Affinity-chromatography purification of alkaline phosphatase from calf intestine.

A crude preparation of alkaline phosphatase (EC 3.1.3.1) from calf intestinal mucosa was purified by affinity chromatography on Sepharose-bound derivatives of arsanilic acid, which was found to be a competitive inhibitor of the enzyme. Three biospecific adsorbents were prepared for the chromatography, and the best results were obtained with a tyraminyl-Sepharose derivative coupled with the diazonium salt derived from 4-(p-aminophenylazo)phenylarsonic acid. Alkaline phosphatase was the only enzyme retained by the affinity column in the absence of Pi. The enzyme eluted by phosphate buffer had a specific activity of about 1200 units per mg of protein at pH 10.0, with 5.5mM-p-nitrophenyl phosphate as the substrate.     

Studies on alkaline phosphatase. Phosphorylation of calf intestinal alkaline phosphatase by 32P-labelled pyrophosphate

1. A purified preparation of alkaline phosphatase from calf-intestinal mucosa was phosphorylated by (32)P-labelled PP(i) at a serine residue on the enzyme. Under the conditions employed, up to 0.15mum-labelled sites were obtained from 1mum-[(32)P]PP(i). 2. The phosphorylated enzyme was labile, the rate of dephosphorylation being similar to the overall rate of substrate hydrolysis. 3. A stopped-flow technique was used to determine the number of phosphomonoesterase active sites, which agreed with the number of (32)P-labelled sites. 4. It is concluded that calf-intestinal alkaline phosphatase is both a phosphomonoesterase and a pyrophosphatase.     

Alkaline phosphatase of the calf intestine hydrolyzes phospholipids

Pure alkaline phosphatase of the calf intestine is able to hydrolyze phosphatidylinositol 4,5-diphosphate (TPI) to phosphatidylinositol and Pi and to dephosphorylate phosphatidic acid. This phosphomonoesterase activity shows a considerably high specific activity when an incubation medium at neutral pH containing 3 mM deoxycholate is used. The activity is inhibited by low concentrations of Ca2+. The enzyme has no detectable phosphodiesterase activity under the conditions tested.     

Phosphatidylserine modulates calf intestine alkaline phosphatase activity towards low and high molecular weight substrate

Pretreatment of calf intestine alkaline phosphatase with phosphatidylserine resulted in an inhibition of the phosphatase activity towards low - (p-nitrophenylphosphate) and high (phosphohistone) molecular weight substrate. Phosphatidylcholine, irrespectively of the substrate used did not cause enzyme modulation. 12-O-tetradecanoylphorbol-13-acetate, 1,2-diolein as well certain retinoids, known to effect phosphatidylserine-sensitive enzyme systems (Castagna, M. et al. 1982, J. Biol. Chem. 257, 7847-7851; Gmeiner, B. 1986, Biochim. Biophys. Acta 856, 392-394) had no influence on the modulated phosphatase. The lipid interacting drug trifluoperazine inhibited the enzyme activity towards phosphohistone, but not towards p-nitrophenylphosphate as a substrate. The results indicate that acidic phospholipid may play a role in activity modulation of calf intestine membranous alkaline phosphatase activity.     

Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.

Alkaline phosphatase is anchored to the membrane via glycosylphosphatidylinositol (GPI). Mannose residues of the GPI glycan are suggested to be derived from dolichol-P-mannose. In the present study we examined the effect of 2-fluoro-2-deoxy-D-glucose (F-Glc), an inhibitor of dolichol-P-mannose synthesis, on the biosynthesis and processing of alkaline phosphatase in JEG-3 cells. In control cells, a proform precursor (64.5 kDa) with a hydrophobic peptide domain at the COOH terminus was immediately processed into an intermediate form (63 kDa) by proteolytic removal of the COOH-terminal extension and replacement with the GPI anchor, and then to a mature form (66 kDa) by terminal glycosylation of its N-linked oligosaccharides. In contrast, when cells were treated with F-Glc (1 mM), the protein was synthesized as a proform of 61 kDa. The reduction in its molecular mass was mostly due to the inhibition in maturation of N-linked oligosaccharides by F-Glc. The 61-kDa proform identified by antibodies to the COOH-terminal peptide was detectable even at 3 h after the synthesis, and was gradually processed to doublet forms of 58-59 kDa which were finally secreted into the medium. None of these forms were labeled with [3H]ethanolamine and [3H]stearic acid, components of the GPI anchor, and expressed on the cell surface as a membrane-bound form. Taken together, these results suggest that the inhibition of the GPI synthesis causes a prolonged accumulation of the proform, which is then gradually processed into secretory forms by proteolytic removal of the COOH-terminal hydrophobic peptide.     

