scholarly journals Plant protein phosphatases. Subcellular distribution, detection of protein phosphatase 2C and identification of protein phosphatase 2A as the major quinate dehydrogenase phosphatase

1991 ◽  
Vol 273 (3) ◽  
pp. 733-738 ◽  
Author(s):  
C MacKintosh ◽  
J Coggins ◽  
P Cohen
Keyword(s):  
Oilseed Rape ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Soluble Fraction ◽  
Protein Phosphatases ◽  
Leaf Extracts ◽  
Carrot Cells ◽  
Rapid Inactivation ◽  
Wheat Leaf ◽  
Wheat Leaves

Protein phosphatases 1 and 2A (PP1 and PP2A) were identified in a variety of plant cells and found to be particulate or soluble depending on the species. In extracts prepared from oilseed-rape seeds these enzymes were associated with microsomes and more rapidly sedimenting fractions, whereas in wheat leaf extracts they were largely microsomal, the remainder being present in the soluble fraction. In pea leaf and carrot cell extracts PP1 and PP2A were almost entirely soluble. No PP1 or PP2A activity was associated with the membranes or stroma of chloroplasts in oilseed-rape seeds, pea leaves and wheat leaves. An Mg2(+)-dependent okadaic acid-insensitive protein phosphatase that resembles protein phosphatase 2C (PP2C) was detected in carrot cells, pea leaves and wheat leaves, but not in oilseed-rape seeds. In wheat leaf extracts PP2C was mostly present in the soluble fraction, a different location from PP1 or PP2A. The rapid inactivation of the cytosolic enzyme quinate dehydrogenase (QDH) in a fraction prepared from light-grown carrot cells was completely blocked by either okadaic acid or microcystin (two potent and specific inhibitors of PP1 and PP2A), whereas inhibitor 2 (a specific inhibitor of PP1) inhibited inactivation by only about 10%. Addition of the purified PP2A catalytic subunit from mammalian skeletal muscle increased the rate of QDH inactivation, whereas addition of mammalian PP1 did not. It is concluded that PP2A is the major enzyme responsible for dephosphorylating (inactivating) QDH in carrot cells. These observations indicate that okadaic acid and microcystin may be useful for identifying other plant processes that are controlled by phosphorylation/dephosphorylation mechanisms. Okadaic acid did not prevent the rapid inactivation of phosphoribulokinase or activation of glucose-6-phosphate dehydrogenase in a fraction prepared from light-grown pea leaves, and addition of the purified catalytic subunits of PP1 and PP2A did not accelerate either process. These observations, in conjunction with the absence of PP1 and PP2A activity in chloroplasts, suggest that these phosphatases are not involved in the regulation of chloroplast metabolism.

scholarly journals Insulin-receptor phosphotyrosyl-protein phosphatases

1988 ◽  
Vol 256 (3) ◽  
pp. 893-902 ◽  
Author(s):  
M J King ◽  
G J Sale
Keyword(s):  
Insulin Receptor ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Protein Phosphatase ◽  
Egf Receptor ◽  
Soluble Fraction ◽  
Protein Phosphatases ◽  
Egf Receptors ◽  
Cell Extracts ◽  
Dependent Protein

