The Na(+)-F(1)F(0)-ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8- and 18-kda.
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Eight genes (atpI, atpB, atpE(1), atpE(2), atpE(3), atpF, atpH, and atpA) upstream of and contiguous with the previously described genes atpG, atpD, and atpC were cloned from chromosomal DNA of Acetobacterium woodii. Northern blot analysis revealed that the eleven atp genes are transcribed as a polycistronic message. The atp operon encodes the Na(+)-F(1)F(0)-ATPase of A. woodii, as evident from a comparison of the biochemically derived N termini of the subunits with the amino acid sequences deduced from the DNA sequences. The molecular analysis revealed that all of the F(1)F(0)-encoding genes from Escherichia coli have homologs in the Na(+)-F(1)F(0)-ATPase operon from A. woodii, despite the fact that only six subunits were found in previous preparations of the enzyme from A. woodii. These results unequivocally prove that the Na(+)-ATPase from A. woodii is an enzyme of the F(1)F(0) class. Most interestingly, the gene encoding the proteolipid underwent quadruplication. Two gene copies (atpE(2) and atpE(3)) encode identical 8-kDa proteolipids. Two additional gene copies were fused to form the atpE(1) gene. Heterologous expression experiments as well as immunolabeling studies with native membranes revealed that atpE(1) encodes a duplicated 18-kDa proteolipid. This is the first demonstration of multiplication and fusion of proteolipid-encoding genes in F(1)F(0)-ATPase operons. Furthermore, AtpE(1) is the first duplicated proteolipid ever found to be encoded by an F(1)F(0)-ATPase operon. (+info)
Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
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The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex. (+info)
Characterization of an endothelin ET(B) receptor in the gill of the dogfish shark Squalus acanthias.
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Endothelins (ETs) are potent vasoconstrictive peptides that are secreted by the vascular endothelium and other tissues in vertebrates. Previous studies have demonstrated that ETs are expressed in a variety of fish tissues and contract various blood vessels. In order to determine if receptors for ET are expressed in fish gill tissue, we examined the binding kinetics of (125)I-labeled, human ET-1 to membrane fragments isolated from the gill of the dogfish shark, Squalus acanthias. (125)I-ET-1 bound at a single site, with a dissociation constant (K(d)) and binding site number (B(max)) very similar to those described in a variety of mammalian blood vessels. ET-1 and ET-3 competed equally with (125)I-ET-1, suggesting that the receptor was ET(B), which has been shown in mammalian systems to bind to both ligands equally. The ET(B)-specific agonists sarafotoxin S6c, IRL-1620, and BQ-3020 also competed against (125)I-ET-1 at a single site, supporting this hypothesis. We conclude that the shark gill expresses an ET(B) receptor with substantial homology to the mammalian receptor and that ET may play an important role in modulating such vital gill functions as gas exchange, ion regulation, acid-base balance, and excretion of nitrogen. (+info)
Non-adrenergic binding of [3H]atipamezole in rat kidney--regional distribution and comparison to alpha2-adrenoceptors.
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1 Atipamezole (4-(2-ethyl-2,3-dihydro-1H-inden-2-yl)-1H-imidazole) was first introduced as a potent and specific alpha2-adrenoceptor antagonist, but in some tissues [3H]atipamezole identifies an additional population of binding sites, distinct from both classical alpha2-adrenoceptors and I1- and I2-imidazoline receptors identified with [3H]para-aminoclonidine or [3H]idazoxan. 2 In the present study we have characterized [3H]atipamezole binding sites in rat kidney by receptor autoradiography and membrane binding assays and determined whether they are pharmacologically identical with the previously described binding sites for [3H]para-aminoclonidine and [3H]idazoxan. [3H]RX821002 and [3H]rauwolscine were used to compare the regional distribution of alpha2-adrenoceptors to that of non-adrenergic binding sites of [3H]atipamezole. 3 Comparative autoradiographic experiments demonstrated the differential localisation of [3H]atipamezole, [3H]RX821002 and [3H]rauwolscine binding sites in rat kidney. The pattern of distribution of non-adrenergic [3H]atipamezole binding sites is clearly distinct from that of alpha2-adrenoceptors. 4 The non-adrenergic binding of [3H]atipamezole in rat kidney does not fall into any of the previously identified three classes of imidazoline receptors studied with [3H]para-aminoclonidine, [3H]idazoxan and [3H]RX821002. 5 Atipamezole had no inhibitory effect on MAO-A or MAO-B activity in renal membranes, which speaks against the involvement of MAOs in the observed radioligand binding. (+info)
Identification of the G-protein-coupled ORL1 receptor in the mouse spinal cord by [35S]-GTPgammaS binding and immunohistochemistry.
