Atrazine binding

A. Bronshtein, N. Aharonson, D. Avnir, A. Turniansky, and M. Altstein, Sol-gel matrixes doped with atrazine antibodies atrazine binding properties. Chem. Mater. 9, 2632-2639 (1997). 

Fig. 5. Example of competitive and displacement assays. (A) Competitive assay of atrazine, binding curves obtained for mixtures of MAb (ascites fluid, 1,000x diluted) preincubated for 15 min with variable concentrations of atrazine. (B) Displacement of antibody from the sensor in the presence of increasing concentrations of the analyte -peptide representing a surface epitope of the antigen. The antigen-modified sensor was preincubated with antibody (not shown), thus a constant amount of immunocomplexes (fa 350 Hz) was present before starting each of the measurements.

Fig. 3 Sensorgrams obtained from direct atrazine SPR biosensor for atrazine binding to the heavy-subunit-histidine-tagged photosynthesis reaction center (HHisRC) immobilized on the sensor chip atrazine concentrations 1-100 p.gmL [26]...

These data indicate that the membrane became more compact and less ion-permeable at lower pH and this permeability decreased even more as a result of atrazine binding. These results can be explained by protein conformation changes upon interaction with herbicide. It is noteworthy that the responses to atrazine were noticeable in the acid media only and that these responses were irreversible. Because of poorly controllable properties and disordered structure of the cross-linked BSA matrix, reproducibility of sensor performance was insufficient. 

Figure 2A. Comparison of data derived from studies of C atrazine binding for thylakoid membranes incubated with 2 /xg of trypsin/mL for various times (0-20 min) with that obtained from direct analysis of electron transport capacity (H2O...

Figure 9 Graph of B/T vs. amount of polymer for " C-atrazine binding to an atrazine MIP in 25 mM citrate buffer pH 6.0/EtOH (9 1 v/v) [24], Circles, MIP squares, control polymer...

The discrimination between wild type and mutant thylakoids shown by both the benzylamino (I) and N-methyl anilino (II) cyanoacrylates (Table 1) suggest that they, like atrazine, bind to the peptide in the wild type close to the serine2 4 residue. Likewise, the lack of discrimination shown by the thiolate salt (III) implies that it binds away from the modified amino acid region. 

Therefore, we concluded that i) in PSII preparations depleted in extrinsic polypeptides plus part of Mn + addition of exogenous Mn2+ enhances atrazine binding probably contributing partially the native conformation to the PSII core complex and ii) in preparation where 16 and 23 kDa polypeptides are depleted and Ca and Cl" are disorganized, addition of exogenous CaCl2 reduces atrazine inhibition, probably producing barrier in atrazine accessibility to its sites of action in the vicinity of the PSII complex. From the overall discussion... 

Photoinhibition at 20 C caused a significant loss of atrazine binding sites. This degradation of D1-protein was strongly inhibited at 0 C compared to 20 C (Fig.l). probably due to the inhibition of a membrane bound protease activity through lowering the temperature. The functional properties of reaction centre II. i.e. PSII-photochemistry (H2O —> SiMo)... 

FIGURE 1. The time course of photoinhibition at 20 C and 0 C. Symbols represent ( , ) atrazine binding sites,... 

FIGURE 3. The decline of electron transport capacity H2O -> Fecy) (a) and atrazine binding sites (b) in the presence of i) SOD/catalase ), ii) glutathione and ascorbate (a) or a combination of i) and il) (a) (O) represents controls without additions. 

Attached leaves of pumpkin (Cucurbita pepo L.) were photoinhibited at 750, 1500 or 2500 umol PAR m 2s either at room temperature (RT) or at 1 0, in saturated humidity. After the treatment, different photosynthetic parameters were measured either from leaf discs (fluorescence induction at 77 K, apparent quantum yield of oxygen evolution), or from thylakoids isolated from treated leaves (electron transfer activities, fluorescence induction in the presence of DCMU or FeCN, atrazine binding). 

The low-temperature inhibition was further characterized by an extensive study of thylakoid functions. When the light treatment was given at lOQ, the whole-chain (WC) activity was inhibited much more than PSII electron transfer to PPBQ (Fig. 2.a.). At RT, PPBQ-dependent oxygen evolution was slightly more inhibited than WC-activity (Fig. 2. a.). After 2-h treatments at 1500 umol PAR m 2s , the number of atrazine-binding sites had decreased from U.5 nmol atrazine bound per mg chi to U.U at room temperature and to 3.8 at I C. Thus, much faster... 

The absolute content of PSII-core complexes was determined as the concentration of atrazine-binding sites. The concentration of PSI-core complexes was estimated from quantitative measurements of the light induced absorbance change at 700 nm. 

In order to confirm directly, whether the content of PSII core Chla-protein complex varies, depending on the light conditions, the amount of Qb was quantified by determination of the number of atrazine binding sites. As seen in Table 5, low light thylakoids have a lower content of Qb than ordinary light thylakoids. This fits well with the results from gel electrophoresis. The other electron-transfer components, such as Cytf also become less abundant for low light thylakoids. On the contrary, the amount of P-700 per protein increased slightly. 

Comparison of chlorophyll a/b ratio, yield, the concetration of core Chla-proteins of PSI and PSII and Cytf content between"low light" and "ordinary light" membrane preparations. Chla/b Yield P-700/prot. Atrazine-bind prot. Cytf... 

Thylakoids have 400 Chl/atrazine binding site (measured in our laboratory) and 85 Z PSIIa and 15 Z VSllfi ... 

With the assumptions above and the distribution of chlorophyll between the photosystems (Table 2) we can calculate Chl/P700 or atrazine binding site. Table 3. Note that in Alternative B the antenna size of PSIa is larger than VSlfi which is an additional evidence for the heterogeneity of PSI. 

TABLE 3. Antenna sizes of the 4 different photosystems expressed as moles Chl/P700 (PSI) or Chl/atrazine binding site (PSII)... 

Measurements of photoinhibition of PS2 The ratio of variable to maximal fluorescence, Fv/Fm, was determined using a modulated fluorimeter. Leaves were dark-adapted for 15 min before being exposed to the weak, yellow modulated beam (PPFD 2 pmol m" -1) that produced Fq. White actinic light (PPFD 2500 jjmol m -l), which was saturating for Fv, was used to determine Fm. The atrazine-binding capacity of isolated thylakoids was determined using a previously described method (9). Thylakoids were isolated from mesophyll cells as described previously (10). 

Atrazine (up to 0.2 yM) did not bind to R-TM. In all other cases, herbicide incorporation increased linearly up to 0.1 yM, then decreased progressively (saturable binding). DCMU incorporation was slightly higher in S- than in R-TM. In S-TM, atrazine binding displayed a biphasic pattern, which was conserved... 

Therefore, PL would favour the binding of the two herbicides to the 32 Kd H-B protein in both S- and R-TM. In contrast, GL would facilitate atrazine binding but hinder that of DCMU. Thus GL and PL would cooperate for atrazine binding whereas they would behave as antagonists for DCMU binding. 

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