Isozymes of bovine intestinal alkaline phosphatase

Alkaline phosphatases from calf and bovine small intestines have been isolated in homogeneous form from both mucosa and luminal contents. The detergent-solubilized calf enzyme resolves into two peaks of activity, C-1 and C-2, on chromatofocusing. Only one of these activity peaks is present in the enzyme from the adult animal. Amino acid compositions, N-terminal sequences, and tryptic peptide maps show that C-1 and C-2 are isozymes of differing primary structure and that the adult form of the enzyme is identical to C-2. The developmentally controlled expression of the two isozymes reported here suggests a molecular basis for the previous indications that functional changes in intestinal alkaline phosphatase occur with tissue maturation. The sugar composition of the carbohydrate chains of these isozymes has been determined and enzymatic deglycosylation with endo-beta-N-acetylglucosaminidase-F indicates two N-linked and one or more O-linked glycoconjugates/monomer.     

Effect of metal ions on the secondary structure and activity of calf intestine phosphatase

Cobalt is an essential microelements in many biological processes involving enzymatic activity. We found that Zn2+ and Mg2+, which are in the active site of native calf intestine alkaline phosphatase (CIP), can be replaced by Co2+ directly in solution. The effect of Co2+ concentration on the substitution reaction was examined at ratios of [Co2+]/[CIP] from 0:1 to 8:1. The quantity of Zn2+ in CIP decreased progressively as the ratio was increased, but the amount of Mg2+ changed in irrregular fashion. A series of active site models of the reaction mechanism of CIP are proposed. Low pH was found to promote the replacement of Mg2+ by Co2+. To understand how the substitution affects the enzyme, we also solved the secondary structure of CIP after reaction with Co2+ in different conditions.     

Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.     

The effect of metal ions on the alkaline phosphatase of Rhizobium leguminosarum

The alkaline phosphatase (EC 3.1.3.1.) from Rhizobium leguminosarum WU235 has been purified. The enzyme is a non-specific phosphomonoesterase, has a molecular weight of 78,500 and a sub-unit molecular weight of 39,400. Magnesium and zinc ions are implicated in the structure of the enzyme; atomic absorption analysis gave 1.9 g-atoms Mg²⁺ and 1.9–5.1 g-atoms Zn²⁺ per mole of enzyme. In addition high concentrations of Mg²⁺ markedly stimulate the enzyme. The phosphatase is inhibited by Li⁺ and Na⁺ and stimulated by K⁺, Rb⁺ and Cs⁺, which suggests that the enzyme is K⁺ activated.     

金属离子和脲对白蜡虫碱性磷酸酶的影响

各种金属离子及脲对白蜡虫Ericerus pela (Chavannes)碱性磷酸酶的活性有不同的影响。从白蜡虫雌成虫中分离纯化得到碱性磷酸酶,加入各种不同浓度的金属离子及脲测定酶的活力。一价金属离子Na⁺、K⁺、Li⁺对酶活力没有影响。碱土金属离子Ca²⁺、Mg²⁺、Ba²⁺对酶有激活作用,激活作用的大小顺序依次为Ca²⁺、Ba²⁺、Mg²⁺。第一过渡金属离子中,Mn²⁺、Co²⁺、Ni²⁺对酶有激活作用,而Zn²⁺、Cu²⁺有抑制作用。重金属离子Cd²⁺、Pb²⁺对酶有抑制作用。Ca²⁺激活作用表现为非竞争性激活效应。Cu²⁺抑制作用表现为非竞争性抑制效应。脲对碱性磷酸酶有变性失活作用,按脲浓度可分为低于3 mol/L和高于3 mol/L两种类型。低浓度的脲对白蜡虫碱性磷酸酶的活性抑制的动力学表现为混合型效应。     

Activation of Pyrococcus furiosus alkaline phosphatase by divalent metal ions

Treatment of a hyperthermophilic enzyme, alkaline phosphatase from Pyrococcus furiosus (PfuAP), with EDTA completely deactivated PfuAP, indicating that the presence of one or more divalent metal ions is essential for its catalytic activity. Subsequent addition of various divalent metal ions to the apoprotein recovered the enzymatic activity and, in particular, the addition of Co(II) resulted in an over 50-fold increase in activity compared with PfuAP before EDTA treatment. Intriguingly, PfuAP with Co(II) exhibited weaker stability toward heat treatment, suggesting that Co²⁺ destabilizes the tertiary structure of PfuAP at high temperature.     

Elucidation of the quaternary structure of reversibly immobilized alkaline phosphatase derivatives.

