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At least 19 recordsLinked to original sources

PubMed1976

The role of the carbodiimide-reactive component of the adenosine-5'-triphosphatase complex in the proton permeability of Escherichia coli membrane vesicles.

Membrane vesicles isolated from wild-type and dicyclohexylcarbodiimide-resistant strains of Escherichia coli exhibit identical respiration-dependent transport activities, and in both cases, this activity is abolished by extraction of the vesicles with 1.0 M guanidine-HCl. Transport activity of extracted wild-type vesicles is completely restored by exposing the vesicles to lipophilic or water-soluble carbodiimides, while transport activity of the mutant vesicles is not restored by exposure to lipophilic carbodiimides. Strikingly, however, complete reactivation of transport in mutant vesicles is observed with water-soluble carbodiimides. Similarly, the Ca2+, Mg2+-stimulated ATPase activity of wild-type vesicles is inhibited by both classes of carbodiimides, while the ATPase activity of mutant vesicles is inhibited by water-soluble carbodiimides, but resistant to inhibition by lipophilic carbodiimides. The carbodiimide-reactive component of the membraneous Ca2+, Mg2+-stimulated ATPase complex in wildtype vesicles is readily labeled with N,N'-dicyclohexyl[14C]-carbodiimide, while the analogous component in mutant vesicles is not reactive. Alternatively, when vesicles are treated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide [14C]methiodide, a water-soluble carbodiimide, the carbodiimide-reactive component is labeled to a similar degree in both preparations. The results suggest that the altered carbodiimide-reactive proteolipid in the dicyclohexylcarbodiimide-resistant mutant is specifically defective in its ability to react with lipophilic carbodiimides. In addition, these and other findings indicate that the increase in proton permeability observed on extraction of isolated membrane vesicles with chaotropic agents is due exclusively to an effect on the carbodiimide-reactive component of the Ca2+, Mg2+-stimulated ATPase complex.

Adenosine Triphosphatases↗

PubMed1976

Purification of the carbodiimide-reactive protein component of the ATP energy-transducing system of Escherichia coli.

The ATP-energy transducing system in membranes of Escherichia coli is inhibited by dicyclohexylcarbodiimide. The protein component of this complex with which carbodiimides covalently react to inhibit function was previously identified by labeling wild type and dicyclohexylcarbodiimide-resistant mutants with dicyclohexyl[14C]carbodiimide (Fillingame, R. H. (1975) J. Bacteriol. 124, 870-883). This specific carbodiimide-reactive protein has now been purified. The protein was extracted from the membrane with chloroform:methanol and chromatographed on DEAE-cellulose and hydroxypropyl Spehadex G-50 in this sulvent mixture. The resultant 700-fold purification yielded a protein that was homogeneous on dodecyl sulfate-acrylamide gel electrophoresis and virtually free of phospholipid. It remained soluble in neutral chloroform:methanol throughout the purification procedure. The amino acid composition of the purified protein was extraordinary in that only 16% of the amino acids present could be considered polar. Histidine, serine, cysteine, and tryptophan were not found. Abnormally high contents of methionine, glycine, alanine, and leucine were present. One mole of lysine and threonine were found/mole of dicyclohexyl[14C]carbodiimide bound. The minimum molecular weight based on the amino acid composition was 8400. The specific carbodiimide-reactive protein has also been purified without prior modification by dicyclohexylcarbodiimide. The unmodified protein eluted from DEAE-cellulose at a higher salt concentration than the dicyclohexylcarbodiimide-modified form, which suggested that the reaction with the carbodiimide neutralized the negative charge. Only one-third of the total carbodiimide-reactive protein in the membrane was modified by dicyclohexylcarbodiimide under conditions which maximally inhibited adenosine triphosphatase activity. These results rais the possibility that the carbodiimide-reactive protein may be present as an oligomer in the energy-transducing complex. The purification of the unmodified carbodiimide-reactive protein should permit assessment of tis biological function, particularly its role in the protein-translocation process that is catalyzed by this energy-transducing complex.

Adenosine Triphosphate↗

PubMed1975

Carbodiimide enhancement of complement-dependent antibody-mediated tumor cell lysis in vitro and antitumor activity in vivo.

