The role of binding in inhibition of hepatic microsomal metabolism by parathion and malathion

Parathion, malathion and their oxygenated analogs bind to the reduced form of cytochrome P-450 from rats and mice producing spectra with a maximum at 421 nm and a minimum at 450 nm. Determinations of microsomal heme suggest that the Soret at 421 nm is not associated with conversion of cytochrome P-450 to cytochrome P-420. $\text{In vivo}$ pretreatment with C¹⁴-parathion indicates that this insecticide covalently binds to mouse hepatic microsomal protein. These findings suggest that the mechanism by which these insecticides and their analogs inhibit hepatic microsomal metabolism is identical to their mode of inhibiting esterases, that is, covalent binding to catalytic site.     

A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTE_Ar), and are efficient enzymes with broad substrate ranges. The turnover rate of PTE_Ar for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pk_a of its leaving group. In this study, we rationalise the turnover rate of PTE_Ar for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTE_Ar binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTE_Ar variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k _cat/K _M for malathion. X-ray crystal structures for the PTE_Ar Ser308Leu\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue.     

Relative Efficacy of Some Insecticides Against Brinjal Fruit and Shoot Borer,Leucinodes orbonalis Guen., and Their Impact on Fruit Yield

Six insecticides and their eight combinations were tested for their efficacy against brinjal fruit and shoot borer, Leucinodes orbonalis. Endosulfan + deltamethrin (0.07%, 0.0025%) and endosulfan + fenvalerate (0.07% + 0.005%) were highly effective against fruit borer that recorded only 13.3% damage as compared to 69.8% in control. The other promising treatments which significantly reduced the fruit damage over the control were in the order: carbaryl + fenvalerate = dichlorvos + fenvalerate (14.9%) > malathion + fenvalerate (16.4%) > fenvalerate + deltamethrin (16.6%) > dichlorvos = carbaryl + deltamethrin = malathion = dichlorvos + deltamethrin = malathion + deltamethrin (18.3%) > endosulfan (20.0%) > carbaryl (21.6%) with mean percentage of damage 14.9, 16.4, 18.3, 20.0, 21.6 and 69.8%, respectively. Carbaryl was least effective, but its combinations with pyrethroids were proved superior over carbaryl alone. Cost - benefit ratio ranged from a minimum of 1: 5.10 (carbaryl) to a maximum of 1: 20.44 (fenvalerate). Dichlorvos + fenvalerate combination gave the highest yield of 263.45 q/ha, whereas carbaryl was least effective giving 225.7 q/ha. with a net gain of Rupees 42,443.00 (US$ 886.00) and 28,141.00 (US$ 587.49), respectively. The other treatments were intermediate between the two insecticide regimes. However, all the treatments were superior over the control which produced 113.58 q/ha with a net gain of Rupees 340.00 only.     

Use of mobility ratios to estimate binding constants of ligands to proteins in affinity capillary electrophoresis

This work evaluates the use of mobility ratios (M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (ΔM) over the concentration of ligand [L] versus ΔM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility Δμ over [L] versus Δμ in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the μ form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.     

Pharmacodynamics of malathion and carbaryl in susceptible and multiresistant German cockroaches (Dictyoptera: Blattellidae)

Studies with malathion and carbaryl were done to compare toxicity; absorption, metabolism, internal accumulation, and excretion; and in vivo inhibition of acetylcholinesterase (AChE) after topical applications to adult male susceptible (S, Orlando normal) or multiresistant (R, HRDC) German cockroaches, Blattella germanica (L.). Compared with the S strain, R cockroaches were highly resistant to malathion (about 33-fold) and only moderately resistant or tolerant to carbaryl (about 5-fold). Tests with topically applied 14C-labeled malathion and carbaryl indicated that both compounds penetrated rapidly and radioactive products were readily excreted. Rates of absorption or excretion in S and R strains did not differ significantly. Both insecticides were extensively metabolized; each yielded the same array and similar concentrations of metabolites in insects from either strain. In contrast, metabolic detoxification of malathion and carbaryl was significantly greater in R cockroaches when the insects were treated by injection. Strains did not differ significantly in the in vitro inhibition of brain AChE by either malaoxon or carbaryl. However, dramatic differences were observed between strains in the in vivo inhibition of AChE during a 6-h test period after topical treatment with malathion, and moderate but significant differences occurred between strains in the in vivo inhibition of AChE by carbaryl. These data suggest that the strong resistance to malathion and moderate resistance or tolerance to carbaryl in R cockroaches is probably a result of enhanced capability for metabolic detoxification.     

