J.H. Anstee

Effects of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera

Soybean Kunitz trypsin inhibitor (SBTI) and soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) were incorporated into artificial diet and fed to newly moulted 3rd-instar larvae of Helicoverpa armigera, the corn earworm. 1. 1. Neither SBTI nor SBBI affected larval survival for 14 days. After this time, many of the larvae fed diets with high levels of inhibitor suddenly died. This effect was most marked with SBTI diets. 2. 2. When compared with the effect of the control diet (no inhibitor), inhibitors in the diet reduced total larval biomass and mean larval weight. This effect on larval growth was much greater with dietary SBTI than with SBBI. The relative effectiveness of the inhibitors in vitro is in agreement with previous data showing that SBTI was a more effective inhibitor of the H. armigera trypsin-like gut protease in vitro. 3. 3. The presence of inhibitor in the diet reduced the mean larval weight at moult, suggesting a possible response of ecdysis to semi-starvation. This effect was greater with SBTI than with SBBI. 4. 4. Faecal output was used as a measure of food intake. By day 10 of the feeding trials, the mean faecal output/larva was similar for larvae fed the control and SBBI diets, but it was significantly lower for larvae fed dietary SBTI. However, when faecal output was corrected to account for developmental states and weights of larvae, no differences were found between the effects of control and inhibitor diets. 5. 5. Feeding larvae a diet containing 0.234 mM SBTI significantly reduced the trypsin-like enzyme activities found in their gut contents when compared with the levels found in larvae fed the control diet. The possible mechanisms of action of SBTI and SBBI in vivo are discussed, as are their relative potentials for protecting crop plants against damage by larval H. armigera.

Endoproteases from the midgut of larval Spodoptera littoralis include a chymotrypsin-like enzyme with an extended binding site

Abstract Endoproteases from the lumen of the midgut of larvae of Spodoptera littoralis were characterised with respect to their substrate specificity and pH optimum. Alkaline serine protease activities corresponding to trypsin and chymotrypsin were detected. Cysteine, aspartic or metalloprotease activities were not found, nor was serine protease activity associated with microbes. Chymotrypsin hydrolysed SA 2 PPpNA, SA 2 PLpNA, BTEE, SA 3 pNA and casein, but not BTpNA, ALpNA or SPpNA, indicating an extended binding site. It was effectively inhibited by chymostatin, PMSF, antipain, SBTI, BBTI, LBTI, CEOI and TEW, but unaffected by TPCK. Trypsin hydrolysed BApNA, TGPLpNA, TAME and BAEE. It was effectively inhibited by TLCK, benzamidine, leupeptin, chymostatin, antipain, CEOI, aprotinin, pCMPS and E-64. Chymostatin, antipain and CEOI were the only three inhibitors which were effective against both endopeptidases. Trypsin and chymotrypsin have a molecular weight of 24,000 and 19,000, respectively, as determined by gel filtration. The optimal pH for both enzymes was pH 10.0 using p-nitroanilide substrates, but lower for esterase substrates (between pH 8.0 and 9.5) and higher for casein hydrolysis by chymotrypsin (pH 11.0).

Relationship of Na+K+-activated ATPase to fluid production by malpighian tubules of Locusta migratoria

Abstract The presence of a Na+K+-activated, Mg2+-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na+K+ activated ATPase ‘pump’ is involved in fluid transport across the walls of the tubules.

Fluid and cation secretion by the Malpighian tubules of Locusta

Abstract In vitro preparations of Locusta Malpighian tubules are able to transport K+ against its concentration gradient. The ‘urine’ is slightly hyper-osmotic with respect to the bathing solution and the rate of secretion is inversely dependent on the osmotic pressure of the latter. The rate of fluid secretion increases with increasing temperature; being maximal at approx 40°C. The ionic composition of the secreted fluid, as indicated by Na+/K+ ratios, is altered by the presence of 1 mM ouabain in the bathing solution. Fluid secretion is inhibited by 1 mM ouabain. In addition, oxygen consumption by the Malpighian tubules is inhibited by either the presence of 1 mM ouabain or the absence of K+ in the bathing solution. The relationship between respiration, active transport and the Na+K+-activated ATPase is discussed.

Microelectrode studies on Malpighian tubule cells of Locusta migratoria: Effects of external ions and inhibitors

Abstract Basal and apical membrane potentials were recorded from Malpighian tubules of Locusta using intracellular microelectrodes. Ion substitution experiments, involving Na + , K + and Cl − in the bathing media, indicated that the basal membrane was more permeable to K + than Na + or Cl − . Two different electrical responses to high [K + ] salines were noted and these probably reflect distinct physiological states of basal membrane permeability. Experiments with ouabain and orthovanadate suggested that whilst (Na + + K + )-ATPase activity was not significantly electrogenic, asymmetric ionic distribution across the basal membrane was partly maintained by this enzyme. The actions of furosemide and bumetanide indicated that chloride might cross the basal membrane by a cation co-transport mechanism. In addition, data from ion substitution experiments suggest that Cl − entry across the basal membrane is K + -dependent. The responses to Na + -free and Cl − -free salines bring into question the role of Na + in the entry of chloride across the basal membrane by co-transport.