The water-soluble carbodiimide salt 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide. HCl (EDCI-HCl) has been shown to increase the complement-dependent lysis of cultured mouse neuroblastoma C1300 cells by two types of antibody: (1) natural antibodies in the sera of normal (nonimmunized) rabbits, and (b) serum antibodies from snygeneic tumor-bearing A/HeJ mice. In the latter case, both the level of serum antibodies and the extent of carbodiimide enhancement of immune lysis were demonstrated in vitro to be substantially greater with sera from mice bearing 21-day-old tumors relative to 4-day-old tumors. The carbodiimide EDCI-HCl has also been found to increase the complement-dependent lysis of cultured TA3 carcinoma cells by serum antibodies from isogeneic LAF1/J mice bearing ascites tumors in advanced stages of growth. Finally, it has been shown that EDCI-HCl exerts an antitumor activity in vivo that is significantly greater against 21-day-old than against 4-day-old neuroblastoma c1300 tumors. The increase in EDCI-HCl activity with tumor age is contrary to the response that would be expected if this drug were serving as an antimetabolite. This is evidenced by data showing that the antimetabolite 6-thioguanine is most effective against young rapidly growing neuroblastoma C1300 tumors. The correlation between carbodiimide antitumor activity and host production of cytotoxic antibodies suggests that EDCI-HCl may operate in vivo by an immunological mechanism comparalbe to that demonstrated in vitro.

Adenocarcinoma↗

PubMed1977

Effects of chemical modification of antibodies on their clearance from the circulation. Addition of simple aliphatic compounds by reductive alkylation and carbodiimide-promoted amide formation.

Anti-hapten antibodies from the ascitic fluid of inbred mice were purified by immunoadsorption and characterized immunochemically for in vivo studies of their plasma clearance rates and organ distributions after chemical modification. Following sodium borohydride-promoted reductive methylation and carbodiimide-promoted amide linkage of glycine and several other simple aliphatic compounds, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into syngeneic animals. Using double radioiodine labels, it was possible to show that stoichiometric combinations of additive and chemical reactant did not alter antibody survival time in the circulation or antigen-binding capabilities. However, modifications involving excess reagent (carbodiimide or sodium borohydride) resulted in significant decreases in both circulatory longevity and ligand binding capacities. Excess carbodiimide treatments resulted in immunoglobulin cross-linkage which could be detected by molecular sieve chromatography. Increased kidney localization found with carbodiimide-treated antibodies was apparently due to trapping of the cross-linked aggregates. Elevated clearance of highly methylated antibodies could not be attributed to a single organ. However, sodium borohydride-catalyzed reductive methylation resulted in antibody populations which were localized primarily in the liver and spleen. Results are evaluated in terms of the concept, developed in this paper, of essential groups for the circulatory longevity of glycoproteins.

Alkylation↗

PubMed1975

The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts.

1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding.

Calcium↗

PubMed1979

Carbodiimide-reactive carboxyl groups at the active site of an insect midgut trehalase.

Carbodiimide modification of the Rhynchosciara americana midgut trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) at different pH values revealed the existence of two essential groups (pKa 5.28 and pKa 7.74) for the trehalos activity. Those groups must be carboxyl groups since the alternative possibilities (sulfhydryl and phenol groups) have been discarded by selective modification and attempts to reactivate the modified enzyme with hydroxylamine. Furthermore, the increase of the pKa values of carbodiimide-reactive groups in the presence of dioxane supports further evidence that they are carboxyls. The results suggest the pKa 5.28 carboxyl is in the active site, while the pKa 7.74 carboxyl is in its neighborhood buried in the enzyme molecule. The possible role for the carbodiimide-reactive carboxyl groups in catalysis is discussed.

Animals↗

PubMed1977

Radioactive labeling of protein carboxyl groups on factor VIII: use of carbodiimides for nuclear medicine.

Carbodiimides have been used to study macromolecular structure and to produce immunologically active antigens. We have used this method to label a labile coagulation protein, factor VIII, with 14C-glycine-ethyl-ester. No discernible chemical change, loss of biologic function in vitro, or alteration of the plasma disappearance of factor VIII resulted. The carbodiimide labeling method has potentially broad application because many biologic molecules contain carboxyl groups that are generally not critical to their chemical or immunologic character. This method can be used to incorporate short-lived positron emitters, such as 11C and 13N, into biologic compounds, or to attach ligands to useful antibodies for subsequent chelation to radioactive metals, such as 111In. Carbodiimides are especially useful for radionuclidic labeling of labile proteins because of the mild conditions, rapid reaction, and firmly attached label.

Carbodiimides↗

PubMed1977

The enzymatic conversion of L-histidine to urocanic acid by whole cells of Micrococcus luteus immobilized on carbodiimide activated carboxymethylcellulose.

