Tricarboxylic Acid Metabolite Imbalance in Rats with Acute Thioacetamide-Induced Hepatic Encephalopathy Indicates Incomplete Recovery.

Exposure to the toxin thioacetamide (TAA) causes acute hepatic encephalopathy (HE), changes in the functioning of systemic organs, and an imbalance in a number of energy metabolites. The deferred effects after acute HE development are poorly understood. The study considers the balance of the tricarboxylic acid (TCA) cycle metabolites in the blood plasma, liver, kidneys, and brain tissues of rats in the post-rehabilitation period. The samples of the control (n = 3) and TAA-induced groups of rats (n = 13) were collected six days after the administration of a single intraperitoneal TAA injection at doses of 200, 400, and 600 mg/kg. Despite the complete physiological recovery of rats by this date, a residual imbalance of metabolites in all the vital organs was noted. The results obtained showed a trend of stabilizing processes in the main organs of the animals and permit the use of these data both for prognostic purposes and the choice of potential therapeutic agents.

Residual Amino Acid Imbalance in Rats during Recovery from Acute Thioacetamide-Induced Hepatic Encephalopathy Indicates Incomplete Healing.

The delayed consequences of the influence of hepatic encephalopathy (HE) on the metabolism of animals have not been studied enough. We have previously shown that the development of acute HE under the influence of the thioacetamide (TAA) toxin is accompanied by pathological changes in the liver, an imbalance in CoA and acetyl CoA, as well as a number of metabolites of the TCA cycle. This paper discusses the change in the balance of amino acids (AAs) and related metabolites, as well as the activity of glutamine transaminase (GTK) and ω-amidase enzymes in the vital organs of animals 6 days after a single exposure to TAA. The balance of the main AAs in blood plasma, liver, kidney, and brain samples of control (n = 3) and TAA-induced groups (n = 13) of rats that received the toxin at doses of 200, 400, and 600 mg/kg was considered. Despite the apparent physiological recovery of the rats at the time of sampling, a residual imbalance in AA and associated enzymes persisted. The data obtained give an idea of the metabolic trends in the body of rats after their physiological recovery from TAA exposure and may be useful for prognostic purposes when choosing the necessary therapeutic agents.

Kinetic and Regulatory Properties of Yarrowia lipolytica Aconitate Hydratase as a Model-Indicator of Cell Redox State under pH Stress.

This paper presents an analysis of the regulation activity of the partially purified preparations of cellular aconitate hydratase (AH) on the yeast Yarrowia lipolytica cultivated at extreme pH. As a result of purification, enzyme preparations were obtained from cells grown on media at pH 4.0, 5.5, and 9.0, purified by 48-, 46-, and 51-fold and having a specific activity of 0.43, 0.55 and 0.36 E/mg protein, respectively. The kinetic parameters of preparations from cells cultured at extreme pH demonstrated: (1) an increase in the affinity for citrate and isocitrate; and (2) a shift in the pH optima to the acidic and alkaline side in accordance with the modulation of the medium pH. The regulatory properties of the enzyme from cells subjected to alkaline stress showed increased sensitivity to Fe2+ ions and high peroxide resistance. Reduced glutathione (GSH) stimulated AH, while oxidized glutathione (GSSG) inhibited AH. A more pronounced effect of both GSH and GSSG was noted for the enzyme obtained from cells grown at pH 5.5. The data obtained provide new approaches to the use of Y. lipolytica as a model of eukaryotic cells demonstrating the development of a stress-induced pathology and to conducting a detailed analysis of enzymatic activity for its correction.

Preparative Biocatalytic Synthesis of α-Ketomethylselenobutyrate-A Putative Agent for Cancer Therapy.

Biomedical studies of the role of organic selenium compounds indicate that the amino acid derivative of L-selenomethionine, α-ketomethylselenobutyrate (KMSB), can be considered a potential anticancer therapeutic agent. It was noted that, in addition to a direct effect on redox signaling molecules, α-ketoacid metabolites of organoselenium compounds are able to change the status of histone acetylation and suppress the activity of histone deacetylases in cancer cells. However, the wide use of KMSB in biomedical research is hindered not only by its commercial unavailability, but also by the fact that there is no detailed information in the literature on possible methods for the synthesis of this compound. This paper describes in detail the procedure for obtaining a high-purity KMSB preparation (purity ≥ 99.3%) with a yield of the target product of more than 67%. L-amino acid oxidase obtained from C. adamanteus was used as a catalyst for the conversion of L-selenomethionine to KMSB. If necessary, this method can be used as a basis both for scaling up the synthesis of KMSB and for developing cost-effective biocatalytic technologies for obtaining other highly purified drugs.

The metabolic importance of the glutaminase II pathway in normal and cancerous cells.

In rapidly dividing cells, including many cancer cells, l-glutamine is a major energy source. Utilization of glutamine is usually depicted as: l-glutamine → l-glutamate (catalyzed by glutaminase isozymes; GLS1 and GLS2), followed by l-glutamate → α-ketoglutarate [catalyzed by glutamate-linked aminotransferases or by glutamate dehydrogenase (GDH)]. α-Ketoglutarate is a major anaplerotic component of the tricarboxylic acid (TCA) cycle. However, the glutaminase II pathway also converts l-glutamine to α-ketoglutarate. This pathway consists of a glutamine transaminase coupled to ω-amidase [Net reaction: l-Glutamine + α-keto acid + H2O → α-ketoglutarate + l-amino acid + NH4+]. This review focuses on the biological importance of the glutaminase II pathway, especially in relation to metabolism of cancer cells. Our studies suggest a component enzyme of the glutaminase II pathway, ω-amidase, is utilized by tumor cells to provide anaplerotic carbon. Inhibitors of GLS1 are currently in clinical trials as anti-cancer agents. However, this treatment will not prevent the glutaminase II pathway from providing anaplerotic carbon derived from glutamine. Specific inhibitors of ω-amidase, perhaps in combination with a GLS1 inhibitor, may provide greater therapeutic efficacy.

A novel efficient producer of human ω-amidase (Nit2) in Escherichia coli.

Nit2/ω-amidase catalyzes the hydrolysis of α-ketoglutaramate (KGM, the α-keto acid analogue of glutamine) to α-ketoglutarate and ammonia. The enzyme also catalyzes the amide hydrolysis of monoamides of 4- and 5-C-dicarboxylates, including α-ketosuccinamate (KSM, the α-keto acid analogue of asparagine) and succinamate (SM). Here we describe an inexpensive procedure for high-yield expression of human Nit2 (hNit2) in Escherichia coli and purification of the expressed protein. This work includes: 1) the design of a genetic construct (pQE-Nit22) obtained from the previously described construct (pQE-Nit2) by replacing rare codons within an 81 bp-long DNA fragment "preferred" by E. coli near the translation initiation site; 2) methods for producing and maintaining the pQE-Nit22 construct; 3) purification of recombinant hNit2; and 4) activity measurements of the purified enzyme with KGM and SM. Important features of the hNit2 gene within the pQE-Nit22 construct are: 1) optimized codon composition, 2) the presence of an N-terminus His6 tag immediately after the initiating codon ATG (Met) that permits efficient purification of the end-product on a Ni-NTA-agarose column. We anticipate that the availability of high yield hNit2/ω-amidase will be helpful in elucidating the normal and pathological roles of this enzyme and in the design of specific inhibitors.

Preparative Biocatalytic Synthesis of α-Ketoglutaramate.

α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations.

Changes of Coenzyme A and Acetyl-Coenzyme A Concentrations in Rats after a Single-Dose Intraperitoneal Injection of Hepatotoxic Thioacetamide Are Not Consistent with Rapid Recovery.

Small biomolecules, such as coenzyme A (CoA) and acetyl coenzyme A (acetyl-CoA), play vital roles in the regulation of cellular energy metabolism. In this paper, we evaluated the delayed effect of the potent hepatotoxin thioacetamide (TAA) on the concentrations of CoA and acetyl-CoA in plasma and in different rat tissues. Administration of TAA negatively affects liver function and leads to the development of hepatic encephalopathy (HE). In our experiments, rats were administered a single intraperitoneal injection of TAA at doses of 200, 400, or 600 mg/kg. Plasma, liver, kidney, and brain samples were collected six days after the TAA administration, a period that has been suggested to allow for restoration of liver function. The concentrations of CoA and acetyl-CoA in the group of rats exposed to different doses of TAA were compared to those observed in healthy rats. The results obtained indicate that even a single administration of TAA to rats is sufficient to alter the physiological balance of CoA and acetyl-CoA in the plasma and tissues of rats for an extended period of time. The initial concentrations of CoA and acetyl-CoA were not restored even after the completion of the liver regeneration process.

Real-time multiparameter study of mitochondrial functions: Instrumental and analytical approaches.

