Assay of acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.

Imidazolinones and acetohydroxyacid synthase from higher plants: properties of the enzyme from maize suspension culture cells and evidence for the binding of imazapyr to acetohydroxyacid synthase in vivo.

Acetohydroxyacid synthase has been purified from maize (Zea mays, var Black Mexican Sweet) suspension culture cells 49-fold by a combination of ion exchange chromatography, gel filtration, and hydroxyapatite chromatography. Use of the nondenaturing, zwitterionic detergent 3-([3-cholamidopropyl]dimethyl-ammonio)-1-propanesulfonate was necessary to dissociate the enzyme from the heterogeneous, high molecular weight aggregates in which it appears to reside in vitro. The solubilized maize acetohydroxyacid synthase had a relative molecular mass of 440,000. The purified enzyme was highly unstable. Acetohydroxyacid synthase activities in crude extracts of excised maize leaves and suspension cultured cells were reduced 85 and 58%, respectively, by incubation of the tissue with 100 micromolar (excised leaves) and 5 micromolar (suspension cultures) of the imidazolinone imazapyr prior to enzyme extraction, suggesting that the inhibitor binds tightly to the enzyme in vivo. Binding of imazapyr to maize acetohydroxyacid synthase could also be demonstrated in vitro. Evidence is presented which suggests that the interaction between imazapyr and the enzyme is reversible. Imazapyr also exhibited slow-binding properties when incubated with maize cell acetohydroxyacid synthase in extended time course experiments. Initial and final K(i) values for the inhibition were 15 and 0.9 micromolar, respectively. The results suggest that imazapyr is a slow, tight-binding inhibitor of acetohydroxyacid synthase.

Effects of phenylamide herbicides on the physical properties of phosphatidylcholine membranes.

A number of phenylamide herbicides are observed to uncouple electron transport in isolated chloroplasts and mitochondria and alter the H+ permeability of artificial liposomes. Several of these phenylamides were incorporated into phosphatidylcholine multilamellar and small unilamellar vesicles to measure their effects on the physical properties of membranes. X-ray diffraction analysis of the multilamellar vesicles revealed that the herbicides partitioned into the hydrocarbon chain region of the bilayer, but caused only minimal perturbations on hydrocarbon chain packing. 31P-NMR spectroscopy of these multilamellar vesicles showed both a broadening and lowering of the phase transition temperature of the bilayer lipids upon addition of the herbicides. 13C-NMR spectroscopy of small, unilamellar phosphatidylcholine vesicles was performed to measure the effects of the phenylamides on the chemical shifts and the spin-lattice relaxation times of the individual phosphatidylcholine carbon atoms. None of the added compounds had any measurable effect on the 13C-NMR chemical shifts of the phosphatidylcholine. However, the herbicides significantly modified spin-lattice relaxation times of certain of the lipid carbon atoms. These results generally indicate that the herbicides orient in the lipid bilayers such that the hydrocarbon chains of the phenylamides associate with the hydrocarbon chains of the lipid, whereas the phenyl moiety resides in the polar region of the bilayer.

On the localization of ubiquinone in phosphatidylcholine bilayers.

The location of ubiquinone-10 in phospholipid bilayers was analyzed using a variety of physical techniques. Specifically, we examined the hypothesis that ubiquinone localizes at the geometric center of phospholipid bilayers. Light microscopy of dipalmitoylphosphatidylcholine at room temperature in the presence of 0.05-0.5 mol fraction ubiquinone showed two separate phases, one birefringent lamellar phase and one phase that consisted of isotropic liquid droplets. The isotropic phase had a distinct yellow color, characteristic of melted ubiquinone. [13C]NMR spectroscopy of phosphatidylcholine liposomes containing added ubiquinone indicated a marked effect on the 13C-spin lattice relaxation times of the lipid hydrocarbon chain atoms near the polar head region of the bilayer, but almost no effect on those atoms nearest the center of the bilayer. X-ray diffraction experiments showed that for phosphatidylcholine bilayers, both in the gel and liquid-crystal-line phases, the presence of ubiquinone did not change either the lamellar repeat period or the wide-angle reflections from the lipid hydrocarbon chains. In electron micrographs, the hydrophobic freeze-fracture surfaces of bilayers in the rippled (P beta') phase were also unmodified by the presence of ubiquinone. These results indicate that the ubiquinone which does partition into the bilayer is not localized preferentially between the monolayers, and that an appreciable fraction of the ubiquinone forms a separate phase located outside the lipid bilayer.

Imidazolinones: potent inhibitors of acetohydroxyacid synthase.

The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K(i) for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides.

C Nuclear Magnetic Resonance Studies of Crassulacean Acid Metabolism in Intact Leaves of Kalanchoë tubiflora.

