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Results for “Transitional endoplasmic reticulum ATPase”

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PubMed1997

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Animals↗
PubMed2007

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells.

A sustained increase in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca(2+) stores by thapsigargin (TG), induced an increase in the [Ca(2+)](i) and cell death. However, the TG-induced death was not related to the [Ca(2+)](i) increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca(2+)-dependent anti-apoptotic mechanism. After TG treatment, Ca(2+)-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca(2+)-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca(2+)](i) increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca(2+)-dependent MAPK activation counteracts death signaling; removal of Ca(2+) activated a second caspase-independent death pathway.

Animals↗
PubMed2001

SERCA activity is required for timely progression through G1/S.

Changes in intracellular Ca2+ correlate with specific events in the cell cycle. Here we investigated the role of Ca2+ in the G1 phase. HEK 293 cells were arrested in mitosis and subjected to short-term treatments that alter Ca2+ homeostasis prior to their release into G1. Treatment with thapsigargin (TG), an irreversible inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) lengthened the G1 phase. Moreover, TG treatment also resulted in a dramatic alteration in cellular morphology and attachment and in the reduction of MAPK activity and lower levels of cyclin D1 and cyclin E proteins. Treatments with reagents that transiently increase or decrease cytosolic Ca2+ or that temporarily inactivate SERCA did not alter any of the above parameters. Cells expressing a TG-resistant form of SERCA progressed normally through the G1/S transition after TG treatment. These results suggest that long-term SERCA inactivation affects cell cycle-dependent events and compromises progression through G1/S.

Calcium↗
PubMed1997

Characterization of the Golgi complex cleared of proteins in transit and examination of calcium uptake activities.

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were > 99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

Adenosine Triphosphatases↗
PubMed2005

Nucleotide dependent motion and mechanism of action of p97/VCP.

The AAA (ATPases associated with a variety of cellular activities) family of proteins bind, hydrolyze, and release ATP to effect conformational changes, assembly, or disassembly upon their binding partners and substrate molecules. One of the members of this family, the hexameric p97/valosin-containing protein p97/VCP, is essential for the dislocation of misfolded membrane proteins from the endoplasmic reticulum. Here, we observe large motions and dynamic changes of p97/VCP as it proceeds through the ATP hydrolysis cycle. The analysis is based on crystal structures of four representative ATP hydrolysis states: APO, AMP-PNP, hydrolysis transition state ADP x AlF3, and ADP bound. Two of the structures presented herein, ADP and AMP-PNP bound, are new structures, and the ADP x AlF3 structure was re-refined to higher resolution. The largest motions occur at two stages during the hydrolysis cycle: after, but not upon, nucleotide binding and then following nucleotide release. The motions occur primarily in the D2 domain, the D1 alpha-helical domain, and the N-terminal domain, relative to the relatively stationary and invariant D1alpha/beta domain. In addition to the motions, we observed a transition from a rigid state to a flexible state upon loss of the gamma-phosphate group, and a further increase in flexibility within the D2 domains upon nucleotide release. The domains within each protomer of the hexameric p97/VCP deviate from strict 6-fold symmetry, with the more flexible ADP state exhibiting greater asymmetry compared to the relatively rigid ADP x AlF3 state, suggesting a mechanism of action in which hydrolysis and conformational changes move about the hexamer in a processive fashion.

Adenosine Triphosphatases↗
PubMed2005

Managing the manganese: molecular mechanisms of manganese transport and homeostasis.

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.

Biological Transport, Active↗
PubMed2003

Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein.

