Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.     

Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa (BIAP) is an homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion-chelating agents, exhibited a dramatic decrease of its hydrolase activity, concomittant to conformational changes in its quaternary structure. By rate-zonal centrifugation and electrophoresis, we demonstrated, for the first time, that the loss of divalent ions leads to some monomerization process for a metal-depleted alkaline phosphatase. Divalent ions are also involved in the secondary and tertiary structures. Metal-depletion induced more exposure of some Trp residues and hydrophobic regions to the solvent (as proved by intrinsic and ANS fluorescences). These changes might correspond to the disappearance of alpha-helices and/or turns with a concomittant appearance of unordered structures and beta-sheets (as probed by FTIR spectroscopy). For BIAP, three steps of temperature-induced changes were exhibited, while for apoBIAP, only one step was exhibited at 55 degrees C. Our work on BIAP showed two main differences with alkaline phosphatase from Escherichia coli. The loss of the divalent ions induces protein monomerization and the total recovery of enzyme activity by divalent ion addition to apoBIAP was not obtained.     

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.     

Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

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.     

Effect of metal ions on the structure and activity of calcium-activated neutral protease (CANP)

To clarify the mechanism of activation of calcium activated neutral protease (CANP, or mCANP: active at mM Ca2+), the structure of mCANP was examined by measuring CD spectra and by titration of SH groups in the presence of Mn2+. Mn2+ significantly increases the sensitivity of CANP to Ca2+ but CANP is not active with Mn2+ alone. The structural changes induced by Mn2+ were compared with those induced by Ca2+, and the structure of muCANP, which is active at microM Ca2+, was also examined for comparison. Mn2+ and Ca2+ induced the same structural changes of CANP. However, specific activation of the active site SH group by Ca2+ was not observed with Mn2+. Six moles of calcium bound to mCANP and the average dissociation constant of Ca2+ was 150 microM. The structure of muCANP was similar to that of mCANP in terms of the CD spectra. The titration of SH groups of muCANP indicated that the structure of muCANP was looser or SH groups were more exposed than in the case of mCANP. A model which can explain the activation of mCANP is proposed and the mechanism of activation is discussed based on the proposed model. The role of Ca2+ can be explained in terms of conformational change and activation of the active-site SH group of CANP.     

Influence of metal ions on structure and catalytic activity of papain

Papain is an endoprotease belonging to cysteine protease family. The catalytic activity of papain in presence of two different metal ions namely zinc and cadmium has been investigated. Both the metal ions are potent inhibitors of the enzyme activity in a concentration dependent manner. The enzyme loses 50% of its activity at 2 x 10(-4) M of CdCl2 and 4 x 10(-4) M of ZnCl2. It is completely inactivated above 1 x 10(-3) M concentration of either ZnCl2 or CdCl2. Of the two metal ions zinc with a ki value of 5 x 10(-5) M is a more potent inhibitor than cadmium which has a ki value of 8 x 10(-5) M. Both the metal ions have higher affinity for active site than the substrate. At concentrations above 1 x 10(-2) M of metal ions the inhibition is not reversible. Calorimetric studies showed decreased thermal stability of papain upon binding of these metal ions. Far UV circular dichroic spectral data showed only small changes in the beta-structure content upon binding of these metal ions. These data are also supported by decrease in the apparent thermal transition temperature of papain by 5 degrees C upon binding of metal ions indicating destabilization of the papain molecule. The mechanism of both partial and complete inactivation of papain in presence of these two metal ions both at lower and higher concentration has been explained.     

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.     

Effects of zinc and other metal ions on the stability and activity of Escherichia coli alkaline phosphatase

Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn(2+), Co(2+), Cu(2+) or Cd(2+) also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Evidence for glycosylphosphatidylinositol anchoring of intralumenal alkaline phosphatase of the calf intestine.

1. Considerable amounts of intestinal alkaline phosphatase (AP) were found intralumenally in all animal species investigated, i.e. calf, pig, goat, rat, mouse, guinea pig, hen and carp. The ratios between the total activity of AP found intralumenally and the total intestinal activity vary considerably. Calves and pigs show the highest, i.e. 0.77 and 0.44, respectively, while rodents have much lower ratios. Only 20-34% of the intralumenal alkaline phosphatase (IAP) of the calf and pig is soluble and not within the sediment after centrifugation at 135,000 x g for 60 min. whereas the IAP of rodents is soluble in the range of 60-72% of the total IAP. 2. For the IAP of the mucosa and chyme of calf, all criteria were found which are generally used, indicating a glycosylphosphatidylinositol (GlcPtdIns) anchor as proved by strong hydrophobicity using Triton X-114 phase partitioning, phenyl-Sepharose binding and enzyme aggregation, and the susceptibility to phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and papain digestion. 3. More than 80% of the mucosa alkaline phosphatase (MAP) of the proximal part of the intestine and of the particulate fraction of IAP exhibit these criteria indicating the presence of the GlcPtdIns-anchor structure, whereas the anchor content of the soluble intralumenal enzyme decreases from the pylorus to the ileocecal junction. 4. MAP partially purified to a specific activity of 1747 IU/mg retains the anchor structure. 5. The results presented indicate that the release of large amounts of AP into the chyme is realized without splitting the GlcPtdIns anchor. The possible intralumenal function of this form of AP is discussed.     

Effect of pH on the modulation of rat osseous plate alkaline phosphatase by metal ions.

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.     

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+).     

Effect of simultaneous administration of ibuprofen and ethanol on the alkaline phosphatase activity in the small intestine

Using the interferometric technique the authors studied the effect of simultaneous administration of ibuprofen (Polfa) and ethanol on the activity of alkaline phosphatase in the proximal jejunum. Both ibuprofen and ethanol cause in the digestive tract functional and morphological changes. The used technique of quantitative determination of alkaline phosphatase activity at the site of its primary location made possible an assessment of changes in the activity of the enzyme caused by the administered agents. It was found that after 60 days of the experiment both agents caused a statistically greater changes were observed in the rats receiving both these agents simultaneously. The obtained results suggest the conclusion that simultaneous administration of ibuprofen and ethanol causes in the mucosal gland in the proximal small intestine development of an interaction of the additive.     

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.     

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.