• Proceedings of the Korean Biophysical Society Conference
• /
• 1997.07a
• /
• pp.39-39
• /
• 1997

Native strain of inhibitory SERPINS (Serine protease inhibitors) is thought to be used in the facile conformational switch to play biological regulation. Many heat stable variants of $\alpha$$_1$-antitrypsin, a prototype of inhibitory serpins, increased their stability by reducing the native strain.(omitted)

• BMB Reports
• /
• v.33 no.5
• /
• pp.379-384
• /
• 2000

The native conformation of serpins (serine protease inhibitors) is strained. Upon cleavage of the reactive center loop of serpins by a protease, the amino terminal portion of the cleaved loop is inserted into the central ${\beta}-sheet$, A sheet, as the fourth strand, with the concomitant release of the native strain. We questioned the role of protease in this conformational switch from the strained native form into a stable relaxed state. Chemical cleavage of the reactive center loop of ${\alpha}_1-antitrypsin$, a prototype serpin, using hydroxylamine dramatically increased the stability of the serpin. A circular dichroism spectrum and peptide binding study suggests that the amino terminal portion of the reactive center loop is inserted into the A sheet in the chemically-cleaved ${\alpha}_1-antitrypsin$, as in the enzymatically-cleaved molecule. These results indicate that the structural transformation of a serpin molecule does not require interaction with a protease. The results suggest that the serpin conformational switch that occurred during the complex formation with a target protease is induced by the cleavage of the reactive center loop per se.

• Proceedings of the Korean Biophysical Society Conference
• /
• 1998.06a
• /
• pp.23-23
• /
• 1998

To understand structural and functional basis of loaded energy in the metastable native structure of inhibitory serpins (serine protease inhibitors), we characterized mutations that decreased the loaded energy of ${\alpha}$$_1$-antitrypsin and simultaneously influenced its inhibitory activity. Various folding defects such as side-chain locking, buried polar groups in unfavorable hydrophobic environment, and cavities were found as the structural basis of the metastability of ${\alpha}$$_1$-antitrypsin in a region presumably directly involved in the formation of complex between the inhibitor and a target protease.(omitted)

• Proceedings of the Korean Biophysical Society Conference
• /
• 2001.06a
• /
• pp.31-31
• /
• 2001

The native strain of serpins (serine protease inhibitors) has been recognized as a mechanism of biological regulation. Indeed, some stabilizing single residue mutations of human $\alpha$$_1$-antitrypsin, a prototype serpin, relieved local strain and caused the loss of inhibitory activity. The native strain of $\alpha$$_1$-antitrypsin is distributed throughout the whole molecule, but the strain that regulates the function directly is highly localized in the regions that appear to be mobilized during complex formation with a target protease.(omitted)

• Journal of Animal Science and Technology
• /
• v.51 no.6
• /
• pp.527-536
• /
• 2009

A potent protein degrading bacterium was isolated from soil samples of different environments. Polyphasic taxonomic studies and phylogenetic 16S rRNA sequence analyses led to identify the isolate IB No. 11 as a strain of Bacillus subtilis. The isolated strain was recognized to produce protease constitutively, and the maximum production (1.64 units/ml) was attained in a shake flask culture when the isolate was grown at $40^{\circ}C$, for 32 h in basal medium supplemented with starch (0.25%) and gelatin (1.25%) as sole carbon and nitrogen source, respectively. The optimum pH and temperature for the protease activity were determined to be pH 7.0 and $50^{\circ}C$, respectively. $Ca^{2+}$ and $Mn^{2+}$ enhanced remarkably the protease activity but neither showed positive effect on the protease's thermal stability. In addition, it was observed that the protease was fairly stable in the pH range of 6.5-8.0 and at temperatures below $50^{\circ}C$, and it could be a good candidate for an animal feed additive. The inhibition profile of the protease by various inhibitors indicated that the enzyme is a member of serine-proteases. A combination of UV irradiation and NTG mutagenesis allowed to develop a protease hyper-producing mutant strain coded as IB No. 11-4. This mutant strain produced approximately 3.23-fold higher protease activity (6.74 units/mg) than the parent strain IB No. 11 when grown at $40^{\circ}C$ for 32h in the production medium. The protease production profile of the selected mutants was also confirmed by the zymography analysis.

• Bulletin of the Korean Chemical Society
• /
• v.34 no.4
• /
• pp.1212-1220
• /
• 2013

The human coagulation factor XI (FXI) is a serine protease that plays a significant role in blocking of the blood coagulation cascade as an attractive antithrombotic target. Selective inhibition of FXIa (an activated form of factor XI) disrupts the intrinsic coagulation pathway without affecting the extrinsic pathway or other coagulation factors such as FXa, FIIa, FVIIa. Furthermore, targeting the FXIa might significantly reduce the bleeding side effects and improve the safety index. This paper reports on a docking-based three dimensional quantitative structure activity relationship (3D-QSAR) study of the potent FXIa inhibitors, the chloro-phenyl tetrazole scaffold series, using comparative molecular field analysis (CoMFA) and comparative molecular similarity analysis (CoMSIA) methods. Due to the characterization of FXIa binding site, we classified the alignment of the known FXIa inhibitors into two groups according to the docked pose: S1-S2-S4 and S1-S1'-S2'. Consequently, highly predictive 3D-QSAR models of our result will provide insight for designing new potent FXIa inhibitors.

