• The Korean Journal of Physiology
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• v.10 no.2
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• pp.1-10
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• 1976

The action of acetylcholine on the sodium plus potassium activated ATPase activity in the rabbit red cell membrane has been investigated and the experiments were also designed to determine the mechanism of action of acetylcholine on the ATPase activity. The following results were observed. 1. The activity of the NaK ATPase from red cell membrane is inhibited by acetylcholine. 2. The ratio of inhibition of NaK ATPase by acetylcholine is decreased by raising the potassium concentration, and is increased by raising the sodium concentration. 3. The ATPase activity is increased by small amounts of calcium but inhibited by larger amounts. The ratio of inhibition of the enzyme by acetylcholine is increased by raising the calcium concentration. 4. The inhibitory action of acetylcholine on the NaK ATPase activity was not related to the sulfhydryl group of cysteine, the hydroxyl group of threonine, or the carboxyl group of aspartic acid. 5. The inhibitory action of acetylcholine on the ATPase activity is due to amino group of the enzyme of NaK ATPase.

• Journal of the Korean Electrochemical Society
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• v.2 no.1
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• pp.27-30
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• 1999

Self-Assembled monolayers of carboxyl-terminated alkanethiols, which is negatively charged in pH 7.0, were usually used to facilitate the electron transfer between the positively charged protein and the electrode. In case of L-cysteine, as it has both positive and negative group, it can be a candidate for a new modifier to facilitate positively charged protein or negatively charged protein. Our investigation of L-cysteine shows that the electron transfer occurs successfully to both cytochrome c (cyt. c) and pyrroloquinoline quinone (PQQ). By using 1-ethyl-3-(3-dime-thlyaminopropyl) carbodiimide (EDC), we made a covalent bond between cyt. c and monolayer. Then PQQ was electrostatically adsorbed to the same monolayer. Cyclic voltammograms show that both molecules do not interfere each other and electron transfer is appreciable.

• Journal of Microbiology and Biotechnology
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• v.14 no.5
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• pp.1075-1080
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• 2004

Dextransucrase (DSRB742) from Leuconostoc mesenteroides NRRL B-742CB is a glucosyltransferase that catalyzes the synthesis of dextran using sucrose, or the synthesis of oligosaccharides when acceptor molecules, like maltose, are present. The DSRB742 gene (dsrB742) was cloned and the properties were characterized. In order to identify critical amino acid residues, the DSRB742 amino acid sequence was aligned with glucosyltransferase sequences, and three amino acid residues reported as sucrose binding amino acids in Streptococcus glucosyltransferases were selected for site-directed mutagenesis experiments. Asp-533, Asp-536, and His-643 were independently replaced with Ala or Asn. D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether. Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities. However, modifications at Asp-533 or at His-643 retained acceptor reaction activities in the range of 8.4% to 21.3% of DSRB742 acceptor reaction activity. Thus at least two carboxyl groups of Asp-533 and Asp-536, and His-643 as a proton donor, are essential for the catalysis process.

• BMB Reports
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• v.43 no.8
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• pp.541-546
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• 2010

We utilized a mammalian expression system to purify and characterize autotaxin (ATX)/lysophospholipase D, an enzyme present in the blood responsible for biosynthesis of lysophosphatidic acid. The human ATX cDNA encoding amino acids 29-915 was cloned downstream of a secretion signal of CD5. At the carboxyl terminus was a thrombin cleavage site followed by the constant domain (Fc) of IgG to facilitate protein purification. The ATX-Fc fusion protein was expressed in HEK293 cells and isolated from conditioned medium of a stable clone by affinity chromatography with Protein A sepharose followed by cleavage with thrombin. The untagged ATX protein was further purified to essential homogeneity by gel filtration chromatography with a yield of approximately 5 mg/liter medium. The purified ATX protein was enzymatically active and biologically functional, offering a useful tool for further biological and structural studies of this important enzyme.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.592-597
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• 2001

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• BMB Reports
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• v.38 no.4
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• pp.432-439
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• 2005

$Ca^{2+}$/calmodulin transduction pathways have been implicated in mediating stress response and tolerance in plants. Here, three genes encoding calmodulin (Cam) members of the EF-hand family of $Ca^{2+}$-binding proteins were identified from Oryza sativa L. databases. Complementary DNA for each of the calmodulin genes, OsCam1, OsCam2, and OsCam3 were sequenced. OsCam1 and OsCam2 encode a conventional 148-amino acid calmodulin protein that contains four characteristic $Ca^{2+}$-binding motifs. OsCam3 encode a similar protein with a 38-amino-acid extension containing a putative prenylation site (CVIL) at the carboxyl terminus. RT-PCR showed that each of the genes is expressed in leaves and roots of 2-week old rice seedlings. By RNA gel blot analysis, OsCam1 mRNA levels strongly increased in response to NaCl, mannitol and wounding treatments. In contrast, OsCam2 mRNA levels were relatively unchanged under all conditions investigated. NaCl treatment and wounding also increased the OsCam3 mRNA level, but in a more transient manner. Our results indicate that although the expression of genes encoding different calmodulin isoforms is ubiquitous, they are differentially regulated by various stress signals. In addition, we have demonstrated that the calcium-channel blocker lanthanum chloride inhibited the induction of OsCam1 gene expression by both NaCl and mannitol treatments. These results suggest that osmotic stress induced expression of OsCam1 gene requires the $[Ca^{2+}]_{cyt}$ elevation that is known to occur in response to these stimuli.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.807-812
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• 2002

