scholarly journals Inhibitory effect of okadaic acid on protein phosphatases: characteristics as a tightly binding inhibitor.

1991 ◽  
Vol 31 (1) ◽  
pp. 13-17
Author(s):  
Akira TAKAI
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatases ◽  
Inhibitory Effect

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 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 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 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.

Inhibitors of Protein Phosphatases (Okadaic Acid and Tautomycin) Block Sea Urchin Development

1995 ◽  
Vol 189 (2) ◽  
pp. 201-201 ◽  
Author(s):  
Walter Troll ◽  
Naoko Sueoka ◽  
Eisaboro Sueoka ◽  
Jeffrey D. Laskin ◽  
Diane E. Heck
Keyword(s):  
Sea Urchin ◽  
Okadaic Acid ◽  
Protein Phosphatases

Ca2+ and calmodulin-dependent protein phosphatase from Leishmania donovani

Parasitology ◽  
1999 ◽  
Vol 118 (6) ◽  
pp. 567-573 ◽  
Author(s):  
C. BANERJEE ◽  
D. SARKAR ◽  
A. BHADURI
Keyword(s):  
Cyclosporin A ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Leishmania Donovani ◽  
Protein Phosphatases ◽  
Cross Reactivity ◽  
Calmodulin Antagonists ◽  
Type Protein ◽  
Complete Obliteration ◽  
Dependent Protein

A protein phosphatase exclusively dependent upon micromolar amounts of Ca2+ and calmodulin has been identified and partially purified from Leishmania spp. Complete obliteration of its activity is observed in the presence of calmodulin antagonists such as trifluoperazine, fluphenazine and calmidazolium. Relative insensitivity to okadaic acid and lack of activation in the absence of Ca2+ and calmodulin distinguishes this enzyme from PP1, PP2A and PP2C-type protein phosphatases. Cross-reactivity of the enzyme was observed with antibodies that recognize both the A and B chains of calcineurin, a PP2B type Ca2+ and calmodulin-dependent phosphatase from brain. FK506, an immunosuppresive drug that inhibits the enzyme from other sources inhibited the enzyme only in the presence of exogenous FK binding protein, whereas Cyclosporin A inhibited the enzyme in crude preparations. Taken together these results reveal the presence of a Ca2+ and calmodulin-dependent phosphatase from Leishmania. This is the first report of the presence of a PP2B-type protein phosphatase from a pathogenic protozoa.

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