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

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.
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PMID:Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells. 358 24

Interactions of several divalent cations (Mn2+, Ca2+, Co2+, Sr2+, and Zn2+) with EGTA-inhibitable adenylate cyclase were investigated in washed membranes (particles) isolated from the gray matter of rat cerebral cortex. The EGTA-inhibitable (called sensitive) enzyme activity was assayed in the presence of Triton X-100 since this detergent caused a marked increase (up to 20-fold) in the enzyme activity. The effects of various divalent metals (all added as chloride salt) indicated the presence of two distinct sites called site I and site II. At low concentrations (less than micromolar) Mn2+, Co2+, and Ca2+ increased (up to 10-fold) the enzyme activity to the same extent and appeared to act via binding to site I (high affinity site). The rank order of affinity was Mn2+ greater than or equal to Co2+ greater than Ca2+. Zn2+ showed the highest affinity and Sr2+ the lowest towards binding to site I; both these metals increased the enzyme activity to lesser extents than Mn2+, Co2+, or Ca2+. GTP was not required for the stimulation of this enzyme by low concentrations of Ca2+. The interaction of Mn2+ with site II (low affinity site) caused further increase in the enzyme activity, whereas Co2+, Ca2+, and Sr2+ were inhibitory at concentrations greater than 10 microM. Isolated fraction contained loosely and tightly associated pools of calmodulin. Myelin basic protein, but not calcineurin, inhibited the EGTA-sensitive adenylate cyclase activity. The EGTA-insensitive enzyme activity was increased by norepinephrine by mechanisms that depended on GTP and was inhibited by Ca2+. The stimulation of the EGTA-insensitive enzyme modulated the Mg2+ requirement such that Mg2+ binding to the low affinity site (site II) apparently occurred with higher affinity. The likely significance of these results is discussed with regard to (i) the presence of two classes of adenylate cyclase in rat cerebral cortex gray matter and (ii) the regulation of their activities by calmodulin-requiring and GTP-requiring mechanisms.
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PMID:EGTA-sensitive and -insensitive forms of particulate adenylate cyclase in rat cerebral cortex: regulation by divalent cations and GTP. 393 3

Four phosphoprotein phosphatases, with the ability to act upon hydroxymethylglutaryl (HMG)-CoA reductase, phosphorylase, and glycogen synthase have been purified from rat liver cytosol through a process that involves DEAE-cellulose, aminohexyl-Sepharose-4B, and Bio-Gel A 1.5 m chromatographies. Protein phosphatase II (Mr 180,000) was the major enzyme (68%) with a very broad substrate specificity, showing similar activity toward the three substrates. Phosphatases I1 (Mr 180,000) and I3 (Mr 250,000) accounted for only 12 and 15% of the total activity, respectively, and they were also able to dephosphorylate the three substrates. In contrast, phosphatase I2 (Mr 200,000) showed only phosphorylase phosphatase activity with insignificant dephosphorylating capacity toward HMG-CoA reductase and glycogen synthase. Upon ethanol treatment at room temperature, the Mr of all phosphatases changed; protein phosphatases I2, I3, and II were brought to an Mr of 35,000, while phosphatase I1 was reduced to an Mr of 69,000. Glycogen synthase phosphatase activity was decreased in all four phosphatases. There was also a decrease in phosphatase I1 activity toward HMG-CoA reductase and phosphorylase as substrates. The HMG-CoA reductase phosphatase and phosphorylase phosphatase activities of phosphatases I2, I3, and II were increased after ethanol treatment. Each protein phosphatase showed a different optimum pH, which changed depending on the substrate. The four phosphatases increased their activity in the presence of Mn2+ and Mg2+. In general, Mn2+ was a better activator than Mg2+, and phosphatase I1 showed a stronger dependency on these cations than any other phosphatase. Phosphorylase was a competitive substrate in the HMG-CoA reductase phosphatase and glycogen synthase phosphatase reactions of protein phosphatases I1, I3, and II. HMG-CoA reductase was also able to compete with phosphorylase and glycogen synthase for phosphatase activity. Glycogen synthase phosphatase activity presented less inhibition in the low-Mr forms. A comparison has been made with other protein phosphatases previously reported in the literature.
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PMID:Modulation of rat liver hydroxymethylglutaryl-CoA reductase by protein phosphatases: purification of nonspecific hydroxymethylglutaryl-CoA reductase phosphatases. 397 May 34

Calcineurin was dissociated into subunits A and B by SDS and the dissociated subunits were separated by Sephadex G-100 column chromatography in SDS. The phosphatase activity was associated with the A subunit and was detected only in the presence of MnCl2 of the various divalent cations tested. The Mn2+-dependent phosphatase of A subunit was stimulated (4-5-fold) by calmodulin. The subunit B increased only modestly Mn2+ stimulated phosphatase activity of subunit A but markedly increased it when assay also contained calmodulin. These results support the view that subunit B plays an important role in Mn2+/calmodulin regulation of subunit A phosphatase activity. They also lend further support to our earlier postulate ([1984] FEBS Lett. 169, 251-255) that Mn2+ is a powerful regulator of calcineurin phosphatase.
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PMID:Resolution of bovine brain calcineurin subunits: stimulatory effect of subunit B on subunit A phosphatase activity. 404 87

