scholarly journals Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship

1992 ◽  
Vol 284 (2) ◽  
pp. 539-544 ◽  
Author(s):  
A Takai ◽  
M Murata ◽  
K Torigoe ◽  
M Isobe ◽  
G Mieskes ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Structural Changes ◽  
Protein Phosphatases ◽  
Inhibitory Effect ◽  
Chemical Processes ◽  
Hydroxyl Group ◽  
Confidence Limits ◽  
X Ray ◽  
Structural Modifications

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

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.

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 Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics

1988 ◽  
Vol 256 (1) ◽  
pp. 283-290 ◽  
Author(s):  
C Bialojan ◽  
A Takai
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Marine Sponge ◽  
Protein Phosphatases ◽  
Kinetic Studies ◽  
High Specificity ◽  
Inhibitory Effect ◽  
Acid Type ◽  
Nitrophenyl Phosphate

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.

Pleiotropic effect of okadaic acid on maturing mouse oocytes

Development ◽  
1991 ◽  
Vol 112 (4) ◽  
pp. 971-980 ◽  
Author(s):  
H. Alexandre ◽  
A. Van Cauwenberge ◽  
Y. Tsukitani ◽  
J. Mulnard
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Chromatin Condensation ◽  
Polar Body ◽  
Germinal Vesicle ◽  
Inhibitory Effect ◽  
Negative Control ◽  
Germinal Vesicle Breakdown ◽  
Mouse Oocytes

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.

scholarly journals Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish.

1989 ◽  
Vol 109 (6) ◽  
pp. 3347-3354 ◽  
Author(s):  
A Picard ◽  
J P Capony ◽  
D L Brautigan ◽  
M Dorée
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Germinal Vesicle ◽  
Histone H1 ◽  
Specific Inhibition ◽  
Factor Activity ◽  
Microtubule Network ◽  
Germinal Vesicle Breakdown ◽  
M Phase

Specific inhibition of types 1 and 2A protein phosphatases by microinjection of okadaic acid (OA) into starfish oocytes induced germinal vesicle breakdown and activation of M phase-promoting factor (MPF) and histone H1 kinase. The effects were evident in immature oocytes arrested at first meiotic prophase as well as in fully mature oocytes arrested at the pronucleus stage. In addition, MPF and histone H1 kinase were stabilized for several hours and protected from inactivation by inhibition of type 1 protein phosphatases with either OA or specific anti-phosphatase antibodies. Microinjection of okadaic acid was associated with unusual changes of the microtubule network, including the disappearance of spindles and extension of the cytoplasmic array of microtubules. MPF activation after OA injection was associated with dephosphorylation of phosphothreonine and phosphoserine residues in cdc2, showing that neither type 1 nor 2A protein phosphatases catalyzes these dephosphorylations. The effects of OA on MPF activation and inactivation appeared to involve the cyclin subunit. OA did not induce MPF activation in the absence of protein synthesis and it prevented degradation of cyclin. Therefore protein phosphatases types 1 and 2A appear to be involved in activation and inactivation of MPF involving mechanisms that operate after cyclin synthesis and before its degradation.

Structural Modifications in Thin Films Caused by Gamma Radiation

2005 ◽  
Vol 480-481 ◽  
pp. 13-20 ◽  
Author(s):  
Khalil Arshak ◽  
Olga Korostynska ◽  
John Henry
Keyword(s):  
Thin Films ◽  
Gamma Radiation ◽  
Structural Changes ◽  
X Ray Diffraction ◽  
Thermal Vacuum ◽  
X Ray ◽  
Structural Modifications ◽  
Thin Oxide Films ◽  
Radiation Induced ◽  
Induced Changes

This paper reports on the gamma radiation-induced changes in thin oxide films deposited by thermal vacuum technique. Structures of various oxides thin films, such as In2O3, SiO and TeO2 and their mixtures in different proportions were studied. The influence of gamma radiation on In2O3/SiO films has resulted in significant changes in the microstructure of this film. Some kind of agglomerations with variable sizes in the range 0.5-3 µm has occurred. After a dose of 8160 µSv an evidence of partial crystallisation was observed with X-ray diffraction. Structural changes in TeO2 thin film were explored by means of Raman spectroscopy. After they have been exposed to g- radiation, a strong peak appeared at 448.83 cm-1, indicating further transformation to g-TeO2 modification.