The hepatitis B virus X protein (HBx) is a multifunctional protein, acting on different targets (e.g. transcription factors, cytoplasmic kinases, and mitochondrial proteins) and exerting cellular effects as diverse as stimulation of cell proliferation and apoptosis. In its biological effects, the modulation of cellular Ca2+ signals has been proposed to be involved, but the direct assessment of Ca2+ homeostasis in HBx-transfected cells has not been carried out yet. In this work, we have employed for this purpose aequorin-based recombinant probes specifically targeted to intracellular organelles and microdomains. Using these probes, we observed that overexpression of HBx enhanced agonist-evoked cytosolic Ca2+ signals in HepG2 and HeLa cells, without affecting either the steady state of endoplasmic reticulum Ca2+ concentration or the kinetics of Ca2+ release. Rather, caspase-3-dependent cleavage of the plasma membrane Ca2+ ATPase could be demonstrated, and larger rises were detected in the cytoplasmic rim beneath the plasma membrane. In mitochondria, major morphological (fragmentation and swelling) and functional (reduced Ca2+ uptake) alterations were detected in HBx-expressing cells. As to the cellular consequences, we observed that HBx-induced apoptosis was markedly reduced when the alterations in Ca2+ signaling (e.g. by loading a Ca2+ chelator or preventing PMCA cleavage) or the downstream effects (e.g. by inhibiting mitochondrial permeability transition) were prevented. Overall, these results indicate that HBx perturbs intracellular Ca2+ homeostasis, acting on the extrusion mechanisms, and that this effect plays an important role in the control of HBx-related apoptosis.

Apoptosis↗
PubMed2005

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC.

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.

Animals↗
PubMed2000

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices.

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.

Actins↗
PubMed1985

Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1.

In rats treated orally with a single dose of aflatoxin B1 (5 mg/kg body weight) characteristic focal and nodular liver lesions developed which differed in their fine structure, enzyme histochemical pattern and growth behaviour from other types of carcinogen-induced hepatic foci and nodules described earlier. The foci were composed of a distinct cell population which showed specific structural changes of the cytoplasm. Typically, unusually large and abundant basophilic bodies consisting of highly ordered stacks of cisternae of the rough endoplasmic reticulum (ER) were arranged in long, striped bands and stood out against an acidophilic background which was due to hypertrophy of the smooth ER. We propose the descriptive terms 'tigroid cells', and 'tigroid cell foci' for this population of altered hepatocytes. Correlative cytochemical investigations on the tigroid cell foci revealed characteristic changes in carbohydrate metabolism, such as a decrease in the activity of glycogen synthetase and glycogen phosphorylase and an increase in the activity of glucose-6-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The activity of glucose-6-phosphatase and ATPase was normal (or partially reduced) and that of the gamma-glutamyl-transpeptidase was always lacking. A progressive increase in the number and size of the tigroid cell foci and transitions from tigroid cell foci to neoplastic nodules with similar morphological and cytochemical features were observed during the time period of 104 weeks. The mitotic index within tigroid cell foci and nodules was approximately 100 times higher than that of the surrounding hepatic tissue or the liver parenchyma of untreated control animals. The important question whether the tigroid cell foci represent a specific pre-neoplastic or early neoplastic cell population requires further investigations.

Aflatoxin B1↗
PubMed2000

Clomiphene, an ovulation-inducing agent, mobilizes intracellular Ca2+ and causes extracellular Ca2+ influx in bladder female transitional carcinoma cells.

BACKGROUND/METHODS: The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of BFTC human bladder cancer cells was explored by using fura-2 as a Ca2+ indicator. RESULTS: Clomiphene at concentrations between 10 and 75 microM increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 50 microM. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about 40-50% in maximum [Ca2+]i. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 50 microM clomiphene in Ca2+-free medium, suggesting that clomiphene induced capacitative Ca2+ entry. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and CCCP (to uncouple mitochondria) inhibited 85% of clomiphene-induced intracellular Ca2+ release. Conversely, pretreatment with 50 microM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin, thapsigargin or CCCP. The intracellular Ca2+ release was unaltered by inhibiting formation of inositol-1,4,5-trisphosphate (IP3) with 2 mM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; a phospholipase C inhibitor). CONCLUSION: The [Ca2+]i increase induced by 50 microM clomiphene was not affected by 10 microM of nifedipine, verapamil or diltiazem. Collectively, the results suggest that clomiphene releases intracellular Ca2+ in an IP3-independent manner and also activates extracellular Ca2+ influx.

