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)

To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulation of human platelet Ca(2+)-ATPase and Ca2+ restoration by calpain. 839 39

The actin binding and ATPase properties, as well as the functional domain structure of chick brain myosin-V, a two-headed, unconventional myosin, is reported here. Compared to conventional myosin from skeletal muscle, brain myosin-V exhibits low K-EDTA- and Ca-ATPase activities (1.8 and 0.8 ATP/s per head). The physiologically relevant Mg-ATPase is also low (approximately 0.3 ATP/s), unless activated by the presence of both F-actin and Ca2+ (Vmax of 27 ATP/s). Ca2+ stimulates the actin-activated Mg-ATPase over a narrow concentration range between 1 and 3 microM. In the presence of saturating Ca2+ and 75 mM KCl, surprisingly low concentrations of F-actin activate the Mg-ATPase in a hyperbolic manner (KATPase of 1.3 microM). Brain myosin-V also binds with relatively high affinity (compared to other known myosins) to F-actin in the presence of ATP, as assayed by cosedimentation. Digestion of brain myosin-V with calpain yielded a 65-kDa head domain fragment that cosediments with actin in an ATP-sensitive manner and a 80-kDa tail fragment that does not interact with F-actin. The 80-kDa fragment results from cleavage one residue beyond the proline-, glutamate-, serine-, threonine-rich region. Our data indicate that the Mg-ATPase cycle of brain myosin-V is tightly regulated by Ca2+, probably via direct binding to the calmodulin light chains in the neck domain, which like brush border myosin-I, results in partial (approximately 30%) dissociation of the calmodulin associated with brain myosin-V. The effect of Ca2+ binding, which appears to relieve suppression by the neck domain, can be mimicked by calpain cleavage near the head/neck junction.
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PMID:Enzymatic characterization and functional domain mapping of brain myosin-V. 866 47

We studied the activity of plasma membrane (Ca+Mg)ATPase from erythrocytes of Milan hypertensive rat strain (MHS) and Milan low calpastatin rat strain (MLCS), that show an activity level of the specific calpain inhibitor, calpastatin, about five fold reduced in comparison with the Milan normotensive rat strain (MNS), while the protease activity level is similar. This imbalance of calpain:calpastatin ratio leads to a decrease of the erythrocyte plasma membrane (Ca+Mg)ATPase activity and to the appearance of 124 kDa fragments, which are the typical products of proteolytic calpain action on the 136 kDa (Ca+Mg)ATPase native form.
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PMID:Association between plasma membrane (Ca+Mg) ATPase and calpain/calpastatin system in rat erythrocytes. 867 Feb 46

Calpains are Ca-activated neutral proteases present in all cells together with an endogenous inhibitor, calpastatin. Proposed substrates are; cytoskeletal proteins like microtubules and actin, protein kinases such as PKC and membrane-bound enzymes like Ca-ATPase and the Ca-channel. In lenses from different species calpains have been detected in decreasing amounts from the epithelium to the cortex to the nucleus. Several substrates for calpain in the lens have been demonstrated: crystallins, vimentin, actin, beaded filaments and MP26 among others. Both studies on animal models and capsulorhexis indicate that calpains are mainly involved in cortical cataract.
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PMID:Calpains in the human lens: relations to membranes and possible role in cataract formation. 872 65

Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.
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PMID:Endoplasmic reticulum Ca(2+)-ATPase in microsomal vesicles isolated from bovine retinae. 874 9

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca(2+)-dependent protease calpain. The effects of the Ca(2+)-ATPase inhibitors thapsigargin, cyclopiazonic acid and 2.5-di-(t-butyl)-1,4-benzohydroquinone were compared with those of the Ca2+ ionophore A23187. Whereas all three Ca(2+)-ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca2+ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca(2+)-free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca2+ stores by A23187. Incubation of fluo-3-loaded plateters with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 microM Ca2+ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca2+] reached 3-8 microM. To assess the physiologic significance of these results, the subpopulation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin-V-coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca2+] that is brought about by influx across the plasma membrane.
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PMID:Calcium influx is a determining factor of calpain activation and microparticle formation in platelets. 877 8

