• Journal of the Korean Chemical Society
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• v.29 no.3
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• pp.295-303
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• 1985

Carboxypeptidase B from Salmo Salar eggs was purified by CM-cellulose, 0.5 ammonium sulfate saturation, DEAE-cellulose, and Sephadex G-75 gel filtration and its enzymatic properties were investigated. Optimum temperature was 55$^{\circ}C$, pH optima were 4.0 and 7.0 at 37$^{\circ}C$, and the enzyme was stable at pH 2.0∼3.0 and 5.5∼7.0 for 1.5h. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-arginine. Its K$_m$ values was 0.21mM, and the enzyme activity was stimulated by Cu$^{2+}$ and Fe$^{3+}$, while inhibited by Zn$^{2+}$. The lysine was found to be competitive inhibitor and its K$_i$ value was determined to be 4.3mM. Molecular weight of this enzyme was determined to be 36,400 daltons by SDS-PAGE and the enzyme was monomeric protein composed of 19 kinds of amino acid residues.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.25-32
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• 2021

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the anti-endotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

• Korean Journal of Soil Science and Fertilizer
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• v.22 no.1
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• pp.31-38
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• 1989

A series of experiments was conducted to determine the contents and distribution of amino acids in the hydrolysates of humic acids extracted from 3 plant materials. Wild grass hay, and leaves of forest trees were used in this study. Seventeen amino acids were analyzed and their amounts determined. Results obtained from the experiments are summarized in the following: 1. Contents and distribution of hydrolyzable amino acids in the humic acid fractions depend on the kind of plant materials and the allowed time for humification. 2. Neutral amino acids was the largest part of the total amino acids, followed by acidic amino acids, and basic amino acids. 3. The total amounts of amino acids in the hydrolyzable humic acid fractions of well humified residues were in the following order: wild grass hay > leaves of deciduous trees > leaves of coniferous trees 4. In general the relative amounts of lysine increased with humification progressing. S. Glycine and glutamic acid were the two major amino acids in common for the hydrolysate of humic acids extracted from well decomposed residues of plant materials. Alanine, glutamic acid, glycine, aspartic acid and leucine were the five major amino acid in common in raw materials without exception. 6. Arginine appeared to be absent in any of the hydrolysates of humic acids from well humified plant materials. 7. Phenylalanine and tyrosine were present in all hydrolysates and their relative contents increased with the humification of plant materials.

• Journal of Microbiology and Biotechnology
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• v.22 no.4
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• pp.501-509
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• 2012

A 990 bp full-length gene (xynAHJ2) encoding a 329-residue polypeptide (XynAHJ2) with a calculated mass of 38.4 kDa was cloned from Bacillus sp. HJ2 harbored in a saline soil. XynAHJ2 showed no signal peptide, distinct amino acid stretches of glycoside hydrolase (GH) family 10 intracellular endoxylanases, and the highest amino acid sequence identity of 65.3% with the identified GH 10 intracellular mesophilic endoxylanase iM-KRICT PX1-Ps from Paenibacillus sp. HPL-001 (ACJ06666). The recombinant enzyme (rXynAHJ2) was expressed in Escherichia coli and displayed the typical characteristics of low-temperature-active enzyme (exhibiting optimum activity at $35^{\circ}C$, 62% at $20^{\circ}C$, and 38% at $10^{\circ}C$; thermolability at ${\geq}45^{\circ}C$). Compared with the reported GH 10 low-temperature-active endoxylanases, which are all extracellular, rXynAHJ2 showed low amino acid sequence identities (<45%), low homology (different phylogenetic cluster), and difference of structure (decreased amount of total accessible surface area and exposed nonpolar accessible surface area). Compared with the reported GH 10 intracellular endoxylanases, which are all mesophilic and thermophilic, rXynAHJ2 has decreased numbers of arginine residues and salt bridges, and showed resistance to $Ni^{2+}$, $Ca^{2+}$, or EDTA at 10 mM final concentration. The above mechanism of structural adaptation for low-temperature activity of intracellular endoxylanase rXynAHJ2 is different from that of GH 10 extracellular low-temperature-active endoxylanases. This is the first report of the molecular and biochemical characterizations of a novel intracellular low-temperature-active xylanase.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2002.07a
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• pp.113-113
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• 2002

