Amino acid transport in insects.

Most insect cell membranes seem to contain uniporters that facilitate the diffusion of amino acids into and out of the cells. In addition to these passive diffusion systems, all but one of the insect tissues studied to date seem to contain at least one amino acid-cation symport system that allows their cells to accumulate certain amino acids from the extracellular medium. cDNAs encoding three such symporters have very recently been cloned and sequenced. The deduced amino acid sequence of each insect symporter was determined to be homologous to that of symporters mediating the transport of the same or related substrates in mammalian tissues.

The Bacillus thuringiensis Cry1Ac toxin-induced permeability change in Manduca sexta midgut brush border membrane vesicles proceeds by more than one mechanism.

Aminopeptidase N purified from whole Manduca sexta midgut binds the Cry1Ac insecticidal toxin from Bacillus thuringiensis and this binding is inhibited by N-acetylgalactosamine (GalNAc). We have examined the membrane permeabilising activity of the Cry1Ac toxin using brush border membrane vesicles (BBMV) prepared from the anterior (A-BBMV) and posterior (P-BBMV) subregions of the M. sexta midgut. A toxin mixing assay demonstrated a faster rate of toxin activity on P-BBMV than on A-BBMV. In the presence of GalNAc this rapid activity on P-BBMV was reduced to the rate seen with A-BBMV. GalNAc had no effect on the rate of A-BBMV permeabilisation by Cry1Ac. Aminopeptidase N assays of A- and P-BBMV demonstrated that this Cry1Ac binding protein is concentrated in the posterior midgut region of M. sexta. It therefore appears that there are two mechanisms by which Cry1Ac permeabilises the M. sexta midgut membrane: a GalNAc-sensitive mechanism restricted to the posterior midgut region, probably involving aminopeptidase N binding, and a previously undetected mechanism found in both the posterior and anterior regions.

Site-directed mutations in the third domain of Bacillus thuringiensis delta-endotoxin CryIAa affect its ability to increase the permeability of Bombyx mori midgut brush border membrane vesicles.

A series of mutant Bacillus thuringiensis CryIAa delta-endotoxin proteins was prepared by replacing the first, second, and last arginine residues of the conserved third-domain sequence, R-521 YRVRIR-527, with other amino acids. The stable mutant proteins were bioassayed against Bombyx mori larvae and found to all be approximately half as active as wild-type CryIAa. The toxins were also tested by means of a light-scattering assay for their ability to increase permeability of larval B. mori midgut brush border membrane vesicles. Three of the mutant toxins were as active as the wild-type toxin in the vesicle permeability assay.

Bacillus thuringiensis delta-endotoxin and larval Manduca sexta midgut brush-border membrane vesicles act synergistically to cause very large increases in the conductance of planar lipid bilayers.

Brush-border membrane vesicles prepared from midguts of Manduca sexta larvae were incorporated into planar phospholipid bilayers. Addition of Bacillus thuringiensis delta-endotoxin to the buffered salt solutions bathing these bilayers resulted in large irreversible increases in conductance. At pH 9.6, the smallest toxin-dependent increase in bilayer conductance observed was 13 nS. Similar conductance increases were never observed in the absence of delta-endotoxin or in delta-endotoxin-treated bilayers not containing components of insect brush-border membranes.

Substrate structure and amino acid/K+ symport in brush-border membrane vesicles from larval Manduca sexta midgut.

