Human neutrophils employ chlorine gas as an oxidant during phagocytosis.

Reactive oxidants generated by phagocytes are of central importance in host defenses, tumor surveillance, and inflammation. One important pathway involves the generation of potent halogenating agents by the myeloperoxidase-hydrogen peroxide-chloride system. The chlorinating intermediate in these reactions is generally believed to be HOCl or its conjugate base, ClO-. However, HOCl is also in equilibrium with Cl2, raising the possibility that Cl2 executes oxidation/ halogenation reactions that have previously been attributed to HOCl/ClO-. In this study gas chromatography-mass spectrometric analysis of head space gas revealed that the complete myeloperoxidase-hydrogen peroxide-chloride system generated Cl2. In vitro studies demonstrated that chlorination of the aromatic ring of free L-tyrosine was mediated by Cl2 and not by HOCl/ClO-. Thus, 3-chlorotyrosine serves as a specific marker for Cl2-dependent oxidation of free L-tyrosine. Phagocytosis of L-tyrosine encapsulated in immunoglobulin- and complement-coated sheep red blood cells resulted in the generation of 3-chlorotyrosine. Moreover, activation of human neutrophils adherent to a L-tyrosine coated glass surface also stimulated 3-chlorotyrosine formation. Thus, in two independent models of phagocytosis human neutrophils convert L-tyrosine to 3-chlorotyrosine, indicating that a Cl2-like oxidant is generated in the phagolysosome. In both models, synthesis of 3-chlorotyrosine was inhibited by heme poisons and the peroxide scavenger catalase, implicating the myeloperoxidase-hydrogen peroxide system in the reaction. Collectively, these results demonstrate that myeloperoxidase generates Cl2 and that human neutrophils use an oxidant with characteristics identical to those of Cl2 during phagocytosis. Moreover, our observations suggest that phagocytes exploit the chlorinating properties of Cl2 to execute oxidative and cytotoxic reactions at sites of inflammation and vascular disease.

Photoaffinity labelling of submitochondrial membranes with the 3-azido analogue of 9-amino-3-chloro-7-methoxyacridine.

9-Amino-3-azido-7-methoxyacridine has been synthesized and shown to be a suitable photoaffinity probe for the site(s) of interaction of 9-amino-3-chloro-7-methoxyacridine with submitochondrial membranes. Both the excitation and emission spectra of the azido analogue covalently bound to membranes in the energized state display distinctive differences from the spectra of labelled, non-energized membranes (i.e., in the absence of oxidizable substrate, or its presence when uncoupler (FCCP) is also present during photolysis). Enzymatic analyses indicate that the probe interacts with the ATPase and the respiratory chain enzymes; energization appears to afford some protection against inactivation. Electrophoresis of the labelled membranes and isolation of their lipid and protein components indicate that the spectral differences are attributable to differing interactions with the lipid components of energized, relative to non-energized, membranes. Similar results have been obtained with the 3-azido analogue of quinacrine (Mueller, D.M., Hudson, R.A. and Lee, C.P. (1982) Biochemistry 21, 1445-1453), which differs significantly, however, in the extent of its interactions with the enzymes of the respiratory chain and the ATPase. These results indicate that the energy-linked fluorescence responses of 9-aminoacridines with submitochondrial membranes arise from direct interactions with membrane components and may involve redistribution of the probe molecules and/or alteration of their microenvironments upon energization.

Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media.

Two independent cold-sensitive pet mutants in the gene (ATP5) coding for the oligomycin sensitivity conferring protein (OSCP) have been isolated in the yeast Saccharomyces cerevisiae. The mutations in both strains alter the initiating methionine codon in the ATP5 gene: ATG to ATA (Ile) and AAG (Lys). Western blot analysis of total yeast protein after the cells were grown at 18 degrees C, 30 degrees C, and 37 degrees C, indicates that the level of OSCP decreased 80% relative to the wild type strain. In addition, the level of the oligomycin-sensitive ATPase decreased 85% relative to the wild type strain, after growth at 30 degrees C. These findings indicate that for S. cerevisiae, the level of oxidative phosphorylation can decrease 85% without showing a large growth defect on media containing glycerol at 30 degrees C, but not at 18 degrees C.

Increased expression of F1ATP synthase subunits in yeast strains carrying point mutations which destabilize the beta subunit.

In yeast strains (S. cerevisiae) carrying a point mutation of the ATP2 gene, which destabilizes the beta subunit of F1 ATP synthase in vitro, the growth rate was reduced significantly, demonstrating that the mutation is also deleterious in vivo. Immunoblots showed that levels of the mutated beta, but also of the wild-type alpha subunit were increased in the mutated strains, together with levels of the corresponding mRNAs (approximately 1.6-fold). Northern analysis showed that this was due to both the appearance of new transcript species as well as upregulation of the cognate transcripts, strongly indicating that the increase was probably due to activation of transcription. Levels of other mitochondrial proteins, e.g. cytochrome c oxidase, were unaffected. We conclude that a specific signal communicates the actual performance of the ATP synthase inside the mitochondria to the nuclear genes encoding its subunits.

