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Biokemistri
Nigerian Society for Experimental Biology
ISSN: 0795-8080
Vol. 17, Num. 2, 2005, pp. 129-136

Biokemistri, Vol. 17, No. 2, Dec, 2005, pp. 129-136

Regulatory effect of divalent cations on rat liver alkaline phosphatase activity: How Mg2+ activates (and inhibits) the hydrolysis of p-nitrophenylphosphate

Rotimi O. ARISE1, Femi F. BOLAJI1,4, Olalekan A. JIMOH1,4, Joseph O. ADEBAYO2, Femi J. OLORUNNIJI1,3* and Sylvia O. MALOMO1

1. Department of Biochemistry, Faculty of Science, University of Ilorin, P.M.B 1515, Ilorin, Nigeria.
2. Department of Biochemistry & Physiology, Faculty of Health Sciences, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria.
3. Division of Molecular Genetics, Institute of Biomedical and Life Science, Anderson College, University of Glasgow, Glasgow, G11 6NU, United Kingdom.
4. Deceased
*Author to whom all correspondence should be addressed.  E-mail: femijohn@gmail.comF.Olorunniji@bio.gla.ac.uk Tel: +44 1413 571866

Received 19 April 2005

Code Number: bk05018

Abstract

The concentration-dependent stimulation of rat liver alkaline phosphatase (ALP) catalyzed hydrolysis of para- nitrophenylphosphate (pNPP) was studied. ALP displayed some activity even in the absence of exogenous Mg2+. Kinetic analyses show that activation by Mg2+ is exerted at the Vmax   level without necessarily enhancing the affinity of the enzyme for the ion.  However, the hyperbolic activation operates only within the optimal level of 0 to 5mM concentrations of the metal ion.  Higher concentrations were actually inhibitory in a pure non-competitive manner. Mg2+, either as an activator (optimal concentrations) or inhibitor (supra-optimal levels) exerts its action via a Vmax effect with only negligible effect on Km for the substrate.

Key words: Magnesium ion, Alkaline phosphatase, Supra optimal regulation

INTRODUCTION

Despite intensive efforts towards understanding the biochemical nature of alkaline phosphatase, the role of this enzyme in biological processes is still not completely understood1,2. Although, a variety of evidence points towards diverse mechanisms of action of this enzyme, it is well known that alkaline phosphatase is a zinc metalloenzyme that can be activated by magnesium ion3. On the other hand, excess zinc has been reported to inhibit several alkaline phosphatase by replacing magnesium ions on the enzyme molecule4.

Various divalent metal ions have been shown to have both activating and inhibitory effects on mammalian alkaline phosphatases5,6, but the question of how the mechanisms of activation and inhibition is accomplished, however, remains unanswered. This is in spite of the different effects that have been described in the presence of various divalent metal ion7.

It has been shown that alkaline phosphatase have three classes of metal binding sites, which have been designated to perform catalytic, regulatory and structural roles. The alkaline phosphatase of E. coli is a dimer composed of two identical subunits with four atoms of zinc and two atoms of magnesium8. Removal of the zinc ions leads to loss of catalytic activity while replacement of the zinc ions by other divalent cations resulted in lower maximal activity. Three metal ions (two Zn2+ and one Mg2+) in ALP active sites are essential for enzymatic activity8. The inhibition of alkaline phosphatase by excess Zn2+ has been proposed to be due to the replacement of Mg2+ by Zn2+ at one site of the enzyme5. In an earlier report we showed that vanadate and L-phenylalanine display positive synergistic interactions in their inhibition of rat liver alkaline phosphatase9.

This report describes an attempt to examine the effect of low (optimal) and high (supra-optimal) magnesium ion concentrations on rat liver alkaline phosphatase, with a view to understanding how the metal ion performs the dual role.

MATERIALS AND METHODS 

P-nitrophenylphosphate (pNPP) was obtained from sigma chemical company, St. Louis, US. Magnesium salt of chloride was a product of British Drug House, Poole, UK. All other chemicals used in this study were of high quality research grade.

