scholarly journals Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis

1992 ◽  
Vol 287 (1) ◽  
pp. 101-106 ◽  
Author(s):  
A Takai ◽  
Y Ohno ◽  
T Yasumoto ◽  
G Mieskes
Keyword(s):  
Okadaic Acid ◽  
Rate Constants ◽  
Protein Phosphatase ◽  
Time Course ◽  
Dissociation Constants ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Substrate Complex ◽  
Igg1 Monoclonal Antibody ◽  
Nitrophenyl Phosphate

As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.

scholarly journals Inhibitory effect of okadaic acid on the p-nitrophenyl phosphate phosphatase activity of protein phosphatases

1991 ◽  
Vol 275 (1) ◽  
pp. 233-239 ◽  
Author(s):  
A Takai ◽  
G Mieskes
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Enzyme Concentration ◽  
Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Type 1 Phosphatase

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.

scholarly journals Identification, characterization and purification to near-homogeneity of a novel 67 kDa phosphotyrosyl protein phosphatase associated with pig lung annexin extract

1991 ◽  
Vol 278 (2) ◽  
pp. 435-440 ◽  
Author(s):  
P Vicendo ◽  
J Fauvel ◽  
J M F Ragab-Thomas ◽  
H Chap
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Protein Phosphatase ◽  
Protein Tyrosine Kinase ◽  
Growth Factor Receptor ◽  
Inhibitory Effect ◽  
Immunological Reactivity ◽  
Anionic Phospholipids ◽  
Nitrophenyl Phosphate ◽  
Annexin Vi ◽  
Epidermal Growth

During the purification of annexin VI from pig lung, we previously reported the isolation of another 67 kDa protein (protein 67E) differing from the former by immunological reactivity, amino acid composition, inability to interact with anionic phospholipids in the presence of Ca2+ and inability to inhibit phospholipase A2 [Fauvel, Vicendo, Roques, Ragab-Thomas, Granier, Vilgrain, Chambaz, Rochat, Chap & Douste-Blazy (1987) FEBS Lett. 221, 397-402]. Attempts to phosphorylate protein 67E by the protein tyrosine kinase of epidermal-growth-factor receptor revealed a dramatic inhibition of receptor autophosphorylation, which was also observed with insulin receptor. This inhibitory effect was found to be supported by a phosphatase active towards p-nitrophenyl phosphate, phosphotyrosine, [32P]phosphotyrosyl histones and [32P]phosphotyrosyl poly(Glu,Tyr), but inactive towards phosphoserine, phosphothreonine and [32P]phosphoseryl histones. Although not purified to complete homogeneity, the enzyme was purified 273-fold over EGTA extracts from pig lung and corresponded to a monomeric protein displaying an apparent molecular mass of 67 kDa. With [32P]phosphotyrosyl poly(Glu,Tyr) as substrate, the purified enzyme displayed Km and Vmax. values of 10 microM and 1.93 mumol/min per mg respectively, which compare reasonably well with other recently described phosphotyrosyl protein phosphatases. From these data and from its sensitivity to various inhibitors, it is concluded that protein fraction 67E contains a novel phosphotyrosyl protein phosphatase, the association of which with annexin extract might offer a clue to the understanding of its possible targeting to membrane substrates.

Inhibitory effect of calyculin A, a Ser/Thr protein phosphatase type I inhibitor, on renin secretion

1998 ◽  
Vol 275 (5) ◽  
pp. F664-F670 ◽  
Author(s):  
Chun Sik Park ◽  
Mi Hyun Kim ◽  
Chae Hun Leem ◽  
Yeon Jin Jang ◽  
Hae Won Kim ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain ◽  
Renin Secretion ◽  
Type I ◽  
Dependent Manner ◽  
Calyculin A ◽  
Selective Inhibitors ◽  
Intracellular Ca

