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At least 19 recordsLinked to original sources
PubMed1984

Influence of pH on the rheologic and antisickling effects of dimethyl adipimidate.

Dimethyl adipimidate, a bifunctional imidoester, is an effective inhibitor of sickling in vitro. We determined the reaction conditions necessary to inhibit sickling without impairing cell deformability. Although imidoesters are routinely used at alkaline pH, we found that reaction of red blood cells with dimethyl adipimidate at pH 8.4 shortened the survival of rat red blood cells and impaired the deformability of human red blood cells. These adverse effects were eliminated by changing the pH of the reaction from 8.4 to 7.4. Furthermore, treatment of sickle cells with dimethyl adipimidate at pH 7.4 inhibited sickling, as demonstrated by dose-dependent reductions in the percentage of sickle forms, viscosity, and potassium efflux. Dimethyl adipimidate appeared to inhibit sickling by several mechanisms: namely, it increased deoxygenated sickle hemoglobin solubility, oxygen affinity, and cell hydration. We conclude that dimethyl adipimidate at pH 7.4 can inhibit sickling without impairing the rheologic properties of the red blood cell.

Anemia, Sickle Cell↗
PubMed1984

Mechanism of inhibition of sickling by dimethyl adipimidate. Effects of intertetramer cross-linking.

Dimethyl adipimidate (DMA), an effective antisickling agent in vitro, reacts with free amino groups producing chemically modified and cross-linked molecules. In this report, we have investigated the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization. Since the extent of cross-linking is pH-dependent, we first compared the solubilities of deoxygenated hemolysates prepared from sickle cells previously treated with dimethyl adipimidate at either pH 7.4 or 8.4. The solubility of the hemolysate increased from 18.6 +/- 0.8 g/dl in the untreated sample to 20.9 +/- 1.5 g/dl (pH 7.4) and to 25.4 +/- 3.0 g/dl (pH 8.4) after dimethyl adipimidate treatment. Removal of cross-linked hemoglobin tetramers from hemolysate obtained from dimethyl adipimidate-treated cells abolished part of this effect; at pH 7.4, the solubility decreased from 20.9 +/- 1.5 to 19.4 +/- 0.2 and at pH 8.4 from 25.4 +/- 3.0 to 21.0 +/- 1.5. However, the ratio of [14C]DMA-labelled hemoglobin in the sol phase to that in the gel phase in the unfractionated hemolysate was 1.17 at pH 7.4 and 1.25 at pH 8.4, suggesting that part of the cross-linked hemoglobin tetramers was incorporated into the gel. In order to further investigate the effect of cross-linked hemoglobin tetramers on sickle hemoglobin polymerization, we separated cross-linked hemoglobin tetramers on a gel-filtration column, prepared mixtures of untreated sickle hemoglobin and cross-linked hemoglobin tetramers and studied the polymerization of these mixtures. The Csat of the untreated hemolysate increased progressively from 18.6 +/- 0.8 to 22.5 +/- 0.8 g/dl with 33% cross-linked hemoglobin tetramers. The hemoglobin concentration in the gel decreased from 43 +/- 1.0 to 33.8 +/- 1.0 g/dl with 33% cross-linked hemoglobin tetramers, while the pellet volume fraction, phi p, increased with and almost approached 1 at 50% cross-linked hemoglobin tetramers. In addition, the sol phase contained a higher molecular weight distribution of cross-linked hemoglobin tetramers than the gel phase. These observations suggest that a loose polymer was formed in the gel phase with a hemoglobin concentration much lower than that of the control. Thus, polymerization of sickle hemoglobin is inhibited by: (1) exclusion of higher molecular weight cross-linked hemoglobin tetramers from the gel, and (2) loose incorporation of cross-linked hemoglobin tetramers into the gel, perhaps preventing lateral packing and formation of tightly ordered fibers.

Chromatography, Gel↗
PubMed1979

Modification of hemoglobin A with dimethyl adipimidate. Contribution of individual reacted subunits to changes in oxygen affinity.

