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Enzyme Kinetics Studies of Nucleoside Diphosphate Kinase in Human Erythrocytes and Frequency Distribution in Healthy Subjects and Transplant Recipients in Chinese Han Population

Abstract

Nucleoside diphosphate kinase (NDPK), as a house-keeping protein, involves in various molecular processes including signal transduction, energy and drug metabolism. The main objective was to investigate NDPK kinetics in human erythrocytes and to monitor the frequency distribution of NDPK activity levels in Chinese healthy subjects and transplant recipients. METHODS: NDPK activity in erythrocytes was detected by a validated ion-pair high-performance liquid chromatogram method. NDPK kinetics studies were carried out systematically. NDPK activity levels were determined in 500 healthy subjects, 250 kidney and 250 liver transplant recipients in Chinese Han population. RESULTS: Thermal and pH stability studies indicated NDPK was relatively stable at temperature 30-45ºC and pH 6.0-9.0. In substrate dependency study, the apparent Michaelis-Menten constant (Km) and maximum velocity of enzymatic reaction (Vmax) increased with concentration of substrates. Meanwhile, in product inhibition study, with the increasing concentration of dATP, the Vmax of dADP decreased with constant Km and Km of dGTP increased with constant Vmax. NDPK activity levels revealed a 7-fold variability and were not normally distributed in all groups. NDPK activity levels were significantly (P<0.05) higher in transplant group than those in health group. Additionally, much higher NDPK activity levels had been shown (P<0.001) in liver transplant recipients when compared to kidney transplant cases. CONCLUSIONS: NDPK kinetics studies indicated substrate dependency of NDPK and a “ping-pong” mechanism for production inhibition. Skewness distributions of NDPK activity levels were shown in the study population. The transplant recipients showed higher NDPK activity levels when compared to healthy subjects.

Rufei Shen, Chunxiao Yang, Xiaomei Luo, Tingyu Yang, Jiali Zhou, Yani Liu, Shaojun Shi*

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