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At least 19 recordsLinked to original sources
PubMed1979

A fluorescence stopped-flow study on troponin labeled with N-ethyl maleimide and N-(p-(2-benzimidazolyl)phenyl) maleimide.

The kinetics of the conformational change of the troponin-C (TN-C) subunit in N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM)-N-ethyl maleimide (NEM)-labeled troponin induced by calcium binding or removal were studied with the fluorescence stopped-flow method. The kinetic process of the conformational change was biphasic, the rate constants of the two phases were determined as a function of the free calcium ion concentration of the protein solution. The kinetic behaviour of the conformational change of TN-C in BIPM-NEM-labeled troponin was explained by a simple molecular kinetic mechanism: (Formula: see text) This molecular kinetic mechanism is different from that of the isolated TN-C which we found in the previous work (1). That is, formation of a complex of TN-C with troponin-I (TN-I) and troponin-T (TN-T) modifies the molecular kinetic mechanism of the conformational change of TN-C.

Animals↗
PubMed1982

Reversed-phase ion-pair high-performance liquid chromatography of mercaptoacetate and N-acetylcysteine after derivatization with N-(1-pyrene)maleimide and N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide.

We have developed a high-performance liquid chromatographic system capable of resolving mercaptoacetate and N-acetylcysteine as their N-(1-pyrene)maleimide (PM) and N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide (DACM) derivatives. Good resolution was obtained by ion pairing with tetramethylammonium hydroxide and chromatography on reversed phase. The detection limits for the thiols were about 50 fmol as their DACM derivatives and about 400 fmol as their PM derivatives. The method is illustrated by chromatography of urinary thiols which indicates that the derivatization and chromatography procedures should be well applicable in bioanalytical work.

Acetylcysteine↗
PubMed1996

Design, synthesis, and biochemical evaluation of N-substituted maleimides as inhibitors of prostaglandin endoperoxide synthases.

N-(Carboxyalkyl)maleimides are rapid as well as time-dependent inhibitors of prostaglandin endoperoxide synthase (PGHS). The corresponding N-alkylmaleimides were only time-dependent inactivators of PGHS, suggesting that the carboxylate is critical for rapid inhibition. Several N-substituted maleimide analogs containing structural features similar to those of the nonsteroidal anti-inflammatory drug aspirin were synthesized and evaluated as inhibitors of PGHS. Most of the aspirin-like maleimides inactivated the cyclooxygenase activity of purified ovine PGHS-1 in a time- and concentration-dependent manner similar to that of aspirin. The peroxidase activity of PGHS was also inactivated by the maleimide analogs. The cyclooxygenase activity of the inducible isozyme, i.e., PGHS-2, was also inhibited by these compounds. The corresponding succinimide analog of N-5-maleimido-2-acetoxy-1-benzoic acid did not inhibit either enzyme activity, suggesting that inactivation was due to covalent modification of the protein. The mechanism of inhibition of PGHS-1 by N-(carboxyheptyl)maleimide was investigated. Incubation of apoPGHS-1 with 2 equiv of N-(carboxyheptyl)[3,4-14C]maleimide led to the incorporation of radioactivity in the protein, but no adduct was detected by reversed-phase HPLC, suggesting that it was unstable to the chromatographic conditions. Furthermore, hematin-reconstituted PGHS-1, which was rapidly inhibited by N-(carboxyheptyl)maleimide, displayed spontaneous regeneration of about 50% of the cyclooxygenase and peroxidase activities, suggesting that the adduct responsible for the inhibition breaks down to regenerate active enzyme. ApoPGHS-1, inhibited by N-(carboxyheptyl)maleimide, did not display regeneration of enzyme activity, but addition of hematin to the inhibited apoenzyme led to spontaneous recovery of about 50% of cyclooxygenase activity. These results suggest that addition of heme leads to a conformational change in the protein which increases the susceptibility of the adduct toward hydrolytic cleavage. ApoPGHS-1, pretreated with N-(carboxyheptyl)maleimide, was resistant to trypsin cleavage, suggesting that the carboxylate functionality of the maleimide binds in the cyclooxygenase channel. A model for the interaction of N-(carboxyheptyl)maleimide in the cyclooxygenase active site is proposed.

