inhibit [125I]TID photolabeling of the AChR as a function of drug concentration, both as a measure of the ability of these drugs to desensitize the AChR, and to characterize the [125I]TID binding site. To localize the site(s) of drug action, experiments were performed in the absence and presence of saturating concentrations of alpha-bungarotoxin (BgTx), to block drug binding to the agonist binding site. On the basis of the concentration dependence of their effects, which was not altered by the presence of BgTx, tetracaine and dibucaine appeared to block [125I]TID incorporation competitively, suggesting that the high-affinity [125I]TID binding site is the non-competitive blocker binding site presumed to exist in the interior of the AChR ion channel. Procaine, chlorpromazine, lidocaine and phencyclidine blocked [125I]TID incorporation at lower concentrations in the absence of BgTx than in its presence, suggesting that these drugs block incorporation by inducing desensitization when bound to their high-affinity non-competitive blocker binding sites and that BgTx countered the drug effect by allosterically stabilizing the resting state.

Anesthetics, Local↗
PubMed1995

Snake venom toxins, unlike smaller antagonists, appear to stabilize a resting state conformation of the nicotinic acetylcholine receptor.

Previous studies have shown that the pattern and degree of 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) photoincorporation into the nicotinic acetylcholine receptor (nAChR) can be used as a sensitive measure of nAChR conformation. Upon desensitization by prolonged exposure to agonists, certain drugs and detergents, or reconstitution into desensitizing lipids, the levels of [125I]TID incorporation into the subunits of the nAChR are dramatically reduced. In this study, we characterized the effects of the snake venom proteins alpha-bungarotoxin and alpha-cobrotoxin, as well as the smaller antagonists tubocurarine and gallamine, on [125I]TID incorporation into the subunits of both partially-purified nAChR in native lipids, or affinity-purified nAChR reconstituted into different combinations of lipids. Unlike all other compounds previously tested, alpha-bungarotoxin and alpha-cobrotoxin reproducibly increased the level of [125I]TID incorporation into all four subunits of nAChR reconstituted into dioleoylphosphatidylcholine, dioleoylphosphatidic acid and cholesterol. Gallamine had little or no effect on [125I]TID incorporation at any concentration tested (0.1 microM-5 mM). Tubocurarine had no effect on [125I]TID incorporation at low concentrations, but at higher concentrations reduced the level of [125I]TID labeling. The snake venom proteins may shift the population of nAChR, which exists as a mixture of resting state and desensitized conformations, entirely to the resting state. However, the binding of the snake venom toxins does not appear sufficient to induce the resting state conformation in nAChR which have been desensitized by other means, such as solubilization in desensitizing detergents or reconstitution in densitizing lipids.

Affinity Labels↗
PubMed1977

Bovine serum albumin as a catalyst. VI. Specificity of several nucleophilic groups in the protein for N-dansylaziridine.

The reaction of N-dansylaziridine with serum albumin (both bovine and human) results in incorporation of about 3 mol of covalently bound dansyl label per mol protein. This indicates that a number of nucleophilic groups in these proteins (in addition to the free sulfhydryl group) will react with this reagent. The reaction has been studied in detail for bovine serum albumin and the results suggest that one of the sites labelled by the reagent may be at the unusual "catalytic site" responsible for the enzyme-like activity of bovine serum albumin recently described (Taylor, R.P., Chau, V., Bryner, C. and Berga, S. (1975) J. Am. Chem. Soc. 97, 1934-1942). The reaction of N-dansylaziridine with a variety of other proteins indicates a pattern of labelling consistent with high specificity for the sulfhydryl group. The explanation for the unexpected excess reactivity of albumin with the "sulfhydryl specific" reagent N-dansylaziridine must be related to the three-dimensional structure in albumin which enables a number of specific residues to manifest unusually high degrees of nucleophilic reactivity.

Aziridines↗