Nitrogen dioxide radical generated by the myeloperoxidase-hydrogen peroxide-nitrite system promotes lipid peroxidation of low density lipoprotein.

Myeloperoxidase, a heme protein secreted by activated phagocytes, is present and enzymatically active in human atherosclerotic lesions. In the current studies, we explored the possibility that reactive nitrogen species generated by myeloperoxidase promote lipid peroxidation of low density lipoprotein (LDL) -- a modification that may render the lipoprotein atherogenic. We found that myeloperoxidase, an H2O2-generating system and nitrite (NO2-) peroxidized LDL lipids. The process required NO2- and each component of the enzymatic system; it was inhibited by catalase, cyanide and ascorbate, a potent scavenger of aqueous phase radicals. LDL peroxidation did not require chloride ion, and it was little affected by the hypochlorous acid scavenger taurine. Collectively, these results suggest that lipid peroxidation is promoted by a nitrogen dioxide radical-like species. These observations indicate that myeloperoxidase, by virtue of its ability to form reactive nitrogen intermediates, may promote lipid peroxidation and atherogenesis.

Mass spectrometric quantification of 3-chlorotyrosine in human tissues with attomole sensitivity: a sensitive and specific marker for myeloperoxidase-catalyzed chlorination at sites of inflammation.

Oxidative modification of proteins has been implicated in a variety of processes ranging from atherosclerosis to aging. Identifying the underlying oxidation pathways has proven difficult, however, due to the lack of specific markers for distinct oxidation pathways. Previous in vitro studies demonstrated that 3-chlorotyrosine is a specific product of myeloperoxidase-catalyzed oxidative damage and that the chlorinated amino acid may thus serve as an index of phagocyte-mediated tissue injury in vivo. Here we describe a highly sensitive and specific analytical method for the quantification of 3-chlorotyrosine content of tissues. The assay combines gas chromatography with stable isotope dilution mass spectrometry, and it detects attomole levels of 3-chlorotyrosine in a single determination. Furthermore, the method is highly reproducible, with inter- and intra-sample coefficients of variance of < 3%. The specificity, sensitivity, and reproducibility of 3-chlorotyrosine determination should make this method useful for exploring the role of myeloperoxidase in catalyzing oxidative reactions in vivo.

Partial assembly of the yeast mitochondrial ATP synthase.

The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation of ADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 different peptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, one of which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor. Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis that the yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, of the polypeptides that are thought to comprise the rotor. However, the enzyme complex assembled in the absence of the rotor is thought to be uncoupled, allowing protons to freely flow through F0 into the mitochondrial matrix. Left uncontrolled, this is a lethal process and the cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose or delete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encoding essential components of F0. Recent biochemical studies in yeast, and prior studies in E. coli, have provided support for the assembly of a partial ATP synthase in which the ATP synthase is no longer coupled to proton translocation.

Site directed mutagenesis of the beta-subunit of the yeast mitochondrial ATPase.

Site directed mutagenesis has been performed on the gene coding for the beta-subunit of the yeast mitochondrial F1-ATPase. Two different regions were studied. First, the corresponding yeast amino acid, Tyr-344, which was affinity labeled in the bovine enzyme was changed to Phe-344 and Ala-344. The Phe-344 enzyme was completely active and less sensitive to the affinity reagent, 4-chloro-7-nitrobenzofurazan. In contrast, the in vivo level of the Ala-344 enzyme was greatly diminished and apparently inactive. The second region studied is in the glycine rich region homologous in nucleotide binding proteins. Five different replacements were made and all mutations but one completely eliminated the biological activity and reduced the in vivo level of the mutant peptides. These results support the importance of these amino acids in the function of the ATPase.

A mutation altering the kinetic responses of the yeast mitochondrial F1-ATPase.

Nucleotide-binding proteins, including the mitochondrial F1-ATPase, the ras proteins, and the G-proteins, contain a homologous glycine-rich sequence that is thought to constitute part of the active site. This study reports the effects of a single amino acid replacement of Thr197 to Ser197, which is located at the hinge region of this putative loop, in the yeast Saccharomyces cerevisiae F1-ATPase. This replacement resulted in a 3-fold increase in the specific activity of the enzyme, eliminated the stimulatory effects of oxyanions, and modulated the effects of the inhibitor NaN3 while having little effect on the uni-site ATPase. These results indicate a role of the glycine-rich loop in many of the kinetic responses of the F1-ATPase.

Arginine 328 of the beta-subunit of the mitochondrial ATPase in yeast is essential for protein stability.