Preparation of rat liver alkaline phosphatase

Crude homogenates of rat liver was concentrated for ALP activity using ammonium sulphate according to a modified procedure of Hung and Melnykovych10. Rat liver was homogenized in 0.25M sucrose solution at 4°C and the crude homogenate was centrifuged at 5000 rpm for 20 minutes at the same temperature. To the supernatant fraction was added a 0.55g/ml-(4.17M) solution of (NH4)2SO4 gradually with stirring until 30% saturation was achieved. The precipitate was collected by centrifugation at 5000 rpm for 20 minutes and re-dissolved in 0.1M carbonate-bicarbonate buffers, pH10.1. The crude preparation was further fractionated on a Sephadex G-100 column to obtain a rich and highly active alkaline phosphatase fraction.  The activities of the ALP prepared this way and used in this study were highly reproducible and gave linear results with a correlation level sufficient for kinetic work9.

Determination of alkaline phosphatase hydrolysis of pNPP

Alkaline phosphatase activity was measured by the hydrolysis of p-nitrophenylphosphate (pNPP) at 25°C in the presence of a previously added magnesium chloride in 0.1M Na2CO3/ NaHCO3 buffers, pH 10.1 as previously described11. Enzyme activity is expressed as the μM of p-nitrophenol released per minute. The concentrations of Mg2+ investigated were 0, 0.2 and 1.0mM at pNPP concentrations ranging from 0.63-5.1mM. Protein concentration was determined using the Folin-phenol method of Lowry et. Al.12 with Bovine Serum Albumin (BSA) as standard. Spectrophotometric readings were taken on spectronic-21 UV-VIS spectrophotometer.

Investigation of the effect of Mg2+ concentration on alkaline phosphatase activity

In order to determine the concentration dependent effect of Mg2+ within optimal levels, the reaction medium was set up essentially as described by Wright and Plummer13 but Mg2+ concentration was varied in the range of 0.1-3.8mM. Investigations on the effect of supra-optimal levels of Mg2+ on ALP activity were carried out at concentrations ranging from 12.5-25.4mM. The concentration range of pNPP was kept constant (0.63-5.1mM) for all Mg2+ concentrations studied and incubation was allowed for 10 minutes before stopping the reaction by the addition of 0.1M NaOH. The absorbance was monitored at 400nm against a blank of the buffered substrate and the corresponding activities were recorded. 

Analysis of inhibition data

Cornish-Bowden14 and Cortes et. al15 proposed a relationship between inhibition constants, inhibitor concentrations for 50% inhibition (i0.5)and types of inhibition. This ‘higher resolution’ kinetic procedure allows for detailed and non-confounding analysis of inhibition mechanism. The quantitative expressions of i0.5 were derived by Cheng and Prusoff16 for competitive, uncompetitive and mixed inhibition.  When a straight line is obtained by plotting a function of the rate ‘v’ against the inhibitor concentration ‘i0.5’ whether this is the reciprocal17 rate ‘’ or the reciprocal rate multiplied by the substrate18 concentration ‘a’ that is ‘’; the intercept of the extrapolated line on the ‘i’ axis is -i0.5.This provides a simple and accurate way of estimating i0.5. This is however easily demonstrated by reference to the expression15

Where ‘v’ is the limiting rate, ‘Km’ is Michaelis constant, ‘Kic’ is the competitive inhibition constant and ‘Kiu’ is the uncompetitive inhibition constant. A plot of against is a secondary plot, which gives a straight line according to the following expression15

The combination of the two plots discriminates clearly between all the different types of linear inhibition, and supplies the values of both inhibition constants; furthermore, it provides a direct link between the biochemical characteristics of the inhibition. The plots of [pNPP]/v versus [Mg2+] for the Cornish-Bowden analysis and the secondary plot were made accordingly.