Animals↗
PubMed1991

Frequency-domain fluorescence spectroscopy resolves the location of maleimide-directed spectroscopic probes within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum.

We have used fluorescence spectroscopy to characterize three covalently bound spectroscopic maleimide derivatives with respect to their location within the tertiary structure of the Ca-ATPase of sarcoplasmic reticulum (SR). These derivatives include (1) 2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid, (2) 4-(dimethylamino)azobenzene-4'-maleimide, and (3) fluorescein 5'-maleimide. Biochemical assays demonstrate that modification with any of these three derivatives results in the same functional effects, observed following derivatization of cysteines 344 and 364 by N-ethylmaleimide [Saito-Nakatsuka et al. (1987) J. Biochem. (Tokyo) 101, 365-376]. These residues bracket the ATPase's phosphorylation site (Asp 351) and thus may provide spectroscopic probes of the protein's conformation in this essential region. In agreement with sequencing results, SDS-polyacrylamide gels show that maleimide-modified SR exhibits fluorescence exclusively on the A1 tryptic fragment of the Ca-ATPase. Extensive tryptic digestion followed by centrifugation demonstrates essentially all of the fluorescence was associated with the soluble rather than insoluble (membrane-associated) peptides, confirming the predicted extramembranous location of these residues. Utilizing frequency-domain fluorescence spectroscopy, we were able to recover the transient effects associated with a distribution of donor-acceptor distances. We find from these fluorescence resonance energy transfer measurements that covalently bound maleimide probes are 36 A apart, independent of whether a discrete distance is assumed or a distance distribution model is utilized, in which the conformational variability of the protein is taken into account. While a unimodal distance distribution is adequate to describe the intensity decay associated with maleimide-directed donor-acceptor pairs, a bimodal distribution of distances is necessary to describe the frequency response associated with the energy transfer between maleimide-directed chromophores and other covalently bound probes on the Ca-ATPase, consistent with the large spatial separation observed between maleimides. We recover mean distances of 42 and 77 A between maleimide sites and bound FITC (Lys 515) and mean distances of 28 and 37 A between the maleimide- and the iodoacetamide-directed probes (Cys 670 and 674, whose close proximity approximates a single locus). The measured distances are presented in a model and have permitted us to describe a unique arrangement of these covalently bound probes within both the secondary and tertiary structure of the Ca-ATPase. The resolution inherent in the frequency-domain fluorescence technique to multiple donor-acceptor distances should be generally applicable to a wide range of biological systems in which specific labeling of single unique donor-acceptor sites is not feasible.

Amino Acid Sequence↗
PubMed1995

N-ethyl maleimide stimulates arachidonic acid release through activation of the signal-responsive phospholipase A2 in endothelial cells.

Treatment of bovine endothelial cells with the alkylator N-ethyl maleimide results in arachidonic acid mobilization. N-ethyl maleimide-stimulated arachidonic acid release was dose and time dependent and maximum release was achieved after 10-15 min with 50 microM N-ethyl maleimide, N-ethyl maleimide-stimulated arachidonic acid release could be prevented by pretreating the cells with the phospholipase A2 inhibitor quinacrine. Based on the finding that N-ethyl maleimide was not able to release oleic acid from oleic acid-preloaded cells, it was clear that the effect of N-ethyl maleimide was limited to an arachidonic acid-specific phospholipase. The effect of N-ethyl maleimide does not appear to be dependent on calcium, as shown by the observation that N-ethyl maleimide was not able to increase intracellular calcium concentration in FURA2-loaded cells. Pretreatment of the cells with staurosporine totally inhibited N-ethyl maleimide-stimulated arachidonic acid liberation. The tyrosine kinase inhibitor genistein was also able to significantly inhibit arachidonic acid release. It is concluded from the results obtained in this study that N-ethyl maleimide stimulates arachidonic acid release by stimulating the activity of a specific, signal-responsive phospholipase A2. Furthermore this activation is not mediated by intracellular calcium fluxes but by a stimulation of intracellular kinase activity which eventually leads to the activation of this signal-responsive phospholipase A2.

