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PubMed1986

17O electron nuclear double resonance characterization of substrate binding to the [4Fe-4S]1+ cluster of reduced active aconitase.

To characterize the binding of substrate to aconitase, we have made 17O electron nuclear double resonance (ENDOR) measurements on reduced active ([4Fe-4S]1+) beef heart aconitase, both in H216O and H217O, in the presence of substrate and the inhibitors, tricarballylate, trans-aconitate, and 1-hydroxy-2-nitro-1, 3-propanedicarboxylate, referred to here as nitroisocitrate; the hydroxyl of the latter also was isotypically labeled with 17O. The hydroxyl oxygen of citrate and isocitrate is exchanged with solvent water by aconitase, but the hydroxyl of nitroisocitrate is not. Thus, the isotopic composition of nitroisocitrate can be chemically controlled, allowing direct identification of any 17O ENDOR signal associated with it. 17O ENDOR signals were observed from Hx17O (mean = 1 or 2) bound to the [4Fe-4S]1+ cluster in samples prepared with trans-aconitate and unlabeled nitroisocitrate. 17O-Labeled nitroisocitrate in H216O bound to the cluster showed a signal from the 17OH group; in H217O it showed 17O ENDOR resonances due to both Hx17O and 17OH of substrate. This result demonstrates that the cluster participates in substrate binding and can simultaneously coordinate the hydroxyl of a substrate (or analogue) and water (or hydroxyl). The sample with citrate in H217O showed only the Hx17O signal, although aconitase exchanges the hydroxyl of substrate with solvent water. The mechanism of action of aconitase is discussed in light of this observation. Comparison shows the ENDOR study to be in agreement with previous Mössbauer and EPR spectroscopic results.

Aconitate Hydratase↗
PubMed1977

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo. II. Early ultrastructural modifications correlated to the biochemical lesion induced by fluoroacetate.

Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slighly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.

Aconitate Hydratase↗
PubMed1975

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.

Aconitate Hydratase↗
PubMed1976

Phthalonic acid, an inhibitor of alpha-oxoglutarate transport in mitochondria.

Phthalonic acid is a powerful inhibitor of alpha-oxoglutarate transport in mitochondria. This conclusion is based on the following observations: 1. Phthalonic acid inhibits the oxidation of alpha-oxoglutarate but has no effect on the oxidation of glutamate or cis-aconitate. 2. With arsenite present, phthalonic acid inhibits the oxidation of glutamate plus malate and of cis-aconitate plus malate. Under these conditions alpha-oxoglutarate accumulates inside the mitochondria. With glutamate plus malate as substrates the inhibition is competitive with malate with a Ki value of 20 muM. 3. Phthalonic acid inhibits the oxidation of intramitochondrial NAD(P)H by alpha-oxoglutarate plus ammonia. The inhibition is competitive with respect to alpha-oxoglutarate with a Ki of 30 muM. 4. Phthalonic acid inhibits the exchange between extramitochondrial alpha-oxoglutarate and intramitochondrial malate.

Aconitic Acid↗
PubMed1996

Preparation and characterization of conjugates of (modified) human serum albumin and liposomes: drug carriers with an intrinsic anti-HIV activity.

