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Publisher Summary Purified preparations of synaptic plasma membranes contain more than 25 polypeptides susceptible to phosphorylation by resident kinases that are dependent on a variety of activators such as CAMP, Ca 2+ at low concentration enhanced by CaM and Ca 2+ at high concentration. Among the more prominent phosphoproteins are ones with M app = 44 x 10 3 and two different proteins with M app = 54 x 10 3 , one formed in a Ca 2+ and another in a CAMP-requiring reaction, that can be separated on 2D gels. The 44 × 10 3 protein has been identified as the a subunit of pyruvate dehydrogenase and is localized in both cell body and intraterminal mitochondria. The protein phosphorylated in the presence of Ca 2+ is identical with phosphoprotein B-50 and appears to exhibit a localization confined to the interior surface of presynaptic membranes. The protein phosphorylated in the CAMP-dependent reaction has been identified as the regulatory, brain-specific, type II subunit of CAMP-dependent protein kinase and is located on pre- as well as postsynaptic membranes. Treatment of synaptic and postsynaptic membranes with low concentrations of nonionic detergents appears to facilitate interaction of membrane-bound kinases with potential substrates, resulting in an extensive phosphorylation of many membrane proteins, some of them protected from this modification in intact membranes.

The polypeptide gene products expressed in and by the mitochondria of respiration-competent eucaryotic cells appear to be few in number and conserved in kind: They consist of three subunits of cytochrome oxidase, of cytochrome b of the respiratory chain, and a more variable number (≥2) of subunits of the membrane-integrated (F.) portion of the ATP synthase (oligomycin-sensitive ATPase). In spite of this apparent invariance, the resident genophore and its means of expression appear to have been - and perhaps still are - subject to profound and relatively rapid evolutionary changes. A particularly instructive example is provided by various yeast strains of the species Saccharomyces cerevisiae and carlsbergensis in which the mtDNA, although topologically and functionally homologous, can vary in size by as much as 12 kbp (15% of the total). These insertions/deletions can be as large as 900-3000 bp and are located in specific gene regions. The organization and expression of the mitochondrial gene for apocytochrome b , investigated in several laboratories, is highly complex: four coding regions (exons) appear to be interspersed among at least three non-coding regions (introns). This sequence organization resembles that found in nuclear and viral eucaryotic genes, but in the mitochondrial case mutations in introns can readily be isolated. They have been shown to result in i) the appearance of novel polypeptides not present in the wild type and ii) regulation of the expression of a second, unlinked mt gene, i.e. the one responsible for the specification of the largest subunit of cytochrome oxidase. This gene probably also exhibits a complex sequence organization. However, other mt genes may not share this property. Thus the mt genophore in Saccharomyces is unusual in several respects, among them i) an unusually large proportion and length of AT-rich sequences, ii) non-contiguity of genes for rRNA precursors, and iii) the simultaneous presence of genes exhibiting both a pro- and a eucaryotic pattern of sequence organization.

Publisher Summary This chapter discusses the exon–intron structures of some mitochondrial genes, examines the mitochondrial intervening sequences, and surveys their characteristic properties. Mitochondrial DNA is a physically and biochemically discrete entity, which is distinct and readily separable from its classical nuclear counterpart. Large mitochondrial genomes that are more representative of ancestral forms than are the more streamlined versions found in animals exhibit architecture both within and among their genes that are analogous to those found in large nuclear genomes. A number of similarities and additional features of mitochondrial genes and their expressions (such as codons, tRNAs, codon utilization, transcription, and processing of transcripts), which diverge from their prokaryotic counterparts, show that a mitochondrial ancestor is unlikely to have borne any resemblance to a contemporary Eubacterium.

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.

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.

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.

Publisher Summary Much of current thinking concerning the biogenesis of organelles of energy conversion rests on a series of assumptions, with some of them explicit and based on sound experimental facts, others implicit and based only on inference and analogy. The chapter presents assumptions for the case of mitochondria. Mitochondria or mitochondria-like bodies that function as mitochondrial analogues or precursors are found in all unicellular eucaryotes cells. Mitochondria arise neither de novo from non-membranous constituents nor do they originate from other non-mitochondrial organelles; instead, they originate by accretion of precursors to pre-existing mitochondria resulting in the growth and division. This continuity and persistence of mitochondrial phenotype depends on and is, at least partially, caused by continuity and persistence of mitochondrial genotype. Individual mitochondria constitute discrete and distinct functional and genetic entities. Their persistence in successive mitochondrial and cellular generations requires the simultaneous participation and mutual interaction of both, their own and the nucleo-cytoplasmic system of gene duplication and expression.

This chapter discusses about the diaphorases. The assay methods involved MB reduction. The DPNH is generated by the interaction of substrate, DPN, and a dehydrogenase and either the Thunberg technique or manometric experiments is used; the disappearance of DPNH spectrophotometrically is followed, using MB as the immediate and O 2 as the final electron acceptor. The method employs measurement of 2,6-di-chlorophenolindophenol reduction at 600 mμ. The method as outlined is directly applicable to use with crude extracts or homogenates provided that (1) the turbidity introduced by the preparation is not so great as to make accurate spectrophotometry impossible and (2) the assay mixture is made 0.001 M with respect to CN – . Under these conditions, the method measures not only diaphorase, if any, but also dye reduction due to DPNH cytochrome reductase. The steps described in the purification procedure are: (1) preparation of homogenate; (2) precipitation at pH 4.6; (3) alcohol extraction; (4) treatment with alumina; (5) heat treatment and first ammonium sulfate fractionation; (6) denaturation by dialysis; and (7) second heat treatment and ammonium sulfate fractionation. The soluble, purified enzyme is specific for DPNH .

Publisher Summary This chapter discusses the DPNH cytochrome c reductase for animal. The reduction of ferricytochrome c by DPNH in the presence of the enzyme is measured spectrophotometrically at 550 mμ. The unit of enzyme activity is defined as that amount of enzyme which yields an "initial rate" of 1.00 per minute at 22°. Specific activity is defined as the number of units per milligram of protein. Protein is determined by the biuret reaction. The assay method is applicable to the determination of reductase activity in homogenates or crude tissue extracts, provided that the following modifications and precautions are observed: (1) the turbidity introduced by the enzyme preparation should not be so great as to make accurate spectrophotometry impossible; (2) the reaction is carried out in 0.05 M phosphate buffer, pH 7.4, in the presence of 0.001 M KCN. The steps described in the purification procedure are: (1) preparation of lyophilized extract, (2) acid ammonium sulfate fractionation, (3) ammonium sulfate fractionation in phosphate at pH 8.0, and (4) refractionation with ammonium sulfate. The purified enzyme is best stored as a solution, 1% in enzyme protein and 1 to 2 % with respect to albumin, in the frozen state at –10°.