Detecting ACCase-targeting herbicides effect on ACCase activity utilizing a malachite green colorimetric functional assay

Weed Science ◽  
2021 ◽  
pp. 1-18
Author(s):  
Suma Basak ◽  
Douglas Goodwin ◽  
Jahangir Alam ◽  
James Harris ◽  
Jinesh D. Patel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Malachite Green ◽  
Amino Acid Substitution ◽  
Dose Response ◽  
Colorimetric Assay ◽  
Functional Assay ◽  
Acetyl Coenzyme A ◽  
Order Of Magnitude ◽  
Acetyl Coenzyme A Carboxylase

Research was conducted to evaluate acetyl-Coenzyme A carboxylase (ACCase) enzyme activity using a functional malachite green colorimetric assay previously identified as resistant to sethoxydim, and select aryloxyphenoxypropionate (FOPs) herbicides, fenoxaprop, and fluazifop. Two resistant southern crabgrass [Digitaria ciliaris (Retz.) Koeler] biotypes, R1 and R2, containing an Ile-1781-Leu amino acid substitution and previously identified as resistant to sethoxydim, pinoxaden, and fluazifop but not clethodim was utilized as the resistant chloroplastic ACCase source compared to known susceptible (S) ACCase. Dose-response studies with sethoxydim, clethodim, fluazifop-p-butyl, and pinoxaden (0.6 to 40 µM) were conducted to compare the ACCase enzyme-herbicides interaction of R1, R2, and S using the malachite green functional assay. Assay results indicated that R biotypes required more ACCase-targeting herbicides to inhibit ACCase activity compared to S. IC50 values of all four herbicides for R biotypes were consistently an order of magnitude greater than S. No sequencing differences in the carboxyltransferase domain was observed for R1 and R2, however, R2 IC50 values were greater across all herbicides. These results indicate the malachite green functional assay is effective in evaluating ACCase enzyme activity of R and S biotypes in the presence of ACCase-targeting herbicides, which can be used as a replacement for the 14C-based radiometric functional assay.

Frost Reduces Clethodim Efficacy in Clethodim-Resistant Rigid Ryegrass (Lolium rigidum) Populations

Weed Science ◽  
2016 ◽  
Vol 64 (2) ◽  
pp. 207-215 ◽  
Author(s):  
Rupinder Kaur Saini ◽  
Jenna Malone ◽  
Christopher Preston ◽  
Gurjeet S. Gill
Keyword(s):  
Dose Response ◽  
Coenzyme A ◽  
Resistance Mechanism ◽  
Lolium Rigidum ◽  
Weed Species ◽  
Susceptible Population ◽  
Acetyl Coenzyme A ◽  
Survival Percentage ◽  
Rigid Ryegrass ◽  
Acetyl Coenzyme A Carboxylase

Rigid ryegrass, an important annual weed species in cropping regions of southern Australia, has evolved resistance to 11 major groups of herbicides. Dose–response studies were conducted to determine response of three clethodim-resistant populations and one clethodim-susceptible population of rigid ryegrass to three different frost treatments (−2 C). Clethodim-resistant and -susceptible plants were exposed to frost in a frost chamber from 4:00 P.M. to 8:00 A.M. for three nights before or after clethodim application and were compared with plants not exposed to frost. A reduction in the level of clethodim efficacy was observed in resistant populations when plants were exposed to frost for three nights before or after clethodim application. In the highly resistant populations, the survival percentage and LD50were higher when plants were exposed to frost before clethodim application compared with frost after clethodim application. However, frost treatment did not influence clethodim efficacy of the susceptible population. Sequencing of the acetyl coenzyme A carboxylase (ACCase) gene of the three resistant populations identified three known mutations at positions 1781, 2041, and 2078. However, most individuals in the highly resistant populations did not contain any known mutation in ACCase, suggesting the resistance mechanism was a nontarget site. The effect of frost on clethodim efficacy in resistant plants may be an outcome of the interaction between frost and the clethodim resistance mechanism(s) present.

