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At least 127 records · Page 7Linked to original sources
PubMed1978

Fluorescence energy transfer between epsilon-ATP at the nucleotide binding site and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide at Cys-373 of G-actin.

The method of fluorescence energy transfer is used to measure the distance from the nucleotide binding site to Cys-373 of G-actin. The fluorescent ATP analogue 1-N6-ethenoadenosine 5'-triphosphate was used as donor and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide was used as acceptor. From the measurements of the efficiency of fluorescence energy transfer by both static and time resolved fluorometries, the distance between nucleotide binding site and Cys-373 residue of G-actin was calculated to be about 30 A.

Actins↗
PubMed1990

Interaction of the catalytic subunits of protein phosphatase-1 and 2A with inhibitor-1 and 2: a fluorescent study with sulfhydryl-specific pyrene maleimide.

The catalytic subunits of protein phosphatase-1 and 2A were covalently modified in their reactive sulfhydryl groups with N-(3-Pyrene) maleimide resulting in fluorescent labeling of the proteins to an extent of 0.85 and 0.9 mole dye/mole enzyme, respectively. The reaction of the sulfhydryl group led to the partial inactivation of both phosphatase-1 and 2A. Inhibitor-1 and inhibitor-2 increased markedly the fluorescence intensity of the dye-phosphatase-1 conjugate implying that the labeled enzyme retained its ability to bind these proteins. In contrast, inhibitor-1 or inhibitor-2 had no influence on the fluorescence of the dye-phosphatase-2A conjugate. No change in either the fluorescence intensity or polarization of labeled phosphatase-1 and 2A was observed in the presence of thiophosphorylase a, suggesting a lack of interaction of these enzyme forms with the substrate after modification of the reactive sulfhydryl group.

Carrier Proteins↗
PubMed1983

Effect of maleimide derivatives, sulfhydryl reagents, on stimulation of neutrophil superoxide anion generation with concanavalin A.

Using maleimide derivatives with relatively rare side reactions, their effect on stimulation of O-2 production by guinea-pig neutrophils with Con A was studied. Showdomycin, a very slowly penetrating agent, did not affect O-2 production whereas NEM, a rapidly penetrating agent, markedly inhibited O-2 production without interference with binding of Con A to cells. Particulate fractions from neutrophils stimulated with Con A showed markedly increased NADPH-dependent O-2 production compared with fractions from unstimulated cells. Treatment of neutrophils with NEM before exposure to Con A inhibited the enhancement of NADPH-dependent O-2 production of particulate fractions by Con A. However, particulate fractions from Con A-stimulated and unstimulated cells hardly exhibited reduced NADPH oxidase activity after direct exposure to NEM. Treatment of neutrophils with NEM after activation by Con A had no effect on NADPH-dependent O-2 production of particulate fractions. These results indicate that NEM inhibits the activation process of the O-2-generating enzyme, probably NADPH oxidase with Con A.

Animals↗
PubMed1986

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent.

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.

Animals↗
PubMed1996

N-(2-ferrocene-ethyl)maleimide: a new electroactive sulphydryl-specific reagent for cysteine-containing peptides and proteins.

We report the synthesis and application of a specific electroactive label, N-(2-ferrocene-ethyl)maleimide, which provides new redox properties to organic compounds and proteins possessing sulphydryl groups. Its reaction conditions with the cysteine-containing peptide, glutathione, and a terminal monooxygenase enzyme, cytochrome P450cam are presented. The labelled peptide and enzyme acquired reversible electrochemical properties due to the attached ferrocene moiety.

Bacillus cereus↗
PubMed1983

N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM): an alternative label for fluorescence tracing.

N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM), a fluorescent thiol reagent, has a high molar extinction coefficient and a high quantum yield when it reacts with protein sulphhydryl bonds. DACM proved to be effective as a fluorescent probe when used for labelling rabbit immunoglobulin G anti-human gamma-chain (IgG). DACM-labelled rabbit immunoglobulin G anti-human gamma-chain conjugates (DACM-IgG) can be purified efficiently by Sephadex G-50 gel chromatography. The immunological properties of the IgG are not altered appreciably by conjugation. As a label DACM has advantages with regard to ease of preparation, specific immunofluorescence and strong photoresistance.

Animals↗
PubMed1991

Determination of urinary Tamm-Horsfall protein by ELISA using a maleimide method for enzyme-antibody conjugation.