Human serum albumin (HSA) derivatized with cis-aconitic anhydride (Aco-HSA) that was earlier shown to inhibit replication of human immunodeficiency virus type 1 (HIV-1), was covalently coupled to conventional liposomes, consisting of phosphatidylcholine, cholesterol and maleimido-4-(p-phenylbutyryl)phosphatidylethanolamine, using the heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA). The amount of HSA that could be coupled to the liposomes depended on derivatization of the HSA and ranged from 64.2 +/- microgram HSA/micromol total lipid for native HSA to 29.5 +/- 2.7 microgram HSA/micromol total lipid for HSA in which 53 of the epsilon amino groups of lysine were derivatized with cis-aconitic anhydride (Aco53-HSA). Incorporation of 3.8 mol% of total lipid of a poly(ethylene glycol) derivative of phosphatidylethanolamine (PEG-PE) in the liposomes resulted in a lower coupling efficiency of Aco-HSA. The elimination and distribution of the liposomal conjugates in rats in vivo was largely dependent on the modification of the HSA coupled to the liposomes. With native HSA-liposomes, more than 70% of the conjugate was still found in the blood plasma 30 min after i.v. injection in rats, while at this time Aco-HSA-liposomes were completely cleared from the circulation. The rapid clearance of conventional Aco-HSA-liposomes was due to a rapid uptake into the liver and could be considerably decreased by incorporating PEG-PE in the liposomal bilayer. After 3 h 60% of Aco-HSA-PEG-liposome conjugates were found in the blood. In an in vitro anti-HIV-1 assay, the 50% inhibitory concentrations (IC50) for Aco39-HSA-liposomes and Aco53-HSA-liposomes expressed as protein weight, were 2.87 microgram/ml and 0.154 microgram/ml, respectively. When PEG-PE was incorporated, the Aco53-HSA-liposomes retained anti HIV-1 activity (IC50:3.13 microgram/ml). The possibility to modulate the residence time in the bloodstream of Aco-HSA-liposomes and the potent anti-HIV-1 activity of these conjugates, may allow the development of an intrinsically active drug carrier system. By incorporating anti HIV-1 drugs such as AZT into such liposomes a drug delivery system can be designed that might act simultaneously on the virus/cell binding by virtue of the coupled Aco-HSA and on the RNA/DNA transcription of the HIV-1 replication cycle through the nucleoside analogue.

Aconitic Acid↗
PubMed2005

An iron regulatory-like protein expressed in Plasmodium falciparum displays aconitase activity.

Plasmodium falciparum iron regulatory-like protein (PfIRPa) has homology to both mammalian iron regulatory proteins and aconitases and is capable of binding RNA iron response elements. We examined the subcellular localization of PfIRPa and its enzymatic properties at low oxygen tension. Differential digitonin permeabilization of isolated trophozoites with subsequent Western blot analysis suggests that the localization of PfIRPa is predominantly in the membranous compartments of the parasite, such as the mitochondrion. Immunofluorescence analysis showed that PfIRPa colocalizes with heat shock protein 60 (Hsp60), a mitochondrial marker, and is also present in the parasitic cytosol/food vacuole. Under conditions favoring the formation of an iron-sulfur cluster, recombinant PfIRPa (rPfIRPa) had aconitase activity as detected by a colorimetric NADPH-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. As assessed by the hydration of cis-aconitate spectrophotometrically at 240 nm, rPfIRPa had high affinity for cis-aconitate (Km=3.5 microM) but a low turnover number (Kcat= 3.3 s(-1)). The overall catalytic efficiency (Kcat/Km) of rPfIRPa was similar in magnitude to human cytosolic IRP1/aconitase and human mitochondrial aconitase. PfIRPa immunoprecipitated from parasite lysates also had aconitase activity, as assessed by an MTT-based assay. Our results provide evidence that PfIRPa localizes in the mitochondrion and in the cytosol/food vacuole and is able to demonstrate aconitase activity. Further understanding of the role of PfIRPa/aconitase in the regulation of P. falciparum homeostasis may contribute towards the development of novel antimalarial strategies against plasmodial species.

Aconitate Hydratase↗
PubMed2002

The influence of charge clustering on the anti-HIV-1 activity and in vivo distribution of negatively charged albumins.

