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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↗
PubMed1986

Binding of DNA to albumin and transferrin modified by treatment with water-soluble carbodiimides.

N-Acylurea derivatives of albumin and transferrin prepared with the water-soluble carbodiimides N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide and N-ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide have been found to bind different types of DNA. The two proteins were reacted with varying amounts of carbodiimide in water at pH 5.5 for 36-60 hr at 20 degrees, and then purified. In the case of iron-loaded transferrin, reactions with carbodiimides were in phosphate-buffered saline (pH 7.5) to prevent loss of iron from the protein. [3H]N-Ethyl-N'-(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the modified proteins, and gel electrophoresis for changes in charge and possible aggregation through cross-linking. Binding of DNA to N-acylurea proteins was studied by means of gel electrophoresis and nitrocellulose filter binding. N-Acylurea albumin and N-acylurea transferrin at low concentrations retarded the migration of lambda-Pstl restriction fragments, pBR322 plasmid and M13 mp8 single-stranded DNA on agarose gels, while at higher concentrations of modified protein the N-acylurea protein-DNA complexes were unable to enter the gel. Nitrocellulose filter assays showed that binding pBR322 DNA and calf thymus DNA to N-acylurea proteins is rapid and dependent on protein concentration and the ionic strength of the medium. N-Acylurea albumins prepared with each each of the two carbodiimides gave comparable plots for DNA bound versus protein concentration. On the other hand, binding of DNA by N-acylurea transferrins differed according to the carbodiimide used in the synthesis. N-Acylurea CDI-tkransferrin (prepared with tertiary carbodiimide) was less effective than either of the two N-acylurea albumins in binding DNA. In contrast with these results, N-acylurea Me+-CDI-transferrin (prepared with quaternary carbodiimide) was far more effective in binding DNA and in this respect was similar to the N-acylurea albumins. On the basis of experiments in which N-acylurea protein-DNA complexes were treated with heparin, two types of binding could be distinguished. These were a weak binding occurring in the initial stages of interaction and a tight binding which developed on further incubation of the complexes. These studies show that binding of DNA by N-acylurea proteins is a reversible process dependent on ionic strength; interaction appears to be electrostatic in nature, although other forms of binding might be involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Preparation and identification of a population of antibodies that recognize carbodiimide-modified heparin.

Protein-heparin complexes, prepared by a water-soluble carbodiimide coupling technique, were used to produce anti-heparin antibodies in rabbits. Antiserums that recognized carbodiimide-treated heparin, but not untreated heparin, were obtained. Carbodiimide-treated heparan sulfate exhibited 10% to 20% cross-reactivity compared with a similarly treated heparin, whereas there was no cross-reactivity with five other carbodiimide-treated mucopolysaccharides. 3H-1-ethyl-3-(3-trimethylammoniumpropyl) carbodiimide iodide was used to demonstrate that carbodiimide forms a stable adduct with heparin and other mucopolysaccharides. Using an antibody fraction that eluted from 1-ethyl-3-(3-trimethylammoniumpropyl) carbodiimide iodide-treated heparin-Sepharose with 2 mol/L KI, it was demonstrated that, for the antibody population studied, the addition of one carbodiimide per heparin molecule resulted in complete epitope expression without loss of anticoagulant activity. The addition of up to eight additional carbodiimide molecules to heparin did not increase the extent of epitope formation, although anticoagulant activity was lost. Except for heparan sulfate, the addition of radiolabeled carbodiimide to other mucopolysaccharides did not result in epitope formation. These data demonstrate that antibodies to an epitope derived from heparin can be formed, that the epitope is fully expressed while anticoagulant activity is present, and that the antibody is specifically directed against an altered portion of the polysaccharide.

Animals↗
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↗
PubMed1986

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation.

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.

Adenosine Triphosphate↗
PubMed1986

Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification.

