• Journal of Microbiology
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• v.38 no.3
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• pp.160-168
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• 2000

A-factor I a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. to identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by Streptomyces griseus IFO 13350 and its A-factor deficient mutant strain, Streptomyces griseus HH1, as well as Streptomyces griseus HH1 transformed with the afsA gene were sturdied. In general Streptomyces griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of Streptomyces griseus IFO 13350 was greatly enhanced more than twice compared with that of Streptomyces griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of Streptomyces griseus HH1 was greatly enhanced more than twice compared with that of Streptomyces griseus IFO 13350, and this observation was reversed in the presence of thiostreptione, However, Streptomyces griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between Streptomyces griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-amionpeptidase activity was 2 times higher in Streptomyces griseus HH1 than in strain IFO 13350 . Streptomyces griseus HH1 harboring afsA showed a similar level of enzyme activity , however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morpholofical differentiation, the formation of aerial meycelium and spores was delayed by two or three days.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.46 no.2
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• pp.119-128
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• 2013

A protease inhibitor was fractionated from fish eggs using methods based on protein solubility. Fractionation efficiency was evaluated with regard to percent recovery and total inhibitory activity (U). The fractionation of protease inhibitor (PI) from egg extracts of skipjack tuna (ST, Katsuwonus pelamis), yellowfin tuna (YT, Thunnus albacores), and Alaska pollock (AP, Theragra chalcogramma) was performed by precipitation with cold acetone or ammonium sulfate (AS). Fractions exhibiting the strongest inhibitory activity contained 20-40% (v/v) cold acetone or 40-60% saturated AS fractions. AS fractionation was more effective in isolating PI than was precipitation with acetone. The total inhibitory activity and percent recovery of fraction obtained with AS 40-60% toward trypsin and $N{\alpha}$-benzoyl-L-arginine-p-nitroanilide (BAPNA) were 4,976 U and 24.2% for ST, 3,331 U and 38.1% for YT, and 4,750 U and 43.8% for AP, respectively. In comparisons against six commercial proteases, 40-80% AS fractions, made by combining the 40-60% and 60-80% AS fractions from fish egg extract, exhibited the strongest inhibition of trypsin when using a casein substrate. These results suggest that fish eggs act as serine protease inhibitors and may be useful for protease inhibition in foodstuffs.

• Mycobiology
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• v.35 no.4
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• pp.219-225
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• 2007

A keratinolytic enzyme secreted by Aspergillus flavus K-03 cultured in feather meal basal medium (FMBM) containing 2% (w/v) chicken feather was purified and characterized. Keratinolytic enzyme secretion was the maximal at day 16 of the incubation period at pH 8 and $28^{\circ}C$. No relationship was detected between enzyme yield and increase of fungal biomass. The fraction obtained at 80% ammonium sulfate saturation showed 2.39-fold purification and was further purified by gel filtration in Sephadex G-100 followed by ion exchange chromatography on DEAE-Sephadex A-50, yielding an active protein peak showing 11.53-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms indicated that the purified keratinase is a monomeric enzyme with 31 kDa molecular weight. The extracellular keratinase of A. flavus was active in a board range of pH ($7{\sim}10$) and temperature ($30^{\circ}C{\sim}70^{\circ}C$) profiles with the optimal for keratinase activity at pH 8 and $45^{\circ}C$. The keratinase activity was totally inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), iodoacetic acid, and ethylenediaminetetraacetate (EDTA) while no reduction of activity by the addition of dithiothreitol (DTT) was observed. N-terminal amino acid sequences were up to 80% homologous with the fungal subtilisins produced by Fusarium culmorum. Therefore, on the basis of these characteristics, the keratinase of A. flavus K-03 is determined to be subtilisins-like.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11b
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of a 1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of a 1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of a 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Electrical & Electronic Materials
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• v.14 no.12
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• pp.3-6
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• 2001

In common globular proteins, the native form is n its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, ad internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of $\alpha$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of $\alpha$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of $\alpha$1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11a
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these Proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of ${\alpha}$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of ${\alpha}$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of e 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

• BMB Reports
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• v.32 no.3
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• pp.247-253
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• 1999

Chicken egg ovalbumin is a non-inhibitory member of the serpin (serine protease inhibitors) family whose members share a common tertiary fold. In the present study, we succeeded in high-level production of a disulfide-free form of refolded recombinant ovalbumin. Conformational characterization of the recombinant ovalbumim revealed that it is well-folded, following two-state unfolding transition with the midpoint of transition at 4.7 M at $25^{\circ}C$. This value is very close to that of the reduced form of authentic ovalbumin. The recombinant ovalbumin can serve as a model molecule of non-inhibitory serpins in comparative studies with inhibitory members of the serpin family.

• Journal of Microbiology
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• v.41 no.4
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• pp.335-339
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• 2003

Many genetic variants of ${\alpha}_1$-antitrypsin have been associated with early onset emphysema and liver cirrhosis. However, the detailed structural basis of pathogenic ${\alpha}_1$-antitrypsin molecules is rarely known. Here we found that a recombinant $M_{malton}$ variant (Phe52-deleted) lost inhibitory activity by spontaneous conformational conversion into a more stable, inactive form under physiological conditions. Biochemical and spectroscopic data suggested that the variant converts into a reactive center loop-inserted conformation, resembling the latent form of plasminogen activator inhibitor-1.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.15-22
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• 2009

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 $A_2(cPLA_2)$. The ATP depletion-dependent arachidonic acid release was inhibited by $cPLA_2$ specific inhibitor $AACOCF_3$. ATP depletion-induced alterations in $Na^+$-dependent phosphate uptake and cell viability were prevented by $AACOCF_3$. 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 $cPLA_2$ 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.