Calcium↗
PubMed1998

Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Animals↗
PubMed2006

Metal transportome of Neurospora crassa.

Neurospora crassa has been the model filamentous fungus for the study of many fundamental cellular mechanisms of transport and metabolism. The recently completed genome sequence of N. crassa has over 10,000 genes without significant matches for a large number of genes (41%) in the sequence databases, indeed presents many challenges for new discoveries. Using transporter database and BLAST searches a total of 65 open reading frames for putative cation transporter genes have been identified in N. crassa. These were further confirmed by characteristic features of the family like transmembrane domains (TOPPRED 2), conserved motifs (Clustal W) and phylogenetic analysis (TREETOP). In Neurospora cation transporter genes constitute nearly 18.3% of the total membrane transport systems, which is higher than E. coli (8.8%), S. cerevisiae (13.7%), S. pombe (17.2%), A. fumigatus (10.1%), A. thaliana (16.8%) and H. sapiens (15.6%). We refer to the complete complement of metal ion transporter genes as "Metal Transportome". There are a total of 33 putative transporters for alkali and alkaline earth metals constituting 18 for calcium (P-ATPase, VIC, CaCA, Mid1), 7 for sodium (P-ATPase, CPA1, CPA2), 4 for potassium (Trk, VIC, KUP), and 4 for magnesium (MIT). Transition metal ion transporters account for 32 transporters including 7 for zinc (ZIP), 6 for copper (Ctr2, Ctr1), 2 each for manganese (Nramp), iron (OFeT), arsenite (ArsAB, ACR3) and other metal ions (ABC and P-ATPase) and 1 each for nickel (NiCoT) and chromate (CHR). N. crassa has 7 linkage groups of which LGI harbors 21 of metal ion transporters and in contrast LGVII has only 2. Studies on metal transportomes of different organisms will help to unravel the role of metal ion transporters in homeostasis.

Animals↗
PubMed2003

Differential degradation of the Na+/K+-ATPase subunits in the plasma membrane.

The cell-surface expression of the Na(+)/K(+)-ATPase is tightly regulated at the ER/Golgi transit level. The assembly of the alpha- and beta-subunits is a prerequisite for the maturation of the enzyme and its exit from the ER. At present, the fate of the functional ATPase, once it reaches the plasma membrane, is obscure, although endocytosis, recycling, and degradation in the lysosome are known to be involved. In this study, we shall demonstrate the differential degradation of the Na(+)/K(+)-ATPase subunits in the plasma membrane: that is, lysosomal degradation of alpha-subunits and proteasomal degradation of beta-subunits, and propose the possibility of the subunit dissociation in the plasma membrane.

Animals↗
PubMed2005

Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene.

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.

Adenosine Triphosphatases↗
PubMed2002

Dissection of the functional differences between sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) 1 and 3 isoforms by steady-state and transient kinetic analyses.

Steady-state and transient-kinetic studies were conducted to characterize the overall and partial reactions of the Ca(2+)-transport cycle mediated by the human sarco(endo)plasmic reticulum Ca(2+)-ATPase 3 (SERCA3) isoforms: SERCA3a, SERCA3b, and SERCA3c. Relative to SERCA1a, all three human SERCA3 enzymes displayed a reduced apparent affinity for cytosolic Ca(2+) in activation of the overall reaction due to a decreased E(2) to E(1)Ca(2) transition rate and an increased rate of Ca(2+) dissociation from E(1)Ca(2). At neutral pH, the ATPase activity of the SERCA3 enzymes was not significantly enhanced upon permeabilization of the microsomal vesicles with calcium ionophore, indicating a difference from SERCA1a with respect to regulation of the lumenal Ca(2+) level (either an enhanced efflux of lumenal Ca(2+) through the pump in E(2) form or insensitivity to inhibition by lumenal Ca(2+)). Other differences from SERCA1a with respect to the overall ATPase reaction were an alkaline shift of the pH optimum, increased catalytic turnover rate at pH optimum (highest for SERCA3b, the isoform with the longest C terminus), and an increased sensitivity to inhibition by vanadate that disappeared under equilibrium conditions in the absence of Ca(2+) and ATP. The transient-kinetic analysis traced several of the differences from SERCA1a to an enhancement of the rate of dephosphorylation of the E(2)P phosphoenzyme intermediate, which was most pronounced at alkaline pH and increased with the length of the alternatively spliced C terminus.