Irreversible inhibition of Na,K-ATPase and K+-dependent p-nitrophenylphosphatase activities was produced by incubation of purified Na,K-ATPase enzyme with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EPC). Inhibition was time and [EPC] dependent and displayed first order kinetics with respect to time. The [EPC] to reduce the enzyme velocity by 50% for Na,K-ATPase and phosphatase activities was 1.6 and 2.2 mM, respectively. Analysis of the kinetics of inhibition by EPC indicated that reaction at one site was sufficient to produce inhibition. Inhibition was greatly reduced by the presence of Mg2+, Na+, K+, choline, or Tris (decreasing order of effectiveness); ATP was without effect. This suggests that cation-bound enzyme forms were less reactive with the carbodiimide than free enzyme; ATP-bound enzyme was as reactive. Apparently the cations Na+, Mg2+, Tris, and choline stabilize E1 forms of the enzyme which are different from the E1 form stabilized by ATP. Addition of [14C]glycine ethyl ester (Gly-OEt) resulted in incorporation of radioactivity into both alpha and beta subunits that was dependent upon the presence of EPC, and the incorporation was reduced by the cations which reduced the inhibition due to EPC. Simultaneous addition of Gly-OEt with EPC prevented inhibition, although 14C incorporation still took place. If Gly-OEt addition was delayed the initial inactivation was not affected, but little subsequent inactivation occurred. The protection against inactivation by EPC occurs on the addition of other exogenous nucleophiles, such as aminoethane or ethylenediamine. Dicyclohexylcarbodiimide, a more potent hydrophobic carbodiimide inhibitor, shows similar effects; the inhibition due to dicyclohexylcarbodiimide is also prevented by the simultaneous presence of a nucleophile. After treatment with a carbodiimide and exogenous nucleophile the Na,K-ATPase has modified carboxyl residues but is not inhibited. Thus, modification of the cation-protectable carboxyl groups does not by itself cause inhibition. It seems likely that the inhibition of activity due to carbodiimide alone is not due to the modification of a carboxyl group per se but to the formation of an intramolecular bond between the carbodiimide-activated carboxylic acid and an endogenous nucleophile. The formation of such bonds suggests the close juxtaposition of amine and carboxyl groups in the secondary structure of the enzyme.

Animals↗
PubMed1980

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria.

1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.

Adenosine Triphosphatases↗
PubMed1990

The effect of Ca2+ and calmodulin on the inhibition of Ca2(+)+Mg2(+)-ATPase in erythrocyte ghost membranes by nonpolar and polar carbodiimides.

N,N'-dicyclohexylcarbodiimide (DCCD) and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide (CMCD) inhibited calmodulin-dependent Ca2(+)+Mg2(+)-ATPase activity in erythrocyte ghost membranes. The extent of the inhibition caused by carbodiimides strongly depended on their hydrophobicity. Hydrophobic DCCD was a more potent inhibitor then hydrophilic CMCD. Calmodulin (CaM) protected the enzyme against the former carbodiimide, whereas Ca2+ did the same against the latter. In contrast to previous observations made by Villalobo et al., on the purified enzyme, neither carbodiimide affected the calmodulin-independent ATPase activity in ghost membranes. Inhibition of the calmodulin-dependent ATPase activity was due to a decrease of the maximum activity, whereas the Km value for Ca2+ remained unchanged. Titration of erythrocyte ghost membranes with CaM revealed a biphasic response of ATPase to this activator. Two affinity constants were found for CaM, 0.64 nM and 14 nM. DCCD affected the interaction with CaM at high- and low-affinity binding sites in a competitive manner. CMCD acted as a noncompetitive inhibitor for CaM low-affinity sites, whereas it behaved in a competitive way against CaM interaction with high-affinity sites. In E2 form (stabilized by vanadate and EGTA) ATPase was more sensitive to carbodiimides than in E1 form (induced by La3+).

Animals↗
PubMed2003

Crosslinking of biological tissues using genipin and/or carbodiimide.

