Biochemical characterization of alkaline phosphatase in guinea pig thymus.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) in guinea pig thymus was extracted optimally in 10 mM Tris - HCl buffer at pH 8.0 containing 5 g/l Triton X-100. 2. alpha-Glycerophosphate, beta-glycerophosphate and phenolphthalein monophosphate were hydrolyzed by thymus extract with a pH optimum at 9.8-10.0, whereas p-nitrophenylphosphate and alpha-naphthylphosphate were hydrolyzed with pH optima at 10.7-10.8 and beta-naphthylphosphate at pH 11.2. P-Nitrophenylphosphate and phenolphthalein monophosphate proved to be the most suitable substrates. 3. Alkaline phosphatase was effectively inhibited by EDTA, Zn2+, histidine and urea therefore resembling the inhibition characteristics of alkaline phosphatase in the placenta and kidney, but not that in the liver and intestine, which differed markedly. 4. DEAE-cellulose chromatography and polyacrylamide disc electrophoresis revealed three enzyme peaks which did not differ in their substrate specificities and modifier characteristics. 5. Polyacrylamide disc electrophoresis of thymus, serum, placenta, kidney, liver, bone and intestine revealed no alkaline phosphatase bands definitely unique to thymus.     

The inhibition of enzymes by beryllium

1. The action of beryllium on the following enzymes has been examined: alkaline phosphatase (Escherichia coli and kidney), acid phosphatase, phosphoprotein phosphatase, apyrase (potato), adenosine triphosphatase (liver nuclei, liver mitochondria, brain microsomes), glucose 6-phosphatase, polysaccharide phosphorylases a and b, phosphoglucomutase, hexokinase, phosphoglyceromutase, ribonuclease, A-esterase (rabbit serum), cholinesterase (horse serum), chymotrypsin. Alkaline phosphatase and phosphoglucomutase are inhibited by 1mum-beryllium sulphate whereas the other enzymes are largely unaffected by 1mm-beryllium sulphate. 2. Possible mechanisms for the inhibition of phosphoglucomutase and alkaline phosphatase are discussed.     

In vivo inhibition by ethyloxanilates of alkaline phosphatases of the cestode Echinococcus multilocularis

Alkaline phosphatases (E.C.3.1.3.1), membranous enzymes of the cestode Echinococcus multilocularis have been studied in the parasite and in the experimental host liver. Synthetic inhibitors interaction with metacestode alkaline phosphatases is reported. In regard to the alkaline phosphatases inhibition, the ethyloxanilate 2 is more efficient in the cestode itself than in the host liver.     

Mammalian Brain Alkaline Phosphatase: Expression of Liver/Bone/Kidney Locus Comparison of Fetal and Adult Activities

The alkaline phosphatases (EC 3.1.3.1) are determined by at least three gene loci, which can be sharply distinguished one from another by their sensitivity to inhibition with various amino acids and peptides and by ther-mostability. Alkaline phosphatase is present in the brains of guinea pig, rat, mouse, hamster, squirrel, rabbit, cat, sheep, cow, tamarin, baboon, and man. The gene locus coding for alkaline phosphatase in all these brains is the liver/ bone/kidney locus, as indicated by thermostability studies and by inhibition studies with L-phenylalanine, L-homoarginine, and L-phenylalanylglycylglycine. The average brain alkaline phosphatase activity is about 35% of the average for the livers and only 7.2% and 4.4% of the average kidney and placental activities, respectively. During growth and development, brain alkaline phosphatase activity decreases in the mammals studied. The amount of change is tissue- and species-dependent.     

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.     

Comparative biochemical study of alkaline phosphatase isozymes in fish, amphibians, reptiles, birds and mammals

Alkaline phosphatases from the liver, kidney and intestine in various vertebrates were strongly inhibited by beryllium, 2-mercaptoethanol, potassium cyanide and EDTA. The enzymes showed various sensitivities to the inhibition by zinc and to heat denaturation at 56 degrees C for 5 min at pH 7.0. The liver and kidney enzymes showed higher sensitivity to the inhibition by L-homoarginine than by L-phenylalanine. The intestinal enzymes in higher vertebrates were more sensitive to the inhibition by L-phenylalanine than by L-homoarginine, whereas the intestinal ones in lower vertebrates showed quite similar sensitivities to both amino acids.     

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.     

Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase

Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.     

Phosphomonoesterases in the male genital system of some mammals.

The various male genital organs of the experimental animals used in this investigation (rat, guinea pig, rabbit, cat and dog) showed a widely different localizations of alkaline and acid phosphatases. Alkaline phosphatase was presented as secretory, stromal, nuclear and vascular, while with acid phosphatase, distinction was made only between secretory and nuclear phosphatases. Although the morphological distributions of both enzymes were sometimes overlapping, they were not identical.     

Calix[4]arene methylenebisphosphonic acids: the features of alkaline phosphatases inhibition

The inhibition of alkaline phosphatases by calix[4]arenes functionalysed at the macrocyclic upper rim by one or two methylenebisphosphonic acid fragments has been investigated. It is established, that calix[4]arene bismethylenebisphosphonic acid displayed stronger inhibition of alkaline phosphatase from bovine intestine mucosa than calix[4]arene methylenebisphosphonic acid. At the same time, the both inhibitors showed almost similar levels of inhibitory activities in respect of bovine kidney alkaline phosphatase or E. coli alkaline phosphatase. The tested compounds were docked computationally to the active site of the E. coli alkaline phosphatase. On the basis of results obtained the possible binding modes of inhibitors were analysed.     

