Acta physiologica Scandinavica. Supplementum最新文献

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.

The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.

The gene defective in cystic fibrosis encodes a Cl- channel named CFTR, which belongs to the family of transport proteins identified by their cytoplasmic domains that bind and hydrolyse ATP. CFTR channels require phosphorylation by protein kinase A at one or more serine residues in the large central regulatory domain before they will open. Severl findings argue that hydrolysis of ATP at the N-terminal nucleotide binding domain is the rate-limiting step for opening a phosphorylated CFTR channel. Although AMP-PNP the non-hydrolysable, but close structural, analog of ATP fails to open phosphorylated CFTR channels, once a channel has been opened, AMP-PNP can bind tightly to the channel and "lock" it into the open conformation for several minutes. This tight binding of AMP-PNP presumably occurs at CFTR's C-terminal nucleotide binding domain. Because it structurally resembles AMP-PNP, ATP must also bind tightly there, which suggests that hydrolysis of that ATP normally prompts channel closing. That conclusion is supported by the finding that free [Mg2+] level controls the rate of CFTR channel closure. A normal closed-open-closed gating cycle of a CFTR channel thus seems to involve hydrolysis of one ATP molecule to open it, and hydrolysis of a second ATP to close it. Stabilization of an active state by tight binding of a nucleotide, and termination of that state by hydrolysis of the nucleotide, are characteristics reminiscent of G proteins. Indeed, CFTR's nucleotide binding domains share with G proteins not only this functional similarity, but also some sequence homology, at least in certain highly conserved motifs.

The two Ca2+ pumps of higher eucaryotes are strictly targeted to different membrane systems: the plasma membrane (PMCA) and the sarco(endo)plasmic reticulum (SERCA). Chimeric constructs of the two pumps expressed in COS-7 cells have revealed a strong signal for endoplasmic reticulum retention in the N-terminal cytosolic portion of the SERCA pump: the signal is contained in a stretch of 28 amino acids that follows the N-terminus. A second, but masked, endoplasmic reticulum retention signal is contained in a cytosolic C-terminal sequence immediately preceding the calmodulin-binding domain of the Ca2+ pump. Selective mutations on the SERCA pump have led to the conclusion that 5 conserved residue membrane domains (TM)4, 5, and 6 form the Ca2+ channel through the pump protein. A comparative sequence inspection has failed to reveal any of these residues in TM5 of the PMCA pump. Mutation of the conserved residue in TM4 and of two in TM6 abolished the ability of the pump to form the Ca(2+)-dependent phosphoenzyme. However, one of the mutations (N979, TM6) also caused retention of the PMCA pump in the reticulum, suggesting structural alterations. Of the four basic isoforms of the pump, two (1, 4) are ubiquitously expressed, two (2, 3) are essentially brain specific. Isoform 2 has the highest calmodulin affinity. Primary cultures of cerebellar granule cells from newborn rats did not express isoforms 2 and 3 at plating time. Incubation of the cells in depolarizing concentrations of KCl, which promote Ca2+ influx, promoted the expression of isoforms 2 and 3, and of a brain specific spliced variant of isoform 1. Incubation of the cells in L-type Ca2+ channel blockers abolished the upregulation of the pump genes.

We have used a combination of site-directed mutagenesis and spectroscopic techniques to investigate electron-transfer reactions between hemes a and a3 in cytochrome c oxidase. A state of the enzyme was prepared in which heme a/CuA are oxidized and heme a3/CuB are reduced with CO bound to heme a3, which stabilizes the reduced state of the binuclear center. In addition, in this state the pKs of protonatable groups in the vicinity of the binuclear center, interacting electrostatically with heme a3, are larger than with oxidized heme a3. Upon flash photolysis of CO from the two-electron reduced enzyme electrons at heme a3 equilibrate rapidly with heme a. In the R. sphaeroides enzyme the electron-transfer rates from heme a to a3 and from heme a3 to a were, deconvoluted and were found to be approximately 1.5.10(5) s-1 and approximately 1.4.10(5) s-1, respectively. After this rapid electron equilibration between hemes a and a3, protons are released from groups interacting electrostatically with heme a3, which is associated with additional electron transfer from heme a3 to heme a. The proton-coupled electron transfer displays a pH dependent extent and rate. In addition, it displays a deuterium-isotope effect of a factor of about three. The reaction sequence is compatible with the three-dimensional cytochrome c oxidase structure, which shows that more protonatable groups are found around heme a3 than around heme a and supports the involvement of the binuclear center in proton pumping. Proton uptake/release upon reduction/oxidation of heme a3 takes place through a proton pathway including residues Thr(I-359) and Lys(I-362) (K-pathway), but not through the pathway including residues Asp(I-132) and Glu(I-286) (D-pathway). During reaction of the reduced enzyme with O2, both substrate and pumped protons are taken up through the D-pathway.

