Aspergillus flavus K-03 isolated from poultry forming soil in Korea was studied for its ability to produce extracellular proteases on basal medium containing 2% (w/v) chicken feathers. The fungus was observed to be a potent producer of such enzymes. Keratinolytic enzyme secretion was the best at 15 days of incubation period at pH 9 and temperature 40degrees C. No relationship existed between the enzyme yield and increase of biomass. Enzyme production was suppressed by exogenous sugars in descending order arabinose>maltose>mannose>fructose. But glucose did not influence the enzyme activity. The keratinolytic enzyme released by the fungus demonstrated the ability to decompose keratin substrates as chicken feather when exogenous glucose was present. The keratinolytic activity was inhibited by HgCl2 and serine-protease inhibitors such as phenymethylsulfonyl fluoride (100%), chymostain (88%), crystalline soybean trypsin inhibtor (80%), antipain (45%) and aprotinin (40%), and was not by cystein-protease and aspartyl-protease inhibitors. The enzyme activity is only partially inhibited by metallo-protease inhibitor. Thus, the enzyme secreted by A. flavus K-03 belongs to the alkaline serine-type protease.
Animals ; Antipain ; Aprotinin ; Aspergillus flavus* ; Aspergillus* ; Biomass ; Carbohydrates ; Chickens ; Crystallins ; Feathers ; Fluorides ; Fungi ; Glucose ; Hydrogen-Ion Concentration ; Korea ; Mercuric Chloride ; Peptide Hydrolases ; Poultry ; Soil ; Soybeans ; Trypsin

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na+-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na+-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na+-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na+ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na+-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na+-dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na+-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.
Adenosine Triphosphate ; Amino Acids ; Antipain ; Arachidonic Acid ; Arachidonic Acids ; Catalase ; Cell Death ; Cell Survival ; Cytosol ; Diminazene ; Hydrogen Peroxide ; Hydroxyl Radical ; Iodoacetic Acid ; Membranes ; Ouabain ; Peptide Hydrolases ; Phospholipases A2 ; Potassium Cyanide ; Protease Inhibitors ; Reactive Oxygen Species ; RNA, Messenger ; Trypan Blue