Effects of Synergistic Inhibition on α-glucosidase by Phytoalexins in Soybeans

To determine the mechanism of action of the effects of phytoalexins in soybeans, we analyzed α-glucosidase inhibition kinetics using Michaelis–Menten plots and Lineweaver–Burk plots. The results showed that the type of inhibition with glyceollin was competitive, that of genistein was noncompetitive, that of daidzein was uncompetitive, and luteolin showed a mixed mode of action. The Ki values were determined using a Dixon plot as glyceollin, 18.99 μM; genistein, 15.42 μM; luteolin, 16.81 μM; and daidzein, 9.99 μM. Furthermore, potential synergistic effects between glyceollin and the three polyphenols were investigated. A combination of glyceollin and luteolin at a ratio of 3:7 exhibited synergistic effects on α-glucosidase inhibition, having a combination index (CI) of 0.64244, according to the CI–isobologram equation. Collectively, these results showed that a combination of glyceollin and luteolin has the potential to inhibit α-glucosidase activity via a synergistic mode of inhibition.

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.

Enzyme-Kinetic Studies on the Interaction of Norflurazon with Phytoene Desaturase

Bleaching herbicides inhibit carotene biosynthesis in photosynthetic organisms. The interaction of norflurazon [4-chloro-5-methylamino-2-(3-trifluoromethylphenyl)-pyridazin-3(2H)one] with its target enzyme phytoene desaturase, has been characterized by enzyme-kinetic studies. A Lineweaver-Burk plot showed a non-competitive m anner for norflurazon inhibition. Binding of norflurazon to phytoene desaturase was reversible as dem onstrated by complete replacem ent of bound 14C-labeled herbicide by unlabeled norflurazon. In a linear Dixon plot the ki value for norflurazon was determ ined as 0.09 μм . With the in vitro system from Anacystis used in this study it was possible to perform structure-activity studies with selected m -phenyl-substituted pyridazinones. A linear relationship between inhibitory properties of these compounds and their lipophilicity could be established.

Characterisation of the Enzyme Intermediates of the Sarcoplasmic Transport ATPase by the Use of Inhibitors

1. Based on a detailed reaction scheme of the phosphorylation process of the sarcoplasmic transport ATPase the inhibition mechanisms of benzoctamine, DIO 9, AMP-PNP and of Ca2+-ions at relatively high concentrations (1 ~ 100 μᴍ) were determined. 2. The inhibition mechanisms were analyzed by measuring the γ-phosphate exchange between ATP and ADP and evaluated by applying conventional and an extended Dixon plot procedures. 3. The kinetic patterns of the inhibition were shown to be compatible with the assumed reaction scheme. 4. Each inhibitor combines with definite intermediates: Benzoctamine with the interm ediate species ~ P; DIO 9 with E and MgE and Ca2+ at relatively hign concentrations with E. 5. The central interm ediate blocked by benzoctamine can partially exist as -benzoctamine which is detected as phosphoprotein after acid denaturation

Biological Systems to Assay Herbicidal Bleaching

Two strains of Scenedesmus acutus were found useful to study the influence of bleaching agents on either the greening process or the fully pigmented algal cell during growth. Both physiological conditions exhibit high sensitivity to bleaching herbicides. With this new assay, contrasting bleach­ing effects with the same compound can be found allowing differentiation of the herbicidal action of bleaching agents which apparently is a multifunctional one. Furthermore, the I50 can be determined rather rapidly in a simple graphical method by a Dixon plot. A subsequent application of bleaching herbicides to cultures of the fungus Phycomyces blakesleeanus rules out a possible specific action of the compounds assayed on chlorophyll or photosynthetic redox carriers. This latter assay can show whether or not the herbicides synthesis as is the case with difunon or SAN 9789.