The difference in substrate selectivity and inhibitor sensitivity among human alkaline phosphatases

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  • ヒトアルカリ性ホスファターゼの基質選択性と阻害剤に対する感受性の相違

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Abstract

The human alkaline phosphatase (ALP), is homologous to not only other mammal ALP but also bacterial ALP. However, the substrate and function of ALP in vivo have not been cleared. Recently, we obtained human ALP as a chemical agent. In this study, we examined the differences in substrate selectivity and inhibitor sensitivity among several types of human ALP. We used 4 types of human ALP from bone, intestine, placenta, and liver. Also used were p -nitrophenyl phosphate (p -NPP), sodium pyrophosphate (Na-PPi), adenosine triphosphate (ATP) or pyridoxal phosphate (PLP) for substrate. The amount of inorganic phosphate as a result of hydrolysis by ALP was measured by Chifflet methods under different pH or substrate concentration. To examine the inhibitory effect on activity, levamisole or vanadate were used. The substrate concentration exhibiting 50% activity and the concentration exhibiting 50% inhibition were proposed as K0.5 and Ki0.5 respectively. When Na-PPi, the substrate of bone ALP, was used, the optical pH and K0.5 did not show differences among each of the ALP types (optical pH : 8.8 ～ 9.1, K0.5 : 3 ～ 5 mM). In the case of PLP, the substrate of intestine ALP, the optical pH and K0.5 did not show differences among each of the ALP types (optical pH : 9.9 ～ 10.4, K0.5 : 1.1 ～ 1.6 mM), although the optical pH shifted to the alkaline side. Additionally, the optical pH and K0.5 on p -NPP, which is not in the human body, but many researchers use it, did not show differences among any of the ALP types. However, in the case of PLP, Ki0.5 of levamisole was different among each of the ALP types, although Ki0.5 of levamisole or vanadate did not show any differences among the ALP types. We found that human ALP showed different inhibitor sensitivity among several ALP types, although the substrate selectivity was not determined.

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