Evolution of structure and function of V-ATPases

Proton pumping ATPases/ATPsynthases are found in all groups of present-day organisms. The structure of V- and F-type ATPases/ATP synthases is very conserved throughout evolution. Sequence analysis shows that the V- and F-type ATPases evolved from the same enzyme already present in the last common ancestor of all known extant life forms. The catalytic and noncatalytic subunits found in the dissociable head groups of the V/F-type ATPases are paralogous subunits, i.e., these two types of subunits evolved from a common ancestral gene. The gene duplication giving rise to these two genes (i.e., encoding the catalytic and noncatalytic subunits) predates the time of the last common ancestor.Mapping of gene duplication events that occurred in the evolution of the proteolipid, the noncatalytic and the catalytic subunits, onto the tree of life leads to a prediction for the likely subunit structure of the encoded ATPases. A correlation between structure and function of V/F-ATPases has been established for present-day organisms. Implications resulting from this correlation for the bioenergetics operative in proto-eukaryotes and in the last common ancestor are presented. The similarities of the V/F-ATPase subunits to an ATPase-like protein that was implicated to play a role in flagellar assembly are evaluated.Different V-ATPase isoforms have been detected in some higher eukaryotes. These data are analyzed with respect to the possible function of the different isoforms (tissue specific, organelle specific) and with respect to the point in their evolution when these gene duplications giving rise to the isoforms had occurred, i.e., how far these isoforms are distributed.     

Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.     

Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study

Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c₂ were studied by¹H–¹⁵N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe⁸² to Asp⁸⁷ are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His¹⁷, Leu³⁷, Arg⁴³, Ala⁴⁵, Gly⁴⁶, Ile⁵⁷, Val⁵⁸, Leu⁶⁰, Gly⁶¹ and Leu¹⁰⁰ suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR nuclear magnetic resonance - HMQC homonuclear multiple quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 shows dynamic not structural differences. A 1H and 15N NMR study

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild-type and mutant Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 are indistinguishable. The ring resonances of Phe-51 and Tyr-53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly-34 and the NpiH of His-17 increase by approximately 10(2) in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3JchiNH 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Increased sensitivity of the rapid hydrophobic grid membrane filter enzyme-labeled antibody procedure for Escherichia coli O157 detection in foods and bovine feces.

Several strains of Escherichia coli O157:H7 artificially inoculated into vegetables and dairy products were recovered on hydrophobic grid membrane filters and enumerated by an enzyme-labeled antibody assay. The mean of the recoveries from 12 fresh vegetables was 108.8%, whereas that from 10 dairy products was 93.2%. Modified tryptic soy broth at 43 degrees C with shaking at 100 rpm provided optimum recovery of the organism from meat, with a sensitivity of less than or equal to 1 CFU/g, which is 10 times more sensitive than direct plating. The method performed equally well with vegetable and dairy products. Tryptic soy broth, however, under the same conditions gave the best results for fecal samples. Of 22 asymptomatic dairy cattle, reported as having positive Brucella titers when assayed with polyclonal antibodies, eight were found to contain E. coli O157 in their feces as demonstrated by the enzyme-labeled antibody assay by using monoclonal antibodies. This finding may explain some of the false-positive Brucella tests.     

Leaf resin flavonoids of Diplacus aurantiacus

Two previously unknown flavonoids, diplacone and diplacol, have been isolated from the leaf resin of Diplacus aurantiacus (Scrophulariaceae). Diplacone is 6-geranyl-eriodictyol, and diplacol is the corresponding dihydroflavonol. The resin is produced at high levels (up to 30% of leaf dry wt) and apparently composed primarily of these two compounds and their methyl ethers.     

Preparation of two dinor-PGI2 metabolites from 6-keto-PGF1α by mycobacterium rhodochrous

The transformation of 6-keto-PGF_1α to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF_1α (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF_1α (II) by $\text{Mycobacterium rhodochrous}$ UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF_1α to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ¹³ reductase enzyme systems.