Substrate specifity in Amoeba proteus

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

A comparative study of alkaline phosphatases among human placenta, bovine milk, hepatopancreases of shrimp Penaeus monodon (Crustacea: Decapoda) and clam Meretrix lusoria (Bivalvia: Veneidae): to obtain an alkaline phosphatase with improved characteristics as a reporter

1. Alkaline phosphatases were purified from human placenta, bovine milk, shrimp and clam with a final spec. act. of 67,000, 32,000, 22,000 and 15,000 U/mg of protein respectively. 2. The alkaline phosphatase from Meretrix lusoria is unique with its thermostability at 65 degrees C for 30 min; whereas the remaining enzymes studied, including the human placental alkaline phosphatase, are inactivated and have negligible activities. 3. The alkaline phosphatase from Penaeus monodon can be differentiated by its pH optimum at 9.0; the remaining enzymes studied have their optimal pH at 10.0. 4. The alkaline phosphatases from shrimp and clam are proposed to be applied as "reporters" in the study of mammalian cells.     

Phosphatase activity in Amoeba proteus at pH 9.0

In the free-living amoeba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as a substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called "fast", "intermediate", and "slow" phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be both acid phosphatase and protein tyrosine phosphatase (PTP). Based on data of inhibitor analysis, large substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, other than in the fast and intermediate phosphatases localization in the amoeba cell, it is concluded that only slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

Phosphatase activity in <Emphasis Type="Italic">Amoeba proteus</Emphasis> at pH 9.0

In the free-living ameba Amoeba proteus (strain B), after PAAG disk-electrophoresis of the homogenate supernatant, at using 1-naphthyl phosphate as substrate and pH 9.0, three forms of phosphatase activity were revealed; they were arbitrarily called “fast,” “intermediate,” and “slow” phosphatases. The fast phosphatase has been established to be a fraction of lysosomal acid phosphatase that preserves some low activity at alkaline pH values. The question as to which particular class the intermediate phosphatase belongs to has remained unanswered: it can be either acid phosphatase, or protein tyrosine phosphatase. Based on data of inhibitor analysis, broad substrate specificity, results of experiments with reactivation by Zn ions after inactivation with EDTA, and another localization in the ameba cell than of the fast and intermediate phosphatases, it is concluded that only the slow phosphatase can be classified as alkaline phosphatase (EC 3.1.3.1).     

Resolution, purification and characterization of the orthophosphate releasing activities from fracture callus calcifying cartilage.

Callus calcifying cartilage alkaline phosphatase was resolved by DEAE-cellulose column chromatography into two distinct phsophatase activities. The phosphatase activity which was eluted first from the column, (phosphatase I), was active towards a variety of phosphate esters, sodium pyrophosphatase and several linear polyphosphates, while the second phosphatase activity , (phosphatase II), was active toward simple phosphate esters but not towards sodium pyrophosphate and linear oligo or polyphosphates. All the phosphate esters, sodium pyrophosphate and polyphosphates at higher concentrations were inhibitory for phosphatase I. The modulating effects of magnesium, calcium, zinc and other phosphatase modulators have been investigated. Both phosphatases from callus calcifying cartilage were found to be substrates of neuraminidase with sialic acid as the product. Besides the difference in their specificity, the phosphatases were found to be immunologically different and to have different molecular weights, strong indication that they are different enzymes.     

Mutationally altered rate constants in the mechanism of alkaline phosphatase

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.     

Phosphatase of Chlamydomonas reinhardi: biochemical and cytochemical approach with specific mutants.

The unicellular alga Chlamydomonas reinhardi produces two constitutive acid phosphatases and three depressible phosphatases (a neutral and two alkaline ones) that can utilize napthyl phosphate as a substrate. Specific mutants depressible phosphatase were used to investigate biochemical properties and the cytochemical localization of these enzymes. The two constitutive phosphatases show similar pH optima (about 5.0) and Km values (2 x 10(-3) to 3.3 x 10(-3) M) but differ in their heat sensitivity and affinity for glycerophosphate.     

