Purified Alkaline Phosphatase Research Articles

Effect of purified alkaline phosphatase from Bacillus licheniformis on growth of Zea mays L.

Some soil microbes have the capability to solubilize mineral phosphate into organic phosphorous and used as biofertilizer to improve crop productivity in agricultural field. In this study, phosphate solubilization assay was carried out onto media plates containing calcium phsophate precipitated nutrient agar media for bacterial strains like Bacillus megaterium MTCC 453, Bacillus subtilis MTCC 1134, Bacillus licheniformis MTCC 2312, Pseudomonas aeruginosa MTCC 424, Escherichia coli MTCC 570. Among these bacterial strains, B. licheniformis MTCC 2312 showed largest clear zone of phosphate solubilzation and maximum activity of alkaline phosphatase. The enzyme alkaline phosphatase was purified from B. licheniformis MTCC 2312 with purification fold 3.52 and specific activity 295.89 Unit/mg protein using DEAE-sepharose chromatography. This enzyme showed molecular weight as 60 KD, thermostability upto 50?C, pH stability up to 8.5 and Michaelis constant (Km) and maximum activity (Vmax) as 2.30 mM and 2223 U/ml respectively. The lyophilized powder of this enzyme was further supplemented with media components for the growth of Zea mays for carrying tissue culture experiment. The sterilized soil supplemented with alkaline phosphatase improved the total height, dry weight, % phosphate content in the stem and root of Zea mays by 3.07, 3.15, 2.35 and 1.76 fold respectively compared to control set. This enzyme could be used at large extent as effective biofertilizer for the agricultural industry.

Arsanilic acid modified superparamagnetic iron oxide nanoparticles for Purification of alkaline phosphatase from hen's egg yolk.

Recent studies of magnetic carrier technology have focused on its applications in separation and purification technologies, due to easy separation of the target from the reaction medium by applying an external magnetic field. In the present study, Fe3O4 superparamagnetic nanoparticles were prepared to utilize a chemical co-precipitation method, then the surfaces of the nanoparticles were modified with arsanilic acid derivatives which were used as the specific nanocarriers for the affinity purification of alkaline phosphatase from the hen's egg yolk. The six different types of magnetic nanocarriers with varied lengths of the linkers were obtained. All samples were characterized step by step and validated using FTIR, SEM, EDX, VSM and XRD analysis methods As the results were shown, the use of inflexible tags with long linkers on the surface of the nanocarrier could lead to better results for separation of alkaline phosphatase from the hen's egg yolk with 76.2% recovery and 1361.7-fold purification. The molecular weight of the purified alkaline phosphatase was estimated to be 68kDa by SDS-PAGE. The results of this study showed that the novel magnetic nanocarriers were capable of purifying alkaline phosphatase in a practically time and cost effective way.

Alkaline phosphatase activity of a phosphate solubilizing Alcaligenes faecalis, isolated from Mangrove soil

Microorganisms are capable of converting insoluble phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-26, was isolated from mangrove of the Mahanadi delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Alcaligenes faecalis. The maximum phosphate solubilizing activity of the strain was found to be 48μg/ml with decrease in pH of the growth medium from 7.0 to 3.2. During phosphate solubilization, various organic acids, such as oxalic acid (289mg/L), citric acid (0.2mg/L), malic acid (0.3mg/L), succinic acid (0.5mg/L) and acetic acid (0.4mg/L) produced in the broth culture were detected through HPLC analysis. Crude alkaline phosphatase activity of the strain was determined by p-nitrophenyl phosphate assay and optimized with different growth parameters to obtain maximum enzyme production. Under optimized sets of conditions, maximum alkaline phosphatase activity of 93.7U/ml was observed. Partially purified alkaline phosphatase exhibited three protein bands of sizes approximately 45kDa, 25kDa and 17kDa. Partially purified alkaline phosphatase during characterization showed maximum activity at pH 9.0 (96.53U/ml), temperature of 45°C (97.99U/ml) and substrate concentration of 1.75mg/ml (96.51U/ml). The effect of the bacterium on growth of Arabidopsis thaliana plant showed that inoculation of bacterial culture exhibited better growth in comparison to the control. Hence the phosphate solubilizing and alkaline phosphatase production activity of the bacterium may have probable use for future biotechnological application.