REACTION OF FORMALDEHYDE AND THIOFORMALDEHYDE WITH MERCURIPAPAIN

1965 ◽  
Vol 43 (7) ◽  
pp. 1171-1177 ◽  
Author(s):  
W. A. Darlington ◽  
L. Keay
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Functional Groups ◽  
Tertiary Structure ◽  
Colorimetric Assay ◽  
Amino Groups ◽  
Full Activity ◽  
Amino Acid Side Chains ◽  
Lysine Residues ◽  
Secondary And Tertiary Structure

In a colorimetric assay with benzoyl-DL-arginine p-nitroanilide, acetylated and benzoylated papains retain full activity. Thus the ε-amino groups of the lysine residues are not required for enzyme activity. Intramolecular crosslinking of an enzyme could in theory stabilize secondary and tertiary structure and oppose denaturation. Thioformaldehyde is much more reactive with mercuripapain than formaldehyde, incorporating much more readily into the enzyme at equivalent concentrations. Incorporation is extensive, however, on reactive functional groups on the amino acid side chains, since acetylation decreased the incorporation markedly. In no case was there evidence of heat stabilization.

scholarly journals VIM-15 and VIM-16, Two New VIM-2-Like Metallo-β-Lactamases in Pseudomonas aeruginosa Isolates from Bulgaria and Germany

2008 ◽  
Vol 52 (8) ◽  
pp. 2977-2979 ◽  
Author(s):  
Ines Schneider ◽  
Emma Keuleyan ◽  
Rudolf Rasshofer ◽  
Rumyana Markovska ◽  
Anne Marie Queenan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Class I ◽  
Class I Integrons

ABSTRACT Two Pseudomonas aeruginosa urine isolates from Bulgaria and Germany produced two new VIM-2 variants. VIM-15 had one amino acid substitution (Tyr218Phe) which caused a significant increase in hydrolytic efficiency. The substitution Ser54Leu, characterizing VIM-16, showed no influence on enzyme activity. Both genes were part of class I integrons located in the chromosome.

scholarly journals Electrogenic Sodium–Sodium Exchange Carried Out by Na,k -Atpase Containing the Amino Acid Substitution Glu779ala

2000 ◽  
Vol 116 (1) ◽  
pp. 61-74 ◽  
Author(s):  
R. Daniel Peluffo ◽  
José M. Argüello ◽  
Jerry B Lingrel ◽  
Joshua R. Berlin
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
High Capacity ◽  
Outward Current ◽  
Α Subunit ◽  
High Affinity ◽  
High Level ◽  
Kinetics Of ◽  
Insight Into

Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the α subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+–Na+ exchange activityin the absence of K +. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 ± 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K +. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K +. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K +-free solutions. Electrogenic Na+–Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+o) concentration. At all Na+o concentrations tested (3–148 mM), exchange current was maximal at negative membrane potentials (VM), but decreased as VM became more positive. Analyzing this current at each VM with a Hill equation showed that Na+–Na+ exchange had a high-affinity, low-capacity component with an apparent Na+o affinity at 0 mV (K 00.5) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a K 00.5 of 120 ± 13 mM (n = 17). Both high- and low-affinity exchange components were VM dependent, dissipating 30 ± 3% and 82 ± 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+–Na+ exchange could be explained by Na+o acting as a low-affinity K + congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+o binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K -ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47–59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K -ATPase.

scholarly journals A New World Monkey Resembles Human in Bitter Taste Receptor Evolution and Function via a Single Parallel Amino Acid Substitution

2021 ◽  
Author(s):  
Hui Yang ◽  
Songlin Yang ◽  
Fei Fan ◽  
Yun Li ◽  
Shaoxing Dai ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
New World ◽  
Bitter Taste ◽  
World Monkey ◽  
Taste Receptor ◽  
Feeding Preference ◽  
Functional Assay ◽  
New World Monkey ◽  
Dietary Preference

Abstract Bitter taste receptors (Tas2Rs) serve as a vital component in the defense system against toxin intake by animals, and the family of genes encoding these receptors has been demonstrated, usually by family size variance, to correlate with dietary preference. However, few systematic studies of specific Tas2R to unveil their functional evolution have been conducted. Here, we surveyed Tas2R16 across all major clades of primates, which represent diverse feeding ecologies, and observed a rare case of a convergent change to increase sensitivity to β-glucopyranosides in human and a New World monkey, the white-faced saki (Pithecia pithecia). We combined evolutionary, 3D modeling and functional assay analyses to demonstrate that a parallel amino acid substitution (K172N) shared by these two species is responsible for this functional convergence of Tas2R16. Considering the specialized feeding preference of the white-faced saki, the K172N change likely played an important adaptive role in its early evolution to avoid potentially toxic cyanogenic glycosides, as suggested for the human TAS2R16 gene.

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