Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

Three dimensional typological studies using scanning electron microscopy for characterization of Termitomyces pellets obtained from submerged growth conditions

There are gaps in existing understanding of fungal pellet growth dynamics. We used scanning electron microscopy (SEM) for morphological characterization of the biomass organization of Termitomyces pellets for seven species: T. microcarpus (TMI1), T. albuminosus (TAL1, TAL2), T. striatus (TSTR), T. aurantiacus (TAUR), T. heimii (THE1, THE2), T. globulus (TGLO) and T. clypeatus (TCL1, TCL2, TCL3, TCL4, TCL5). We assessed the utility of SEM for morphological and structural characterization of Termitomyces spp. in three dimensional (3D) pellet form to identify ideal pellet morphology for industrial use. Typological classification of Termitomyces species was based on furrows, isotropy, total motifs and fractal dimensions. The pellets formed were entangled and exhibited highly compacted mycelial mass with microheterogeneity and microporosity. The mean density of furrows of Termitomyces species was between 10,000 and 11,300 cm/cm², percentage isotropy was 30?80 and total motifs varied from 300 to 2500. TGLO exhibited the highest furrow mean density, 11243 cm/cm², which indicated a compact, cerebroid structure with complex ridges and furrows, whereas TAL2 exhibited the lowest furrow density. TMI1a exhibited a high percentage isotropic value, 74.6, TSTR exhibited the lowest, 30.9. Total motif number also was used as a typological classification parameter. Fractal values were 2.64?2.78 for various submerged conditions of Termitomyces species. TAL1 exhibited the highest fractal dimension and TAL2 the lowest, which indicates the complexity of branching patterns. Three-dimensional SEM image analysis can provide insight into pellet micromorphology and is a powerful tool for exploring topographical details of pellets.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Purification of all thirteen polypeptides of bovine heart cytochrome c oxidase from one aliquot of enzyme. Characterization of bovine fetal heart cytochrome c oxidase

A protocol has been worked out for separating all thirteen different polypeptides in the beef heart cytochrome c oxidase complex from a single aliquot of enzyme. This involves an initial separation of polypeptides by gel filtration on a Biogel P-60 column in SDS, a step which purifies subunits CIV and CVIII and gives mixtures of CV + CVI, ASA, AED and STA, as well as CVII, CIX and IHQ. These mixtures are then resolved by reverse-phase high-performance liquid chromatography. The separation procedures have been applied to fetal heart cytochrome c oxidase of gestation between 100 and 200 days. No differences were found in the N-terminal sequences of any of the cytoplasmically made subunits or in the entire sequence of CIX between late fetal and adult forms of the enzyme.     

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.     

Mitochondrial myopathy involving ubiquinol-cytochrome c oxidoreductase (complex III) identified by immunoelectron microscopy

The distribution of respiratory chain complexes in bovine heart and human muscle mitochondria has been explored by immunoelectron microscopy with antibodies made against bovine heart mitochondrial proteins in conjunction with protein A-colloidal gold (12-nm particles). The antibodies used were made against NADH-coenzyme Q reductase (complex I), ubiquinol cytochrome c oxidoreductase (complex III), cytochrome c oxidase, core proteins isolated from complex III and the non-heme iron protein of complex III. Labeling of bovine heart tissue with any of these antibodies gave gold particles randomly distributed along the mitochondrial inner membrane. The labeling of muscle tissue from a patient with a mitochondrial myopathy localized by biochemical analysis to complex III was quantitated and compared with the labeling of human control muscle tissue. Complex I and cytochrome c oxidase antibodies reacted to the same level in myopathic and normal muscle samples. Antibodies to complex III or its components reacted very poorly to the patient's tissue but strongly to control muscle samples. Immunoelectron microscopy using respiratory chain antibodies appears to be a promising approach to the diagnosis and characterization of mitochondrial myopathies when only limited amounts of tissue are available for study.     

Structure-function relationships of the Escherichia coli ATP synthase probed by trypsin digestion

Trypsin cleavage has been used to probe structure-function relationships of the Escherichia coli ATP synthase (ECF1F0). Trypsin cleaved all five subunits, alpha, beta, gamma, delta, and epsilon, in isolated ECF1. Cleavage of the alpha subunit involved the removal of the N-terminal 15 residues, the beta subunit was cleaved near the C-terminus, the gamma subunit was cleaved near Ser202, and the delta and epsilon subunits appeared to be cleaved at several sites to yield small peptide fragments. Trypsin cleavage of ECF1 enhanced the ATPase activity between 6- and 8-fold in different preparations, in a time course that followed the cleavage of the epsilon subunit. This removal of the epsilon subunit increased multisite ATPase activity but not unisite ATPase activity, showing that the inhibitory role of the epsilon subunit is due to an effect on cooperativity. The detergent lauryldimethylamine oxide was found to increase multisite catalysis and also increase unisite catalysis more than 2-fold. Prolonged trypsin cleavage left a highly active ATPase containing only the alpha and beta subunits along with two fragments of the gamma subunit. All of the subunits of ECF1 were cleaved by trypsin in preparations of ECF1F0 at the same sites as in isolated ECF1. Two subunits, the beta and epsilon subunits, were cleaved at the same rate in ECF1F0 as in ECF1 alone. The alpha, gamma, and delta subunits were cleaved significantly more slowly in ECF1F0.(ABSTRACT TRUNCATED AT 250 WORDS)