Integumental phosphatase isoenzymes from white puparia of Ceratitis capitata: isolation and characterization

1991 ◽  
Vol 69 (10-11) ◽  
pp. 731-735 ◽  
Author(s):  
K. Bourtzis ◽  
V. J. Marmaras
Keyword(s):  
Alkaline Phosphatase ◽  
Ceratitis Capitata ◽  
Divalent Cations ◽  
Deae Cellulose ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Isolation And Characterization ◽  
Two Peaks ◽  
Ph Curve ◽  
Hydrolysis Of

Two specific alkaline phosphatase forms were identified in the integument of wild-type Ceratitis capitata during transition of larvae to pupae. The separation was achieved by DEAE-cellulose chromatography; alkaline phosphatase 1 and alkaline phosphatase 2 were eluted in 0.1 and 0.4 M KC1, respectively. Both isoenzymes have a molecular weight of approximately 180 000. The pH curve reveals two peaks for both alkaline phosphatases: one at 9.4 and the other at 11.0. The two isoenzymes at both pH optima catalyze the hydrolysis of phosphotyrosine and β-glycerophosphate, but not phosphoserine, phosphothreonine, ATP, or AMP. However, at pH 9.4, alkaline phosphatase 1 is more effective than ALPase 2 and exhibits a preference for phosphotyrosine. The divalent cations Mn2+, Mg2+, and Ba2+ activate the enzymes, while Cu2+ and Zn2+ are inhibitors for both isoenzymes. Both isoenzymes are inactivated by EDTA. The effect of amino acids on enzyme activity was also tested. Alkaline phosphatase 1 is inhibited by L-tyrosine, while alkaline phosphatase 2 is unaffected. L-Phenylalanine has no effect on either isoenzyme. Both isoenzymes are inhibited by urea and 2-mercaptoethanol. Simultaneous addition of urea and 2-mercaptoethanol reveals that ALPase 1 is more sensitive to these inhibitors than ALPase 2.Key words: enzyme, Diptera, integument, phosphatase, isoenzymes, phosphotyrosine.

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).

scholarly journals Characterization of a Highly Thermostable Alkaline Phosphatase from the Euryarchaeon Pyrococcus abyssi

2001 ◽  
Vol 67 (10) ◽  
pp. 4504-4511 ◽  
Author(s):  
Sébastien Zappa ◽  
Jean-Luc Rolland ◽  
Didier Flament ◽  
Yannick Gueguen ◽  
Joseph Boudrant ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Disulfide Bonds ◽  
Metal Ion ◽  
Divalent Cations ◽  
E Coli ◽  
Isolation And Characterization ◽  
Pyrococcus Abyssi ◽  
Increased Activity ◽  
Metal Ion Chelators

ABSTRACT This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of theE. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far,P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition,P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

scholarly journals The influence of metal ions on the orthophosphatase and inorganic pyrophosphatase activities of human alkaline phosphatase

1969 ◽  
Vol 112 (5) ◽  
pp. 699-701 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ions ◽  
Competitive Inhibitor ◽  
Inorganic Pyrophosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Low Concentrations ◽  
Pyrophosphatase Activity ◽  
Hydrolysis Of ◽  
Pyrophosphate Hydrolysis

1. The differential effects of adding Zn2+ and Mg2+ on the orthophosphatase and inorganic pyrophosphatase activities of human intestinal alkaline phosphatase were studied. 2. In the presence of excess of Zn2+, inorganic pyrophosphatase activity is inhibited. At higher concentrations of pyrophosphate, hydrolysis of this substrate takes place, but is inhibited competitively by the Zn2+–pyrophosphate complex. This complex also acts as a competitive inhibitor of orthophosphate hydrolysis. 3. Excess of Mg2+ also inhibits pyrophosphatase action by removal of substrate; at low concentrations, this ion activates pyrophosphatase, as is the case with orthophosphatase. 4. It is concluded that, when interactions between metal ions and pyrophosphate are taken into account, the effects of these ions are consistent with the view that alkaline phosphatases possess both orthophosphatase and inorganic pyrophosphatase activities.

scholarly journals Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid

1974 ◽  
Vol 141 (1) ◽  
pp. 293-298 ◽  
Author(s):  
Kunio Hiwada ◽  
Ernst D. Wachsmuth
Keyword(s):  
Alkaline Phosphatase ◽  
Brush Border Membrane ◽  
Deae Cellulose ◽  
Multiple Forms ◽  
Alkaline Phosphatases ◽  
Acetylneuraminic Acid ◽  
Pig Kidney ◽  
Ph Dependency ◽  
Polypeptide Chains

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg2+ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of Km and Vmax. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.

scholarly journals Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in IdentifyingStreptococcus agalactiae Secreted Proteins

1999 ◽  
Vol 181 (18) ◽  
pp. 5790-5799 ◽  
Author(s):  
Martin H. Lee ◽  
Aphakorn Nittayajarn ◽  
R. Paul Ross ◽  
Cynthia B. Rothschild ◽  
Derek Parsonage ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Protein ◽  
Enterococcus Faecalis ◽  
Signal Sequence ◽  
Subcellular Location ◽  
Lactose Permease ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Gram Positive ◽  
Gram Positive Bacteria

ABSTRACT We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression ofphoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme’s subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZΔss, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria.

scholarly journals Potential inhibitors of rat tooth alkaline phosphatase studied by means of different histochemical techniques.

