Dimethylmaleic Anhydride Research Articles

Reversible modification of 50S ribosomal subunits with dimethylmaleic anhydride: protein-deficient particles.

The reversible modification of protein amino groups with dimethylmaleic anhydride, which had already been used to dissociate proteins from the 70S ribosomes of Escherichia coli (Pintor-Toro, J. A., et al. (1979) Biochemistry 18, 3219) was applied to the preparation of protein-deficient particles from the 50S subunits. Three successive cycles of treatment with dimethylmaleic anhydride, separation of dissociated proteins and regeneration of the modified amino groups produce partially inactivated ribosomal 'cores' lacking proteins L7, L11 and L12, and having very small amounts of L1, L6 and L10. Incubation of these 'cores' with the corresponding split proteins is accompanied by complete reactivation of the polypeptide synthesizing activity as compared with control 50S subunits.

Some reactions of phenolic maleamic acids relevant to carboxypeptidase catalysis

In neutral aqueous acetonitrile, N- o -hydroxybenzyl-2,3-dimethylmaleamic acid is rapidly converted to N- o -hydroxybenzyl-2,3-dimethylmaleimide, while N-propyl-N- o -hydroxybenzyl-2,3-dimethylmaleamic acid is converted to dimethylmaleic anhydride; both reactions are catalyzed by the phenolic groups.

Dissociation of proteins from Escherichia coli ribosomes after dimethylmaleic anhydride treatment : Effects of elongation factor G and antibiotics

FEBS LettersVolume 135, Issue 1 p. 21-24 Full-length articleFree Access Dissociation of proteins from Escherichia coli ribosomes after dimethylmaleic anhydride treatment Effects of elongation factor G and antibiotics José Antonio Pintor-Toro, José Antonio Pintor-Toro Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorAbelardo López-Rivas, Abelardo López-Rivas Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorFrancisco Hernández, Francisco Hernández Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorEnrique Palacián, Enrique Palacián Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this author José Antonio Pintor-Toro, José Antonio Pintor-Toro Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorAbelardo López-Rivas, Abelardo López-Rivas Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorFrancisco Hernández, Francisco Hernández Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this authorEnrique Palacián, Enrique Palacián Instituto de Bioquímica de Macromoléculas, Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Canto Blanco, Madrid-34, SpainSearch for more papers by this author First published: November 30, 1981 https://doi.org/10.1016/0014-5793(81)80935-0Citations: 2AboutPDF 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 onFacebookTwitterLinked InRedditWechat Citing Literature Volume135, Issue1November 30, 1981Pages 21-24 ReferencesRelatedInformation

Protein-deficient ribosomal particles obtained by reversible modification with dimethylmaleic anhydride

Preparation of protein-deficient ribosomal particles from Escherichia coli ribosomes by reversible modification of protein amino groups with dimethylmaleic anhydride ( J. A. Pintor-Toro, D. Vázquez, and E. Palacián, 1979, Biochemistry 18, 3219 ) is accompanied by degradation of r-RNA and reagent-independent inactivation. Alternative conditions to regenerate the modified amino groups have been found, which reduce the time needed to prepare the ribosomal “cores” from 9 to 3 days, and prevent RNA degradation and inactivation. The ribosomal particles obtained from 70 S ribosomes and 50 S subunits by this modified procedure show no extensive degradation of RNA and very little reagent-independent inactivation, which allow good recovery of the polypeptide synthesizing activity when incubated with the corresponding split proteins.

Dissociation of the protein components from chromatin by reversible modification with dimethylmaleic anhydride.

Modification of calf thymus chromatin with the protein reagent dimethylmaleic anhydride is accompanied by dissociation of histones and non-histone proteins. Gel electrophoresis of the released proteins and of those bound to the residual chromatin showed that histone H1 is dissociated more easily than the core histones (H2A, H2B, H3 and H4). These are apparently released in the proportion in which they are present in chromatin. After regeneration of the modified amino groups by incubation at pH 6.0, the released proteins are able to bind to the residual chromatin, under two different sets of reconstitution conditions, to form nucleosome-like structures.

