Compartmentalization of proteinases and amylases in Nauphoeta cinerea midgut.
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.
Elpidina
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EN
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia. elp@genebee.msu.su
Vinokurov
K S
KS
Gromenko
V A
VA
Rudenskaya
Y A
YA
Dunaevsky
Y E
YE
Zhuzhikov
D P
DP
D000681:Amylases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002850:Chromatography, Gel; D002940:Circadian Rhythm; D003058:Cockroaches / Q000201:enzymology* / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry; D006863:Hydrogen-Ion Concentration
D002364:Caseins; D004791:Enzyme Inhibitors; C026018:azocasein; D000681:Amylases; D010450:Endopeptidases