RESULTS

Activation of alkaline phosphatase with optimal concentrations of Mg2+

Figure 1 shows the effect of Mg2+ concentration on the kinetics of ALP-catalyzed hydrolysis of pNPP. The stimulation was Mg2+ ion concentration dependent in the range of 0.1-3.8mM (panel A). Within this range, the activation was hyperbolic, an indication of a saturating process characteristic of Michaelis-Menten kinetics describing the saturating nature of the variation of reaction velocity with substrate concentration. This suggests that essentially all the enzyme molecules present were combined with the metal ion in the reaction solution. It is likely that the Mg2+ site in the protein is saturable in a way that influences catalysis. A double reciprocal transformation of the activation data is shown in panel B. This allows for estimations of Michaelis constant and maximum rates of 1.16mM and 2.19s-1 respectively. While the binding of Mg2+ to the enzyme is not a true enzyme-substrate phenomenon, the good fit of the data to the Michaelis-Menten equation affords a simple way of analyzing the data.

Figure 2A shows the hydrolysis of various concentrations of pNPP by alkaline phosphatase in the presence of 0, 0.2 and 1.0mM Mg2+. Within the range of substrate concentration examined, the hydrolysis of pNPP by alkaline phosphatase obeyed Michaelis-Menten kinetics.                                                            

Values of Vmax and Km for pNPP at the three levels of Mg2+ are derived from the double reciprocal transformations shown in Figure 2B. It is apparent that ALP displayed activity even in the absence of exogenous Mg2+, though the activity was low. This finite activity is evident that the preparatory procedures employed in obtaining the enzyme did not lead to exhaustive loss of the bound Mg2+ in the enzyme. Stimulation of ALP hydrolysis of pNPP was also dependent on Mg2+ concentration as it was observed that ALP activity was higher at 1.0mM Mg2+ compared with 0.2mM.

Higher concentrations of Mg2+ inhibits ALP activity

The hyperbolic and concentration-dependent activation of ALP by Mg operates only at the optimal level of bout 0 to 5mM concentrations of the metal ion. Further experiments were carried out to study the kinetic pattern of this inhibition. The strategy used was to analyze how Mg2+ at 12.5, 16.7, 21.1 and 25.4mM affect the kinetics of pNPP hydrolysis within the substrate concentration range of 0.63 to 5.1mM. The basic pattern is shown in Figure 3 along with activity at an optimal (2mM) concentration of Mg. The presence of these higher concentrations of Mg2+ suppressed the hydrolysis of pNPP. It is noteworthy however that ALP-catalyzed hydrolysis of pNPP at these inhibitory levels of Mg2+ still obeys Michaelis-Menten kinetics. The double reciprocal transformation (Figure 3B) gave a series of linear fits characteristic of a pure non-competitive mechanism for the inhibition of pNPP hydrolysis by highest concentration of Mg2+. This was further confirmed by subjecting

The plots of [pNPP]/v versus [Mg2+] for the Cornish-Bowden analysis and the secondary plot against are shown in Figures 4A and 4B respectively. When these were compared with the classical patterns of Cornish-Bowden transformations, they gave a pure non-competitive mechanism, within the limits of experimental error.  Figure 5A is a plot of against Mg2+ concentration while 5B is a plot of Km/Vmax against Mg2+ concentration. These plots evaluate the inhibition mechanism while showing the inhibitory effect of Mg2+ at high concentrations on the kinetic parameters of Km and Vmax. The plots reveal a systematic curvature, which is indicative of hyperbolic inhibition14. This provisionally happens when the ‘EX’ complex is less reactive than the free enzyme, in this instance; ‘X’ is the normal substrate or cofactor. This is mechanistically different from the ‘EI’ complex of competitive inhibition since the ‘EX’ is still catalytically reactive, albeit at a slower rate14. It is interesting to note that Mg2+ as an activator (optimal concentration) and as an inhibitor (at higher concentration) exerted its action as a Vmax effect with only negligible effect on Km for the substrate.