Animals↗
PubMed1976

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent.

N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins.

Binding Sites↗
PubMed1992

Kinetic and inhibition studies on catechol-O-methyltransferase affinity labelling by N-(3,4-dihydroxyphenyl)maleimide.

Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies.

Affinity Labels↗
PubMed1987

Probing of sulfhydryl groups in the adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier by maleimide spin-labels.

Binding of spin-labeled maleimides to the mitochondrial ADP/ATP carrier was investigated both in mitochondria and in the detergent-solubilized carrier protein. In mitochondria, spin-label binding to the carrier was evaluated by preincubation with the inhibitor carboxyatractyloside. The membrane sidedness of SH groups in the carrier molecule was determined by chemical reduction of nitroxides on the cytosolic membrane surface by Fe2+ or by pretreatment of the mitochondria with impermeant SH reagents. These experiments suggest that each subunit of the dimeric carrier incorporates one spin-labeled maleimide. Roughly half of the carrier-bound spin-labels were found on either side of the mitochondrial membrane. The detergent-solubilized carrier protein was labeled with a series of maleimide derivatives containing a spacer of increasing length between the maleimide and nitroxide moieties. A total spin-label binding of 2-3 mol/mol of protein dimer, depending on the spin-label length, was found. The electron spin resonance spectra of the spin-labeled protein invariably showed strongly and weakly immobilized components. Increasing the distance of the nitroxide from the maleimide ring resulted in a strong increase of the contribution of the weakly immobilized component. These observations led to the conclusions that the geometrical constraint of spin-label mobility changes at a distance of about 10 A from the maleimide binding site.

Animals↗
PubMed1988

Inhibition of glucocorticoid receptor transformation, subunit dissociation, and temperature-dependent inactivation by various N-substituted maleimides.

A series of N-substituted maleimides were synthesized, and their effect on the activation to the DNA binding state of the rat liver glucocorticoid receptor was studied. Unactivated (preincubated at 0 degrees C) cytosolic [3H]triamcinolone acetonide-receptor complexes were pretreated with various N-alkylmaleimides at 0 degrees C and then heated at 25 degrees C and assayed for DNA-cellulose binding. No inhibition of the DNA binding activity was observed with either N-ethylmaleimide or N-substituted maleimides bearing an ionizable substituent, like N-(omega-carboxyalkyl)maleimides and N-[2-(trimethylammonio) ethyl]maleimide. On the contrary, treatment with long-chain alkylmaleimides like N-heptylmaleimide resulted in significant inhibition. The highest inhibition was obtained with N-benzylmaleimide and, to a lesser extent, N-(ethylphenyl)-maleimide, whereas N-benzylsuccinimide was ineffective. Treatment of cytosol containing unactivated glucocorticoid complexes at 3 degrees C with N-benzymaleimide also prevents the temperature-mediated conversion of 8S receptor to 4S. Moreover, N-benzylmaleimide was able to inhibit the inactivation of the receptor steroid-binding activity caused by heat. N-Benzylmaleimide shares with molybdate ions the ability to inhibit glucocorticoid receptor activation, dissociation, and inactivation. However, their respective mechanisms of action are probably distinct, since their effects on receptor inactivation appear additive. It is suggested from the comparison of the various maleimides tested that the sulfhydryl groups essential for receptor activation and dissociation lie in a rather nonpolar environment including aromatic amino acid(s).

Animals↗
PubMed1988

Inhibition of hexose transport and labelling of the hexose carrier in human erythrocytes by an impermeant maleimide derivative of maltose.