The pyrolytic behavior of ethylenetetracarboxylic acid

The controlled pyrolysis of ethylenetetracarboxyclic acid at 220°C followed by methylation of the pyrolyzate results in the formation of methyl propiolate as the major product along with lesser amounts of methyl fumarate. Evidence is presented which indicates that the methyl fumarate probably arises from some precursor in the pyrolyzate during the methylation reaction. Extended heating of the tetra acid at its melting point produces, in addition to the substances cited above, ethylenetetracarboxylic dianhydride and dicarboxymaleic anhydride. The high-temperature (300–400°C) pyrolysis of the tetraacid gives l0–20% yields of dimethylmaleic anhydride.

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: Comparison of reconstituted vesicles with native membrane vesicles

A Na +-specific and Na +-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na + could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K +, Li +, and choline +. The electrochemical effect of the lipophilic anion, SCN −, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl − or SO 4 2−. The Na +-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K m of 2 m m. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na +-specific and Na +-stimulated concentrative α-aminoisobutyric acid uptake.

Stereospecific d-glucose transport in mixed membrane and plasma membrane vesicles derived from cultured chick embryo fibroblasts

Mixed membrane vesicles prepared from cultured chick embryo fibroblasts possess a stereospecific d-glucose transport system, the properties of which are identical to those of the system in intact cells. Uptake of d-glucose proceeds without chemical alteration. The rate of stereospecific uptake of d-glucose into the mixed vesicles is 70% greater than that of the homogenate and uptake is directly proportional to membrane protein concentration. Stereospecific d-glucose uptake appears linear for 0.3 min, reaches a maximum at 2–5 min, and declines to zero by 5 h as l-glucose enters the vesicles. Uptake is osmotically sensitive and inhibited by cytochalasin B ( K i = 0.13 μM) and the structural analogues of d-glucose : d-mannose, 2-deoxy- d-glucose, 3- O- methyl- d- glucose , d-galactose and maltose, but not by sucrose or l-glucose. Uphill counterflow can be demonstrated and the apparent activation energy displays a transition from 47.7 kcal/mol below 11°C to 18.1 kcal/mol above 11°C. Stereo-specific uptake rates of mixed vesicles prepared from Rous sarcoma virus-transformed cells are increased 30% over control values, and are increased 66% in vesicles derived from cells incubated for 24 h in glucose-free medium. Plasma membrane vesicles prepared from these cells by a dextran cushion centrifugation procedure display a 9-fold increase in the specific activity of stereospecific d-glucose uptake relative to the homogenate. Extraction of these membranes with dimethylmaleic anhydride (5 mg/mg protein) results in substantial or complete removal of major polypeptides of molecular weight 40 000, 55 000, 75 000, 78 000 and 200 000 with no loss in total uptake activity. Following extraction, major polypeptides of molecular weight 28 000, 33 000 and 68 000 remain in the membrane residue.

ChemInform Abstract: MOLECULAR COMPLEXES OF SUPEROXIDE ION WITH MALEIC ANHYDRIDE AND ITS ANALOGS

AbstractDie γ‐Bestrahlung von Maleinsäureanhydrid in einem O2‐enthaltenen Glas aus 2‐ Methyl‐tetrahydrofuran bei 77 K und nachfolgender Erwärmung auf 98 K führt zur Bildung von 1:1‐ bzw. 1 c2‐Komplexen aus dem Anion O; und dem Anhydrid.

Studies on ketene and its derivatives. XCVII. Photoreactions of diketene with maleic anhydride and dimethylmaleic anhydride.

The photoreactions of diketene with maleic anhydride (4) and dimethylmaleic anhydride (6) gave 2-oxo-1-oxaspiro [3.3] heptane-cis-5, 6-dicarboxylic anhydride (5a, b) and 5, 6-dimethyl-2-oxo-1-oxaspiro [3.3] heptane-cis-5, 6-dicarboxylic anhydride (7a, b), respectively. Treatment of compounds 5 and 7 with hydrogen chloride in ethanol and methanol afforded the corresponding dialkyl 5-alkoxycarbonyl-3-oxoheptanedioate derivatives (8 and 10) and (9 and 11), respectively.