Detection and identification of sulfhydryl conjugates of p-benzoquinone in microsomal incubations of benzene and phenol

The glutathione and cysteine conjugates of p-benzoquinone are detected and conclusively identified in microsomal incubations of benzene and phenol using liquid chromatography/electrochemistry (LCEC). Identification of the compounds is based on retention time, electrochemical behavior and acid hydrolysis. The fact that both of these compounds can be detected easily in a benzene incubation provides further evidence that p-benzoquinone or the corresponding semiquinone is a product of benzene metabolism in vivo. The conjugation of p-benzoquinone with glutathione is predominantly a nonenzymatic process. This is illustrated by the fact that the addition of cytosolic glutathione-S-transferases do not significantly increase the amount of glutathione conjugate produced in a phenol incubation containing glutathione.The kinetic constants for phenol metabolism to hydroquinone by microsomal protein are calculated. As suspected, the rate of metabolism of phenol is significantly higher than the rate of benzene metabolism. The V_max for phenol metabolism was calculated to be 7.1 nmol/min/mg protein and the K_M was found to be 0.38 mM.The further oxidation of hydroquinone to p-benzoquinone appears to be primarily an enzymatic process. Incubations of just hydroquinone with glutathione at 37°C produced only a small amount of the glutathione conjugate. The addition of cytosolic protein increases the amount of p-benzoquinone produced about 10-fold. This could be due to the peroxidases found in that medium. The addition of microsomal protein and NADPH increases the amount of glutathione conjugate produced to over 100-fold of that produced nonenzymatically. This indicates that a microsomal enzyme is responsible for the oxidation of hydroquinone to p-benzoquinone in vitro and the subsequent covalent binding to macromolecules.     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.     

Comparison of the effects of inhibitors of cytochrome P-450-mediated reations on human platelet aggregation and arachidonic acid metabolism

Metyrapone and SKF-525A, together with amphenone B, a structural analogue of metyrapone, which are all inhibitors of cytochrome P-450-mediated reactiors, were shown to inhibit the arachidonic acid-induced aggregation of human platelets. Amphenone B, like metyrapone, exhibited a type II (ligand) binding spectrum with rat liver microsomal cytochrome P-450, in contrast to SKF 525A which is a type I (substrate) binding agent. Independently of their type of binding spectra and of their maximum spectral change, however, the affinity of the three compounds for rat liver cytochrome P-450 showed a close proportional correlation with their platelet aggregation inhibitory potency. All three compounds inhibited the formation of [1?¹⁴C]thromboxane B₂ from [1?¹⁴C]arachidonic acid by human platelets aggregated with collagen. The effect of metyrapone on the remaining labelled products suggested that it is a selective thromboxane synthesis inhibitor, while amphenone B exhibited activity reminiscent of cyclo-oxygenase inhibitors. SKF 525A produced complex effects possibly attributable to cyclo-oxygenase inhibition and enhanced lipid peroxidation, since it also enhanced platelet malonaldehyde formation, which the other two compounds inhibited. These data provide further support for a role of cytochrome P-450 in thromboxane synthesis and platelet aggregation.     

