Recognition of glycoconjugates byHelicobacter pylori: an apparently high-affinity binding of human polyglycosylceramides,a second sialic acid-based specificity

Helicobacter pylori has been reported to agglutinate erythrocytes and to bind to various other cells in a sialic acid-dependent way. The binding was inhibited by sialyllactose or fetuin and other sialylated glycoproteins. The specificity apparently requires bacterial growth on agar, since we found that it was lost after growth in the nutrient mixture Ham's F12. Instead, the bacteria bound with high affinity and in a sialic acid-dependent way to polyglycosylceramides of human erythrocytes, a still incompletely characterized group of complex glycolipids.Bacteria grown in F12 medium were metabolically labelled with³⁵S-methionine and analysed for binding to glycolipids on thin-layer chromatograms and to glycoproteins on blots after electrophoresis, with human erythrocyte glycoconjugates in focus. There was no binding to simpler gangliosides including GM3 or sialylparagloboside, or to a mixture of brain gangliosides. In contrast, polyglycosylceramides of human erythrocyte membranes bound at a pmol level. The activity was eliminated by mild acid treatment, mild periodate oxidation or sialidase hydrolysis. Erythrocyte proteins as well as a range of reference glycoproteins did not bind, except band 3, which was weakly active. However, this activity was resistant to periodate oxidation.These results indicate a second and novel sialic acid-recognizing specificity which is expressed independently of the previously described specificity. Abbreviations: PGCs, polyglycosylceramides; TLC, thin-layer chromatography; C, chloroform; M, methanol; EI/MS, electron impact ionization mass spectrometry, SDS PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; BSA, bovine serum albumin. The carbohydrate and glycosphingolipid nomenclatures are according to recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (Lipids (1977)12: 455–68;J Biol Chem (1982)257: 3347–51 andJ Biol Chem (1987)262: 13–18).This paper is dedicated to Professor S.-i. Hakomori and is paper no. 1 from our research onHelicobacter pylori.     

Screening for the presence of polyglycosylceramides in various tissues: partial characterization of blood group-active complex glycosphingolipids of rabbit and dog small intestines

Twenty different human and animal tissues were investigated for the presence of polyglycosylceramides. The glycolipids were isolated by peracetylation of dry tissue residues left after conventional lipid extraction, followed by extraction with chloroform and subsequent Sephadex LH-20, Sephadex LH-60 and silica gel chromatography. In most of the cases only trace amounts of complex glycolipids were found. Distinct bands of glycosphingolipids migrating on TLC plates in a region of brain gangliosides and below were observed in bovine erythrocytes, human leukocytes and human colon mucosa. Definite fractions of polyglycosylceramides were isolated from rabbit small intestine, dog small intestine, human placenta and human leukocytes. The polyglycosylceramides of dog and rabbit intestine were characterized by colorimetric analysis, methylation analysis, mass spectrometry and immunological assays. The dog material contained branched carbohydrate chains with repeated fucosylated N-acetyllactosamine units. Rabbit intestine polyglycosylceramides resembled rabbit erythrocyte polyglycosylceramides with Hex-Hex- terminal determinants but were more complex in respect of sugar composition and structure. The material isolated from dog intestine showed A, H, Lex and Ley blood group activities. Polyglycosylceramides of human erythrocytes, placenta and leukocytes showed strong binding affinity for Helicobacter pylori, while polyglycosylceramide fractions from rabbit and dog intestine were receptor-inactive for this bacterium or displayed only weak and poorly reproducible binding. Abbreviations: C, chloroform; M, methanol; Hex, hexose; HexNAc, N-acetylhexosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolylneuraminic acid; TLC-thin layer chromatography; FAB/MS, fast atom bombardment mass spectrometry; GC/MS, gas chromatography-mass spectrometry; PGCs, polyglycosylceramides; EI/MS, electron impact ionization mass spectrometry; PBS, phosphate-buffered saline; BSA, bovine serum albumin. The carbohydrate and glycosphingolipid nomenclatures are according to recommendations of IUPAC-IUB Commission on Biochemical Nomenclature (Lipids (1977) 12:455–68; J Biol Chem (1982) 257:3347–51; and J Biol Chem (1987) 262:13–18) This revised version was published online in November 2006 with corrections to the Cover Date.     

