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The activities of three related Bacillus thuringiensis delta-endotoxins, designated CryIA(a), CryIA(b), and CryIA(c), as inhibitors of K(+)-dependent amino acid transport into membrane vesicles prepared from the anterior and posterior portions of Bombyx mori larval midgut were measured. Under experimental conditions similar to those occurring in vivo (membrane potential approximately -100 mV, inside negative; pH 7.2in/8.8out; an inwardly directed K+ gradient) CryIA(a) toxin produced a clear dose-dependent inhibitory effect on leucine uptake by both the anterior and the posterior gut membrane vesicles, giving half-maximal inhibition constants (IC50) of 2.6 +/- 0.3 and 2.1 +/- 0.2 microgram toxin/mg membrane protein, respectively. The other two delta-endotoxins were practically inactive. A dose-dependent inhibition of amino acid transport by CryIA(a) toxin was also observed on the carrier-mediated K(+)-independent component, i.e., the leucine-only form. This result strongly indicates that the activity of the K+/amino acid cotransporter is directly affected after binding of delta-endotoxin to the brush border membrane. When the extravesicular pH was lowered to pH 7.2, the interaction of CryIA(a) toxin with the brush border appeared more complex, as suggested by the Hill coefficient of the dose-response curves higher than 1. In conclusion, our data indicate that (i) CryIA(a) toxin specifically inhibited K+/leucine symport along the length of the midgut; (ii) the interaction between cotransporters and toxin is affected by the pH of the medium; and (iii) the K+/leucine cotransporter or a strictly associated protein may serve as membrane receptor for CryIA(a) delta-endotoxin in the B. mori larval midgut.

1. Activated delta-endotoxins from Bacillus thuringiensis strains toxic to lepidopteran larvae inhibit K(+)-dependent accumulation of amino acids into brush border membrane vesicles (BBMV) from the midgut of the susceptible species Bombyx mori. 2. The activated toxins interfere with the K(+)-dependent uptake of histidine into BBMV only if they are effectively active in vivo. 3. To calculate IC50 values (the toxin concentration which determines 50% of the effect), dose-response curves were performed for each toxin. The values obtained correlate well with the LD50 determined by bioassay. 4. This amino acid inhibition test could represent a rapid (3-6 hr, compared to 3-4 days for bioassay) and sensitive method for the screening of larvicidal activity of known or new recombinant delta-endotoxins.

The mechanism of uptake of the sympathomimetic drug broxaterol by Chang liver and HepG2 cell lines was investigated. When cells were incubated in the presence of low concentrations of broxaterol cell cultures take up (t1/2 = 5-10 min) and, depending on the experimental conditions, accumulate the drug such that the intracellular concentration is over 200-1000 times that in the incubation medium. The uptake was saturable and influenced by the presence of sodium and variations in external pH. These data may represent a model for tissue uptake in vivo in an attempt to investigate that selective uptake of broxaterol is involved in withdrawal of the molecule during drug therapy.

The kinetics of K(+)-leucine cotransport in the midgut of lepidopteran larvae was investigated using brush border membrane vesicles. Initial rate (3 s) of leucine uptake was determined under experimental conditions similar to those occurring in vivo, i.e. in the presence of delta psi much greater than 0 (inside negative) and a delta pH of 1.4 units (7.4in/8.8out). Leucine and K+ bind to the carrier according to a sequential mechanism, and the binding of one substrate changed the dissociation constant for the other substrate by a factor of 0.15. Both trans-K+ and trans-leucine were mixed-type inhibitors of leucine uptake. Moreover, a portion of total leucine uptake was K+ independent, and it was competitively inhibited by trans-leucine. We interpret the trans inhibitory effects to mean that the partially loaded K+ only form is virtually unable to translocate across the membrane, whereas the binary complex carrier, leucine, can isomerize from the trans to the cis side of the membrane. However, the K(+)-independent leucine uptake occurs with a Keq greater than 1, i.e. the efflux route through the partially loaded leucine only form is slower than the rate of isomerization of the unloaded carrier from trans to cis side. Taken together, these results suggest a model in which transport occurs by an iso-random Bi Bi system. Since K+ does not act as a pure competitive activator, this model is different from that proposed for most of the Na(+)-linked solutes transport agencies and may be related to the broadening of the cation specificity of the amino acid transporters in lepidopteran larvae.

