Roles of peritrophic membranes in protecting herbivorous insects from ingested plant allelochemicals

Four mechanisms by which peritrophic membranes (PMs) potentially protect herbivorous insects from ingested allelochemicals are reviewed: adsorption, ultrafiltration, polyanion exclusion, and the capacity of PMs to act as antioxidants. Most of the research on the protective roles of PMs against ingested allelochemicals has focused on their impermeability to tannins. Adsorption of tannins by the PMs in grasshoppers may limit their permeability, but ultrafiltration of tannin complexes in the caeca is an alternative explanation. Polyanion exclusion does not explain the impermeability of caterpillar PMs to tannins (polyphenolate anions). Ultrafiltration remains the most likely mechanism by which tannins, and other tested allelochemicals, are retained in the endoperitrophic space. Although the pores in PMs are too large to impede the passage of most free allelochemicals, large allelochemical complexes are retained. Such complexes form in the gut fluid of caterpillars between tannic acid, proteins, lipids, and polyvalent metal cations, and also in the gut fluid of grasshoppers (Melanoplus sanguinipes) between some amphiphilic allelochemicals (digitoxin) and surfactant micelles. Further work is needed to examine the role of PMs as antioxidants in vivo, such as their potential to bind catalytically-active metal ions.     

Decreased Response to Feeding Deterrents Following Prolonged Exposure in the Larvae of a Generalist Herbivore, Trichoplusia ni (Lepidoptera: Noctuidae)

We investigated the role of experience with several antifeedants on the feeding behavior of a generalist herbivore, Trichoplusia ni. Second-, third-, and fifth-instar larvae of T. ni were examined for their feeding responses to plant extracts (Melia volkensii, Origanum vulgare) and individual plant allelochemicals (cymarin, digitoxin, xanthotoxin, toosendanin, and thymol), after being exposed to them continually beginning as neonates. All tested instars of T. ni were capable of showing a decreased antifeedant response following prolonged exposure to most of the antifeedants tested compared with their naive conspecifics. Cardenolides (digitoxin and cymarin) were the exceptions. The response to oregano was affected as a result of previous exposure to different concentrations of oregano, unlike M. volkensii, leading us to conclude that T. ni sensitivity varies between stimuli and cannot be generalized. To demonstrate that decreased deterrence following prolonged exposure to M. volkensii was the result of learned habituation, three aversive stimuli were used. A high concentration of the particular antifeedant, xanthotoxin, acted as a noxious stimulus and dishabituated (reversed) the decrement in the antifeedant response to M. volkensii. Cold shock or CO₂ was marginally effective in dishabituating the response. The fact that the decrease in antifeedant response can be dishabituated has implications for pest management.     

Morphology of lipid micelles containing lysolecithin.

Mixtures of lysolecithin with various phospholipids were studied by electron microscopy using negative staining. Mixtures of dipalmitoyllecithin and lysolecithin produced disc-shaped structures which were stacked in aggregates with a 6.0--6.4 nm repeat. The disc were 10--50 nm in diameter. The disc-shaped structures were best observed in equimolar mixtures of dipalmitoyllecithin and lysolecithin. When dipalmitoyllecithin was replaced by dimyristoyllecithin, the structures were rather different from those observed in the system containing dipalmitoyllecithin; a cylindrical micellar phase was predominant. Equimolar mixtures of egg lecithin and lysolecithin formed the more usual smectic, concentric lamellae (liposomes) and elongated rod-like micelles which might be bimolecular fragments of spherules. The radius of the rod-like micelles was about 6 nm. Structures of rod-like micelles were observed more frequently in the samples after incubation at room temperature and then further incubation at 0 degrees C. Equimolar mixtures of didecanoyllecithin and lysolecithin produced large amounts of elongated rod-like micelles. Beef brain sphingoymyelin showed disc-shaped structures when mixed with lysolecithin. Incorporation of cholesterol into the mixtures of dipalmitoyllecithin and lysolecithin changed the morphological structure; the size of the disc became larger and eventually liposomes were formed with an increase of cholesterol content. The structures observed in mixtures of dipalmitoyllecithin or sphingomyelin and lysolecithin closely resembled those observed in complexes of apolipoprotein and lipid.     