Maltose-maleimide was synthesized as a potential affinity label for the facilitative hexose carrier with selectivity for exofacial sulphydryl groups. This reagent, although probably a mixture of isomers, did not significantly penetrate the plasma membrane of human erythrocytes at concentrations below 5 mM at 37 degrees C. When allowed to react to completion, it irreversibly inhibited the uptake of 3-O-methylglucose, with a half-maximal response at about 1.5-2.0 mM-reagent. The rate of transport inactivation was a saturable function of the maltose-maleimide concentration. Studies of reaction kinetics and effects of known transport inhibitors demonstrated that irreversible reaction occurred on the exofacial outward-facing carrier, although not at a site involved in substrate binding. Reaction of intact erythrocytes with [14C]maltose-maleimide resulted in labelling of a broad band 4.5 protein of Mr (average) 45,000-66,000 in electrophoretic gels. This protein was very likely the hexose carrier, since its labelling was inhibited by cytochalasin B. Exofacial band 4.5 labelling was stoichiometric with respect to transport inhibition, yielding an estimated 300,000 carriers/cell. These results suggest that the exofacial sulphydryl which reacts with maltose-maleimide is distinct from the substrate binding site on the hexose carrier, but that it confers substantial labelling selectivity to impermeant maleimides. Additionally, the high efficiency of carrier labelling obtained with maltose-maleimide is useful in quantifying numbers of carriers in whole cells.

Biological Transport↗
PubMed1976

Impermeant maleimides. Oriented probes of erythrocyte membrane proteins.

Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.

Blood Protein Electrophoresis↗
PubMed1985

Adenine nucleotide and phosphate transport systems of mitochondria. Relative location of sulfhydryl groups based on the use of the novel fluorescent probe eosin-5-maleimide.

Eosin-5-maleimide is impermeable to the inner mitochondrial membrane, exhibiting essentially no reactivity with matrix glutathione or with beta-hydroxybutyrate dehydrogenase located on the matrix surface of the inner membrane. In intact mitochondria, eosin-5-maleimide is unreactive with the ADP/ATP antiporter even under conditions which promote maximal labeling by N-[3H]ethylmaleimide (i.e., ADP present). However, eosin-5-maleimide readily labels the ADP/ATP antiporter in "inverted" inner membrane vesicles even in the presence of N-ethylmaleimide. Labeling is prevented if the vesicles are prepared from mitochondria pretreated with carboxyatractyloside. In contrast to the ADP/ATP antiporter, essential sulfhydryl groups of the Pi/H+ symporter are accessible to eosin-5-maleimide in intact mitochondria with optimal inhibition of phosphate transport being observed at 25 degrees C. Eosin-5-maleimide also prevents labeling of the Pi/H+ symporter by N-[3H]ethylmaleimide. These results show that essential sulfhydryl groups of the ADP/ATP antiporter and the Pi/H+ symporter have differing reactivities and locations in functionally intact mitochondria. With respect to eosin-5-maleimide, sulfhydryl groups of the ADP/ATP carrier occur in two distinct classes, both of which are inaccessible in intact mitochondria. Only one class, depending on conditions, can be exposed in submitochondrial particles. In contrast, sulfhydryl group(s) of the Pi/H+ symporter behave as a single reactive class which is readily accessible in mitochondria at 25 degrees C.

Animals↗
PubMed1980

Demonstration of sulfhydryl and disulfide groups by a fluorescent maleimide procedure.

Several fluorescent maleimide compounds were evaluated as possible substitutes for N-(4-aminophenyl)maleimide in the histochemical procedures developed by Sippel (1973, 1978a, b, 1980) for the demonstration of sulfhydryl and disulfide groups. The brightest and most selective fluorescence was obtained by using N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), although both eosin-5-maleimide and fluorescein-5-maleimide could also be used if adequate control preparations were made.

Animals↗
PubMedDate not supplied

Introduction of the maleimide function onto resin-bound peptides: a simple, high-yield process useful for discriminating among several lysines.

Incorporation of epsilon-Adpoc-lysine as a residue in solid phase peptide synthesis allows selective deprotection of this residue on the resin-bound peptide relative to other acid labile groups such as Boc. Premature resin cleavage is avoided. A maleimide group, a useful thiol-capture reagent, was readily introduced by reacting the liberated amino function with an acylating agent containing the maleimide functionality. Acidic cleavage from the resin, with an appropriate scavenging system, afforded peptides that are derivatized with a maleimide functionality on a specific lysine. This is advantageous for producing peptide-carrier conjugates of defined specificity, useful as immunogens, by maleimide-thiol coupling. The derivatization and resin removal chemistries appear to proceed in excellent yield with respect to the maleimide group. The structures were confirmed by tandem mass spectrometry.