The study was to investigate the crosslinking characteristics, mechanical properties, and resistance against enzymatic degradation of biological tissues after fixation with genipin (a naturally occurring crosslinking agent) and/or carbodiimide. Fresh tissue was used as a control. It was found that both genipin and carbodiimide are effective crosslinking agents for tissue fixation and genipin crosslinking is comparatively slower than carbodiimide crosslinking. Additionally, tissue fixation in genipin and/or carbodiimide may produce distinct crosslinking structures. Carbodiimide may form intrahelical and interhelical crosslinks within or between tropocollagen molecules, whereas genipin may further introduce intermicrofibrillar crosslinks between adjacent collagen microfibrils. The stability (denaturation temperature and resistance against enzymatic degradation) of the fixed tissue is mainly determined by its intrahelical and interhelical crosslinks. In contrast, intermicrofibrillar crosslinks significantly affect the mechanical properties (tissue shrinkage during fixation, tensile strength, strain at break, and ruptured pattern) of the fixed tissue. Moreover, the degree of enzymatic degradation of the fixed tissue may be influenced by three factors: the availability, to the enzyme, of recognizable cleavage sites, the degree of crosslinking, and the extent of helical integrity of tropocollagen molecules in tissue.

Animals↗
PubMed1986

Calcium-dependent inhibition of the erythrocyte Ca2+ translocating ATPase by carbodiimides.

The ATP hydrolytic activity of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte plasma membrane was strongly inhibited by the nonpolar compound, N,N'-dicyclohexylcarbodiimide, both in the presence and in the absence of calmodulin. However, the more water-soluble carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide had little inhibitory effect on the enzyme. The inhibitory effect of N,N'-dicyclohexylcarbodiimide was most pronounced at acid pH, and declined sharply at alkaline pH values. In addition, the optimum pH for the enzyme activity also shifted to more alkaline values in the presence of the carbodiimide. Calcium ion appears to favor the inhibition induced by the carbodiimide, in contrast to the observed protection by Ca2+ in the sarcoplasmic reticulum Ca2+-translocating ATPase. N,N'-Dicyclohexylcarbodiimide also dramatically decreased the stimulatory effect of calmodulin on the activity of the enzyme.

Adenosine Triphosphate↗
PubMed1983

Interaction between cytochrome c and ubiquinone-cytochrome c oxidoreductase: a study with water-soluble carbodiimides.

The role of carboxyl groups on the interaction between ubiquinone-cytochrome c oxidoreductase (Complex III) and cytochrome c has been probed using the two water-soluble carbodiimides EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) and CMC (1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide metho-p-toluensulphonate). The results suggest that: 1) carboxyl groups present on both cytochrome c1 and subunit VIII are modified. Some of these residues are shielded by cytochrome c. 2) The enzyme activity decreases during the carbodiimide treatment and the extent of inhibition is larger in the presence of cytochrome c. 3) Cytochrome c, equimolar with the enzyme, cross-links to cytochrome c1 and subunit VIII via the carbodiimide-activated carboxyl groups. The two subunits appear to be in contact in the isolated enzyme.

Animals↗
PubMed2006

Preparation of carbodiimide-terminated dithiolane self-assembly monolayers as a new DNA-immobilization method.

A carbodiimide derivative having a dithiolane part at its terminus was designed and synthesized for use to construct carbodiimide-coated self-assembly monolayers (SAMs) on a gold surface with 6-mercaptohexanol (6MH). When treated with poly(dT), poly(dA), or poly(dA)poly(dT), only poly(dT) was immobilized on the surface of the SAMs through a specific reaction of the free imino moiety of thymine (T) with the carbodiimide moiety. The carbodiimide-covered SAM treated with probe DNA was tested in hybridization with sample DNA. Its hybridization efficiency was estimated by ferrocenylnaphthalene diimide (FND), described previously and the result revealed that the carbodiimide-covered SAM electrode can immobilize a DNA probe through the thymine moiety not involved in base pairing. The resulting electrode was capable of hybridizing with the target DNA, as proven by an increased current response of FND.

Carbodiimides↗
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↗
PubMed1991

Effect of carbodiimides on the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal membranes.

1. The hydrophobic N,N'-dicyclohexylcarbodiimide (DCCD) inhibits the activity of Mg(2+)-ATPase of slow-twitch muscle microsomal fraction. 2. The inhibition is dependent on time and concentration, with half-maximal inhibition occurring at 0.4 mM concentration of carbodiimide after a 0.5 hr incubation at room temperature. 3. ATP does not protect against the inhibition. 4. Two water-soluble carbodiimides, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide (CMCD) and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCD), are not inhibitory. 5. Inhibition of Mg(2+)-ATPase activity by DCCD is accompanied by covalent incorporation of the radioactive agent into the partially purified enzyme preparation.

