Dimethylmaleic Anhydride Research Articles

Yeast nucleosomal particles: structural and transcriptional properties

Yeast nucleosomal core particles have been characterized by thermal denaturation, circular dichroism, and digestion with DNase I and with trypsin. Practically all nucleosomal DNA melts in one transition centered at 70 degrees C, and the circular dichroism spectrum is displaced to lower wavelengths as compared to that corresponding to chicken nucleosomal cores. The susceptibility of yeast nucleosomal particles to dissociation by salt is significantly higher than that of chicken nucleosomal cores, a substantial dissociation being observed at 0.5 M NaCl. Treatment of yeast nucleosomal particles with the amino group reagent dimethylmaleic anhydride is accompanied by selective release of histones H2A and H2B. The results indicate not large but significant structural differences between yeast and chicken nucleosomal cores. However, the in vitro transcription properties of complete and H2A.H2B-deficient nucleosomal cores are similar in the two kinds of particles: the histone octamer blocks RNA synthesis, this block being eliminated in part by the partial loss of histones H2A and H2B.

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH. In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of diassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles. Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups. The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride. Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides. With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride. Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride.

Enhanced activity of immobilized dimethylmaleic anhydride-protected poly- and monoclonal antibodies

The effect of reversible protection of the free amino groups of poly- and monoclonal antibodies by dimethylmaleic anhydride on their binding activity following immobilization onto various carriers was studied. The treatment with dimethylmaleic anhydride resulted in a 1.6–1.8-fold increase in the activity of immobilized goat anti-mouse immunoglobulin antibody immobilized onto different epoxy containing carriers and a 3–10.7-fold increase in the activity of immobilized monoclonal antibodies specific for carboxypeptidase A. The increase in activity was most pronounced at low antigen to carrier loads and over a wide range of modifier to protein ratios. The application of reversible protection of antibodies may permit the development of highly active immobilized antibody preparations for use in immunoaffinity purification.

Reconstitution of rat liver 60S ribosomal subunits following disassembly by dimethylmaleic anhydride

Modification of 60S ribosomal subunits from rat liver with dimethylmaleic anhydride (60 mumols/ml) is accompanied by release of 35% of the protein. The acidic ribosomal proteins, as well as 9 basic proteins, are selectively liberated from the ribosomal subunits. Reconstitution of the protein-deficient particles with the corresponding split proteins is accompanied by substantial recovery of the original polyphenylalanine synthetic activity. The described reconstitution procedure can be used to investigate the roles played by the released proteins and the functional similarities of proteins from different sources. Hybrid reconstitution of residual ribosomal particles from rat liver or yeast with the corresponding heterologous split proteins produces subunits which have incorporated heterologous proteins but are inactive in polyphenylalanine synthesis.

Pyrrolo[1,2-b]isoquinoline-5,10-diones and indolizine-5,8-diones

The synthesis of pyrroloisoquinolinediones from phthalic anhydride and pyrrolylmagnesium halides has been improved. A new synthesis, by cyclization of the novel 1-(2-carboxybenzoyl)pyrroles, was generally preferable. From pyrrolylmagnesium bromide and dimethylmaleic anhydride, followed by cyclization, 6,7-dimethylindolizine-5,8-dione was obtained. The process failed with maleic anhydride. Pyrrolo[1,2-b]isoquinoline-5,10-dione could be dinitrated in the pyrrolo ring (with some oxidation to isoquinoline-1,3,4-trione), reduced to 10-hydroxy-3,5-dihydropyrrolo[1,2-b]isoquinolin-5-one by zinc and acetic acid, and hydrogenated over palladium on carbon to 10-hydroxy-1,2,3,5-tetrahydropyrrolo[l,2-b]isoquinolin-5-one. The dihydro derivative reverted in air to the dione.

Dimethyl cis- and trans-1,2-Dimethylcyclohexane-1,2-dicarboxylate

1-Methoxy-3-trimethylsilyloxybuta-1,3-diene adds readily to dimethylmaleic anhydride, and the product can be converted into dimethyl cis-1,2-dimethylcyclohex-4-ene-1,2-dicarboxylate (3). The several steps required mean that this route compares unfavourably with direct addition of butadiene to the anhydride. The best route to the cis-diester (3) is cyclialkylation of the dianion from dimethyl 2,3-dimethylbutanedioate with (Z)-1,4-dichlorobut-2-ene. A bismethylation reaction is used to prepare imide (II), which can be converted into the cis-diester (1) only with considerable difficulty, and bismethylation of the dianion formed from dimethyl cis-cyclohex-4-ene-1,2- dicarboxylate gives mainly the trans-diester (8). 13C n.m.r. spectra were useful in distinguishing the isomeric pairs (1) and (2), and (3) and (8).

Disassembly and reconstitution of yeast 60S ribosomal subunits.