A method for enzyme-antibody conjugation using a new maleimide derivative as coupling reagent has been developed. Since a monomeric conjugate of horseradish peroxidase and Fab' antibody could be readily prepared with high efficiency and reproducibility, the enzyme activity and antigen-binding activity were well preserved and nonspecific staining was greatly reduced. The conjugate is suitable for use in both ELISA procedures and immunohistochemistry. Using both methods we examined the pathophysiological significance of Tamm-Horsfall protein (THP) and the present study describes the ELISA method to quantify urinary THP using the new method with rabbit anti-THP antibody. A low concentration (0.04 M) of urea added to the urine samples increased the linearity of the standard curve and the sensitivity of the assay, permitting the detection of as little as 20 ng/ml THP. Freezing and thawing the urine resulted in variable or lower values of THP concentration. THP concentrations in urine as determined by ELISA were stable for at least one month after -70 degrees C storage, but not after -30 degrees C storage. There was no correlation between THP concentrations in 24 h urine samples and the morning urine of the same patient. These results suggest that it is essential to use fresh or -70 degrees C stored 24 h urine samples with added urea (0.04 M) for the determination of THP concentrations in urine by the present enzyme-antibody conjugation method. The THP concentration in normal 24 h urine of young children was found to be less than 51.8 mg/g Cr.

Adult↗
PubMed1993

Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei.

The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CBH I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.

Cellulose 1,4-beta-Cellobiosidase↗
PubMedDate not supplied

Use of rabbit Fab'-peroxidase conjugates prepared by the maleimide method for detecting plant viruses by ELISA.

In contrast to antibodies conjugated to enzyme with glutaraldehyde or by the periodate method, monomeric Fab' fragments conjugated to enzyme by means of a maleimide compound are not adversely affected by the conjugation procedure. We used such Fab'-enzyme conjugates prepared with antibody to tobacco mosaic virus (TMV), to TMV coat protein and to rabbit IgG for the detection of different tobamoviruses by direct and indirect double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Compared to conjugates prepared by other methods, the sensitivity of TMV detection with Fab'-enzyme conjugates by direct DAS-ELISA was markedly increased. However, because of their monomeric nature, these FAb'-enzyme conjugates did not cross-react with serologically related tobamoviruses. Anti-globulin Fab'-enzyme conjugate was found to be the most efficient anti-globulin conjugate for detecting TMV by indirect DAS-ELISA. Because of their high sensitivity and serotype specificity, FAb'-enzyme conjugates are useful for detecting low amounts of contaminating viruses present in crude viral preparations.

Animals↗
PubMed1988

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence.

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.

Animals↗
PubMed1993

Photoreversible change in the conformation of phytochrome as probed with a covalently bound fluorescent sulfhydryl reagent, N-(9-acridinyl)maleimide.

A fluorescent SH reagent, N-(9-acridinyl)maleimide (NAM), was used as a fluorescent probe to detect changes in the conformation of phytochrome from pea (Pisum sativum) seedlings during photoconversion between the red-light absorbing form (Pr) and the far-red-light absorbing form (Pfr). NAM-Cys conjugates emitted weaker fluorescence in non-polar solvent than in polar solvent. The fluorescence intensity (FI) of NAM-phytochrome conjugates depended on the absorbing form of phytochrome: the FI of NAM-Pr was greater than that of NAM-Pfr, indicating that the Cys residues modified by NAM were in more hydrophobic environment in Pfr than in Pr. The FI of the conjugate prepared from a red-light-irradiated sample of phytochrome was greater than that for the conjugate prepared from Pr, indicating that more Cys residues were modifiable in Pfr than Pr. The fluorescence polarization of the conjugate (0.0251) indicates that the modified Cys residues may be located at the surface of the phytochrome molecule. The FI of phytochrome conjugates with 8-anilinonaphthalene-1-sulfonate (ANS) did not change with the photoconversion of phytochrome. The FI of a mixture of ANS and phytochrome increased upon the first photoconversion of Pr to Pfr. However, it did not change upon subsequent photoconversion between Pr and Pfr. These results suggest that the initial increase in FI may have resulted from the binding of additional ANS to Pfr and that the microenvironment of bound ANS may not be influenced by the photoconversion of phytochrome.

Fabaceae↗
PubMed1991

Influence of N-(o-methoxyphenyl)-maleimide on 5-fluorouracil cytotoxicity in Ehrlich (ascites) carcinoma.

N-(o-methoxyphenyl)-maleimide (I), an intermediate obtained during the synthesis of pyrrolidinedione-N-mustards, did not exhibit antitumor activity against Ehrlich (ascites) carcinoma. The effect of co-administration of (I), with established anticancer drugs was studied against P388 leukemia, S180 (ascites) and Ehrlich (ascites) carcinoma. A significant potentiation in the activity of 5-Fluorouracil (5-FU) against Ehrlich (ascites) carcinoma by (I) was observed. The possible mechanisms responsible for potentiation of the activity of 5-FU are presented.

Animals↗
PubMed1987

High-performance liquid chromatography-fluorometry for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl) phenyl]-maleimide.

The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.

Animals↗
PubMed1994

Increased SS bonds in chronic solar elastosis: a study with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM) stain.