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

An alkaline phosphatase was purified from the gut of sea cucumber Stichopus japonicus by n-butyl alcohol extract, ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl cellulose, gel filtration chromatography with Sephacryl S-200 and preparative electrophoresis with polyacrylamide gel electrophoresis. The native enzyme was estimated to be 166 ± 9 kDa and produced a single predominant band corresponding to active enzyme on nondenaturing electrophoresis, but showed 2 bands of 97 and 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme is composed of two dissimilar subunits. The enzyme displayed maximum activity at pH 11 and 40 °C, showing narrow pH stability (pH 10–12) and thermal instability at temperature higher than 30 °C. The activity of the purified alkaline phosphatase was enhanced by Mg2+, whereas inhibited by Zn2+, Ca2+ and EDTA at 1 and 10 mM, suggesting its activity is in a magnesium ion-dependent manner. The product-analog WO42− and product HPO42− showed strong inhibitory effects on the enzyme activity. Using p-nitrophenyl phosphate as substrate, the Vmax and Km values were 24.45 μmol/L min and 5.76 mM, respectively.

Hydrolysis of Dinitrobenzamide Phosphate Prodrugs: The Role of Alkaline Phosphatase

Phosphate prodrugs which undergo hydrolysis in vivo have been used to improve the solubility and pharmacokinetic properties of a number of drugs. Dinitrobenzamide mustards (DNBM) are examples of such drugs. We investigated the ability of purified alkaline phosphatase isoforms to dephosphorylate three DNBM phosphate prodrugs. In addition, the relative rate of dephosphorylation of these phosphate prodrugs in a number of tissues was determined. These phosphate prodrugs are indeed substrates for alkaline phosphatase, with time dependent formation of the hydrolysis product. Intestinal alkaline phosphatase (IAP) and placental alkaline phosphatase (PLAP) had the highest activity for these substrates and compound P2 was the most rapidly metabolised. Similarly, compound P2 had the shortest half life in mouse serum (t1/2 = 1.15 h) compared with P1 (t1/2 = 13.34 h) and P3 (t1/2 = 4.4 h). However, serum has very low dephosphorylase activity for these substrates compared with intestine and liver homogenates. In addition, there is little or no difference in the relative rate of dephosphorylation of each of the three compounds in mouse tissues in contrast to the pattern observed with purified alkaline phosphatase and mouse serum. Hence additional phosphatase enzymes may be involved in the metabolism of phosphate prodrugs in vivo.

An essential tryptophan residue in alkaline phosphatase from pearl oyster (Pinctada fucata)

Alkaline phosphatases are ubiquitous enzymes found in most species including the pearl oyster, Pinctada fucata, where it is presumably involved in nacreous biomineralization processes. In the present study, we have purified alkaline phosphatases from the pearl oyster and modified the tryptophan residues using N-bromosuccinimide (NBS). We show that the resulting inactivation of purified alkaline phosphatase by NBS is dependent on modification of only one of five tryptophan residues in the enzyme. Substrate protection experiments showed that the tryptophan residue was not located at the substrate-binding site but was involved in the catalytic activity.

Purification and biochemical characterization of a mycelial alkaline phosphatase without DNAase activity produced byAspergillus caespitosus

Biochemical properties of a termostable alkaline phosphatase obtained from the mycelium extract of A. caespitosus were described. The enzyme was purified 42-fold with 32% recovery by DEAE-cellulose and concanavalin A-Sepharose chromatography. The molar mass estimated by Sephacryl S-200 or by 7% SDS-PAGE was 138 kDa and 71 kDa, respectively, indicating a homodimer. Temperature and pH optima were 80 degrees C and pH 9.0. This enzyme was highly glycosylated (approximately 74% saccharide content). The activity was enhanced by Mg2+ (19-139%), NH4+ (64%), Na+ (51%) and Mn2+ (38%). 4-Nitrophenyl phosphate (4-NPP) was preferentially hydrolyzed, but glucose 1-phosphate (93%), UTP (67%) and O-phosphoamino acids also acted as substrates. V(lim) and K(m) were 3.78 nkat per mg protein and 270 micromol/L in the absence of Mg2+ and 7.35 nkat per mg protein and 410 micromol/L in the presence of Mg2+, using 4-NPP as substrate. The purified alkaline phosphatase removed the 5'-phosphate group of a linearized plasmid without showing DNAase activity, indicating its potential for recombinant DNA technology.