Calmodulin-dependent protein phosphatase has been proposed to be an important phosphotyrosyl-protein phosphatase. The ability of the enzyme to attack autophosphorylated insulin receptor was examined and compared with the known ability of the enzyme to act on autophosphorylated epidermal-growth-factor (EGF) receptor. Purified calmodulin-dependent protein phosphatase was shown to catalyse the complete dephosphorylation of phosphotyrosyl-(insulin receptor). When compared at similar concentrations, 32P-labelled EGF receptor was dephosphorylated at greater than 3 times the rate of 32P-labelled insulin receptor; both dephosphorylations exhibited similar dependence on metal ions and calmodulin. Native phosphotyrosyl-protein phosphatases in cell extracts were also characterized. With rat liver, heart or brain, most (75%) of the native phosphatase activity against both 32P-labelled insulin and EGF receptors was recovered in the particulate fraction of the cell, with only 25% in the soluble fraction. This subcellular distribution contrasts with results of previous studies using artificial substrates, which found most of the phosphotyrosyl-protein phosphatase activity in the soluble fraction of the cell. Properties of particulate and soluble phosphatase activity against 32P-labelled insulin and EGF receptors are reported. The contribution of calmodulin-dependent protein phosphatase activity to phosphotyrosyl-protein phosphatase activity in cell fractions was determined by utilizing the unique metal-ion dependence of calmodulin-dependent protein phosphatase. Whereas Ni2+ (1 mM) markedly activated the calmodulin-dependent protein phosphatase, it was found to inhibit potently both particulate and soluble phosphotyrosyl-protein phosphatase activity. In fractions from rat liver, brain and heart, total phosphotyrosyl-protein phosphatase activity against both 32P-labelled receptors was inhibited by 99.5 +/- 6% (mean +/- S.E.M., 30 observations) by Ni2+. Results of Ni2+ inhibition studies were confirmed by other methods. It is concluded that in cell extracts phosphotyrosyl-protein phosphatases other than calmodulin-dependent protein phosphatase are the major phosphotyrosyl-(insulin receptor) and -(EGF receptor) 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.

scholarly journals Identification of Novel Serine/Threonine Protein Phosphatases inTrypanosoma cruzi: a Potential Role in Control of Cytokinesis and Morphology

2000 ◽  
Vol 68 (3) ◽  
pp. 1350-1358 ◽  
Author(s):  
George A. Orr ◽  
Craig Werner ◽  
Jun Xu ◽  
Marcia Bennett ◽  
Louis M. Weiss ◽  
...  
Keyword(s):  
Cell Division ◽  
Okadaic Acid ◽  
Blot Analysis ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Growth Arrest ◽  
Human Protein ◽  
Protein Phosphatase 1 ◽  
Calyculin A ◽  
Metacyclic Trypomastigote

ABSTRACT We cloned two novel Trypanosoma cruzi proteins by using degenerate oligonucleotide primers prepared against conserved domains in mammalian serine/threonine protein phosphatases 1, 2A, and 2B. The isolated genes encoded proteins of 323 and 330 amino acids, respectively, that were more homologous to the catalytic subunit of human protein phosphatase 1 than to those of human protein phosphatase 2A or 2B. The proteins encoded by these genes have been tentatively designated TcPP1α and TcPP1β. Northern blot analysis revealed the presence of a major 2.3-kb mRNA transcript hybridizing to each gene in both the epimastigote and metacyclic trypomastigote developmental stages. Southern blot analysis suggests that each protein phosphatase 1 gene is present as a single copy in the T. cruzi genome. The complete coding region for TcPP1β was expressed inEscherichia coli by using a vector, pTACTAC, with thetrp-lac hybrid promoter. The recombinant protein from the TcPP1β construct displayed phosphatase activity toward phosphorylasea, and this activity was preferentially inhibited by calyculin A (50% inhibitory concentration [IC50], ∼2 nM) over okadaic acid (IC50, ∼100 nM). Calyculin A, but not okadaic acid, had profound effects on the in vitro replication and morphology of T. cruzi epimastigotes. Low concentrations of calyculin A (1 to 10 nM) caused growth arrest. Electron microscopic studies of the calyculin A-treated epimastigotes revealed that the organisms underwent duplication of organelles, including the flagellum, kinetoplast, and nucleus, but were incapable of completing cell division. At concentrations higher than 10 nM, or upon prolonged incubation at lower concentrations, the epimastigotes lost their characteristic elongated spindle shape and had a more rounded morphology. Okadaic acid at concentrations up to 1 μM did not result in growth arrest or morphological alterations to T. cruziepimastigotes. Calyculin A, but not okadaic acid, was also a potent inhibitor of the dephosphorylation of 32P-labeled phosphorylase a by T. cruzi epimastigotes and metacyclic trypomastigote extracts. These inhibitor studies suggest that in T. cruzi, type 1 protein phosphatases are important for the completion of cell division and for the maintenance of cell shape.

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.