Escherichia coli alkaline phosphatase has been reversibly immobilized on Sepharose CL-4B through two different methods, both based on a disulfide linkage, under conditions selected to favour the coupling of the enzyme to the solid support through one covalent linkage. The quaternary structure of the reversibly immobilized subunit, produced by dissociation of the matrix-bound dimer, was examined by cross-linking with the bifunctional reagent dimethyl suberimidate. Following release from the solid support, the protein was analysed by sodium dodecyl sulfate gel electrophoresis demonstrating the presence of a sufficient amount of dimeric structures in the immobilized subunit preparation to account for all the enzyme activity observed in this sample. These results suggest that the subunit of alkaline phosphatase may be catalytically inactive. This approach to studying the quaternary structure of immobilized subunit derivatives offers the opportunity to directly determine the homogeneity and structure of matrix-bound 'monomer' preparations and is particularly useful in determining if low levels of catalytic activity observed in some immobilized subunit populations are due to the presence of contaminating oligomeric structures.     

Refolding and reactivation of calf intestinal alkaline phosphatase with excess magnesium ions

It is well known that Mg(2+) is an essential component in many biological processes. This research investigated the courses of both the reactivation and the refolding in the absence and presence of Mg(2+) ions. Calf intestinal alkaline phosphatase (CIP) was extensively denatured in 3 M guanidine hydrochloride (GdnHCl) solution for 2 h. Under suitable renaturation conditions, about 60-70% of the activity was recovered in the absence and presence of different magnesium ion concentrations. The refolding processes followed two-phase courses, whereas the reactivation processes were monophasic after dilution in proper solutions with or without Mg(2+). The magnesium ions affected both the reactivation and the refolding courses of unfolded CIP. A comparison of rate constants for the refolding of unfolded CIP with those for recovery of enzyme activity at different Mg(2+) concentrations showed that they were not synchronized. The activity recovery was speeded up due to the presence of Mg(2+) ions; while the refolding course of unfolded CIP was somewhat inhibited by the excess Mg(2+).     

ATPase and alkaline phosphatase activities of chick and rat small intestinal mucosa.

The alkaline phosphatase and (Ca2+ +Mg2+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of chick and rat small intestine have been investigated. The same pH optimum was found for membrane-bound and solubilized alkaline phosphatase, whereas those of the corresponding ATPases differed. The solubilised ATPases had inhibition and activation characteristics similar to those of alkaline phosphatase but markedly different from those of the membrane-bound ATPase. These results suggest that membrane-bound alkaline phosphatase and ATPase are not the same enzyme.     

Chemical modification and composition of tetrameric isozyme K of alkaline phosphatase from harp seal intestinal mucosa

1. The carbohydrate content of isozyme K of alkaline phosphatase (EC 3.1.3.1) from harp seal intestinal mucosa was examined. The presence of N-acetylglucosamine, N-acetylgalactosamine and considerable amounts of mannose residues was shown. 2. The amino acid content of seal alkaline phosphatase was determined. A high extent of homology (85%) between bovine and seal alkaline phosphatases was demonstrated. 3. By chemical modification lysine, dicarboxylic acids, arginine and tyrosine residues of tetrameric seal alkaline phosphatase are located near or at the active site. By contrast, the modification of either thiol or imidazole groups resulted in no alterations of the enzyme activity. 4. It has been demonstrated that inorganic phosphate is an inhibitor of alkaline phosphatase and entirely prevents the enzyme inactivation with succinic anhydride.     

The active-site and amino-terminal amino acid sequence of bovine intestinal alkaline phosphatase

The active site of bovine intestinal alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) was labeled with [32P]Pi, a radioactive CNBr peptide was isolated and the amino acid sequence was determined. The sequence of the active-site peptide has limited homology (26%) with the active-site sequence of Escherichia coli alkaline phosphatase except for the ten residues immediately flanking the active-site serine (70%). A possible amino acid sequence deduced from the amino acid composition of an active-site tryptic peptide from human placental alkaline phosphatase is very similar to the bovine intestinal active-site sequence. The amino-terminal sequence of bovine intestinal alkaline phosphatase is homologous (69%) with the human placental enzyme but not with the E. coli phosphatase.     

Monoclonal antibody to alkaline phosphatase from the intestinal mucosa of the harp seal, Phoca groenlandica.

1. Hybridoma secreting a monoclonal antibody APP.1 to the harp seal alkaline phosphatase (A1Ph) was obtained by fusing murine myeloma Sp 2/0 cells with the splenocytes of BALB/c mice immunized with purified isozyme K. 2. The antibody has no effect on the enzyme activity and shows a high affinity for harp seal A1Ph (KD = 8.5 x 10(-10) M). The antibody has similar affinities for the AlPh of harp seal, fur seal, common seal and deer. 3. The antibody APP.1 was coupled to Sepharose and employed in chromatographic purification of the harp seal intestinal AlPh. Alkaline phosphatase isolated on this immunosorbent has a spec. act. of 20,800 units per mg of protein. 4. The antibody-enzyme complex gives an excellent immunocytochemical labeling of tissue sections, cell cultures and smears.