The substitution of human serum albumin with negatively charged molecules, such as succinic acid (Suc-HSA) or aconitic acid (Aco-HSA), resulted in proteins with potent anti-HIV activities, by binding to viral gp120 (V3 loop). The aim of the present study was to investigate whether the distribution of negative charges on the albumin backbone influences the anti-HIV activity. Therefore, we prepared albumins with clusters of negatively charged groups by coupling of heparin. The effects of this substitution on anti-HIV activity, in vivo distribution and the protein structure as compared to random succinylation were assessed. In vitro studies indicated that HSA-modified with heparin 6 or 13 kD displayed anti-HIV activity (IC50=660 and 37 nM, respectively) and exhibited affinity for gp120-V3 loop, although the activity was lower than that of Suc-HSA. Combined derivatization of HSA with heparin 13 kD and aconitic acid groups resulted in significantly increased inhibitory actions (IC50=2.8 nM). Structural analysis showed that modification of HSA with heparin did not lead to extensive unfolding of the protein, meaning that these modified proteins were still globular in structure. In contrast, succinylation of HSA resulted in a highly randomly coiled conformation. Dynamic light scattering experiments revealed that, at neutral pH, the heparin fragments attached to the protein were wrapped around the molecule rather than sticking out into the solution. In conclusion, coupling of sufficient clustered negative charges, by coupling of Hep-fragments, on HSA resulted in a clear anti-HIV activity of the protein. Yet, random distribution of anionic groups in the albumin seemed more optimal for in vitro anti-HIV activity. The higher plasma and lymphatic concentrations of Hep-HSA compared to Suc-HSA seemed more favorable for an anti-HIV activity in vivo.

Aconitic Acid↗
PubMed1997

Cell density triggers slender to stumpy differentiation of Trypanosoma brucei bloodstream forms in culture.

Differentiation from replicating slender forms to non-dividing stumpy bloodstream forms of T. brucei limits the parasite population size in the mammalian host in addition to and independently of the antibody response. Using a culture system for pleomorphic strains of T. brucei we show that slender forms very efficiently differentiate to stumpy forms in vitro and that the induction of differentiation is correlated to cell density. Differentiation in the host and in culture were compared using a battery of markers including cell morphology and volume, cell cycle position, the kinetics of the differentiation, expression of NADH dehydrogenase (diaphorase), expression of several differentially regulated transcripts and the kinetics of transformation to replicating procyclic forms after induction with cis-aconitate. By all available criteria, differentiation in culture reflects the natural process in the mammalian host. Time course experiments reveal a very tight temporal correlation between cell cycle arrest of bloodstream forms, appearance of a stumpy differentiation marker and the competence of a bloodstream form population to initiate transformation to procyclic forms in response to cis-aconitate. Our results show that induction of bloodstream form differentiation can occur independently of host-derived cues. We suggest a density sensing mechanism which induces differentiation to the non-dividing stumpy stage and thereby enables the parasite population to autoregulate its proliferation.

Aconitic Acid↗
PubMed2005

IRP1 Ser-711 is a phosphorylation site, critical for regulation of RNA-binding and aconitase activities.

In iron-starved cells, IRP1 (iron regulatory protein 1) binds to mRNA iron-responsive elements and controls their translation or stability. In response to increased iron levels, RNA-binding is inhibited on assembly of a cubane [4Fe-4S] cluster, which renders IRP1 to a cytosolic aconitase. Phosphorylation at conserved serine residues may also regulate the activities of IRP1. We demonstrate that Ser-711 is a phosphorylation site in HEK-293 cells (human embryonic kidney 293 cells) treated with PMA, and we study the effects of the S711E (Ser-711-->Glu) mutation on IRP1 functions. A highly purified preparation of recombinant IRP1(S711E) displays negligible IRE-binding and aconitase activities. It appears that the first step in the aconitase reaction (conversion of citrate into the intermediate cis-aconitate) is more severely affected, as recombinant IRP1(S711E) retains approx. 45% of its capacity to catalyse the conversion of cis-aconitate into the end-product isocitrate. When expressed in mammalian cells, IRP1(S711E) completely fails to bind to RNA and to generate isocitrate from citrate. We demonstrate that the apparent inactivation of IRP1(S711E) is not related to mutation-associated protein misfolding or to alterations in its stability. Sequence analysis of IRP1 from all species currently deposited in protein databases shows that Ser-711 and flanking sequences are highly conserved in the evolutionary scale. Our results suggest that Ser-711 is a critical residue for the control of IRP1 activities.