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1 Although the ORL1 receptor is clearly located within the spinal cord, the functional signalling mechanism of the ORL1 receptor in the spinal cord has not been clearly documented. The present study was then to investigate the guanine nucleotide binding protein (G-protein) activation mediated through by the ORL1 receptor in the mouse spinal cord, measuring the modulation of guanosine-5'-o-(3-[35S]-thio) triphosphate ([35S]-GTPgammaS) binding by the putative endogenous ligand nociceptin, also referred as orphanin FQ. We also studied the anatomical distribution of nociceptin-like immunoreactivity and nociceptin-stimulated [35S]-GTPgammaS autoradiography in the spinal cord. 2 Immunohistochemical staining of mouse spinal cord sections revealed a dense plexus of nociceptin-like immunoreactive fibres in the superficial layers of the dorsal horn throughout the entire length of the spinal cord. In addition, networks of fibres were seen projecting from the lateral border of the dorsal horn to the lateral grey matter and around the central canal. 3 In vitro [35S]-GTPgammaS autoradiography showed high levels of nociceptin-stimulated [35S]-GTPgammaS binding in the superficial layers of the mouse dorsal horn and around the central canal, corresponding to the areas where nociceptin-like immunoreactive fibres were concentrated. 4 In [35S]-GTPgammaS membrane assay, nociceptin increased [35S]-GTPgammaS binding of mouse spinal cord membranes in a concentration-dependent and saturable manner, affording maximal stimulation of 64.1+/-2.4%. This effect was markedly inhibited by the specific ORL1 receptor antagonist [Phe1Psi (CH2-NH) Gly2] nociceptin (1 - 13) NH2. None of the mu-, delta-, and kappa-opioid and other G-protein-coupled receptor antagonists had a significant effect on basal or nociceptin-stimulated [35S]-GTPgammaS binding. 5 These findings suggest that nociceptin-containing fibres terminate in the superficial layers of the dorsal horn and the central canal and that nociceptin released in these areas may selectively stimulate the ORL1 receptor to activate G-protein. Furthermore, the unique pattern of G-protein activation in the present study provide additional evidence that nociceptin is distinct from the mu-, delta- or kappa-opioid system. (+info)
Proton-pumping-ATPase-targeted antifungal activity of a novel conjugated styryl ketone.
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NC1175 (3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinyle ne]-1- ethyl-4-piperidinol hydrochloride) is a novel thiol-blocking conjugated styryl ketone that exhibits activity against a wide spectrum of pathogenic fungi. Incubation of NC1175 with various concentrations of cysteine and glutathione eliminated its antifungal activity in a concentration-dependent fashion. Since NC1175 is a lipophilic compound that has the potential to interact with cytoplasmic membrane components, we examined its effect on the membrane-located proton-translocating ATPase (H(+)-ATPase) of yeast (Candida albicans, Candida krusei, Candida guilliermondii, Candida glabrata, and Saccharomyces cerevisiae) and Aspergillus (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus nidulans) species. The glucose-induced acidification of external medium due to H(+)-ATPase-mediated expulsion of intracellular protons by these fungi was measured in the presence of several concentrations of the drug. NC1175 (12.5 to 50 microM) inhibited acidification of external medium by Candida, Saccharomyces, and Aspergillus species in a concentration-dependent manner. Vanadate-inhibited hydrolysis of ATP by membrane fractions of C. albicans was completely inhibited by 50 microM NC1175, suggesting that the target of action of NC1175 in these fungi may include H(+)-ATPase. (+info)
Microfibril assembly by granules of chitin synthetase.
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Purified preparations of chitin synthetase (EC 2.4.1.16; UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase), capable of forming microfibrils in vitro, were isolated from yeast cells of Mucor rouxii. Chitin synthetase was obtained either by substrate-induced liberation of bound enzyme (54,000 x g pellet) or by isolation of unbound enzyme present in the 54,000 x g supernatant of a cell-free extract. Both preparations contained ellipsoidal granules from about 350 to 1000 A diameter. Many granules exhibited a marked depression. No typical unit membrane profiles appeared in thin sections of glutaraldehyde/OsO4-fixed samples. Upon incubation with substrate and activators, chitin microfibrils were produced. The microfibrils were often found intimately associated with granules. The most common configurations were: a microfibril with a granule at one end, or two microfibrils "arising" from the same granule. These findings lend support to the granule hypothesis for the elaboration of cell wall microfibrils by end-synthesis. (+info)
Network formation of lipid membranes: triggering structural transitions by chain melting.
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Phospholipids when dispersed in excess water generally form vesicular membrane structures. Cryo-transmission and freeze-fracture electron microscopy are combined here with calorimetry and viscometry to demonstrate the reversible conversion of phosphatidylglycerol aqueous vesicle suspensions to a three-dimensional structure that consists of extended bilayer networks. Thermodynamic analysis indicates that the structural transitions arise from two effects: (i) the enhanced membrane elasticity accompanying the lipid state fluctuations on chain melting and (ii) solvent-associated interactions (including electrostatics) that favor a change in membrane curvature. The material properties of the hydrogels and their reversible formation offer the possibility of future applications, for example in drug delivery, the design of structural switches, or for understanding vesicle fusion or fission processes. (+info)