THE ENZYMICALLY CATALYZED OXIDATION OF INDOLEACETIC ACID

1956 ◽  
Vol 34 (6) ◽  
pp. 905-926 ◽  
Author(s):  
E. R. Waygood ◽  
Ann Oaks ◽  
G. A. Maclachlan
Keyword(s):  
Indoleacetic Acid ◽  
Maleic Hydrazide ◽  
Organic Peroxide ◽  
Horse Radish Peroxidase ◽  
Reaction Sequence ◽  
Leaf Extracts ◽  
Naturally Occurring ◽  
Wheat Leaf ◽  
Wheat Leaves ◽  
Catalyzed Oxidation

Dialyzed wheat leaf extracts, catalase, and horse-radish peroxidase catalyze the decarboxylation and oxidation of indoleacetic acid at pH 5.0–6.0 in the presence of critical concentrations of manganese and monohydric phenols or resorcinol. The equivalent of 1 mole of carbon dioxide is liberated and 1 mole of oxygen consumed per mole of substrate. Manganic ions formed by a phenol–peroxidase–peroxide system initiate the decarboxylation and oxidation. A naturally occurring ether soluble factor from wheat leaves, and maleic hydrazide, can substitute for the active phenols. Catechol, hydroquinone, pyrogallol, seopoletin, and riboflavin, etc. competitively inhibit the oxidation. The nature of the active peroxide is discussed and a reaction sequence involving an organic peroxide or radical rather than hydrogen peroxide is submitted as being a possibility.

QUALITATIVE AND QUANTITATIVE DETERMINATION OF THE WHEAT LEAF CARBOHYDRATES

1950 ◽  
Vol 28c (6) ◽  
pp. 754-779 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Reducing Sugars ◽  
Degradation Products ◽  
Reducing Power ◽  
Insoluble Residue ◽  
Leaf Extracts ◽  
Wheat Leaf ◽  
Leaf Carbohydrates ◽  
Wheat Leaves ◽  
Reducing Substances ◽  
Fructose Determination

The nonreducing sugar in wheat leaves is probably entirely sucrose. It is the only abundant sugar. Free reducing sugars are absent, or almost absent from wheat leaves grown under the conditions described. The reducing power in the cleared alcoholic extracts of the leaves is, at least, partly due to degradation products of ascorbic acid. Other nonsugar reducing substances also are apparently present. The alcohol insoluble residue from wheat leaves contains little or no fructosan, dextrin, or starch at the two and one-half week old stage. Satisfactory methods for extracting and determining the sucrose are described. The following methods gave satisfactory results with wheat leaf extracts: the reducing power methods of Hanes and Somogyi after acid or preferably invertase hydrolysis, Ost's solution for fructose residues, the method of Neuberg and Strauss, the colorimetric resorcinol method. The latter method gives only approximate values for fructose residues unless the sugar concentration is high; the method is then fairly reliable. The following methods did not give satisfactory results: Hanes and Somogyi methods for free reducing sugars initially present in the extract, the hypoiodite titration for aldose sugars, and Sieben's method for fructose determination.

scholarly journals Systemic inflammation suppresses spinal respiratory motor plasticity via mechanisms that require serine/threonine protein phosphatase activity

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Arash Tadjalli ◽  
Yasin B. Seven ◽  
Raphael R. Perim ◽  
Gordon S. Mitchell
Keyword(s):  
Systemic Inflammation ◽  
Motor Neurons ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Mitogen Activated Protein Kinase ◽  
Diaphragm Muscle ◽  
Sprague Dawley ◽  
Mitogen Activated Protein ◽  
Key Enzyme

Abstract Background Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system—a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)—a key enzyme necessary for the expression of phrenic motor plasticity. Methods To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. Results We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 μg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. Conclusions This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Role of protein phosphatases in hypoxic preconditioning

2002 ◽  
Vol 283 (3) ◽  
pp. H1092-H1098 ◽  
Author(s):  
Yury Ladilov ◽  
Hagen Maxeiner ◽  
Christopher Wolf ◽  
Claudia Schäfer ◽  
Karsten Meuter ◽  
...  
Keyword(s):  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Diastolic Pressure ◽  
Protein Phosphatases ◽  
Hypoxic Preconditioning ◽  
Protective Mechanism ◽  
Rat Cardiomyocytes ◽  
Adult Rat ◽  
Rat Hearts ◽  
Developed Pressure