The effect of ethylisopropyl-amiloride (EIPA) and phenamil on sodium uptake in renal brush border membrane vesicles from prehypertensive rats of the Milan strain (MHS) and their normotensive controls (MNS) was investigated. In the presence of both a membrane potential and a pH gradient a differential effect of EIPA and phenamil was evidenced between the two rat strains. In the absence of a pH gradient, but in the presence of a membrane potential, EIPA was about two-fold more potent than phenamil in inhibiting sodium transport in both rat strains, excluding the presence of epithelial sodium channels in our BBMV preparations. Taken together these results support the hypothesis that a structurally different Na+/H+ exchanger located on the brush border membrane may be involved in the increased tubular sodium reabsorption observed in vivo in hypertensive rats.

The effect of ethanol on sodium and glucose transport in rabbit renal brush border membrane vesicles was examined. When membrane vesicles were preincubated in the presence of ethanol the sodium-dependent D-glucose uptake was significantly inhibited. This effect, as suggested by O'Neill et al. (1986) FEBS Lett. 194, 183-188, may be due to a faster collapse of the Na+ gradient. As a matter of fact, the amiloride-insensitive sodium pathway was increased by ethanol in our brush border membrane preparation. However, sodium/D-glucose cotransport was inhibited by ethanol, although to a lesser degree, also in the absence of a sodium gradient. In addition, ethanol inhibited glucose-dependent sodium uptake, suggesting that a direct interaction with the translocator was involved. This conclusion was also supported by kinetic measurements showing a decrease of Vmax and an increase in Km for glucose in membrane vesicles treated with ethanol. Moreover, ethanol influenced the interaction of phlorizin with the cotransporter: uptake experiments performed in the presence of the two inhibitors demonstrated that phlorizin and ethanol behave as not mutually exclusive inhibitors of D-glucose transport. These data indicate that in rabbit renal brush border membranes ethanol not only affects the 'passive pathway', i.e. the sodium permeability, but it also directly interferes with carrier functions.

The (Na+,K+)-ATPase activity from the kidney cortex of the Milan hypertensive rat strain (MHS) and the corresponding normotensive control (MNS) was measured both in active solubilized enzyme preparations and in isolated basolateral membrane vesicles. Kinetic analysis of the purified enzyme showed that the Vmax value was significantly higher in MHS rats. The difference between MHS and MNS was not linked to a different number of sodium pumps, but was related to the molecular activity of the enzyme. Using basolateral membrane vesicles, an increased ATP-dependent ouabain-sensitive sodium transport was also demonstrated in MHS rats. These results support the hypothesis that a higher tubular sodium reabsorption may be involved in the pathogenesis of hypertension in this rat 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.

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.

Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.

We have developed a package program for the estimation of Michaelis-Menten parameters for enzymes that conform to different kinetic mechanisms. Data from different experimental schemes can be fitted with appropriate weighing factors to any of 6 mathematical models, corresponding to 5 kinetic mechanisms: ordered bi-bi, Theorell-Chance, rapid equilibrium random bi-bi, rapid equilibrium ordered bi-bi and ping pong bi-bi. The program also performs a significance test to discriminate between different candidate models. To illustrate the performance of the program, real data from kinetic experiments with glucose 6-phosphate from Leuconostoc mesenteroides have been fitted to different mathematical models, and the results are discussed. The program can be easily implemented for the fitting of kinetic data to any other model.

The computer program MIR previously described (R. Bianchi, G.M. Hanozet and M. Pilone Simonetta, Comput. Prog. Biomed. 16 (1983) 189) that fits trial rate laws to enzyme and transport steady-state kinetic data by the least-squares method has been enhanced. The new version MIR II is an interactive program and it consists of five major routines and a larger number of smaller program elements to perform the linear (three different functional forms) and non-linear (eleven mathematical models) regression analysis of kinetic data from enzyme and transmembrane transport experiments, also in the presence of inhibitors. Other features of the new program include a set of statistics and tests useful for the model building process, for the development of the mathematical model and for its validation and maintenance. An algorithm for fitting a straight line taking into account errors in both x and y is also provided.

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.

