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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ultraviolet (280-nm) irradiation of bovine brain calmodulin results in calcium-dependent changes in its fluorescence emission spectrum. These consist of a decline in the intrinsic tyrosine fluorescence of the protein and the appearance of a new emission maximum at 400 nm. Chromatography of irradiated calmodulin, using Ultrogel AcA 54 and phenyl-agarose columns, yields several distinctive fractions. One of these, representing 2.8% of the total recovered protein and 53% of the total fluorescence emission at 400 nm, was selected for detailed characterization. Analyses performed on acid hydrolysates reveal the presence of dityrosine, a derivative of tyrosine known for its fluorescence near 400 nm, at the level of 0.59-0.89 mol per 16,700 g of protein. Sodium dodecyl sulfate gel electrophoresis experiments demonstrate two components of apparent molecular weights 14,000 (80%) and 16,000 (20%). Observations on the effects of UV irradiation on the thrombic fragments of calmodulin and on related calcium binding proteins (rabbit skeletal muscle troponin C, bovine cardiac troponin C, and parvalbumin) support the interpretation that dityrosine formation in calmodulin results from the intramolecular cross-linking of Tyr-99 and Tyr-138. The dityrosine-containing photoproduct of calmodulin is unable to stimulate the p-nitrophenyl phosphatase activity of calcineurin under standard assay conditions. Fluorescence titrations show a generally weakened interaction with calcium ion occurring in two stages. The pKa of the derivative is considerably higher than that of free dityrosine and is calcium dependent, decreasing from 7.88 to 7.59 on the addition of 3 mM CaCl2. Smooth muscle myosin light chain kinase binds the derivative about 280-fold less effectively than it binds native calmodulin. Of several metal ions tested, only Cd2+ approaches Ca2+ in its ability to promote the appearance of the 400-nm emission band during UV irradiation of calmodulin. Mn2+ and Cu2+ appear to inhibit dityrosine formation. Ascorbic acid, dithiothreitol, and glutathione are also inhibitory.
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PMID:Dityrosine formation in calmodulin. 356 41

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.
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PMID:Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase. 375 57

Stimulus-response coupling mediated by calmodulin involves several steps: a transitory increase in calcium concentration from 0.1 to 10 microM, induced by external stimuli; interaction of calcium with calmodulin, accompanied by stepwise structural transitions; the coordinated interaction with and activation of the many calmodulin-regulated enzymes and proteins. The binding of calcium to calmodulin is a cooperative and selective process that is modulated by magnesium. At physiological ionic strength, and only in the presence of magnesium, a large difference is seen between the affinities of sites III and IV (0.09 X 10(6) M-1) and sites I and II (0.0007 X 10(6) M-1) for calcium. This difference, together with the positive cooperativity previously observed, explains the stepwise conformational changes induced by calcium. The interaction of calmodulin with its target proteins requires the integrity of different portions of the calmodulin molecule. Calmodulin-regulated enzymes can be divided into three classes according to their abilities to bind with and to be activated by calmodulin fragments: enzymes which are activated by the C-terminal fragment, such as the Ca2+-ATPase and phosphorylase kinase; enzymes which require both halves of the molecule, such as cyclic AMP phosphodiesterase and myosin light chain kinase; and enzymes whose interaction with calmodulin fragments is too weak to be detected by activation, such as calcineurin and the multiprotein kinase. Thus different enzymes may be activated by different calmodulin conformers and the stepwise changes exhibited by calmodulin at different calcium levels can be used to regulate different metabolic pathways.
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PMID:Regulation of the calcium signal by calmodulin. 379 36

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity. 623 85

In this study, we examined the role of insulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade in activation of protein phosphatase-1 (PP-1) by using three complementary approaches. First, differentiated L6 cells were acutely exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 400 nM) to activate PKC. In these cells, TPA caused 32% stimulation of PP-1 activity. The PP-1 stimulation by TPA was comparable to stimulation by insulin (t1/2 = 1 min and EC50 = 5 nM) with a maximum effect in 5 min. The effects of insulin and TPA were not additive. Insulin and TPA also stimulated MAPK (> 2-fold increase over basal, with myelin basic protein as a substrate). ML-9, a myosin light chain kinase inhibitor, blocked the effects of insulin and TPA on both MAPK and PP-1 activation. In the second approach, PKC was down-regulated by chronic treatment with TPA. In these cells subsequent effects of insulin on MAPK and PP-1 activation were blocked, without an effect on basal enzyme levels. In the third approach, two selective inhibitors of PKC, calphostin and chelerythrine chloride, were used to inhibit PKC. These inhibitors completely prevented insulin and TPA stimulation of MAPK and PP-1 and blocked insulin-induced translocation of PKC to the plasma membranes. We conclude that PKC plays an important role in insulin stimulation of PP-1 via the activation of MAPK cascade.
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PMID:Stimulation of protein phosphatase-1 activity by phorbol esters. Evaluation of the regulatory role of protein kinase C in insulin action. 751 82

