• Bulletin of the Korean Chemical Society
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• v.26 no.8
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• pp.1225-1228
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• 2005

C-terminal fragments of APP (APP-CTs), that contain A$\beta$ sequence, are found in neurotic plaques, neurofibrillary tangles and the cytosol of lymphoblastoid cells obtained from AD patients. CT26, Thr639-Asp664 (TVIVITLVMLKKKQYTSIHH GVVEVD) includes not only the transmembrane domain but also the cytoplasmic domain of APP. This sequence is produced from cleavage of APP by caspase and $\gamma$-secretase. In this study, the solution structure of CT26 was investigated using NMR spectroscopy and circular dichroism (CD) spectropolarimeter in various membrane-mimicking environments. According to CD spectra and the tertiary structure of CT26 determined in TFE-containing aqueous solution, CT26 has an α-helical structure from $Val^{2}\;to\;Lys^{11}$ in TFE-containing aqueous solution. However, according to CD data, CT26 adopts a $\beta$-sheet structure in the SDS micelles and DPC micelles. This result implies that CT26 may have a conformational transition between $\alpha$-helix and $\beta$-sheet structure. This study may provide an insight into the conformational basis of the pathological activity of the C-terminal fragments of APP in the model membrane.

• Journal of Microbiology and Biotechnology
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• v.8 no.2
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• pp.158-164
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• 1998

Crystal structural analyses of the API-TLCK complex revealed that the ${\epsilon}$-amino group (NZ) of the lysyl part of TLCK forms hydrogen bonds with OD1 of $Asp^225$ which is a substrate specificity determinant of API, OG of $Ser^214$, O of $Ser^214$, OG1 of $Thr^189$, and O of $Thr^189$ l89/. The ${\beta}$-carboxyl oxygen of $Asp^225$ forms hydrogen bonds with the NE1 of $Trp^182$. From these observations, it is thought that besides $Asp^225$, $Thr^189$, $Ser^214$, and $Trp^182$ may also contribute to the steric specificity for lysine and high proteolytic activity of API. The side-chain hydroxyl groups of $Thr^189$ and $Ser^214$ were removed to elucidate the role of these hydrogen bonds in the $S_1$-pocket. The $k_{cat}$/$K_m$ of T189V, S214A, and T189V.S214A were decreased to 1/4, 1/3, and 1/46, respectively, of the value for native API. The decreased activities were mainly due to the increase of $K_m$. The CD and fluoroscence spectra of the three mutants were similar to those of wild-type API. With regards to the kinetic parameters ($K_i\;and\;k_2$) of mutants for the reaction involving TLCK and DFP, $k_2$decreased by increase of $K_1$ only. These results suggest that the decreased catalytic activity of these mutants is caused by the partial loss of the hydrogen bond network in the $S_1$-pocket. On the other hand, the similarity of enzymatic properties between W182F and the native enzyme suggests that the hydrogen bond between OD2 of $Asp^225$ and NE1 of $Trp^182$ is not directly related to the reaction of $Asp^225$ with the substrate.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.303-309
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• 1994

The analysis of membrane receptors for hormones and neurotransmitters has progressed considerably by pharmacological and biochemical means and more recently through the use of specific antibodies. Two kinds of antibodies could be produced, one is from synthetic peptides and the other from proteins such as purified receptor. Anti-peptide antibodies gave some advantages; epitope is evident and also receptor purification in quantity is not prerequisite. It can be also applied to the study of receptor structure-activity relationship. The purpose of the present study was 1) to produce and characterize a polyclonal antibody against a synthetic $\beta$2-adrenergic receptor peptide(Phe-Gly-Asn-Phe-Trp-Cys-Phe-Trp-Thr-Ser-Ile-Asp-Val-Leu) and 2) to determine the effects of this antibody on the $\beta$-adrenergic receptor ligand interaction. The peptide sequence contains an amino acid residue such as Asp-113 which was identified as one of important component for receptor-ligand interaction in site-directed mutagenesis studies. Production of antibody was performed by immunization of rabbits through popliteal lymph node with the peptide coupled with Keyhole Limpet Hemocyanin (KLH). The titer of antibody against this peptide was 1 : 1000. The anti-peptide antibody was able to detect a 67 kDa protein band in western blot corresponding to the molecular weight of the $\beta$-adrenergic receptor in partially purified receptor fraction derived from guinea pig lung. The antisera inhibited the specific binding of [$^3$H]dihydroalprenolol to $\beta$-adrenergic receptor in a concentration-dependent manner. The results from this study suggest that the peptide sequence selected in the present study is important for the receptor ligand interaction.