Whole cells of Micrococcus luteus (formerly Sarcina lutea ATCC 9341) have been covalently linked to a carboxymethylcellulose support system, with the retention of histidine ammonia-lyase activity. The dependence of the rate of urocanic acid formation on pH, temperature, and added surfactant concentration was similar for the free and the immobilized cells. The immobilization procedure used is based on the carbodiimide activation of carboxymethylcellulose and has been optimized for the histidine ammonia-lyase activity of the immobilized cells on a given weight of cellulose. In a column reactor at 23 degrees C and superficial velocity of 0.044 cm/min, 5 g of cellulose with bound cells gave a 35% conversion of an L-histidine solution (0.25M, pH 9.0) to urocanic acid for 16 days of continuous operation. The scope of this carbodiimide assisted immobilization procedure has been investigated for a series of microorganisms and a variety of carboxylate functionalized supports.

Ammonia-Lyases↗

PubMed1978

The effects of carbodiimides on functions associated with the energy-conservation mechanism in beef heart sub-mitochondrial particles.

N,N'-di-n-propyl-, N,N'di-n-butyl-, N,N'-di-n-pentyl-, N,N'di-n-hexyl-, N,N'di-n-octoyl, N,N'-dibenzhydryl-, and N,N'-dibenzhydrylcarbodiimides were synthesized. They were all effective inhibitors (2 nmoles carbodiimide per milligram protein) of the ATP-driven reduction of NAD by succinate and the ATP-driven transhydrogenase activities catalyzed by beef heart submitochondrial particles (SMP). They had no effect on the nonenergy-linked transhydrogenase and stimulated the succinate-driven aerobic transhydrogenase activity of beef heart SMP. It was concluded that they exert their effects by reacting with the N,N'-dicyclohexylcarbodiimide-binding protein. Water-soluble carbodiimides were not effective inhibitors.

Adenosine Triphosphate↗

PubMed1977

Reexamination of carbodiimide as a possible affinity label for the acetylcholine receptor at the frog neuromuscular junction.

The effects of water-soluble carbodiimide were examined at the frog neuromuscular junction. Acetylcholine sensitivity was measured using a fluid electrode technique and intracellular recording of miniature end-plate potentials. The carbodiimide blocked synaptic sensitivity by a reversible, curare-like action. Irreversible blockade was also observed, probably due to covalent binding. The conditions of reaction and irreversibility suggest that several different residues may be attacked. The inability of cholinergic antagonists to protect the receptor from attack indicates that nonspecific sites, and not the acetylcholine binding site, are involved.

Acetylcholine↗

PubMedDate not supplied

Blocking of catecholamine activation of adenylate cyclase by N, N'dicyclohexyl carbodiimide in turkey erythrocytes.

Incubation of erythrocytes or their isolated membranes with N, N'dicyclohexyl carbodiimide (DCC) blocked isoproterenol activation of the adenylate cyclase. Fluoride activation remained unaffected. L-epinephrine and DL-propranolol partially and transiently protected the system against DCC. D-Epinephrine and dopamine did not protect. The enzyme system preactivated by isoproterenol plus Gpp(NH)p was no longer sensitive to DCC. In contrast to the water insoluble DCC, a water soluble carbodiimide acted only at high concentration and blocked fluoride as well as catecholamine activation of the adenylate cyclase. The findings indicate that DCC attacks a group on, or near, the beta-adrenergic receptor and that this group is located in a hydrophobic region of the cell membrane. It is argued that a low nominal concentration of DCC in the aqueous suspension of erythrocytes actually represents a very high concentration of DCC in the hydrophobic region of the cell membranes, near the beta-adrenergic receptor. The reaction with DCC may prove to be a useful tool in future analyses of beta-adrenergic receptor function.

Adenylyl Cyclases↗

PubMed1979

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.

Bacteriorhodopsins↗

PubMed1979

Binding of ovalbumin to mouse spleen cells with and without carbodiimide.

We studied the binding of ovalbumin (OVA) to mouse spleen cells. In the presence or absence of carbodiimide (ECDI), uptake increased with greater cell numbers, increasing OVA concentration and increasing time up to 60 min. Between 60 and 210 microgram OVA could be coupled with ECDI to 2 X 10(8) cells. In the absence of ECDI, however, OVA uptake was still appreciable, but appeared to be of low avidity as it did not occur in the presence of competing fetal calf serum. These experiments provide a basis for the use of protein coupled to autologous cells in the induction of immunologic tolerance.

Animals↗

PubMed1976

Grafting of enzymes on collagen films using Woodward's reagent "K" and a water-soluble carbodiimide derivative.