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.     

Comparative effects of fluoride on three enzymes, hydrolyzing pyrophosphate - acid and alkaline phosphatases and inorganic pyrophosphatase

The effects of fluoride on the activities of acid phosphatase (EC 3.1.3.2) from potato and alkaline phosphatase (EC 3.1.3.1) from E. coli during pyrophosphate and p-nitrophenylphosphate hydrolysis and on the activities of inorganic pyrophosphatase (EC 3.6.1.1) from baker's yeast during pyrophosphate hydrolysis were compared. For both phosphatases the type of interaction was found to be independent on the nature of substrate. For acid phosphatase and inorganic pyrophosphatase the inhibition was of non-competitive and uncompetitive types, respectively. In the case of alkaline phosphatase fluoride increased the rate of p-nitrophenol release during p-nitrophenylphosphate hydrolysis at pH greater than or equal to 7.9 without affecting the rate of phosphate release, which is indicative of fluorophosphate formation in the course of the transphosphorylation reaction. The data obtained suggest the existence of essential differences in the mechanisms of fluoride effects on the three enzymes under study.     

Enzyme Evolution in the Enterobacteriaceae

An immunological approach has been used for the study of alkaline phosphatase evolution in bacteria of the family Enterobacteriaceae. Antisera were prepared against alkaline phosphatase from Escherichia coli and Klebsiella aerogenes and tested against the unpurified alkaline phosphatases of 32 strains of enterobacteria by double diffusion and quantitative micro-complement fixation. The immunological relationships detected among the alkaline phosphatases of enterobacteria agree approximately with those reported for five other enzymes, as well as with the tryptic peptide pattern similarities found for two other enzymes, and with the relationships detected by interspecific deoxyribonucleic acid hybridization tests.     

Characterization of two different alkaline phosphatases in mouse teratoma: Partial purification,electrophoretic, and histochemical studies

Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent ³²PO₄-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

The inhibition of alkaline phosphatase by L-p-bromotetramisole.

A levamisole analogue, the L-p-bromotetramisole is introduced as a potent inhibitor of non-specific alkaline phosphatase. Complete inhibition is achieved cytochemically at a concentration of 0.1 mM in various rat tissues except the intestine, which is not affected. The D-p-bromotetramisole does not influence the alkaline phosphatase activities. Since no effect of the inhibitor is seen on the activities of specific phosphatases, this drug is recommended also as an additive for specific phosphatase media in order to yield the specific activity only.     

A study of the enzymic dephosphorylation of beta-casein and a derived phosphopeptide.

beta-Casein, and the phosphate containing peptide derived from it by tryptic digestion, have been dephosphorylated by the action of two phosphatases. Escherichia coli alkaline phosphatase (EC 3.1.3.1) has been shown to remove the phosphates from these substrates in two distinct stages. Substrate molecules retaining three of the original phosphoseryl residues accumulate during the reaction and are resistant to further dephosphorylation at low enzyme concentrations. In contrast bovine spleen phosphoprotein phosphatase (EC 3.1.3.16) achieves complete dephosphorylation of these substrates sequentially without any of the intervening species showing resistance to the action of the enzyme. The phosphopeptide has been partially dephosphorylated by the action of the two phosphatases and the resultant peptides containing three phosphoseryl residues compared in their reactivity toward the E. coli alkaline phosphatase. The results obtained are discussed in relation to the mode of action of the two enzymes.     

Purification and characterization of alkaline phosphatase from rat kidney.

Alkaline phosphatase [EC 3.1.3.1.] was purified about 250-fold from rat kidney, and its enzymological properties were studied. Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8.X10(4) and 1.0X10(5), as determined by SDS-polyacrylamide gel electrophoresis. Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.     

Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics.

The inhibitory effect of a marine-sponge toxin, okadaic acid, was examined on type 1, type 2A, type 2B and type 2C protein phosphatases as well as on a polycation-modulated (PCM) phosphatase. Of the protein phosphatases examined, the catalytic subunit of type 2A phosphatase from rabbit skeletal muscle was most potently inhibited. For the phosphorylated myosin light-chain (PMLC) phosphatase activity of the enzyme, the concentration of okadaic acid required to obtain 50% inhibition (ID50) was about 1 nM. The PMLC phosphatase activities of type 1 and PCM phosphatase were also strongly inhibited (ID50 0.1-0.5 microM). The PMCL phosphatase activity of type 2B phosphatase (calcineurin) was inhibited to a lesser extent (ID50 4-5 microM). Similar results were obtained for the phosphorylase a phosphatase activity of type 1 and PCM phosphatases and for the p-nitrophenyl phosphate phosphatase activity of calcineurin. The following phosphatases were not affected by up to 10 microM-okadaic acid: type 2C phosphatase, phosphotyrosyl phosphatase, inositol 1,4,5-trisphosphate phosphatase, acid phosphatases and alkaline phosphatases. Thus okadaic acid had a relatively high specificity for type 2A, type 1 and PCM phosphatases. Kinetic studies showed that okadaic acid acts as a non-competitive or mixed inhibitor on the okadaic acid-sensitive enzymes.     

Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.