The antigenic structure of poliovirus

1989 ◽  
Vol 323 (1217) ◽  
pp. 467-478 ◽  
Keyword(s):  
Monoclonal Antibodies ◽  
Virus Particle ◽  
Structural Changes ◽  
Three Dimensional ◽  
Antigenic Structure ◽  
X Ray ◽  
Antigenic Sites ◽  
Type 3 ◽  
Insight Into

We have solved the structure of the Mahoney strain of type 1 and the Sabin (attenuated vaccine) strain of type 3 poliovirus by X -ray crystallographic methods. By providing a three-dimensional framework for the interpretation of a wealth of experimental data, the structures have yielded insight into the architecture and assembly of the virus particle, have provided information regarding the entry of virus into susceptible cells, and defined the sites on the virus particle that are recognized by neutralizing monoclonal antibodies. Thus locating mutations in variants selected for resistance to neutralizing monoclonal antibodies has defined three antigenic sites of the surface of the virion, and provided clues as to the mechanisms by which viruses escape neutralization. Finally, comparison of the structures of the two strains, together with analysis of sequences of many poliovirus strains, have begun to define the structural changes associated with serotypic differences between polioviruses.

Crystallographic and transport studies on AsF5 intercalated graphite from 4.2 to 295 K. II. Effects of structural transformations and demixing on basal plane and c-axis electrical resistivity

1988 ◽  
Vol 3 (1) ◽  
pp. 97-104 ◽  
Author(s):  
E. McRae ◽  
M. Lelaurain ◽  
J. F. Marêché ◽  
G. Furdin ◽  
A. Hérold ◽  
...  
Keyword(s):  
Basal Plane ◽  
Structural Changes ◽  
Structural Data ◽  
Structural Transformations ◽  
Structural Modifications ◽  
Intercalated Graphite ◽  
Transport Studies ◽  
Conductivity Models

Part I of this study has shown that first stage AsF5 intercalated graphite samples can be classified into two types of compounds, depending upon the nature of the structural modifications they undergo upon lowering the temperature from 295 to 4.2 K. These structural changes are related to demixing of the species contained within the intercalate resulting in the formation of phases rich in AsF5, AsF−6 − AsF5, or AsF3 depending on the degree to which the AsF5 has been converted into AsF−6 and AsF3. Resistivity studies have been carried out in the basal plane [ρa (T)] and along the c axis [ρc (T)]. The type 1 compounds, in which the AsF5 has undergone little conversion, manifest a ρa (T) transition related to the incommensurate-to-commensurate (I⇉C) transformation of the AsF5 in-plane unit cell and a rapid, structureless 300 to 200 K decrease in ρc (T). The type 2 compounds involving a greater degree of conversion of AsF5 into AsF3 and AsF6 yield more complex ρc (T) behavior from 300 to 200 K attributed to the more involved ordering phenomena; no anomalies are seen in ρa (T). In the case of the stage 2 compounds, changes in ρc (T) are seen down to lower temperatures in accord with structural data indicating a downshift of the I⇉C transformation by ∼ 70 K. The transport results are discussed in the light of the crystallographic data and the low-temperature results are analyzed within the framework of proposed conductivity models.

scholarly journals Crystallization and preliminary X-ray characterization of the dephospho-CoA kinase fromLegionella pneumophila

2014 ◽  
Vol 70 (5) ◽  
pp. 608-610 ◽  
Author(s):  
Dongmin Yu ◽  
Xiaofang Chen ◽  
Zhongdong Xu ◽  
Honghua Ge
Keyword(s):  
Legionella Pneumophila ◽  
Structural Changes ◽  
Hydroxyl Group ◽  
Diffusion Method ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Phosphate Donor ◽  
Molecular Replacement Method

Dephospho-CoA kinases (DPCKs) are members of the kinase family that catalyze the final step in CoA biosynthesis. Their function is phosphorylation of the 3-hydroxyl group of the ribose using ATP as a phosphate donor. Structural changes induced by ATP binding play an important role during the DPCK catalytic cycle. In this work, DPCK fromLegionella pneumophilawas overexpressed inEscherichia coli. The purification, crystallization and preliminary X-ray analysis of crystals of this protein are described. The protein was crystallized in space groupP21212, with unit-cell parametersa= 36.29,b= 82.20,c= 81.80 Å, using the hanging-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method.

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