1,721,191 research outputs found
Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification
No full textTumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) has been known to induce tumor-specific apoptosis and to share the structural and functional characteristics with he proteins of TNF family. Recently, he crystal structure of human TRAIL showed that TRAIL is a homotrimeric protein whose subunits contain mainly beta-sheets. We characterized the structural changes of recombinan human TRAIL induced by acidification and the biological implication of he structural characteristics a acidic pH in the interaction with the lipid bilayer. A acidic pH below pH 4.5, TRAIL resulted in substantial structural changes to a molten globule (MG)-like state. Far-UV CD spectrum of TRAIL indicated hat he acidification induced a helices that are absent in he native state. TRAIL a acidic pH exhibited significant change of tertiary structures as reflected in the near-UV CD spectrum. Thermal transition curve indicated hat here was less cooperation at acidic pH than at neutral pH in he thermal denaturation of TRAIL. Moreover, TRAIL at the MG-like state not only enhanced the binding ability to liposomes, but also increased he release rate of a fluorescent dye, calcein, encapsulated in liposomes. The binding assay with anilinonaphthalene-8-sulfonic acid revealed hat he surface hydrophobicity of TRAIL was increased while tryptophan residues became more exposed to solvent as judged by blue shift of he maximum fluorescence wavelength. Taken together, our results demonstrate hat he acidification of human TRAIL induces the MG-like state in vitro and makes he membrane permeable through the favorable interaction of TRAIL with the membrane, implicating hat general intrinsic properties such as TRAIL, TNF-alpha and lymphotoxin are shared by TNF family members.X1114sciescopu
IDENTIFICATION OF ACTIVE-SITE RESIDUES BY SITE-DIRECTED MUTAGENESIS OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-PUTIDA BIOTYPE-B
No full textIn order to assess the roles of specific amino acid residues in the Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B during catalysis, we replaced aspartic acid 40 with asparagine (D40N) and tyrosine 16 with phenylalanine (Y16F) in the enzyme by site directed mutagenesis. Both purified mutant enzymes resulted in profound decreases in catalytic activities, 10(3.3)-fold in the Y16F mutant and 10(6.2)-fold in the D40N mutant. Aspartic acid 40 and tyrosine 16 of the enzyme are the corresponding amino acids in the active site of the homologous enzyme from Comamonas testosteroni. Our results indicate that active-site residues of the two homologous enzymes are similar. This is opposite to the previous identification of a cysteine in an active site-directed photoinactivation study of the enzyme.open1135sciescopu
KINETIC-STUDIES OF AN ACTIVE-SITE DOUBLE MUTANT OF DELTA(5)-3-KETOSTEROID ISOMERASE FROM PSEUDOMONAS-PUTIDA BIOTYPE-B
No full textDelta(5)-3-Ketosteroid isomerase (KSI) promotes the highly efficient isomerization of Delta(5)-3-ketosteroids to Delta(4)-3-ketosteroids by means of a direct and stereospecific transfer of the 4 beta-proton to the 6 beta-position in order to compare the catalytic mechanisms of Pseudomonas putida isomerase with Comamonas testosteroni isomerase, well-conserved putative active-site amino acids in the P. putida KSI were replaced by site-directed mutagenesis: Asp-40 to asparagine and Tyr-16 to phenylalanine (D40N + Y16F). The double mutant isomerase was purified to homogeneity and characterized. The molecular weight of the purified double mutant was 14,517 (calculated, 14,519) as determined by electrospray mass spectrometry. While C. testosteroni D38N + Y14F double mutant showed no catalytic activity [Kuliopulos, A., Talalay, P., and Mirdvan, A. S. (1990) Biochemistry 29, 10271-10280], P. putida D40N + Y16F double mutant had some detectable activity. The k(cat) value of the double mutant enzyme is 10(6.6)-fold lower than that of the wild-type enzyme and K-m value is similar to that of wild-type enzyme. Although active-site residues of the two homologous enzymes are well conserved and of similar properties, kinetic properties of P. putida double mutant isomerase are not similar to those of the C. testosteroni double mutant. This result suggests that the precise mechanism of both homologous enzymes are somewhat different.X118sci
