The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.
Bacillus subtilis ; classification ; enzymology ; Enzyme Activation ; Enzyme Stability ; Peptide Hydrolases ; chemistry ; isolation & purification ; Species Specificity ; Substrate Specificity

Mulberry longicorn beetle, Apriona germari, has been reported to produce two endo-beta-1,4-glucanases or AgEGases (accession Nos. Q6SS52 and Q5XQD1). AgEGase sequence contains catalytic motif (amino acid residues 37-48), which is the characteristic of family Glycohydrolase 45 and is identified as the substrate binding site. The application of bioinformatics approaches includes sequence analysis, structural modeling and inhibitor docking to relate the structure and function of AgEGases. We have dissected the sequence and structure of AgEGase catalytic motif and compared it with crystal structure of Humicola insolens endoglucanases V. The results show an involvement of sulfur containing amino acid residues in the active site of the enzyme. Cys residues and position of disulfide bonds are highly conserved between the two structures of endoglucanases of A. germari. Surface calculation of AgEGase structure in the absence of Cys residues reveals greater accessibility of the catalytic site to the substrate involving Asp42, a highly conserved residue. For the inhibition study, tannin-based structure was docked into the catalytic site of AgEGase using ArgusLab 4.0 and it resulted in a stable complex formation. It is suggested that the inhibition could occur through formation of a stable transition state analog-enzyme complex with the tannin-based inhibitor, as observed with other insect cellulases in our laboratory.
Animals ; Catalysis ; Coleoptera ; enzymology ; Endo-1,3(4)-beta-Glucanase ; chemistry ; metabolism ; Enzyme Activation ; Enzyme Stability ; Morus ; parasitology

BACKGROUND: Interleukin-2 receptor (IL-2) is expressed and released predominantly activated T lymphocyte. Increased serum levels of soluble IL-2R have been noted in a variety of autoimmune diseases and in conditions associated with T lymphocyte activation. OBJECTIVE: We aimed to examine whether the T lymphocyte activation has any association with the pathogenesis of Behçet's disease. METHOD: We have measured the serum level of soluble IL-2R in serum samples obtained from 67 patients with Behçet's disease and 30 healthy people as a control group, using a double-antibody sandwich enzyme-linked immunosorbent assay technique. RESULTS: Serum soluble IL-2R levels were found to be significantly elevated in the group of Behçet's disease as compared with the control group. No significant differences were found within clinical subtypes of Behçet's disease. CONCLUSION: These findings suggest the presence of an ongoing T lymphocyte activation in this disease process.
Autoimmune Diseases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-2* ; Lymphocyte Activation ; Lymphocytes ; Methods ; Receptors, Interleukin-2*

BACKGROUND: Interleukin-2 receptor (IL-2) is expressed and released predominantly activated T lymphocyte. Increased serum levels of soluble IL-2R have been noted in a variety of autoimmune diseases and in conditions associated with T lymphocyte activation. OBJECTIVE: We aimed to examine whether the T lymphocyte activation has any association with the pathogenesis of Behçet's disease. METHOD: We have measured the serum level of soluble IL-2R in serum samples obtained from 67 patients with Behçet's disease and 30 healthy people as a control group, using a double-antibody sandwich enzyme-linked immunosorbent assay technique. RESULTS: Serum soluble IL-2R levels were found to be significantly elevated in the group of Behçet's disease as compared with the control group. No significant differences were found within clinical subtypes of Behçet's disease. CONCLUSION: These findings suggest the presence of an ongoing T lymphocyte activation in this disease process.
Autoimmune Diseases ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-2* ; Lymphocyte Activation ; Lymphocytes ; Methods ; Receptors, Interleukin-2*

Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.
Cell Division ; Enzyme Activation ; Isoenzymes/metabolism* ; Phospholipase C/metabolism* ; Second Messenger Systems ; T-Lymphocytes