Effects on drug metabolism of carbaryl and 1-naphthol in the mouse

The effects of carbyl and 1-naphthol on hepatic microsomal drug-metabolizing enzyme systems were investigated. The agents were fed at a level of 25 mmol/kg of feed to groups of young male Swiss-Webster mice for 14-day periods. Body weight was depressed by carbaryl, but not by 1-naphthol. The rates of $\text{in vitro}$ metabolism of aniline and benzphetamine were greater than control rates in livers of mice fed carbaryl, but the rate of $\text{in vivo}$ hydrolysis of the carbamate insecticide Zectran was decreased by carbaryl feeding. Administration of 1-naphthol did not change the rates of $\text{in vitro}$ metabolism of either aniline or benzphetamine. Hepatic microsomal concentrations of cytochromes P-450 and b₅ were increased by carbaryl, but feeding of 1-naphthol did not affect levels of either cytochrome. Radiolabeled pentobarbital disappeared from the blood of carbaryl-fed mice more rapidly than from the blood of control animals, and carbaryl-fed mice slept a shorter period of time than controls following pentobarbital administration. The LD50 of an acute oral dose of carbaryl was increased two-fold by feeding carbaryl for 14 days. It was concluded that carbaryl is a weak inducer of hepatic microsomal drug-metabolizing activity, and that the effects observed are not likely due to 1-naphthol.     

Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs

$\text{In}$$\text{vitro}$ inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate.     

Contact toxicity of some chemical and biological pesticides to several insect parasitoids and predators

Eight pesticides; methyl parathion, malathion (organo-phosphates), toxaphene (chlorinated hydrocarbon), carbaryl (carbamate), pyrethrin (plant derivative),Bacillus thuringiensis, nuclear polyhedrosis virus (Heliothis) (microbial insecticides), and 2,4-DB (postemergence herbicide) were evaluated at the minimum recommended field dose and reduced dosages for contact toxicity toBrachymeria intermedia (Hymenoptera: Chalcididae), Campoletis sonorensis (Hymenoptera: Ichneumonidae), Chelonus blackburni (Hymenoptera: Braconidae), Meteorus leviventris (Braconidae), Voria ruralis (Diptera: tachninidae), Chrysopa carnea (Neuroptera: Chrysopidae), andHippodamia convergens (Coleoptera: Coccinellidae). At minimum field dosages, percent mortality of parasitoids and predators was>27%, for the chemical insecticides. Mortality from pyrethrin was <31%, in all cases and 0% for 5 of the 8 species tested. Mortality of parasitoids and predators exposed toB. thuringiens is and NPV was<4% while mortality from 2,4-DB was<7%. The toxicity of chemical insecticides to parasitoids and predators at reduced dosages in increasing order of toxicity was malathion > carbaryl > toxaphene > methyl parathion.     

Use of meta‐analysis to predict degradation of carbaryl and malathion in freshwater for exposure assessment

Carbaryl (1‐naphthyl methylcarbamate) and malathion (diethyl mercaptosuccinate, S‐ester with O, O‐dimethyl phosphorodithioate) are insecticides used to control grasshopper infestations on rangeland. Insecticides used to control grasshopper infestations pose a hazard to aquatic organisms because although no‐spray buffer zones are observed around aquatic habitats, pesticide may be deposited by drift or mobilized from upland areas by runoff. A number of processes may affect the fate of carbaryl and malathion in the aquatic environment, but no method is available for estimating degradation over the range of conditions that occur in the field. We used results of published studies in meta‐analyses to estimate degradation models that predict half‐life of carbaryl and malathion in freshwater over temperature and pH ranges relevant to western grasshopper‐management programs. Estimated degradation models were:

In (half‐life carbaryl) = 24.3 ‐ 2.36(pH) ‐ 0.0788(t)

and In (half‐life malathion) = 5.98 + 2.84(pH) ‐ 0.326(pH ²) ‐ 0.202(t) + 0.00135(t ²)

where half‐life has units of hours, and temperature (t) has units °C. Both models accounted for a significant amount of total variation (P<0.0001) and had r²>0.97. Accuracy of these degradation models was evaluated by comparing predicted degradation of carbaryl and malathion to field and laboratory data. We suggest that use of these degradation models be restricted to conditions where water has 7 ≤. pH ≤. 10 for carbaryl, and 7 ≤ pH ≤ 8.2 for malathion.     