Production of insulinomimetic antibodies against rat adipocyte membranes by hybridoma cells.

SJL mice were injected intraperitoneally with adipocyte plasma membranes or with intrinsic membrane proteins obtained by extraction of plasma membranes with dimethylmaleic anhydride. Three days after the boost injection, the spleens were removed and fused with NS-a, a thioguanine-resistant myeloma cell line derived from P3X63 Ag8 (Balb/c). Following selection for hybrids with hypoxanthine, aminopterin, and thymidine, medium of the hybrid cells was tested for its ability to bind to the plasma membrane of the adipocyte and to stimulate the oxidation of D-(1-14C) glucose to 14CO2. Approximately 40% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with plasma membranes produced immunoglobulin that bound to adipocyte plasma membranes. About 30% of these mimicked the ability of insulin to stimulate the oxidation of D-(1-14C) glucose to 14CO2 in adipocytes. Media from 51% of the wells containing hybridomas derived from splenocytes of SJL mice immunized with intrinsic membrane proteins produced immunoglobulin that bound to the plasma membrane and 48% of those stimulated glucose oxidation. The bioactivity of the hybrid cell media could be blocked by adsorption with intrinsic membrane proteins or by the removal of immunoglobulins using formalin-fixed Staphylococcus aureus. The hybrids generated in this study can be divided into three categories: 1) hybrids that secrete antibodies that can bind to plasma membranes and mimic insulin action of glucose transport; 2) hybrids that secrete antibodies that bind to plasma membranes but do not stimulate the oxidation of D-(1-14C) glucose to 14CO2; and 3) hybrids that produce no antimembrane antibodies. The data suggest that interaction of immunoglobulins with specific membrane proteins is essential in mimicking the action of insulin on glucose transport and oxidation in the rat adipocyte.

Cycloadditionen von 3,4‐Dimethoxyfuran an Dienophile

On cycloaddition reactions with 3,4‐dimethoxyfuranWe describe new cycloadditions of 3,4‐dimethoxyfuran (3,4‐DF, 1) with various dienophiles. In contrast to furan, the reaction of 3,4‐DF with maleic anhydride gives exo‐ and endo‐adducts at approximately the same rate. The thermodynamically more stable product is again the exo‐product. Less active dienophiles lead also to mixtures of exo‐ and endo‐adducts, except for citraconic anhydride (methylmaleic anhydride) which forms only the adduct with an endo‐methyl group. Dimethylmaleic anhydride could not be reacted with 3,4‐DF.All adducts with 3,4‐DF contain a hidden 1,2‐dicarbonyl group in the form of an endiolether. Its pronounced nucleophilic character allows a series of further additions. Noteworthy are the stereospecific cis‐additions of halogens and the preparation of several acetals. Ozonolysis of the enolether in 4 allows the preparation of the hitherto unknown 2r, 3trans, 4trans, 5cis‐tetrahydrofuran tetracarboxylic acid and derivatives thereof (5 and 6).

Protein composition and structure of human placental microvillous membrane: External surface, sialic acid containing, extrinsic and intrinsic components

The microvillous membrane of human placenta is in direct contact with maternal blood and thus plays a vital role in many essential functions of the placenta. As an initial step in understanding the membrane proteins, and their relationship to these functions and to the structure of the membrane, we have investigated an isolated membrane preparation. Ten major peptide bands and an approximately equal number of minor bands were seen with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Sialoglycoproteins were labeled with periodate (PA- 3H) borohydride and external surface components with lactoperoxidase-[ 125I] (LP- 125I). One principal (69 000 mol. wt) and several minor (100 000, 45 000, and 38–40 000 mol. wt) bands were labeled as Sialoglycoproteins and found to be exposed on the surface of the membrane. Approx. 50% of the membrane protein and all of the sialic acid was tightly bound to membrane lipid and resistant to extraction with dimethyl maleic anhydride (DMMA). Electron microscopy demonstrated extraction by DMMA of microfilaments presumptively identified as actin and other electron dense components from the villous core. The extracted supernate and the residual pellet differed markedly in protein composition. The supernatant contained bands of 180 000, 115 000, 85 000, 70–72 000, 45 000, and 38–40 000 mol. wt whereas the lipid pellet contained components of 200 000, 150 000, 100 000, 69 000, and 64 000 mol. wt. The lipid matrix with which these proteins were associated contained phosphatidyl choline and sphingomyelin and was similar in composition to other plasma membranes. Thus by using a variety of experimental approaches the proteins of the human placental microvillous membrane can be divided into groups based on their sialic acid content, exposure on the external surface, tightness of binding to the membrane lipid, and relation to membrane structure.