Permeability of the Peritrophic Envelopes of Herbivorous Insects to Dextran Sulfate: a Test of the Polyanion Exclusion Hypothesis

We tested the hypothesis that the permeability of the peritrophic envelope in herbivorous insects is greatly reduced for polyanions as a result of an extensive network of anionic sites in the proteoglycans of the matrix. 14C-Dextran sulfate (polyanionic, 8000 M(w)) and fluorescein isothiocyanate-labeled (FITC) dextran (monoanionic, 9400 M(w)) were introduced together into the endoperitrophic space of the midguts of Orgyia leucostigma (Lepidoptera) larvae and Melanoplus sanguinipes (Orthoptera) adults. In all cases more of the 14C-dextran sulfate permeated the peritrophic envelope than the FITC-dextran, the opposite of the result predicted by the polyanion exclusion hypothesis. We conclude that polyanion exclusion is not a mechanism that contributes significantly to the permeability properties of the peritrophic envelopes of these two species, or that explains the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Chitinolytic enzymes from Streptomyces albidoflavus expressed in tomato plants: effects on Trichoplusia ni

Tomato (Lycopersicon esculentum ) cultivars were transformed with genes that encode bacterial chitinolytic enzymes (i.e., endochitinase and chitobiosidase) from Streptomyces albidoflavus. Transgenic tomato plants producing these enzymes were found to have enhanced resistance to cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae), consistently reducing the growth rates of larvae. Mortality was significantly increased in two of three feeding trials. Ingestion of endochitinase and chitobiosidase not only affected development of larval T. ni from neonate to ultimate instar, but they also caused mortality and decreased insect weight when exposure began during the third instar. The results of this study provide some insight into the mode of action of the chitinolytic enzymes, by supporting the hypothesis that ingested chitinolytic enzymes damage the chitin component of the peritrophic envelope, leading to increased permeability. The size of marker molecules (FITC-dextrans) that permeated the peritrophic envelopes of T. ni feeding on transgenic plants were 50% larger than those permeating the peritrophic envelopes of T. ni feeding on the control plants. Further research is needed to more clearly identify the sites and modes of action of these chitinolytic enzymes, and the potential for synergy between these enzymes and pathogens, allelochemicals, and other environmental factors.     

Metabolism and elimination of ingested allelochemicals in a holometabolous and a hemimetabolous insect

Experiments were conducted to test the hypothesis that hemimetabolous insects such as Orthoptera utilize physical barriers in the gut to provide protection from ingested toxins, whereas holometabolous Lepidoptera metabolize such toxins more efficiently. Migratory grasshoppers, Melanoplus sanguinipes Fab. (Acrididae), eliminated the thiophene -terthienyl in their feces more quickly and metabolized it to a lesser degree than did variegated cutworms, Peridroma saucia Hübner (Noctuidae). Xanthotoxin, a phototoxic linear furanocoumarin, and digitoxin, a cardiac glycoside, underwent similar metabolic modification in both species. Rates of fecal elimination of xanthotoxin were similar, whereas digitoxin was voided more rapidly by P. saucia. Overall, our results fail to provide convincing evidence in support of the test hypothesis because the two species investigated do not differ markedly in the manner in which they metabolize and eliminate the allelochemicals utilized in these studies.     

Evidence for an intracellular site of action in the heart for two hydrophobic cardiac steroids

Although cardiac steroids (CS) have long been used to treat cardiac insufficiency, the mechanism(s) of action of these agents remain open to question. While many results indicate that inhibition of Na+,K+-ATPase underlies both the therapeutic and toxic actions of CS, other studies suggest that actions on the SR membrane system may be important. We used two experimental approaches and measurements of left ventricular diastolic pressure (LVDP) in isolated guinea pig hearts to test whether CS had an intracellular site of action. In the first approach, we compared the inotropic effects of a hydrophilic CS, ouabain, and a hydrophobic CS, digitoxin, after the activity of the Na+ pump was reduced by perfusing hearts with solutions maintained at 5 degrees C. Under these conditions, exposure of hearts to 1 microM ouabain for 60 min did not increase LVDP above control levels. In contrast, an equi-effective concentration of digitoxin (0.3 microM) increased LVDP by 40 +/- 8.5% (p < 0.01) over pre-drug control levels. In the second experimental approach, we compared the inotropic effects of ouabain and digitoxin in the presence of rapid-cooling contractures (RCC), which result in the release of SR Ca2+. Hearts were perfused with Tyrode solution or Tyrode solution containing either digitoxin (0.3 microM) or ouabain (1 microM) for 180 sec, rapidly cooled and the RCC responses were analyzed. Compared to RCC elicited in Tyrode solution alone, or in Tyrode solution containing ouabain, RCC in the presence of digitoxin reached peak amplitudes more rapidly, but elicited reduced peak amplitude values. Based on these findings, we suggest that: 1) the ability of the hydrophobic CS, digitoxin, but not the hydrophilic CS, ouabain, to produce a positive inotropic effect at 5 degrees C, when the activity of the Na+ pump is markedly reduced, is consistent with a mechanism other than Na+ pump inhibition and involves an intracellular location; and 2) the diminished RCC observed in the presence of the hydrophobic CS, digitoxin, indicate that this alternative mechanism may involve effects on the SR Ca2+ release channel.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

