To many investigators, these observations suggested the existence of a molecular “pump” mechanism responsible for the maintenance of the ionic gradients. Extensive research over the last twenty years has resulted in the assignment of this role to the magnesium-dependent, sodium- and potassium-stimulated adenosine triphosphatase, first reported by Skou in 1957. This membrane-bound enzyme is the subject of the research presented in this thesis.

P1B-type ATPases transport a number of monovalent and divalent heavy metals (Cu+, Cu2+, Ag+, Zn2+, Cd2+, Pb2+ and Co+2) across biological membranes. These ATPases are found in archea, bacteria and eukaryotes and are one of the key elements required for maintaining metal homeostasis. Plants have an unusually high number of P1B-type ATPases with distinct metal selectivity compared to other eukaryotes that usually have one or two Cu+-ATPases. Higher plants are the only eukaryotes where Zn2+-ATPases have been identified. Towards understanding the physiological roles of plant Zn2+-ATPases, we characterized Arabidopsis thaliana HMA2. We expressed HMA2 in yeast and measured the metal dependent ATPase activity in membranes. We showed that HMA2 is a Zn2+-ATPase that is also activated by Cd2+. Zn2+ transport determinations showed that this enzyme drives the efflux of metal from the cytoplasm. Analysis of HMA2 mRNA levels showed that the enzyme is present in all plant organs. We analyzed the effect of removal of HMA2 full-length transcript in whole plants by gene knock out. Although hma2 mutants did not show a different visible phenotype from the wild type plants, we observed increased levels of Zn2+ or Cd2+. The observed phenotype of hma2 mutants and plasma membrane location of HMA2, mainly in vasculature (Hussain et al., 2004), indicates that this ATPase might have a central role in Zn2+ uploading into the phloem. P1B-type ATPases have cytoplasmic regulatory metal binding domains (MBDs) in addition to transmembrane metal binding sites (TMBDs). Plant Zn2+-ATPases have distinct sequences in both their N- and C-termini that might contribute to novel metal binding sites. These ATPases contain long C-terminal sequences rich in CC dipeptides and His repeats. Removal of the C-terminus (C-MBD) of HMA2 leads to a 50% reduction in the enzyme turnover suggesting a regulatory role for this domain. Atomic Absorption Spectroscopy (AAS) analysis showed that Zn2+ binds to C-MBD with a stoichiometry of three (3 Zn/C-MBD). Chemical modification studies and Zn K-edge X-ray Absorption Spectroscopy (XAS) of Zn-C-MBD showed that Zn2+ is likely coordinated by His in two sites and the third site slightly differs from the others involving a Cys together with three His. All plant Zn2+-ATPases lack the typical CXXC signature sequences observed in Cu+-ATPases and some bacterial Zn2+-ATPases N-terminus metal binding domains (N-MBDs). Instead, these have conserved CCXXE sequences. Truncation of HMA2 N-MBD results in a 50% decrease in enzyme Vmax suggesting that N-MBD is also a regulatory domain. The results indicate that the N-MBD binds Zn2+ with a stoichiometry of one (1 Zn/N-MBD). Metal binding analysis of individual N-MBD mutants Cys17Ala, Cys18Ala and Glu21Ala/Cys prevented Zn+2 binding to HMA2 N-MBD suggesting the involvement of all these residues in metal coordination. ATPase activity measurements with HMA2 carrying the mutations Cys17Ala, Cys18Ala and Glu21Ala/Cys showed a reduction in the enzyme activity similar to that observed the truncated protein indicating that the enzyme activity reduction observed in the N-terminus truncated forms of the enzyme is related to the removal of the metal binding capability. Summaryzing, these studies show the central role of HMA2 in plant Zn2+ homeostasis. They also describe the mechanism and direction of Zn2+ transport. Finally, they establish the presence of novel metal binding domains in the cytoplasmic portion of the enzyme. Metal binding to these domains is required for full enzymatic activity.

Qi Wei. / "November 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 158-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

[Truncated abstract] Osteoclasts are multinucleated giant cells responsible for the resorption of the mineralized bone matrix during the process of bone remodelling. During activation towards bone resorption, polarization of the osteoclast results in the formation of a unique plasma membrane, the ruffled border, the actual resorptive organelle of the osteoclast. Through this domain protons are actively pumped into the resorption lacuna creating an acidic microenvironment that favours the dissolution of the mineralized bone matrix. The polarised secretion of protons is carried out by the action of the vacuolar-type (H+)-ATPase (V-ATPase), composed of functionally and structurally distinct subunits of the V1 and V0 domains. The general structure of the V-ATPase complex is highly conserved from yeast to mammals, however, multiple isoforms for specific V-ATPase subunits do exist exhibiting differential subcellular, cellular and tissue-specific localizations. This study focuses on the molecular identification and characterization of V-ATPase accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit in osteoclasts. Using the techniques of cDNA Subtractive Hybridization and DNA Micro-Array analyses respectively, the accessory subunit Ac45 and the d2 isoform of the V0 domain d subunit were identified in RAW264.7-cells derived OcLs. ... Using web-based computational predictions, two possible transmembrane domains, an N-terminus 'signal anchor' sequence and a C-terminus dilysine- like endoplasmic reticulum (ER) retention signal were identified. By confocal microscopy, EYFP-tagged e was found to localize to the perinuclear region of transfected COS-7 cells in compartments representing the ER and Golgi apparatus with some localization in late endosomal/lysosomal-like vesicles. ER truncation of e did not alter its subcellular localization but exhibited significantly weaker association with Ac45 compared to the wild-type as depicted by BRET analyses. Association with the other V0 subunits remain unaffected. This may hint at a possibility that Ac45 may play a role in the masking of the ER signal of e following it's incorporation into the V0 domain. Although no solid evidence for a role in the assembly of the mammalian VATPase have been established, subunit e still represents a potential candidate whose role in the V-ATPase complex requires further investigation. Collectively, the data presented in this thesis has provided further insight into the composition of the osteoclast V-ATPase proton pump by: 1) identifying an accessory subunit, Ac45 which shows promise as a potential candidate for the regulation and/or targeting of the V-ATPase complex in osteoclasts and truncation of its targeting signal impairs osteoclastic bone resorption; 2) identification and preliminary characterization of the d2 isoform of the V0 domain d subunit whose exact role in the V-ATPase complex and in osteoclasts remains to be determined, although its has been implicated to be essential for osteoclastic function; and 3) Preliminary characterization of subunit-e, a potential assembly factor candidate for the mammalian V-ATPase V0 domain.

Two enzymatic systems from the lactic acid bacterium Oenococcus oeni, isolated from wine, have been studied. The first one is the H+-ATPase for which the activity was characterized under various conditions of growth. The activity gradually increased by l.6 to 1.9-fold upon inoculation at pH 3.5. The H+-ATPase activity did not vary significantly in function of the growth rate or with and without malic acid. However, acidification of the medium in the absence of malic acid induced the activity by 1.5 to 2.2-fold depending on the initial pH. The partially cloned H+-ATPase genes shared high homologies with those from other bacterial F0F1-ATPases. A mRNA of about 7 kb was detected by Northern blot and its size suggests that the genetic organization of O. oeni atp operon is similar to most F0F 1-ATPases. Furthermore, the amount of atp mRNA was shown to increase in acidic conditions. O. oeni H +-ATPase activity was pH-inducible and regulation of the expression seems to occur at the level of mRNA synthesis. Thus, the results confirmed the proposed role of the H+-ATPase in acid tolerance in O. oeni. / The second system studied was a chromosome-encoded P-type ATPase (CopB) and its putative transcriptional regulator (CopR). The copB gene encodes a protein showing great similarities with other Cu2+-ATPases of the CPx-type family of heavy-metal ATPases like Enterococcus hirae copB. Another gene (copR) was found 250 bp upstream of copB and displays great similarities with proteins of the MecI/BlaI family of transcriptional regulators, including En. hirae CopY repressor. O. oeni was shown to be highly resistant to copper and growth occurred in up to 30 mM CuSO4. Northern blot analyses indicated that the amount of copB mRNA increased upon a 0.2 to 4.0 mM copper stress suggesting that expression of the enzyme might be regulated at the level of mRNA synthesis. Whether CopR is involved in this regulation remains to be determined, but the results suggest that copRB genes might be involved in copper resistance in O. oeni.