Effects of dimethylmaleic anhydride and a polyaldehyde fixative on the fertilization of the sea urchin egg, Paracentrotus lividus

Two agents, dimethylmaleic anhydride and a polyaldehyde, capable of reacting with amino groups at the surface of the eggs of sea urchin do not prevent fertilization.

Biological properties of antibodies against rat adipocyte intrinsic membrane proteins. Dependence on multivalency for insulin-like activity.

Antisera from rabbits injected with rat adipocyte plasma membranes or intrinsic proteins from such membranes, obtained by a dimethylmaleic anhydride extraction step, mimicked the action of insulin on both glucose transport and lipolysis in intact adipocytes. Biological activity in both types of antisera was mediated by immunoglobulin binding to one or more intrinsic proteins of the adipocyte plasma membrane since fat cells were unresponsive to all antisera absorbed with dimethylmaleic anhydride-extracted membranes. Acid treatment of immunoprecipitates released antibodies which activated glucose uptake and reacted with solubilized adipocyte membranes on immunodiffusion plates. The biologically active immunoglobulin preparations failed to form immunoprecipitin lines when tested against membranes from brain, liver, lung, muscle, kidney, and spleen. Insulin-sensitive glucose uptake in rat soleus muscle did not respond to the antisera. The antibodies activated hexose uptake into fat cells and reacted with solubilized adipocyte membranes on immunodiffusion plates when rat or mouse adipocytes were studied, but not when monkey fat cells were used. The anti-membrane antibody preparations readily activated hexose uptake in trypsinized fat cells which had lost the capacity to bind or respond to insulin. These data are consistent with the concept previously proposed (Pillion, D.J., and Czech, M.P. (1978) J. Biol. Chem. 253, 3761-3764) that the anti-membrane immunoglobulins do not interact with the insulin binding site of the insulin receptor. Monovalent Fab fragments of the biologically active antisera, prepared by papain digestion of the native anti-membrane immunoglobulins, were ineffective in enhancing glucose uptake in adipocytes. However, biological activity of the anti-membrane Fab fragments was restored by the addition of goat anti-rabbit Fab antisera to cells treated with the Fab fraction. Anti-rabbit Fab antisera alone or in combination with Fab fragments prepared from control rabbit sera exhibited no biological activity. These results demonstrate that the ability of anti-membrane antisera to mimic the biological activity of insulin on isolated fat cells is critically dependent on immunoglobulin binding to one or more intrinsic plasma membrane proteins and the multivalent nature of immunoglobulin structure.

Studies on the Multi‐Enzyme Complex of Yeast Fatty‐Acid Synthetase

1. The multi-enzyme complex of fatty acid synthetase, Mr 2300,000, was dissociated by acylation with dimethyl maleic anhydride under conditions which lead to an acylation of about 30% of the epsilon amino groups of lysine. The complete dissociation into the subunits alpha and beta is demonstrated by analytical ultracentrifugation as well as disc gel electrophoresis. 2. This dissociation is reversible. Hydrolysis of the resulting protein dicarboxylic acid monoamides under mildly acidic conditions leads to the unmodified subunits, which can be reconstituted to form a complex displaying about 60% of the original activity. 3. The subunits were isolated by sucrose-density-gradient centrifugation and studied for the different partial enzyme activities involved in long-chain fatty acid synthesis: malonyl, palmitoyl and acetyl transferase, enoyl reductase and dehydratase were shown to be exclusive functions of the beta chains of the complex, confirming a pentafunctional role of this subunit.