• Journal of Life Science
• /
• v.16 no.4
• /
• pp.598-604
• /
• 2006

The $Ca^{2+}-activated$ neutral protease calpain induced proteolysis has been suggested to play a role in certain cell growth regulatory proteins. Cyclin proteolysis is essential for cell cycle progression. D-type cyclins, which form an assembly with cyclin-dependent kinases (cdk4 and cdk6), are synthesized earlier in G1 of the cell cycle and seem to be induced in response to external signals that promote entry into the cell cycle. Here we show that cyclin D3 protein levels are regulated at the posttranscriptional level by calpain protease. Treatment of human breast carcinoma MDA-MB-231 cells with lovastatin and actinomycin D resulted in a loss of cyclin D3 protein that was completely reversible by the peptide aldehyde calpain inhibitor, LLnL. The specific inhibitor of the 26S proteasome, lactacystin, the lysosome inhibitors, ammonium chloride and chloroquine, and the serine protease inhibitor, phenylmethylsulfonylfluoride (PMSF), did not block the degradation of cyclin D3 by lovastatin and actinomycin D. Results of in vitro degradation of cyclin D3 by purified calpain showed that cyclin D3 protein is degraded in a $Ca^{2+}-dependent$ manner, and the half-life of cyclin D3 protein was dramatically increased in LLnL treated cells. These data suggested that cyclin D3 protein is regulated by the $Ca^{2+}-activated$ protease calpain.

• Applied Biological Chemistry
• /
• v.35 no.4
• /
• pp.237-241
• /
• 1992

The protease from Halomonas sp. ES 10 was purified by methanol precipitation, gel filtration on Sephadex G-150 and G-200, and ion exchange chromatography on DEAE-Sephadex A-50. The purified enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The specific activity of purified enzyme was 1,014 units/mg protein, and the yield of the total activity from the culture filtrate was 7%. The optimal temperature and pH for the enzyme activity were $35^{\circ}C$, and pH 11.0, respectively. And the enzyme was stable in the range of $pH\;7.5{\sim}11.0$. The residual activity of the enzyme was 70%, when the enzyme was incubated at $50^{\circ}C$ for 40 min. The Km value of the enzyme was 7.4 mg/ml to milk casein. $Li^+$, $Ca^{2+}$, SDS and Tween 80 were appeared to activators, whereas $Hg^{2+}$ and EDTA to inhibitors. The addition of DFP and PMSF showed the relative enzyme activities of 63% and 107%, respectively, suggesting that the enzyme may not belong to serine type protease. When the alkaline protease was treated with 0.5 M and 1 M NaCl, the relative enzyme activities were 95% and 65%, respectively. This enzyme showed 20% and 15% higher enzyme activity than that of Aspergillus oryzae (Sigma Chemical Company product, P4755) in the presence of 0.5 M and 1 M NaCl.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.12
• /
• pp.2190-2198
• /
• 2017

Thermoactinomyces sp. strain YT06 was isolated from poultry compost and observed to degrade integral chicken feathers completely at $60^{\circ}C$, resulting in the formation of 3.24 mg/ml of free amino acids from 50 ml of culture containing 10 g/l chicken feathers. Strain YT06 could grow and secrete keratinase using feather as the only carbon and nitrogen sources without other supplement, but complementation of 10 g/l sucrose and 4 g/l $NaNO_3$ increased the production of the keratinolytic enzyme. The maximum protease activity obtained was 110 U/ml and for keratinase was 42 U/ml. The keratinase maintained active status over a broad pH (pH 8-11) and temperature ($60-75^{\circ}C$). It was inhibited by serine protease inhibitors and most metal ions; however, it could be stimulated by $Mn^{2+}$ and the surfactant Tween-20. A reductive agent (${\beta}$-mercaptoethanol) was observed to cleave the disulfide bond of keratin and improve the access of the enzyme to the keratinaceous substrate. Zymogram analysis showed that strain YT06 primarily secreted keratinase with a molecular mass of approximately 35 kDa. The active band was assessed by MALDI-TOF mass spectrometry and was observed to be completely identical to an alkaline serine protease from Thermoactinomyces sp. Gus2-1. Thermoactinomyces sp. strain YT06 shows great potential as a novel candidate in enzymatic processing of hard-to-degrade proteins into high-value products, such as keratinous wastes.

• Archives of Pharmacal Research
• /
• v.28 no.7
• /
• pp.816-822
• /
• 2005

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gin Asp Phe Val Glu Glu lie Leu. CPM-2 was inactivated by $Cu^{2+}\;and\;Zn^{2+}$ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and ${\alpha}_1$-antitrypsin. However, EDTA, EGTA, cysteine, $\beta$-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved $A{\alpha}-$ and $B{\beta}$-chains of fibrinogen and fibrin, and y-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.