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Publisher Summary This chapter discusses the evolutionary origin of the mitochondria. Most prokaryotes and essentially all eukaryotes are adapted to life in the presence of free oxygen. The two fundamental biochemical adaptations for such life are the possession of two enzymes, superoxide dismutase and catalase, which protect cellular components from autooxidation, and an electron transport system that allows cells to use atmospheric oxygen as a terminal electron acceptor for energy yielding biochemical oxidations. Evolutionary rates of amino acid or nucleotide substitution have been computed for several macromolecules. Such rates are generally quite constant for any particular molecular phylogeny. This has been illustrated by Dickerson, who correlated sequence changes in cytochromes from diverse organisms with absolute times of phylogenetic divergence obtained from classic paleontological data. According to the symbiotic model, eukaryotic cytoplasm should show evidence of a fundamentally anaerobic nature, since the anaerobic protoeukaryote acquired its oxygen-mediating systems from the aerobic symbiont. Mitochondria contain their own genetic system. This is composed of a specific mitochondrial DNA (mtDNA), as well as the means for its replication and, at least potentially, expression. Thus the organelles possess a DNA-dependent RNA polymerase and a system for protein synthesis, consisting of ribosomes, mRNA(s), tRNAs, aminoacyl-tRNA ligases, and the three classes of protein factors (initiation, elongation, and termination factors) required for its function.

Synaptic vesicles prepared from bovine corpus striatum exhibit an ATPase activity that is insensitive to ouabain and specific inhibitors of mitochondrial ATPase, but that is stimulated by proton ionophores and inhibited by sulfhydryl reagents. Low concentrations of orthovanadate, DCCD and tributyl tin are also ineffective as inhibitors of the vesicle-associated activity. The properties of the synaptic vesicle enzyme suggest that this ATPase may be similar to that of clathrin-coated vesicles, and to one of the activities described in preparations of adrenal chromaffin granule membranes.

—The rate of degradation of acetylcholinesterase [EC 3.1.1.7] in mature rat cerebral cortex was determined from the time course of the label introduced into the protein by one intraventricular injection of l-leucine-l-14C. The half life of the enzyme was 2.84 ± 013 days.

We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l -malate, l -glutamate, and l 8-isocitrate. Cytochromes b, c1, and aa3 are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected. We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa3, b, and c1; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression. The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.

Protein synthesis was studied in slices from rat cerebral cortex and localized in various purified subcellular membrane fractions isolated after incubation with L-leucine. A synaptosomal fraction isolated from a several times washed crude mitochondrial pellet showed very little contamination by free membranes, and the synaptic membrane fraction isolated from it was estimated to c +ain about 50% of this component. Leucine incorporation into all fractions was highly sensitive (greater than 95%) to emetine except for the cell and synaptic mitochondrial subfractions. They were only 60 to 70% inhibited by emetine and showed 10 to 20% inhibition by chloramphenicol which probably was due to those proteins synthesized in situ by the mitochondria. The net rate of incorporation of labeled protein into the synaptosomal and synaptic membrane subfractions was low, and axonal flow was excluded as a significant source of this label. On the basis of autoradiography it was concluded that the majority of the protein label was contributed by free and membrane-enclosed ribosome-containing contaminants in these fractions. Unlabeled and labeled subcellular fractions were also analyzed by electrophoresis in the presence of sodium dodecyl sulfate. The staining profiles of the microsomal and synaptic membrane subfractions were nearly identical, whereas those of synaptic mitochondria and the soluble proteins of the cell bodies were unique. The labeling of all proteins was blocked by emetine, except for the synaptic mitochondria (cell mitochondria were not examined). These products of mitochondrial protein synthesis exhibited three labeling peaks, the major one with a molecular weight of approximately 38,000. In the absence of emetine, slices incorporated amino acids into soluble and microsomal proteins with high specific activity. The labeled proteins of the soluble fraction were more highly concentrated in the range of molecular weights smaller than 50,000 than were those in the microsomal fraction, and it was concluded that a considerable portion of the protein-synthesizing machinery of the brain is geared to form soluble proteins. The gel patterns of labeled microsomal and synaptic membranes were consistent with contamination by the former contributing most of the protein label in the latter.

