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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↗

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↗

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↗

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↗

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↗

PubMed1991

Inhibition of interleukin-2 production in the human T cell line JURKAT by nonpolar maleimides.

The immunosuppressive properties of polar and nonpolar maleimides were studied by measuring their ability to inhibit mitogen-induced interleukin-2 (IL-2) production by JURKAT T cells. The nonpolar maleimides N-ethylmaleimide (NEM) and N-phenylmaleimide (NPM) inhibited IL-2 production by 85-99%, but only when added to JURKAT cells prior to the mitogen. The polar maleimides N-hydroxymaleimide (NHM) and 4-maleimidosalicylic acid (M84) did not suppress IL-2 production significantly, even though NHM reacted with more cellular thiols (12%) than did NPM (8%). Both NEM and NPM suppressed IL-2 production at doses that did not affect proliferation. NEM inhibited IL-2 production induced by PHA, anti-CD3 (alpha CD3) monoclonal antibodies or PMA, and A23187, but did not interfere with the binding of alpha CD3 to the cells. NEM inhibited IL-2 production at concentrations that did not interfere with the PHA-induced increase in intracellular free calcium [( Ca]i). Neither NPM nor NHM inhibited the rise in [Ca]i, even at the highest concentrations tested. Although JURKAT T cells require both PMA and A23187 to induce IL-2 production, we found that cells pretreated with PMA could respond to A23187 added 18 hr later. PMA-treated cells were not resistant to the immunosuppressive effects of NEM or NPM. However, PMA-pretreated cells became resistant to the inhibitory effects of NEM upon the addition of A23187, suggesting that nonpolar maleimides inhibit activation events induced by the rise in [Ca]i.

Antibodies, Monoclonal↗

PubMed1990

Saturation transfer electron spin resonance of Ca2(+)-ATPase covalently spin-labeled with beta-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels.

The Ca2(+)-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide beta-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the alpha-carbon, to enhance further the electrophilic character of the beta-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2(+)-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the linewidths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer lineheight ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the lineheight ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the lineheight ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2(+)-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.

Animals↗

PubMed1977

Inhibition of bleomycin-induced [3H] thymidine 5'-triphosphate incorporation into liver and hepatoma nuclei by N-ethyl maleimide and daunomycin.

The addition of bleomycin to a nuclear incorporating system results in an increased incorporation of 3H-thymidine 5'-triphosphate (3H-TTP) into the DNA of liver and hepatoma nuclei. Bleomycin added to the nuclear incorporating system also produces scissions of DNA as determined by sucrose density gradient centrifugation of the extracted DNA. The action of bleomycin is dependent on the presence of sulfhydryl agents in the incubation mixture. Two compounds, N-ethyl maleimide and daunomycin, inhibit the bleomycin-induced incorporation of 3H-TTP preferentially. N-Ethyl maleimide inhibits bleomycin-induced activity in liver and hepatoma 7777 nuclei equally. Lower levels of daunomycin inhibit the bleomycin-induced activity in the hepatoma 7777 nuclei than are required to inhibit the activity in liver nuclei. The two compounds inhibit the bleomycin effect by different mechanisms. The addition of N-ethyl maleimide to bleomycin in the incubation system prevents bleomycin from causing breaks in the DNA. The addition of daunomycin, despite inhibition of bleomycin-induced 3H-TTP incorporation, does not affect the bleomycin-produced breaks in the DNA. N-Ethyl maleimide acts by binding to the DNA and by competing with a sulfhydryl agent for bleomycin-sensitive sites on the DNA. Daunomycin apparently inhibits a repair enzyme that is responsible for the increased incorporation following bleomycin treatment.

Animals↗

PubMedDate not supplied

Comparison of immunohistochemical staining of a mitochondrial protein, lipoamide dehydrogenase, with Fab'-peroxidase conjugates prepared by maleimide or periodate.

IgG-maleimide peroxidase, Fab'-maleimide peroxidase, polymer and monomer types of Fab'-periodate peroxidase were prepared from an antibody against rat lipoamide dehydrogenase, a component of the pyruvate dehydrogenase complex which is located in mitochondria. They were examined for immunohistochemical staining of the rat kidney. Fab'-maleimide peroxidase was the best for staining mitochondrial protein. IgG-maleimide peroxidase and the monomer type of Fab'-periodate peroxidase had the same intensity of staining. The polymer type of Fab'-periodate peroxidase could not stain the lipoamide dehydrogenase.

Animals↗

PubMed1979

Chemical modification of lactose repressor protein using N-substituted maleimides.

Lactose repressor protein has been modified with N-ethylmaleimide, two N-maleimide spin labels, and an N-maleimide fluorophore. The reaction with repressor cysteine residues has been characterized. Approximately 2 of the 3 eq of cysteine/repressor monomer are reactive toward these reagents. Repressor cysteines are reactive toward these reagents in the order cysteine 140 greater than or equal to cysteine 107 greater than cysteine 281. The reaction is sulfhydryl-specific. Comparison of chemical modification data obtained in this laboratory using a variety of sulfhydryl-specific reagents has been used to assess chemical features of individual cysteine environments. Effects of the maleimide reagents on biological activity have been determined. Only the fluorophore N-(3-pyrene)maleimide has significant effect; this agent selectively perturbs repressor's ability to bind to operator DNA. This result suggests that regions of protein structure surrounding 1 or more of the cysteine residues possess determinants required for normal operator DNA binding.

Cysteine↗

PubMed1978

New protein reagents. Synthesis and properties of halogenated maleimides and isomaleimides.

The title compounds were investigated as examples of potential hetero-bifunctional protein reagents having an electrophilic moiety in addition to a maleimide or isomaleimide ring. The synthesis of such products by cyclization of the corresponding maleamic acids gives lower yields in the maleimide than in the isomaleimide series, especially when the second function is labile, owing to the more drastic reaction conditions necessary to form the symmetric isomer. On the other hand, a comparison of the isomeric imides showed that despite some differences in reactivity, isomaleimides can be used instead of maleimides for chemical modification of proteins. Therefore, if the synthesis of new maleimide reagents containing a second fragile functional group is difficult, they can be designed in the isoimide form.

Cyclization↗

PubMed1976

Impermeant maleimides. Identification of an exofacial component of the human erythrocyte hexose transport mechanism.

The facilitated diffusion of D-glucose across human erythrocyte membranes requires an exofacial (outer surface) sulfhydryl group which can be alkylated by the impermeant reagents glutathione-maleimide and dextran-maleimide. The irreversible inhibition produced by these reagents is asymmetric; inhibition of glucose efflux considerably exceeds that of influx when transport is assayed in the absence of glucose on the opposite side of the membrane. Both D-glucose and cytochalasin B protect the exofacial transport site from alkylation by the impermeant maleimides. This masking effect provides the basis for a two-step procedure for differential labeling of the outer transport site with radioactive glutathione-maleimide. The method labels clearly and consistently a component of the membrane proteins which migrates in sodium dodecyl sulfate-polyacrylamide gels between Coomassie brilliant blue-stained Bands 4.2 and 5, corresponding to an apparent molecular weight of 65,000 to 70,000. Transport studies after inhibition with N-ethylmaleimide suggest that the hexose mechanism also requires a second sulfhydryl group which is not accessible at the cell surface.

Binding Sites↗