We have recently shown that several putative selective inhibitors of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK), such as ML-9 [1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine], reversibly stimulate renin secretion [C. S. Park, S.-H. Chang, H. S. Lee, S.-H. Kim, J. W. Chang, and C. D. Hong. Am. J. Physiol. 271 ( Cell Physiol. 40): C242–C247, 1996]. We hypothesized that Ca2+ inhibits renin secretion, via phosphorylation of 20-kDa myosin light chain (MLC20), by activating MLCK. In the present studies, we have investigated the types of protein phosphatase (PP) involved in the control of renin secretion through inhibition of MLC dephosphorylation using inhibitors of various types of serine/threonine-specific protein phosphatases. Cyclosporin A, a putative inhibitor of PP type 2 (calcineurin), was without effect. Calyculin A and okadaic acid, putative selective inhibitors of both PP type 1 (PP1) and type 2A (PP2A), significantly inhibited renin secretion under control conditions. Calyculin A had inhibitory effects at least 10-fold more potent than okadaic acid, suggesting that PP1, rather than PP2A, is involved in the control of renin secretion. Furthermore, calyculin A blocked the reversal of renin secretion preinhibited by raised intracellular Ca2+ concentrations in a concentration-dependent manner. Calyculin A (10−6 M) significantly inhibited renin secretion stimulated by lowering intracellular Ca2+ concentrations and blocked the stimulatory effect of ML-9 on renin secretion. Taking all of these results into consideration, we hypothesize that dephosphorylation of MLC20 by Ca2+-independent PP1 stimulates renin secretion, whereas phosphorylation of MLC20 by Ca2+-calmodulin-dependent MLCK inhibits it. This hypothesized regulatory model of renin secretion predicts that the rate of renin secretion at a given time is determined by the ratio of phosphorylated to dephosphorylated MLC20, which is, in turn, determined by the dynamic balance between activity of MLCK and MLC phosphatase.

scholarly journals A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction

1993 ◽  
Vol 294 (1) ◽  
pp. 211-213 ◽  
Author(s):  
H B Brooks ◽  
V L Davidson
Keyword(s):  
Electron Transfer ◽  
Rate Constants ◽  
Kinetic Data ◽  
Transfer Reaction ◽  
Dissociation Constants ◽  
Accurate Method ◽  
Electron Transfer Reaction ◽  
Stopped Flow ◽  
Methylamine Dehydrogenase ◽  
Substrate Complex

The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.

Propofol Inhibits Phosphorylation of N -methyl-d-aspartate Receptor NR1 Subunits in Neurons

2006 ◽  
Vol 104 (4) ◽  
pp. 763-769 ◽  
Author(s):  
Seth Kingston ◽  
Limin Mao ◽  
Lu Yang ◽  
Anish Arora ◽  
Eugene E. Fibuch ◽  
...  
Keyword(s):  
Glutamate Receptors ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Cortical Neurons ◽  
Protein Phosphatase 2A ◽  
Ionotropic Glutamate Receptors ◽  
Dependent Manner ◽  
Calyculin A ◽  
General Anesthetic ◽  
Phosphatase 2A

Background Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-D-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation. Methods The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons. Results Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits. Conclusions Together, these data indicate an inhibitory effect of a general anesthetic propofol on NMDAR NR1 subunit phosphorylation in neurons. This inhibition was mediated through a signaling mechanism involving activation of protein phosphatase 2A.

scholarly journals Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic β-cells

1977 ◽  
Vol 162 (1) ◽  
pp. 9-18 ◽  
Author(s):  
L Å Idahl ◽  
Å Lernmark ◽  
J Sehlin ◽  
I B Täljedal
Keyword(s):  
Plasma Membranes ◽  
Islet Cells ◽  
Protective Action ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Nitrophenyl Phosphate ◽  
Cation Pump ◽  
Ion Pumping ◽  
Hydrolysis Of

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.

scholarly journals Regulation of acrosome reaction of fowl spermatozoa: evidence for the involvement of protein kinase C and protein phosphatase-type 1 and/or -type 2A