Ankyrin links the fodrin-based cytoskeleton to membrane proteins such as Na+/K(+)-ATPase, thereby maintaining cellular integrity. Immunoblotting by antibody raised against erythrocyte ankyrin demonstrated the proteolysis of ankyrin, which was highly correlated with postmortem interval (0-24 h). Proteolysis in the postmortem brain generated the 160-kDa fragment with an identical size as the fragment formed after in vitro proteolysis by calpain. Although microM Ca2+ induced the proteolysis in the homogenate, the presence of mu-calpain was not demonstrated by immunoblotting using the antibody that reacts with large subunits both of mu- and m-calpains. Na+/K(+)-ATPase was also proteolyzed in the postmortem brain.
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PMID:Proteolysis of ankyrin and Na+/K(+)-ATPase in postmortem rat brain: is calpain involved? 915 85

We have shown previously that intracellular Ca+2 chelation and calpain inhibitors block the influx of extracellular Ca+2 and Cl- during the late phase of cell injury in renal proximal tubules (RPT) exposed to the mitochondrial inhibitor antimycin A. Since the endoplasmic reticulum (ER) is the major intracellular Ca+2 storage site, ER Ca+2 release/depletion may mediate the Ca+2 influx and cell death. Treatment of RPT suspensions with thapsigargin, an ER Ca+2-ATPase inhibitor, increased cytosolic free Caf+2 (Ca+2) levels from 122 +/- 7 to 322 +/- 55 nM within 10 sec of addition followed by a return to control levels within 3 min. A 5-min pretreatment of RPT suspensions with thapsigargin blocked antimycin A- and hypoxia-induced influx of extracellular Ca+2 and Cl- and the resulting cell death/lysis. These data suggest that ER Ca+2 release/depletion during cell injury may trigger a signaling cascade that causes extracellular Ca+2 influx followed by Cl- influx, cell swelling, and ultimately cell death/ lysis.
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PMID:Depletion of endoplasmic reticulum calcium stores protects against hypoxia- and mitochondrial inhibitor-induced cellular injury and death. 936 81

White matter of the brain and spinal cord is susceptible to anoxia and ischemia. Irreversible injury to this tissue can have serious consequences for the overall function of the CNS through disruption of signal transmission. Myelinated axons of the CNS are critically dependent on a continuous supply of energy largely generated through oxidative phosphorylation. Anoxia and ischemia cause rapid energy depletion, failure of the Na(+)-K(+)-ATPase, and accumulation of axoplasmic Na+ through noninactivating Na+ channels, with concentrations approaching 100 mmol/L after 60 minutes of anoxia. Coupled with severe K+ depletion that results in large membrane depolarization, high [Na+]i stimulates reverse Na(+)-Ca2+ exchange and axonal Ca2+ overload. A component of Ca2+ entry occurs directly through Na+ channels. The excessive accumulation of Ca2+ in turn activates various Ca(2+)-dependent enzymes, such as calpain, phospholipases, and protein kinase C, resulting in irreversible injury. The latter enzyme may be involved in "autoprotection," triggered by release of endogenous gamma-aminobutyric acid and adenosine, by modulation of certain elements responsible for deregulation of ion homeostasis. Glycolytic block, in contrast to anoxia alone, appears to preferentially mobilize internal Ca2+ stores; as control of internal Ca2+ pools is lost, excessive release from this compartment may itself contribute to axonal damage. Reoxygenation paradoxically accelerates injury in many axons, possibly as a result of severe mitochondrial Ca2+ overload leading to a secondary failure of respiration. Although glia are relatively resistant to anoxia, oligodendrocytes and the myelin sheath may be damaged by glutamate released by reverse Na(+)-glutamate transport. Use-dependent Na+ channel blockers, particularly charged compounds such as QX-314, are highly neuroprotective in vitro, but only agents that exist partially in a neutral form, such as mexiletine and tocainide, are effective after systemic administration, because charged species cannot penetrate the blood-brain barrier easily. These concepts may also apply to other white matter disorders, such as spinal cord injury or diffuse axonal injury in brain trauma. Moreover, whereas many events are unique to white matter injury, a number of steps are common to both gray and white matter anoxia and ischemia. Optimal protection of the CNS as a whole will therefore require combination therapy aimed at unique steps in gray and white matter regions, or intervention at common points in the injury cascades.
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PMID:Anoxic and ischemic injury of myelinated axons in CNS white matter: from mechanistic concepts to therapeutics. 942 2

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.
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PMID:Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation. 969 25


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