Characterization of midgut exopeptidase activity from larval Spodoptera littoralis

Abstract The presence of aminopeptidases and carboxypeptidases was investigated in the midgut of Spodoptera littoralis . Leucine aminopeptidase, cystenyl aminopeptidase and dipeptidyl aminopeptidase IV were found in the midgut tissue only and showed optimum activity between pH 7.5–8.5. The leucine aminopeptidase was partially purified and characterised with respect to a variety of inhibitors and substrates. It preferentially cleaved N-terminal methionine, leucine and alanine, but also cleaved other N-terminal amino acid substrates (aliphatic: Val, Gly; basic: Arg, Lys; aromatic: Phe; heterocyclic: Pro); hydrolysis of GlupNA was extremely slow. The molecular weights of leucine and cystenyl aminopeptidases were estimated to be about 116 kDa. Leucine aminopeptidase activity was inhibited by bestatin, 1,10 phenanthroline, Tris and several metal ions, notably copper (II), lead (II) and zinc (II). The ion-chelator EDTA, magnesium (II) and manganese (II) ions had relatively little effect on activity. Activity towards the carboxyesterase A substrate, HPLA, was present with an optimum activity at about pH 9.0. This activity was not inhibited by DSS. No carboxypeptidase activity could be detected using either HPA (carboxypeptidase A) or HA (carboxypeptidase B) in the presence or absence of an activator, DSS.

Effects of frontal ganglion removal and starvation on activity and distribution of six gut enzymes in Locusta

Abstract A study has been made on the effects of frontal ganglion removal and starvation on the activities and distribution of α- and β-glucosidase, α- and β-galactosidase, trehalase, and ‘trypsin’ in various regions of the alimentary canal of adult locusts. Both treatments resulted in a reduction in the amount of enzyme activity. In addition, the distribution of enzyme activity was changed by comparison with the operated control insects; the foregut of starved and operated animals showing a smaller proportion of the overall gut enzyme activity. The results are discussed in relation to the control of enzyme secretion.

Phenoloxidase and its zymogen from the haemolymph of larvae of the lepidopteran Spodoptera littoralis (Lepidoptera: Noctuidae)

Abstract Haemolymph serum phenoloxidase from larvae of the noctuid moth Spodoptera littoralis is present as an inactive proenzyme, prophenoloxidase. Partially purified serum prophenoloxidase was activated by methanol, but not by laminarin, lipopolysaccharides, bovine trypsin or chymotrypsin. Phenoloxidase activity was optimal between pH 7.0 and 7.5 for the oxidation of l -DOPA, with an apparent K m of 1.35 mM for this substrate. Both Mg 2+ and Ca 2+ stimulated phenoloxidase activity compared with controls and maximal stimulation was observed at about 30 mM for both ions. EDTA had little effect on activity even at high concentrations. Phenoloxidase activity was inhibited by dithiothreitol (50% inhibition at 20 μM) and kojic acid (50% inhibition at 135 μM, inhibition constant of 69 μM).

Studies on the subcellular distribution of (Na+ + K+)-ATPase, K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na+ + K+)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K+-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na+ + K+)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K+-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na+ + K+)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K+-stimulated ATPase was not associated with the basolateral plasma membrane is discussed. Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na+ + K+)-ATPase but do not support the K+-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.

Studies on ouabain-binding to (Na+ + K+)-ATPase from Malpighian tubules of the locust, Locusta migratoria L.

Abstract A study has been made on the binding of [ 3 H]ouabain to (Na + + K + )-ATPase in microsomal preparations from Malpighian tubules of Locusta migratoria . The rate constants at 30°C were 1.5 · 10 3 ± 3.5 · 10 2 M −1 · s −1 and 3.7 · 10 −3 ± 0.6 · 10 −1 for the association and dissociation of the ouabian and the receptor, respectively. This yielded a dissociation constant of 2.5 · 10 −6 M. Scatchard plots indicate heterogeneity of ouabain binding. These have been analysed on the basis that binding occurrd at two classes of independent sites. High-affinity sites were characterized by a dissociation constant of 0.2 ± 0.1 μ M and low capacity ( B max = 11.0 ± 1.2 pmol/mg protein). Low-affinity sites were characterized by a dissociation constant of 4.2 ± 1.3 μ M and B max equal to 25.9 ± 2.5 pmol/mg protein. K d for the low-affinity site was not significantly different from the I 50 value of 1.12 μM, suggesting that this class of site may be associated with inhibition of (Na + + K + )-ATPase activity. Comparison of (Na + + K + )-ATPase activity and amount of ouabain bound indicate a turnover of 2645 ATP hydrolysed/site per min. It is estimated that there are in excess of 3.4 · 10 6 high-affinity sites and 8.1 · 10 6 low-affinity sites per cell (i.e., a total of 1.15 · 10 7 sites/cell). This total site density value, taken in conjunction with the turnover number, predicts rates of metabolic demand and cation translocation which are consistent with observed values.