The mechanism of catabolite inactivation of three gluconeogenetic enzymes, fructose-1,6-bisphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase, has been studied in the yeast Saccharomyces cerevisiae. The glucose-induced inactivation of the three enzymes is remarkably retarded by preincubation of the cells with different caffeine concentrations; however, a full conservation of activity has never been obtained, even at the highest drug concentration. Caffeine modifies the metabolic effects produced in the yeast cell by exposure to glucose. It reduces the consumption rate of glucose; changes the glycolytic intermediate pattern, giving rise to a crossover point at the level of the phosphofructokinase/fructose-bisphosphatase cycle; and increases the ATP level and the energy charge. Moreover, it substantially reduces the peak of intracellular cAMP content that immediately follows glucose entry; the magnitude of this effect is dependent on the drug concentration. The effect on the change of intracellular cAMP level appears, among all metabolic effects determined by caffeine, the only plausible one to explain the interference with catabolite inactivation of enzymes. Actually a strong negative correlation between residual activity of each of the three investigated enzymes and intracellular cAMP level has been demonstrated. The existence of a common mechanism of action of cAMP, as the mediating factor for catabolite inactivation of all three enzymes, is proposed.

Electrophoretically homogeneous phosphoenolpyruvate carboxykinase (EC 4.1.1.49) from Saccharomyces cerevisiae was obtained in high yields by means of a two-step purification procedure consisting of ion-exchange chromatography and affinity chromatography on adenosine 5'-monophosphate-Sepharose 4B. In the latter step the binding of the enzyme to the resin specifically required the presence of Mn2+. The enzyme was eluted when Mn2+ was removed by addition of ethylenediaminetetraacetate to the elution buffer. Homogeneity, molecular weight, and subunit composition of phosphoenolpyruvate carboxykinase were checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. A factor which caused an underestimation of the enzyme activity in crude extracts was identified as adenylate kinase. Finally, a method is proposed for the enzymatic assay of bicarbonate using a purified phosphoenolpyruvate carboxykinase preparation.

To investigate a possible correlation between selective modification and degradation of enzymes, the susceptibility to intracellular yeast proteinases A and B of yeast enzymes treated with fatty acids was tested. Enzymes used were glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-phosphoglycerate kinase (EC 2.7.2.3), which are sensitive to the denaturing modification caused by fatty acids, and alcohol dehydrogenase (EC 1.1.1.1) which is insensitive. Proteinases and substrate enzymes were all pure preparations. Without modification by fatty acids, at neutral pH, the three enzymes are remarkably resistant to degradation by both proteinases. Treatment with myristic or oleic acid definitely enhances the susceptibility to proteolysis of the sensitive glucose-6-phosphate dehydrogenase and 3-phosphoglycerate kinase, whereas it leaves negligible that of the insensitive alcohol dehydrogenase. The selective effect of fatty acids on the degradation is pH-dependent: with proteinase A it was lost at acidic pH. Since intracellular levels of free fatty acids near or even higher than 1 mM were actually measured in yeast cells, it is possible that free fatty acids, in some cellular conditions, affect yeast enzyme composition. However, the control of specific enzyme degradation in yeast is still an open question.

1. The effect of bile acids on the activity of liver alcohol dehydrogenase (L-ADH, EC 1.1.1.1) from different mammalian organisms is species dependent. 2. The kinetic behaviour of purified L-ADH from rat and rabbit liver in presence of deoxycholic acid and with ethanol as substrate shows two rather different patterns: for rabbit enzyme deoxycholic acid acts as a full competitive inhibitor, while for rat enzyme an activation effect is observed, with an increase of both Km and Vmax. Similar patterns are obtained with the steroid substrate 3 beta-hydroxy-5 beta-androstane-17one. 3. These results show that in some species, including man, L-ADH activity can be regulated by bile acids, that could control both ethanol oxidation and their own biosynthesis since L-ADH is involved in both metabolic pathways in liver cell.

The denaturation of eight purified yeast enzymes, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, alcohol dehydrogenase, beta-fructosidase, hexokinase and glucose-6-phosphate isomerase, promoted under controlled conditions by the free fatty acids myristic and oleic, is selective. Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1 oxidoreductase, EC 1.1.1.49) is extremely sensitive to destabilization and was studied in greater detail. Results show that chain length and degree of unsaturation of fatty acids are important to their destabilizing effect, and that ligands of the enzyme can afford protection. The denaturation process results in more than one altered form. These results can be viewed in the perspective of the possibility that amphipathic substances, and in particular free fatty acids, may play a role for enzyme degradation in vivo, by initiating steps of selective denaturation.