DISCUSSION 

McCracken and Meighen19 established the existence of three classes of metal binding sites in ALP. These are designated “catalytic” “structural” and “regulatory”. Zn2+ occupies the catalytic and structural sites, while Mg2+ ions are bound at the regulatory site20,21.  Brunel and Cathala7 in explaining the role of metal ions in metal-activated enzymes systems proposed that the metal ion can act as a bridge between the enzyme and it’s substrate or it can induce conformational changes and thereby convert an inactive or partially activated form of an enzyme into a catalytically active or more active form.

These considerations show the intimate role of Mg2+ in the general structure and function of ALP. Accordingly, it is expected on the basis of simple chemical considerations that changes in the level of the metal ion will sensitively affect the performance of the enzyme at structural and functional levels. It is conceivable that Mg2+ performs more of a structural role by inducing a conformational change in ALP as observed here. This may serve as a basis for its activating role in ALP assay. Occupation of a site on the enzyme molecule could induce a conformational change that brings about activation of the enzyme molecule. It is also possible that a Mg2+- pNPP complex is the true substrate of the enzyme; hence, explaining the hyperbolic behavior. The effect of Mg2+ on pNPP hydrolysis by ALP as observed in this study is via the Vmax effect and did not essentially affect km. This is in conformity with reports from other workers such as Brunnel and Cathala7. The fact that Mg2+ concentration may not affect Km indicate that Mg2+ induces its activation effect on rat ALP through its binding to the enzyme-substrate complex rather than by an action on the free enzyme14. Mechanistically, the action of Mg2+ on the Vmax may be to enhance the breakdown of the ES complex to form the free enzyme and product. The variable effect of Mg2+ on Vmax values points to a substrate binding – independent activation pattern in which the cofactor, Mg2+ binds only to the ES complex to form the free enzyme and product.

The inhibition observed at higher Mg2+ concentration may be as a result of unproductive binding between ALP and excess Mg2+ as the same “agent” transits from an activator to an inhibitor - an interesting mechanistic puzzle. A positive explanation is that the excess Mg2+, via a simple mass action effect, displaced Zn2+ from its site in the enzyme with a corresponding decrease in activity. The intact ALP requires four zinc and two magnesium atoms for catalytic activity and substrate binding8. Since both metal ions can bind to the same site, it is therefore possible for excess Mg2+ to displace some structural and catalytic Zn2+ with a consequent down – regulation of activity. In the reconstitution experiment of Zhang et al.22, while low concentrations of Mg2+ stimulated the refolding of ALP, high concentration actually inhibited the reconstitution of active ALP. This offers another perspective on the role of Mg2+ as found in this study: Stabilization and destabilization of the catalytically active structure at low and high concentrations respectively23.

The ‘Vmax effects’ of Mg2+ as an activator at optimal concentration and, inhibitor at supra optimal concentration as observed in this study is consistent with the work of Park et al.24 where it was shown that activation of ALP is time dependent and not a very rapid process. Generally, at optimal Mg2+ concentrations, Vmax increased steadily while at inhibitory levels, the Vmax was found to degenerate at a gradual rate. Non-competitive inhibitors bind to both free enzymes and the enzyme-substrate complex with equal affinity i.e. Kic=Kiu. This, however may tend to conflict with earlier explanations about the binding  of Mg2+ to the enzyme but this can be substantiated by the allostery ascribed to mammalian alkaline phosphatases25, where they concluded that mammalian ALP are allosteric enzymes in which both monomers act independently, especially when both ALP subunits are completely metalated.

In conclusion, if the primary mechanistic role of Mg2+ in ALP is structural as assumed from earlier studies, the Vmax effect of the ion as observed in this study prescribes that the Mg2+ dependent structural role does not control substrate binding. Rather, Mg2+ hypothetically activates structural features needed for catalysis independent of substrate binding. 

REFERENCES 

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© 2005 Nigerian Society for Experimental Biology.

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