Reproduction ◽  
2006 ◽  
Vol 131 (6) ◽  
pp. 1017-1024 ◽  
Author(s):  
K Ashizawa ◽  
G J Wishart ◽  
S Katayama ◽  
D Takano ◽  
A R A H Ranasinghe ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Acrosome Reaction ◽  
Dependent Manner ◽  
Kinase C ◽  
Protein Phosphatase Type 1 ◽  
Protein Phosphatase Type

The signal transduction pathways involved in the regulation of the acrosome reaction and motility of fowl spermatozoa were investigated. The motility and acrosomal integrity of fowl spermatozoa in TES/NaCl buffer, with or without homogenised inner perivitelline layers (IPVL), prepared from laid fowl eggs, was almost negligible at 40 °C. In the presence of 2 mmol CaCl2/l at 40 °C, motility became vigorous and the acrosome reaction was stimulated when IPVL was added. In the absence of Ca2+, motility was stimulated by the addition of calyculin A and okadaic acid, both specific inhibitors of protein phosphatase-type 1 (PP1) and -type 2A (PP2A), but Okadaic acid, which is a weaker inhibitor of PP1, did not completely restore motility at 40 °C. However, the acrosome reaction was significantly and equally stimulated in a dose-dependent manner by both inhibitors in the range of 10–1000 nmol/l, when spermatozoa were incubated with IPVL but without Ca2+. These inhibitors did not stimulate the acrosome reaction in the absence of IPVL. The vigorous motility of spermatozoa, stimulated by the addition of Ca2+, was reduced gradually as the concentrations of SC-9, a selective activator of protein kinase C (PKC), were increased and a similar SC-9-induced inhibition was observed in the acrosome reaction in the presence of Ca2+ and IPVL. These results confirm that IPVL is necessary for the activation of the acrosome reaction in fowl spermatozoa and that Ca2+ plays an important role in the stimulation of motility and acrosomal exocytosis. Furthermore, it appears that the intracellular molecular mechanisms for the regulation of acrosome reaction of fowl spermatozoa are different from those for the restoration of motility, i.e., protein dephosporylation involving PP1 and/or PP2A in the former, and PP1 alone in the latter case. In addition, the activation of PKC may contribute to a decrease in the flagellar movement and acrosome reaction of fowl spermatozoa.

Effects of okadaic acid and intracellular Cl- on Na(+)-K(+)-Cl- cotransport

1995 ◽  
Vol 269 (4) ◽  
pp. C878-C883 ◽  
Author(s):  
A. A. Altamirano ◽  
G. E. Breitwieser ◽  
J. M. Russell
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Giant Axon ◽  
Subsequent Treatment ◽  
Squid Giant Axon ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Intracellular Atp ◽  
Phosphatase Inhibitor

The Na(+)-K(+)-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cli-) in a concentration-dependent manner ([Cl-]i > or = 200 mM completely inhibits the cotransporter). In the present study we address the question of whether inhibition of cotransport by Cli- is due to a Cl(i-)-induced increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under conditions of [Cl-]i at 0, 150, or 300 mM during measurement of cotransporter-mediated unidirectional Cl- influx into axons. At 0 mM [Cl-]i, the application of 250 nM OKA had no effect on the cotransport-mediated Cl- influx when axons were dialyzed with the normal intracellular ATP concentration ([ATP]i = 4 mM). Reduction of [ATP] to 50 microM resulted in a significant decrease of the bumetanide-sensitive CL- influx, which could be partially reversed by OKA treatment. Similarly, in ATP-limited axons with [Cl-]i at 150 mM, cotransporter influx was partially stimulated by treatment with OKA. However, axons dialyzed with 300 mM [Cl-]i ([ATP]i = 50 microM) had no measurable cotransport influx, nor was subsequent treatment with OKA able to induce a cotransport-mediated Cl- influx. We conclude that the inhibition of cotransport caused by Cli- is not the result of an increase in the OKA-sensitive protein phosphatase activity.

scholarly journals Bacterial Lipopolysaccharide Inhibits Dengue Virus Infection of Primary Human Monocytes/Macrophages by Blockade of Virus Entry via a CD14-Dependent Mechanism

