Structural,immunocytochemical and initial biochemical characterization of NAOH-extracted gap junctions from an insect,Heliothis virescens

Subcellular fractions enriched in gap junctions with an ultrastructure similar to those in intact insect tissue have been obtained by extracting crude membranes from the tobacco budworm Heliothis virescens (Lepidoptera: Noctuidae) with 2.5 mM NaOH. n-Octyl--d-glucopyranoside (OG) was used to further purify integral membrane proteins in the NaOH-extracted fractions. A polyclonal antibody (R16) is described that specifically labels nonextracted and NaOH-extracted gap junctions in cell fractions by electron microscope immunocytochemistry. R16 immunostaining of sectioned Heliothis testis at the light-microscope level yields a pattern of immunoreactivity consistent with the distribution of gap junctions in the tissue. R16 identifies a 40-kDa protein as a candidate gap junction protein on immunoblots of crude membrane, NaOH-extracted and NaOH/OG-extracted fractions.     

Immunocytochemical,electrophoresis, and immunoblot analysis of Heliothis virescens gap junctions isolated in the presence and absence of protease inhibitors

Gap junction-enriched fractions were prepared from larvae of the tobacco budworm Heliothis virescens using the NaOH procedure in the presence or absence of protease inhibitors and were analyzed by SDS-PAGE, immunoblotting and EM immunocytochemistry. Protease inhibitor fractions contained a 48-kDa protein in addition to the 10 proteins in fractions with and without inhibitors. Three polyclonal antibodies were used as probes for gap junction plaques and proteins: R16, against an 40-kDa candidate gap junction protein from Drosophila melanogaster; R17, against the 40-kDa candidate gap junction protein from H. virescens; and R18AP, an affinity purified antibody against a consensus sequence of N-terminal amino acids 2–21 of the H. virescens 40-kDa protein. R16, R17, and R18AP stain the 40- and 48-kDa proteins, R16 and R18AP stain a 64-kDa protein, and R16 stains an 30-kDa protein in the absence of inhibitors. Inclusion of protease inhibitors had no effect on gap junction ultrastructure. R16 and R17 label gap junction plaques in crude membrane and NaOH fractions, whereas R18AP exhibits only a low level of reactivity with gap junctions in crude membrane fractions and none with gap junctions in NaOH fractions. The results show that the 30-, 40-, 48- and 64-kDa proteins are immunologically related and are associated with gap junctions in H. virescens, the N-terminus of the 40-kDa protein is relatively inaccessible or easily lost, and the 48-kDa protein is protease-sensitive.     

Differences in the midgut proteolytic activity of two Heliothis virescens strains,one susceptible and one resistant to Bacillus thuringiensis toxins

The development of resistance to Bacillus thuringiensis toxic proteins is a growing concern because it could threaten both conventional and gene transfer use of this environmentally safe biological insecticide. The most common mechanism of resistance involves changes in binding affinity of toxin receptors in the insect midgut membrane. This has not been the case in Heliothis virescens. We have investigated changes in midgut proteolytic activity as a possibility to explain the resistance observed in this insect species. We have developed an improvement of known methods to demonstrate proteolytic activity in crude extracts. Using this method we have found differences in the proteolytic activity profile of midgut extracts of a susceptible and a resistant H. virescens strain. We also have studied the in vitro processing of CrylA(b) toxin and protoxin by midgut contents of both strains. SDS-PAGE of the in vitro degradation products showed differences between the strains. The resistant strain degrades protoxin more slowly and processes the active toxin more quickly than the susceptible strain. © 1996 Wiley-Liss, Inc.     

4-Azidophlorizin,a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes

A new phlorizin derivative ($\text{2′-O-(β-}\text{d}\text{-}\text{glucopyranosyl}\text{)-4-}\text{azidophloretin}$, 4-azidophlorizin) has been synthesized and its affinity for the d-glucose, Na⁺ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) K′_i, the constant for fully-competitive inhibition of (Na⁺, Δψ)-dependent d-glucose uptake, (2) K′_d, the dissociation constant of 4-azido[³H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) K″_i, the constant for fully-competitive inhibition of the specific ((Na⁺, Δψ)-dependent, d-glucose protectable) high-affinity [³H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (K′_i, K′_d and K″_i) were essentially equal and ranged between 3.2 and 5.2 μM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its K′_i values are some 15–20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244–248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.     

