Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8

Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage.     

Characterization of the (Ca2+-Mg2+)ATPase purified by calmodulin-affinity chromatography from bovine aortic smooth muscle

A calmodulin inhibitor, trifluoperazine, suppresses ATP-dependent Ca2+ uptake into microsomes prepared from bovine aortic smooth muscle. From this microsomal preparation which we expected to contain calmodulin-dependent Ca2+-transport ATPase [EC 3.6.1.3], we purified (Ca2+-Mg2+)ATPase by calmodulin affinity chromatography. The protein peak eluted by EDTA had calmodulin-dependent (Ca2+-Mg2+)ATPase activity. The major band (135,000 daltons) obtained after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) accounted for about 80% of the total protein eluted. This major band was phosphorylated by [gamma-32P]ATP in a Ca2+-dependent manner. All the 32P incorporated into the major band was released by hydroxylaminolysis. The ATPase reconstituted in soybean phospholipid liposomes showed ATP, calmodulin-dependent Ca2+ uptake. The affinity of the ATPase for Ca2+, Km, was 7 microM and the maximum ATPase activity was 1.4 mumol/mg/min. These values were changed to 0.17 microM and 3.5 mumol/mg/min, respectively by the addition of calmodulin. The activity of the purified (Ca2+-Mg2+)ATPase was inhibited by orthovanadate, and the concentration required for half-maximal inhibition was about 1.8 microM which is close to that of plasma membrane ATPases. Judging from the effect of orthovanadate and the molecular weight, the purified (Ca2+-Mg2+)ATPase was considered to have originated from the plasma membrane not from the sarcoplasmic reticulum.     

Manganese selectivity of pmr1, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6. Residue Asp778 is essential for cation transport

We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca(2+)- and Mn(2+)-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment M6 of Pmr1. In phenotypic screens of Ca(2+) chelator and Mn(2+) toxicity reported separately (Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., and Rao, R. (2000) J. Biol. Chem. 275, XXXX-XXXX), D778A was a loss-of-function mutant apparently defective for transport of both Ca(2+) and Mn(2+), whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn(2+) transport. We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP. However, reverse phosphorylation from P(i) is preserved but is insensitive to inhibition by Ca(2+) or Mn(2+) ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca(2+) can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 micrometer, essentially identical to that of wild type (0.07 micrometer). Mn(2+) activation of ATP hydrolysis was half-maximal at 0.02 micrometer in wild type, establishing a normal selectivity profile of Mn(2+) > Ca(2+). Strikingly, Mn(2+)-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 micrometer. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln(783) in M6 may form a critical hydrophobic interaction with Val(335) in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells.

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.     

The Ca2+/Mn2+ pumps in the Golgi apparatus

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Phostoxin-induced biochemical and pathomorphological changes in rabbits

The effect of chronic phostoxin administration on some tissue ATPases, hematology and tissue histopathology was investigated using a combination of gravimetric, enzymatic, colorimetric and histological procedures in New Zealand White rabbits after 2 weeks administration of 0.8mg phostoxin/kg body weight/day, po. The phostoxin treatment led to significant decreases in Na(+)-K+ ATPase activities in renal, hepatic and cardiac tissues. Similar decreases were obtained in the activities of Ca(2+)-ATPase and Mg(2+)-ATPase in liver. In addition, the phostoxin-toxified rabbits manifested significant decreases in hematocrit, red blood cell count, hemoglobin and platelets. Histological examination of the tissues revealed pronounced degenerative changes in liver, heart and kidney.     

Calmodulin stimulates both adenosine 5'-triphosphate hydrolysis and synthesis catalyzed by a cardiac calcium ion dependent adenosinetriphosphatase

A Ca2+-dependent ATPase purified from a rabbit heart membrane preparation was compared to the Ca2+-dependent ATPase purified from skeletal muscle sarcoplasmic reticulum. The two ATPases display an identical electrophoretic pattern and an identical Ca2+-concentration dependence. However, only the cardiac preparation exhibits a 2-3-fold activation by calmodulin. This effect is best observed when the molar concentrations of calmodulin and ATPase are equivalent and in the presence of high Ca2+ (approximately 10(-5) M) and ATP (approximately 10(-3) M) concentrations. It is demonstrated for the first time that calmodulin stimulates the rate of ATP synthesis, as revealed by an increased production of Pi and a faster ATP in equilibrium Pi exchange, as well as the rate of ATP hydrolysis. It is also demonstrated that calmodulin activation is expressed with purified and detergent-solubilized enzyme in addition to membrane-bound systems. These findings indicate that the effect of calmodulin is an acceleration of the enzyme turnover, due to direct interaction of calmodulin with the enzyme.     

Involvement of the L6-7 loop in SERCA1a Ca2+-ATPase activation by Ca2+ (or Sr2+) and ATP

Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

The plant Ca2+ -ATPase repertoire: biochemical features and physiological functions

Ca(2+)-ATPases are P-type ATPases that use the energy of ATP hydrolysis to pump Ca(2+) from the cytoplasm into intracellular compartments or into the apoplast. Plant cells possess two types of Ca(2+) -pumping ATPase, named ECAs (for ER-type Ca(2+)-ATPase) and ACAs (for auto-inhibited Ca(2+)-ATPase). Each type comprises different isoforms, localised on different membranes. Here, we summarise available knowledge of the biochemical characteristics and the physiological role of plant Ca(2+)-ATPases, greatly improved after gene identification, which allows both biochemical analysis of single isoforms through heterologous expression in yeast and expression profiling and phenotypic analysis of single isoform knock-out mutants.     

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+ and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca(2+)] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca(2+)or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca(2+) and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (deltapsi) at 37 degrees C. Addition of exogenous ATPase (300 mlU.ml(-1))]ATP (3.4 mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and deltapsi were reduced. Subsequent ATPase additions (1.3IU.ml(-)) were made with varying amounts of Ca(2+)(0 to 535 nM) to simulate step increases in cardiac work. Ca(2+) additions increased mVo2 and depolarized deltapsi, and were consistent with an activation of Fo/F1)ATPase. In contrast, Ca(2+) reduced the [NADH] response to the ATPase addition, consistent with Ca(2+)-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \2-fold in the presence of Ca(2+). The addition of 172nM free Ca(2+)] ATPase increased mVo2 by 300% (P<0.05, n=8) while deltapsi decreased by 14.9+/-0.1 mV without changes in [NADH] (P > or =0.05, n=8), consistent with working heart preparations. The addition of Ca(2+) and ATPase combined increased the mitochondrial ATP production rate with changes in deltapsi, NADH and [ADP], consistent with an activation of CaDH and F o /F(1)ATPase activity. These balancing effects of ATPase activity and [Ca(2+)] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity

CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.     

Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli

The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10His-tagged fusion proteins. Both proteins exhibit a mainly alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na(+),K(+)-ATPase and Ca(2+)-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.