Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata
Abstract
:1. Introduction
2. Results
2.1. Cloning and Sequence Analysis of the Fifth Putative Ace Gene from P. pseudoannulata
2.2. Recombinant Expression and Enzyme Activity Assay
2.3. Substrate Hydrolysis Kinetics of PpAChE5
2.4. Inhibition Kinetics of PpAChE5
3. Discussion
4. Materials and Methods
4.1. Spiders, Expression Vector, Cell Lines and Chemicals
4.2. Cloning and Homology Analysis of the Putative Ace Gene
4.3. Expression and Biochemical Properties Assaying of the Putative AChE
4.4. Data Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sample Availability: Not available. |
Subsite | T. californica | T. urticae | PpAChE1 | PpAChE2 | PpAChE3 | PpAChE4 | PpAChE5 |
---|---|---|---|---|---|---|---|
Catalytic triad | S200 | S | S | S | S | S | S |
E327 | E | E | E | D | D | E | |
H440 | H | H | H | H | H | H | |
Oxyanion hole | G118 | G | G | G | G | G | G |
G119 | S | G | G | G | A | G | |
A201 | A | A | A | A | A | A | |
Choline binding site | W84 | W | W | W | W | W | W |
Y130 | Y | Y | Y | F | Y | H | |
F330 | Y | Y | F | T | L | F | |
F331 | F | F | Q | L | I | Q | |
Acyl pocket | F288 | F | F | R | R | R | R |
F290 | F | F | F | Y | T | F | |
V400 | F | F | L | R | R | L | |
Peripheral anionic site | Y70 | V | I | Y | Y | F | Y |
Y121 | W | Y | S | R | V | F | |
W279 | E | W | N | S | S | S | |
Wall of the gorge | W114 | W | W | W | F | W | W |
W233 | W | W | L | D | D | L | |
Y334 | Y | Y | T | K | N | T | |
W432 | W | W | W | W | F | W | |
Y442 | E | D | E | D | D | S |
KM (µM) | Vmax (nmol/mg·min) | Vmax/KM (mL/mg·min) | Vmax Ratio | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
ATC | BTC | PTC | ATC | BTC | PTC | ATC | BTC | PTC | ATC vs. BTC | ATC vs. PTC | BTC vs. BTC | |
PpAChE1 | 536.8 ± 63.4 c | 858.8 ± 103.2 c | 1148.6 ± 133.5 c | 112.6 ± 8.8 c | 95.4 ± 12.8 b | 76.2 ± 9.2 c | 209.8 | 111.1 | 66.3 | 1.18 | 1.48 | 1.25 |
PpAChE2 | 4413.5 ± 533.7 a | 4542.2 ± 326.0 a | 4496.3 ± 318.8 a | 253.9 ± 20.4 b | 233.9 ± 16.5 a | 156.8 ± 21.3 a | 57.5 | 51.5 | 34.9 | 1.09 | 1.62 | 1.49 |
PpAChE3 | 42.9 ± 5.8 e | 198.6 ± 22.3 e | 503.6 ± 86.8 d | 124.7 ± 11.5 c | 40.3 ± 5.6 c | 106.2 ± 15.1 b | 2906.8 | 202.9 | 210.9 | 3.09 | 1.17 | 0.38 |
PpAChE4 | 61.6 ± 7.5 d | 269.4 ± 35.5 d | 279.3 ± 35.2 e | 86.6 ± 9.7 d | 105.5 ± 8.9 b | 49.7 ± 8.4 d | 1405.8 | 391.6 | 177.9 | 0.82 | 1.74 | 2.12 |
PpAChE5 | 1303.5 ± 162.9 b | 1528.9 ± 118.4 b | 1548.2 ± 129.1 b | 428.4 ± 29.2 a | 91.6 ± 11.3 b | 59.0 ± 7.2 cd | 328.7 | 59.9 | 38.1 | 4.68 | 7.26 | 1.55 |
On ATC Hydrolysis | On BTC Hydrolysis | ||
---|---|---|---|
BW284C51 | Eserine | ISO-OMPA | |
PpAChE1 | 5.12 ± 0.76 d | 11.67 ± 2.06 d | >10,000 |
PpAChE2 | 254.17 ± 21.40 a | 186.42 ± 25.11 a | 2165.39 ± 325.67 |
PpAChE3 | 3.68 ± 0.53 e | 6.52 ± 1.80 e | >10,000 |
PpAChE4 | 7.36 ± 1.05 c | 24.50 ± 3.94 c | >10,000 |
PpAChE5 | 47.10 ± 6.93 b | 116.28 ± 15.71 b | >10,000 |
Compound | ki (×10−7M−1min−1) |
---|---|
Eserine | 6.24 ± 1.72 b |
Fenobucarb | 18.46 ± 3.85 a |
Carbaryl | 16.79 ± 2.90 a |
Paraoxon | 15.50 ± 3.32 a |
Diazoxon | 16.43 ± 3.58 a |
Primers | ||
---|---|---|
RACE primers | 3′outer primer | TTACAACAAGCAACCCCGACC |
3′inner primer | CCATTTCAGCGAAGCGGCATT | |
5′outer primer | GGCTTCAGTCTCAACTCCAACGT | |
5′inner primer | GAAGCGCCATAGCATCAACACCT | |
Expression primers | Sense primer: | TAGTGCGGCCGCTTTCGAATATGGCTTTTCTTTCCTTAGA |
Anti-sense primer | CTCGAGACTGCAGGCTCTAGTCAGAATCCGAAATACGGGC |
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Meng, X.; Xu, X.; Bao, H.; Wang, J.; Liu, Z. Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata. Molecules 2017, 22, 1118. https://doi.org/10.3390/molecules22071118
Meng X, Xu X, Bao H, Wang J, Liu Z. Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata. Molecules. 2017; 22(7):1118. https://doi.org/10.3390/molecules22071118
Chicago/Turabian StyleMeng, Xiangkun, Xixia Xu, Haibo Bao, Jianjun Wang, and Zewen Liu. 2017. "Characterization of the Fifth Putative Acetylcholinesterase in the Wolf Spider, Pardosa pseudoannulata" Molecules 22, no. 7: 1118. https://doi.org/10.3390/molecules22071118