The effects of amino acid sidechain length, substituent position and c chirality on amino acid/K+ symport have been examined in rapid filtration experiments on brush-border membrane vesicles prepared from larval Manduca sexta midgut. Cis-inhibition and trans-stimulation protocols were used to examine the effects of amino acid analogs on the uptake of alanine, phenylalanine, leucine and lysine, which are cotransported with K+ by a zwitterionic symporter at the high pH characteristic of the midgut in vivo. The symporter was found to translocate both L- and D-stereoisomers of alanine, leucine and lysine, but only the L-form of phenylalanine. Alterations to substrate structure that leave the charge distribution unchanged do not affect symport. Thus, moving the methyl group from C-3 to C-5 in the sequence isoleucine, leucine and norleucine has no effect on their ability to inhibit leucine symport. Increasing sidechain length among alanine homologs has little effect on their ability to inhibit alanine uptake, but increasing the sidechain length of lysine homologs from 1 to 3 methylene groups enhances cis-inhibition and trans-stimulation of lysine symport. The substantial difference in molecular charge distribution among aminobutanoic acid isomers has a large impact on alanine symport with only alpha- (or 2-) aminobutanoic acid functioning as an alanine analog. Only those changes in substrate structure that are coupled to the molecular charge distribution seem to affect symport. The tolerance of the symporter may reflect a balance mandated by the conflicting demands of selectivity and throughput.

Anomalous glutamate/alkali cation symport in larval Manduca sexta midgut.

Rapid filtration assays were used to characterize glutamate/cation uptake in brush-border membrane vesicles from the larval midgut of the lepidopteran Manduca sexta. At pH 10.5, which is close to the physiological pH in the midgut of M. sexta, an inwardly directed K+ gradient stimulated glutamate uptake, suggesting that glutamate was symported. Gradients of Na+ or Li+ were less effective. Neither Rb+ nor Cs+ stimulated glutamate uptake. Anion-specificity was less pronounced: the accumulation maximum was only slightly higher with thiocyanate (SCN-) than with Cl-, although initial uptake was noticeably faster with thiocyanate. A distinct set of amino acids that would cis-inhibit or trans-elicit glutamate uptake was not found. Even L-glutamate itself did not elicit accumulations of labeled glutamate. Taken together, these results suggest that a glutamate-specific symporter may not be present. Moreover, because glutamate symport was found to be electroneutral in vitro whereas amino acid uptake is electrophoretic in vivo, we infer that symport with K+ may not be an important mechanism of glutamate translocation by M. sexta midgut.

Cation-dependent leucine, alanine, and phenylalanine uptake at pH 10 in brush-border membrane vesicles from larval Manduca sexta midgut.

Using the rapid filtration technique, cation gradient driven leucine, alanine and phenylalanine uptake by brush-border membrane vesicles (BBMV) from the highly studied model insect, Manduca sexta, is characterized at the physiological pH of 10. The vesicles are sealed and nonspecific binding is small. Almost identical initial time courses of leucine uptake are obtained whether the vesicles are osmotically balanced initially or at equilibrium. The maximum accumulation values are also similar and the equilibrium values are identical with either treatment. Equilibrium is reached by 60 min. Amino acid accumulation is cation gradient dependent and is abolished by 18 microM valinomycin. Uptake of all three amino acids occurs over a broad pH range with maximum rates at approximately pH 10 and lower rates at pH 7.5. The cation selectivity of phenylalanine and alanine uptake changes with pH; the sequence is K+ > Na+ > Cs+ >> Rb+ = Li+ at pH 10.0, whereas K+ = Na+ at pH 8.0; the selectivity of leucine uptake is K+ = Na+ > Cs+ >> Rb+ = Li+ at pH 10. Maximum K+ driven accumulation of all three amino acids decreases with anions in the order: SCN- > NO3- > Cl- = CO(3)2- = So(4)2- = HPO(4)2- > gluconate-.Vmax values are similar for all three amino acids. There are large differences in initial uptake rates (leucine > phenylalanine = alanine), and maximum accumulation values (leucine > phenylalanine > alanine).

Neutral amino acid symport in larval Manduca sexta midgut brush-border membrane vesicles deduced from cation-dependent uptake of leucine, alanine, and phenylalanine.