In the presence of Ca2+ and calmodulin (CM), purified smooth muscle myosin light chain kinase (MLCKase) was found to undergo autophosphorylation at a rate that was about 200-fold slower than its catalytic activity. Up to 1.7 mol of phosphate were incorporated per mole of kinase. Lower levels of incorporation could be correlated with the presence of an endogenous protein phosphatase which could be inhibited with okadaic acid or Microcystin-LR. The major autophosphorylation site was identified as Thr-863 or Thr-865 and was located on the 24-kDa C-terminal fragment of the kinase. In addition, there was a relatively low and variable contribution of a Ca/CM-independent autophosphorylation at Ser-814 or Ser-815. The initial autophosphorylation rates and maximal incorporation levels were highest at a molar ratio of 2 MLCKase to 1 CM and were inhibited at higher CM levels. This indicated that binding of one molecule of the kinase apoenzyme by a CM-kinase complex was necessary for the reaction to occur. Kinetic analysis of the autophosphorylation reaction was consistent with this interpretation and indicated a second-order intermolecular process that included MLCKase dimerization or oligomerization. In contrast, the low Ca/CM-independent contribution was of intramolecular type since it did not depend on the kinase concentration. The autophosphorylation appeared to be involved in a relatively slow modification of the oligomeric properties of the kinase leading to a 2-4-fold amplification of the kinase catalytic activity which followed its activation by CM. Oligomerization and dimerization of the kinase was independently demonstrated by light scattering measurements.
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PMID:Calmodulin-dependent autophosphorylation of smooth muscle myosin light chain kinase: intermolecular reaction mechanism via dimerization of the kinase and potentiation of the catalytic activity following activation. 754 20

Thrombin-induced cultured bovine endothelial cell (EC) gap formation and albumin permeability is initiated by contraction, which is dependent upon myosin light chain kinase-mediated myosin light chain (MLC) phosphorylation. MLC are then rapidly dephosphorylated (J. G. N. Garcia, H. W. Davis, and C. E. Patterson, J. Cell. Physiol. 163: 510-522, 1995), suggesting a role for MLC dephosphorylation in regulation of EC barrier function. Therefore, we studied the effect of semiselective protein phosphatase (PPase) inhibitors, calyculin A and okadaic acid, on MLC phosphorylation status, myosin-associated PPase activity, and EC monolayer permeability. Calyculin A (0.1-10 nM), but not okadaic acid (1-100 nM) produced significant dose-dependent enhancement of both MLC phosphorylation (three- to four-fold) and EC permeability (eightfold). EC homogenates were utilized to assess Ser/Thr PPase activities using either [32P]phosphorylase A or 32P-labeled skeletal MLC as substrates. Calyculin A at 5 nM (sufficient to inhibit type 1 and type 2A PPase) produced approximately 95% inhibition of all EC PPase activity against both substrates, whereas 2 nM okadaic acid (selective for PPase 2A) only partially inhibited EC PPase activity (40-60%). Fractionation of EC homogenates produced a supernatant fraction containing < 10% of total myosin and a pellet fraction with > 90% of total myosin. PPase activity in the myosin-enriched pellet was insensitive to 2 nM okadaic acid (0% inhibition) but sensitive to 5 nM calyculin (> 95% inhibition). Immunoreactive PPase 1 was present in both fractions, whereas PPase 2A was present only in the myosin-depleted fraction. We conclude that a type 1 myosin-associated PPase is involved in regulation of EC contractility and barrier function.
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PMID:Regulation of endothelial cell gap formation and barrier function by myosin-associated phosphatase activities. 763 21

