scholarly journals [Phosphotyrosine]protein phosphatase in rat brain. A major [phosphotyrosine]protein phosphatase is a 23 kDa protein distinct from acid phosphatase

1986 ◽  
Vol 239 (1) ◽  
pp. 155-162 ◽  
Author(s):  
M Okada ◽  
K Owada ◽  
H Nakagawa
Keyword(s):  
Acid Phosphatase ◽  
Molecular Mass ◽  
Rat Brain ◽  
Column Chromatography ◽  
Protein Phosphatase ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Sodium Vanadate ◽  
Nitrophenyl Phosphate ◽  
Phosphotyrosine Protein Phosphatase

A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5′-AMP, 2′-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.

scholarly journals Isolation and characterization of a γ-type phosphoinositide-specific phospholipase C (PLC-γ2)

1990 ◽  
Vol 269 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Y Homma ◽  
Y Emori ◽  
F Shibasaki ◽  
K Suzuki ◽  
T Takenawa
Keyword(s):  
Phospholipase C ◽  
Column Chromatography ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Isolation And Characterization ◽  
Delta Type ◽  
Hydrolysis Of ◽  
Cellulose Column

A novel bovine spleen phosphoinositide-specific phospholipase C (PLC) has been identified with respect to immunoreactivity with four independent antibodies against each of the PLC isoenzymes, and purified to near homogeneity by sequential column chromatography. Spleen contains three of the isoenzymes: two different gamma-types [gamma 1 and gamma 2, originally named as PLC-gamma [Rhee, Suh, Ryu & Lee (1989) Science 244, 546-550] and PLC-IV [Emori, Homma, Sorimachi, Kawasaki, Nakanishi, Suzuki & Takenawa (1989) J. Biol. Chem. 264, 21885-21890] respectively] and delta-type of the enzyme, but PLC-gamma 1 is separated from the PLC-gamma 2 pool by the first DEAE-cellulose column chromatography. Subsequently, PLC-delta is dissociated on the third heparin-Sepharose column chromatography. The purified enzyme has a molecular mass of 145 kDa on SDS/polyacrylamide-gel electrophoresis and a specific activity of 12.8 mumol/min per mg with phosphatidylinositol 4,5-bisphosphate as substrate. This enzyme activity is dependent on Ca2+ for hydrolysis of all these phosphoinositides. None of the other phospholipids examined could be its substrate at any concentration of Ca2+. The optimal pH of the enzyme is slightly acidic (pH 5.0-6.5).

Inhibition of Bovine Kidney Low Molecular Mass Phosphotyrosine Protein Phosphatase by Uric Acid

2002 ◽  
Vol 17 (5) ◽  
pp. 345-350 ◽  
Author(s):  
José Mauro Granjeiro ◽  
Carmen Verissima Ferreira ◽  
Paulo Afonso Granjeiro ◽  
Cinthia Celestino Da Silva ◽  
Eulázio Mikio Taga ◽  
...  
Keyword(s):  
Uric Acid ◽  
Molecular Mass ◽  
Protein Phosphatase ◽  
Bovine Kidney ◽  
Phosphotyrosine Protein Phosphatase

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

A quantitative study of the fixation of acid phosphatase by formaldehyde and its relevance to histochemistry

1974 ◽  
Vol 186 (1083) ◽  
pp. 137-164 ◽  
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Relative Activity ◽  
Buffer System ◽  
Inhibitory Effects ◽  
Fixed Tissue ◽  
Apparent Loss ◽  
Nitrophenyl Phosphate ◽  
Enzyme Specific Activity ◽  
Tissue Nitrogen

Fixatives reduce the activities of most enzymes in sections and blocks of tissue, but pre­vious studies do not agree by how much. In this paper it is shown that the disagreement is due principally to the use of inappropriate measurement parameters and to variations in the way tissues are prepared. Some new approaches to this problem are reported. They are illustrated by measurements on the activities of acid phosphatase (against p -nitrophenyl phosphate) remaining in blocks and cryostat-cut sections of hamster kidney after fixation in buffered solutions of formaldehyde. Whole fresh kidneys were cut into 1 mm 3 cubes. Two or more ‘lots’ of 9-12 cubes were chosen at random, fixed, washed, homogenized and assayed for residual enzyme specific activity which was expressed in terms of tissue nitrogen content or frozen-dried mass rather than protein content. The activity apparently remaining after fixation depends on, it was found, the time and temperature of fixation, the buffer salt in the fixative, the length of the post-fixation wash, and the duration and temperature of homogenization. For example, the relative activity (that is, specific activity expressed as a percentage of that of unfixed tissue) of acid phos­phatase in cubes assayed immediately after fixation is 46%; after a 24 h post-fixation wash in cold buffer it rises to 84%. Fixation causes only a 2% loss of nitrogenous material from the cubes. A further 2.7% is lost during the 24 h wash. Thus, much of the apparent loss of enzyme specific activity in fixed tissue is due to the inhibitory effects of fixative remaining in the tissue. Similar inhibition effects were observed in cryostat-cut sections, although sections frozen-dried on to coverslips showed a greater tolerance than fresh ones. For example, after 5 min fixation and without a post-fixation wash, the activity of acid phosphatase in fresh, cryostat-cut, mounted sections falls by about 30%, but that in frozen-dried ones is un­affected. After 25 min fixation, however the activity in the frozen-dried sections drops to about 84% of that of the unfixed tissue. A nitrogen-free buffer system, 5-norbornene-2, 3-dicarboxylic acid-NaOH, was used as the fixative vehicle for much of this study. A higher acid phosphatase activity is retained in tissues fixed or stored in this buffer than in either cacodylate or phosphate.

