Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography

Dissing, J.; Dahl, Otto; Svensmark, Ole

doi:10.1016/0005-2744(79)90051-2

Cited by 45 publications

(24 citation statements)

References 40 publications

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“…Because such peptides are prepared by kinasephosphorylation with ATP␥S, the application depends on whether a particular kinase is available or not. As an alternative, the phosphonic acid mimetic, the so-called C-P compound, can be chemically synthesized and is nonhydrolyzable (33). Moesin kinases were unknown at the time when we started to purify moesin phosphatases from platelets, and we therefore employed the synthetic phosphonic acid peptide, KYKcpTLR as the affinity ligand for purification.…”

Section: Discussionmentioning

confidence: 99%

Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Hishiya¹,

Ohnishi²,

Tamura³

et al. 1999

Journal of Biological Chemistry

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During activation of platelets by thrombin phosphorylation of Thr558 in the C-terminal domain of the membrane-F-actin linking protein moesin increases transiently, and this correlates with protrusion of filopodial structures. Calyculin A enhances phosphorylation of moesin by inhibition of phosphatases. To measure this moesin-specific activity, a nonradioactive enzymelinked immunosorbent assay method was developed with the synthetic peptide Cys-Lys 555 -Tyr-Lys-Thr(P)-Leu-Arg 560 coupled to bovine serum albumin as the substrate and moesin phosphorylation state-specific polyclonal antibodies for the detection and quantitation of dephosphorylation. Calyculin A-sensitive and -insensitive protein-threonine phosphatase activities were detected in platelet lysates and separated by DEAE-cellulose chromatography. The calyculin A-sensitive enzyme was identified as a type 1 protein phosphatase. The calyculin A-insensitive enzyme activity was purified to homogeneity by phenyl-Sepharose, protamine-, and phosphonic acid peptide-agarose chromatography and characterized biochemically and immunologically as a 53-kDa protein(s) and a type 2C protein phosphatase (PP2C). Phosphorylation of Thr 558 is necessary for Factin binding of moesin in vitro. The purified enzyme, as well as bacterially made PP2C␣ and PP2C␤, efficiently dephosphorylate(s) highly purified platelet phosphomoesin. This reverses the activating effect of phosphorylation, and moesin no longer co-sediments with actin filaments. In vivo, regulation of these phosphatase activities are likely to influence dynamic interactions between the actin cytoskeleton and membrane constituents linked to moesin.

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Section: Discussionmentioning

confidence: 99%

Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Hishiya¹,

Ohnishi²,

Tamura³

et al. 1999

Journal of Biological Chemistry

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“…26,45) Two low molecular weight acid phosphatases from fish liver have been purified by following the protocol of Manao et al 17) based on (NH 4 ) 2 SO 4 precipitation, SP-Sephadex C-50 chromatography, gel filtration on Sephadex G-75 and affinity chromatography on p-aminobenzyl phosphonic acid derivative of agarose column which was found to be critical for enzyme homogeneity as well as for resolution into two isoenzymes. This affinity gel was also successfully used for the purification of human erythrocytes low molecular weight acid phosphatase 49) and other low molecular weight acid phosphatases from bovine brain, 21) rat liver, 17) chicken liver 27) and chicken heart 48) that are said to be pure enzymes, homogeneous with molecular weight of 18 kDa.…”

Section: Discussionmentioning

confidence: 99%

Acid Phosphatases from the Liver of Labeo rohita: Purification and Characterization