Yeast 60S ribosomal subunits have been dissociated by reversible modification with dimethylmaleic anhydride. Treatment with 40 mumol reagent/ml releases 35% of the protein, producing core particles inactive in polyphenylalanine synthesis, which are totally or highly deficient in 17 different proteins. This preparation of residual particles recovers 45% of the original activity upon incubation with the released proteins. The reconstituted particles can be isolated by centrifugation without loss of activity, having the protein composition of the original subunits.

Dimethylmaleic anhydride, a specific reagent for protein amino groups

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.

Reactions of cyclic anhydrides XVI: A novel approach to angular oxygenated pyrrolobenzoxazinomes

O-Carboxymaleanilic acids IIIa-g when treated with sodium acetate-acetic anhydride underwent double cyclisation leading to pyrrolobenzoxazinones Va-g carrying an angular acetate. A one-flask reaction of dimethylmaleic anhydride and phthalic anhydride with anthranilic acid furnished the angular hydroxy benzoxazinones IVh and IVi respectively, which were converted to the corresponding acetates Vh and Vi. The acetates Va, Vc, Vf, Vg and Vi underwent solvolysis to the corresponding methyl ethers (VII) on refluxing with anhydrous methanol.

Preparation and structural characterization of nucleosomal core particles lacking one H2A.H2B dimer

Nucleosomal core particles lacking one H2A.H2B dimer, (H2A.H2B)1 (H3.H4)2/DNA (146 bp), have been prepared by treatment of nucleosomal cores with dimethylmaleic anhydride, a reversible reagent for protein amino groups. The preparative procedure is simple, produces quantitative conversion of nucleosomal cores into dimer-deficient cores without formation of other subnucleosomal particles, and can be applied to the preparation of different H2A.H2B-deficient mono and oligonucleosomal particles. The structural properties of the dimer-deficient cores and complete nucleosomal cores reconstituted from the deficient particles and H2A.H2B dimers have been studied by DNase I digestion, thermal denaturation and circular dichroism.

ChemInform Abstract: Photocycloadducts of Dimethylmaleic Anhydride with Unsaturated Acids and Esters.

ChemInformVolume 19, Issue 43 Heterocyclic Compounds ChemInform Abstract: Photocycloadducts of Dimethylmaleic Anhydride with Unsaturated Acids and Esters. M. L. WINZENBURG, M. L. WINZENBURG Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorE. K. FIELDS, E. K. FIELDS Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorD. P. SINCLAIR, D. P. SINCLAIR Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorM. E. WRIGHT, M. E. WRIGHT Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorH. K. JUN. HALL, H. K. JUN. HALL Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorG. J. RAY, G. J. RAY Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this author M. L. WINZENBURG, M. L. WINZENBURG Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorE. K. FIELDS, E. K. FIELDS Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorD. P. SINCLAIR, D. P. SINCLAIR Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorM. E. WRIGHT, M. E. WRIGHT Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorH. K. JUN. HALL, H. K. JUN. HALL Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this authorG. J. RAY, G. J. RAY Res. Dev. Dep., Amoco Chem. Corp., Naperville, IL 60566, USASearch for more papers by this author First published: October 25, 1988 https://doi.org/10.1002/chin.198843174Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume19, Issue43October 25, 1988 RelatedInformation

Reconstitution of the active rat liver 60 S ribosomal subunit from different preparations of core particles and split proteins

Proteins extracted from the 60 S rat liver ribosomal subunit with 50% ethanol/0.5 M KCl produced only a partial reactivation of the corresponding core particles. In contrast, the same split proteins were able to reactivate the core particles prepared with dimethyl-maleic anhydride (DMMA) to the same level as that observed using the DMMA-split proteins, i.e. 60–80% of the control according to the catalytic activities tested. Comparative analysis of the two split protein fractions showed only four common proteins: P1–P2, which alone restored part of the activities, especially the EF-2-dependent GTPase one, and L10a, L12, which must be responsible for the additional reactivation. The poor ability of the ethanol/KCl core particles to be reactivated was shown to be probably related to a conformational alteration which destabilized the 5 S RNA-protein complex. Proteins present in the ethanol/KCl wash of Saccharomyces cerevisiae 60 S subunits were found to be partly active in subunit reconstitution using rat liver DMMA core particles.

Structural changes of nucleosomal particles and isolated core-histone octamers induced by chemical modification of lysine residues.

Treatment of nucleosomal particles and isolated core-histone octamers with dimethylmaleic anhydride, but not with acetic anhydride, is accompanied by a biphasic release of the two H2A.H2B dimers, the first dimer being more easily released than the second. With both kinds of particles, 50% of histones H2A and H2B are released for modification of approximately 35% of the histone amino groups. The similar behavior of nucleosomal particles and isolated core-histone octamers is consistent with the same structure of the histone octamer in the nucleosomal particle and in the free octamer in 2 M NaCl. The described release of H2A.H2B dimers allows the preparation of nucleosomal particles deficient in one H2A.H2B dimer and of the histone hexamers H2A.H2B.(H3.H4)2. For more extensive modifications, both reagents, acetic and dimethylmaleic anhydrides, cause the dissociation of nucleosomal particles with liberation of double-stranded DNA, which suggests that lysine amino groups are involved in the binding of histones to DNA. The modified nucleosomal particles are more sensitive to ionic strength than those untreated, and the presence of salt (NaCl) increases the extent of DNA release. The histones corresponding to the liberated DNA, except H2A and H2B released with dimethylmaleic anhydride, are apparently bound to the DNA-containing particles as extra histones.