Isolation of alkaline phosphatase enzyme from cactus plant Aloe Vera and study its properties

The research was concerned with isolation of alkaline phosphatase (ALP) from the aqueous extract of Aloe Vera using gel filtration technique. It was shown that, using gel filtration chromatography on Sephadex G-100, the solution of the proteinous precipitate produced by ammonium sulphate saturation, contained two proteinous peaks. The two peaks possessed a variable activity of alkaline phosphatase, where maximum specific activity was obtained in the first peak with higher molecular weight which showed (28) folds of purification. Furthermore, the comparative molecular weight of the partially purified alkaline phosphatase (first proteinous peak) using gel filtration was found to be (147353) Dalton. The research was also concerned with finding the optimum conditions of alkaline phosphatase. Maximum activity was obtained using sodium carbonate- bicarbonate buffer (100 mM) at pH ( 9.7 ), (50ºC) and (30 mM) of di-sodium phenyl phosphate as a substrate. Using linweaver-Burk plot, it was found that the Vmax and Km have the values of (185.18 enzyme unit/dl of proteinous solution) and (3.9mM) respectively. The results also predicted that the activity of alkaline phosphatase decreased gradually and reached 76% and 50% from the original value when stored at 4ºC and at room temperature (33-40ºC) respectively for 30 days.

Echinococcus multilocularis: immunity response to purified alkaline phosphatase in BALB/c mice

The study of purified alkaline phosphatase and crude extract antigen immunogenicity from Echinococcus multilocularis was carried out on BALB/c mice. The animals were immunized, then infected with E. multilocularis metacestode. The immune response against purified alkaline phosphatase was studied. Flow cytometry analysis of the CD4+ and CD8+ lymphocyte populations showed a predominance of CD4+ populations in infected immunized mice. The specific humoral response to purified alkaline phosphatase was analyzed by enzyme-linked immunosorbent assay method. We noted a stimulation of an immunoglobulin IgG response. The isotypic profile showed a prevalence of IgG1 and IgG3 in immunized infected mice compared to IgG2a and IgG2b. In addition, analysis of the profiles of the in vitro secreted cytokines, after stimulation of the splenocytes from immunized mice, was performed. The cytokine profile was a mix of Th1/Th2 types in the infected and uninfected immunized mice. The results of this study suggest a humoral mixed Th1/Th2 response, with a high predominance of Th2 response. A similar study was conducted in mice immunized with crude total antigen. The comparison of the immune response showed an important immune response in mice immunized with purified alkaline phosphatase compared to mice immunized with the crude total antigen.

Cyclic GMP-dependent Protein Kinase Regulates CCAAT Enhancer-binding Protein β Functions through Inhibition of Glycogen Synthase Kinase-3

The CCAAT enhancer-binding protein (C/EBPbeta) plays an important role in the regulation of gene expression during cell proliferation, differentiation, and apoptosis. We previously showed that C/EBPbeta participates in cGMP-regulated transcription of c-fos in osteoblasts (Chen, Y., Zhuang, S., Cassenaer, S., Casteel, D. E., Gudi, T., Boss, G. R., and Pilz, R. B. (2003) Mol. Cell. Biol. 23, 4066-4082). In the present work, we show that cGMP/cGMP-dependent protein kinase (PKG) induced dephosphorylation and activation of C/EBPbeta by inhibiting glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta on Ser9 negatively regulates the enzyme activity, and we found that PKG phosphorylated this site both in vitro and in vivo; the in vivo phosphorylation occurred rapidly and preceded C/EBPbeta dephosphorylation. Previous studies with GSK-3 inhibitors suggest that GSK-3beta is a C/EBPbeta kinase in resting cells. We determined that GSK-3beta phosphorylated C/EBPbeta in vitro on Thr189, Ser185, Ser181, and Ser177; C/EBPbeta was phosphorylated on these same sites in intact, unstimulated osteoblasts, and phosphorylation was decreased in cGMP-treated cells. Mutation of the GSK-3 phosphorylation sites in C/EBPbeta prevented C/EBPbeta phosphorylation in resting cells, enhanced C/EBPbeta DNA binding, and led to increased target gene transactivation, mimicking the stimulatory effects of cGMP on C/EBPbeta. cGMP regulation of C/EBPbeta was disrupted by a mutant GSK-3beta(Ala9) resistant to cGMP/PKG phosphorylation and inhibition. We conclude that cGMP increases the DNA binding potential of C/EBPbeta by preventing the negative effects of GSK-3 phosphorylation.