Characterization of KB cell alkaline phosphatase. Evidence of similarity to placental alkaline phosphatase.

The alkaline phosphatase from KB cells was purified, characterized, and compared to placental alkaline phosphatase, which it resembles immunologically. Two nonidentical nonomeric subunits of the KB phosphatase were found. The two subunits, which have apparent molecular weights of 64,000 and 72,000, can be separated on polyacrylamide gels containing sodium dodecyl sulfate. The Mr = 64,000 KB subunit appears to be identical in protein structure to the monomer of placental alkaline phosphatase. The Mr = 72,000 KB subunit, while differing in the NH2-terminal amino acid, appears also to be very similar to the placental alkaline phosphatase monomer. Both KB phosphatase subunits bind (32P)phosphate, and bind to Sepharose-bound anti-placental alkaline phosphatase. Native KB phosphatase is identical to the placental isozyme in isoelectric point, pH optimum, and inhibition by amino acids, and has a very similar peptide map. The data presented support the hypothesis that the Mr = 64,000 KB phosphatase subunit may the the same gene product as the monomer of placental alkaline phosphatase. This paper strengthens the evidence that the gene for this fetal protein, normally repressed in all cells but placenta, is derepressed in the KB cell line. In addition, this paper presents the first structural evidence that there are two different subunit proteins comprising the placental-like alkaline phosphatase from a human tumor cell line.     

Study of refolding of calf intestinal alkaline phosphatase

Calf intestinal alkaline phosphatase (CIP) was denatured in 3.0 M guanidine hydrochloride for 2 h at 25 degrees C, before being diluted 20-fold with 0.1 M, pH 8.0, Tris-HCl buffer solution containing various effector molecules such as Mg2+, Zn2+, and nucleotide phosphate. The reactivation courses of the enzyme were investigated by the level of activity recovery, the recovery rate constant, and the relative standard deviation of the data. In the presence of effectors, the courses under reducing and nonreducing conditions of disulfide bonds of protein were compared. It was concluded that for CIP, Mg2+ is a more efficient inducer of reconstitution of the active site and appears to play a specific role. In addition, the present study discusses the differences in the refolding effectors between bacterial and mammalian enzymes.     

Evidence for the retinoid control of urothelial alkaline phosphatase

The amount of alkaline phosphatase activity per μg of DNA in the urothelium (transitional epithelium) of the rat urinary bladder, organ-cultured in chemically-defined serum-free medium, decreased greater than 70% during a 13 day culture period. This decrease in enzyme activity corresponded inversely with the increase in cell number in the urothelium indicating that enzyme synthesis did not accompany growth. Alkaline phosphatase activity was increased back to values approaching normal enzyme levels during a 3 day culture period by the addition of 10 μM retinoic acid. Retinol also increased enzyme activity but it was only half as effective as retinoic acid. A significant increase in enzyme activity was initiated by 1 μM retinoic acid, however the most effective concentration was at 10 μM.     

