Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human muscle sarcoplasmic reticulum (SR) yields three major protein bands. The percent distribution of the mean values of the bands from 15 normal human muscles was 55.4, 14.6, and 30.0 for the 100, 55, and 45-kDa mass proteins, respectively. A mean distribution similar to that in normal muscle SR was found in preparations from 7 patients with polymyositis and from 7 patients with myotonic dystrophy. In 12 preparations from patients with Duchenne dystrophy, the protein distribution differed from that of preparations from normal muscle. The 100-kDa mass protein band was decreased, whereas the 55- and 45-kDa mass bands were increased. Protease inhibitors pepstatin A, antipain, and leupeptin, as well as ethyleneglycol-bis(aminoethyl ether)-N,N,N',N'-tetraacetic acid or ethylenediaminetetraacetic acid, significantly reduced this change. However, some of the changes cannot be prevented by the addition of inhibitors and must be expressed in vivo. Neither protease inhibitors nor chelators affected SR preparations from normal muscle. We found a five- to ten-fold increase in calcium-activated neutral protease activity in Duchenne dystrophic muscles that degraded the calcium-adenosinetriphosphatase of SR. The active protease was identified as the cytoplasmic calpain II. The increased activity in Duchenne muscles may explain many reported abnormalities.
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PMID:Membrane defects in Duchenne dystrophy: protease affecting sarcoplasmic reticulum. 352 39

The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4-benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+)-induced calpain activation and a fusion process inhibited by annexin V.
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PMID:Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: a study using Ca2+-ATPase inhibitors. 754 94

Rat myocardium expresses the 240- and 235-kD polypeptides antigenically related to alpha- and beta-subunits of brain calspectin (nonerythroid spectrin or fodrin), respectively. In the subcellular fractions of the myocardium, alpha-calspectin was found in the 600g, 10,000g, and 100,000g pellets, whereas beta-calspectin was localized to the 10,000g pellet. On the basis of the Na+,K(+)-ATPase activity and the contents of a gap junction protein, the sarcolemma was distributed to the 10,000g and 100,000g pellets, and the intercalated disks were enriched in the 10,000g pellet. Both alpha- and beta-calspectin were proteolyzed by calpain in vitro. The two subunits were also proteolyzed in vivo, when the rat hearts underwent 10 to 60 minutes of global ischemia followed by 30 minutes of reperfusion. The reperfusion following the ischemia induced the proteolysis of alpha-calspectin in the 10,000g and 100,000g pellets, producing the 150-kD fragment. A synthetic calpain inhibitor, calpain inhibitor-1, suppressed the degradation of calspectin in vivo, which indicates that calpain is responsible for the reperfusion-induced proteolysis of calspectin. The inhibitor also improved myocardial stunning. Immunohistochemical study revealed that the proteolysis of alpha-calspectin occurs at the intercalated disks and the sarcolemma after postischemic reperfusion, in accord with the biochemical data. These results suggest that degradation of calspectin partly accounts for the contractile failure of the myocardium after postischemic reperfusion by disrupting the membrane skeleton and the intercalated disks.
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PMID:Reperfusion of rat heart after brief ischemia induces proteolysis of calspectin (nonerythroid spectrin or fodrin) by calpain. 764 30

A calmodulin-binding motif is a common structural feature of a number of calpain substrates (1). Since a calmodulin-like domain has been identified in both subunits of the calpain molecule, the proposal was made that the domain(s) would recognize the calmodulin-binding motifs of the substrates prior to the enzymatic modification by calpain. In keeping with the proposal, a successful attempt to purify mu-calpain from human erythrocytes was made by using an affinity chromatography approach in which the synthetic peptide C49, containing the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase, was coupled to a Sepharose matrix. The calmodulin-like domain of the catalytic subunit of human mu-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column. Both mu-calpain and the calmodulin-like domain interacted with C49 in a Ca(2+)-dependent way and were eluted from the column by Ca(2+)-chelating agents. The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca(2+)-ATPase and the calmodulin-like domain of mu-calpain. Experiments were performed to establish whether irreversibly inactivated mu-calpain or its expressed C-terminal portion containing the calmodulin-like domain could activate the hydrolysis of ATP by the plasma membrane Ca2+ pump, in keeping with evident ATPase stimulation of the same pump by calmodulin. A stimulation was observed, but it was much weaker than that induced by calmodulin.
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PMID:Purification of mu-calpain by a novel affinity chromatography approach. New insights into the mechanism of the interaction of the protease with targets. 778 21

Two versions of the calmodulin binding domain of the plasma membrane Ca2+ ATPase, a 24-amino acid peptide, C24W (Q-I-L-W-F-R-G-L-N-R-I-Q-T-Q-I-R-V-V-N-A-F-R-S-S-NH2), and the corresponding phosphothreonine containing peptide, C24W-P (Q-I-L-W-F-R-G-L-N-R-I-Q-T(phospho)-Q-I-R-V-V-N-A-F-R-S-S-NH2), were synthesized. They were used to investigate the effect of threonine phosphorylation by protein kinase C on the binding of calmodulin by the calmodulin binding domain and on the inhibitory role of the domain on the activity of the Ca2+ pump. The phosphopeptide C24W-P was obtained after global phosphorylation of the free Thr side chain on the protected resin bound peptide. The phosphorylated calmodulin binding domain failed to bind calmodulin; this was shown by gel shift experiments, by fluorescence energy transfer studies and by competition experiments against calmodulin stimulation of the pump. The inhibition of the Ca2+ pump activity by the calmodulin binding domain in the absence of calmodulin was also affected by the phosphorylation of the threonine; the inhibition of the fully active calpain-truncated pump by the phosphothreonine containing peptide was lower than that by the unphosphorylated synthetic domain.
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PMID:Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site. 792 86