1979 ◽  
Vol 27 (5) ◽  
pp. 982-988 ◽  
Author(s):  
A Larsson ◽  
G Hasselgren
Keyword(s):  
Alkaline Phosphatase ◽  
Phosphatase Activity ◽  
Sodium Fluoride ◽  
Alkaline Phosphatase Activity ◽  
Staining Pattern ◽  
Alkaline Phosphatases ◽  
Phosphate Ion ◽  
Potential Inhibitors ◽  
Lead Pyrophosphate ◽  
Hydrolysis Of

Two histochemical methods for demonstration of alkaline phosphatase activity, a lead pyrophosphate- anda naphtholphosphate technique, were compared. Since different results may be due to methodological differences as well as different enzyme activities, the enzymatic hydrolysis of the naphtholphosphate was visualized both by means of an azo-dye coupler and by lead-capturing of the liberated phosphate ion. Various potential inhibitors of alkaline phosphatase activity (diphosphonate, D-penicillamine, and sodium fluoride) were also tested. The use of diphosphonate and D-penicillamine resulted in inhibited or reduced staining, which could mainly be explained by an interference by these compounds with components in the incubation media rather than with the enzyme itself. The addition of sodium fluoride had no effect on the naphtholphosphate staining pattern irrespective of capturing method, whereas the odontoblastic pyrophosphate splitting alkaline phosphatase appeared to be sensitive to sodium fluoride, suggesting the presence of two alkaline phosphatases in odontoblasts.

Kinetic and electrophoretic studies on acid and alkaline phosphatases of the metacercariae of Clinostomum complanatum (Trematoda: Digenea)

1982 ◽  
Vol 56 (1) ◽  
pp. 17-22 ◽  
Author(s):  
Afzal A. Siddiqui ◽  
Wajih A. Nizami
Keyword(s):  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Arsenate ◽  
Alkaline Phosphatases ◽  
Acid And Alkaline Phosphatases ◽  
Nitrophenyl Phosphate ◽  
Two Peaks ◽  
Hydrolysis Of ◽  
Clinostomum Complanatum

ABSTRACTHomogenates of the metacercariae of Clinostomum complanatum showed two peaks for the hydrolysis of p-nitrophenyl phosphate at pH 4·5–5·0 and 11·0; specific activity at pH 5·0 was 0·017 µmol p-nitrophenol/mg protein/min and 0·0049 µmol p-nitrophenol/mg protein/min at pH 11·0 at 37°C. Maximal activities of acid and alkaline phosphatases were observed at 50° and 40°C respectively. Both enzymes reached maximal rates after 50 min of incubation. Linearity in enzyme activity was noticed with increasing homogenate concentrations for both enzymes. The Km for p-nitrophenyl phosphate was 1·1 mM with a Vmax of 0·018 units/mg protein for acid phosphatase and 1·6 mM with a Vmax of 0·0052 units/mg protein for alkaline phosphatase. Sodium arsenate and sodium fluoride inhibit and Mg++ and Co++ stimulate both enzymes. Polyacrylamide gel electrophoresis showed one cathodic band each for both the enzymes.

scholarly journals Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Lorena Morales ◽  
Natalia Gutiérrez ◽  
Vanessa Maya ◽  
Carmen Parra ◽  
Eleazar Martínez-Barajas ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Divalent Cations ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Phosphate Deficiency ◽  
Ionic Exchange ◽  
Sds Page ◽  
Hydrolysis Of

Two phosphatase isoforms from roots of the common bean (<em>Phaseolus vulgaris</em> L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-PAGE and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of pH 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phosphoenolpyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg<sup>2+</sup>.

scholarly journals Alkaline Phosphatase Mutants of Bacillus subtilis

1975 ◽  
Vol 28 (3) ◽  
pp. 323 ◽  
Author(s):  
A RGlenn
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Control System ◽  
Phosphate Starvation ◽  
Wild Type ◽  
Alkaline Phosphatases ◽  
Wild Type Enzyme ◽  
Vegetative Cells ◽  
Specific Alkaline Phosphatase ◽  
Low Levels

Alkaline phosphatases from vegetative and sporulating cells of B. subtilis have been shown previously to be identical in all criteria examined. Despite this, 15 mutants producing low levels of the phosphatase during phosphate starvation of vegetative cells have been shown to produce high levels of the sporulation-specific alkaline phosphatase. It has been shown by imrnunochemical means that seven of these mutants when starved of phosphate produce low levels of normal wild-type enzyme. The sporulation form of the enzyme from one mutant (P-l00) has been shown to be identical with the phosphatases from vegetative and sporulating cells of the wild type. It is proposed that all the mutants have regulatory defects in the control of the alkaline phosphatase from vegetative cells but nevertheless retain an intact structural gene for the enzyme and the control system for the phosphatase during sporulation.

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