CLONING, SEQUENCING AND EXPRESSION OF THE GENE ENCODING A MAJOR ALLOTYPIC PREPROCARBOXYPEPTIDASE-A FROM BOVINE PANCREAS
No full textA cDNA encoding one of two major allotypic forms of bovine pancreatic preprocarboxypeptidase A (preproCPA) has been cloned, and its entire nucleotide (nt) sequence determined. The cloned cDNA contains a 26-nt T-noncoding region, a 1260-nt open reading frame and a 51-nt 3'-noncoding region. The amino acid (aa) sequence deduced from the gene sequence contains Ile(179), Ala(228) and Val(305) at allotypic aa residues. The procarboxypeptidase A (proCPA) was produced in yeast and secreted into the medium. Upon treatment with trypsin, proCPA generated an active enzyme with the same size as the mature carboxypeptidase A (CPA), as analyzed by use of anti-CPA polyclonal antibody.X116sciescopu
beta-synuclein exhibits chaperone activity more efficiently than alpha-synuclein
No full textbeta-Synuclein exhibits high sequence homology and structural similarity with alpha-synuclein, a protein implicated in the pathogenesis of Parkinson's disease. We investigated the chaperone function of beta-synuclein and its anti-fibrillar activity in comparison with alpha-synuclein. beta-Synuclein suppressed the heat-induced aggregation of aldolase, alcohol dehydrogenase, and citrate synthase, and its anti-aggregative activity was remarkably higher than that of alpha-synuclein. Heat-induced inactivation of citrate synthase was significantly protected by beta-synuclein. Moreover, beta-synuclein inhibited the amyloid formation of both Abeta(1-40) and alpha-synuclein. It is, therefore, suggested that beta-synuclein can prevent abnormal protein aggregations more effectively than alpha-synuclein by acting as a molecular chaperone. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.X112325sciescopu
Fluorometric assay of turnip mosaic virus NIa protease
No full textTurnip mosaic virus (TuMV) NIa protease cleaves the viral polyprotein at seven distinct junctions out of nine. The amino acid sequences of the seven cleavage sites have three conserved amino acids, V, H, Q in positions P4, P2, P1, respectively. Small molecules as well as conjugated peptides were tested for proteolytic activity of the enzyme. None of small molecules tested, such as methylumbelliferyl-p-guanidinobenzoate, p-nitrophenyl-p'-guanidinobenzoate, p-nitrophenyl acetate, and methylumbelliferyl-N-acetylglutamate, were hydrolyzed, Ac-V-Y-H-Q-Mca was also not hydrolyzed, Intramolecularly quenched fluorogenic substrates Dns-P-V-Y-H-Q-A-W-NH2 and Dns-P-V-Y-H-Q-W-NH2 emitted fluorescence after addition of TuMV NIa protease. The proteolysis rate of Dns-P-V-Y-H-Q-A-W-NH2 was comparable to that of the tetradecapeptide with an optimum sequence, but Dns-P-V-Y-H-Q-WNH, was hydrolyzed at a slower rate, which was confirmed independently by HPLC analysis. These results suggest that intramolecularly quenched fluorogenic substrates can be used for the continuous assay of TuRMV NIa protease. (C) 2000 Academic Press.X114sciescopu
A triple mutant (Y14F/Y30F/Y55F) of ketosteroid isomerase is more stable than its wild type through hydrophobic interaction as well as aromatic-aromatic interaction.
No full textX11sciescopu
An overview of biotechnology in Korea
No full textDuring the 1980s and 1990s, Korea has invested heavily in biotechnology research and development, with the aim of becoming a leading nation in this field. It is important to understand the historical progress of this development in order to appreciate the current state of biotechology in Korea. However, it is equally important to know what research is currently being planned and carried out, and by whom.X111sciescopu
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