NADPH-cytochrome c reductase in outer membrane of kidney mitochondria. Purification and properties.

NADPH-cytochrome c reductase of vitamin D₃-deficient chick kidney mitochondria has been purified approximately 1100-fold to a specific activity of 788 nmol cytochrome c reduced/min/mg protein. Analytical gel electrophoresis of the purified enzyme revealed two bands when stained for protein, but only the more anodic band demonstrated NADPH-tetrazolium reductase activity. The relative ease of solubilization of the reductase without the use of lipases, proteases, or detergents was the first line of evidence that suggested a novel mitochondrial localization for this previously unreported NADPH-linked cytochrome c reductase. The apparent properties of the reductase suggest that the enzyme is a component of kidney mitochondrial outer membrane. The kinetic determination of Michaelis constants with respect to NADPH, cytochrome c, and NADH gave the following values: K_mNADPH = 1.7 μM, K_mcytc = 3.4 μM, and K_mNADH = 20 mM. These constants were different from those of the intact kidney microsomal reductase: K_mNADPH = 5.5 μM, K_mcytc = 10.5 μM, and K_mNADH = 13.3 μM. The mitochondrial as well as the intact microsomal reductases exhibited a ping-pong kinetic mechanism for NADPH-mediated cytochrome c reduction. Spectrofluorometric measurements demonstrated the presence of equimolar amounts of FAD and FMN. The oxidized enzyme has absorption maxima at 280 and 450 nm with a shoulder at 415 nm. Upon reduction with NADPH a distinct loss in the 450-nm absorption was observed. Ouchterlony immunodiffusion studies with rabbit antiserum to chick renal mitochondrial ferredoxin did not reveal cross-reactivity when the purified reductase was the antigen. This excludes the involvement of a ferredoxin-type iron-sulfur protein in the NADPH-mediated reduction of cytochrome c by the purified reductase.     

Contributions of three-site mutations in acetylcholinesterase and cytochrome P450 to genetic variation in susceptibility to organophosphate insecticides within a natural population of Drosophila melanogaster

In this study, we attempted to elucidate the two resistance factors conferring resistance to organophosphates within the Katsunuma population of Drosophila melanogaster (Meigen), one of which has been mapped on the second chromosome and the other on the third chromosome. With regard to the second chromosome factor, we tested susceptibility to malathion of 54 recombinant inbred lines with recombination between ltd and vg. Analyses of variance (ANOVAs) showed highly significant variation in susceptibility to malathion between recombinant lines. In addition, susceptibility of the second-chromosome resistant line to malathion was increased with additional application of piperonyl butoxide, suggesting a member of the Cyp gene family located between ltd and vg. With regard to the third-chromosome factor, we conducted inhibition assays of acetylcholinesterase (AChE) with respect to fenitroxon and carbaryl, to evaluate the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. I ₅₀ values of resistant lines, isolated from this population, were about 15 times higher for fenitroxon, and about two times higher for carbaryl, than those of susceptible lines, suggesting the contribution of mutated AChE to organophosphate resistance within the Katsunuma population. We further investigated the genetic variation in the acetylcholinesterase (Ace) gene within the newly collected Katsunuma population, by using the allele-specific polymerase chain reaction (PCR) approach, and revealed that within this population there were high frequencies of resistant-type mutations at three sites in the Ace gene, which play critical roles in altering sensitivity of AChE to organophosphate and carbamate insecticides.     

Lethal and Sublethal Effects of Insecticides on the Biological Attributes of <Emphasis Type="Italic">Zygogramma bicolorata</Emphasis> Pallister (Coleoptera: Chrysomelidae): a Biocontrol Agent of <Emphasis Type="Italic">Parthenium hysterophorus</Emphasis> L.