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg2+-ATPase; (b) an ATPase active at high concentrations of Ca2+ in the absence of Mg2+ (CaH-ATPase); (c) a Ca2+ requiring Mg2+-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca2+-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and CaH-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.

Using 3 H-Quinuclinidyl Benzilate ( 3 H-QNB) as a high affinity ligand for quantitative studies of specific binding to muscarinic cholinergic receptors we have demonstrated the presence of such receptors in homogenates of goldfish retina. Only one set of binding sites could be detected with an apparent dissociation constant of 1.9 × 10 −10 M and a density of 53.5 fentomoles per mg of protein. The receptor sites become saturated at a QNB concentration of 1.2 nM. The pharmacological profile of the specific binding is similar to those described for homogenates of beef and chick retina, as well as for rodent brain and smooth muscle.

We have reviewed here the genetic methods used for isolating and manipulating nuclear and mitochondrial mutants of bakers' yeast that affect the function and biogenesis of complex III of the mitochondrial respiratory chain. All the methods have been used with success in the past, and it is hoped that this compilation will aid biochemists in using these techniques to study electron transfer.

When a number of wild-type strains of Saccharomyces cerevisiae—all capable of utilizing the three sugars galactose, maltose, and α-methyl- d -glucoside for growth—were converted by ethidium bromide (EtdBr) mutagenesis to stable cytoplasmic petite (rho−) mutants, the latter lost the ability to grow on one or more of these sugars. The actual pattern of retention (or loss) or sugar utilization by these mutants depended on the wild-type strain, but was independent of the length of exposure to EtdBr during mutagenesis. This treatment varied from 0.5 to 24 h, by which time the majority of the mutants must have been of the mitochondrial (mt) DNA-deficient rho0 type. Furthermore, with one exception—involving the ability of one set of mutants to utilize α-methyl-glucoside—all rho− mutants derived from the same wild type exhibited the same, discrete pattern of sugar utilization. Respiration-deficient mutants with defined lesions in their mtDNA (mit− mutants) exhibited the same pattern of sugar utilization as did the petite mutants of the same strain. Diploid petite strains also exhibited discrete, but less stringent, patterns of sugar utilization. For any one genotype this pattern was identical whether the mutant was generated by crossing two haploid rho− strains, themselves derived by EtdBr mutagenesis, or by EtdBr mutagenesis of the diploid obtained from a haploid wild-type × wild-type cross. In such mutant diploids the sugar-positive phenotype was usually dominant, but there were indications in some instances of modulation of this effect by virtue of nuclear gene interactions. Various respiration-deficient mutants incapable of utilizing α-methylglucoside also were unable to form α-glucosidase, but were able to do so after being rendered permeable by exposure to dimethyl sulfoxide. Arguments are advanced that respiring mitochondria generate an entity—probably not directly related to ATP production—required for the expression of nuclear genes or their products, some of which may be necessary for plasma membrane function.

Summary We have investigated the major 3′:5′-adenosine monophosphate (cAMP) binding proteins associated with the plasma membrane of Saccharomyces cerevisiae. At least four proteins with apparent molecular weights of 25,000, 34,000, 46,000 and 58,000 have been identified using the photo-affinity probe 8-azidoadenosine[32P]3′:5′-monophosphate (8-N3-[32P]cAMP). Together, they constitute a homogeneous population of binding sites interacting with the photoaffinity ligand in a non-cooperative fashion.

