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An international journal published by the

Nigerian Society for Experimental Biology
Printed in Nigeria

Cofactor interactions in the activation of tissue non-specific alkaline phosphatase: Synergistic effects of Zn2+ and Mg2+ ions

Femi J. OLORUNNIJI*, Adedoyin IGUNNU, Joseph O. ADEBAYO, Rotimi O. ARISE and Sylvia O. MALOMO

Department of Biochemistry, University of Ilorin, P.M.B. 1515 Ilorin, Nigeria

Received 19 March 2007

MS/No BKM/2007/028, © 2007 Nigerian Society for Experimental Biology. All rights reserved. ---------------------------------------------------------------------------------------------------------------------------------------

Abstract The interactions of Mg2+ and Zn2+ ions in the activation of non-specific tissue alkaline phosphatase were investigated using crude extracts of rat kidney. Activation of alkaline phosphatase by the metal ions was accompanied by changes in the kinetic parameters of pnitrophenylphosphate hydrolysis. The results suggest some synergistic interactions between Mg2+ and Zn2+ ions in promoting the hydrolysis of p-nitrophenylphosphate by alkaline phosphatase. The results show that assays of alkaline phosphatase activity in homogenised tissue samples will give better responses if both Mg2+ and Zn2+ ions are included in the reactions Keywords: Alkaline phosphatase; kinetics; Enzyme-cofactor interaction; synergism * corresponding author. Email: femijohn@gmail.com

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INTRODUCTION The roles of metal ions in metalloenzymes include direct participation in catalysis, stabilization of protein structure and regulation of enzymatic activity. Membrane alkaline phosphatase (ALP) is a metal-containing enzyme that serves as a good model for the study of metal ion interactions in enzyme catalysis. Native E. coli ALP contains three metal ion binding sites (two Zn2+ sites and one Mg2+ site), and studies on their roles and interrelationships have provided some insights into the mechanism of the enzyme1. E. coli ALP is a zinc metalloenzyme, which can be activated by magnesium ion2. Removal of the Zn2+ leads to loss of catalytic activity while its replacement by other divalent cations (Mn2+, Co3+, Cd2+, and Cu2+) resulted in lower maximal activity2. It has been shown that while low concentrations of Mg2+ stimulated the refolding of E. coli ALP, high concentration actually inhibited its reconstitution into the active form3. This suggests that Mg2+ mediates stabilization and destabilization of the catalytically active structure of ALP at low and high concentrations respectively4,5. In E. coli ALP, Mg2+ is thought to have a regulatory effect on the expression of catalytic activity and maintenance of structural integrity of the enzyme1. The specific binding of Mg2+ to apo-ALP depends on both pH and the cooperative effects of Zn2+ binding. Mg2+ alone does not confer catalytic activity on ALP, but it does regulate the Zn2+-induced restoration of activity and perhaps, structural integrity of the metal-binding loci1,6. Mammalian ALPs are glycoproteins that are present as different isoenzymes in several tissues including bone, intestine, kidney, and placenta. Like their E. coli counterpart, they are zincmetalloenzymes that can be activated by Mg2+ ions; both ions being essential for catalysis and structural stability. The human genome, like other mammalian systems contains four ALP loci, one coding for the tissue nonspecific ALP (TNAP) expressed in a variety of organs such as liver, bone, kidney, etc. and three tissue-specific ALP (TSAP) genes coding for the intestinal AP (IAP), placental ALP (PLAP), and germ cell ALP (GCAP). TNAP shows approximately 50% sequence similarity with the TSAP isozymes.

Mammalian ALPs show 25-35% sequence identity with the E. coli enzyme in those regions of the protein assuming α-helix and β-strand secondary structures, and critical for catalysis. The catalytic residues, i.e. Asp91, Ser92, Arg166, and ligands coordinating the divalent metal ions (Zn2+ and...
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