scholarly journals Epitope mapping of a monoclonal antibody to human glutathione transferase P1–1 the binding of which is inhibited by glutathione

1997 ◽  
Vol 321 (2) ◽  
pp. 531-536
Author(s):  
Takenori TAKAHATA ◽  
Shigeki TSUCHIDA ◽  
Masashi OOMURA ◽  
Takashi MATSUMOTO ◽  
Junichi AZUMI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Epitope Mapping ◽  
Glutathione Transferase ◽  
Three Dimensional ◽  
Cysteine Residue ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Steric Configuration ◽  
Terminal Amino

Although the three-dimensional structure of human glutathione transferase (GST) P1Ő1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1Ő1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently inhibited the activity of GST P1Ő1 but did not affect the activities of either GST A1Ő1 or M1Ő1. On immunoblotting, the antibody reacted strongly with GST P1Ő1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1Ő1. When GST P1Ő1 and the antibody were incubated in the presence of 60 ƁM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 ƁM. The binding of GST P1Ő1 to antibody adsorbed to Protein AŐSepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1Ő1 were resolved by HPLC and a peptide that reacted with the antibody was detected by absorption experiments. N-Terminal amino acid sequencing revealed the peptide to be in the C-terminal portion of the enzyme, stretching from amino acid residues 198 to 208. A synthetic peptide of this sequence also absorbed the antibody. These results suggest that both GSH bound to the active site and N-ethylmaleimide bound to the cysteine residue repress antibody binding to the C-terminal region. Thus this antibody may be useful for examining the steric configuration of the C-terminal and other regions of GST P1Ő1 in the absence of GSH.

Characteristics of Two Forms of α-Amylases and Structural Implication

1999 ◽  
Vol 65 (10) ◽  
pp. 4652-4658 ◽  
Author(s):  
Kohji Ohdan ◽  
Takashi Kuriki ◽  
Hiroki Kaneko ◽  
Jiro Shimada ◽  
Toshikazu Takada ◽  
...  
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Amylase Gene ◽  
Raw Starch ◽  
Raw Starch Binding ◽  
Terminal Amino ◽  
Carboxyl Terminal

ABSTRACT Complete (Ba-L) and truncated (Ba-S) forms of α-amylases fromBacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the α-amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same α-amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as α-amylase.

Location of amino acid residues important for cobra venom factor function mapped on the three-dimensional structure of complement components C3 and C3c

2007 ◽  
Vol 44 (1-3) ◽  
pp. 172 ◽  
Author(s):  
David C. Fritzinger ◽  
Brian E. Hew ◽  
Bert J.C. Janssen ◽  
Piet Gros ◽  
Carl-Wilhelm Vogel
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Cobra Venom ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Complement Components ◽  
Cobra Venom Factor ◽  
Three Dimensional Structure ◽  
Factor Function

scholarly journals Efficacy of Dideoxynucleosides against Human Foamy Virus and Relationship to Its Reverse Transcriptase Amino Acid Sequence and Structure

2001 ◽  
Vol 75 (15) ◽  
pp. 7184-7187 ◽  
Author(s):  
Anne Yvon-Groussin ◽  
Pierre Mugnier ◽  
Philippe Bertin ◽  
Marc Grandadam ◽  
Henri Agut ◽  
...  
Keyword(s):  
Amino Acid ◽  
Reverse Transcriptase ◽  
Foamy Virus ◽  
Three Dimensional ◽  
Retroviral Vectors ◽  
Dimensional Structure ◽  
Human Foamy Virus ◽  
Amino Acid Residues ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human foamy virus (HFV), a retrovirus of simian origin which occasionally infects humans, is the basis of retroviral vectors in development for gene therapy. Clinical considerations of how to treat patients developing an uncontrolled infection by either HFV or HFV-based vectors need to be raised. We determined the susceptibility of the HFV to dideoxynucleosides and found that only zidovudine was equally efficient against the replication of human immunodeficiency virus type 1 (HIV-1) and HFV. By contrast, zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), and didanosine (ddI) were 3-, 3-, 30-, and 46-fold less efficient against HFV than against HIV-1, respectively. Some amino acid residues known to be involved in HIV-1 resistance to ddC, 3TC, d4T, and ddI were found at homologous positions of HFV reverse transcriptase (RT). These critical amino acids are located at the same positions in the three-dimensional structure of HIV-1 and HFV RT, suggesting that both enzymes share common patterns of inhibition.