Eine decarboxylative Dimerisierung von Maleinsäureanhydrid zu Dimethylmaleinsäureanhydrid

Decarboxylative Dimerization of maleic Anhydride to Dimethylmaleic AnhydrideWe have found a simple synthesis of dimethylmaleic anhydride (1). Treatment of maleic anhydride (2) in boiling acetic acid in presence of 2‐aminopyridine gives, with decarboxylation, 1 in a one‐pot reaction in good yield.

Sodium-stimulated active transport of aminoisobutyric acid by reconstituted vesicles from partially purified plasma membranes of mouse fibroblasts transformed by simian virus 40.

Plasma membrane fractions isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40 were partially purified by treatment with dimethylmaleic anhydride followed by extraction with 2% cholate. The extracted proteins were combined with exogenous phospholipids and eluted through a Sephadex G50 column. Reconstituted vesicles thus obtained were shown to possess the ability of Na+-stimulated transport of alpha-aminoisobutyric acid. The simultaneous addition of NaSCN and alpha-aminoisobutyric acid to these vesicles produced a transient accumulation above the equilibrium level (overshoot, active transport). The Na+-stimulated transport of alpha-aminoisobutyric acid was sensitive to the accompanying anion and to the temperature of incubation. The results demonstrate that partially purified membrane proteins of mouse fibroblast cells can be incorporated into the liposomes that have the characteristics of Na+-stimulated and electrochemically sensitive active transport of alpha-aminoisobutyric acid.

Antibodies against intrinsic adipocyte plasma membrane proteins activate D-glucose transport independent of interaction with insulin binding sites.

Plasma membrane vesicles from rat adipocytes were treated with dimethylmaleic anhydride to remove extrinsic proteins and then used to immunize rabbits. Immunodiffusion experiments performed in agarose containing Triton X-100 (0.1%) revealed a precipitin reaction between anti-membrane serum and the detergent-solubilized proteins from either intact adipocyte plasma membranes or membranes extracted with dimethylmaleic anhydride. Anti-membrane serum caused cytolysis of intact adipocytes and this effect could be eliminated by incubating the serum at 56 degrees C for 30 min to inactivate complement. Heat-inactivated anti-membrane serum caused a significant increase in 14CO2 production from D-[1-14C]glucose in intact fat cells and the partially purified immunoglobulin fraction from anti-membrane serum markedly stimulated 3-O-methylglucose transport. Maximum activation of transport occurred at a 1:5 dilution and was not additive to that achieved by a maximal dose of insulin. Under these conditions, the anti-membrane immunoglobulin fraction had no effect on the binding of 125-i-insulin to fat cells at all concentrations of the hormone tested. The data are consistent with the hypothesis that activation of hexose transport in these studies results from the binding of immunoglobulin directly to the transport system, although an indirect action is also possible.

Retention of insulin-stimulated D-glucose transport activity by adipocyte plasma membranes following extraction of extrinsic proteins.

Plasma membrane vesicles prepared from adipocytes incubated with insulin exhibited accelerated D-glucose transport activity characteristic to insulin action on intact fat cells. Both control and insulin-stimulated D-glucose transport activities were inhibited by cytochalasin B and thiol reagents. Extraction of plasma membranes with dimethylmaleic anhydride eluted 80% of the protein from plasma membrane vesicles. The two major glycoprotein bands (94,000 and 78,000 daltons) and small amounts of a 56,000-dalton band were retained in dodecyl sulfate gels of the extracted membranes. Both control and insulin-activated D-glucose transport activities were retained by plasma membrane vesicles extracted with dimethylmaleic anhydride. Cytochalasin B binding activity was also retained by extracted membrane vesicles and D-glucose uptake into extracted vesicles derived from untreated or insulin-treated fat cells was inhibited by cytochalasin B. These results suggest that the modification of the adipocyte hexose transport system elicited by insulin action is not altered by a major purification step which involves quantitative extraction of extrinsic membrane proteins.