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, alpha and beta subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the alpha-treated (alpha t beta c) or the beta-treated subunits (alpha c beta t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated alpha and beta globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of alpha subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of beta subunits has no effect. However, treatment of beta subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the alpha and beta subunits contributes to regulating the oxygen affinity of human hemoglobin.

Circular Dichroism↗
PubMed1976

Correction of the permeability defect in hereditary stomatocytosis by dimethyl adipimidate.

The effect of imidoesters on the extraordinarily increased cation permeability of hereditary stomatocytes was evaluated in erythrocytes from a splenectomized boy with persistent hemolytic anemia. Reaction of stomatocytes with dimethyl adipimidate reduced ouabain-associated potassium loss from 15 to 1.7 and sodium gain from 22 to 2.5 meq per liter of red cells per hours. Red-cell volume, cation concentration, and deformability, previously abnormal, rapidly became normal after stomatocytes were reacted with dimethyl adipimidate. Instead of stomatocytes, normal red cells and target cells were noted. The survival (half-time) of stomatocytes treated with 51Cr-labeled dimethyl adipimidate infused into rats rendered tolerant to human erythorocytes by pretreatment with ethyl palmitate and cobra-venom factor was double that of untreated stomatocytes. Chemical modification of the defect in vitro allowed stomatocytes to regain many properites of normal erythrocytes and favorably influenced the subsequent survival of these cells in vivo.

Adenosine Triphosphate↗
PubMed1984

Prolongation of sickle cell survival by dimethyl adipimidate is compromised by immune sensitization.

The effect of dimethyl adipimidate (DMA), an amino-reactive crosslinking reagent with demonstrated antisickling properties in vitro, on the survival of 51Cr-labeled autologous sickle cells was evaluated in five adult males with sickle cell anemia. The survival of cells pretreated with 5 mmol/L DMA (pH 7.4), normal (t1/2 28-33 days) in four subjects and near-normal (t1/2 20 days) in the fifth, was considerably longer than that usually observed in sickle cell disease. In fact, the effect of DMA on the survival of sickle cells in vivo equals or exceeds that of any other agent tested to date. In three subjects, the survival of a second infusion of DMA-treated red cells was much shorter (t1/2 1.8, 3, 4.5 days) than in the initial study. An antibody was detected in the serum of these subjects that was directed to DMA-treated red cells. Modification of the immunogenicity of treated cells will be required before further consideration of DMA for use in the therapy of sickle cell anemia.

Adult↗
PubMed1975

Dimethyl adipimidate: a new antisickling agent.

A new approach to the prevention of sickling in vitro by use of the bifunctional crosslinking reagent, dimethyl adipimidate, is described. Prior treatment of sickle erythrocytes with dimethyl adipimidate will inhibit sickling in completely deoxygenated erythrocytes. Treated erythrocytes do not demonstrate the potassium loss and viscosity increase that usually accompany sickling. The oxygen affinity of hemoglobin in these cells is increased independently from changes in the concentration of 2,3-diphosphoglycerate. The hemoglobin obtained from treated erythrocytes contains a high-molecular-weight component as well as additional positively charged components. The relative degree to which chemical modification and/or crosslinking is an essential part of the antisickling properties of the material is not known.

Adipates↗
PubMed2002

Preparation of well-defined bovine polyhemoglobin based on dimethyl adipimidate and glutaraldebyde cross-linkage.

A well-defined bovine polyhemoglobin was prepared by dimethyl adipimidate (DMA) and glutaraldehyde double cross-linkage method. DMA was used to block some amino groups of hemoglobin, followed by further polymerization with glutaraldehyde. The amino modification degree of hemoglobin was 32% when DMA reacted with hemoglobin at the molar ratio of 200. The bovine polyhemoglobin with narrow molecular weight distribution (mainly 128 kDa) was obtained when glutaraldehyde reacted with DMA-modified hemoglobin. The P(50) and the Hill coefficient for DMA-modified hemoglobin were 19.4 mm Hg and 2.28, respectively, while those for the bovine polyhemoglobin were 15.1 mm Hg and 1.70, respectively. The number of Bohr protons released for DMA-modified hemoglobin and the polyhemoglobin was 0.86 and 0.56 H/tetramer, respectively.

