Tetrazolium salts have become some of the most widely used tools in cell biology for measuring the metabolic activity of cells ranging from mammalian to microbial origin. With mammalian cells, fractionation studies indicate that the reduced pyridine nucleotide cofactor, NADH, is responsible for most MTT reduction and this is supported by studies with whole cells. MTT reduction is associated not only with mitochondria, but also with the cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane. The net positive charge on tetrazolium salts like MTT and NBT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential. However, second generation tetrazolium dyes that form water-soluble formazans and require an intermediate electron acceptor for reduction (XTT, WST-1 and to some extent, MTS), are characterised by a net negative charge and are therefore largely cell-impermeable. Considerable evidence indicates that their reduction occurs at the cell surface, or at the level of the plasma membrane via trans-plasma membrane electron transport. The implications of these new findings are discussed in terms of the use of tetrazolium dyes as indicators of cell metabolism and their applications in cell biology.

Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction.

Publisher Summary This chapter describes structural features and evolution of metazoan mitochondrial DNA (mtDNA) molecules. Throughout the evolution of metazoa, gene content of mitochondria-genomes is highly conserved, as has the close packing of genes. Most of the occasional sequence expansions that have occurred, by way of either repeated or noncoding unique sequences, are found in the control or putative control region, rather than being dispersed between genes. Of the 13 open reading frames recognized in the human mtDNA molecules, four (COI, COII, COIII, and Cyt b) are originally identified in regard to the proteins they encode, from similarities of their predicted amino acid sequences to known amino acid sequences of bovine proteins, and predicted amino acid sequences of yeast mt-protein genes. Among mtDNAs of vertebrates and higher invertebrates, there are genes that overlap. Some overlaps are among the 3′ ends of two genes that are encoded in opposite strands of the molecule. The extent of size reduction within metazoan mitochondrial-transfer RNA (mt-tRNA) gene sets strongly correlates with the degree to which the more variable secondary structure element-forming regions of mt-rRNA genes are lost.

Animal mitochondrial DNA: structure and evolution.

The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule ofDrosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A+T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtDNAs that is associated with initiation of second-strand DNA synthesis is not present inD. yakuba mtDNA. Introns are absent fromD. yakuba mitochondrial genes and there are few (0–31) intergenic nucleotides. The genes found inD. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although theD. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs.D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochrondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In theD. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense codon are used in theD. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C-A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in theD. yakuba mtDNA molecule where these nucleotides are compatible with function.

The mitochondrial DNA molecular of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code.

The mitochondrial genome: structure, transcription, translation and replication

Metabolism and function of coenzyme Q

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae

We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.

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The specificity of mitochondrial complex I for ubiquinones.

The universality of bioenergetic disease: the role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase.

Oxidation of 2,2,5-trimethyl-5-phenylseleno-1,3-dioxan-4,6-dione with m-chloroperbenzoic acid in methylene chloride gives a solution containing 2,2-dimethylspiro[1,3-dioxan-5,2'-oxiran]-4,6-dione and the unstable 2,2-dimethyl-5-methylene-1,3-dioxan-4,6-dione, which forms stable adducts with cyclohexadiene and cyclopentadiene. Flash vacuum pyrolysis of the cyclopentadiene adduct over the temperature range 460- 570° and detection of the components present in the pyrolysate by mass spectrometry showed that cyclopentadiene, acetone, carbon dioxide and methyleneketene (CH2=C=C=O) are formed; the last breaks down into acetylene and carbon monoxide at higher temperatures (520-570°). Infrared measurements on the pyrolysis products kept at liquid nitrogen temperature showed absorption near 2100 cm-1 which is attributed to methyleneketene. Reaction of the pyrolysate with aniline vapour or methanol vapour yielded acrylanilide and methyl acrylate respectively. Pyrolysis in the absence of trapping agents gave a glassy solid on an uncooled glass surface. This solid is considered to be formed by addition of methyleneketene to 2,2-dimethyl-5-methylene-1,3-dioxan-4,6- dione. Methanolysis of the solid and esterification with diazomethane gave dimethyl 2-methoxycarbonyl-4-methylenepentanedioate.

Methyleneketenes and methylenecarbenes. VII. Evidence for the pyrolytic generation of methyleneketene (propadienone)

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specificin vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH∶ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH∶ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group or groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.

Gabrielle L. McMullen

Papers

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae

The specificity of mitochondrial complex I for ubiquinones.

The universality of bioenergetic disease: the role of mitochondrial mutation and the putative inter-relationship between mitochondria and plasma membrane NADH oxidoreductase.

Methyleneketenes and methylenecarbenes. VII. Evidence for the pyrolytic generation of methyleneketene (propadienone)

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators