The sarcoplasmic reticulum Ca²⁺ -pumping ATPase is the primary system responsible for the removal of calcium from the sarcoplasm during relaxation of skeletal and cardiac muscles. Since the rat heart SR is used frequently in our laboratory to study the Ca²⁺ -transport defects in disease states, the Ca²⁺ - ATPase activity of this system was characterized. Calmodulin (CaM) and cAMP-dependent protein kinase (cAMP-PK) are known to regulate the dog cardiac SR Ca²⁺ -pump. The effects of these regulators on the rat heart SR Ca²⁺ -pump were studied. Studies were also carried out to investigate the effects of Triton X-100 on SR Ca²⁺ -ATPase activity and the regulation of this activity by CaM. The rat heart SR Ca²⁺-ATPase was stimulated in a concentration-dependent manner by both Ca²⁺ and Mg²⁺ in the complete absence of the other cation. Magnesium produced a concentration-dependent increase in the basal ATPase activity without affecting the maximal ATPase activity. This appeared to result in a gradual disappearance of the Ca²⁺ dependency of the ATPase activity. Addition of 100µM CDTA (trans-1,2-diaminocyclo- hexane-N,N,N',N'-tetraacetic acid), in the absence of added magnesium, produced no effect on Ca²⁺ stimulation of ATPase activity. The results appear to indicate the presence of a low affinity non-specific divalent cation-stimulated ATPase. At a constant Mg: ATP ratio, ATP simulated the SR Ca²⁺-ATPase activity in a concentration-dependent manner. Double-reciprocal plots of the data suggest that the true substrate for rat heart SR Ca²⁺-ATPase may be ATP and not Mg.ATP. In the crude SR, CaM did not stimulate total or Ca²⁺-stimulated ATPase activity over a range of Ca²⁺ and Mg²⁺ concentrations. CaM also failed to stimulate membrane phosphorylation over a range of Mg²⁺ concentrations. Furthermore, CaM did not produce a significant effect on calcium transport into SR vesicles. The catalytic subunit of cAMP-dependent protein kinase was also ineffective in stimulating membrane phosphorylation and Ca²⁺ -ATPase activity. Two CaM antagonists, trifluperazine and compound 48/80, did not affect the rat heart SR ATPase activity. The ATPase activity in Triton-washed SR membranes appeared to be increased at low Triton concentrations. This effect was probably due to the removal of non-intrinsic proteins, leaky vesicles or altered membrane fluidity. At higher Triton X-100 concentrations, the ATPase activity was lost, probably due to loss of the phospholipid environment. When SR membranes phosphorylated under conditions similar to those used for the ATPase assay were analysed by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) followed by autoradiography, a single phosphorylated protein of 7,500-9,000 dalton was observed. This protein may represent the monomeric form of phospholamban. CaM, however, appeared to have no effect on the phosphorylation of this 7,500-9,000 dalton protein in either untreated or Tritan-washed SR membranes. It is speculated that the rat heart SR contains tightly bound CaM which cannot be removed by treatment with Triton X-100. / Pharmaceutical Sciences, Faculty of / Graduate

Lee Hing Ken. / "November 2001." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 154-159). / 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.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 124-135).

The plasma membrane calcium-transport ATPase plays a major role in maintaining the low cytosolic calcium concentrations required for normal cellular function. Calcium, magnesium, calmodulin and lanthanum have been shown to alter the activity of the calcium-stimulated, magnesium-dependent ATPase activity in human erythrocytes. In an attempt to examine the reaction sequence of the (Ca²⁺ + Mg²⁺)-ATPase, the effects of these agents on the kinetics of calcium dependent phosphoprotein formation, the first step in the partial reaction sequence, were examined. Calmo-dulin-depleted erythrocyte membranes were prepared by hypotonic lysis in the presence of EDTA, according to the method of Carafoli et al (1980). Calcium-dependent formation of the phosphorylated intermediate was biphasic; the high calcium-affinity component was associated with low levels of E.Ca.P and a shallow response to changing calcium concentrations, whereas in the region of the low calcium-affinity component, E.Ca.P rose sharply in response to increasing calcium concentrations. The low affinity component of E.Ca.P lies in the range of calcium concentrations which inhibit (Ca²⁺ + Mg²⁺)-ATPase activity. When analyzed on LiDS acid PAGE, both components of calcium-dependent phosphoprotein formation were due to hydroxylamine-sensitive phosphorylation of a 135,000-145,000 dalton protein. Hence, the low calcium-affinity component of phosphoprotein formation and calcium-dependent inhibition of (Ca²⁺ + Mg²⁺)-ATPase activity were likely due to calcium-inhibition of dephosphorylation. Kinetic studies of calcium-dependent phosphoprotein formation, at two different calcium concentrations (1.0 μM, 0.4 mM), indicated that a steady-state was reached much sooner at higher calcium concentrations. Lanthanum, which is known to block dephosphorylation of the intermediate complex, increased both the apparent rate of formation and the steady-state level of the phosphorylated intermediate. Calmodulin, which has previously been shown to increase both the maximum velocity and the calcium affinity of the (Ca²⁺ + Mg²⁺)-ATPase, did not affect either calcium-dependent inhibition of (Ca²⁺ + Mg²⁺ )-ATPase activity or the biphasic nature of calcium-dependent phosphoprotein formation. At low calcium concentrations, calmodulin increased the apparent rate of phosphoprotein formation, whereas at higher calcium concentrations (0.4 mM) calmodulin reduced the steady-state level of the phosphoprotein; the apparent rate of formation was unaffected. In the presence of lanthanum, calmodulin increased both the apparent rate of formation and steady-state level of the phosphoprotein, suggesting that the true rate of formation was increased by calmodulin at higher calcium concentrations, but this was normally hidden by a simultaneous increase in the rate of dephosphorylation. Removal of endogenous magnesium, using trans-1,2-diamino-cyclohexane tetraacetic acid (CDTA) did not alter the calcium sensitivity or rate of formation of the phosphorylated intermediate, however turnover of the intermediate was markedly reduced. In the absence of free magnesium, both the velocity and calcium sensitivity of the (Ca²⁺ + Mg²⁺)-ATPase were also found to be lower. The low calcium-affinity component of calcium-dependent phosphoprotein formation, which Schatzmann (1982) has attributed to an action of calcium at a "magnesium-specific" site, was not affected by magnesium concentrations as high as 1 mM. Furthermore, this phosphoprotein could be dephosphorylated along either the forward or reverse pathways. These results indicate that the transformation from E₁.Ca.P to E₂.Ca.P may not be the site of the calcium-dependent inhibition of dephosphorylation. Calmodulin-depleted membrane fragments were prepared from the erythrocytes of cystic fibrosis patients as well as age- and sex-matched controls. Under conditions in which dephosphoryla-tion is inhibited, phosphoprotein formation and (Ca²⁺ + Mg²⁺)-ATPase activities were determined. Both (Ca²⁺ + Mg²⁺)-ATPase activity and phoshoprotein formation were found to be significantly reduced in the preparations derived from patients with cystic fibrosis. Turnover of the phosphorylated intermediate did not differ significantly between the two groups. A reduction in (Ca²⁺ + Mg²⁺)-ATPase activity and phosphoprotein formation suggests that there may be fewer active calcium-pumping sites in the erythrocyte membranes of cystic fibrosis patients compared to normal subjects. / Pharmaceutical Sciences, Faculty of / Graduate

The mechanism of regulation of the Ca²⁺ pump ATPase of the human erythrocyte membrane by calmodulin, cyclic AMP and the anion channel was studied using membrane fragments, resealed "ghosts", inside-out vesicles and a Triton X-100 solubilized enzyme preparation. The (Ca²⁺ + Mg²⁺ )-ATPase activity in erythrocyte membranes or a Triton X-100 solubilized enzyme preparation showed biphasic (high and low affinity) Ca²⁺ activation kinetics. The anionic calcium binding protein, calmodulin, increased both the calcium sensitivity (Kca²⁺) and the maximum velocity (Vmax ) of the enzyme. Certain polyanionic agents (poly-L-aspartic acid, poly-L-glutamic acid), alicyclic sulfonic acids (HEPES,N-2-hydroxyethylpiperazine-N¹-2-ethanesulfonic acid, MES,2-N- (morpholinoethanesulfonic acid)), and aromatic carboxylic acids (benzoic and salicylic acids) increased the Kca²⁺ but not the Vmax of (Ca²⁺ + Mg²⁺ )-ATPase in erythrocyte membranes and Triton X-100 solubilized enzyme preparations. Trifluoperazine (30 μM) antagonized activation of the enzyme by calmodulin and poly-L-aspartic acid, but not by sodium-HEPES or sodium-MES. Limited trypsin proteolysis of (Ca²⁺ + Mg²⁺ )-ATPase in the erythrocyte membrane abolished activation by calmodulin, poly-L-aspartic acid and sodium-HEPES. These results suggest that the modulation of the Ca²⁺ sensitivity of (Ca²⁺ + Mg²⁺ )-ATPase by calmodulin may be associated with the anionic properties of this protein, and that this property can be mimicked by some other anions, probably by interacting at an anion-regulatory site on the enzyme. Cyclic AMP (5 μM) was found to inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity (approx. 20%) in erythrocyte membranes, probably via endogenous cyclic AMP protein kinase, since this effect could be blocked by cyclic AMP protein kinase inhibitor (PKI) from the rabbit skeletal muscle, By contrast, bovine heart PKI stimulated (Ca²⁺ + Mg²⁺ )-ATPase activity (approx. 100%) by increasing the Kca²⁺ but not the Vmax of the enzyme in membrane or Triton X-100 solubilized preparations. At a low calcium concentration the stimulation by bovine heart PKI and saturating levels of calmodulin was additive, suggesting that the two effectors acted by distinct mechanisms. The stimulation of (Ca²⁺ + Mg²⁺ )-ATPase activity by bovine heart PKI was not solely due to its antagonism of the protein kinase because a) modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP protein kinase, but had no effect on the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase; b) trifluoperazine (20 μM) antagonized the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. It is suggested that different mechanisms are involved in the inhibition of cyclic AMP protein kinase and the stimulation of (Ca²⁺ + Mg²⁺ )-ATPase by bovine heart cyclic AMP PKI. Next, the role of anion channel blockers on the (Ca²⁺ + Mg²⁺ )- ATPase was studied. The photolabeling reagent N-(4-azido-2-nitrophenyl)- 2 aminoethylsulfonate (NAP-taurine) was found to inhibit the (Ca²⁺+ Mg²⁺ )-ATPase of fragmented red cell membranes. Half maximal inhibition occurred between 25 μM and 50 μM. At these concentrations Mg²⁺ -ATPase and (Na⁺ + K⁺)-ATPase activities in the membranes were not affected. The reversible inhibition of (Ca²⁺ + Mg²⁺ )-ATPase produced by NAP-taurine in the dark became irreversible after photolysis in the presence of this reagent. Incubation of the membranes with Ca²⁺ , Mg²⁺ , ATP or calmodulin, prior to photolysis in the presence of NAP-taurine, did not protect the enzyme from Inhibition. Limited trypsin proteolysis of (Ca²⁺ + Mg²⁺ )-ATPase in fragmented membranes, which abolished activation by calmodulin, did not affect the inhibition by NAP-taurine. NAP-taurine was found to Inhibit the (Ca²⁺ + Mg²⁺ )-ATPase activity from the cytoplasmic side of the membrane, as determined from the following experiments. Addition of NAP-taurine (50 μM) to resealed erythrocyte ghosts inhibited less than 5% of the (Ca²⁺ + Mg²⁺ )-ATPase activity, compared to 50-60% Inhibition in ghosts resealed in the presence of 50 μM NAP-taurine. Furthermore, NAP-taurine inhibited ATP-dependent Ca²⁺ - transport into inside-out vesicles at a similar concentration (50 μM). The inhibition of the (Ca²⁺ + Mg²⁺ )-ATPase activity of membranes by NAP-taurine appeared to be a direct action on the enzyme, rather than through inhibition of the anion channel, as (Ca²⁺ + Mg²⁺ )-ATPase activity was not inhibited in membranes made from red blood cells reacted irreversibly with 50 μM NAP-taurine or the anion channel blocker 4,4'-diisothiocyano- 2,2' stilbene disulfonate (DIDS) (5 μM) or in membranes assayed in the presence of another anion channel blocker, probenecid (125 μM). This is the first reported selective antagonist of the Ca²⁺ pump, and it is suggested that NAP-taurine could be a useful tool for studying the Ca²⁺- transport ATPase in a variety of cells. / Pharmaceutical Sciences, Faculty of / Graduate

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xiii, 134 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 119-128).

Thesis (Ph. D.)--University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Includes bibliographical references (p. 95-106).

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xv, 181 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 101-112).

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