• Tuberculosis and Respiratory Diseases
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• v.45 no.1
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• pp.77-89
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• 1998

Background: Genetic and environmental factors are known to affect the incidence and severity of asthma. Stimulation of $\beta_2$-Adrenergic Receptor ($\beta_2$AR) results in smooth muscle relaxation, leading to decrease in resistance of airflow. The gene encoding the $\beta_2$AR has recently been seguenced. The $\beta_2$AR genotype at the polymorphic loci of codons 16, 27, 34, and 164 was known to cause changes in the amino acids. The relationships between the structure of the $\beta_2$AR and its functions are being elucidated. Purpose : The gene encoding the $\beta_2$AR was carried out to assess the frequency of polymorphisms in bronchial asthma, to determine wheather these polymorphisms have any relation to the severity, or nocturnal symptoms in bronchial asthma. Methods: The subjects studied were 103 patients with bronchial asthma, which consisted of 30 mild episodic, 32 mild persistent, 17 moderate, and 24 severe asthma patients. The polymorphisms of the $\beta_2$AR gene were detected by mutated allele specific amplification (MASA) method at the codons 16,27,34, and 164. Results: The most frequent polymorphism was arginine 16 to glycine. The other two polymorphisms, valine 34 to methionine and glutamine 27 to glutamic acid occured in 11 and 6 patients respectively. The polymorphism of threonine 164 to isoleucine was not found in our enrolled patients. The homozygous polymorphism of $\beta_2$AR gene was found in only arginine 16 to glycine (12.6%). The heterozygous polymorphisms of $\beta_2$AR gene were in arginine 16 to glycine, valine 34 to methionine, and glutamine 27 to glutamic acid, as 65.1 %,10.7%, and 5.8% respectively in asthma patients. The presence of agrginine 16 to glycine heterozygous or/and homozygous polymorphism was associated in severe asthma (p=0.015), but there was no association between the other three polymorphisms and the severity of asthma. The frequency of the $\beta_2$AR gene polymorphisms was no relation in nocturnal asthma as compared with non-nocturnal asthma. Conclusion: The arginine 16 to glycine polymorphism of the $\beta_2$AR gene is the most frequently found in asthma patients and association with severe asthma. But there was no association between the polymorphism of the $\beta_2$AR gene and nocturnal asthma.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1291-1299
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• 2007

Two ${\beta}$-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS 1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were $^1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr^{15}$-for DI and $^1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys^{15}$-for DIII. The apparent sequences exhibited high sequence similarities with other bacterial ${\beta}$-1,4-glucanases as well as ${\beta}$-1,4-xylanases.

• BMB Reports
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• v.44 no.10
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• pp.653-658
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• 2011

Sclerotinia sclerotiorum fungus has three endoxylanases induced by wheat bran. In the first part, a partial xylanase sequence gene (90 bp) was isolated by PCR corresponding to catalytic domains (${\beta}5$ and ${\beta}6$ strands of this protein). The high homology of this sequence with xylanase of Botryotinia fuckeliana has permitted in the second part to amplify the XYN1 gene. Sequence analysis of DNA and cDNA revealed an ORF of 746 bp interrupted by a 65 bp intron, thus encoding a predicted protein of 226 amino acids. The mature enzyme (20.06 kDa), is coded by 188 amino acid (pI 9.26). XYN1 belongs to G/11 glycosyl hydrolases family with a conserved catalytic domain containing $E_{86}$ and $E_{178}$ residues. Bioinformatics analysis revealed that there was no Asn-X-Ser/Thr motif required for N-linked glycosylation in the deduced sequence however, five O-glycosylation sites could intervene in the different folding of xylanses isoforms and in their secretary pathway.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.2
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• pp.121-124
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• 2009

We investigated sericin extracted from sericinjam, which was inbred at National Academy of Agricultural Science, Suwon, Korea. Sericinjam sericin is composed of 5 fractions: 250 kDa, 120 kDa, 90 kDa, 70 kDa and 40 kDa. Amino acid analysis showed that the major amino acids of sericinjam sericin were Ser, Gly, Asp, Glu, Thr and Ala. Infrared spectra showed that sericinjam sericin has $\beta$-sheet structure. Thermal property of sericin was investigated using DSC and then they showed characteristic degradation peak at around $215{\sim}240^{\circ}C$.