Identification of the gene for the monomeric alkaline phosphatase of Vibrio cholerae serogroup O1 strain

Alkaline phosphatase (APase) of Vibrio cholerae is the first monomeric alkaline phosphatase reported [Roy, N.K., Ghosh, R.K., Das, J., 1982a. Monomeric alkaline phosphatase of V. cholerae. J. Bacteriol. 150, 1033–1039.]. The gene ( phoA VC ) encoding this enzyme is not identified in the published genome sequence of the V. cholerae serogroup O1 El Tor strain N16961 [Heidelberg et al., 2000, DNA sequence of both the chromosome of cholera pathogen V. cholerae. Nature 406, 477–484.]. However two genes ( phoB VC and phoR VC ) regulating the synthesis of alkaline phosphatase in this organism, equivalent to phoB and phoR of Escherichia coli, are located in tandem on chromosome I of V. cholerae. We have identified the phoA VC gene on the N16961 genome sequence by amino acid sequence analysis of the purified alkaline phosphatase of V. cholerae classical strain 569B followed by BLAST search. The gene was found to be located on the hypothetical protein locus VCA0033 of chromosome II. The identity of the gene was confirmed by expressing the cloned VCA0033 locus in phoA mutant E. coli E15 and JC9223 cells and isolating V. cholerae monomeric alkaline phosphatase. Insertional inactivation of the gene also resulted in complete loss of the phenotype. Unlike in E. coli where phoB, phoR and phoA are closely linked, phoA VC is not linked to phoB VC and phoR VC .

Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

Glycosylphosphatidylinositol-anchored proteins may be concentrated in membrane microdomains (lipid rafts) that are also enriched in cholesterol and sphingolipids. The glycosyl anchor of these proteins is a specific, high affinity receptor for the channel-forming protein aerolysin. We wished to determine if the presence of rafts promotes the activity of aerolysin. Treatment of T lymphocytes with methyl-beta-cyclodextrin, which destroys lipid rafts by sequestering cholesterol, had no measurable effect on the sensitivity of the cells to aerolysin; nor did similar treatment of erythrocytes decrease the rate at which they were lysed by the toxin. We also studied the rate of aerolysin-induced channel formation in liposomes containing glycosylphosphatidylinositol-anchored placental alkaline phosphatase, which we show is a receptor for aerolysin. In liposomes containing sphingolipids as well as glycerophospholipids and cholesterol, most of the enzyme was Triton X-100-insoluble, indicating that it was localized in rafts, whereas in liposomes prepared without sphingolipids, all of the enzyme was soluble. Aerolysin was no more active against liposomes containing rafts than against those that did not. We conclude that lipid rafts do not promote channel formation by aerolysin.

Characterization of a conidial alkaline phosphatase from the thermophilic fungusHumicola grisea var.thermoidea

An extracellular alkaline phosphatase (EC 3.1.3.1) was purified from conidia of Humicola grisea var. thermoidea. The molecular mass of the purified native enzyme was estimated as 126 kDa by gel filtration. SDS-PAGE revealed a single polypeptide band of 63 kDa, suggesting that the native enzyme was a dimer of identical subunits. The pI of the enzyme was about 3.0. Optima of pH and temperature were 9.0 and 70 °C, respectively. In Tris-HCl buffer, pH 9.0, the enzyme was activated by MgCl2 and strongly inhibited by ZnCl2, CoCl2, Al2Cl3, HgCl2, EDTA and β-mercaptoethanol. In contrast, in glycine buffer and at the same pH the enzyme was activated by ZnCl2 and CoCl2, as well as by MgCl2. The enzyme was fully stable when incubated up to one hour at 65 °C without magne-sium labilized the enzyme incubated at 65 °C or 70 °C. p-Nitrophenyl phosphate was the best substrate, but AMP, ADP, ATP and β-glycerophosphate also served as substrates. The remarkable thermostability exhibited by the purified alkaline phosphatase of H. grisea may be of interest for practical applications.

Antibodies against Echinococcus multilocularis alkaline phosphatase as markers for the specific diagnosis and the serological monitoring of alveolar echinococcosis.