Phylogenetically conserved Bcl-2 family proteins play a pivotal role in the regulation of apoptosis from virus to human. Members of the Bcl-2 family consist of antiapoptotic proteins such as Bcl-2, Bcl-xL, and Bcl-w, and proapoptotic proteins such as BAD, Bax, BOD, and Bok. It has been proposed that anti- and proapoptotic Bcl-2 proteins regulate cell death by binding to each other and forming heterodimers. A delicate balance between anti- and proapoptotic Bcl-2 family members exists in each cell and the relative concentration of these two groups of proteins determines whether the cell survives or undergoes apoptosis. Mcl-1 (Myeloid cell :leukemia-1) is a member of the Bcl-2 family proteins and was originally cloned as a differentiation-induced early gene that was activated in the human myeloblastic leukemia cell line, ML-1 . Mcl-1 is expressed in a wide variety of tissues and cells including neoplastic ones. We recently identified a short splicing variant of Mcl-1 short (Mcl-IS) and designated the known Mcl-1 as Mcl-1 long (Mcl-lL). Mcl-lL protein exhibits antiapoptotic activity and possesses the BH (Bcl-2 homology) 1, BH2, BH3, and transmembrane (TM) domains found in related Bcl-2 proteins. In contrast, Mcl-1 S is a BH3 domain-only proapoptotic protein that heterodimerizes with Mcl-lL. Although both Mc1-lL and Mcl-lS proteins contain BH domains fecund in other Bcl-2 family proteins, they are distinguished by their unusually long N-terminal sequences containing PEST (proline, glutamic acid, serine, and threonine) motifs, four pairs of arginine residues, and alanine- and glycine-rich regions. In addition, the expression pattern of Mcl-1 protein is different from that of Bcl-2 suggesting a unique role (or Mcl-1 in apoptosis regulation. Tankyrasel (TRF1-interacting, ankyrin-related ADP-related polymerasel) was originally isolated based on its binding to TRF 1 (telomeric repeat binding factor-1) and contains the sterile alpha motif (SAM) module, 24 ankyrin (ANK) repeats, and the catalytic domain of poly(adenosine diphosphate-ribose) polymerase (PARP). Previous studies showed that tankyrasel promotes telomere elongation in human cells presumably by inhibiting TRFI though its poly(ADP-ribosyl)action by tankyrasel . In addition, tankyrasel poly(ADP-ribosyl)ates Insulin-responsive amino peptidase (IRAP), a resident protein of GLUT4 vesicles, and insulin stimulates the PARP activity of tankyrase1 through its phosphorylation by mitogen-activated protein kinase (MAPK). ADP-ribosylation is a posttranslational modification that usually results in a loss of protein activity presumably by enhancing protein turnover. However, little information is available regarding the physiological function(s) of tankyrase1 other than as a PARP enzyme. In the present study, we found tankyrasel as a specific-binding protein of Mcl-1 Overexpression of tankyrasel led to the inhibition of both the apoptotic activity of Mel-lS and the survival action of Mcl-lL in mammalian cells. Unlike other known tankyrasel-interacting proteins, tankyrasel did not poly(ADP-ribosyl)ate either of the Mcl-1 proteins despite its ability to decrease Mcl-1 proteins expression following coexpression. Therefore, this study provides a novel mechanism to regulate Mcl-1-modulated apoptosis in which tankyrasel downregulates the expression of Mcl-1 proteins without the involvement of its ADP-ribosylation activity.

• Journal of Life Science
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• v.26 no.2
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• pp.164-173
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• 2016

Isotocin (IT), a nonapeptide homolog of oxytocin in mammals, has been suggested to be involved in physiological processes including social behaviors, stress responses, and osmoregulation in teleost fish. To study its structure and function, the gene encoding the IT precursor was cloned from the genomic DNA and brain cDNA of the cinnamon clownfish, Amphiprion melanopus. The IT precursor gene consists of three exons separated by two introns, and encodes an open reading frame of 156 amino acid (aa) residues, comprising a putative signal peptide of 19 aa, a mature IT protein of 9 aa, a proteolytic processing site of 3 aa, and 125 aa of neurophysin. Tissue-specific analysis of the IT precursor transcript indicated its expression in the brain and gonads of A. melanopus. To examine its osmoregulatory effects, the salinity of the seawater (34 ppt) used for rearing A. melanopus was lowered to 15 ppt. Histological analysis of the gills indicated the apparent disappearance of an apical crypt on the surface of the gill lamella of A. melanopus, as pavement cells covered the surface upon acclimation to the lower salinity. The level of Na⁺/K^+-ATPase activity in the gills was increased during the initial stage of acclimation, followed by a decrease to its normal level, suggesting its involvement in osmoregulation and homeostasis. The only slight increase in the level of IT precursor transcript in the A. melanopus brain upon low-salinity acclimation suggested that IT played a minor role, if any, in the process of osmoregulation.

• Journal of Life Science
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• v.23 no.1
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• pp.15-23
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• 2013

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9- and a 2.5-fold higher $k_{cat}$, respectively, and a 2.1-1.9-fold lower $K_m$, respectively. This resulted in a 3.8- and a 4.7-fold increase in catalytic efficiency ($k_{cat}/K_m$), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at $60^{\circ}C$. The half-lives of AprE51-7 and AprE51-8 at $50^{\circ}C$ were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.