The preparation of a four-subunit enzyme from yeast, capable of catalyzing the oxidation of ferrocytochrome c, is described. It is derived from proteins containing seven or five subunits by means of recycling exclusion chromatography in the presence of 0.1% sodium dodecyl sulfate. Its catalytic properties are similar to those of the parent enzyme. Gel electrophoresis of this preparation in the presence of sodium dodecyl sulfate reveals three bands, migrating with RF values that correspond to molecular weights of 14.6, 12.3, and 10.6 X 10(3), with the largest exhibiting an apparent 2:1 stoichiometry relative to the other two. Visible spectra in the region of 390 to 630 nm do not show any detectable difference from that of the parent cytochrome oxidase, while its heme a and copper content are raised to values around 20 nmol or ng atoms/mg of protein, respectively, corresponding to minimal molecular weights of 50 X 10(3). The molecular weight determined by physical means equals 107 X 10(3). Thus the enzyme probably contains two copies of each subunit. After extensive dialysis to remove as much as possible of the sodium dodecyl sulfate used in its preparation, this enzyme remains in solution in phosphate buffer in the absence of any added detergent, while under similar conditions the seven-subunit complex precipitates completely. A similar preparation can also be obtained from beef heart. The significance of these findings is discussed with respect to the role of the large subunits in the function as well as the biogenesis of the mitochondrial cytochrome oxidase complex.

We have reviewed here the genetic methods used for isolating and manipulating nuclear and mitochondrial mutants of bakers' yeast that affect the function and biogenesis of complex III of the mitochondrial respiratory chain. All the methods have been used with success in the past, and it is hoped that this compilation will aid biochemists in using these techniques to study electron transfer.

These studies describe the properties of three mit- mutants designated EM17, EM25, and PZ1, all mapping at two closely linked sites near one of the boundaries of the region of the mitochondrial genome concerned with the specification of cytochrome b. They all exhibit complex phenotypes affecting cytochrome b, cytochrome aa3, and additional polypeptides not found in the wild type. In the case of EM 17 this complexity can be ascribed to the presence of two mutations induced in the course of the initial mutagenic treatment: one, the cob2 mutation proper, is responsible for the loss of cytochrome b which is replaced by an altered, functionally inactive polypeptide, cytochrome b. This polypeptide can be further modified, or even eliminated, by the controlled introduction of another mutation in the cob1 segment of the cob region. The reduction in cytochrome oxidase subunit I, responsible for the effects on cytochrome aa3 and enzymatic activity in EM17, is due to a second (not mit-) mutation that has been located in the par1-proximal segment of the oxi3 region. This second mutation as well as the cob mutation can be overcome, and the respective aspect of wild type function restored to EM17, by recombination with rho- strains retaining the appropriate segment(s) of the wild type genome. The phenotype of the other two mutants is due to a single mutagenic event. This conclusion is confirmed by their ability to restore wild type functions by reversion. The mutation in EM25 appears to be due to a frameshift, which has led to premature chain termination, producing a polypeptide of Mr = 15,000 related to apocytochrome b. This change is accompanied by a decrease in the amount of subunit I of cytochrome oxidase. Revertants fall into three classes: on galactose two produce a polypeptide indistinguishable from apocytochrome b, but vary in its amount, while the third fails to increase apocytochrome b above mutant levels. Production of subunit I is increased but fails to reach wild type levels. Complete restoration of wild type functions can, however, be obtained by recombination of EM25 with rho- (cob2+) strains. Mutation PZ1 results in a complete absence of any polypeptide related to apocytochrome b and of cytochrome oxidase subunit I. These cells produce a novel polypeptide with a Mr = 45,000 not found in the wild type, and unrelated to all its normal polypeptides. Reversion or recombination with rho- (cob2+) strains results in virtually complete restoration of all wild type functions and the elimination of the novel polypeptide.

Intraoperative real-time sonography was used to monitor surgical reduction of acute thoracolumbar burst fractures in eight patients. Real-time sonography was performed after laminectomy through a saline-filled operative field using either a 5.0 or a 7.5 MHz transducer. Sonography was performed during Harrington rod distraction and during fracture reduction to confirm adequacy of decompression of the spinal canal. This technique is useful to monitor surgery and in defining adequate fracture reduction.