Isolated erythroglycans have a high-affinity interaction with wheat germ agglutinin but are poorly accessible in situ

The sugar and cell specificities of wheat germ agglutinin have been studied extensively. In particular, it is well established that wheat germ agglutinin will interact with highly sialylated glycoconjugates of the type carried by the erythrocyte glycoprotein, glycophorin (Adair, W.L. and Kornfeld, S. (1974) J. Biol. Chem. 249, 4696-4704). We have found that polylactosamines isolated from adult and fetal erythrocytes can have a high-affinity interaction with immobilized wheat germ agglutinin. In fact, this interaction is much stronger than the sialic acid-dependent interaction. Using flow microfluorimetry in conjunction with various serological and enzymatic pretreatments, we have measured the extent to which polylactosamines contribute to wheat germ agglutinin binding. We have found that most of the neuraminidase-resistant receptors on erythrocytes are polylactosamine in nature. However, this residual binding of wheat germ agglutinin to neuraminidase-treated erythrocytes is of much lower apparent affinity than the sialic acid-dependent interaction. The lower reactivity of polylactosamines at the erythrocyte surface suggests that these large glycans are actually poorly accessible.     

Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures

Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation.     

SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.     

Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori.

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of 相似文献   

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: “2nd trimester” (14–19 weeks), “3rd trimester” (29–37 weeks), “perinatal period” (38–40 weeks) , “delivery at term” (39–41 weeks) and “post date pregnancy” (41–43 weeks). In the “3rd trimester” the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by α2-6-linked sialic acid (p < 0.00002) and by α1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked α1-3 (p < 0.02). At the perinatal period the relative amount of α2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the “3rd trimester” group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.     

Investigation of the lectin-like binding domains in pertussis toxin using synthetic peptide sequences. Identification of a sialic acid binding site in the S2 subunit of the toxin.

Synthetic peptides corresponding to selected sequences in the S2 and S3 subunits of pertussis toxin were prepared and evaluated for their ability to inhibit the binding of biotinylated pertussis toxin and three biotinylated sialic acid specific plant lectins to fetuin and asialofetuin. The screening results indicated that two regions in the S2 subunit corresponding to amino acids 78-98 and 123-154 inhibited pertussis toxin binding to fetuin at submillimolar concentrations, while S3 sequences corresponding to amino acids 87-108 and 134-154 inhibited pertussis toxin-biotin binding to asialofetuin albeit with lower affinity. These results confirm earlier findings, which suggest that the S2 subunit is responsible for binding sialylated glycoconjugates. This was further confirmed by the ability of S2 peptides to inhibit the binding of the lectins from Maackia amurensis and wheat germ to fetuin. Two additional peptides from the S2 subunit of pertussis toxin corresponding to sequences 9-23 and 1-23 were found to contain within their sequences a 6-amino acid fragment which has strong homology with a sequence in wheat germ agglutinin that has been shown to be a component of the sialic acid binding site as determined by x-ray crystallography. One of these sequences from S2 (9-23) was biotinylated and evaluated for its ability to bind to carbohydrate. Through a series of experiments using fetuin, asialofetuin, asialoagalactofetuin, and simple saccharides, the biotinylated peptide was shown to bind with high affinity to sialic acid-containing glycoconjugates indicating that these sequences within the S2 subunit of pertussis toxin also play an important role in binding sialic acid.     

Structural differences between the putative carbohydrate-recognition domains of human IL-1 alpha, IL-1 beta and IL-1 receptor antagonist obtained by in silico modeling

In a previous report (Cebo et al. J Biol Chem 276 (2001) 5685–5691), it was established that biologically active recombinant human IL-1α and IL-1β had different carbohydrate-binding properties. IL-1α recognized a di-antennary N-glycan with two α2-3-linked sialic acid residues, whereas IL-1β recognized the GM₄, a α2-3-linked sialylated glycosphingolipid. These different carbohydrate-binding properties of two interleukins binding to the same receptor (IL-1R) could explain why these molecules had different biological effects and cell specificities. Molecular modeling of the ligands and in silico docking experiments defined putative carbohydrate-recognition domains localized in the same area of the two molecules, a domain different from that defined as the type I IL-1R binding domain. The calculated pattern of hydrogen bonding and of van der Waals interactions fulfilled the essential features observed for calcium-independent lectins (mammalian, viral or bacterial). The analysis of the same domain of the third members of this family of molecules, the IL-1R-antagonist, indicated it did not fulfill the criteria for carbohydrate-recognition domains. It is proposed that its role as a pure antagonist is due to the absence of lectin activity and consequently explained its inability to associate IL-1R with other surface molecular complexes necessary for signaling.     

