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)

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.
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PMID:Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart. 170 50

The delta-subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue isolated form receptor purified in the absence of protein phosphatase inhibitors contains a total of four phosphate groups. Three of these are shown to represent phosphoserine groups. The fourth possible represents phosphotyrosine. The phosphate groups are localized within the primary structure: We found phosphoserine in positions delta S361 and delta S377, the predicted sites phosphorylated by PKA and PKC, respectively. In addition, we found that position delta S362 is also phosphorylated. Phosphorylation experiments with the synthetic peptide delta L357-delta K368 show that phosphorylation of this novel site can be catalyzed by PKA and by PKC. It is concluded that the delat-subunit of the acetylcholine receptor is stably and not transiently phosphorylated. Implications for the physiological functions of receptor phosphorylation are discussed.
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PMID:Phosphorylation sites of the nicotinic acetylcholine receptor. A novel site detected in position delta S362. 170 13

The most prominent tyrosyl-phosphorylated protein in maturing sea star oocytes was identified as the 44 kDa myelin basic protein (MBP) kinase p44mpk. Immunoblotting studies with anti-phosphotyrosine PY-20 antibody and phosphoamino acid analysis of in vivo [32P]phosphate-labelled p44mpk showed that the tyrosyl phosphorylation of the kinase correlated with a greater than 10-fold stimulation of its MBP phosphotransferase activity. The activation of p44mpk was reversed almost completely by purified preparations of the protein-tyrosyl phosphatases CD45 and 1B. Purified p44mpk has previously been shown to undergo autophosphorylation in vitro on seryl residues and this was associated with further enhancement of its MBP phosphorylating activity (Sanghera et al. (1991) J. Biol. Chem. 266, 6700-6707). p44mpk also underwent seryl phosphorylation during oocyte maturation, and the protein-seryl/threonyl phosphatase 2A reversed partially the maturation-associated stimulation of its MBP kinase activity. The properties of p44mpk resemble the murine 42 kDa mitogen-activated protein kinase (p42mapk). While p44mpk may feature the phosphorylatable tyrosyl residue that is critical for activation in p42mapk, it lacks the upstream threonyl phosphorylation site that is also required for p42mapk activity (Payne et al. (1991) EMBO J: 10, 885-892). These findings indicate partial differences in the regulatory mechanisms that govern the activities of these isozymes.
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PMID:Tyrosyl phosphorylation and activation of the myelin basic protein kinase p44mpk during sea star oocyte maturation. 171 36

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.
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PMID:A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts. 171 69

A convenient synthesis is reported for the preparation of the phosphite ester of tyrosine methyl ester. By use of calcineurin, at 30 degrees C, a phosphite ester was hydrolyzed with a VM value [119 nmol/(min.micrograms of E)] approximately 500 times greater than that obtained with tyrosine phosphate [0.23 nmol/(min.microgram of E)] as substrate, but with similar KM values (12 mM for Tyr-PH ME, 11 mM for Tyr-P). Acid phosphatase, on the other hand, hydrolyzed the phosphite ester with a VM and KM value lower than those obtained with tyrosyl phosphate. The temperature dependence of the kinetic parameters (KM and VM) was evaluated, and the activation parameters were obtained with both substrates. The entropy of activation associated with the enzymatic hydrolysis of tyrosine phosphate agrees with the entrophy change for the hydrolysis of the monoanion of phosphate monoesters. The energy of activation for both substrates was in agreement with the energy change for hydrolysis of the oxygen-phosphorous linkage of phosphate monoester monoanions and phosphite esters. These results are consistent with a scheme of general acid catalysis in the action of calcineurin.
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PMID:Calcineurin-catalyzed reaction with phosphite and phosphate esters of tyrosine. 184 82

The phosphatase activities of type 2A, type 1 and type 2C protein phosphatase preparations were measured against p-nitrophenyl phosphate (pNPP), a commonly used substrate for alkaline phosphatases. Of the three types of phosphatase examined, the type 2A phosphatase exhibited an especially high pNPP phosphatase activity (119 +/- 8 mumol/min per mg of protein; n = 4). This activity was strongly inhibited by pico- to nano-molar concentrations of okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases that has been shown to have no effect on alkaline phosphatases. The dose-inhibition relationship was markedly shifted to the right and became steeper by increasing the concentration of the enzyme, as predicted by the kinetic theory for tightly binding inhibitors. The enzyme concentration estimated by titration with okadaic acid agreed well with that calculated from the protein content and the molecular mass for type 2A phosphatase. These results strongly support the idea that the pNPP phosphatase activity is intrinsic to type 2A protein phosphatase and is not due to contamination by alkaline phosphatases. pNPP was also dephosphorylated, but at much lower rates, by type 1 phosphatase (6.4 +/- 8 nmol/min per mg of protein; n = 4) and type 2C phosphatase (1.2 +/- 3 nmol/min per mg of protein; n = 4). The pNPP phosphatase activity of the type 1 phosphatase preparation shows a susceptibility to okadaic acid similar to that of its protein phosphatase activity, whereas it was interestingly very resistant to inhibitor 2, an endogenous inhibitory factor of type 1 protein phosphatase. The pNPP phosphatase activity of type 2C phosphatase preparation was not affected by up to 10 microM-okadaic acid.
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PMID:Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases. 185 Feb 39