An antiserum against highly purified cytochrome oxidase from the yeast Saccharomyces cerevisiae ( Shakespeare and Mahler (1971) J. Biol. Chem. 246 , 7649) has been prepared. Addition of this antiserum to reaction mixtures containing either purified cytochrome oxidase or mitochondrial membranes results in inhibition of the oxidation of reduced cytochrome c competitive to the latter: K m for cytochrome c equals 95 μ m with both preparations; in the presence of 5 μg of antiserum this value is raised to 210 and 250 μ m , with the purified enzyme and the membranes, respectively. The antibody preparation has been labeled with 3 H by reductive methylation (condensation with HCHO followed by reduction) and used to study and compare binding to mitochondrial membranes from controls with those from cells in which all cytochrome oxidase synthesis has been stopped by growth in the presence of ethidium bromide. The binding isotherms for the two preparations were quantitatively similar. Thus the two types of membranes appear to contain roughly equivalent amounts of material (s) able to interact with an antibody preparation, itself capable of blocking the cytochrome c binding sites of cytochrome oxidase. The use and limitations of studies with antibodies designed to probe the function and biogenesis of cytochrome oxidase are discussed.

Mixtures of protoporphyrin, deuteroporphyrin, haematoporphyrin, pemptoporphyrin and mesoporphyrin can be separated as free acids on talc thin-layer chromatography plates by developing them with a mixture of ethanol-2,6-lutidine-water (30:3:67) in tanks containing an atmosphere saturated with ammonia vapour. Monoacrylic monopropionic deuteroporphyrin (“S/411” porphyrin) can be separated from coproporphyrin using this system. The same mixture of two carboxyl porphyrins can also be separated on Whatman No. 1 chromatography paper by developing with pyridine-0.2 M sodium borate buffer pH 8.6 (1:9) by either ascending or descending techniques.

1. 1. Efficiency of conversion of uninfected or T3-infected E. coli to protoplasts and lysates by means of lysozyme and Versene has been studied and found to be essentially unaffected by bacteriophage infection. 2. 2. The stability of uninfected or infected E. coli protoplasts to exposure to RNase and DNase in various media is such as to make studies of the effects of these agents profitable. 3. 3. Brief exposure in appropriate media of T3-infected protoplasts to RNase during early stages of phage development, greatly and consistently inhibits this process. Similar effects, although far less consistent, have also been obtained by DNase pretreatment. 4. 4. The addition of DNase or RNase singly or in combination to the medium throughout growth of bacteriophage T3 leads to almost complete inhibition of this process.

Publisher Summary This chapter discusses the life cycle of T2 bacteriophage, which is one of a somewhat an arbitrary family of phages capable of attacking Escherichia coli , strain B. The physical properties, morphology, biochemical make-up, and life cycles of these phages are well known. T2 is the archetype for virulence among viruses because of its ability to commandeer the metabolism of the host cell in the interests of its own reproduction. Hence, it is, in many ways, an ideal model for a study of the processes of cell control. Chemically and metabolically inert by itself, T2 consists externally of a complex of some eight proteins. Its head seems composed of a single protein whose function is to protect and stabilize, both chemically and mechanically, the contained genetic apparatus of the virus. The proteins of T2 in their native state, such as those of viruses in general, are remarkably stable to proteolytic enzymes. When the virus and host cell are mixed in liquid suspension at adequate concentrations, the virus collides with the cell as the result of random Brownian motion. The structural information of the invading phage particle must be transmitted not only heterocatalytically or functionally––that is, into directions for the synthesis of specific proteins––but also autocatalytically––that is, into the replication to form new phage particles.