To find a protein kinase C (PKC)-independent preconditioning mechanism, hypoxic preconditioning (HP; i.e., 10-min anoxia and 10-min reoxygenation) was applied to isolated rat hearts before 60-min global ischemia. HP led to improved recovery of developed pressure and reduced end-diastolic pressure in the left ventricle during reperfusion. Protection was unaffected by the PKC inhibitor bisindolylmaleimide (BIM; 1 μmol/l). It was abolished by the inhibitor of protein phosphatases 1 and 2A cantharidin (20 or 5 μmol/l) and partially enhanced by the inhibitor of protein phosphatase 2A okadaic acid (5 nmol/l). In adult rat cardiomyocytes treated with BIM and exposed to 60-min simulated ischemia (anoxia, extracellular pH 6.4), HP led to attenuation of anoxic Na+/Ca2+ overload and of hypercontracture, which developed on reoxygenation. This protection was prevented by treatment with cantharidin but not with okadaic acid. In conclusion, HP exerts PKC-independent protection on ischemic-reperfused rat hearts and cardiomyocytes. Protein phosphatase 1 seems a mediator of this protective mechanism.

scholarly journals Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin

1998 ◽  
Vol 334 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Sucharita J. MISTRY ◽  
Heng-Chun LI ◽  
George F. ATWEH
Keyword(s):  
Cell Cycle ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Protein Phosphatases ◽  
Specific Protein ◽  
G1 Phase ◽  
Major Increase ◽  
Protein Phosphatase Type

Stathmin is a major cytosolic phosphoprotein that regulates microtubule dynamics during the assembly of the mitotic spindle. The activity of stathmin itself is regulated by changes in its state of phosphorylation during the transition from interphase to metaphase. For a better understanding of the regulation of stathmin activity during the cell cycle, we explored the mechanism(s) responsible for the decrease in the level of phosphorylation of stathmin as cells complete mitosis and enter a new G1 phase. We show that stathmin mRNA and protein are expressed constitutively throughout the different phases of the cell cycle. This suggests that the non-phosphorylated stathmin that predominates during G1 is not generated by degradation of phosphorylated stathmin in mitosis and synthesis of new unphosphorylated stathmin as cells enter a new G1 phase. This suggested that protein phosphatases might be responsible for dephosphorylating stathmin as cells enter a new cell cycle. Okadaic acid-mediated inhibition of protein phosphatases in vivoshowed a major increase in the level of phosphorylation of stathmin. Dephosphorylation studies in vitro showed differential patterns of site-specific dephosphorylaton of stathmin to protein phosphatase type 1, protein phosphatase type 2A and protein phosphatase type 2B. Thus stathmin might be a target for okadaic acid-sensitive protein phosphatase(s), and its activity in eukaryotic cells might be modulated by the sequential activity of specific protein kinases and phosphatases.

THE ISOLATION AND IDENTIFICATION OF SUCROSE FROM THE SEEDLING WHEAT LEAF

1953 ◽  
Vol 31 (1) ◽  
pp. 75-80 ◽  
Author(s):  
G. M. Ward
Keyword(s):  
Chromatographic Analysis ◽  
Soluble Sugar ◽  
Analytical Techniques ◽  
Barium Salt ◽  
X Ray Diffraction ◽  
Leaf Extracts ◽  
Isolation And Identification ◽  
X Ray ◽  
Wheat Leaf ◽  
Wheat Leaves

Crystalline sucrose has been isolated from cleared alcoholic extracts of mature wheat leaves by precipitation as the barium salt. The crystals were positively identified by melting point, X-ray diffraction analysis, and glucose–fructose ratio as well as by conventional analytical techniques. The evidence for the presence of sucrose in the wheat leaf has been corroborated by chromatographic analysis of leaf extracts. It is concluded that sucrose forms the bulk of the soluble sugar reserve of this tissue.

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