1999 ◽  
Vol 73 (4) ◽  
pp. 2650-2657 ◽  
Author(s):  
Yun-Chi Chen ◽  
Sheng-Yuan Wang ◽  
Chwan-Chuen King
Keyword(s):  
Dengue Virus ◽  
Time Course ◽  
Time Lag ◽  
Inhibitory Effect ◽  
Bacterial Lipopolysaccharide ◽  
Target Cells ◽  
Dependent Manner ◽  
Necrosis Factor Alpha ◽  
Cytokines And Chemokines ◽  
Viral Adsorption

ABSTRACT Monocytes/macrophages (MO/Mφ) are the major target cells for both dengue virus (DV) and bacterial lipopolysaccharide (LPS), and the aim of this study was to define their interactions. We had found that LPS markedly suppressed DV infection of primary human MO/Mφ when it was added to cultures prior to or together with, but not after, viral adsorption. The inhibitory effect of LPS was direct and specific and was not mediated by LPS-induced secretion of cytokines and chemokines such as tumor necrosis factor alpha, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, alpha interferon, MIP-1α, and RANTES. In fact, productive DV infection was not blocked but was just postponed by LPS, with a time lag equal to one viral replication cycle. Time course studies demonstrated that LPS was only effective in suppressing DV infection of MO/Mφ that had not been previously exposed to the virus. At various time points after viral adsorption, the level of unbound viruses that remained free in the culture supernatants of LPS-pretreated cultures was much higher than that of untreated controls. These observations suggest that the LPS-induced suppression of DV replication was at the level of virus attachment and/or entry. Blockade of the major LPS receptor, CD14, with monoclonal antibodies MY4 or MoS39 failed to inhibit DV infection but could totally abrogate the inhibitory effect of LPS. Moreover, human serum could significantly enhance the LPS-induced DV suppression in a CD14-dependent manner, indicating that the “binding” of LPS to CD14 was critical for the induction of virus inhibition. Taken together, our results suggest that LPS blocked DV entry into human MO/Mφ via its receptor CD14 and that a CD14-associated cell surface structure may be essential for the initiation of a DV infection.

scholarly journals Affinity of okadaic acid to type-1 and type-2A protein phosphatases is markedly reduced by oxidation of its 27-hydroxyl group

1994 ◽  
Vol 298 (2) ◽  
pp. 259-262 ◽  
Author(s):  
K Sasaki ◽  
M Murata ◽  
T Yasumoto ◽  
G Mieskes ◽  
A Takai
Keyword(s):  
Okadaic Acid ◽  
Dissociation Constants ◽  
Protein Phosphatases ◽  
Three Dimensional ◽  
Inhibitory Effect ◽  
Hydroxyl Group ◽  
Hydroxyl Groups ◽  
Intramolecular Bonding ◽  
Free Energy Of Binding

Okadaic acid (OA), a potent inhibitor of type-1 and type-2A protein phosphatases (PP1 and PP2A), has four hydroxyl groups at 2, 7, 24 and 27 positions (see Figure 1). By chemical treatment of OA we synthesized a derivative, in which the 27-hydroxyl group was specifically oxidized (27-dehydro-OA). The inhibitory effect of this OA derivative was examined on the activities of PP1 and PP2A, which were inhibited by intact OA with dissociation constants (Ki) of 150 nM and 32 pM respectively. We found that the affinity of OA was decreased 40-fold (Ki = 6 microM) with PP1 and 230-fold (Ki = 7.3 nM) with PP2A after oxidation of the 27-hydroxyl group. According to the model of the three-dimensional conformation of OA on the basis of X-ray analyses, the 27-hydroxyl group appears to be present in a position relatively free from intramolecular bonding formation, in comparison with the other three hydroxyl groups. The marked increases in the Ki values for PP1 and PP2A, which indicate the reduction of the absolute values of the free energy of binding by 9 kJ/mol and 14 kJ/mol respectively, may imply that the 27-hydroxyl group serves as a binding site with the phosphatase molecules.

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