Smooth muscle contraction is regulated primarily by the reversible phosphorylation of myosin by myosin light chain kinase. Secondary mechanisms that might modulate contractility are phosphorylation-dephosphorylation of myosin light chain kinase and thin-filament proteins, caldesmon and calponin. Purification of several protein phosphatases that are active toward myosin light chains and (or) myosin and heavy meromyosin from smooth muscles has been reported. All the cytosolic turkey gizzard smooth muscle phosphatases, termed SMP-I, -II, -III, and -IV, dephosphorylate myosin light chains rapidly, but only SMP-III and -IV are active toward myosin and heavy meromyosin, suggesting that SMP-III and -IV might be directly involved in the relaxation of smooth muscle. SMP-III and -IV exhibit properties typical of type 1 protein phosphatases following tryptic digestion. These enzymes appear to share structural similarity with myofibrillar phosphatase PP1M. Purified calponin phosphatase and caldesmon phosphatase from chicken gizzards are structurally and immunologically identical with SMP-I, a type 2A protein phosphatase. SMP-I dephosphorylates calponin faster than it does caldesmon, and has much higher activity toward these substrates than SMP-II, -III, and -IV. Thus, one role for SMP-I might be to regulate the activities of caldesmon and calponin. Since SMP-I is active toward myosin light chain kinase, it might also modulate this enzyme.
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PMID:Smooth muscle phosphatases: structure, regulation, and function. 776 89

The sites of action of many chemical agents that modify the contraction of smooth muscle are in the smooth muscle membrane. However, a few agents, such as calmodulin inhibitors and protein kinase inhibitors, interact directly with contractile elements of the actomyosin system so as to modify smooth muscle contraction. Here, we describe experimental procedures that are applicable for the screening of smooth muscle relaxants with this mode of action. Myosin B was extracted from chicken gizzard smooth muscle. Because myosin B was a crude preparation of smooth muscle actomyosin, it consisted of regulatory proteins of calmodulin, myosin light chain kinase and protein phosphatase in addition to the contractile proteins of actin and myosin. Interaction of chemical agents with these proteins could be detected by measuring the Mg-ATPase activity of the myosin B preparation. Then we examined whether the agents that altered the ATPase activity was associated with changes in phosphorylation of myosin light chain. If the levels are altered, the agents may interact with the regulatory protein(s). If not, the site of their action was in the contractile proteins. The analysis with these respective proteins will be also described.
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PMID:[Studies on agonists and antagonists of smooth muscle contraction by the use of an actomyosin preparation]. 782 22

Q10 values of the protein phosphatases that can dephosphorylate the regulatory light chain of smooth muscle myosin were determined. Six phosphatases were examined, i.e. skeletal muscle protein phosphatase 1c; protein phosphatase 2Ac; smooth muscle phosphatases (SMP) I, II, and IV; and myosin-associated protein phosphatase (MAP phosphatase). Among them, SMP-IV and MAP phosphatase, which can dephosphorylate intact smooth muscle myosin, showed extremely high Q10 values (5.3 and 5.2, respectively). On the other hand, the Q10 values of other tested phosphatases were within the range of the normal enzyme reaction (Q10 = 2.0). The rate of dephosphorylation of the myosin light chain in alpha-toxin-skinned strips was measured at different temperatures. The results provided a Q10 of 5.1, which was quite similar to those values obtained for SMP-IV and MAP phosphatase. These results suggest that the physiological myosin light chain phosphatases are SMP-IV and/or MAP phosphatase, i.e. type 1 protein phosphatases. The temperature dependence of maximum force, the steady-state extent of myosin light chain phosphorylation, and the relaxation rate of alpha-toxin-permeabilized rabbit portal vein smooth muscle strips were measured. Both maximum force and the extent of myosin light chain phosphorylation were significantly higher at lower temperature (15 degrees C) than at higher temperature (25 degrees C) under all pCa conditions tested, i.e. > 8, 6.3, and 5. The temperature dependence of the relaxation rate was much steeper (decreased 4 times by lowering the temperature from 25 to 15 degrees C) than that of the initial rate of increase in force development (decreased 1.4 times by lowering the temperature from 25 to 15 degrees C). These results are consistent with the Q10 values of myosin light chain phosphatases (Q10 = 5) and myosin light chain kinase (Q10 = 1.7) and further show that the smooth muscle type 1 phosphatases are responsible for the dephosphorylation of smooth muscle myosin in situ.
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PMID:Correlation between high temperature dependence of smooth muscle myosin light chain phosphatase activity and muscle relaxation rate. 811 26


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