The mitochondrial ATPase is rapidly inactivated by the arginine selective reagent phenylglyoxal. Recently, the purported major reacting residue has been reported for the chloroplast enzyme (Viale, A. M., and Vallejos, R. H. (1985) J. Biol. Chem. 260, 4958-4962) corresponding to Arg-328 in the beta-subunit of the yeast Saccharomyces cerevisiae mitochondrial ATPase, a highly conserved residue in the ATPase. This arginine residue was concluded to be in the active site of the ATPase and possibly involved in the binding of nucleotides. To test this hypothesis, site-directed mutagenesis of the yeast enzyme has been used to replace Arg-328 with alanine and lysine. The modified genes were transformed into a yeast strain, DMY111, which contained a null mutation in the gene coding for the beta-subunit of the ATPase. Both of the substitutions were functional in vivo as demonstrated by the ability of yeast transformants to grow on a nonfermentable carbon source. The water soluble F1-ATPase with Ala-328 and Lys-328 were extremely unstable, but could be stabilized with glycerol. The rate of enzymatic decay followed first order kinetics with half-lives of 1.1 and 4.0 min for the mutants with Ala-328 and Lys-328 in 10% and 5% glycerol, respectively, while the wild type enzyme was stable even in the absence of glycerol. Kinetic analysis of both ATPase and GTPase has been determined. The wild type enzyme had two observable apparent Km and Vmax values for ATPase which were 0.056 mM-1 and 67 units/min/mg and 0.140 mM-1 and 100 units/min/mg. The mutant enzyme containing Lys-328 showed similar kinetic values of 0.066 mM-1 and 23 units/min/mg and 0.300 mM-1 and 43 units/min/mg. The mutant enzyme containing Ala-328, however, only demonstrated a single site with values of 0.121 mM-1 and 45 units/min/mg. In contrast to ATPase activity, kinetic values for GTPase were nearly identical for the wild type and mutant enzymes. Opposite to predicted results, the mutant enzymes were more sensitive to the reagent phenylglyoxal. These results indicate that Arg-328 is important for protein stability, but not involved in catalysis.

Steady state analysis of mitochondrial RNA after growth of yeast Saccharomyces cerevisiae under catabolite repression and derepression.

The steady state levels of mitochondrial rRNAs, 5 tRNAs, the 9 S RNA, and the RNA products from the genes coding for subunits 6 and 9 of the ATP synthase, cytochrome b, and subunit 1 of cytochrome oxidase have been determined after growth of yeast under conditions of respiratory repression or derepression. The analysis indicates that the mitochondrial rRNAs are present in 2000 or 9000 copies/cell in repressed or derepressed yeast, respectively. The levels of the other RNAs also differed to a similar extent, with the exception of the level of the tRNAfMet which differs by only 1.7-fold. The levels of the individual protein coding RNAs varied from 480 copies/cell for the Oli-1 RNA to 100 copies/cell for the Oli-2 RNA under derepressive conditions and from 130 copies/cell to 33 copies/cell for the same RNAs in glucose repressive conditions. The levels of the tRNAs varied even more markedly, ranging from 4200 copies/cell for the tRNAPhe to 240 copies/cell for the tRNACys after growth in derepressive conditions and from 800 copies/cell for the tRNAfMet to 30 copies/cell for the tRNACys of glucose repressed yeast. These results indicate that glucose repression uniformly decreases the levels of the individual mitochondrial RNAs studied. This decrease is related to a lower synthesis of mitochondrial RNA in the glucose repressed cells as compared to derepressed cells.

Transcriptional regulation of the mitochondrial genome of yeast Saccharomyces cerevisiae.

The relative rates of transcription of several classes of the mitochondrial genes of the yeast Saccharomyces cerevisiae have been determined. The rates were measured by pulse labeling whole yeast with [32P]O4, isolating the total RNA, hybridization to single-stranded M13 DNA probes containing segments of the gene of interest, digestion with RNase A or T1, and separation of the protected fragment by gel electrophoresis. This analysis indicated that, among the genes analyzed, transcriptional promoters varied in strength by 20-fold while the rates of transcription varied by more than 50-fold. The strengths of the promoters of the genes were ordered: tRNAMetf greater than tRNAPhe greater than 14 S rRNA greater than 21 S rRNA greater than tRNAGlu greater than Oli-1 much greater than tRNACys. In addition, transcription rates were measured within polygenic transcription units. This analysis indicated that there was transcriptional attenuation within all the polygenic transcription units with the greatest attenuation factor being as much as 17-fold, occurring after the tRNAGlu and tRNAMetf genes. This analysis indicated that regulation of the rates of transcription in the yeast mitochondrial genome occurs by two distinct mechanisms, modulation of the rate of transcriptional initiation and attenuation of transcriptional elongation.

Direct determination of the specific activity of RNA uniformly labeled with 32P.

