Giorgio M. Hanozet

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

Abstract 1. 1. Brush border membrane vesicles (BBMV) were prepared from midguts of Pieris brassicae larvae by Mg/EGTA precipitation and differential centrifugation. 2. 2. Their morphology, polypeptide composition, and marker enzyme enrichment was similar to BBMV from other larval lepidopteran midguts. 3. 3. In the presence of an inwardly directed KSCN gradient, these BBMV transiently accumulated alanine, phenylalanine, histidine, lysine, or gultamic acid. 4. 4. Initial uptake rates for neutral and basic amino acids were similar in the presence of either a potassium or a sodium gradient. 5. 5. Initial uptake of glutamic acid was much more efficient in the presence of a sodium gradient.

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,

Intestinal amino acid absorption in lepidopteran larvae

The characteristics of K+-l-phenylalanine cotransport across brush border membrane vesicles isolated from the midgut of two lepidopteran larvae were studied. The amino acid is cotransported mainly with K+ and Na+, whereas other alkali metal cations are much less effective. The amino acid uptake displayed saturation kinetics with respect to external K+ concentration and with respect to external amino acid concentration. In the latter case a free diffusion component was evident. The activation by K+ involved an increase in Jmax and a decrease in Km. The involvement of alkali cations in amino acid absorption was also confirmed in the isolated midgut, by replacing K+ with other monovalent cations in the luminal side only or on both sides. A possible model for amino acid absorption in the midgut of lepidopteran larvae, with K+ as cotransported cation, is proposed and discussed.

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

Membrane vesicles prepared from the midguts of Philosamia cynthia larvae (Lepidoptera) show a concentrative uptake of phenylalanine in the presence of salt gradients. Unlike mammalian intestines, the highest accumulation of the amino acid occurs with a potassium salt gradient. Glucose is very poorly permeable across the vesicles.

Glucose-stimulated CAMP increase may be mediated by intracellular acidification in Saccharomyces cerevisiae

It has been reported that addition of glucose to cells of Saccharomyces cerevisiae grown on a sugar‐free medium causes a peak of intracellular cAMP levels. Also, it has been proposed that this effect might be mediated by plasma membrane depolarization. However, here, we observed a hyperpolarizing effect of glucose in S. cerevisiae and, in addition, no change in cAMP levels when depolarization was induced by valinomycin in the presence of K+. In contrast, treatments that induced a rapid intracellular acidification such as addition of the protonophore carbonyl cyanide p‐trifluoromethoxyphenylhydrazone at pH 5.5 but not at pH 8.0, extracellular pH shift from 8.5 to 3.5, and glucose itself, also increased the cyclic nucleotide. Thus, our data strongly support the hypothesis that intracellular acidification mediates the effect of glucose on cAMP levels.

Induction of d-Amino-acid Oxidase by d-Alanine in Rhodotorula gracilis Grown in Defined Medium

SUMMARY: The obligate aerobe yeast Rhodotorula gracilis was grown in batch culture on a chemically defined, pH-controlled medium containing glucose or d-alanine as carbon sources, ammonium or d-alanine as nitrogen sources, and d-alanine as a sole carbon and nitrogen source. Under these conditions, d-alanine induced the synthesis of d-amino-acid oxidase (EC 1.4.3.3) to an extent depending on the nutrients, the highest specific activity of the enzyme [up to 0.6 U (mg protein)−1] being detected when both d-alanine and glucose were present in the growth medium. In contrast, enzyme activity was negligible when both ammonium and glucose were present in the growth medium, even in the presence of d-alanine. The racemic mixture dl-alanine was also utilized as a source of both carbon and nitrogen for the growth of R. gracilis, but the enzyme activity appeared only after the depletion of l-alanine from the medium. Data on transmembrane transport of d-alanine in the presence of different nutrients clearly indicated that the l-isomer prevented induction by d-alanine through inhibition of the transport of the d-amino acid into cells. However, such an effect was not exerted by ammonium, indicating that this compound probably acts at the level of enzyme synthesis.

Presence of a potential-sensitive Na+ transport across renal brush-border membrane vesicles from rats of the Milan hypertensive strain

Sodium transport was measured in brush-border membrane vesicles prepared from kidney cortex of the Milan hypertensive strain (MHS) rats and the corresponding normotensive controls. In the presence of an outwardly directed proton gradient, 22Na was transiently accumulated in the vesicles. When a transmembrane electrical potential was imposed across membrane vesicles, both the accumulation ratio and the initial uptake were increased, indicating the presence of an electrogenic pathway for sodium in these membranes. The potential-dependent sodium uptake was significantly higher in MHS rats. Kinetic analysis give simple Michaelis Menten curves in the presence and in the absence of a membrane potential. In both conditions Jmax was significantly increased in MHS rats, whereas Km was the same for the two rat strains. Sodium uptake was inhibited by amiloride at concentrations that inhibit Na+-H+ exchange. The presence of the higher, potential-sensitive, sodium uptake in MHS is in agreement with studies on renal physiology which support the hypothesis that an increase in tubular sodium reabsorption may be the primary cause for the development of hypertension in this rat strain.

L- and D-alanine transport in brush border membrane vesicles from lepidopteran midgut: evidence for two transport systems.

SummaryIn brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when Kout>Kin and Kout=Kin.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.

Electrogenic K+-Basic Amino-Acid Cotransport in the Midgut of Lepidopteran Larvae

SummaryExperiments performed on isolated midgut demonstrate that the model proposed for the absorption of neutral amino acids in the K+-transporting intestinal epithelium of lepidopteran larvae applies also to the transport of the basic amino acids histidine and lysine. The characteristics of these K+-basic amino-acid cotransports have been studied in brush-border membrane vesicles. Histidine and lysine are transported by different transport agencies, which share, to a different degree, a high sensitivity to transmembrane electrical potential difference. Kinetic analysis showed thatKm for histidine and lysine increased 10-fold and three-fold, respectively, whereasVmax was only slightly modified when the electrical potential difference was abolished. The relationship between potassium concentration and histidine uptake indicates a cooperative binding of more than one potassium to the transporter. Countertransport experiments with glutamine as elicitor show that histidine and glutamine are transported through the same system.

Na-dependent uptake of phenylalanine in the midgut of a cockroach (Blabera gigantea)

SummaryA net absorption of sodium ions and ofl-phenylalanine, in the absence of chemical gradients, occurs across the isolated midgut of the cockroachBlabera gigantea. Both sodium and amino acid net fluxes were abolished by the haemolymphatic addition of the Na−K ATPase inhibitor ouabain, at a concentration 1 mM. A purified fraction of brush border membranes, prepared from the midgut tissue by Ca-precipitation, was used to investigate the occurrence of a cotransport system in the luminal membrane of the cockroach enterocyte. An inwardly directed Na gradient (100 mM outside the vesicles, 0 mM inside) drives the uphill movement of phenylalanine into the vesicular space, whilst other monovalent cations fail to induce the concentrative uptake of the amino acid. Moreover, the amino acid uptake seems to be dependent on the transmembrane potential, since inwardly directed gradients of different sodium salts determine a decreasing rate of phenylalanine uptake in agreement with the presumptive permeabilities of Na counterions. These data suggest the presence of a Na-phenylalanine cotransport system located on the brush border membrane ofB. gigantea midgut.