Acute toxicity and sublethal effects of six commonly used insecticides, i.e., malathion, carbaryl, imidacloprid, cypermethrin, dimethoate, and monocrotophos, were evaluated through biological and life table parameters of Zygogramma bicolorata Pallister in laboratory. Concentration of these insecticides was within the minimum ranges of recommended field rate. Among the insecticides tested for acute toxicity, monocrotophos and imidacloprid caused the highest mortality of third instars and prolonged the development time of treated larvae. Fecundity and egg viability were also reduced in monocrotophos-treated group. Sublethal toxicity (carryover effect) of insecticides was evaluated through life table analysis of F₁ progenies developed from surviving third instars treated for acute toxicity experiment. Survivorship was prolonged to 117 days in carbaryl treated group. Monocrotophos prolonged the overall immature development time compared to other insecticidal treatments and untreated control. Moreover, the lowest female survival, and the lowest value of life indices parameters, i.e., m _x , R ₀, r _m, and λ, was evident in monocrotophos-exposed groups compared to that in other tested insecticides. However, mean generation time (T _c) and doubling time (DT) were significantly prolonged in the insecticide-treated groups compared to those in the untreated group. A significantly greater number of females were produced in control groups than those in treated with malathion and sex ratio (proportion of male) was computed as 0.34 and 0.37, respectively. Based on the present study, it can be concluded that none of the tested insecticides can be classified as safe to Z. bicolorata. However, comparisons among the tested insecticides showed that malathion was less toxic compared to other insecticides tested.     

The lethal and sublethal effects of three pesticides on the striped lynx spider (Oxyopes salticus Hentz)

The striped lynx spider, Oxyopes salticus Hentz, is found in high abundances in agricultural fields where it forages on many agricultural pests. Pesticides are applied to these fields and can therefore impact these natural pest predators. Researchers have examined the effects of a number of pesticides on this spider and other pest predators, but many of these studies only examine how these pesticides affect mortality. More recently, researchers have begun to examine the sublethal effects of these chemicals. We examined both the lethal and sublethal effects of three common pesticides with the active ingredients bifenthrin, carbaryl and malathion. We found that only malathion significantly reduced the post‐exposure lifespan of these spiders; however, each pesticide had sublethal effects on behaviour. Exposure to malathion reduced jumping, likely an important foraging and escape behaviour. Spiders exposed to bifenthrin spent increased time grooming, which can reduce the time spent performing other important behaviours. Finally, spiders that were exposed to carbaryl surprisingly increased their prey capture rate. We show here that pesticides can not only directly affect the lifespan of the striped lynx spider but that each pesticide can cause different sublethal effects that likely impact the survival and ecology of these important pest predators.     

Soluble and membrane-bound Drosophila melanogaster CYP6G1 expressed in Escherichia coli: Purification,activity, and binding properties toward multiple pesticides

Cytochrome P450 CYP6G1 has been implicated in the resistance of Drosophila melanogaster to numerous pesticides. While in vivo and in vitro studies have provided insight to the diverse functions of this enzyme, direct studies on the isolated CYP6G1 enzyme have not been possible due to the need for a source of recombinant enzyme. In the current study, the Cyp6g1 gene was isolated from D. melanogaster and re-engineered for heterologous expression in Escherichia coli. Approximately 460 nmol L^?1 of P450 holoenzyme were obtained in 500 mL cultures. The recombinant enzyme was located predominantly within the bacterial cytosol. A two-step purification protocol using Ni-chelate affinity chromatography followed by removal of detergent on a hydroxyapatite column produced essentially homogenous enzyme from both soluble and membrane fractions. Recombinant CYP6G1 exhibited p-nitroanisole O-dealkylation activity but was not active against eleven other typical P450 marker substrates. Substrate-induced binding spectra and IC₅₀ values for inhibition of p-nitroanisole O-dealkylation were obtained for a wide selection of pesticides, namely DDT, imidacloprid, chlorfenvinphos, malathion, endosulfan, dieldrin, dicyclanil, lufenuron and carbaryl, supporting previous in vivo and in vitro studies on Drosophila that have suggested that the enzyme is involved in multi-pesticide resistance in insects.     

Studies on erythrocruorin. IV. Circular dichroism of earthworm erythrocruorin.