Two new methods of activation were developed to graft enzymes on collegen films. They involved chemical modifications of surface groups of collagen either by Woodward's reagent "K" or by EDC, a water-soluble derivative of carbodiimide. EDC was a better coupling agent and a detailed study was conducted with this agent. It could be used either in a global method of activation and coupling, or in a two-step procedure of activation of collagen, followed by spontaneous coupling of enzyme. All enzymes tested were successfully bound: malate dehydrogenase, lactate dehydrogenase, aspartate aminotransferase, urease, creatine kinase, hexokinase. The influence on the yield of grafted enzyme, of pretreatment of films, time and temperature of EDC activation, concentration of EDC and enzyme, protecting agents was studied. Stability of enzyme activity on storage was greatly increased after grafting. A co-grafted dual system creatine kinase/heoxkinase, was achieved which exhibited a good efficiency. A striking renaturing process at 0-4degreesC after thermal denaturation, was observed with hexokinase.

Aspartate Aminotransferases↗

PubMed1977

Carbodiimide modification of superhelical PM2 DNA: considerations regarding reaction at unpaired bases and the unwinding of superhelical DNA with chemical probes.

Superhelical PM2 DNA I can be modified with N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethyl carbodiimide (CMC). The transition of the sedimentation coefficient uncorrected for buoyant density change (S20,*) vs. % reactivity in terms of base pairs shows the following characteristics. The S20,* increases by 4.5 S units upon 1% modification. There is a plateau in S20,* between 1 and 4% reactivity. The extent of reactivity was determined by buoyant density and 14C radioactive CMC binding measurements. Further reactivity was not explored since Pulleyblank and Morgan's (22) data of S20,* vs. % reactivity from 6 to 34% was previously published. The initial results obtained in this study are complementary to the cited results of the above authors. Consequently, both sets of data taken together represent a complete description of S20,* vs. % reactivity with CMC. It is shown that the model in which superhelical DNA is proposed to contain small intrastrand hairpin regions can be extended to account for the observed transitions in S20,* vs. reactivity.

Carbodiimides↗

PubMed1977

Inhibition of transcription of supercoiled PM2 DNA by carbodiimide modification.

PM2 superhelican DNA (form I), which as been reacted with the single strand specific reagent, N-cyclohexyl-N'-beta-(methylmorpholinium)ethyl carbodiimide (CMC) is more than 95% inhibited in its ability to support transcription with E. coli B RNA polymerase in vitro. Almost complete inhibition of transcription was achieved after 2 hours of reaction with FI when only 1% of the bases were modified. A large increase in S20,* (from 26.8 S to 33.6 S) of FI DNA was observed during the course of reaction. Rifampicin resistant transcription is more susceptible to inhibition by CMC than total transcription, suggesting that the CMC is preferentially binding at promoter sites. These results clearly are in accord with the observation that supercoiled DNA contains localized regions of unpaired bases. The promotor sites for E. coli RNA polymerase in FI PM2 DNA appear to be located at or near these unpaired sites.

Carbodiimides↗

PubMed1978

Modification of rabbit muscle phosphorylase b by a water-soluble carbodiimide.

Glycogen phosphorylase b from rabbit muscle was rapidly inactivated by incubation with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC) at pH 5.1. The inactivation was pH-dependent and was not restored by treatment with hydroxylamine. The addition of glycine ethyl ester or N-(2,4-dinitrophenyl)-ethylenediamine (DNP-EDA) markedly increased the rate of inactivation. Of the various amino analogs of glucose tested, only glucosyl amine accelerated the inactivation, although they are all bound to the glucose 1-phosphate site of the enzyme. In the absence of amines, incorporation of about 3 mol of [metho-14C]CMC per protein monomer was observed on complete inactivation. In the presence of DNP-EDA, however, only 2 mol of [metho-14C]CMC and 1 mol of DNP-EDA were incorporated before the activity was completely lost. The treatment of phosphorylase b with CMC did not change the Km values of the enzyme for glucose 1-phosphate and AMP, in spite of the 56% inactivation. It is suggested that, in the phosphorylase-catalyzed reaction, an essential carboxyl group of the enzyme plays a role in the protonation of the glucosidic oxygen of glucose 1-phosphate.

Adenosine Monophosphate↗

PubMed1975

Peptide synthesis with carbodiimide.

An isotope dilution assay for racemization during acid couplings is described and applied to carbodiimide-mediated synthesis. Using the coupling of t-BOC-L-phenylalamine with glycine derivatives as a model, racemization in solution falls in the 0.01 to 0.1% range and it is a function of reagent concentrations. A typical coupling using the solid phase method shows 0.03% racemate.

Carbodiimides↗