Gonadotropin-Releasing Hormone↗
PubMed1995

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.

Adenosine Triphosphate↗
PubMed1976

Reaction of N-(3-pyrene)maleimide with thiol groups of reticulocyte ribosomes.

The reaction of N-(3-pyrene)maleimide with thiol groups of rabbit reticulocyte ribosomes offers a possible fluorescent probe for studying ribosomal structure and conformation. At relatively low concentrations of N-(3-pyrene)maleimide a group of 30-40 readily reactive sulfhydryl residues is derivatized. The major ribosomal proteins containing these thiol groups are identified as S2 + S3, S5, S7, S8, S29, S31, S32, L1, L5, L6, L10 + L14, L15, L18 + L19, and L36. Ribosomal activity, as measured by the nonenzymic binding of phenylalanyl-tRNA and polyphenylalanine synthesis, is inhibited by this degree of reaction with N-(3-pyrene)maleimide. The inhibition is relieved by the prior binding of polyuridylic acid to the ribosomes while the extent of derivatization by N-(3-pyrene)-maleimide is diminished only slightly. The average relative polarization of the fluorescence of the ribosomal bound N-(3-pyrene)maleimide changes significantly with the degree of derivatization of ribosomal thiol groups or with the binding of polyuridylic acid, indicating the value of such a fluorescent thiol-derivatizing agent as a probe of ribosomal structure.

Animals↗
PubMed1994

Prejunctional actions of N-ethyl-maleimide and phenoxybenzamine in rat vas deferens.

In studies of electrically evoked isometric contractions of rat vas deferens, N-ethyl-maleimide (30 microM) pretreatment significantly reduced the prejunctional inhibitory potencies of xylazine and 5-hydroxytryptamine but failed to affect the potency of the alpha 1-adrenoceptor agonist amidephrine. Phenoxybenzamine (1 microM) or N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (10 microM) produced significant shifts in the potency of xylazine and significantly reduced the maximum inhibition, but the combination of phenoxybenzamine or EEDQ and N-ethyl-maleimide (30 microM) produced no further alteration in the effects of xylazine. In displacement studies, N-ethyl-maleimide displaced the binding of [3H]MK 912 ((2S,12bS)1',3'-dimethylspiro- (1,3,4,5',6,6',7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinazoline)- 2,4'- pyrimidin-2'one) to rat renal cortex membranes with a Ki of 466 +/- 133 microM (n = 5), and so does not bind to alpha 2-adrenoceptors in the concentration range in which it affects prejunctional receptor mediated responses. This may suggest that N-ethyl-maleimide has actions other than inactivation of G-proteins or that the irreversible alpha 2-adrenoceptor antagonists phenoxybenzamine and EEDQ inactivate G-proteins sensitive to N-ethyl-maleimide in concentrations at which they bind to alpha 2-adrenoceptors.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed1988

Inhibition and labelling of the mitochondrial 2-oxoglutarate carrier by eosin-5-maleimide.

Unlike hydrophobic maleimides, eosin-5-maleimide and to a lesser extent other relatively polar maleimides inhibit the 2-oxoglutarate carrier of bovine heart mitochondria. The impermeable eosin-5-maleimide labels the 2-oxoglutarate carrier in intact mitochondria but not in submitochondrial particles. 2-Oxoglutarate protects the carrier against inactivation by eosin-5-maleimide and decreases the fluorescence associated with the purified protein. Other anions which are not substrates of the carrier have no protective effect. It is concluded that sulfhydryl groups essential for the activity of the 2-oxoglutarate carrier are located at the cytosolic face of the inner mitochondrial membrane. They appear to be present at the substrate-binding site and located in a hydrophilic environment.

Biological Transport↗