JOURNAL OF NEUROCHEMISTRY
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2018
·Vol. 148
·期 4
·550-560
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IF 4.609
Meisam Rostaminasab Dolatabad,Lu-lu Guo,Peng Xiao,Zhongliang Zhu,Qing-tao He,Du-xiao Yang,Chang-xiu Qu,Sheng-chao Guo,Xiao-lei Fu,Rui-rui Li,Lin Ge,Ke-jia Hu,Hong-da Liu,Yue-mao Shen,Xiao Yu,Jin-peng Sun,Peng-ju Zhang
s Protein Phosphatase Mg 2+ /Mn 2+ -Dependent 1K (PPM1K),also named as PP2Cm or branched-chain α-ketoacid dehydrogenase complex phosphatase, is a member of the metal-dependent phosphatase family and an important metabolic regulator. Single nucleotide polymorphisms (SNPs) in PPM1K contributing to protein functional defects have been found to be associated with numerous human diseases, such as cardiovascular disease, maple syrup urine disease, type 2 diabetes, and neurological disease. PPM1K N94K is an identified missense mutant produced by one of the SNPs in the human PPM1K coding sequence. However, the effects of the N94K mutant on its activity and structural property have not been defined. Here, we performed a detailed enzymological study using steady-state kinetics in the presence of pNPP or phospho-peptide substrates and crystallographic analyses of the wild-type and N94K PPM1K. The PPM1K-N94K significantly impaired its Mg 2+ -dependent catalytic activity and structural analysis demonstrated that the N94K mutation induced a conformational change in the key residue in coordinating the Mg 2+ in the active site. Specifically, three Mg 2+ were located in the active site of the PPM1K N94K instead of two Mg 2+ in the PPM1K wild type. Therefore, our results provide a structure basis for the metal ion-dependent PPM1K-N94K phosphatase activity.
研究领域:
生物化学
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结构生物学
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酶学
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分子生物学
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PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2022
·Vol. 414
·期 9
·2991-3003
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IF 4.478
Rao Honghong,Li Jianying,Luo Mingyue,Zhang Kehui,Gou Hao,Liu Haixia,Xue Zhonghua
Ratiometric electrochemical assays have been demonstrated to be more sensitive and selective in various sensing events, mainly due to their affordable built-in correction and good self-reference capability. But it is known that complicated modification and labeling operations usually are necessary for the construction of ratiometric electrochemical assays, therefore is a hot and important issue needing consideration carefully. We herein report a new yet simple bare electrode-based ratiometric electrochemical bioassay to achieve sensitive and selective analysis of alkaline phosphatase (ALP), using a liquid phase system that contains CoOOH nanozymes and commercially available indicator substrate. This proposed bioassay works based on the ratiometric change of dual electrochemical signals, arising from an exclusive target ALP-triggered hydrolysis of electrochemical substrate p-nitrophenyl phosphate (PNPP). In this design, the two hydrolyzed products of electrochemically active p-nitrophenol (PNP) and electrochemically inactive phosphate anion (PO 4 3− ) are responsible together for the ratiometric electrochemical analysis of ALP. PNP exhibits a straightforward current response toward ALP content; however, PO 4 3− cannot show a direct electrochemical signal thus is rationally designed to offer an alternative response by linking it with the specific CoOOH nanozyme-catalyzed reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2 O 2 , in which the nanozyme-catalyzed product oxTMB shows a direct reduction current at the GCE, and significantly decreases with increasing PO 4 3− species due to the good inhibition of PO 4 3− toward CoOOH nanozyme activity. As a result, a ratiometric electrochemical strategy for ALP analysis with a low limit of detection of 0.366 U/L (S/N = 3) was successfully achieved by integrating the above direct and indirect dual electrochemical responses. This developed bioassay can allow the quantitative diagnosis of ALP activity especially with a label-free and modification-free merit, therefore paving the way for simple, convenient, and portable electroanalytical tools in biosensing design and application. Graphical abstract
研究领域:
分析化学
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生物化学
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电化学
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纳米技术
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生物传感器
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PubMed
高影响因子
Analytical Methods
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2015
·Vol. 7
·期 23
·9984-9991
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IF 1.821
Qingming Zhang,Xue Li,Bingchen Li,Huanshun Yin,Shiyun Ai
A sensitive and selective electrochemical biosensor was fabricated for polynucleotide kinase (PNK) activity assay and inhibitor screening based on phos-tag-biotin mediated double signal amplification, where streptavidin and alkaline phosphatase-conjugated biotin (biotin-ALP) were used as the signal amplification units. After the immobilization of probe DNA on the electrode surface, it was hybridized with complementary DNA containing the 5′-OH terminal, the OH can be phosphorylated under the catalytic effect of PNK in the presence of ATP. The generated phosphate group at the 5′-terminal can be further recognized by phos-tag-biotin, where phos-tag-biotin was not only used as the specific recognition reagent for the phosphate group, but also the capture reagent for streptavidin to induce the further assembly of biotin-ALP. Under the catalytic effect of ALP, the substrate of p-nitrophenyl phosphate disodium salt hexahydrate (PNPP) can be hydrolyzed to produce p-nitrophenol (PNP). According to the relationship between the electrochemical response of PNP and the logarithmic value of PNK concentration, a linear range of 0.01–5 units per mL and a low detection limit of 0.0027 unit per mL were achieved. The developed method showed high selectivity. The inhibition effect of (NH4)2SO4 and Na2HPO4 was also evaluated and the IC50 values were calculated.