Modification of arginine residues in calf intestinal alkaline phosphatase

Calf intestinal alkaline phosphatase is inactivated by 2,3-butanedione and phenylglyoxal. The reaction with either reagent results in a biphasic loss of enzymatic activity. Inactivation by 2,3-butanedione in borate buffer can be reversed after gel-filtration in Tris buffer but no enzyme reactivation is observed after phenylglyoxal treatment. Phosphate, ATP and NADH protect the enzyme from both compounds while no protection is displayed by L-phenylalanine. The selective chemical modification indicates that two differently reacting types of arginines are present in the active site domains of the dimeric enzyme.     

Effect of extraneous zinc on calf intestinal alkaline phosphatase

The effect of extraneous zinc on calf intestinal alkaline phosphatase was studied for quick reversible binding and slow irreversible binding of zinc ions at various concentrations. Under the conditions of slow binding of zinc to CIP increasing Zn²⁺ (less than 1.0 mM, n_M/n_E 1.0 × 10⁶) inhibited enzymatic activity, and further increasing Zn²⁺ resulted in an increase of activity. For quick reversible binding of Zn²⁺, the effect on CIP activity changed at lower concentrations of substrate, indicating a complex cooperativity between Zn²⁺ and pNPP. Both protein intrinsic emission fluorescence and ANS-bound protein fluorescence, as well as circular dichroism spectra have shown that the binding of zinc ions changed the enzyme conformation, which was the reason for the changes in enzyme activity induced by extraneous zinc.     

Stabilization of bovine intestine alkaline phosphatase by sugars

Bovine intestine alkaline phosphatase (BIALP) is widely used as a signaling enzyme in sensitive assays such as enzyme immunoassay. In this study, we evaluated the effects of sugars on the kinetic stability of BIALP in the hydrolysis of p-nitrophenylphosphate (pNPP). The temperatures reducing initial activity by 50% in a 30-min incubation, T(50), of BIALP with 1.0 M disaccharide (sucrose and trehalose) or 2.0 M monosaccharide (glucose and fructose) were 55.0-55.5 °C, 4.7-5.2 °C higher than without sugar (50.3±0.1 °C). The T(50) of BIALP increased to 58.4±0.3 °C when the trehalose concentration was from 1.0 to 1.5 M, but did not change when the glucose concentration was from 2.0 to 3.0 M. Thermodynamic analysis revealed that the stabilization of BIALP by sugars was driven by the increase in the enthalpy change of activation for thermal inactivation of BIALP. No sugars affected the k(cat) of BIALP in the hydrolysis of pNPP. These results suggest that not only trehalose, which is considered the most effective stabilizer of enzymes, but also sucrose, glucose, and fructose can be used as stabilizers of BIALP.     

Evidence for the importance of arginine residues in pig kidney alkaline phosphatase.

The arginine-specific reagents 2,3-butanedione and phenylglyoxal inactivate pig kidney alkaline phosphatase. As inactivation proceeds there is a progressive fall in Vmax. of the enzyme, but no demonstrable change in the Km value for substrate. Pi, a competitive inhibitor, and AMP, a substrate of the enzyme, protect alkaline phosphatase against the arginine-specific reagents. These effects are explicable by the assumption that the enzyme contains an essential arginine residue at the active site. Protection is also afforded by the uncompetitive inhibitor NADH through a partially competive action against the reagents. Enzyme that has been exposed to the reagents has a decreased sensitivity to NADH inhibition. It is suggested that an arginine residue is important for NADH binding also, although this residue is distinct from that at the catalytic site. The protection given by NADH against loss of activity is indicative of the close proximity of the active and NADH sites.     

An improved purification procedure of alkaline phosphatase from calf intestine by applying partition in aqueous two-phase systems and dye-ligand chromatography.