In modern biomedical science, a descriptive study is no longer the major focus of many fields. More researchers are now seeking approaches that will help them obtain maximum information from a single sample or model, which will allow them to make more detailed conclusions than previously about mechanisms that underlie certain phenomena. Clearly, simultaneous measurement of multiple parameters will provide more useful information compared to that which can be assessed through parallel studies with multiple single-parameter measurements. Mitochondria are actively involved in the regulation of a number of biochemical processes that are vitally important for normal cell functioning. Dysregulation of cell metabolism occurs under multiple pathological conditions. While changes in mitochondrial and cellular functioning are related to each other, understanding of the details of most mechanisms underlying these relationships are still unknown. It would be appropriate to have an instrument that will help to uncover sequences of events and temporal links among the parameters that involve functional mitochondrial and cellular integration. The current review is focused on the analysis of these technological limitations, and, based on the combined approach, provides hypothetical suggestion on how possibly to create such an instrument.

Determination of Coenzyme A and Acetyl-Coenzyme A in Biological Samples Using HPLC with UV Detection.

Coenzyme A (CoA) and acetyl-coenzyme A (acetyl-CoA) play essential roles in cell energy metabolism. Dysregulation of the biosynthesis and functioning of both compounds may contribute to various pathological conditions. We describe here a simple and sensitive HPLC-UV based method for simultaneous determination of CoA and acetyl-CoA in a variety of biological samples, including cells in culture, mouse cortex, and rat plasma, liver, kidney, and brain tissues. The limits of detection for CoA and acetyl-CoA are >10-fold lower than those obtained by previously described HPLC procedures, with coefficients of variation <1% for standard solutions, and 1-3% for deproteinized biological samples. Recovery is 95-97% for liver extracts spiked with Co-A and acetyl-CoA. Many factors may influence the tissue concentrations of CoA and acetyl-CoA (e.g., age, fed, or fasted state). Nevertheless, the values obtained by the present HPLC method for the concentration of CoA and acetyl-CoA in selected rodent tissues are in reasonable agreement with literature values. The concentrations of CoA and acetyl-CoA were found to be very low in rat plasma, but easily measurable by the present HPLC method. The method should be useful for studying cellular energy metabolism under normal and pathological conditions, and during targeted drug therapy treatment.

Simultaneous determination of tricarboxylic acid cycle metabolites by high-performance liquid chromatography with ultraviolet detection.

Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.

HPLC determination of α-ketoglutaramate [5-amino-2,5-dioxopentanoate] in biological samples.

α-Ketoglutaramate is an important glutamine metabolite in mammals, plants, and many bacteria. It is also a nicotine metabolite in certain bacteria. Previously published methods for the determination of α-ketoglutaramate in biological samples have considerable drawbacks. Here, we describe a relatively simple high-performance liquid chromatography (HPLC)-based method for measurement of α-ketoglutaramate in plasma and deproteinized tissues that overcomes these drawbacks. Concentrations of α-ketoglutaramate in normal rat liver, kidney, brain, and plasma were found to be approximately 216, 13, 6, and 19 µM, respectively. The HPLC method should be useful for studying the role of α-ketoglutaramate in eukaryotic glutamine metabolism and in bacterial nicotine metabolism.

ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases.

In mammals, two major routes exist for the metabolic conversion of L-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of L-glutamine to L-glutamate catalyzed by glutaminases, followed by the conversion of L-glutamate to α-ketoglutarate by the action of an L-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of L-glutamine to α-ketoglutarate (the glutaminase II pathway) in which L-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of L-asparagine to oxaloacetate. In the most extensively studied pathway, L-asparagine is hydrolyzed to L-aspartate by the action of asparaginase, followed by transamination of L-aspartate to oxaloacetate. However, another pathway also exists for the conversion of L-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, L-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways--ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The review also discusses a potential dichotomous function of ω-amidase as having a role in tumor progression. Finally, the possible role of KGM as a biomarker for hyperammonemic diseases is discussed.

Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate ß-lyase activity and can transaminate L-selenomethionine.

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate ß-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no ß-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate ß-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate ß-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to ß-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the ß-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze ß-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.

Energy Metabolites and Indicative Significance of α-Ketoglutarate and α-Ketoglutaramate in Assessing the Progression of Chronic Hepatoencephalopathy.