研究领域:
生物化学
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分析化学
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电化学
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分子生物学
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2023
·
IF 4.8
Shiyi Chen,Feiyang Tian,Juan Peng,Yan Meng,Yu Wang,Xiaogang He,Qian Wang,Ganghui Chu
Chemical warfare agents, especially organophosphorus warfare agents are a variety of various chemical substances employed in warfare that can massively cause damage to the organism even death. The partial catalytic function of Universitetet i Oslo-66 (UiO-66, a zirconium-based metal–organic framework) produced via de novo synthesis is similar to calf intestinal alkaline phosphatase (CIAP, EC 3.1.3.1) and has a lower usage cost, is expected to be a mimetic enzyme for hydrolyzing organophosphorus warfare agents due to the fact that it has lattice defects. 0.1–5.6 mL of acetic acid was added as a regulator to form different lattice defects. Typically, mimetic enzyme activity tests often require expensive reagents as substrates, and the experiment processes are time-consuming and inconvenient. As a result, a novel approach based on near-infrared spectroscopy (NIRs) was developed in this research for predicting UiO-66 mimetic enzyme activity. p -Nitrophenyl phosphate disodium salt ( p NPP) was selected as a lower toxic organophosphorus warfare agent to conduct the subsequent simulant research. The absorbance of the mimetic enzyme catalytic system was evaluated using Ultraviolet–Visible (UV–Vis) spectroscopy to investigate the amount of its activity and compare it with CIAP activity. The synthesized samples’ enzyme activities ranged from 1.91 to 6.67 U/g and their catalytic costs ranged from 0.65 to 2.26 CNY. The powdered samples' near-infrared spectra were obtained. The sample’s enzyme activity data were linked with each sample’s original NIR spectra to establish the NIR model. A partial least squares (PLS) model of mimetic enzyme activity was developed by coupling spectral preprocessing with a variable screening technique. The root-mean-square error of cross-validation (RMSECV), the correlation coefficient of validation set (R) value, and the ratio of prediction to deviation (RPD) value were employed as assessment model indicators. The near-infrared spectrum model was developed by merging the best 2nd derivative spectral preprocessing with the Competitive Adaptive Reweighted Sampling (CARS) variable screening method. This model's RMSECV was 0.41 U/g, the correlation coefficient of calibration set (R_cv) value was 0.916, the root-mean-square error of prediction (RMSEP) set was 0.52 U/g, R was 0.948, and RPD was 2.51. As a result, the established model could be implemented to quantify the activity of UiO-66 quickly by including more variations of de novo -synthesized samples. The prediction method is simple, rapid, low cost and adaptable to be the theoretical and practical basis for further studying other interdisciplinary research work in biomimetic enzymology and spectroscopy.
研究领域:
化学
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材料科学
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酶学
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光谱学
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毒理学
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高影响因子
FOOD CHEMISTRY
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2025
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IF 9.8
Zizhe Li,Liang Luo,Chaochao Chen,Qiang Li,Qiyuan Zhao,Ghulam Mujtaba Mari,Chen Xing,Xuefeng Xia,Jianzhong Shen,XueZhi Yu,Zhanhui Wang,Yantong Pan
In this study, we synthesized a polydopamine-polyethyleneimine (PDA-PEI) copolymer nanoparticles (NPs) fluorophore by a simply and environment-friendly one-step self-assembly reaction at room temperature to minimize environmental pollution and resource waste caused by the extensive use of organic solvents for material synthesis and sample pretreatment in immunoassay. Based on the inner filter effect between the synthesized PDA-PEI NPs fluorophore and p -nitrophenyl that produced by alkaline phosphatase catalyzed p -nitrophenylphosphate, we proposed one novel and environment-friendly fluorometric/colorimetric dual-mode enzyme-linked immunosorbent assay for the highly sensitive detection of norfloxacin (NOR) in various samples without organic solvents and highly irritating acid-base solutions. After comprehensive optimization, the detection limits of the two modes of immunoassays were 0.02–0.75 μg/kg with recovery rates of 75.38 % ~ 109.50 % in four types of meat and two waters. Finally, the established simple, highly sensitive, and robust immunoassay offers a powerful tool for NOR detection in various food and environmental samples.
研究领域:
环境科学
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分析化学
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生物医学工程
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DOI
PubMed
高影响因子
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