Circular dichroism spectra of Lumbricus erythrocruorin in the absence and in the presence of heme ligands have been analyzed under a variety of experimental conditions in view of the peculiarities in ligand binding displayed by this high molecular weight heme protein (M_r = 3 × 10⁶).The undisaociated molecule exists in a “metastable” form with high cooperativity in oxygen binding, which can be converted into a stable form with low co-operativity either by changes in pH or temperature; circular dichroism spectra of oxyerythrocruorin in the Soret region give direct evidence of a local alteration in the heme environment under the conditions which affect co-operativity in oxygen binding of the undissociated molecule. Similar, although more pronounced changes in the same spectral region are observed in the dissociated molecule of M_r = 270,000, which displays low co-operativity in oxygen binding.Deoxygenation is accompanied by an inversion in the double Soret-Cotton effect, which indicates a substantial rearrangement in the heme environment upon removal of the ligand.The double peak in the Soret region found in all erythrocruorin derivatives can be taken as an indication of a distinctive distribution of the aromatic side-chains interacting with the heme chromophore.     

In silico analyses of major active constituents of fingerroot (Boesenbergia rotunda) unveils inhibitory activities against SARS-CoV-2 main protease enzyme

Boesenbergia rotunda (L.) Mansf., commonly known as fingerroot is a perennial herb in the Zingiberaceae family with anticancer, anti-leptospiral, anti-inflammatory, antioxidant, antiulcer, and anti-herpes viral activities. While the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibitory activity of B. rotunda extract has been recently found, the active compounds contributing to this activity are yet unknown. The main protease (M^pro) enzyme is one of the most well established therapeutic targets among coronaviruses which plays a vital role in the maturation and cleavage of polyproteins during viral replication. The current work aims to identify active phytochemical substances from B. rotunda extract that can inhibit the replication of SARS-CoV-2 by using a combined molecular docking and dynamic simulation approaches. The virtual screening experiment revealed that fifteen molecules out of twenty-three major active compounds in the plant extract have acceptable drug-like characteristics. Alpinetin, Pinocembrin, and Pinostrobin have binding energies of ?7.51 kcal/mol, ?7.21 kcal/mol, and ?7.18 kcal/mol, respectively, and can suppress M^pro activity. The stability of the simulated complexes of the lead compounds with the drug-receptor is demonstrated by 100-ns MD simulations. The binding free energies study utilizing molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) show that the compounds and M^pro enzyme have favourable thermodynamic interactions, which are majorly driven by van der Waals forces. Thus, the selected bioactive phytochemicals from B. rotunda might be used as anti-SARS-CoV-2 candidates that target the M^pro enzyme.     

The binding of phenol red to rabbit renal cortex

The binding of phenol red to the microsomal fraction of rabbit kidney cortex was rapid, reversible and consisted of two independent populations of binding sites: a high affinity and low capacity class which had an association constant of 11.29 · 10³ M^?1 and a binding capacity of 2.41 μmol phenol red bound per g of protein, and a low affinity binding population with an association constant of 0.80 · 10³ M^?1 and a maximal binding capacity of 55.06 μmol per g of protein. Probenecid (0.32 mM) competitively inhibited phenol red binding to only the high affinity binding site, whereas 2,4-dinitrophenol (0.77 mM) competitively inhibited phenol red binding to both the high and the low affinity population of binding sites. The binding of phenol red was highly sensitive of the cationic composition of the medium. The affinity of phenol red to the high and the low affinity binding populations was lowered by decreasing the sodium and potassium concentrations to 19 and 6 mequiv./1, respectively; however, the maximal binding capacity was unchanged. Calcium appeared to have no effect on the phenol red binding to the microsomes. All of these considerations suggest that the high affinity phenol red binding to the microsomal fraction may represent the interaction of phenol red with the physiological receptor necessary for organic acid transport at the peritubular membrane. Phenol red binding to the low affinity binding population may indicate an intracellular binding population which contributes to the intracellular accumulation of weak organic acids.