Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut

Abstract

Compartmentalization of proteinases, amylases, and pH in the midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was studied in order to understand the organization of protein and starch digestion. Total proteolytic activity measured with azocasein was maximal at pH 11.5 both in anterior (AM) and posterior (PM) halves of the midgut, but the bulk of activity (67%) was found in PM. Total AM and PM preparations were fractionated on a Sephadex G-50 column and further analysed by means of activity electrophoresis and specific inhibitors and activators. The major activity in PM was classified as an unusual SH-dependent proteinase with M(r) 24,000 and pH optimum with synthetic substrate BApNA at 10.0. The enzyme was 43-fold activated in the presence of 1 mM DTT, insensitive to synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cysteine (IAA, E-64) proteinases, strongly inhibited by STI, and displayed four active bands on zymograms. In PM, activities of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine proteinases were observed. Aspartic and metalloproteinases were not detected. In AM, activity of unusual SH-dependent proteinase also dominated and activity of chymotrypsin-like proteinase was observed, but their levels were much lower than in PM. Distribution of amylase activity, exhibiting an optimum at pH 6.0, was quite the opposite. The major part of it (67%) was located in AM. Treatment of amylase preparation with proteinases from AM and PM reduced amylase activity twofold. pH of the midgut contents was 6.0-7.2 in AM, 6.4-7.6 in the first and 8.8-9.3 in the second halves of PM. Thus, pH in AM is in good agreement with the optimal pH of amylase, located in this compartment, but the activity of proteinases, including the ability to degrade amylase, in such an environment is low. Active proteolysis takes place in the second half of PM, where pH of the gut is close to the optimal pH of proteinases.

Proteinase inhibitors in Nauphoeta cinerea midgut

“…94% of SI and TI activity were concentrated in AM (Table 1), while the major part of digestive proteinase activities, as shown in Elpidina et al (2001), was localized in PM. Thus, the low proteolytic activity in AM could be due to the effects of proteinase inhibitors.…”

Virulence determination of Beauveria bassiana isolates on a predatory hemipteran, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae)

“…General proteolytic activities of isolates were measured by using 2% azocasein (Sigma-Aldrich, A2765) as substrate described by Elpidina et al (2001). The reaction mixture consisted of 80 μl of Tris-HCl buffer (20 mM, pH 7), 40 μl of azocasein (2%) and 20 μl of enzyme.…”

Serine and cysteine proteases of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) larvae raised on date palms (Phoenix dactylifera)

“…Protease assay using general substrate General proteolysis was carried out according to the methods of Elpidina et al (2001) and Gatehouse et al (1999) using azocasein (0.1% w/v) as substrate with some modifications. The buffer used was glycine-NaOH pH 10.0.…”