Aqueous two-phase partitioning has been elaborated in order to improve the purification of alkaline phosphatase from calf intestine in larger scale. The laborious precipitation and centrifugation steps for the removal of the enzyme from the cell debris and from the bulk protein were replaced by this technique yielding a high recovery (88%) and a significant lower time requirement. For the preparation of 100.000 units (46 mg) of a homogeneous enzyme 2.0 kg of a system containing 200 g PEG 4000 and only 10 g dextran M 70 is necessary. Affinity partitioning in aqueous two-phase systems was used to screen 41 dyes for selecting a suitable ligand for the dye-ligand chromatography of the enzyme. In the case of alkaline phosphatase the results obtained by affinity partitioning coincide with the experimental requirements for the affinity chromatography of the enzyme. Procion Navy HE-R (Blue 171) exhibits a high affinity, selectivity and binding capacity for the enzyme compared with other dyes investigated. The purification procedure provided the same degree in purity (2200 U/mg) and yield (59%) if mucosa or chyme was applied as starting material. In the range of practical use the purified enzyme contains no detectable activities of DNAses (endonucleases) and DNA-nicking activities. The contamination with phosphodiesterase I (EC. 3.1.4.1.) is less than 0.01%.     

Evidence for two structural genes for alkaline phosphatase in Bacillus subtilis.

Two secreted alkaline phosphatase proteins were purified from cultures of Bacillus subtilis JH646MS. The two proteins showed slight differences in subunit molecular weight, substrate specificity, and charge characteristics. A total of 62% of the first 22 amino-terminal amino acids were identical. Both sequences showed conservation of structural features identified in Escherichia coli and human alkaline phosphatases. One alkaline phosphatase was a monomer and the other was a dimer. Southern analysis of genomic DNA with degenerative oligomers based on the amino acid sequences suggest that there are two structural genes for alkaline phosphatase in the genome of B. subtilis.     

Physico-chemical properties of alkaline phosphatase from the seal small intestine

Alkaline phosphatase of the Greenland seal was purified to homogeneity, using immobilized concanavalin A. The specific activity of the enzyme is 1200-1500 mu/mg protein. The molecular mass of alkaline phosphatase as determined by electrophoresis performed under non-denaturating conditions is 260 kD, whereas that determined in the presence of beta-mercaptoethanol and SDS is 70 kD, which points to the tetrameric type of the seal alkaline phosphatase molecule. Using the atomic adsorption method, it was demonstrated that the phosphatase molecule contains four zinc atoms. Some physico-chemical parameters of seal alkaline phosphatase (pH-dependence, effects of temperature and cations on the enzyme activity, pI, thermal stability) were determined.     

Phosphate binding to Escherichia coli alkaline phosphatase. Evidence for site homogeneity.

The reversible, noncovalent binding of inorganic phosphate to Escherichia coli alkaline phosphatase at pH 8 has been examined by equilibrium dialysis at two temperatures and two ionic strengths. Binding occurs with a stoichiometry of two phosphate ions per dimeric enzyme molecule and a single dissociation constant that is not very sensitive to temperature or ionic strength. These results contradict published evidence for anti-cooperative binding of inorganic phosphate to alkaline phosphatase. Reasons are presented for believing that the apparent anti-cooperativity reported by other workers is artifactual.     

Ultrastructural localization of alkaline phosphatase activity in the intestine of developing Rana catesbeiana.

Alkaline phosphatase activity has been localized at the light and electron microscopic levels in the intestine of developing frog, Rana catesbeiana. The intensity of the histochemical reactivity decreases along the intestinal tract. The intracellular localization of the enzymatic activity shows continuous series of organelles loaded with the reaction product from the Golgi zones to the brush border. These results are in agreement with the biochemical observations made on the same material.     

Evidence for synthesis of alkaline phosphatase on membrane-bound polysomes in Escherichia coli.

Polysomes containing nascent chains of alkaline phosphatase have been isolated from a membrane-bound polysome preparation. Indirect immunoprecipitation using conformation-specific antibodies has been employed. This technique provides a good enrichment of these polyribosomes since routinely no more than than 10--15% of non-specific immunoprecipitation was observed. The yield of the procedure is generally 40% but can be increased if higher non-specific immunoprecipitation is tolerated. Antibodies, previously described, directed against uncoiled or folded monomers of alkaline phosphatase can be used as primary antibody to recognize the nascent chains contained in membrane-bound polysomes which suggests that these chains are partially folded.