An ultrastructural study of phosphatases and aryl sulphatase in BHK and CHO cells grown in culture

Summary The ultrastructural distribution of a number of phosphatases and aryl sulphatase has been studied in BHK 21/C 13, BHK21/J 1 and CHO cells grown in culture. In all three cell lines acid -glycerophosphatase and aryl sulphatase appear to be confined to lysosomes and elements of the Golgi apparatus and glucose-6-phosphatase to the endoplasmic reticulum. With thiamine pyrophosphate at pH 7.0 in CHO cells reaction product is present in lysosomes, the Golgi apparatus, the endoplasmic reticulum and on the cell surface. Preincubation at acid pH reduces the reactions in the endoplasmic reticulum but enhances the surface activity. At pH 5.0 and pH 7.0 in CHO cells p-nitrophenylphosphatase is present in lysosomes, the Golgi apparatus and the endoplasmic reticulum and this activity is inhibited by sodium fluoride. p-nitrophenylphosphatase activity is also present on the cell surface of CHO cells and this activity is not inhibited by sodium fluoride. No activity could be demonstrated in any cells at pH 9.O. The significance of these results is discussed with respect to the possible role of surface acid phosphatase in the process of transformation.     

Phosphatase activity in Trypanosoma cruzi. Phosphate removal from ATP, phosphorylated proteins and other phosphate compounds

T. cruzi epimastigotes have a lysosomal acid phosphatase (pH 4.0) and acid and alkaline phosphatases (pH 5.5 and 8.0) localized in the cytosolic fraction. The levels of the lysosomal acid phosphatase increase with the age of the cultures, but the cytosolic phosphatases decline after the logarithmic phase of growth. The lysosomal phosphatase preferentially hydrolyses low mol. wt phosphate esters; whereas, the cytosolic alkaline phosphatases primarily act on phosphorylated proteins, and both the cytosolic acid and alkaline phosphatases on uridine nucleotide derivatives. The parasite also contains a microsomal glucose 6-phosphatase, and ATPases (Mg2+ and Ca2+-activated) derived from plasma membranes and mitochondria.     

The catalytic-centre activity and kinetic properties of bovine milk alkaline phosphatase

1. The phosphorylation of milk alkaline phosphatase was studied under various conditions: maximum incorporation occurred at pH5.0 and 50% incorporation at pH6.6-7.0. 2. The phosphorylation was shown to be specific and the results suggest that the active centre of the enzyme is involved in the process. 3. Phosphoryl-enzyme is rapidly hydrolysed at alkaline pH. at pH7.0 the results suggest that a phosphoryl-enzyme could occur as a transient intermediate in the hydrolysis of phosphate esters by the phosphatase. 4. The catalytic-centre activity of the enzyme was found to be 2700sec.(-1) at pH10.0 and 25 degrees with p-nitrophenyl phosphate as substrate.     

Formation of multiple forms of acid and alkaline phosphatases in relation to the culture age of Aspergillus niger

Acid phosphatase (EC 3.1.3.2 [EC] ) was extracted from mycelia ofAspergillus niger, then separated and purified into four fractions.These acid phosphatases, designated IA, IB, II and III, hadpH optima at 5.0, 4.5–5.0, 4.5 and 2.5, respectively.None required the presence of divalent cations, and all werestrongly inhibited by NaF. They were non-specific acid phosphatasesbut varied in their activities with various substrates. Thealkaline phosphatase (EG 3.1.3.1 [EC] ) of A. niger was also separatedinto two fractions, alkaline phosphatases I and II. Changes in the activity ratios of these acid and alkaline phosphataseswere studied during culture in a peptone medium. The activityof acid phosphatase II was higher than the others when the culturewas young. The activity of acid phosphatase III increased toa maximum in the actively growing phase, then decreased. Thatof acid phosphatase I became highest in the mature culture.In contrast, the activity of alkaline phosphatase I was higherthan the others in young cultures, while alkaline phosphataseII became dominant in the mature culture. Activities of the various acid and alkaline phosphatases indifferent regions of the growing colonies were also studied.The changing patterns of these enzymes in both liquid and surfacecultures were compared. When A. niger was cultured in a medium containing a low concentrationof phosphate, acid phosphatase activity greatly increased afterthe consumption of phosphate, but alkaline phosphatase activitydid not. ¹ The present experiments were carried out, for the most partat the Institute of Applied Microbiology of the University ofTokyo. (Received February 10, 1975; )     

Partial purification and biochemical properties of acid and alkaline phosphatases from Myxococcus coralloides D

F. GONZÁLEZ, M.E. FÁREZ-VIDAL, J.M. ARIAS AND E. MONTOYA. 1994. Acid phosphatase and alkaline phosphatase from vegetative cells of Myxococcus coralloides D were purified by two chromatographic steps. The molecular weights were estimated by gel filtration and SDS-PAGE. Optimum pH, stability, optimum temperature and thermal inactivation studies were made for both enzymes. EDTA and other chelating agents inhibited alkaline but not acid activity. Mg²⁺ activated the alkaline phosphatase, while the acid phosphatase was inhibited by fluoride. Both enzymes degraded a number of phosphomonoesters, but were unable to hydrolyse either polyphosphates or cAMP. The K _m values of the acid and alkaline phosphatases for p -nitrophenylphosphate were 5.0 times 10^-3 mol ***l^-1 and 1.5 times 10^-3 mol l^-1, respectively.     