Animals↗
PubMed2004

Helix-sense selective polymerization of carbodiimides: building permanently optically active polymers from achiral monomers.

The helix-sense selective polymerization of achiral monomers by homochiral catalysts was investigated. Polymerization of chiral carbodiimides (N-(R)-2,6-(dimethylheptyl)-N'-phenylcarbodiimide) by achiral catalysts yields polymers that undergo mutorotation at elevated temperatures, thus illustrating that these chains are formed under kinetic rather than thermodynamic control. Building on this observation, the polymerization of achiral carbodiimides by (S-BINOL)Ti(OiPr)2, I, was studied. Monomers (N-hexyl-N'-(X)carbodiimide, where X = isopropyl (3), hexyl (4) or phenyl (5)), N-methyl-N'-(2-methyl-6-isopropylphenyl)carbodiimide, 6, and N-dodecyl-N'-(1-naphthyl)carbodiimide, 7, were all polymerized with I in good yields (86-95%), and all showed varying degrees of asymmetric induction. Poly-3, -4, and -5 racemized upon heating at elevated temperatures, but poly-6 and poly-7, bearing nonsymmetric phenyl groups, yielded optically active polymers that could not be racemized even at elevated temperatures. Thin films of poly-7 were found to be highly opalescent.

Journal Article↗
PubMed1990

Stability of water-soluble carbodiimides in aqueous solution.

A dimethylbarbituric acid reagent has been used to follow the kinetics of loss of two water-soluble carbodiimides, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and the structurally related 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide (EAC), in aqueous solution as a function of pH and added chemical reagents. In 50 mM 2-(N-morpholino)ethanesulfonic acid at 25 degrees C, EDC has t1/2 values of 37, 20, and 3.9 h at pH 7.0, 6.0, and 5.0, respectively, while the corresponding values for EAC are 12, 2.9, and 0.32 h. Iodide, bromide, or chloride, at 0.1 M, has very little or no effect on carbodiimide stability. However, 0.1 M glycine methyl ester or 0.1 M ethylenediamine causes a significant increase in the rate of loss of EAC and EDC, while the presence of 0.1 M phosphate, 0.1 M hydroxylamine, or 0.01 M ATP decreases the half-lives to less than or equal to 0.4 h at all pH values.

Alkanesulfonates↗
PubMed1988

The mechanisms of inhibition of anion exchange in human erythrocytes by 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide.

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

1-Phenyl-3-trimethylaminopropyl carbodiimide: a new inhibitor of thymidylate synthase.

Thymidylate synthase (EC 2.1.1.45) from methotrexate-resistant Lactobacillus casei was inactivated by 1-phenyl-3-trimethylaminopropyl carbodiimide (PTC), 1-phenyl-3-dimethyl aminopropyl carbodiimide (PDC), and 1-ethyl-3-dimethyl aminopropyl carbodiimide (EDC). In the presence of excess PTC, the inactivation followed pseudo-first order kinetics; the second order rate constant was approximately 200 M-1min-1 at 30 degrees C. The rate of inactivation by PTC was faster than that by either PDC or EDC. Concentrations of the substrate dUMP greater than 0.15 mM, or of the product dTMP greater than 1.6 mM completely protected the enzyme from inactivation by PTC, but 10 mM dUrd provided very little protection. The rate of inactivation of EDC was reduced by only 40% in the presence of 50 mM dUMP. Nucleophiles (sulfanilic acid, glycine methyl ester, or glycine ethyl ester) had no effect on the rate of inactivation by PTC. The complete inactivation of thymidylate synthase by PTC was accompanied by the incorporation of approximately 2 mols of 14C-PTC per mol of enzyme. Although carbodiimides normally modify carboxyl groups in proteins, results from sulfhydryl group titrations and from reversible modification of sulfhydryl groups by methyl methanethiosulfonate suggest that two of the four cysteine residues of thymidylate synthase were modified by PTC.

Carbodiimides↗