Michael G. Wolfersberger

Bacillus thuringiensis toxin inhibits K+‐gradient‐dependent amino acid transport across the brush border membrane of Pieris brassicae midgut cells

The luminal membrane of larval midgut cells is the site of action of insecticidal delta‐endotoxin from Bacillus thuringiensis. At concentrations that correspond to normal effective doses in vivo, the toxin inhibits the uptake of amino acids by brush border membrane vesicles prepared from midguts of Pieris brassicae larvae. The toxin does not interact with the K+‐amino acid symport but rather increases the K+ permeability of the membrane. The toxin does not increase the permeability of lepidopteran midgut brush border membrane to either Na+ or H+ nor does it increase the K+ permeability of brush border membrane vesicles prepared from mammalian small intestine,

ISOLATION OF GOBLET CELL APICAL MEMBRANE FROM TOBACCO HORNWORM MIDGUT AND PURIFICATION OF ITS VACUOLAR-TYPE ATPASE

Publisher Summary This chapter focuses on the isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase. The only known method for isolation of the goblet cell apical membrane from larval lepidopteran midgut is described in this chapter. This chapter also described is the purification of the ATPase associated with this membrane as well as procedures for measuring both membrane-bound and purified solubilized ATPase activity. The chapter also discusses a detailed description of reliable and sensitive colorimetric methods for measurement of inorganic phosphate and protein is included. This chapter presents a discussion on the isolation of the goblet cell apical membrane. The isolated posterior midgut segment is placed lumen side down and the Malpighian tubules are removed.this chapter concludes with the discussion on Purification of the goblet cell apical membrane ATPase.

Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut

Purified goblet cell apical membranes from Manduca sexta larval midgut exhibit a specific ATPase activity approx. 20-fold higher than that in the 100 000 X g pellet of a midgut homogenate. The already substantial ATPase activity in this plasma membrane segment is doubled in the presence of 20-50 mM KCl. At ATP concentrations ranging from 0.1 to 3.0 mM, the presence of 20 mM KCl leads to a 10-fold increase in the enzymes affinity for ATP. ATPase activity is greatest at a pH of approx. 8. In addition to ATP, GTP serves as a substrate, but CTP, ADP, AMP and p-nitrophenyl phosphate do not. Either Mg2+ or Mn2+ is required for activity and cannot be replaced by Ca2+ or Zn2+. The ATPase activity of goblet cell apical membranes is inhibited by neither the typical (Na+ + K+)-ATPase inhibitors, ouabain and orthovanadate, nor by the typical mitochondrial F1F0-ATPase inhibitors, azide and oligomycin. Although 1.5 microM DCCD is ineffective, 150 microM DCCD leads to total inhibition of ATPase activity. The ATPase activity of goblet cell apical membranes is stimulated not only by K+, but also, in order of decreasing effectiveness, by Rb+, Li+, Na+ and even Mg2+. Replacement of Cl- by Br-, F- and HCO3- has less influence than variation of the cations. However, replacement of Cl- by NO3- inhibits strongly this ATPase activity. The ATPase activity described above is characteristic of the alkali metal ion pump containing apical membranes of goblet cells and is not enhanced to a similar degree in other purified midgut epithelial cell plasma membrane segments. Its localization, its broad cation specificity and its insensitivity to ouabain all mimic properties of active ion transport by the lepidopteran midgut and suggest this ATPase as a possible key component of the lepidopteran electrogenic alkali metal ion pump.

Isolation of separate apical, lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

The tissue used in this study was the midgut of the tobacco hornworm larva, Manduca sexta. The midgut epithelium is a single layer of cells resting on a thin basal lamina and underlying discontinuous muscle layer. The epithelial cells are of two main types, goblet and columnar cells, joined together by the septate junctions characteristic of insect epithelia. From this tissue we were able to isolate four distinct plasma membrane fractions; the lateral membranes, the columnar cell apical membrane, the goblet cell apical membrane and a preparation of basal membranes from both cell types. The lateral membranes were isolated by density gradient centrifugation following gentle homogenization of the midgut hypotonic medium, which caused the cells to rupture at their apical and basal surfaces, releasing long segments of lateral membranes still joined by their septate junctions. For isolation of apical and basal membranes the tissue was disrupted by ultrasound, based on the light microscopic observation that carefully controlled ultrasound can be used to disrupt each cell in layers starting at the apical surface. The top layer contained the columnar cell apical membrane, which consists of microvilli forming a brush border covering the lumenal surface of the epithelium. The second layer contained the goblet cell apical membrane, which is invaginated to form a cavity occupying the apical half of the cell, and the third layer contained the basal membranes. As each layer was stripped off the epithelium it was collected and the plasma membrane purified by differential or density gradient centrifugation. For all four membrane fractions, the isolation procedure was designed to preserve the original structure of the membrane as far as possible. This allowed electron microscopy to be used to follow each step in the isolation procedure, and to identify the constituents of each subcellular preparation. Although developed specifically for M. sexta midgut, these techniques could readily be modified for use on other epithelia.

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.

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.

Comparison of brush border membrane vesicles prepared by three methods from larval Manduca sexta midgut

Brush border membrane vesicles (BBMV) were prepared from freshly isolated posterior larval Manduca sexta midguts by differential calcium precipitation, differential magnesium precipitation and differential ultrasonication. BBMV were also prepared from frozen posterior larval M. sexta midguts by differential calcium precipitaion and differential magnesium precipitation. The yield of BBMV by both differential precipitation methods was 5–6 times greater than that by the differential ultrasonification method. Enrichments of the brush border membrane marker enzymes alkaline phosphatase, γ-glutamyl transferase, and aminopeptidase were similar in all preparations. The polypeptide composition of all preparations was also similar. The specific activity of mitochondrial and microsomal marker enzymes was higher in BBMV prepared from freshly isolated midguts by the differential precipitation methods than in BBMV prepared by the ultrasonication method. The specific activity of cytochrome-c oxidase was 2.5–7 times higher in BBMV prepared from frozen midguts than in BBMV prepared from fresh tissue.

Pathogenesis of Bacillus thuringiensis toxins

The mode of action, and hence the histopathological events of all the δ-endotoxins produced as secondary metabolites by the species Bacillus thuringiensis are identical. The protein particles produced during the sporulation process have to be ingested by the target insects. In the intestine they are dissolved and undergo activation by gut juice proteinases. Following receptor mediated binding pores are formed leading to the loss of normal membrane function. The gut epithelial cells swell. Microvilli and other cell organelles are distorted along with the formation of vacuoles. The pathogenesis, the site and mode of action, is unique for an insecticidal metabolite. The process involved in the pathogenesis explains the specificity and the high level of safety of Bt products. The possible origin of the δ-endotoxin, reflecting a close connection between the target insects and the bacterium is discussed.

LOCALIZATION OF AMINO ACID ABSORPTION SYSTEMS IN THE LARVAL MIDGUT OF THE TOBACCO HORNWORM MANDUCA SEXTA

Abstract Brush border membrane vesicles were prepared separately from the three morphologically distinct regions of larval Manduca sexta midguts by Mg/EGTA precipitation and differential centrifugation. Inwardly directed potassium thiocyanate gradients resulted in transient accumulation of arginine, leucine and proline by vesicles prepared from posterior midgut but only arginine by vesicles prepared from anterior midgut.