In order to analyze the cellular proteins which interact with core protein of hepatitis C virus (HCV), a yeast two-hybrid screening technique was employed. A carboxyl terminus truncated core protein, which contained amino acid residues from the 1st to 120th, was used as a bait to screen cellular proteins. The expression library prepared from HeLa cell was screened and 400 positive clones were selected. The 75 clones from the positive clones were sequenced and analyzed by undergoing the Blast search. Interestingly, 7 out of the 75 clones encoded E7 antigen of human papilloma virus (HPV). We studied in detail the Interaction between the truncated version of HCV core and E7 antigen in vitro. The core$_{120}$ protein expressed in chimeric form with G57 was able to bring down the E7 protein of HPV type 18 expressed in bacteria. It is therefore suggested that the core of HCV might affect the interaction between E7 and a normal cellular tumor suppressor, known as Rb protein.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.551-558
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• 1992

The extracellular endoinulase from Streptomyces sp. 556 was purified and characterized, The culture broth was fractionated by ammonium sulfate saturation followed by DEAE-cellulose column chromatography and 5ephadex G-200 gel filtration, The ultimately purified fraction revealed a single band in 7.5% polyacrylamide gel electropherogram. The purified enzyme showed the maximal activity at pH 5.5-6.0 and $50^{\circ}C$, but lost 93% of inulase activity after 30 min incubation at $55^{\circ}C$ . The essen.tial amino acid residue for catalytic activity appeared to be tryptophan. This endo inulase was activated by $Mn^{2+}$, whereas inactivated by $Ag^{+}$, $Hg^{+}$, $Cu^{2+}$, $Zn^{2+}$, $Fe^{3+}$ and $Mo^{6+}$ EDTA and 8-hydroxyquinoline inhibited the enzyme so that the enzyme was considered to be a metalloenzyme. The Km value for inulin was 0.287 mM, and no invertase or $\alpha$-glucosidase activity was found in the enzyme.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.12
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• pp.1942-1949
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• 2019

Objective: Leukocyte cell-derived chemotaxin 2 (LECT2) is associated with several physiological processes including inflammation, tumorigenesis, and natural killer T cell generation. Chicken LECT2 (chLECT2) gene was originally identified as one of the differentially expressed genes in chicken kidney tissue, where the chickens were fed with different calcium doses. In this study, the molecular characteristics and gene expression of chLECT2 were analyzed under the stimulation of toll-like receptor 3 (TLR3) ligand to understand the involvement of chLECT2 expression in chicken metabolic disorders. Methods: Amino acid sequence of LECT2 proteins from various species including fowl, fish, and mammal were retrieved from the Ensembl database and subjected to Insilco analyses. In addition, the time- and dose-dependent expression of chLECT2 was examined in DF-1 cells which were stimulated with polyinosinic:polycytidylic acid (poly [I:C]), a TLR3 ligand. Further, to explore the transcription factors required for the transcription of chLECT2, DF-1 cells were treated with poly (I:C) in the presence or absence of the nuclear factor ${\kappa}B$ ($NF{\kappa}B$) and activated protein 1 (AP-1) inhibitors. Results: The amino acid sequence prediction of chLECT2 protein revealed that along with duck LECT2 (duLECT2), it has unique signal peptide different from other vertebrate orthologs, and only chLECT2 and duLECT2 have an additional 157 and 161 amino acids on their carboxyl terminus, respectively. Phylogenetic analysis suggested that chLECT2 is evolved from a common ancestor along with the actinopterygii hence, more closely related than to the mammals. Our quantitative polymerase chain reaction results showed that, the expression of chLECT2 was up-regulated significantly in DF-1 cells under the stimulation of poly (I:C) (p<0.05). However, in the presence of $NF{\kappa}B$ or AP-1 inhibitors, the expression of chLECT2 is suppressed suggesting that both $NF{\kappa}B$ and AP-1 transcription factors are required for the induction of chLECT2 expression. Conclusion: The present results suggest that chLECT2 gene might be a target gene of TLR3 signaling. For the future, the expression pattern or molecular mechanism of chLECT2 under stimulation of other innate immune receptors shall be studied. The protein function of chLECT2 will be more clearly understood if further investigation about the mechanism of LECT2 in TLR pathways is conducted.

• BMB Reports
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• v.33 no.5
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• pp.391-395
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• 2000

Calexcitin, a calcium-binding protein, was previously cloned and functionally characterized in the squid Loligo pealei. We now report the cloning of a second form of Calexcitin, Calexcitin-2, found in the squid Todarodes pacificus optic lobe. Calexcitin-2 has a significantly different carboxyl terminal region than Calexcitin-1. It lacks the CAAX motif, which is a farnesylation site. The amino acid sequence of Calexcitin-2 shows an 84% identity with Calexcitin-1 and also displays a strong cross immunoreactivity. Western blotting shows that Calexcitin-2 was expressed exclusively in the optic lobe region of squid, but not in other body organs. Regardless of its lack of conserved regions for GTP-binding, Calexcitin-2 shows moderately low affinity GTP-binding and also shows dramatic conformational change induced by GTP-binding. Three possible GTP-binding region mutations, K142A, D144A, and K157A, did not change the G TP binding affinity. This raises the possibility that Calexcitin-2 may have a novel GTP-binding motif.