The effect of selected allelochemicals on germination of conidia and blastospores and mycelial growth of the entomopathogenic fungus,Paecilomyces fumosoroseus (Deuteromycotina: Hyphomycetes)

Selected allelochemicals that protect plants from invasion by plant pathogenic fungi were investigated for their activity against the entomopathogenic fungus, Paecilomyces fumosoroseus. The alkaloids tomatine, solanine, and camptothecin; the furanocoumarin, xanthotoxin; and the phenolic, tannic acid were tested for their effects on germination of conidia and blastospores and growth of mycelia. The LC50 values (corresponding to 50% inhibition of germination) for tomatine, solanine, camptothecin, xanthotoxin and tannic acid were 51.6, 95.9, 55.9, 83.0 and 72.8 mg/l respectively. When blastospores were placed on media containing a concentration of the individual allelochemicals that inhibit germination in approximately 50% of conidia, all but blastospores on tomatine had significantly less germination than did aerial conidia. Growth rates of mycelia were slowest in the camptothecin medium, followed by those of tomatine and xanthotoxin and were not significantly different from controls in the media containing solanine and tannic acid. A multitude of biotic and abiotic factors are responsible for specificity and degree of pathogenicity of entomopathogens. The effect of crop plant chemistry on the efficacy of entomopathogens should be quantified further in order to maximize their potential when used concomitantly with resistant plant varieties. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA is wrapped by the nuclear aggregates of polyamines: the imaging evidence

In the cell nucleus, putrescine, spermidine, and spermine self-assemble with phosphate ions to generate three forms of compounds, named nuclear aggregates of polyamines (NAPs), which may interact with DNA. In an in vitro setting mimicking the cell nucleus milieu, this molecular aggregation occurs within well-defined ratios. Structural and functional analogies exist between the in vitro NAPs (ivNAPs) and their extractive homologues. The present Article reports images of ivNAPs at different resolution levels. Independent of the DNA template, ivNAPs become hierarchically stacked to produce ultimately macroscopic filamentous structures. The ivNAP-DNA complexes arranged in long and repetitive structures that displayed the self-similar features of natural fractals when dehydrated onto glass slides. Atomic force microscopy showed that ivNAPs have a cyclic structure and dispose around the DNA in a tube-like arrangement. Overall, the images indicate that these aggregates envelope the genomic DNA, thus proving that NAPs play a crucial role in DNA compaction and functioning.     

Solubilization of multilamellar liposomes of egg yolk lecithin by the bile salt sodiumtaurodeoxycholate and the effect of cholesterol--a rapid-ultrafiltration study

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.     

Evidence for the existence of the same endogenous digitalis-like factor in several mammalian species

1. Endogenous digitalis-like activity was studied comparatively in four mammalian species: guinea pig, dog, cow and rat. 2. Water extracts were prepared from guinea pig, dog, cow and rat hearts and assayed by ouabain radioreceptorassay, digoxin radioimmunoassay and digitoxin radioimmunoassay. Extracts were further analysed by fractionation by gel permeation chromatography with Sephadex G-25. 3. A similar behaviour was observed with the four species in the three assays. Extracts displaced tritiated ouabain binding to its receptor and labeled digoxin analogue binding to antidigoxin antibodies in a competitive manner. Displacement of labeled digitoxin analogue to antidigitoxin antibodies did not follow Michaelis-Menten kinetics. IC50 ratios between assays were similar for the four species studied. 4. Extracts from the four species exhibited a similar pattern when fractionated with Sephadex G-25. Endogenous digoxin-like immunoreactivity eluted after the salts, suggesting that the active material is of a molecular weight of less than 1000. 5. Results suggest that a similar endogenous factor endowed with digitalis-like characteristics is present in all mammalian species.     