Acute pancreatitis is an inflammatory disorder which is developed by multiple etiologies such as gallstone, alcohol, hypertriglyceridemia, etc. The pathogenesis of acute pancreatitis is not fully clarified yet. However, it is widely accepted that intracellular premature activation of pancreatic digestive enzyme is the earliest initiating event. Intracellular premature enzyme activation is associated with co-localization with lysosomal enzyme cathepasin B and zymogen granule. The autodigestive injured pancreas is subject to inflammatory injury. Over the past few years, significant evidence has been accumulated stating that synthesis and release of proinflammatory cytokines and chemokines are responsible for local pancreatic injury and systemic dispersion of inflammation. Recently, a neurogenic mechanism of inflammation such as substance P and NK-1R has been elucidated in the pathogenesis of acute pancreatitis.
Chemokines ; Cytokines ; Enzyme Activation ; Gallstones ; Hypertriglyceridemia ; Inflammation ; Pancreas ; Pancreatitis* ; Substance P

To introduces the body process of iridoid in gardenia and effect of biological activity of enzymes systematically and discusses the mechanism of these compounds on the basis of the domestic and foreign recent literatures. It also provides a literature basis for the instruction of rational clinical prescription, reform of dosage forms, and development and utilization.
Animals ; Enzyme Activation ; drug effects ; Enzymes ; metabolism ; Gardenia ; chemistry ; Humans ; Iridoids ; metabolism ; pharmacology

Objective: To verify whether the enzymatic activity of kynureninase (KYNU) could be changed by the Arg188Gln (G/A) mutation. Methods: The total RNA of human hepatic tissue was extracted and the KYNU gene cDNA was amplified by RT-PCR. Primers were designed according to the sequences around the site Arg188Gln of KYNU gene and the Arg188Gln (G/A) mutant KYNU cDNA was generated by site-directed mutagenesis. Both the wild-type and mutant-type KYNU genes were subcloned into pcDNA vectors and the recombinant plasmids were constructed. After being transfected into human embryonic kidney 293 (HEK293) cells, the expression of KYNU recombinant plasmids were assessed by Western blot. The enzymatic activities of KYNU were detected by high performance liquid chromatography (HPLC). Results: The KYNU enzyme activities were expressed in both wild and mutant HEK293 cells. Michaelis constants (Km) of the wild and mutant KYNU were (9.833±0.513) μmol/L and (29.900±0.265) μmol/L, respectively (P<0.05). The maximum velocities (Vmax) of the wild and mutant KYNU were (0.700±0.096) nmol·mg^-1·min^-1 and (0.084±0.003) nmol·mg^-1·min^-1, respectively (P<0.05). Conclusion: Arg188Gln (G/A) mutation can decrease the enzymatic activity of KYNU.
Arginine ; genetics ; Enzyme Activation ; genetics ; HEK293 Cells ; Humans ; Hydrolases ; genetics ; metabolism ; Mutation ; Plasmids

Cerebral Infarction ; etiology ; Enzyme Activation ; Humans ; Infant ; Male ; Protein C Deficiency ; complications ; Protein S Deficiency ; complications

A model describing the solubilization of arabinoxylans and degradation by endo-xylanase random attacking during mashing was developed. The model was expected to predict the arabinoxylans concentration in wort at the settings of different initial value and mashing parameters for diminishing the negative effects of arabinoxylans on brewing. Results showed that the modeling errors range for the final concentration of arabinoxylans in wort was -9.5% to +13.6%. The model prediction accuracy for industrial scale mashing process was lower than that in laboratory scale. The errors were given 16.8% and 17.9%, respectively. The simulation results showed that arabinoxylans concentration was increased with the increase of mashing-in temperature, but it was decreased with prolonging the mashing-in time. The effect of initial arabinoxylans in malt on arabinoxylans concentration in wort was more remarkable than that of endo-xylanase activity in grist.
Endo-1,4-beta Xylanases ; metabolism ; Enzyme Activation ; Fermentation ; Models, Chemical ; Solubility ; Xylans ; metabolism