The immunological properties of the purified alkaline phosphatase (pAP) of Echinococcus multilocularis metacestodes have been investigated using alveolar echinococcosis (AE) patient sera in ELISA tests. A comparative study was done with EmC-Ag (crude antigen) and pAP-Ag (purified antigen). When the parasite purified enzyme pAP was used as antigen, the specificity of the ELISA was markedly increased since it reached 100% without any decrease of its sensitivity (100%). The serologic follow-up of AE patients was conducted during several months with these two antigens in three categories of patients: cured, stabilized and aggravated. There was a good correlation between clinical and serologic data when the pAP was used as antigen in ELISA tests. The anti-pAP antibodies titres did change more rapidly than anti-EmC antibodies titres when a recurrence occurred. Modifications of the anti-pAP antibodies levels were also observed during the patient's therapy: mebendazole, albendazole and Isoprinosine. These results suggest that pAP-Ag should be used for the diagnosis and the follow-up of AE patients.

Utilization of the Bone/Liver Alkaline Phosphatase Activity Ratio in Blood Plasma as an Indicator of Ascorbate Deficiency in Salmonid Fish

The goal of this study was to test the hypothesis that the ratio of liver to bone alkaline phosphatase in blood plasma reflects the ascorbate status in scurvy-prone teleost fish (rainbow trout [Oncorhynchus mykiss]). The studies focused on finding a method for distinguishing bone alkaline phosphatase present in blood plasma from other alkaline phosphatase isoforms. We tested temperature optima and thermostability of liver, kidney, gill cartilage, and intestinal alkaline phosphatases. We did not observe differences among liver, bone, and kidney enzymes with respect to temperature optima and thermostability. We partially purified alkaline phosphatase from juvenile rainbow trout vertebrae and liver using n-butanol solubilization and ammonium sulfate fractionation. We found a difference between bone alkaline phosphatase, which precipitated in 0%-20% ammonium sulfate saturation, and liver enzyme, which required 40%-50% ammonium sulfate saturation to precipitation. We conducted a series of urea inactivation studies on partially purified enzymes from liver and vertebrae. Urea differentially inhibited the enzymes with t 1/2 = 1.1 and 0.4 min, for bone and liver, respectively. Subsequently, we subjected blood plasma alkaline phosphatase to urea inhibition, and using regression analysis we calculated the ratio of liver to bone alkaline phosphatase. We found that thus obtained ratios of bone enzyme in blood plasma correlated with liver ascorbate concentration. Bone alkaline phosphatase declined in ascorbate deficiency 10-fold, whereas low ascorbate status resulted in a 3.5-fold decrease. In order to draw a general conclusion on the linearity of the response of blood plasma/bone alkaline phosphatase as an indicator of ascorbate deficiency in fish, further studies must include analysis of individual fish followed in the process of developing avitaminosis.

Phosphorylation of rubisco in Cicer rietinum: Non-phosphoprotein nature of rubisco in Nicotiana tabacun

Leaf discs from Cicer and Nicotiana were incubated in a solution of [ 32P]-orthophosphate (24 hr). RUBISCO was purified from these and electrophoresed on native PAGE. A single protein stained band was observed on gels, thus proving the electrophoretic homogeneity of RUBISCO both in Cicer and Nicotiana. Autoradiography of gels revealed a radioactive band in the zone of protein stained band of Cicer RUBISCO, whereas no radioactivity was detected in the region of the protein stained band of Nicotiana RUBISCO. This suggested that Cicer RUBISCO unlike that of Nicotiana is a phosphoprotein. Further analysis of [ 32P]-labelled purified Cicer RUBISCO on SDS—PAGE showed two protein stained bands corresponding to the large and small subunits of the enzyme. Autoradiography of the denaturing gel showed a radioactive band exclusively in the zone of the small subunit. Chemical characterization of [ 32P]-labelled Cicer RUBISCO by acid hydrolysis showed radioactivity in phosphoserine and phosphotyrosine. Treatment of [ 32P]-labelled Cicer RUBISCO with purified alkaline phosphatase brought about a significant dephosphorylation (∼70%) of the enzyme. The in vitro dephosphorylation of Cicer RUBISCO resulted in the dissociation of the small subunits from the catalytic octameric large subunits. Concomitantly, there was a marked decrease in the catalytic activity of the dephosphorylated Cicer RUBISCO in comparison with the phosphorylated form of RUBISCO. Phosphorylation of the small subunit seems crucial for the assembly of large and small subunits and thus contributes to the regulation of Cicer RUBISCO. By contrast, Nicotiana RUBISCO showed no loss of catalytic activity following its treatment with alkaline phosphatase.