Three toxins,two receptors,one mechanism: Mode of action of Cry1A toxins from Bacillus thuringiensis in Heliothis virescens

Insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) are highly active against Lepidoptera. However, field-evolved resistance to Bt toxins is on the rise. The 12-cadherin domain protein HevCaLP and the ABC transporter HevABCC2 are both genetically linked to Cry toxin resistance in Heliothis virescens. We investigated their interaction using stably expressing non-lytic clonal Sf9 cell lines expressing either protein or both together. Untransfected Sf9 cells are innately sensitive to Cry1Ca toxin, but not to Cry1A toxins; and quantitative PCR revealed negligible expression of genes involved in Cry1A toxicity such as cadherin, ABCC2, alkaline phosphatase (ALP) and aminopeptidase N (APN). Cry1Aa, Cry1Ab or Cry1Ac caused swelling of Sf9 cells expressing HevABCC2, and caused faster swelling, lysis and up to 86% mortality in cells expressing both proteins. No such effect was observed in control Sf9 cells or in cells expressing only HevCaLP. The results of a mixing experiment demonstrated that both proteins need to be expressed within the same cell for high cytotoxicity, and suggest a novel role for HevCaLP. Binding assays showed that the toxin-receptor interaction is specific. Our findings confirm that HevABCC2 is the central target in Cry1A toxin mode of action, and that HevCaLP plays a supporting role in increasing Cry1A toxicity.     

Steady-state fluorescence polarization in planar lipid membranes. Experimental and theoretical analysis of the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-Diphenyl-1,3,5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids

In continuation of earlier work, the steady-state fluorescence polarization in a globally oriented system of planar lipid membranes was analyzed experimentally and theoretically for the fluorophores 8-anilino-1-naphthalenesulfonate, 1,6-diphenyl-1,3, 5-hexatriene, dansyllysine-valinomycin and n-(9-anthroyloxy) fatty acids. The theoretical analyses of experiments were mainly done in terms of the mean orientation of transition moments with respect to the membrane normal, an angle describing the region of hindered rotational diffusion and the coefficients of rotational diffusion perpendicular to the membrane and around the membrane normal. The nonvanishing angle between the moments of absorption and emission was taken into account. In the case of n-(9-anthroyloxy) fatty acids it was found that the orientational disorder increases significantly with the depth of the fluorophore within the membrane. In order to compare with recent results from time-dependent fluorescent polarization in globally isotropic membrane suspensions and with 2H-NMR experiments, the second moment ('order parameter') of the steady-state orientational distribution of absorption dipoles was calculated. For all fluorophores the theoretical analysis indicates a preferred orientation of absorption moments within the membrane plane.     

Kinetics of Ca2+- and ATP-dependent, voltage-controlled anion conductance in the plasma membrane of mesophyll cells of Pisum sativum

Whole-cell patch-clamp techniques were used to measure anion currents through the plasma membrane of protoplasts of mesophyll cells of expanding pea (Pisum sativum L.) leaves. Voltage-induced changes of the currents could be modelled with single exponential activation and deactivation kinetics. The anion currents were activated at negative membrane potentials. The time constant of activation, τ_act, increased from 145 ms at −140 mV to 380 ms at −20 mV. A Boltzmann fit to the activation curve, n_∞ (ΔG_Vm/ΔG_max), yielded a half-activation voltage of +27 mV. Opening and closing rate constants, α and β respectively, were calculated from the values of τ and n_∞. The currents depended on the presence of cytoplasmic Ca²⁺ concentrations higher than 10⁻⁶ M. Including 3 mM MgATP in the intracellular solution resulted in a voltage-dependent inactivation of the anion current. The conductance-voltage relation resulting from the voltage-dependent activation and inactivation had a maximum at about −25 mV. The relations of the current in pea are discussed with respect to the anion currents in guard cells and suspension-cultured tobacco cells, and its possible role in growing leaf cells. Received: 1 March 1996 / Accepted: 16 September 1996     