N-Myristoylation is essential for protein phosphatases PPM1A and PPM1B to dephosphorylate their physiological substrates in cells

2013 ◽  
Vol 449 (3) ◽  
pp. 741-749 ◽  
Author(s):  
Toko Chida ◽  
Masakatsu Ando ◽  
Tasuku Matsuki ◽  
Yutaro Masu ◽  
Yuko Nagaura ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Specific Activity ◽  
Wild Type ◽  
Α Subunit ◽  
Nitrophenyl Phosphate ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Physiological Substrates ◽  
In Cells

PPM [metal-dependent protein phosphatase, formerly called PP2C (protein phosphatase 2C)] family members play essential roles in regulating a variety of signalling pathways. While searching for protein phosphatase(s) that act on AMPK (AMP-activated protein kinase), we found that PPM1A and PPM1B are N-myristoylated and that this modification is essential for their ability to dephosphorylate the α subunit of AMPK (AMPKα) in cells. N-Myristoylation was also required for two other functions of PPM1A and PPM1B in cells. Although a non-myristoylated mutation (G2A) of PPM1A and PPM1B prevented membrane association, this relocalization did not likely cause the decreased activity towards AMPKα. In in vitro experiments, the G2A mutants exhibited reduced activities towards AMPKα, but much higher specific activity against an artificial substrate, PNPP (p-nitrophenyl phosphate), compared with the wild-type counterparts. Taken together, the results of the present study suggest that N-myristoylation of PPM1A and PPM1B plays a key role in recognition of their physiological substrates in cells.

scholarly journals Acid phosphatases and ribonucleases from Dactylis glomerata seeds. I. Chromatographic and electrophoretic heterogeneity of the enzymes

2015 ◽  
Vol 47 (4) ◽  
pp. 441-453 ◽  
Author(s):  
Eleonora Wieczorek ◽  
Janina Wiśniewska ◽  
Bronisława Morawiecka
Keyword(s):  
Acid Phosphatase ◽  
Sodium Acetate ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Dactylis Glomerata ◽  
Molecular Forms ◽  
Acid Phosphatases ◽  
Sodium Acetate Buffer ◽  
Optimal Activity ◽  
Optimum Ph

Acid phosphatase and ribonuclease extracted with 0.1 M sodium acetate buffer, pH 5.1 from Dactylis glomerata seeds, and partially purified by means of 70% ethanol precipitation showed electrophoretic and Chromatographic heterogeneity. After chromatography on DEAE-cellulose acid phosphatase and ribonuclease were separated into four peaks. Nonadsorbing acid phosphatase on DEAE-cellulose (peak I) was separated into four peaks on CM-cellulose. The highest activity (11 units/mg) was found in fraction b (acid phosphatase Ib). The enzyme was activated by Mg<sup>2+</sup>, Ca<sup>2+</sup>, Li<sup>+</sup>, Cs<sup>+</sup>, K<sup>+</sup> ions and inhibited by Cu<sup>2+</sup>, Zu<sup>2+</sup>, F<sup>-</sup> and Mo<sup>-6</sup> at optimum pH 5.0. Strong absorbing ribonuclease on DEAE-cellulose (peak IV) was further separated on G-200 Sephadex into two molecular forms: RN-asa1 and RN-ase2. Ribonuclease l, a thermolabile enzyme with specific activity 807 units/mg, showed an optimal activity at pH 4.8-5.1.

Functional characterization of the low-molecular-mass phosphotyrosine-protein phosphatase of Acinetobacter johnsonii 1 1Edited by M. Yaniv

1998 ◽  
Vol 278 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Christophe Grangeasse ◽  
Patricia Doublet ◽  
Carole Vincent ◽  
Elisabeth Vaganay ◽  
Mylène Riberty ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Protein Phosphatase ◽  
Functional Characterization ◽  
Acinetobacter Johnsonii ◽  
Phosphotyrosine Protein Phosphatase

Isolation and Properties of a Novel Phospholipase a from Rat Brain That Hydrolyses Fatty Acids at sn-1 and sn-2 Positions

1998 ◽  
Vol 35 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Hiroshi Yoshida ◽  
Yosuke Tsujishita ◽  
Françoise Hullin ◽  
Kimihisa Yoshida ◽  
Shun-Ichi Nakamura ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Phosphatidic Acid ◽  
Molecular Mass ◽  
Rat Brain ◽  
Column Chromatography ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Phospholipase A ◽  
Membrane Phospholipids ◽  
Arachidonic Acids

A Ca2+-independent phospholipase A that releases various fatty acids from sn-1 and sn-2 positions was partially purified from rat brain soluble fraction. The enzyme showed an approximate molecular mass of 300 kDa on gel filtration column chromatography. Its enzymatic properties are distinct from those of well characterized phospholipase A2 enzymes; by using a series of synthetic phosphatidylcholines, the enzyme cleaved oleic, linoleic, and arachidonic acids like phospholipase A2, and released palmitic and stearic acids like phospholipase A1. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid were hydrolysed with almost equal efficiencies by this enzyme. These results indicate that the enzyme isolated is a novel Ca2+ -independent intracellular phospholipase A that might be responsible for production of various fatty acids from membrane phospholipids.

scholarly journals Human prostatic acid phosphatase has phosphotyrosyl protein phosphatase activity

1986 ◽  
Vol 235 (2) ◽  
pp. 351-357 ◽  
Author(s):  
M F Lin ◽  
G M Clinton
Keyword(s):  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Acid Phosphatase Activity ◽  
High Performance ◽  
Gel Filtration ◽  
Prostatic Acid Phosphatase ◽  
Peptide Sequence ◽  
Phosphatase Activities ◽  
Nitrophenyl Phosphate

The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein.

Sign in / Sign up

Export Citation Format

Share Document