Siddiqua

Rehmat

Saeed

et al. 2008

Biological & Pharmaceutical Bulletin

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Acid phosphatases (EC 3.1.3.2) are widely distributed in nature and often occur in multiple forms differing in molecular sizes, localization within the cell, substrate specificity and sensitivity to inhibitors. 1) In animals its biological role is not yet clear but it is involved in many biological systems which are linked to energy metabolism, metabolic regulation and cellular signal transduction path ways. 2)Mammalian liver contains two acid phosphatase forms (molecular weight 90-107 kDa and 14-30 kDa) and these can be separated by gel chromatography on Sephadex G-75. [3][4][5][6] The effects of inhibitors, substrates and kinetic parameters also distinguish between the enzymes. 7-9) The acid phosphatase (molecular weight 107 kDa) purified from human liver to homogeneity had been extensively characterized. 5,8) The enzymes from liver of carp, catfish and frog had also been purified [10][11][12] and found to be glycoprotein in nature. Previously, low molecular weight acid phosphatases had been purified from human liver, 9,13) bovine liver, 3,14,15) rat liver, 16,17) porcine liver, 18) bovine heart, 19,20) brain, 21) human placenta 22) and human erythrocytes. 23) In all cases, the enzymes had molecular weights ranging between 14-18 kDa.Two distinct isoforms of low molecular weight acid phosphatase exist in rat liver (AcP1 and AcP2), human erythrocytes (B fast and B slow ) and human placenta (HCPTP-A and HCPTP-B) and these have also been isolated, characterized and sequenced. 17,24,25) The AcP1, B fast and HCPTP-A have been classified as IF-1 while AcP2, B slow , HCPTP-B, bovine heart PTPase and bovine liver PTPase as IF-2. IF-1 and IF-2 differ from each other in type specific sequence of the 40-73 regions, in substrate affinity and in the sensitivity to activators and inhibitors. 26) In our laboratory we have also purified and characterized these enzyme couples from chicken liver 27) and uromastix liver. 28) These differ in pyridoxal-5Ј-PO 4 inhibition, cGMP activation and some kinetic parameters. Both isoenzyme couples possess phosphotyrosine protein phosphatase activity. None of all enzymes (LM-ACP and HM-ACP) need metal ion for their activity.Another class of acid phosphatases called Zn ϩϩ -dependent acid phosphatases, exist in two forms of different molecular weight. 29,30) Zn ϩϩ -dependent acid phosphatases have also been isolated and characterized in the liver of different vertebrates. 31)This paper describes the purification and characterization of LM-ACP and HM-ACP from fish (Labeo rohita) liver and the resolution of the two low molecular weight isoenzymes (IF-1 and IF-2 types) by affinity chromatography on paminobenzyl phosphonic acid-agarose column. MATERIALS AND METHODSMaterials L. rohita (common name Rohu) was captured from Indus river (N.W.F.P. Pakistan) and liver was excised immediately and stored at 4°C for use. para-Nitrophenyl phosphate (pNPP), phenyl phosphate, flavin mononucleotide (FMN), phosphotyrosine and other substrates were purchased from Merk, Fluka and Sigma Chemical Co.; SDS molecular ...

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“…Phosphatase activity assays p-NPP substrate Acid phosphatase activity was determined as described by Dissing et al (1979). Briefly, 0·7 µg of recombinant LmPP2C was incubated in the following buffers: 50 mM MES, pH 5-6·5; 50 mM MOPS, pH 7-7·5; 50 mM HEPES, pH 8; 50 mM TRIS, pH 8·5-9 and 50 mM CAPS, pH 10-11 and added to 10 mM MgCl 2 and 10 mM p-nitrophenyl phosphate [ p-NPP] in a final volume of 100 µL for 60 min at 37°C.…”

Section: Production Of Polyclonal Antiserummentioning

confidence: 99%

Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

et al. 2017

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The main goal of this work consisted in cloning, purifying and characterizing a protein phosphatase 2C (PP2C) from promastigotes of Leishmania major. The gene was cloned and amplified by PCR using specific oligonucleotides and the recombinant protein was purified by affinity chromatography. The peak with maximal protein concentration was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and revealed a protein of 44·9 kDa with PP2C activity. This activity was dependent on divalent cations (Mg +2 and Mn +2 ) and was optimal at pH of 8·5, using phosphothreonine as the substrate. Sanguinarine inhibited the activity of the recombinant LmPP2C, while protein tyrosine phosphatase inhibitors had no effect. The recombinant LmPP2C was used to generate polyclonal antibodies. These antibodies recognized a protein of 44·9 kDa in different Leishmania species; the LmPP2C was localized in the flagellar pocket and the flagellum of promastigotes.

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Phosphonic and arsonic acids as inhibitors of human red cell acid phosphatase and their use in affinity chromatography

Cited by 45 publications

References 40 publications

Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Protein Phosphatase 2C Inactivates F-actin Binding of Human Platelet Moesin

Acid Phosphatases from the Liver of Labeo rohita: Purification and Characterization

Protein phosphatase PP2C in the flagellum of Leishmania major: cloning and characterization

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