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 20, 2009) Includes bibliographical references. Also issued in print.

The synthesis of nanostructured materials, especially metallic nanoparticles, has accrued utmost interest over the past decade owing to their unique properties that make them applicable in different fields of science and technology. The limitation to the use of these nanoparticles is the paucity of an effective method of synthesis that will produce homogeneous size and shape nanoparticles as well as particles with limited or no toxicity to the human health and the environment. The biological method of nanoparticle synthesis is a relatively simple, cheap and environmentally friendly method than the conventional chemical method of synthesis and thus gains an upper hand. The biomineralization of nanoparticles in protein cages is one of such biological approaches used in the generation of nanoparticles. This method of synthesis apart from being a safer method in the production of nanoparticles is also able to control particle morphology. In this study, a comparative biological synthesis, characterization and biomedical effects of metallic nanoparticles of platinum, gold and silver were investigated. Metallic nanoparticles were biologically synthesized using cage-like (apoferritin), barrel-like (GroEL) and non-caged (ribonuclease) proteins. Nanoparticles generated were characterized using common techniques such as UV-visible spectroscopy, scanning and transmission electron microscopy, inductively coupled optical emission spectroscopy, Fourier transform infra-red spectroscopy and energy dispersion analysis of X-rays (EDAX). Nanoparticles synthesised biologically using apoferritin, GroEL and RNase with exhibited similar chemical and physical properties as thoses nanoparticles generated chemically. In addition, the metallic nanoparticles fabricated within the cage-like and barrel-like cavities of apoferritin and GroEL respectively, resulted in nanoparticles with relatively uniform morphology as opposed to those obtained with the non-caged ribonuclease. The enzymatic (ferroxidase) activity of apoferritin was found to be greatly enhanced with platinum (9-fold), gold (7-fold) and silver (54-fold) nanoparticles. The ATPase activity of GroEL was inhibited by silver nanoparticles (64%), was moderately activated by gold nanoparticles (47%) and considerably enhanced by platinum nanoparticles (85%). The hydrolytic activity of RNase was however, lowered by these metallic nanoparticles (90% in Ag nanoparticles) and to a higher degree with platinum (95%) and gold nanoparticles (~100%). The effect of synthesized nanoparticles on the respective enzyme activities of these proteins was also investigated and the potential neurotoxic property of these particles was also determined by an in vitro interaction with acetylcholinesterase. Protein encapsulated nanoparticles with apoferrtin and GroEL showed a decreased inhibition of acetylcholinesterase (<50%) compared with nanoparticles attached to ribonuclease (>50%). Thus, it can be concluded that the cavities of apoferitin and GroEL acted as nanobiofactories for the synthesis and confinement of the size and shape of nanoparticles. Furthermore, the interior of these proteins provided a shielding effect for these nanoparticles and thus reduced/prevented their possible neurotoxic effect and confirmed safety in their method of production and application. The findings from this study would prove beneficial in the application of these nanoparticles as a potential drug/drug delivery vehicle for the prevention, treatment/management of diseases associated with these enzymes/proteins.