In the example of a rat model with chronic hepatoencephalopathy (HE), changes in the organ morphology of rats affect the balance of metabolites of the tricarboxylic acid (TCA) cycle and metabolites of the glutamine-glutamate (Gln-Glu) cycle, namely α-ketoglutarate (αKG) and α-ketoglutaramate (αKGM), as well as the enzymes associated with them, ω-amidase (ωA) and glutamine transaminase (GTK). This model of rats was obtained as a result of 2-22 weeks of consumption by animals of hepatotoxin thioacetamide (TAA) added to drinking water at a concentration of 0.4 g/L. The control (n = 26) and TAA-induced (n = 55) groups of rats consisted of 11 cohorts each. The control cohorts consisted of 2-4 rats, and the TAA-induced cohorts consisted of 4-7 individuals. Every two weeks, samples of blood plasma, liver, kidney, and brain tissues were taken from the next cohort of rats (a total of 320 samples). By the end of the experiment, irreversible morphological changes were observed in the organs of rats: the weight of the animals was reduced up to ~45%, the weight of the kidneys up to 5%, the brain up to ~20%, and the weight of the liver increased up to ~20%. The analysis revealed: (i) a decrease in the activity of ωA and GTK in the tissues of the brain, kidneys, and liver of rats with chronic HE (by ~3, 40, and 65% and ~10, 60, and 70%, respectively); and (ii) the appearance of a significant imbalance in the content of metabolites of the Gln-Glu cycle, αKG, and αKGM. It is indicative that a ~1.5-12-fold increase in the level of αKG in the blood plasma and tissues of the organs of rats with chronic HE was accompanied by a synchronous, ~1.2-2.5-fold decrease in the level of αKGM. The data obtained indicate an essential involvement of the Gln-Glu cycle in the regulation of energy metabolism in rats under conditions of chronic HE. Attention is focused on the significance of the αKG/αKGM ratio, which can act as a potential marker for diagnosing the degree of HE development.

New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase.

Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Chromatomass-Spectrometric Method for the Quantitative Determination of Amino- and Carboxylic Acids in Biological Samples.

A highly sensitive method for the qualitative and quantitative determination of amino- and carboxylic acids, as well as a number of urea and methionine cycle metabolites in the studied solutions, is presented. Derivatives (esterification) were obtained for amino acids by their reaction in a solution of 3 N of hydrochloric acid in n-butanol for 15 min at 65 °C and for carboxylic acids by their reaction with phenol in ethyl acetate with 3 N of hydrochloric acid for 20 min at 65 °C. Experimental work on the determination of individual metabolites was carried out using the HPLC-MS/MS method and included the creation of a library of spectra of the analyzed compounds and their quantitative determination. Multiplex methods have been developed for the quantitative analysis of the desired metabolites in a wide range of concentrations of 3-4 orders of magnitude. The approach to the analysis of metabolites was developed based on the method of the dynamic monitoring of multiple reactions of the formation of fragments for a mass analyzer with a triple quadrupole (QQQ). The effective chromatographic separation of endogenous metabolites was carried out within 13 min. The calibration curves of the analyzed compounds were stable throughout the concentration range and had the potential to fit below empirical levels. The developed methods and obtained experimental data are of interest for a wide range of biomedical studies, as well as for monitoring the content of endogenous metabolites in biological samples under various pathological conditions. The sensitivity limit of the methods for amino acids was about 4.8 nM and about 0.5 µM for carboxylic acids. Up to 19 amino- and up to 12 carboxy acids and about 10 related metabolites can be tested in a single sample.

Synthetic and natural polyanions induce cytochrome c release from mitochondria in vitro and in situ.

A synthetic polyanion composed of styrene, maleic anhydride, and methacrylic acid (molar ratio 56:37:7) significantly inhibited the respiration of isolated rat liver mitochondria in a time-dependent fashion that correlated with 1) collapse of the mitochondrial membrane potential and 2) high amplitude mitochondrial swelling. The process is apparently Ca(2+) dependent. Since it is blocked by cyclosporin A, the process is ascribed to induction of the mitochondrial permeability transition. In mitoplasts, i.e., mitochondria lacking their outer membranes, the polyanion rapidly blocked respiration. After incubation of rat liver mitochondria with the polyanion, cytochrome c was released into the incubation medium. In solution, the polyanion modified by conjugation with fluorescein formed a complex with cytochrome c. Addition of the polyanion to cytochrome c-loaded phosphatidylcholine/cardiolipin liposomes induced the release of the protein from liposomal membrane evidently due to coordinated interplay of Coulomb and hydrophobic interactions of the polymer with cytochrome c. We conclude that binding of the polyanion to cytochrome c renders it inactive in the respiratory chain due to exclusion from its native binding sites. Apparently, the polyanion interacts with cytochrome c in mitochondria and releases it to the medium through breakage of the outer membrane as a result of severe swelling. Similar properties were demonstrated for the natural polyanion, tobacco mosaic virus RNA. An electron microscopy study confirmed that both polyanions caused mitochondrial swelling. Exposure of cerebellar astroglial cells in culture to the synthetic polyanion resulted in cell death, which was associated with nuclear fragmentation.