Insect digestive enzymes: properties, compartmentalization and function

The use of synthetic organic insecticides developed during the last half of this century may pose risks to human health and can cause environmental prob­ lems. Consequently, interest has developed in using alternative strategies for insect pest management. One contemporary approach that has received atten­ tion is the development of Bacillus thuringiensis (Bt) toxins as insecticides. B. thuringiensis, a gram-positive bacteria, produces a proteinaceous paraspor­ al crystalline inclusion during sporulation. Upon ingestion by insects, this crystalline inclusion is solubilized in the midgut, releasing proteins called S-endotoxins. These proteins (protoxins) are activated by midgut proteases, and the activated toxins interact with the larval midgut epithelium causing a disruption in membrane integrity and ultimately leading to insect death. In spite of significant efforts directed towards the study of Bt, its use in pest control is restricted, in part because of the selectivity of Bt and in part because of its moderate efficacy. Attempts to improve these two qualities have met with only moderate success because inadequate effort has been devoted to understanding the molecular basis of selectivity and insecticidal properties of these toxins.

The mode of action of Bacillus thuringiensis endotoxins.

Epidemiological, migration, intervention, and genetic studies in humans and animals provide very strong evidence of a causal link between high salt intake and high blood pressure. The mechanisms by which dietary salt increases arterial pressure are not fully understood, but they seem related to the inability of the kidneys to excrete large amounts of salt. From an evolutionary viewpoint, the human species is adapted to ingest and excrete <1 g of salt per day, at least 10 times less than the average values currently observed in industrialized and urbanized countries. Independent of the rise in blood pressure, dietary salt also increases cardiac left ventricular mass, arterial thickness and stiffness, the incidence of strokes, and the severity of cardiac failure. Thus chronic exposure to a high-salt diet appears to be a major factor involved in the frequent occurrence of hypertension and cardiovascular diseases in human populations.

Links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases.

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.

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The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

K+-dependent phenylalanine uptake in membrane vesicels isolated from the midgut of Philosamia cynthia larvae

Experiments with intestinal brush-border membrane vesicles from lepidopteran larvae disclosed the occurrence of unique cotransporter proteins that use K+ as the driver cation for the transmembrane transfer of amino acids across the luminal border of midgut enterocytes. Six apical membrane amino acid transport systems have been identified. These systems are 1) a neutral amino acid transporter with a broad spectrum of interactions with most neutral amino acids, which is highly concentrative, strongly K+- and electrical potential-dependent, poorly stereospecific, and recognizes histidine, but not proline, glycine, or alpha-(methylamino)isobutyric acid (MeAIB); 2) a specific system for L-proline; 3) a specific system for glycine with a higher affinity for Na+ than for K+; 4) a specific system for L-lysine, which is dependent on membrane potential, is highly sensitive to external K+, and does not interact with L-arginine or neutral amino acids; 5) a specific K+-dependent process for glutamic acid, which does not recognize aspartic acid; and last, 6) an apparently unique K+- driven mechanism for D-alanine, which is potential-dependent and strongly stereospecific.

Amino acid transport systems in intestinal brush-border membranes from lepidopteran larvae.

Sodium transport across luminal membranes of proximal tubules isolated from the kidney cortex of young, prehypertensive rats of the Milan hypertensive strain (MHS) and their corresponding normotensive controls, the Milan normotensive strain (MNS), was measured. A higher sodium uptake was observed in vesicles from MHS, although membrane preparations from both strains behaved similarly as far as enzyme profile and sodium-dependent glucose transport were concerned. In the presence of an inwardly directed sodium gradient, sodium uptake depended on the relative permeability of the counterion: in the presence of 100 mM NaCl, sodium transport in MHS was 26% higher than that in normotensive controls (p less than 0.05). Also, a significantly faster sodium uptake by membrane vesicles from MHS was observed when a pH gradient and an electrical potential difference (inside-negative) were imposed across the membrane. In this condition, sodium uptake by membrane vesicles from MHS was up to 39% higher than that in control MNS (p less than 0.01). Therefore, the difference in sodium transport observed between preparations of luminal membrane from the proximal tubule of MNS and MHS seems to be due to a higher rheogenic sodium pathway in the MHS. The present results are in keeping with previous data showing an increased sodium transport across renal tubules of the MHS and support the hypothesis that the abnormality in sodium and water handling by kidneys from MHS can be related to an alteration in sodium transport across the luminal membrane of the proximal tubule cells.

Sodium and glucose transport across renal brush border membranes of Milan hypertensive rats.

SUMMARY The transepithelial electrical potential difference across the isolated midgut of Bombyx mori larvae is dependent on the presence of potassium and is unaffected by the addition of hexoses to perfusion media, whereas it is enhanced by alanine, aspartic acid, glutamic acid and the corresponding 2oxoacids, glutamine and malate. The midgut enzyme profile indicates that the substrates for the tricarboxylic acid cycle are supplied mainly by amino acid metabolism via transaminases. Accordingly, aminoxyacetate drastically reduces the intestinal transepithelial electrical potential difference stimulated by amino acids. Measurement of the free amino acid concentration in the lumen content, intestinal cells and haemolymph shows that glutamic acid, asparagine and glutamine are accumulated in the cell, whilst the haemolymph is enriched with basic amino acids and with glycine, alanine, serine and tyrosine, the major components of the silk fibroin. Therefore, amino acid metabolism directly related to the tricarboxylic acid cycle seems to be the primary source of energy for the potassium pump activity in B. mori midgut.

G. M. Hanozet

Papers

Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae)

K+-dependent phenylalanine uptake in membrane vesicels isolated from the midgut of Philosamia cynthia larvae

Amino acid transport systems in intestinal brush-border membranes from lepidopteran larvae.

Sodium and glucose transport across renal brush border membranes of Milan hypertensive rats.

Metabolic Activity Related to the Potassium Pump in the Midgut of Bombyx Mori Larvae