We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.
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PMID:Phosphorylation and dephosphorylation of human platelet surface proteins by an ecto-protein kinase/phosphatase system. 185 Mar 5

An inhibitory subunit (P gamma) of cGMP phosphodiesterase from vertebrate rod photoreceptors (frog, toad, and bovine) was phosphorylated by cytosolic protein kinase(s) derived from intact frog rod outer segments. The phosphorylation of frog P gamma was stimulated by phosphatidylinositol but not by cAMP or cGMP. One- and two-dimensional gel electrophoresis revealed that 70-80% of P gamma was phosphorylated with 1 mol of phosphate per frog P gamma under optimal conditions. A peptide that derived from an active domain of bovine P gamma was also phosphorylated. Phosphorylation of frog P gamma was inhibited by addition of the peptide to the reaction mixture. Phosphorylation of frog P gamma was also inhibited by addition of transducin subunits or active (P gamma-less) cGMP phosphodiesterase. Okadaic acid, on the other hand, enhanced P gamma phosphorylation, suggesting the presence of protein phosphatase(s) in the cytosolic fraction. These data suggest another mechanism for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors.
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PMID:Phosphatidylinositol-stimulated phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in vertebrate rod photoreceptors. 185 3

The mechanisms by which glycogen metabolism, glycolysis and gluconeogenesis are controlled in the liver both by hormones and by the concentration of glucose are reviewed. The control of glycogen metabolism occurs by phosphorylation and dephosphorylation of both glycogen phosphorylase and glycogen synthase catalysed by various protein kinases and protein phosphatases. The hormonal effect is to stimulate glycogenolysis by the intermediary of cyclic AMP, which activates directly or indirectly the protein kinases. The glucose effect is to activate the protein phosphatase system; this occurs by the direct binding of glucose to glycogen phosphorylase which is then a better substrate for phosphorylase phosphatase and is inactivated. Since phosphorylase a is a strong inhibitor of synthase phosphatase, its disappearance allows the activation of glycogen synthase and the initiation of glycogen synthesis. When glycogen synthesis is intense, the concentrations of UDPG and of glucose 6-phosphate in the liver decrease, allowing a net glucose uptake by the liver. Glucose uptake is indeed the difference between the activities of glucokinase and glucose 6-phosphatase. Since the Km of the latter enzyme is far above the physiological concentration of its substrate, the decrease in glucose 6-phosphate concentration proportionally reduces its activity. The control of glycolysis and of gluconeogenesis occurs mostly at the level of the interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate under the action of phosphofructokinase 1 and fructose 1,6-bisphosphatase. Fructose 2,6-bisphosphate is a potent stimulator of the first of these two enzymes and an inhibitor of the second. It is formed from fructose 6-phosphate and ATP by phosphofructokinase 2 and hydrolysed by a fructose 2,6-bisphosphatase. These two enzymes are part of a single bifunctional protein which is a substrate for cyclic AMP-dependent protein kinase. Its phosphorylation causes the inactivation of phosphofructokinase 2 and the activation of fructose 2,6-bisphosphatase, resulting in the disappearance of fructose 2,6-bisphosphate. The other major effector of these two enzymes is fructose 6-phosphate, which is the substrate of phosphofructokinase 2 and a potent inhibitor of fructose 2,6-bisphosphatase; these properties allow the formation of fructose 2,6-bisphosphate when the level of glycaemia and secondarily that of fructose 6-phosphate is high.
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PMID:Mechanisms of blood glucose homeostasis. 212 8

In this study a rho-nitrophenyl phosphate (PNPP) phosphatase was purified 476-fold from bovine brain cytosol. The molecular weight of the enzyme is 84,000 as determined by gel filtration. The PNPP phosphatase could also dephosphorylate [32P-Tyr]-casein and -poly (Glu, Tyr). [32P-ser]-casein and -histone were not substrates. The phosphatase activity was found to be totally dependent on divalent metal ions. Mg2+ was the most effective with Ka of 20 microM. Ca2+ was found to be a potent inhibitor of the phosphatase. Using PNPP as a substrate the IC50 for Ca2+ was 0.6 microM. Several known inhibitors of phosphotyrosyl protein phosphatases such as Zn2+, vanadate, and molybdate also inhibited the PNPP phosphatase. The very high sensitivity for inhibition by Ca2+ suggests that the activity of the phosphotyrosyl protein phosphatase may be regulated by fluctuations in the intracellular concentrations of Ca2+.
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PMID:Characterization of a bovine brain magnesium-dependent phosphotyrosine protein phosphatase that is inhibited by micromolar concentrations of calcium. 215 12


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