Digestive proteolytic activity in the gut and salivary glands of the predatory bug Podisus maculiventris (Heteroptera: Pentatomidae); effect of proteinase inhibitors

Abstract

Abstract. Proteinase activity in the midgut of the pentatomid stinkbug Podisus maculiventris was investigated. The optimal pH for adult and nymph proteolysis was pH 6.0 and pH 6.5, respectively. Proteinase activity was characterised using a range of diagnostic inhibitors. Activity of both adult and nymphal gut extracts, detected by the hydrolysis of Z-Phe-Arg-pNA, was inhibited to <20% of control levels by several inhibitors (e.g. E-64 and chicken egg white cystatin) associated with the inhibition of cysteine proteinases. The less specific inhibitor leupeptin reduced proteolytic activity to around 1.0% of the control values. In-gel analysis of the enzymes revealed that proteolytic activity was due to at least four proteinases, of ca. 30, 36, 50 and 110 kDa, which were all susceptible to E-64 inhibition. Salivary gland extracts gave maximal activity at pH 8.0 when tested for general proteolytic activity using fluorescent BODIPY-FL casein substrate, and showed moderate levels of inhibition when incubated with inhibitors of serine-, cysteine-, aspartic-and metallo-proteinases. Leupeptin and PMSF gave the highest levels of inhibition of salivary proteolytic activity, at ca. 50%, whilst the plant-derived inhibitors SKTI, CpTI and OC-1 did not inhibit proteolysis.

Biochemical characterisation of the tissue degrading enzyme, collagenase, in the spined soldier bug, Podisus maculiventris (Hemiptera: Pentatomidae)

“…Our results, however, revealed that the collagenase extracted from the salivary glands of P. maculiventris is sensitive to EDTA and so collagenase is a metalloproteinase. In line with our findings, Bell et al (2005) showed that the salivary gland extract of P. maculiventris is affected by EDTA, implying that there is a presence of metalloproteinase in the salivary glands. A recent study on the closely related species P. nigrispinus, definitely revealed that collagenase is a metalloproteinase enzyme and it is one of the most important proteases in the salivary gland and midgut of the predatory bug (Fialho et al 2012).…”

“…The salivary glands' collagenase clearly is a major factor in the extra-oral digestion process and liquefaction of the internal host body's organs. Collagenase alongside serine proteases (Bell et al 2005) are the main enzymes present in the salivary glands of the P. maculiventris. Optimal pH of total proteolytic activity of the salivary glands is associated with optimum pH of collagenase and serine proteases activity in the P. maculiventris (Bell et al 2005) and other asopin species (Cohen 1995;Oliveira et al 2006).…”

“…Collagenase alongside serine proteases (Bell et al 2005) are the main enzymes present in the salivary glands of the P. maculiventris. Optimal pH of total proteolytic activity of the salivary glands is associated with optimum pH of collagenase and serine proteases activity in the P. maculiventris (Bell et al 2005) and other asopin species (Cohen 1995;Oliveira et al 2006). Although the maximum collagenolytic activity in the midgut occurs at pH 8, the highest proteolytic activity in the different midgut parts corresponds to the activity of cysteine proteases.…”

“…Although the maximum collagenolytic activity in the midgut occurs at pH 8, the highest proteolytic activity in the different midgut parts corresponds to the activity of cysteine proteases. Bell et al (2005) showed that maximum proteolytic activity of the midgut and salivary glands of P. maculiventris using the substrate azocasein, occurs at a pH of 6 and 8.5, respectively. They also emphasised that the major proteolytic activity in the midgut and salivary glands are associated with cysteine, protease and serine proteases, respectively.…”

“…The process of EOD also depends completely on the presence of different digestive enzymes in the saliva. Thus, the presence of hydrolyzing enzymes such as trypsin, chymotrypsin, phospholipase, lipase, amylase, and collagenase in the saliva of these insects seems to be reasonable (Cohen 1995;Bell et al 2005;Oliveira et al 2006;Fialho et al 2012).…”