• BMB Reports
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• v.32 no.6
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• pp.579-584
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• 1999

Mantis egg fibrolase (MEF-3) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, DEAE Affi-Gel blue gel affinity chromatogragphy, and MONO-Q anion-exchange chromatography. This protease had a molecular weight of 35,600 Da as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions and its isoelectric point was 6.0. The N-terminal amino acids sequence was Ala-Thr-Gln-Asp-Asp-Ala-Pro-Pro-Gly-Leu-Ala-Arg-Arg. This sequence was 80% homologous to the serine protease from Tritirachium album. MEF-3 readily digested the ${\alpha}$-and ${\beta}$-chains of fibrinogen and more slowly the ${\gamma}$-chains. It showed strong proteolytic and fibrinolytic activities. Phenylmethanesulfonyl fluoride and chymostatin inhibited its proteolytic activity, while EDTA, EGTA, cysteine, ${\beta}$-mercaptoethanol, elastinal, tosyl-lysine chloromethylketone, and tosyl-amido-2-phenylethyl chloromethyl ketone did not affect its proteolytic activity. Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEF-3 was benzoyl-Phe-Val-Arg-p-nitroanilide. Based on these experimental results, we speculated that MEF-3 is a serine protease with a strong fibrin(ogen)olytic activity.

• Microbiology and Biotechnology Letters
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• v.28 no.4
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• pp.195-201
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• 2000

Glucoamylase of Saccharomyces diastaticus is produced as a large precursor composed of signal peptide (21 amino acid residues), Thr and Ser-rich region and functional glucoamylase. To evaluate the utility of the glucoamylase signal peptide (GSP) for the secretion of foreign proteins, four types of GSP mutants (ml : Pro-18 longrightarrowLeu-18, m2 : Tyr-13 longrightarrowLeu, m3 : Ser-9longrightarrowLeu-9, m4 : Asn-5 longrightarrowPro-5) were constructed and secretion efficiency of each mutant was compared with that of native GSP by the expression and secretion of Bacillus subtilis CMCase under the control of GAP in N-terminal domain and hydrophobic domain. n mutant 4, a polar amino acid was replaced by a helix - breaking Pro residue. CMCase activity assay and Western blot analysis revealed that CMCase secretion by GSP mutants replaced by Leu were increased compared with native GSP. In the case of m2 and m3, the substitution of Leu for Tyr-13 and Ser-9 in the hydrophobic region resulted in a twofold increase in the extracellular CMCase activity.

• Food Science of Animal Resources
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• v.30 no.6
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• pp.923-929
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• 2010

Colostral whey prepared from colostrum (pooled from first six post-partum milkings) was heated for 10 min at $100^{\circ}C$ Heated colostral whey was incubated with 1% enzymes (protein equivalent basis) for 15, 30, 60, 90, and 120 min at $50^{\circ}C$. Papain, pepsin, trypsin, and alcalase produced different degrees of hydrolysis (DH), 10.66%, 12.42%, 10.83%, and 25.31%, respectively, at an incubation time of 120 min. The SDS-PAGE reveals that significant amounts of bovine serum albumin (BSA), ${\beta}$-lactoglobulin (${\beta}$-LG), and ${\alpha}$-lactalbumin (${\alpha}$-LA) survived papain digestion. In contrast, pepsin completely removed BSA but not ${\beta}$-LG present in heated colostral whey. Alcalase completely eliminated BSA, ${\beta}$-LG, and ${\alpha}$-LA. This differential hydrolysis was confirmed by reversed-phase HPLC analysis. Using ion-exchange chromatography, fraction-1 (F-1) was obtained from alcalase hydrolysate at a NaCl gradient concentration of 0.25 M. Reversed-phase HPLC chromatograms of alcalase F-1 showed numerous small peaks, which probably indicate that a variety of new peptides were produced. Iron content of alcalase F-1 was 28.94 ppm, which was the highest among all enzyme fractions, whereas iron content of colostral whey was 36.56 ppm. Main amino acids contained in alcalase F-1 were Thr (15.45%), Glu (14.12%), and Ser (10.39%). Therefore, alcalase can be used to generate good iron-binding peptides in heated colostral whey, and the resulting iron-binding peptides could be suitable as a value-added food ingredient for food supplements.