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Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii
Zhi-Jiang Wang1, Wenming Ma1, Jun-Mo Yang2
1College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, PR China.
Abstract:
To gain insight into the effect of Cu2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii, the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography. We studied Cu2+-mediated inhibition and aggregation of ALP, and found that Cu2+ significantly inactivated ALP activity with an IC50 of 1.47 ± 0.02 mM. We further revealed that Cu2+ reversibly inhibited ALP in a mixed-type manner with Ki = 0.41 ± 0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii.
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