Animals↗
PubMed2000

Effect of dimethyl adipimidate on K+ transport and shape change in red blood cells from sickle cell patients.

Dimethyl adipimidate (DMA) reduces K+ loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (Psickle), the Ca2+-activated K+ channel (or Gardos channel) and the K+-CI- cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca2+ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of Psickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K+ pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.

Anemia, Sickle Cell↗
PubMed1985

The cross-linking of human Met-hemoglobin with [14C]dimethyl adipimidate.

Met-hemoglobin, cross-linked with [14C]dimethyladipimidate (cross-linking span 9 A; 1 A = 0.1 nm), yields four distinct molecular weight products (monomer, dimer, trimer, and tetramer) as observed on sodium dodecyl sulfate - polyacrylamide gels. The dimer species was purified to homogeneity by gel filtration. It was proteolytically degraded using a combination of Staphylococcus aureus V8 protease, pepsin, trypsin, and chymotrypsin. The resultant peptides were fractionated using gel filtration and ion-exchange chromatography methods. Two cross-links were unambiguously identified: (i) alpha 1Lys7-alpha 1Lys11 and (ii) beta 1Lys82-beta 2Lys82. The identified cross-links correlated well with the known structure of hemoglobin. However, attempts to isolate and identify a greater number of cross-linked peptides were unsuccessful owing to the complexity of the peptide mixtures. The complexity was a direct result of the chemical modification of the hemoglobin molecule. Therefore, attempts to employ chemical cross-linking as a means of examining sites of protomer contact within large oligomeric proteins should be approached with caution.

Cross-Linking Reagents↗
PubMed1981

The subunit structure of bovine heart mitochondrial transhydrogenase.

Reaction of purified bovine heart transhydrogenase with bifunctional cross-linking reagents dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, and dithiobis(succinimidyl propionate) results in the appearance of a dimer band on sodium dodecyl sulfate polyacrylamide gels with no higher oligomers formed. Treatment of the enzyme with 6 M urea led to inactivation and prevented cross-linking by dimethyl suberimidate. Transhydrogenase reconstituted into phosphatidylcholine proteoliposomes also yielded a dimer band on cross-linking. These data indicate that soluble and functionally reconstituted transhydrogenase possesses a dimeric structure.

Animals↗
PubMed1985

Chemically attenuated larvae of S. mansoni as a novel tool in schistosomiasis research: 2. Chemical attenuation of cercariae by diimidoesters.

A novel approach to chemically attenuate cercariae of S. mansoni is presented. The method utilizes the biologically active surface proteins/glycoproteins which are essential for the survival of the organism as a target for inactivation. The inactivation was achieved by reaction with 0.01 M dimethyl adipimidate, dimethyl pimelimidate or dimethyl suberimidate at pH 8.5. The cercariae lost their viability, but retained the ability to exclude trypan blue for up to 2 years when stored at 4 degrees C in a manner similar to live cercariae and in contrast to dead cercariae which took up the dye immediately. In addition, the attenuated cercariae reacted with monoclonal and polyclonal antischistosome antibodies in an indirect immunofluorescence assay indicating the retention and preservation of surface antigens after attenuation. The immunochemical reactivity of the attenuated cercariae was preserved after storage for 2 years at 4 degrees C as shown by reaction with antisera from infected mice and rats in IIF assay. Attenuated cercariae revealed the presence of antischistosome antibodies as early as one week after infection in mice and rats. The presence of receptors for the Fc portion of human IgG on the attenuated cercariae interfered in their use as an immunodiagnostic reagent for human schistosomiasis. The attenuated cercariae were also used to screen cultures for monoclonal antischistosome antibodies. Preliminary results indicated that immunization with attenuated cercariae was capable of imparting protective immunity in mice.

Animals↗
PubMed1996

A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.

The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.

Animals↗