Permeability of the peritrophic membranes of some Diptera to labelled dextrans

The permeability of the continuous, tube-like peritrophic membrane of some Diptera was investigated. Dyes, haemoglobin, cytochrome c, horseradish peroxidase, and especially dextran fractions labelled with the fluorescent dye fluorescein isothiocyanate were used as markers. The peritrophic membrane of larvae of Aedes aegypti was permeable only to dextran with a molecular weight of less than 2,400 Daltons; Evans Blue (960.8 Daltons) permeated only slowly through this peritrophic membrane. Labelled dextran with a mol. wt of 32,000 Daltons did not penetrate the peritrophic membrane of larvae of Anopheles stephensi. Dextrans larger than 32,000 Daltons did not permeate through the peritrophic membrane of Culex pipiens, Odagmia ornata, Anisopus (Phryne) cinctus, Sarcophaga barbata and Calliphora erythrocephala. Labelled dextran with mol. wt of 4,000–6,000 Daltons penetrated only slowly, and dextran of 6,200 Daltons did not penetrate the peritrophic membranes of adults of Sarcophaga barbata. The peritrophic membranes of the blowfly, Calliphora erythrocephala, were permeated slowly by dextran of 6,200 Daltons but not by dextran of 17,200 Daltons. Dextrans are readily soluble in water where the long chains form coils of round or oval shape. Charged protein molecules are more compact with smaller radii when compared to a dextran fraction of the same molecular weight. Therefore the results of investigations on permeability have to be compared in terms of the effective radii and not of the molecular weight.     

Functional antagonism by a monoclonal antibody to digoxin in a test system of cultured rat heart myocytes

A monoclonal antibody with a high affinity for digitoxin (K_A = 0.50 nM) and digoxin (K_A = 0.55 nM) was produced by somatic cell fusion. This antibody, designated 2A3(47), displayed little cross reactivity with other glycosides. In cultured rat heart myocytes, 2A3(47), antagonized the positive chronotropic effect exerted by digitoxin but did not alter that of ouabain. Our results suggest that this monoclonal antibody may prove to be useful in treating digoxin and digitoxin intoxication.     

Synthesis and micellar formation of spin-labeled lysolecithin.

A spin-labeled lysolecithin, 1-[12'-(N-oxyl-4",4"-dimethyloxazolidine)-stearoyl]-sn-glycero-3-phosphorylcholine, has been synthesized in which the spin is covalently attached to its fatty acyl chain. The electron spin resonance spectra of this lysolecithin is an aqueous solution generally showed sharp three resonance lines superposed on a broad resonance line. Investigation of changes in the signal intensity of these spectra against the concentration of lysolecithin led to the inference that the sharp lines are due to monomers of lysolecithin while the broad one to micelles. The critical micellar concentration was consistent with that evaluated from the spectral shift of a dye. In the electron spin resonance spectra obtained from spin-labeled lysolecithin solutions with various amounts of dimyristoyllecithin, the line width of broad signal arised from micellar spin-labeled lysolecithin broadened as increase of dimyristoyllecithin. The line-broadening thus observed was briefly discussed.     

Studies on cardioactive steroids. III. Characterization of different cardiac glycosides by their effects on contractility and rhythmicity at different extracellular potassium concentrations.