Aconitate Hydratase↗
PubMed1987

Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy.

The active form of aconitase has a diamagnetic [4Fe-4S]2+ cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mössbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 17O and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H2O or OH- (HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fea of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction.

Aconitate Hydratase↗
PubMed2005

Antiproliferative properties of chemically modified recombinant IFN-alpha2b.

The antiproliferative activity of aconitylated (AIFN) and succinylated (SIFN) derivatives of recombinant interferon- alpha2b (IFN-alpha2b) was examined. Acylation of IFN-alpha2b was performed by succinic and cis-aconitic anhydrides. Antiproliferative properties of AIFN and SIFN were studied in vitro on the CaOv cell line, highly sensitive to IFN, and on the SW-480 cell line, with low sensitivity to IFN-alpha2b. Acylation of one lysine in the IFN-alpha2b molecule with cis-aconitic or succinic anhydride resulted in a 3-3.5-fold increase of its antiproliferative activity on CaOv cells. The highest antiproliferative activity of acylated IFN-alpha2b on SW-480 cells was observed for both AIFNs and SIFNs with three modified lysine residues. In conclusion, aconitylated and succinylated IFNs may be useful antiproliferative agents for cancer treatment.

Aconitic Acid↗
PubMed2004

Cold shock and regulation of surface protein trafficking convey sensitization to inducers of stage differentiation in Trypanosoma brucei.

Transmission of a protozoan parasite from a vertebrate to invertebrate host is accompanied by cellular differentiation. The signals from the environment that trigger the process are poorly understood. The model parasite Trypanosoma brucei proliferates in the mammalian bloodstream and in the tsetse fly. On ingestion by the tsetse, the trypanosome undergoes a rapid differentiation that is marked by replacement of the variant surface glycoprotein (VSG) coat with GPI-anchored EP and GPEET procyclins. Here we show that a cold shock of DeltaT > 15 degrees C is sufficient to reversibly induce high-level expression of the insect stage-specific EP gene in the mammalian bloodstream stages of T. brucei. The 3'-UTR of the EP mRNA is necessary and sufficient for the increased expression. During cold shock, EP protein accumulates in the endosomal compartment in the proliferating, slender, bloodstream stage, whereas the EP is present on the plasma membrane in the quiescent, stumpy, bloodstream stage. Thus, there is a novel developmentally regulated cell surface access control mechanism for a GPI-anchored protein. In addition to inducing EP expression, cold shock results in the acquisition of sensitivity to micromolar concentrations of cis-aconitate and citrate by stumpy but not slender bloodstream forms. The cis-aconitate and citrate commit stumpy bloodstream cells to differentiation to the procyclic stage along with rapid initial proliferation. We propose a hierarchical model of three events that regulate differentiation after transmission to the tsetse: sensing the temperature change, surface access of a putative receptor, and sensing of a chemical cue.

3' Untranslated Regions↗
PubMed1989

2-Oxoglutarate: oxidation and role as a potential precursor of cytosolic acetyl-CoA for the synthesis of acetylcholine in rat brain synaptosomes.

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.

ATP Citrate (pro-S)-Lyase↗
PubMed1981

Aconitase activity, citrate oxidation, and zinc inhibition in rat ventral prostate.

Aconitase activity and citrate oxidation were determined in mitochondrial preparations from rat ventral prostate. Aconitase activity in prostate mitochondria was approximately 10% of the activity observed in kidney preparations. The prostate aconitase activity was completely inhibited by the addition of 1.0 mmol/l zinc and kidney aconitase was inhibited by approximately 50%. The presence of 1.0 mmol/l zinc also markedly inhibited the formation of isocitrate from cis-aconitate. The reduction of NADP by citrate was markedly inhibited (approx. 95%) by 1.0 mmol/l zinc as compared with a 70 and 40% inhibition of aconitate--and isocitrate--stimulated reduction, respectively. These results indicate that zinc might be a strong inhibitor of aconitase activity with an inhibitory effect on isocitrate dehydrogenase also. The effect of 1.0 mmol/l zinc on citrate oxidation was studied by determining 14CO2 production from 6-14C-citrate. 14CO2 was significantly inhibited by zinc. These results further corroborate the conclusion that citrate accumulation in prostate might be the result of limited citrate oxidation which results from a combination of low aconitase activity and possibly zinc inhibition.

