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 were studied in the midgut of Nauphoeta cinerea Oliv. (Blattoptera: Blaberidae) in experimental conditions, excluding their nutritional origin. One trypsin inhibitor (TI) with M(r) 8,000 and two subtilisin inhibitors (SI1 and SI2) with M(r) 13,000 and 8,000 were detected after fractionation of total protein preparation on Sephadex G-50. Ninety-four percent of both types of inhibitors was located in anterior midgut (AM). TI was 120-fold purified by FPLC-chromatography on Mono Q. Its isoelectric point was 4.3. TI lost a large part of activity in acidic and especially in alkaline medium. TI, SI1, and SI2 effectively inhibited activities of endogenous proteinases from posterior midgut (PM) of the cockroach. A search for inhibitor of endogenous unusual SH-dependent proteinase from AM revealed in AM a new inhibitor with M(r) 18,000. It was also inactivated in alkaline medium and was effective against proteinases from PM along with unusual SH-dependent proteinase from AM. A mechanism of regulation of activity of midgut proteinases is proposed based on pH-stability of inhibitors.