Uptake of tritiated leucine, alanine, and phenylalanine was measured at the physiological pH of 10 by rapid filtration in brush-border membrane vesicles from the midgut of the larval tobacco hornworm, Manduca sexta. A 20-fold excess of unlabeled leucine, isoleucine, methionine, valine, alanine, lysine, histidine, phenylalanine, and glutamine inhibited uptake of leucine and phenylalanine, and six of these amino acids inhibited uptake of alanine, by more than 50% both in the presence and absence of a potassium ion gradient. These inhibitory amino acids also drove countertransport of leucine, alanine, and phenylalanine with accumulation ratios exceeding 2. These results are consistent with the hypothesis that leucine, alanine, and phenylalanine share a common uptake system - a broad scope B type symporter - which interacts strongly with half of the commonly occurring amino acids, interacts moderately with an additional quarter of them, but does not interact with cysteine, arginine, glutamate, aspartate, or proline.

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the gypsy moth (Lymantria dispar).

Brush border membrane vesicles were prepared from both fresh and frozen midguts of Lymantria dispar larvae by Mg/EGTA precipitation and differential centrifugation. The vesicles were enriched 10 to 13-fold, relative to the homogenate, in aminopeptidase and gamma-glutamyltransferase activity. No significant difference was found in enzyme enrichment of vesicles prepared from fresh and frozen midguts. Inwardly directed potassium salt gradients resulted in transient accumulation of leucine and lysine but not glutamic acid by the vesicles.

V-ATPase-energized epithelia and biological insect control.

Background is provided for the experimentally detailed contributions concerning the structure, distribution and function of V-ATPase-based ion pumps in insect epithelia. The mode of action of an insecticidal bacterial protein, which is dependent upon the V-ATPase-energized state in larval lepidopteran midgut for activity, is discussed.

Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin.

The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects. Upon dissolution at pH > 9 it yields a protein subunit called protoxin. Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect. It is known that this toxic activity decreases considerably above pH 10. In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties. At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx. 26% alpha-helix and approx. 45% beta-structure. As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased. The net secondary structure did not change significantly at pH values below 10. Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values. The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.

Antibodies to mammalian and plant V-ATPases cross react with the V-ATPase of insect cation-transporting plasma membranes.

In immunobiochemical blots, polyclonal antibodies against subunits of plant and mammalian vacuolar-type ATPases (V-ATPases) cross-react strongly with corresponding subunits of larval Manduca sexta midgut plasma membrane V-ATPase. Thus, rabbit antiserum against Kalanchoe daigremontiana tonoplast V-ATPase holoenzyme cross-reacts with the 67, 56, 40, 28 and 20 kDa subunits of midgut V-ATPase separated by SDS-PAGE. Antisera against bovine chromaffin granule 72 and 39 kDa V-ATPase subunits cross-react with the corresponding 67 and 43 kDa subunits of midgut V-ATPase. Antisera against the 57 kDa subunit of both beet root and oat root V-ATPase cross-react strongly with the midgut 56 kDa V-ATPase subunit. In immunocytochemical light micrographs, antiserum against the beet root 57 kDa V-ATPase subunit labels the goblet cell apical membrane of both posterior and anterior midgut in freeze-substituted and fixed sections. The plant antiserum also labels the apical brush-border plasma membrane of Malpighian tubules. The ability of antibodies against plant V-ATPase to label these insect membranes suggests a high sequence homology between V-ATPases from plants and insects. Both of the antibody-labelled insect membranes transport K+ and both membranes possess F1-like particles, portasomes, on their cytoplasmic surfaces. This immunolabelling by xenic V-ATPase antisera of two insect cation-transporting membranes suggests that the portasomes on these membranes may be V-ATPase particles, similar to those reported on V-ATPase-containing vacuolar membranes from various sources.

The toxicity of two Bacillus thuringiensis delta-endotoxins to gypsy moth larvae is inversely related to the affinity of binding sites on midgut brush border membranes for the toxins.

The delta-endotoxin from Bacillus thuringiensis subspecies kurstaki strain HD1-9 is almost 400 times more potent than the delta-endotoxin from strain HD-73 as a gypsy moth larvicide. The two delta-endotoxins compete for a high-affinity binding site on the brush border membrane of larval gypsy moth midguts. The affinity for the delta-endotoxin from strain HD-73 is much greater than the affinity for the delta-endotoxin from strain HD1-9.

The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides.

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.