In aqueous media of low ionic strength (Na+ = 6 × 10−3 M ) the alkaloid cyclobuxine, a steroidal diamine, exerts a profound effect on the stability of helical polynucleotides (DNA, S-RNA and dAT:dAT). This effect is biphasic in the sense that at low concentrations of the agent (≤ 10−4M) there is stabilization of the native, while at higher concentrations (≥5 × 10−4 M ) there is stabilization of the denatured conformation. In a medium containing 51% methanol only the labilizing interaction persists while at high ionic strength both types of interaction are eliminated. These effects of cyclobuxine are restricted to the reversible order—disorder transition and do not extend to strand separation or re-combination. Cyclobuxine affects the absorption spectrum of DNA and other polynucleotides by producing a generalized hyperchromicity centered at 270 mμ. For low concentrations of the agent the magnitude of this increased absorption falls in the order native DNA > denatured DNA ~ S-RNA > polyribonucleotides » nucleotides.

The recent discovery that the ubiquitous energy-transducing organelle of eukaryotic cells, the mitochondrion, contains its own DNA, distinct from that of the nucleus (for recent reviews, see 1-4), has generated intensive investigations in a number of related areas. They have made accessible possible answers to such fundamental questions as the nature and extent of the genetic and biogenetic autonomy of the organelle; the origin, mode of transmission, and significance of extrachromosomal, non-Mendelian hereditary determinants; and the whole chain of events that must intervene in the course of the expression of the mitochondrial genome. Central to this area is the problem of mitochondrial protein synthesis, both in vivo and in vitro; known now for some 13 years (5,6), it has been raised from its former status of relative obscurity as a laboratory curiosity to one of the fashionable fields of inquiry of contemporary biochemistry and molecular biology. Since the topic has been the subject of several recent and comprehensive reviews (7ߝ11), I shall restrict my presentation, in the main, to our own investigations with the unicellular eukaryote Saccharomyces cerevisiae or baker’s yeast.

A system has been developed for the in vitro synthesis of RNA from nucleoside triphosphates in morphologically intact rat liver nuclei in a medium of low ionic strength. It requires all four nucleoside triphosphates (NTP's), Mg2+ (or possibly Mn2+), and is stimulated by NaF, NH4+, an ATP generating system, and several oligoamines. Spermidine or other oligo-, di-, or monoamines are required for the preservation of nuclear morphology at the incubation temperature; immediate nuclear lysis results from agitation of the assay mixture in their absence. Sucrose, sorbitol, Mg2+, or monovalent ions at higher ionic strength cannot substitute for the amine, while NH4+ shows only a slight stabilizing effect. Incorporation is inhibited by RNase, DNase, actinomycin, Mn2+, deletion of one NTP, Ca2+, and calf thymus histones. Potassium chloride and NaCl have no effect at moderate concentrations but lead to nuclear lysis at high ionic strength. Mercaptoethanol, sonication, trypsinization, or puromycin treatment of the nuclei do not affect the incorporation. The system is highly active (7 mμmoles GMP incorporated/ mg DNA-P in 5 minutes), and functions over a broad pH range from about 7.8 to 9.5. A stimulation of incorporation in vitro is observed if the fluorinated steroid hormone triamcinolone diacetate is injected into the intraperitoneal cavity of normal rats 3–8 hours before sacrifice. This stimulation by hormone is similar in magnitude to one observed by means of an in vivo pulse of 14C-orotate. The optical density profiles obtained from sucrose density gradients of RNA isolated from the nuclei after in vitro incubations and after in vivo pulses are similar, but the radioactivity profiles and double labeling experiments indicate extensive degradatation of RNA with s values greater than 16 during the in vitro incubations. Differences in radioactivity profiles of RNA from control and hormone-treated rats after short pulses of 14C-orotic acid can be interpreted in terms of stimulation of RNA anabolism by the hormone. No stimulation of incorporation has as yet been obtained by in vitro incubation of the nuclei with triamcinolone.