Site-directed mutagenesis studies of the structure and function of the Ca2+ binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase are reviewed. The Ca2+ binding properties of six mutants with alterations to amino acid residues with oxygen-containing side chains in the membrane segments M4, M5, M6, and M8 were investigated. The mutations to Glu309 in M4, Glu771 in M5, Asn796, Thr799, and Asp800 in M6 all disrupted Ca2+ occlusion, suggesting that the side chains of these residues donate oxygen ligands to Ca2+ binding at the high-affinity sites and/or are involved in conformational changes that occlude the sites. Alanine substitution of Glu908 in transmembrane segment M8 did not prevent Ca2+ occlusion, thereby excluding this residue from playing a central role in Ca2+ coordination. Titrations of Ca2+ activation of phosphorylation from ATP and of inhibition by Ca2+ of phosphorylation from Pi allowed us to assign Ca2+ liganding residues separately to the two high-affinity Ca2+ sites. Hence, residues Glu771 and Thr799 are associated with the site binding the first calcium ion in the sequential mechanism ("site 1"), whereas Glu309 and Asn796 are associated with the site binding the second calcium ion ("site 2"), and Asp800 donates Ca2+ ligands to both sites. On this basis we discuss two possible structural models for the Ca2+ sites.

The cysteinyl leukotrienes (leukotriene C4, D4 and E4) have potent biological actions which significantly contribute to the airway obstruction in asthma. Several of these effects are blocked by drugs known as CysLT1-receptor antagonists. However, there are actions of leukotrienes which are not sensitive to these antagonists, suggesting the presence of additional receptor subtypes. It was the aim of this Thesis to extend the knowledge about receptors for cysteinyl leukotrienes. Three different isolated smooth muscle preparations kept in organ baths under non-flow conditions were characterised with respect to responsiveness to cysteinyl leukotrienes and sensitivity to purported CysLT1-receptor antagonists. In addition, the study involved evaluation of a leukotriene E4 analogue, BAY u9773, suggested to inhibit responses which cannot be blocked by CysLT1-receptor antagonists. These responses have provisionally been considered to be mediated by CysLT2-receptors. In the guinea pig ileum, BAY u9773 but not the selective CysLT1 receptor antagonist ICI 198,615 inhibited the contractile response to leukotriene C4 in a fashion suggesting competitive antagonism. In sheep trachealis muscle, BAY u9773 antagonised contractions induced by leukotriene C4 and leukotriene D4 in a similar manner, whereas ICI 198,615 did not. The observations support that leukotriene C4 in guinea pig ileum, and leukotriene C4 as well as leukotriene D4 in sheep trachealis muscle, mediated contractions via activation of CysLT2-receptors. In guinea pig lung parenchyma, the effects of BAY u9773 and conventional cysteinyl leukotriene receptor antagonists (ICI 198,615, FPL 55,712) were more complex. First, BAY u9773 evoked a contraction, which could be inhibited by antagonists of CysLT1- and TP-receptors. This suggested that BAY u9773 acted as an agonist at these two receptors. Second, pretreatment with BAY u9773 inhibited a distinct but relatively small component of the contractile response to leukotriene C4 and D4. The effects of BAY u9773 and ICI 198,615 were similar in guinea pig lung parenchyma. The findings suggest that the receptor mediating the major part of the contractile response to exogenous cysteinyl leukotrienes in guinea pig lung parenchyma was different from the currently defined CysLT2-receptor. Furthermore, the data suggested that BAY u9773 was a partial agonist at cysteinyl leukotriene receptors, which presumably contributed to its profile of activity as a combined CysLT1- and CysLT2-receptor antagonist. In addition to contracting guinea pig lung parenchyma, leukotriene C4 and lipoxin A4 also evoked release of thromboxane A2. This release was sensitive to CysLT1-receptor antagonists and contributed to part of the contractile response. Finally, the investigations included a characterisation of the role of leukotrienes in antigen-induced contractions of lung parenchyma from actively sensitised guinea pigs. Combination of antihistamines with CysLT1-receptor antagonist

Environmental influences on human health include the effects of toxic materials and adverse ecological factors. Natural milieu stressors also affect emotions that may adversely affect cardiovascular function and precipitate or otherwise contribute to complications of cardiovascular diseases. However, although variously hypothesized, there is inadequate evidence that they directly contribute to the pathogenesis of sustained hypertension or coronary atherosclerosis.