The cell surface associated phosphatase activity of Mycobacterium bovis BCG is not regulated by environmental inorganic phosphate

Non-specific phosphomonoesterase activities (alkaline phosphatase (EC 3.1.3.1) and acid phosphatase (EC 3.1.3.2)) were examined at the cell surface of Mycobacterium bovis BCG. Using p-nitrophenylphosphate as the substrate, peaks of phosphatase activity were detected at pH 6.0, pH 10.0 and pH 12.0, suggesting the presence of one acid phosphatase and two alkaline phosphatases with distinct optimum pH values. Contrary to the situation observed in several other microorganisms, the expression of these enzymes is not regulated by the environmental inorganic phosphate concentration.     

Biochemical and histochemical studies of alkaline and acid phosphatases in a digenetic trematode, Pegosomum egretti

The biochemistry and histochemistry of Pegosomum egretti have been studied using standard techniques. Phosphatases were analysed colorimetrically; the optimum pH for acid phosphatase activity was 5.0 and for alkaline phosphatase was 10.0. The results were compared with those of other trematodes. Histochemical localization of acid and alkaline phosphatases revealed differences in enzymes activity in various tissues. These differences in the site and pattern of distribution of the two enzymes have been discussed in relation to transport of raw materials and the metabolism of the cell concerned.     

Modulation of activity of human alkaline phosphatases by Mg2+ and thiol compounds

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.     

Inhibition of phosphatase and sulfatase by transition-state analogues

The inhibition constants for vanadate, chromate, molybdate, and tungstate have been determined with Escherichia coli alkaline phosphatase, potato acid phosphatase, and Helix pomatia aryl sulfatase. Vanadate was a potent inhibitor of all three enzymes. Inhibition of both phosphatases followed the order WO4(2-) greater than MoO4(2-) greater than CrO4(2-). The Ki values for potato acid phosphatase were about 3 orders of magnitude lower than those for alkaline phosphatase. Aryl sulfatase followed the reverse order of inhibition by group VI oxyanions. Phenol enhanced inhibition of alkaline phosphatase by vanadate and chromate but did not affect inhibition of acid phosphatase. Phenol enhanced inhibition of aryl sulfatase by metal oxyanions in all cases following the order H2VO4- greater than CrO4(2-) greater than MoO4(2-) greater than WO4(2-), and N-acetyltyrosine ethyl ester enhanced inhibition of aryl sulfatase by H2VO4- and CrO4(2-) more strongly than did phenol. It is apparent that the effectiveness of metal oxyanions as inhibitors of phosphatases and sulfatases can be selectively enhanced in the presence of other solutes. The relevance of these observations to the effects of transition metal oxyanions on protein phosphatases in vivo is discussed.     

Alkaline fixation-resistant acid phosphatases in human tissues: histochemical evidence for a new type of acid phosphatase in endothelial, endometrial and neuronal sites

The effect of pH during formalin fixation on acid phosphatases in human tissues was studied. Lysosomal-type acid phosphatase was sensitive to alkaline fixation, being completely inactive after fixation at pH 9.0. Prostatic and tartrate-resistant osteoclastic/macrophagic types were alkaline fixation-resistant, as was an acid phosphatase localized in endothelium, endometrial stromal cells and intestinal nerves. The latter activity was further separable into fluoride- and tartrate-sensitive beta-glycerophosphatase and fluoride-sensitive, tartrate-resistant alpha-naphthyl phosphatase. The activities appeared to represent either different, tightly associated enzymes or separate activity centres of a single enzyme. Alkaline fixation-resistant alpha-naphthyl phosphatase at endothelial, endometrial and neuronal sites was also well demonstrated in unfixed or neutral formalin-fixed sections as tartrate-resistant activity similar to classical tartrate-resistant acid phosphatase, but these phosphatases appear to be antigenically different. Alkaline fixation-resistant acid phosphatase showed a restricted tissue distribution both in endothelium (mainly in vessels of abdominal organs) and at neuronal sites (only in intestinal nerves). Alkaline fixation-resistant acid phosphatase appears to represent a previously unknown or uncharacterized enzyme activity whose chemical properties could not be classified as any previously known type of acid or other phosphatases.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.