Bound and purified alkaline phosphatase from rat duodenal and jejunal brush-border membranes: kinetics and magnesium stimulation.

Brush-border membranes from rat duodenal and jejunal mucosa were prepared by differential Ca(2+)-precipitation. Kinetical properties and Mg(2+)-stimulation of alkaline phosphatase were studied for the enzyme either bound to these membranes or purified from these membranes by liquid chromatography. With p-nitrophenylphosphate as substrate, the alkaline phosphatase apparent Km was lower in jejunum (90 microM) as compared with duodenum (160 microM), and lower for the purified enzyme (jejunum: 55 microM; duodenum: 97 microM) as compared to the bound one. In the presence of 5 mM MgCl2, the substrate affinity was in all cases decreased. For the bound enzyme Vmax was 10 times greater in duodenum compared to jejunum. 5 mM MgCl2 tripled the Vmax of the duodenal bound enzyme and increased it by 50% for the jejunal one, but a seven-fold increase was recorded for the purified enzyme at both levels of intestine. The apparent affinity for Mg2+ was similar for the bound and the free enzyme, for duodenum and for jejunum (Mg0.5: +/- 40 microM).

High-Molecular-Weight Alkaline Phosphatase in Serum Has Properties Similar to the Enzyme in Plasma Membranes of the Liver

Partially purified high-molecular-weight alkaline phosphatase from serum was compared with two other forms of the enzyme from the human liver, enzyme in native plasma membranes and purified alkaline phosphatase as a hydrophilic dimer. In a high-molecular-weight form from serum and plasma membranes, and when treated with 1% (v/v) Triton X-100, alkaline phosphatase showed a major band on gradient gel electrophoresis with a mobility equivalent to 400 kD. Nondetergent-treated material from both sources did not enter the gel and was in the voided volume of a gel permeation column. Stimulation of catalytic activity by four different phospholipids and by albumin yielded similar results for high-molecular-weight alkaline phosphatase and for the enzyme in plasma membranes, but these were different from the hydrophilic form. Inhibitors of alkaline phosphatase had similar effects on all forms. Of the three forms of the enzyme, only the hydrophilic dimer did not become incorporated into liposomes or adsorb to octyl-Sepharose after solubilization with Triton X-100 and removal of the detergent. Km (substrate concentration to give half maximal velocity) values with p-nitrophenylphosphate and heat and sodium dodecyl sulfate stabilities were similar for all forms. In the high-molecular-weight form from serum and in plasma membranes, alkaline phosphatase and 5'-nucleotidase showed similar rates of release by phosphatidylinositol phospholipase C. Three preparations of phospholipase D failed to release alkaline phosphatase from either the high-molecular-weight form or from plasma membranes. Based on these similarities, it is probable that the complex of high-molecular-weight alkaline phosphatase in serum most often originates from fragments of hepatic plasma membranes.

Characterization of different molecular forms of alkaline phosphatase in the hepatopancreas from the shrimp Penaeus monodon (Crustacea: Decapoda)

1. 1. Alkaline phosphatase from the hepatopancreas of Penaeus monodon is resolved into three forms, AP-A, AP-B1 and AP-B2, by non-denaturing polyacrylamide gel electrophoresis. 2. 2. After purification, the spec. act. of the purified enzyme is 34,778,4660 and 4990 units/mg of protein for AP-A, AP-B1 and AP-B2, respectively. 3. 3. Upon polyacrylamide gel electrophoresis under denaturing conditions, the purified alkaline phosphatase from shrimp was found to consist of monomers of M r 31,000, 31,000 and 43,000 for AP-A, AP-B1 and AP-B2, respectively, and by gel filtration on Sephacryl S-2-00, the M r of active alkaline phosphatase was estimated to be 200,000 for AP-B1 or AP-B2, and 300,000 for AP-A. 4. 4. All three forms of the enzyme in hepatopancreas are purified as polymers and with the attachment site of phosphatidylinositol. 5. 5. They can be differentiated by inhibitions with EDTA, by inactivation with heating, by K m or by pI. 6. 6. All the three forms of alkaline phosphatases were found to be resistant to the action of bacterial sialidase.

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water ( k 3) or a nucleophile ( k 4) is rate-determining. According to this model, in the presence of diethanolamine, k 3 and k 4 were determined to be 4.44 s −1 M −1 and 1000 s −1 M −1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.