Effect of calcium ions on rat osseous plate alkaline phosphatase activity.
Rat osseous plate alkaline phosphatase is a metalloenzyme with two binding sites for Zn2+ (sites I and III) and one for Mg2+ (site II). This enzyme is stimulated synergistically by Zn2+ and Mg2+ (Ciancaglini et al., 1992) and also by Mn2+ (Leone et al., 1995) and Co2+ (Ciancaglini et al., 1995). This study was aimed to investigate the modulation of enzyme activity by Ca2+. In the absence of Zn2+ and Mg2+, Ca2+ had no effects on the activity of Chelex-treated, Polidocanol-solubilized enzyme. However, in the presence of 10 microM MgCl2, increasing concentration of Ca2+ were inhibitory, suggesting the displacement of Mg2+ from the magnesium-reconstituted enzyme. For calcium-reconstituted enzyme, Zn2+ concentrations up to 0.1 microM were stimulatory, increasing specific activity from 130 U/mg to about 240 U/mg with a K0.5 = 8.5 nM. Above 0.1 microM Zn2+ exerted a strong inhibitory effect and concentrations of Ca2+ up to 1 mM were not enough to counteract this inhibition, indicating that Ca2+ was easily displaced by Zn2+. At fixed concentrations of Ca2+, increasing concentrations of Mg2+ increased the enzyme specific activity from 472 U/mg to about 547 U/mg, but K0.5 values were significantly affected (from 4.4 microM to 38.0 microM). The synergistic effects observed for the activity of Ca2+ plus magnesium-reconstituted enzyme, suggested that these two ions bind to the different sites. A model to explain the effect of Ca2+ on the activity of the enzyme is presented.
Leone
F A
FA
Departamento de Química, Faculdade de Filosofia, Ciências e Letras, Universidade de São Paulo, Ribeirão Preto, Brasil. fdaleone@usp.br
Ciancaglini
P
P
Pizauro
J M
JM
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001842:Bone and Bones / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008956:Models, Chemical; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000737:chemistry / Q000494:pharmacology; D011137:Polystyrenes / Q000494:pharmacology; D011145:Polyvinyls / Q000494:pharmacology; D051381:Rats; D012995:Solubility; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D011137:Polystyrenes; D011145:Polyvinyls; D000077423:Polidocanol; D011092:Polyethylene Glycols; C006960:chelex; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc; D002118:Calcium