(13)C nuclear magnetic resonance spectroscopy of intact leaves of Kalanchoë tubiflora was used to observe Crassulacean acid metabolism in vivo. (13)C signals from C-4 of malate were observed after overnight exposure of leaves to (13)CO(2). Illumination of the labeled leaves resulted in a gradual decrease in the malate signals. After a period of darkness in normal air, (13)C signals were detected in all four carbons of malate in the previously labeled leaves. The (13)C nuclear magnetic resonance spectrum of malate in solution was pH dependent, which allowed an estimation of the vacuolar pH from the whole leaf spectrum. The pH was 4.0 following a 14-hour dark period, but rose to greater than 6.0 after 6 hours of illumination.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : I. FACTORS AFFECTING NET CO(2) UPTAKE IN INTACT LEAVES AND CONTRIBUTION FROM RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE MEASURED IN VIVO AND IN VITRO.

As part of an extensive analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, we have examined the response of leaf gas exchange and ribulose-1,5-bisphosphate (RuBP) carboxylase activity to temperature. Emphasis was placed on elucidating the specific processes which regulate the temperature response pattern. The inhibitory effects of above-optimal temperatures on net CO(2) uptake were fully reversible up to 40 degrees C. Below 40 degrees C, temperature inhibition was primarily due to O(2) inhibition of photosynthesis, which reached a maximum of 65% at 45 degrees C. The response of stomatal conductance to temperature did not appear to have a significant role in determining the overall temperature response of photosynthesis. The intracellular conductance to CO(2) increased over the entire experimental temperature range, having a Q(10) of 1.2 to 1.4. Increases in the apparent Michaelis constant (K(c)) for RuBP carboxylase were observed in both in vitro and in vivo assays. The Q(10) values for the maximum velocity (V(max)) of CO(2) fixation by RuBP carboxylase in vivo was lower (1.3-1.6) than those calculated from in vitro assays (1.8-2.2). The results suggest that temperature-dependent changes in enzyme capacity may have a role in above-optimum temperature limitations below 40 degrees C. At leaf temperatures above 40 degrees C, decreases in photosynthetic capacity were partially dependent on temperature-induced irreversible reductions in the quantum yield for CO(2) uptake.

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : II. CONTRIBUTION FROM ELECTRON TRANSPORT AND PHOTOPHOSPHORYLATION.

As part of an analysis of the factors regulating photosynthesis in Agropyron smithii Rydb., a C(3) grass, the response of electron transport and photophosphorylation to temperature in isolated chloroplast thylakoids has been examined. The response of the light reactions to temperature was found to depend strongly on the preincubation time especially at temperatures above 35 degrees C. Using methyl viologen as a noncyclic electron acceptor, coupled electron transport was found to be stable to 38 degrees C; however, uncoupled electron transport was inhibited above 38 degrees C. Photophosphorylation became unstable at lower temperatures, becoming progressively inhibited from 35 to 42 degrees C. The coupling ratio, ATP/2e(-), decreased continuously with temperature above 35 degrees C. Likewise, photosystem I electron transport was stable up to 48 degrees C, while cyclic photophosphorylation became inhibited above 35 degrees C. Net proton uptake was found to decrease with temperatures above 35 degrees C supporting the hypothesis that high temperature produces thermal uncoupling in these chloroplast thylakoids. Previously determined limitations of net photosynthesis in whole leaves in the temperature region from 35 to 40 degrees C may be due to thermal uncoupling that limits ATP and/or changes the stromal environment required for photosynthetic carbon reduction. Previously determined limitations to photosynthesis in whole leaves above 40 degrees C correlate with inhibition of photosynthetic electron transport at photosystem II along with the cessation of photophosphorylation.

pH effects on light dependence of the stoichiometry of proton influx and efflux to electron transport in spinach chloroplasts.

Initial and steady state rates of proton transport at low light intensity have been measured and compared with steady state rates of electron transport in the pH range of 6.0-7.6 in envelope-free spinach chloroplasts. At pH 6-7, the H(+)/e(-) values computed using the initial rate of proton transport varied with light intensity, from a value of 2 at low light to almost 5 at high light. In contrast, the H(+)/e(-) values computed using the steady state rate of proton transport did not show a dependence on light intensity, having a constant value of 1.7±0.2. Likewise, at pH 7.6, the H(+)/e(-) ratio, computed using either the initial or steady state rates of proton transport did not vary with light intensity but was constant at H(+)/e(-)=1.7±0.1. Analysis of the light dependence of electron and proton transport allowed determination of (a) the quantam requirements of transport, (b) the rates of transport at light saturation, and (c) H(+)/e(-) ratios for initial and steady state proton transport. Extrapolating the initial proton transport to zero light, we found that both H(+)/photon and H(+)/e(-) values were not strongly dependent on pH, approaching a near constant value of 2.0. Using the initial rate of proton transport extrapolated to saturating light intensity we found the H(+)/e(-) ratio to be strongly pH-dependent. We suggest that internal pH controls electron transport at high light intensities. The true stoichiometry is reflected only in measurements taken at low light using the initial proton transport data. Our findings and interpretation reconcile some conflicting data in the literature regarding the pH-dependence of the H(+)/e(-) ratio and support the concept that internal pH controls noncyclic electron transport.