Adenosine Triphosphate↗
PubMed1983

Induction of megamitochondria in the mouse and rat livers by hydrazine.

The correlation between structures of chemicals and their inducibility for megamitochondrial formation was investigated. Since the chemical structure universal to the inducers of megamitochondria previously reported (cuprizone and isonicotinic acid derivatives) is the carbazoyl group (-CONHNH2), semicarbazide (NH2NHCONH2) was tested first. Then, hydrazine (NH2NH2) was tested, replacing the carbazoyl group of semicarbazide by an amino group (-NH2). The present study demonstrates that (1) megamitochondria were induced in mouse and rat hepatocytes by feeding the animals with a diet containing semicarbazide or hydrazine, suggesting that the carbazoyl group was not essential for megamitochondrial formation; (2) hydrazine-induced megamitochondrial formation was a reversible process. Coupling efficiencies and activities of ATPase and cytochrome oxidase of megamitochondria induced by hydrazine were slightly decreased, while the activity of monoamine oxidase was moderately decreased. Freeze-fracture electron microscopy revealed particle-free regions in the outer membranes of megamitochondria fixed with glutaraldehyde at 22 degrees C; the regions disappeared at 25 degrees C, indicating that the temperature of the liquid crystalline to gel state lipid phase transition in the megamitochondrial outer membrane was elevated. It is speculated that chemical structure of inducer of megamitochondria could be simplified to NH2-G (G, substituting group).

Aging↗
PubMed2005

Dissection of the functional differences between human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 1 and 2 isoenzymes by steady-state and transient kinetic analyses.

Human secretory pathway Ca2+/Mn2+-ATPase (SPCA) 2 encoded by ATP2C2 is only expressed in a limited number of tissues, unlike the ubiquitously expressed SPCA1 pump (encoded by ATP2C1, the gene defective in Hailey-Hailey disease). It has not been determined whether there are significant functional differences between SPCA1 and SPCA2 pump enzymes. Therefore, steady-state and transient kinetic approaches were used to characterize the overall and partial reactions of the Ca2+ transport cycle mediated by the human SPCA2 enzyme upon heterologous expression in HEK-293 cells. The catalytic turnover rate of SPCA2 was found enhanced relative to SPCA1 pumps. SPCA2 displayed a very high apparent affinity for cytosolic Ca2+ (K0.5 = 0.025 microm) in activation of the phosphorylation activity but still 2.5-fold lower than that of SPCA1d. Our kinetic analysis traced both differences to the increased rate characterizing the E1 approximately PCa to E2-P transition of SPCA2. Moreover, the reduced rate of the E2 to E1 transition seems to contribute in determining the lower apparent Ca2+ affinity and the increased sensitivity to thapsigargin inhibition, relative to SPCA1d. SPCA2 also displayed a reduced apparent affinity for inorganic phosphate, which could be explained by the observed enhanced rate of the E2-P dephosphorylation. The insensitivity to modulation by pH and K+ concentration of the constitutively enhanced E2-P dephosphorylation of SPCA2 is similar to SPCA1d and possibly represents a novel SPCA-specific feature, which is not shared by sarco(endo)plasmic reticulum Ca2+-ATPases.

Adenosine Triphosphatases↗