The Ca(2+)-pump isolated in rod outer segment disk membranes as aspartylphosphate intermediate E-P has been characterized: the 100 kDa phosphoprotein was completely inhibited by thapsigargin, was not sensitive to digestion by calpain, and displayed a tryptic digestion pattern with the formation of two autophosphorylatable fragments of about 55 and 35 kDa. These results are typical of the calcium pumps of sarcoplasmic or endoplasmic reticulum calcium and differ from those of plasma membrane, such as the Ca(2+)-ATPase of the red blood cells, here shown as controls. The physiological role of calcium pump in disk membranes of vertebrate photoreceptors is discussed in terms of intracellular calcium buffering.
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PMID:Characterization of Ca(2+)-ATPase in rod outer segment disk membranes. 798 May 47

A review on the human erythrocyte Ca pump is presented, discussing general aspects of the catalytic cycle and the effect of some naturally occurring modulators such as calmodulin, acidic phospholipids and polyunsaturated fatty acids, controlled calpain proteolysis and protein kinases. The effect of both ATP, mono and divalent cations and recent experiments by the author and coworkers are also discussed in the same context, pinpointing Ca and Mg as new Ca ATPase modulators.
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PMID:[Possible new modulators of the calcium pump of human erythrocytes]. 808 10

The activation of calpain in normal human erythrocytes incubated in the presence of Ca2+ and the Ca2+ ionophore A23187 led to the decline of the Ca(2+)-dependent ATPase activity of the cells. Preloading of the erythrocyte with an anticalpain antibody prevented the decline. The pump was also inactivated by applied to isolated erythrocyte plasma membranes. The decline of the pump activity corresponded to the degradation of the pump protein and was inversely correlated to the amount of the natural inhibitor of calpain, calpastatin, present in the cells. In erythrocytes containing only 50% of the normal level the degradation started at a concentration of Ca2+ significantly lower than in normal cells. A comparison of the concentrations of Ca2+ required for the degradation of a number of erythrocyte membrane proteins showed that the Ca2+ pump and band 3 were the most sensitive. All other membrane proteins tested were attacked at higher levels of intracellular Ca2+. Thus, the degradation of the Ca2+ pump protein may be a simple and sensitive means to monitor calpain activation in vivo. Furthermore, the results have shown that the calpastatin level correlated directly with the amount of activable calpain and with the concentration of Ca2+ required to trigger the activation process.
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PMID:The plasma membrane calcium pump is the preferred calpain substrate within the erythrocyte. 814 3

Effect of intraerythrocyte Ca2+ elevation on human and rat erythrocyte membrane (Ca(2+)-Mg2+)-ATPase along with that of incubation of the erythrocyte ghosts in their own hemolysates enriched with Ca2+ has been studied. While the membrane (Ca(2+)-Mg2+)-ATPase levels of Ca(2+)-loaded human erythrocytes showed an initial increase and subsequent decline, membranes incubated in their own hemolysate showed a consistent decrease in the enzyme activity. Calmodulin sensitivity was retained by the preparations in contrast to the earlier observations made with washed erythrocyte membranes. Similar changes in (Ca(2+)-Mg2+)-ATPase activity but of greater magnitude were observed in response to Ca2+ in the calpain-rich rat erythrocytes. Considerable crosslinking and proteolysis was observed in case of human and rat erythrocytes exposed to high Ca2+ concentrations. The Ca(2+)-activable transglutaminase, however, did not play any role in the activation of the (Ca(2+)-Mg2+)-ATPase.
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PMID:Ca(2+)-induced alterations in the activity of membrane (Ca(2+)-Mg2+)-ATPase of human and rat erythrocytes. 835 24

This study investigates ischemia-induced degradation of the spectrin-based cytoskeleton in rat brain, heart, and kidney. Spectrin, in conjunction with ankyrin, structurally supports the plasma membrane and sequesters integral membrane proteins. After 60 and 120 min of ischemia, brain tissue displayed both spectrin and ankyrin breakdown. The spectrin fragmentation pattern is similar to previously reported ischemia-induced calpain I proteolysis of spectrin in N-methyl-D-aspartate receptor-containing neurons. Ischemic heart tissue displayed no spectrin or ankyrin degradation. Ischemic renal tissue showed minimal breakdown of spectrin but a major loss of ankyrin (25%/30 min of ischemia) that was essentially complete after 120 min of ischemia. Interestingly, this profound loss of ankyrin in the intact ischemic kidney was not mimicked in three renal cell lines (MDCK, LLC-PK1, and JTC cell lines) exposed to chemical anoxia. Immunocytochemistry showed ankyrin was concentrated in thick ascending limb (cTAL) cells and, although delayed by 30 min, was lost at the same rate as measured by immunoblot analysis. Spectrin and Na(+)-K(+)-ATPase, which complex with ankyrin, were essentially unaffected by ischemia. Ankyrin degradation in cTAL cells correlated with the loss of basal infolding organization. In conclusion, the spectrin-based cytoskeleton is differentially targeted by ischemia-induced degradative processes in different in vivo tissues.
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PMID:Degradation of spectrin and ankyrin in the ischemic rat kidney. 838 46


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