scholarly journals Identification of high levels of type 1 and type 2A protein phosphatases in higher plants

1989 ◽  
Vol 262 (1) ◽  
pp. 335-339 ◽  
Author(s):  
C MacKintosh ◽  
P Cohen
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Higher Plants ◽  
Protein Phosphatases ◽  
Phosphorylase Kinase ◽  
Rabbit Muscle ◽  
Plant Type ◽  
Mammalian Tissues ◽  
Type 1 Phosphatase

Extracts of Brassica napus (oilseed rape) seeds contain type 1 and type 2A protein phosphatases whose properties are indistinguishable from the corresponding enzymes in mammalian tissues. The type 1 activity dephosphorylated the beta-subunit of phosphorylase kinase selectively and was inhibited by the same concentrations of okadaic acid [IC50 (concentration causing 50% inhibition) approximately 10 nM], mammalian inhibitor 1 (IC50 = 0.6 nM) and mammalian inhibitor 2 (IC50 = 2.0 nM) as the rabbit muscle type 1 phosphatase. The plant type 2A activity dephosphorylated the alpha-subunit of phosphorylase kinase preferentially, was exquisitely sensitive to okadaic acid (IC50 approximately 0.1 nM), and was unaffected by inhibitors 1 and 2. As in mammalian tissues, a substantial proportion of plant type 1 phosphatase activity (40%) was particulate, whereas plant type 2A phosphatase was cytosolic. The specific activities of the plant type 1 and type 2A phosphatases were as high as in mammalian tissue extracts, but no type 2B or type 2C phosphatase activity was detected. The results demonstrate that the improved procedure for identifying and quantifying protein phosphatases in animal cells is applicable to higher plants, and suggests that okadaic acid may provide a new method for identifying plant enzymes that are regulated by reversible phosphorylation.

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 Decreased activity and impaired hormonal control of protein phosphatases in rat livers with a deficiency of phosphorylase kinase

1989 ◽  
Vol 264 (2) ◽  
pp. 429-436 ◽  
Author(s):  
B Toth ◽  
M Bollen ◽  
W Stalmans
Keyword(s):  
Phosphatase Activity ◽  
Wistar Rats ◽  
Glycogen Synthase ◽  
Protein Phosphatases ◽  
Hormonal Control ◽  
Lower Amount ◽  
Phosphorylase Kinase ◽  
Specific Substrate ◽  
Liver Cytosol

1. Livers from gsd/gsd rats, which do not express phosphorylase kinase activity, also contain much less particulate type-1 protein phosphatases. In comparison with normal Wistar rats, the glycogen/microsomal fraction contained 75% less glycogen-synthase phosphatase and 60% less phosphorylase phosphatase activity. This was largely due to a lower amount of the type-1 catalytic subunit in the particulate fraction. In the cytosol, the synthase phosphatase activity was also 50% lower, but the phosphorylase phosphatase activity was equal. 2. Both Wistar rats and gsd/gsd rats responded to an intravenous injection of insulin plus glucose with an acute increase (by 30-40%) in the phosphorylase phosphatase activity in the liver cytosol. In contrast, administration of glucagon or vasopressin provoked a rapid fall (by about 25%) in the cytosolic phosphorylase phosphatase activity in Wistar rats, but no change occurred in gsd/gsd rats. 3. Phosphorylase kinase was partially purified from liver and subsequently activated. Addition of a physiological amount of the activated enzyme to a liver cytosol from Wistar rats decreased the V of the phosphorylase phosphatase reaction by half, whereas the non-activated kinase had no effect. The kinase preparations did not change the activity of glycogen-synthase phosphatase, which does not respond to glucagon or vasopressin. Furthermore, the phosphorylase phosphatase activity was not affected by addition of physiological concentrations of homogeneous phosphorylase kinase from skeletal muscle (activated or non-activated). 4. It appears therefore that phosphorylase kinase plays an essential role in the transduction of the effect of glucagon and vasopressin to phosphorylase phosphatase. However, this inhibitory effect either is specific for the hepatic phosphorylase kinase, or is mediated by an unidentified protein that is a specific substrate of phosphorylase kinase.

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.

Regulation of flagellar dynein by an axonemal type-1 phosphatase in Chlamydomonas

1996 ◽  
Vol 109 (7) ◽  
pp. 1899-1907 ◽  
Author(s):  
G. Habermacher ◽  
W.S. Sale
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Double Mutant ◽  
Sliding Velocity ◽  
Wild Type ◽  
Phosphatase Inhibitors ◽  
Radial Spokes ◽  
Gamma S ◽  
Type 1 Phosphatase