The Lmgpi15 gene, encoding a component of the glycosylphosphatidylinositol anchor biosynthesis pathway, is required for morphogenesis and pathogenicity in Leptosphaeria maculans

Random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m20, was analysed in detail here. The mutant phenotype was investigated by microscopic analyses of infected plant tissues and in vitro growth assays. Complementation and silencing experiments were used to identify the altered gene. Its function was determined by bioinformatics analyses, cell biology experiments and functional studies. The mutant was blocked at the invasive growth phase after an unaffected initial penetration stage, and displayed a reduced growth rate and an aberrant hyphal morphology in vitro. The T-DNA insertion occurred in the intergenic region between two head-to-tail genes, leading to a complex deregulation of their expression. The unique gene accounting for the mutant phenotype was suggested to be the orthologue of the poorly conserved Saccharomyces cerevisiae gpi15, which encodes for one component of the glycosylphosphatidylinositol (GPI) anchor biosynthesis pathway. Consistent with this predicted function, a functional translational fusion with the green fluorescent protein (GFP) was targeted to the endoplasmic reticulum. Moreover, the mutant exhibited an altered cell wall and addition of glucosamine relieved growth defects. It is concluded that the GPI anchor biosynthetic pathway is required for morphogenesis, cell wall integrity and pathogenicity in Leptosphaeria maculans.     

Manganese proteins isolated from spinach thylakoid membranes and their role in O2 evolution: I. A 56 kilodalton manganese-containing protein,a probable component of the coupling factor enzyme

The binding of endogenous manganese (Mn) to proteins released from spinach grana-thylakoid membranes by 2% cholate detergent or by osmotic shock is investigated. A mixture of 15–20 proteins is released by cholate and has been separated by isoelectric focusing in a sucrose gradient or by chromatofocusing. Mn coelutes with several proteins, but is lost upon dialysis. A dramatic redistribution of this Mn occurs in proteins released by osmotic shock in the presence of hydrophobic and hydrophilic oxidants. Maintaining an oxidizing solution potential during extraction apparently precludes reduction of the higher oxidation states of Mn to the labile Mn(II) state by reducing agents released from the membranes during lysing. This allows proteins to be separated which bind non-labile Mn ions. Under these extraction conditions, a protein is isolated which has an apparent molecular weight (M_r) of 65 000 or 56 000 on SDS-polyacrylamide gel electrophoresis depending on the sample buffer system used. The nondissociated protein occurs as a monomer of 58 kDa (90%) and an apparent dimer of 112 kDa (10%) by gel filtration. This protein binds little Mn if extracted by cholate and separated by isoelectric focusing. However, extraction by osmotic shock in the presence of oxidants and separation by chromatofocusing results in the retention of 1.9 ± 0.3 Mn ions per monomer. This protein is identical to that reported by Spector and Winget (Spector, M., and Winget, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 957–959). Contrary to their result, this protein does not reconstitute O₂ evolution when added to depleted membranes. Rabbit antibody to this purified protein inhibits O₂ evolution by 20% when incubated with intact grana-thylakoid membranes or 10–20% with partially inverted, French-pressed thylakoids. This inhibition is completely removed by 10^?3 M NH₃Cl as an uncoupler of photophosphorylation. These results support a role in Phosphorylation and a location on the outer surface of the thylakoids. This antibody also selectively binds purified coupling factor, CF₁, the multisubunit phosphorylation enzyme which is located on the outer thylakoid surface and which is known to bind two Mn ions tightly (Hochman, Y. and Carmeli, C. (1981) Biochemistry 20, 6293–6297). Thus the β-subunit of CF₁, which has a molecular weight of 56 kDa, can be identified as the locus of Mn binding in CF₁ and as the Mn protein isolated by Spector and Winget. This protein plays no role on O₂ evolution.     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes,using a rapid immunological procedure for the isolation of plasma membrane

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 μM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a ‘high K_m’ form, whereas the particulate activity had both ‘high K_m’ and ‘low K_m’ components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 μM substrate concentration, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both ‘high K_m’ and ‘low K_m’ phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at μM levels, in the liver. The ‘low K_m’ enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellylar membranes in hepatocytes.The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisiting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.