Topography and stoichiometry of acidic phosphoproteins in rat liver 60 S ribosomal subunit

In reconstitution experiments of active 60 S subunits from inactive core particles obtained by using dimethyl maleic anhydride (DMMA), we observed that the phosphoproteins P1–P2 were extracted from the subunit by DMMA as a complex with other proteins. This complex was separated by electrophoresis and zonal centrifugation and shown, after 125I iodination of its components, to contain L22 and S12 in addition to P1–P2. Results suggest that it contains two copies of P1–P2 for one of L22 and S12.

Photocycloadducts of dimethylmaleic anhydride with unsaturated acids and esters

Cycloaddition sous UV de dihydro-2,5 dimethyl-3,4 furannedione-2,5 avec des acides et esters ethyleniques. Obtention d'oxa-3bicyclo [3.2.0] heptanediones-2,4'

Role of acidic phosphoproteins in the partial reconstitution of the active 60 S ribosomal subunit

We have recently shown that rat liver 60 S ribosomal subunits active in protein synthesis can be reconstituted from inactive core particles lacking 30% of the total proteins, mainly L10a, L12, L22, L24, A33 and the acidic phosphoproteins P1–P2, obtained by treatment of 60 S subunits with dimethylmaleic anhydride [(1987) Eur. J. Biochem. 163, 15–20]. In this study, an ethanol extract of the 60 S subunit which contains only P1–P2 was also shown to be effective in reconstitution with the DMMA-core-particies: it strongly stimulated the EF-2-dependent GTP hydrolysis and, to a lesser extent, polyphenylalanine synthesis; like the DMMA wash it shifted the thermal denaturation curve of the DMMA-core particles towards that of control subunits. Prior dephosphorylation of the ethanol extract by alkaline phosphatase inhibited the reconstruction process.

Partial reassembly of active 60S ribosomal subunits from rat liver following treatment with dimethylmaleic anhydride.

Rat liver 60S ribosomal subunits were treated with dimethylmaleic anhydride, a reagent for protein amino groups, at a 1/15,000 mol/mol ratio. This caused the dissociation of specific proteins, which were separated from the 56S residual core particles by centrifugation and identified by two-dimensional gel electrophoresis. The core particles lacking 30% of the total proteins retained most of the initial activity measured by the puromycin reaction but only small percentages of activities measured by polyphenylalanine synthesis, elongation-factor-2(EF-2)-dependent GTP hydrolysis and EF-2-mediated GDP binding. Upon reconstitution, the complementary amount of split proteins was incorporated into ribosomal particles, which had almost the same catalytic activities and biophysical properties (density, sedimentation coefficient and capability to reassociate to 40S subunits) as the original subunits.

Modification of the amino and hydroxyl groups of lysozyme with carboxylic acid anhydrides: a comparative study

Modification of hydroxyl groups by carboxylic acid anhydrides is an unwanted reaction when these reagents are used to modify protein amino groups. It is important to know the specificity of different anhydrides, and the stability of the modified hydroxyl groups. Lysozyme was treated with acetic, succinic, maleic, monomethylmaleic and dimethylmaleic anhydrides, and the extent of modification of the amino and hydroxyl groups was evaluated. Amino groups were found to be much more reactive than the hydroxyl groups. Of the reagents used, dimethylmaleic anhydride is the most specific for amino groups, with practically no modification of hydroxyamino acid residues. The modified hydroxyl groups are preferentially deacylated at alkaline pH. For acetic, succinic and maleic anhydrides, the half-lives of the modified serine (25–45 min) and threonine (14–30 h) residues were determined at pH 10.0 and 37°C. Under these conditions, the modified hydroxyl groups can be selectively deacylated without practically affecting the modified amino groups.

Modification of 40S ribosomal subunits from yeast with dimethylmaleic anhydride.

Modification of 40S ribosomal subunits from Saccharomyces cerevisiae with dimethylmaleic anhydride (DMMA), a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity and by dissociation of proteins from the particles. The protein-deficient ribosomal particles, originated from 40S subunits by treatment with dimethylmaleic anhydride at a molar ratio of reagent to particle of 250, can partially reconstitute active subunits upon addition of the corresponding released proteins, and regeneration of the modified amino groups.

Peptidyl transferase centres of rat and yeast ribosomes. Different response to modification of protein amino groups

1. 1. Modification of rat liver ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, causes a large stimulation of peptidyl transferase activity assayed by the “fragment” reaction, as well as the inactivation of poly(U)-directed polyphenylalanine synthesis. 2. 2. In contrast to rat ribosomes, the peptidyl transferase of yeast ribosomes is little affected by modification. 3. 3. Although other interpretations are not excluded, these results might be due to differences between the peptidyl transferase centres of mammalian and yeast ribosomes.