The aim of the present investigation was to determine whether a plasma membrane high affinity Ca²+-ATPase plays an integral role in the maintenance of cytoplasmic free Ca²+ in pancreatic acinar cells. To achieve this, the Ca²+-transport and Ca²+-ATPase activities were characterized and their properties compared. Plasma membranes from guinea-pig pancreatic acini were shown to contain an ATP-dependent high affinity Ca²+-pump and a high affinity Ca²+-dependent ATPase activity. In addition, a low affinity ATPase activity was also observed. The high affinity Ca²+-ATPase activity as well as the Ca²+-transport were found to be dependent on Mg²+, whereas the low affinity ATPase activity appeared to be inhibited by Mg²+. The high affinity ATPase activity was 7-fold greater in magnitude than the Ca²+-transport. Whereas the Ca²+-transport was very specific for ATP as a substrate, the high affinity Ca²+-ATPase showed little specificity for various nucleotide triphosphates. These data would suggest that the Ca²+-transport and the high affinity Ca²+-dependent ATPase in guinea-pig pancreatic acinar plasma membranes may be two distinct activities To further investigate whether the two activities were related, we investigated how the Ca²+-transport and Ca²+-ATPase activities were regulated by intracellular mediators. Regulation of the two activities by calmodulin, cyclic AMP-dependent protein kinase, Protein kinase C and inositol phosphates was investigated. Calmodulin failed to stimulate either activity. In addition, calmodulin antagonists, trifluoperazine and compound 48/80 produced a concentration-dependent inhibition of Ca²+-transport. These data suggested the presence of endogenous calmodulin. Both antagonists failed to influence the Ca²+-dependent ATPase activity. Experiments using boiled extracts from guinea-pig pancreatic acinar plasma membranes and erythrocyte plasma membranes Ca²+-ATPase confirmed the presence of endogenous calmodulin. The catalytic subunit of cyclic AMP-dependent protein kinase stimulated Ca²+ transport, suggesting that cyclic AMP may have a role in the regulation of Ca²+-pump-mediated Ca²+ efflux from pancreatic acini. Ca²+-dependent ATPase activity, on the other hand, was not affected by the catalytic subunit. HA 1004, a specific inhibitor of cAMP-dependent protein kinase, failed to inhibit the Ca²+-transport and Ca²+-dependent ATPase activities. Since, this inhibitor was also ineffective at inhibiting the catalytic-subunit-stimulated Ca²+ transport, it may be concluded that HA 1004 is ineffective in blocking the actions of cAMP-dependent protein kinase in pancreatic acinar plasma membranes. In our studies, purified protein kinase C, the phorbol ester TPA and the diacylglycerol derivative, SA-DG, failed to stimulate the Ca²+-uptake activity. However, these agents produced stimulation of the Ca²+-dependent ATPase activity in the presence of phosphatidylserine. CGP 41 251, a potent and selective inhibitor of protein kinase C, did not inhibit the Ca²+-transport or Ca²+-dependent ATPase activities. These observations suggest that protein kinase C may not be involved in the regulation of the plasma membrane Ca²+-pump in guinea-pig pancreatic acinar cells. These results also point to another difference between Ca²+-transport and the Ca²+-ATPase activities in guinea-pig pancreatic acinar plasma membranes. Neither inositol trisphosphate nor inositol tetrakisphosphate produced a statistically significant effect on Ca²+-uptake, suggesting that IP₃- and/or IP₄-mediated Ca²+ releasing pathways may not operate in the isolated guinea-pig pancreatic acinar plasma membrane vesicles. In summary, the results presented here provide evidence to suggest that the high affinity Ca²+-ATPase is not the biochemical expression of plasma membrane Ca²+-transport in panreatic acini. Our results imply a role for calmodulin and cAMP-dependent protein kinase, but not protein kinase C, in the regulation of Ca²+ efflux from pancreatic acinar cells. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

From the standpoint of physiologists, emphasis has been placed on viewing the chemical nature of the membrane as an operational barrier to the free diffusion of ions. This tends to explain the fact that the ionic composition of the cytoplasm of the animal cell differs from its external fluid environment. It is well know that where sodium is the principal cation of the extracellular fluid, potassium has such a role inside the cell. Since there appears to be differential distribution of these ions across the cell membrane, this infers a concentration gradient of these ions. This differential distribution is important for certain life processes, for example, the propagation of nervous impulses mainly is dependent on the changes in concentration of sodium and potassium ions on both sides of the axonal membrane. Much effort has been put into elucidating the mechanism which cells maintain and change such concentration gradients. The enzyme system investigated in this study, namely, the sodium-activated adenosine triphosphatase, might be involved in maintaining this ionic gradient.<\p>