In the present paper, the naturally occurring glycosides digitoxin, gitoxin, 16-acetyl-gitoxin, digoxin, cymarol, ouabain, and proscillaridin, and the semi-synthetic 16-epi-gitoxin and 16-acetyl-16-epi-gitoxin are investigated as to their inotropic action and their effects on rhythmicity at isolated spontaneously beating atria of the guinea-pig heart in dependence on the variation of the potassium concentration of the nutritive fluid ([K+]0: 1.34, 2.68, and 5.36 mM resp.). The major results are as follows. 1. Effects of raising [K+]0 from 1.34 to 2.68 mM: The range of the inotropically effective concentrations as well as the size of the maximum inotropic action are more or less strongly improved with all glycosides. The glycoside concentrations required to get inotropic maximum had to be increased to a high degree with proscillaridin and digoxin. The mean arrhythmia percentage occurring at the inotropic maximum is either decreased (gitoxin, 16-epi-gitoxin, digoxin, proscillaridin), unchanged (digitoxin, 16-acetyl-16-epi-gitoxin) or even increased (16-acetyl-gitoxin, cymarol, ouabain). The inotropic value is improved to a high extent with gitoxin only. 2. Effect of raising [K+]0 from 2.68 to 5.36 mM: The range of the inotropically effective concentrations is extended (digitoxin and cymarol) or diminished (proscillaridin), but remains essentially unchanged with most glycosides. The size of the maximum inotropic effect is increased with digoxin, ouabain and 16-epi-gitoxin, but decreased significantly with digitoxin and proscillaridin. The glycoside concentrations required to produce the inotropic maximum are essentially unchanged with the exception of 16-epi-gitoxin, 16-acetyl-gitoxin and ouabain. The mean arrhythmia percentage at the maximum inotropic effect is dramatically reduced with digoxin, cymarol and proscillaridin. The inotropic value is improved with all glycosides except digitoxin. 3. Evaluation of the various glycosides: When judged on the basis of the range of inotropically effective concentrations, the maximum inotropic effect, the mean arrhythmia percentage at the inotropic maximum and the inotropic value, the best first three glycosides include 16-epi-gitoxin and digoxin. 16-Epi-gitoxin and its 16-acetate show that most favourable relationship between the effect on contractility and rhythmicity. The cause of the differential actions of the structurally-different glycosides on contractility and rhythmicity is hypothesized to be due to divergences in structure and/or conformation of the receptor areas of (Na+ + K+)-ATPase of contractile and excitable cells.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Transport functions of the liver. Lack of correlation between hepatocellular ouabain uptake and binding to (Na+ + K+)-ATPase

Ouabain uptake was studied on isolated rat hepatocytes. Hepatocellular uptake of the glycoside is saturable (Km = 348 mumol/l, Vmax = 1.4 nmol/mg cell protein per min), energy dependent and accumulative. Concentrative ouabain uptake is not present on permeable hepatocytes, Ehrlich ascites tumor cells and AS-30D ascites hepatoma cells. There is no correlation between ouabain binding to rat liver (Na+ + K+)ATPase and ouabain uptake into isolated rat hepatocytes. While ouabain uptake is competitively inhibited by cevadine, binding to (Na+ + K+)-ATPase is not affected by the alkaloid. Although the affinities of digitoxin and ouabain to (Na+ + K+)-ATPase are similar, digitoxin is 10000-times more potent in inhibiting [3H]ouabain uptake as compared to ouabain. That binding to (Na+ + K+)-ATPase appears to be no precondition for ouabain uptake was also found in experiments with plasmamembranes derived from Ehrlich ascites tumor cells and AS-30D hepatoma cells. While tumor cell (Na+ + K+)-ATPase is ouabain sensitive, the intact cells are transport deficient. Hepatic ouabain uptake might be related to bile acid transport. Several inhibitors of the bile acid uptake system also inhibit ouabain uptake.     

Demonstration and Characterization of two Classes of Cardiac Glycoside Binding Sites to Rat Heart Membrane Preparations Using Quantitative Computer Modeling

Abstract

Cardiac glycoside binding to rat heart membrane preparations was measured by rapid filtration technique. The binding data were analyzed using quantitative computer analysis. The experimental results using [³H]-ouabain as the labeled ligand were consistent with a model in which cardiac glycoside specific binding occurs at two independent classes of sites. The high affinity sites were characterized by a dissociation constants of 40 nM, 50 nM, and 61 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 1.3 pmoles/mg protein. The lower affinity sites for ouabain were characterized by dissociation constants of 2.3 µM, 67 nM and 71 nM for ouabain, digoxin and digitoxin, respectively, with a binding capacity of 3 pmoles/mg protein. Potassium ions inhibit [³H]-ouabain binding in a dose dependent manner with an IC₅₀ of 500 µM. Quantitative computer modelling indicated that potassium inhibits ouabain binding at both binding sites.     

Molecular analysis of CYP321A1, a novel cytochrome P450 involved in metabolism of plant allelochemicals (furanocoumarins) and insecticides (cypermethrin) in Helicoverpa zea

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.