1H, 13C and 15N NMR assignments of the C1A and C1B subdomains of PKC-delta

The Protein Kinase C family of enzymes is a group of serine/threonine kinases that play central roles in cell-cycle regulation, development and cancer. A key step in the activation of PKC is translocation to membranes and binding of membrane-associated activators including diacylglycerol (DAG). Interaction of novel and conventional isotypes of PKC with DAG and phorbol esters occurs through the two C1 regulatory domains (C1A and C1B), which exhibit distinct ligand binding selectivity that likely controls enzyme activation by different co-activators. PKC has also been implicated in physiological responses to alcohol consumption and it has been proposed that PKCα (Slater et al. J Biol Chem 272(10):6167–6173, 1997; Slater et al. Biochemistry 43(23):7601–7609, 2004), PKCε (Das et al. Biochem J 421(3):405–413, 2009) and PKCδ (Das et al. J Biol Chem 279(36):37964–37972, 2004; Das et al. Protein Sci 15(9):2107–2119, 2006) contain specific alcohol-binding sites in their C1 domains. We are interested in understanding how ethanol affects signal transduction processes through its affects on the structure and function of the C1 domains of PKC. Here we present the ¹H, ¹⁵N and ¹³C NMR chemical shift assignments for the Rattus norvegicus PKCδ C1A and C1B proteins.     

The potential role of sialoadhesin as a macrophage recognition molecule in health and disease

Sialoadhesin is a macrophage-restricted transmembrane glycoprotein of 185 kDa that mediates cell–cell interactions through recognition of Neu5Acα2,3Gal in glycoconjugates. The extracellular region of sialoadhesin is composed of seventeen immunoglobulin-like domains, of which the amino-terminal two are highly-related structurally and functionally to the amino-terminal domains of CD22, myelin associated glycoprotein and CD33. These proteins, collectively known as the sialoadhesin family, are able to mediate sialic acid-dependent binding with distinct specificities for both the type of sialic acid and its linkage to subterminal sugars. In this review we discuss our recent studies on sialoadhesin and suggest how this molecule may contribute to a range of macrophage functions, both under normal conditions as well as during inflammatory reactions. Abbreviations: Ig, immunoglobulin; CEA, carcinoembryonic antigen; MAG, myelin associated glycoprotein; SMP Schwann cell myelin protein; mAb, monoclonal antibody; Chinese hamster ovary (CHO); UTR, untranslated region This revised version was published online in November 2006 with corrections to the Cover Date.     

Metabolism of diazirine-modified N-acetylmannosamine analogues to photo-cross-linking sialosides

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photo-cross-linking approach in which a diazirine photo-cross-linker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogues in which the length of the linker between the pyranose ring and the diazirine was varied. These analogues were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogues could be incorporated into both α2-3 and α2-6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently cross-linked to their interaction partners. We demonstrate that all three diazirine-modified analogues were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogues were less efficiently metabolized, yet could still function as effective cross-linkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogues to glycans other than sialosides, providing fidelity to selectively incorporate the cross-linker into sialylated molecules. These compounds are nontoxic and display only minimal growth inhibition at the concentrations required for cross-linking studies. This report provides essential information for the deployment of photo-cross-linking analogues to capture and study ephemeral, yet essential, sialic acid-mediated interactions.     

Development of an enzyme-linked glycosorbent method to monitor the inhibition of sialic acid-dependent Helicobacter pylori adhesion

Horseradish peroxidase conjugation with fetuin, which expresses sialic acid-dependent binding specificity to Helicobacter pylori, was used to develop an enzyme-linked glycosorbent method. This method yielded results that were consistent with those from a hemagglutination assay using a microscope and allowed the quantitative analysis of inhibitors of sialic acid-dependent Helicobacter pylori adhesion to host cells. The results of inhibitor screening with carbohydrates, including commercially available polysaccharides and extracted from various sources, displayed not only the relative inhibition potencies among carbohydrates, but also their respective concentration-dependencies.     

Selective binding by Helicobacter pylori of leucocyte gangliosides with 3-linked sialic acid, as identified by a new approach of linkage analysis

The human gastric pathogen Helicobacter pylori has been shown to bind to glycoconjugates of human leucocytes in a sialic acid-dependent way. In order to improve the identification of the binding epitope, a new technique was developed to analyze the ketosidic linkage position between a terminal sialic acid and the consecutive monosaccharide. Permethylation and reduction with LiAlH4 followed by trifluoroacetolysis in 1000:1 trifluoroacetic anhydride:trifluoroacetic acid (24 h, 100 °C) results in the cleavage of glycosidic but not ketosidic bonds. The disaccharide products were analyzed by gas chromatography-mass spectrometry and sialyl-3 or -6 position and NeuAc or NeuGc are identified by their separate retention times and mass spectra. The method was worked out on model saccharides and applied on five-sugar gangliosides (sialylparaglobosides) of human leucocytes. Radiolabeled Helicobacter pylori was shown to bind to the upper part, but not to the lower part, of the five-sugar interval of a mixture of gangliosides separated on a thin-layer chromatogram. Using a membrane blotting procedure the active and inactive bands were isolated and shown to be NeuAcα2-3- and NeuAcα2-6-paraglobosides, respectively.     

Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues.

Sialic acids are critical components of many glycoconjugates involved in biologically important ligand-receptor interactions. Quantitative and structural variations of sialic acid residues can profoundly affect specific cell-cell, pathogen-cell, or drug-cell interactions, but manipulation of sialic acids in mammalian cells has been technically limited. We describe the finding of a previously unrecognized and efficient uptake and incorporation of sialic acid analogues in mammalian cells. We added 16 synthetic sialic acid analogues carrying distinct C-1, C-5, or C-9 substitutions individually to cell cultures of which 10 were readily taken up and incorporated. Uptake of C-5- and C-9-substituted sialic acids resulted in the structural modification of up to 95% of sialic acids on the cell surface. Functionally, binding of murine sialic acid-binding immunoglobulin-like lectin-2 (Siglec-2, CD22) to cells increased after N-glycolylneuraminic acid treatment, whereas 9-iodo-N-acetylneuraminic acid abolished binding. Furthermore, susceptibility to infection by the B-lymphotropic papovavirus via a sialylated receptor was markedly enhanced following pretreatment of host cells with selected sialic acid analogues including 9-iodo-N-acetylneuraminic acid. This novel experimental strategy allows for an efficient biosynthetic engineering of surface sialylation in living cells. It is versatile, extending the repertoire of modification sites at least to C-9 and enables detailed structure-function studies of sialic acid-dependent ligand-receptor interactions in their native context.     

Inhibition of influenza A virus hemagglutinin and induction of interferon by synthetic sialylated glycoconjugates.

Multivalent forms of neoglycoproteins and polyacrylamides containing sialic acid were prepared and shown to be potent inhibitors of influenza A virus (H3N2) hemagglutinin with chick red blood cells. The synthetic sialylated glycoconjugates, although they were neuraminidase substrates, did not suppress viral neuraminidase and did not reduce infectivities in chick embryos. The copolyacrylamide conjugate containing a spacer group of approximately 11 A (1 A = 0.1 nm) between the polymer backbone and the sialic acid residues was the best hemagglutinin inhibitor. Moreover, it exhibited promising interferon-inducing properties.     

The heme environment of mouse neuroglobin: histidine imidazole plane orientations obtained from solution NMR and EPR spectroscopy as compared with X-ray crystallography

The ¹H NMR chemical shifts of the heme methyl groups of the ferriheme complex of metneuroglobin (Du et al. in J. Am. Chem. Soc. 125:8080–8081, 2003) predict orientations of the axial histidine ligands (Shokhirev and Walker in J. Biol. Inorg. Chem. 3:581–594, 1998) that are not consistent with the X-ray data (Vallone et al. in Proteins Struct. Funct. Bioinf. 56:85–94, 2004), and the EPR spectrum (Vinck et al. in J. Am. Chem. Soc. 126:4516–4517, 2004) is only marginally consistent with these data. The reasons for these inconsistencies appear to be rooted in the high degree of aqueous solution exposure of the heme group and the fact that there are no strong hydrogen-bond acceptors for the histidine imidazole N–H protons provided by the protein. Similar inconsistencies may exist for other water-soluble heme proteins, and ¹H NMR spectroscopy provides a simple means to verify whether the solution structure of the heme center is the same as or different from that in the crystalline state.     

Adhesion ofHelicobacter pylori strains toα-2,3-linked sialic acids

Helicobacter pylori is a human pathogen associated with gastritis and peptic ulcer. Adhesion properties ofH. pylori to various structures have been described in the literature, including evidence for sialic acid-binding. To study the specificity and frequency of sialic acid-binding, fourteenH. pylori strains were investigated using haemagglutination with derivatized erythrocytes carrying sialic acids only on defined glycans and using haemagglutination inhibition assays. From these studiesH. pylori strains can be grouped into sialic acid-dependent and sialic acid-independent classes. The sialic acid-dependent strains require -2,3-linked sialic acid for haemagglutination. The potential roles of sialic acid-dependent adhesions forH. pylori-related infections are discussed.Abbreviations Sia sialic acids - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolylneuraminic acid - Neu5Fm N-formylneuraminic acid - Neu5TFA N-trifluoroacetylneuraminic acid - RBC human red blood cells (erythrocytes)     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.