Physiological studies have demonstrated that flagellar radial spokes regulate inner arm dynein activity in Chlamydomonas and that an axonemal cAMP-dependent kinase inhibits dynein activity in radial spoke defective axonemes. These studies also suggested that an axonemal protein phosphatase is required for activation of flagellar dynein. We tested whether inhibitors of protein phosphatases would prevent activation of dynein by the kinase inhibitor PKI in Chlamydomonas axonemes lacking radial spokes. As predicted, preincubation of spoke defective axonemes (pf14 and pf17) with ATP gamma S maintained the slow dynein-driven microtubule sliding characteristic of paralyzed axonemes lacking spokes, and blocked activation of dynein-driven microtubule sliding by subsequent addition of PKI. Preincubation of spoke defective axonemes with the phosphatase inhibitors okadaic acid, microcystin-LR or inhibitor-2 also potently blocked PKI-induced activation of microtubule sliding velocity: the non-inhibitory okadaic acid analog, 1-norokadaone, did not. ATP gamma S or the phosphatase inhibitors blocked activation of dynein in a double mutant lacking the radial spokes and the outer dynein arms (pf14pf28). We concluded that the axoneme contains a type-1 phosphatase required for activation of inner arm dynein. We postulated that the radial spokes regulate dynein through the activity of the type-1 protein phosphatase. To test this, we performed in vitro reconstitution experiments using inner arm dynein from the double mutant pf14pf28 and dynein-depleted axonemes containing wild-type radial spokes (pf28). As described previously, microtubule sliding velocity was increased from approximately 2 microns/second to approximately 7 microns/second when inner arm dynein from pf14pf28 axonemes ws reconstituted with axonemes containing wild-type spokes. In contrast, pretreatment of inner arm dynein from pf14pf28 axonemes with ATP gamma S, or reconstitution in the presence of microcystin-LR, blocked increased velocity following reconstitution, despite the presence of wild-type radial spokes. We conclude that the radial spokes, through the activity of an axonemal type-1 phosphatase, activate inner arm dynein by dephosphorylation of a critical dynein component. Wild-type radial spokes also operate to inhibit the axonemal cAMP-dependent kinase, which would otherwise inhibit axonemal dynein and motility.

scholarly journals Stimulation of membrane serine-threonine phosphatase in erythrocytes by hydrogen peroxide and staurosporine

1998 ◽  
Vol 274 (2) ◽  
pp. C440-C446 ◽  
Author(s):  
Isabel Bize ◽  
Patricia Muñoz ◽  
Mitzy Canessa ◽  
Philip B. Dunham
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Glycogen Phosphorylase ◽  
Kinase Inhibitor ◽  
Indirect Evidence ◽  
Phosphatase Inhibitors ◽  
Low Sensitivity ◽  
Type 1 Phosphatase ◽  
Stimulation Of

Indirect evidence has suggested that K-Cl cotransport in human and sheep erythrocytes is activated physiologically by a serine-threonine phosphatase. It is activated experimentally by H2O2and by staurosporine, a kinase inhibitor. Activation by H2O2and staurosporine is inhibited by serine-threonine phosphatase inhibitors, suggesting that the activators stimulate the phosphatase. The present study shows that sheep and human erythrocytes contain membrane-associated as well as cytosolic serine-threonine phosphatases, assayed from the dephosphorylation of32P-labeled glycogen phosphorylase. In cells from both species, the relatively low sensitivity of the membrane enzyme to okadaic acid suggests it is type 1 protein phosphatase. The cytosolic phosphatase was much more sensitive to okadaic acid. Membrane-associated phosphatase was stimulated by both H2O2and staurosporine. The results support earlier conclusions that the membrane-associated type 1 phosphatase identified here is regulated by phosphorylation and oxidation. The results are consistent with the phosphatase, or a portion of it, being responsible for activating K-Cl cotransport.

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.

scholarly journals Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle

1977 ◽  
Vol 162 (2) ◽  
pp. 423-433 ◽  
Author(s):  
J F Antoniw ◽  
H G Nimmo ◽  
S J Yeaman ◽  
P Cowen
Keyword(s):  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Glycogen Synthase ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Phosphorylase Kinase ◽  
Substrate Specificities ◽  
Phosphatase Activities

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.

scholarly journals Protein phosphatase composition in the smooth muscle of guinea-pig ileum studied with okadaic acid and inhibitor 2

1989 ◽  
Vol 262 (2) ◽  
pp. 617-623 ◽  
Author(s):  
A Takai ◽  
M Troschka ◽  
G Mieskes ◽  
A V Somlyo
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Okadaic Acid ◽  
Total Activity ◽  
Guinea Pig Ileum ◽  
Control Activity ◽  
Triton X ◽  
Type 1 Phosphatase ◽  
Intracellular Structure

Using okadaic acid, a potent inhibitor of type 2A and type 1 protein phosphatases, and inhibitor 2, an intrinsic inhibitory factor of type 1 phosphatase, we characterized the phosphorylated myosin light-chain (PMLC) phosphatase activity in the smooth-muscle extracts of guinea-pig ileum. In the intact fibres the control activity was 254 +/- 13 nmol of Pi/min per g wet wt. (n = 15) against 32P-labelled PMLC (4 microM) from chicken gizzard. The following phosphatase fractions were identified: an inhibitor-2-sensitive (type 1) fraction (fractional activity = 35%), a Mg2+-dependent and okadaic acid-insensitive (type 2C) fraction (17%), and two type 2A-like fractions that had different susceptibility to okadaic acid. The type 2A-like fraction with lower affinity to okadaic acid accounted for 30% of the control activity. After the cell membrane was permeabilized by Triton X-100, more than 60% of this fraction remained and accounted for about 90% of the total activity, whereas the other fractions were nearly abolished. The type 2A-like fraction may be bound to some intracellular structure such as contractile proteins.

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.

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