Aconitate Hydratase↗
PubMed1997

Commitment to differentiation and cell cycle re-entry are coincident but separable events in the transformation of African trypanosomes from their bloodstream to their insect form.

African trypanosomes undergo extensive changes in cellular morphology, biochemistry and surface antigen expression as they differentiate from their bloodstream form to those forms that colonise the midgut of their tsetse fly vector. If initiated with stumpy-form cells, a non-dividing sub-type of the bloodstream parasite, differentiation and cell cycle re-entry occur synchronously in the population and provide a means to dissect the respective controls of proliferation and transformation. We have exploited this synchrony to determine the respective importance and hierarchy of the known triggers for differentiation (cis aconitate, temperature drop) for individual components of both differentiation and the cell cycle. This has revealed the pre-eminence of cis aconitate as a primary trigger for parasite differentiation, and has allowed us to determine that the cellular commitment to both differentiation and cell-cycle re-entry are precisely co-incident processes.

Aconitic Acid↗
PubMed2001

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.

Aconitate Hydratase↗
PubMed1985

Alloxan effects on mitochondria in vitro, studied with regard to inhibition of mitochondrial aconitase.

The recently observed inhibition by alloxan of mitochondrial aconitase was studied with regard to oxygen consumption, accumulated Ca2+ and volume of isolated mitochondria from mouse islets (not used for respiratory experiments), liver and kidneys. Altered respiration, efflux of Ca2+ and volume increase were induced in a manner dependent on the concentration of alloxan, the energetic state of the mitochondria, the addition of Ca2+ and reductant, and the type and concentration of substrate used. State 3 and, less pronounced, state 4 respiration were inhibited, accumulated Ca2+ was released, and the mitochondrial volume was increased at a minimum concentration of alloxan of 10-100 microM when pyruvate, citrate or cis-aconitate were substrates. The alloxan effects were less marked or absent when other substrates were used. The efflux of Ca2+ induced by alloxan in mitochondria respiring on pyruvate, citrate or cis-aconitate was inhibited or reversed by isocitrate. Potentiation of the alloxan effects was seen on increasing the concentration of alloxan, decreasing the concentration of substrate, addition of low concentrations of Ca2+ or reductant, and addition of alloxan before the substrate (non-energized state). The data support the finding of inhibition by alloxan of mitochondrial aconitase.

Aconitate Hydratase↗
PubMed1997

Characterization and In Vitro Cytotoxic Effect of Adriamycin-conjugated Monoclonal Antibody Prepared Against Breast Cancer Cell Line.

We have produced an adriamycin-conjugated monoclonal antibody, AM1, covalently linked by acid-labile cis-aconitic spacer. The immunoconjugate, cis-aconitic adriamycin(cAA)-AM1, was confirmed to retain the binding activity against human breast cancer cell lines by flow cytometry. The immunoconjugate was shown to be internalized into antigen-positive cancer cells by flow cytometry analysis and high performance liquid chromatography. Antitumor effects of cAA-AM1 were assessed on human breast cancer and colon cancer cell lines, with inhibition of (3) H-leucine uptakes to the cells. cAA-AM1 demonstrated a selective cytotoxicity to ZR-75-1 which was reactive with AM1, whereas it showed no antitumor effect on SW1116 cells which did not react with AM1. Free adriamycin demonstrated a non-selective cytotoxicity against both cell lines. AM1 alone and cAA-control IgM did not show any antitumor effect on ZR-75-1. These results suggest that cAA-AM1 retains binding activity and specificity against breast cancer cells in vitro.

Journal Article↗