A simple method for the direct determination of the specific activity of RNA uniformly labeled with 32P is described. The procedure is based on the premise that upon disintegration of 32P to 32S, the phosphodiester bond is broken. Analysis of the rate of decay of the full-length molecule by gel electrophoresis and autoradiography can accurately determine the "intramolecular specific activity" of the RNA. An equation that predicts the relative intensity of the intact RNA molecules remaining as a function of time is presented. These predictions are confirmed using in vitro-synthesized RNA labeled at a known specific activity. This procedure has been used to determine the intramolecular specific activity of RNA labeled in vivo in yeast. It can also be employed to choose the best conditions for experiments utilizing uniformly labeled RNA or single-stranded DNA and requiring the detection of intact molecules.

Structural interactions of the oligomycin sensitivity-conferring protein in the yeast ATP synthase.

The structure/function relationship of oligomycin sensitivity-conferring protein (OSCP), subunit 5 of the mitochondrial ATP synthase, from yeast Saccharomyces cerevisiae has been studied by a combination of genetic and biochemical methods. OSCP was studied by deletion mutagenesis of the N- and C-terminal regions by modifying the gene coding for OSCP. Two deletion mutations were made immediately downstream of the leader peptide of OSCP and five were made at the C-terminus. OSCP was functional with deletions of amino acids 3 to 17 (ND15) and of the last 8 amino acids (CD8), while deletion of amino acids 3 to 31 (ND29) and the last 9 amino acids (CD9) inactivated the ATP synthase, as determined by in vivo analysis. The deletion mutants were expressed in Escherichia coli, purified, and studied by in vitro reconstitution studies. Circular dichroism studies suggested that the mutant proteins, with the possible exception of ND29, were folded in a similar fashion as wild-type OSCP. Mutants ND15 and CD8 were able to reconstitute an oligomycin-sensitive ATPase complex, although not as well as wild-type OSCP, while ND29 and CD9 were completely ineffective. Binding studies of ND29 and CD9 indicate that these mutants in OSCP were unable to bind to the membrane portion of the ATP synthase, F0, and these results were supported by competition binding studies. These results support the hypothesis that the N- and C-terminal regions of subunit 5 interact with F0 and suggest that the central region interacts with F1.

Mutagenesis of beta-V198 in the F1-ATPase of yeast Saccharomyces cerevisiae and its role in binding nucleotide.

Residue beta-V198 of the yeast mitchondrial F1-ATPase abuts the P-loop motif and the side chain is within 3.8 A of the nucleotide as shown in the crystal structure of the bovine ATPase [J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1984) Nature 370,621-628]. This study has made and analyzed 17 replacements of V198 to understand the importance of the side chain in the nucleotide binding site. In addition, a suppressor of V198S, beta-L390F, was studied in the presence of various replacements at position 198. In vivo and in vitro analyses indicate that the Val side chain is critical for forming a stable and active enzyme. Biochemical analysis of mitochondria isolated from the mutant strains indicates that amino acids with hydrophobic side chains are the most effective replacements. In addition, size is important, but a large side chain can be largely compensated for until the size reaches that of the Phe and Trp. A methyl group is the minimal side chain necessary for function, as the beta-subunit is not stable in vivo with Gly at position 198. These results indicate that V198 forms critical hydrophobic interactions with the adenine ring of the nucleotide.

Complementation of deletion mutants in the genes encoding the F1-ATPase by expression of the corresponding bovine subunits in yeast S. cerevisiae.

The F1F0 ATP synthase is composed of the F1-ATPase which is bound to F0, in the inner membrane of the mitochondrion. Assembly and function of the enzyme is a complicated task requiring the interactions of many proteins for the folding, import, assembly, and function of the enzyme. The F1-ATPase is a multimeric enzyme composed of five subunits in the stoichiometry of alpha3beta3gammadeltaepsilon. This study demonstrates that four of the five bovine subunits of the F1-ATPase can be imported and function in an otherwise yeast enzyme effectively complementing mutations in the genes encoding the corresponding yeast ATPase subunits. In order to demonstrate this, the coding regions of each of the five genes were separately deleted in yeast providing five null mutant strains. All of the strains displayed negative or a slow growth phenotype on medium containing glycerol as the carbon source and strains with a null mutation in the gene encoding the gamma-, delta- or epsilon-gene became completely, or at a high frequency, cytoplasmically petite. The subunits of bovine F1 were expressed individually in the yeast strains with the corresponding null mutations and targeted to the mitochondrion using a yeast mitochondrial leader peptide. Expression of the bovine alpha-, beta-, gamma-, and epsilon-, but not the delta-, subunit complemented the corresponding null mutations in yeast correcting the corresponding negative phenotypes. These results indicate that yeast is able to import, assemble subunits of bovine F1-ATPase in mitochondria and form a functional chimeric yeast/bovine enzyme complex.