Effect of membrane moiety and magnesium ions on the inhibition of matrix-induced alkaline phosphatase by zinc ions

1. The inhibition of matrix-induced alkaline phosphatase by zinc ions is due to the displacement of magnesium ions from its binding site. 2. Binding of magnesium ions to alkaline phosphatase induces conformational changes which activate the enzyme. 3. Binding of zinc ions to alkaline phosphatase induces conformational changes which impair the catalytic action of the enzyme. 4. The inhibition of the enzyme by zinc ions is affected by membrane environment and magnesium ions.     

Interaction of immobilized DNA with alkaline metal and ammonium ions

Ion exchangers with various capacities (0.1-0.2 mg-equiv/g of dry gel) are synthesized by means of immobilization of DNA in polyacrylamide gel. Exchanges of alkali metal cations and ammonium are studied on these exchangers and selectively coefficients are determined. The following selectivity series of immobilized DNA in reference to the above-mentioned cations is stated: Li+ greater than or equal to NH4+ greater than or equal to Cs+ greater than Rb+ greater than K+ greater than or equal to Na+. The peculiar properties of Li+ and NH4+ in this series are noted and a possible explanation of this fact is offered. A supposition regarding the reduced activity of water in the polyacrylamide gel containing DNA is made.     

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.     

Potentiometric bioelectronic tongue for the analysis of urea and alkaline ions in clinical samples

Urea biosensors based on urease covalently immobilized on to ammonium and hydrogen ion-selective electrodes were included in arrays together with ammonium, potassium, sodium, hydrogen and generic response to alkaline sensors. Response models based on artificial neural network (ANN) and partial least squares (PLS1) were built, tested and compared for the simultaneous determination of urea, ammonium, potassium and sodium. The results show that it is possible to obtain good ANN and PLS calibration models for simultaneous determination of these four species, but with better prediction capability when the ANN are used. The developed bioelectronic tongue was applied to multidetermination in urine samples. The ANN model showed again better agreement with reference methods, allowing a simple direct determination of urea in the real samples without the necessity of eliminating the alkaline interferences, or compensating endogenous ammonium.     

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.     

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 metal ions and EGTA on the optical properties of concanavalin A at alkaline pH

In our earlier communications, we reported the effect of salts and alcohols on alpha-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.