We have studied the distribution of SH groups and SS linkages in solar elastosis, in comparison with that in aged and juvenile sun-unexposed skin, using N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM) stain. In heavy solar elastosis we found increased fluorescence for SS bonds in the upper and middle reticular dermis in the elastotic masses; these were separated from the epidermis by a network of actinically-damaged, SS-positive elastic fibers, showing irregularly and variously interwoven oxytalan and elaunic fibers. These latter, unlike the elastotic masses, revealed also SH groups. In moderate and weak elastosis, where the thick irregular masses were absent, the fluorescence for SH and SS linkages was present in equal proportions in the altered elastic fibers; collagen bundles revealed only fluorescence for SS bonds. Aged and juvenile sun-unexposed skin showed the same proportions of SH and SS linkages in the changed elastic fibers. Collagen bundles of aged and juvenile skin showed fluorescence for SS bonds; SH groups were present on a few collagen fibers only in aged skin. Our results are consistent with opinion supporting an elastic origin of elastotic degeneration, which could be the consequence of a defect, due to chronic solar exposure, in one of the steps--degradation or synthesis--of metabolic turnover of elastic fibers, resulting in abnormal and excessive polypeptide chains extremely rich in disulfide bonds.

Adolescent↗
PubMed2003

A novel cold-adapted imidase from fish Oreochromis niloticus that catalyzes hydrolysis of maleimide.

In this paper we report the first comparative study of cold-adapted imidase (EC 3.5.2.2) from the fish (Oreochromis niloticus) liver and its thermophilic counterparts taken from pig liver and Escherichia coli (overexpressed recombinant hydantoinase from Agrobacterium radiobacter NRRL B1). Approximately 6000-fold purification and a 40% yield of fish imidase activity were obtained through ammonium sulfate precipitation, octyl, chelating, DEAE, and hydroxyapatite chromatography. This cold-adapted imidase was characterized by a specific activity 10- to a 100-fold higher than those of its thermophilic counterparts below room temperature (25 degrees C or lower) conditions but less stable at elevated temperatures (40 degrees C or higher). A less organized helical structure (compared to those of pig liver and bacterial imidases) was observed by circular dichroism. Furthermore, maleimide was first identified as a novel substrate of all imidases examined, and confirmed by HPLC and NMR analysis. These results constituted a first study to discover a novel cold-adapted imidase with surprising high activity. These findings might be also helpful for industrial application of imidase.

Adaptation, Physiological↗
PubMed2006

Synthesis, SAR studies, and pharmacological evaluation of 3-anilino-4-(3-indolyl) maleimides with conformationally restricted structure as orally bioavailable PKCbeta-selective inhibitors.

Conformationally restricted 3-anilino-4-(3-indolyl)maleimide derivatives were designed and synthesized aiming at discovery of novel protein kinase Cbeta (PKCbeta)-selective inhibitors possessing oral bioavailability. Among them, compounds having a fused five-membered ring at the indole 1,2-position inhibited PKCbeta2 with IC50 of nM-order and showed good oral bioavailability. One of the most potent compounds was found to be PKCbeta-selective over other 6 isozymes and exhibited ameliorative effects in a rat diabetic retinopathy model via oral route.

Administration, Oral↗
PubMed2006

Synthesis and evaluation of peptidic maleimides as transglutaminase inhibitors.

A series of novel transglutaminase inhibitors was prepared, based on the scaffold of a commonly used peptide substrate and bearing an electrophilic maleimide group. These compounds were evaluated in vitro and shown to lead to irreversible inactivation of tissue transglutaminase. Comparison with inhibitors studied previously provides insight into the steric environment of the enzyme active site.

Animals↗
PubMed2006

High performance liquid chromatography analysis of 2-mercaptoethylamine (cysteamine) in biological samples by derivatization with N-(1-pyrenyl) maleimide (NPM) using fluorescence detection.

2-Mercaptoethylamine (cysteamine) is an aminothiol compound used as a drug for the treatment of cystinosis, an autosomal recessive lysosomal storage disorder. Because of cysteamine's important role in clinical settings, its analysis by sensitive techniques has become pivotal. Unfortunately, the available methods are either complex or labor intensive. Therefore, we have developed a new rapid, sensitive, and simple method for determining cysteamine in biological samples (brain, kidney, liver, and plasma), using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent and reversed-phase high performance liquid chromatography (HPLC) with a fluorescence detection method (lambda(ex)=330 nm, lambda(em)=376 nm). The mobile phase was acetonitrile and water (70:30) with acetic acid and o-phosphoric acid (1 mL/L). The calibration curve for cysteamine in serine borate buffer (SBB) was found to be linear over a range of 0-1200 nM (r(2)=0.9993), and in plasma and liver matrix, the r(2) values were 0.9968 and 0.9965, respectively. The coefficients of the variation for the within-run and between-run precisions ranged from 0.68 to 9.90% and 0.63 to 4.17%, respectively. The percentage of relative recovery ranged from 94.1 to 98.6%.

Animals↗