Calcineurin, originally identified as a calmodulin-dependent phosphoprotein phosphatase (Stewart, A.A. et al. (1982) FEBS Lett. 137, 80-84) also uses p-nitrophenyl phosphate and phosphotyrosine as substrates (Pallen, C.J. and Wang, J.H. (1983) J. Biol. Chem. 258, 8550-8553). We have surveyed a wide range of nonprotein phosphocompounds and found that several synthetic aryl phosphocompounds serve as calcineurin substrates. Among more than 20 naturally occurring phosphocompounds tested, only phosphoenol pyruvate possesses significant calcineurin substrate activity. The phosphoenol pyruvate phosphatase activity is dependent on Ni2+ and Mn2+, is stimulated by calmodulin, and is inhibited by a monoclonal antibody to calcineurin, thus indicating that it is an intrinsic property of calcineurin. The results suggest that functional roles of calcineurin may include actions of the enzyme toward nonprotein phosphocompounds.
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PMID:Survey of calcineurin activity towards nonprotein compounds and identification of phosphoenol pyruvate as a substrate. 405 87

To investigate the alterations of phosphoseryl/phosphothreonyl-protein phosphatases in neoplastic tissues, the cytosols of rat liver and AH-13, a strain of rat ascites hepatoma, were chromatographed on DEAE-cellulose and the fractions obtained were assayed for protein phosphatase with glycogen synthase D and phosphorylase alpha as phosphoprotein substrates. While the glycogen synthase phosphatase and phosphorylase phosphatase activities of liver cytosol were largely due to phosphatases IA and II, respectively, as previously reported, these phosphatases were absent or present in only small amounts in AH-13 cytosol, whose glycogen synthase phosphatase and phosphorylase phosphatase activities were due almost wholly to a novel protein phosphatase that appeared to be absent in liver. This phosphatase, termed phosphatase H, was purified further by aminohexyl-Sepharose-4B and Sephadex G-200 chromatography without altering its glycogen synthase D/phosphorylase alpha activity ratio. Purified phosphatase H required Mg2+ or Mn2+ for activity and had a molecular weight of about 330,000. It displayed a substrate specificity broader than that of either phosphatase IA or II.
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PMID:Cytosolic protein phosphatases of rat ascites hepatoma AH-13 as compared with those of rat liver: isolation and characterization of a novel protein phosphatase. 608 36

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.
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PMID:Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+. 608 12

Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.
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PMID:The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme. 608 14

In liver and muscle the major active phosphorylase and synthase phosphatase activity is associated with the glycogen particle. When we examined the effect of the inhibitor-1 and modulator protein on the enzyme present in crude glycogen fractions from dog liver, the phosphorylase phosphatase was not or only slightly affected. Since the enzyme isolated from the glycogen complex by DEAE-cellulose chromatography could be inhibited by inhibitor-1 as well as the modulator protein, it was assumed that an unknown mechanism or factor present in the glycogen fraction was responsible for this reduced sensitivity of the protein phosphatase. This led to the discovery (7) of the deinhibitor protein which has now been extensively purified from dog liver. The deinhibitor protein was shown to be thermostable, ethanol- and trichloroacetic acid-resistant, but non-dialyzable and it was destroyed by pronase or trypsin. The apparent molecular weight was estimated at about 17,500 in gel filtration, 8,300 in sodium dodecyl sulfate polyacrylamide gel electrophoresis and 5,500 in sucrose density gradient centrifugation, behavior which is consistent with the assumption that the deinhibitor protein may have little ordered structure. Glycogen synthesis requires both phosphorylase and glycogen synthase as dephosphorylated enzymes. The interaction of the deinhibitor protein with the protein phosphatase brings about several effects which, when considered together, could all facilitate the dephosphorylation of glycogen synthase and phosphorylase. The protein phosphatase present in a resuspended glycogen pellet dephosphorylates inhibitor-1 in the absence of Mn2+. This ability of the phosphatase, which is lost during purification of the enzyme, can be restored upon addition of the deinhibitor protein. Owing to the association of the deinhibitor protein with the active phosphatase the enzyme becomes insensitive to inhibition by inhibitor-1 and the modulator protein, and more resistant to the conversion into the FA-ATP,Mg-dependent form, brought about by the modulator protein. During the activation of the ATP,Mg-dependent phosphatase under conditions where kinase FA is rate limiting, the deinhibitor protein increases the level without affecting the rate of activation.
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PMID:Regulation of protein phosphatase activity by the deinhibitor protein. 608 15


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