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

Plant glutathione transferases — a decade falls short

2007 ◽  
Vol 85 (5) ◽  
pp. 443-456 ◽  
Author(s):  
Mahesh Basantani ◽  
Alka Srivastava
Keyword(s):  
Glutathione Transferase ◽  
Three Dimensional ◽  
Dehydroascorbate Reductase ◽  
Detoxification Enzymes ◽  
Reduced Form ◽  
Dimensional Structure ◽  
Glutathione Metabolism ◽  
Amino Acid Residues ◽  
Glutathione Peroxidases ◽  
Associated Proteins

The glutathione transferase (GST) superfamily in plants has been subdivided into eight classes, seven of which (phi, tau, zeta, theta, lambda, dehydroascorbate reductase, and tetrachlorohydroquinone dehalogenase) are soluble and one is microsomal. Since their identification in plants in 1970, these enzymes have been well established as phase II detoxification enzymes that perform several other essential functions in plant growth and development. These enzymes catalyze nucleophilic conjugation of the reduced form of the tripeptide glutathione to a wide variety of hydrophobic, electrophilic, and usually cytotoxic substrates. In plants, the conjugated product is either sequestered in the vacuole or transferred to the apoplast. The GSTs of phi and tau classes, which are plant-specific and the most abundant, are chiefly involved in xenobiotic metabolism. Zeta- and theta-class GSTs have very restricted activities towards xenobiotics. Theta-class GSTs are glutathione peroxidases and are involved in oxidative-stress metabolism, whereas zeta-class GSTs act as glutathione-dependent isomerases and catalyze the glutathione-dependent conversion of maleylacetoacetate to fumarylacetoacetate. Zeta-class GSTs participate in tyrosine catabolism. Dehydroascorbate reductase- and lambda-class GSTs function as thioltransferases. Microsomal-class GSTs are members of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily. A plethora of studies utilizing both proteomics and genomics approaches have greatly helped in revealing the functional diversity exhibited by these enzymes. The three-dimensional structure of some of the members of the family has been described and this has helped in elucidating the mechanism of action and active-site amino-acid residues of these enzymes. Although a large amount of information is available on this complex enzyme superfamily, more research is necessary to answer additional questions such as, why are phi- and tau-class GSTs more abundant than GSTs from other classes? What functions do phi- and tau-class GSTs perform in plant taxa other than angiosperms? Do more GST classes exist? Future studies on GSTs should focus on these aspects.

scholarly journals A Membrane-Bound Flavocytochromec-Sulfide Dehydrogenase from the Purple Phototrophic Sulfur Bacterium Ectothiorhodospira vacuolata

2000 ◽  
Vol 182 (11) ◽  
pp. 3097-3103 ◽  
Author(s):  
Vesna Kostanjevecki ◽  
Ann Brigé ◽  
Terrance E. Meyer ◽  
Michael A. Cusanovich ◽  
Yves Guisez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Signal Sequence ◽  
Sulfide Oxidation ◽  
Three Dimensional ◽  
Chromatium Vinosum ◽  
Dimensional Structure ◽  
Northern Hybridization ◽  
Amino Acid Residues ◽  
Membrane Bound

ABSTRACT The amino acid sequence of Ectothiorhodospira vacuolatacytochrome c-552, isolated from membranes withn-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp.thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of ChromatiumFCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosumnumbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.

scholarly journals Amino Acid Residues 4425–4621 Localized on the Three-Dimensional Structure of the Skeletal Muscle Ryanodine Receptor

2000 ◽  
Vol 78 (3) ◽  
pp. 1349-1358 ◽  
Author(s):  
Brenda L. Benacquista ◽  
Manjuli R. Sharma ◽  
Montserrat Samsó ◽  
Francesco Zorzato ◽  
Susan Treves ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Ryanodine Receptor ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure

scholarly journals In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

2002 ◽  
Vol 184 (14) ◽  
pp. 4018-4024 ◽  
Author(s):  
Ulf Olsson ◽  
Annika Billberg ◽  
Sara Sjövall ◽  
Salam Al-Karadaghi ◽  
Mats Hansson
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Three Dimensional Structure ◽  
Activity Measurements

ABSTRACT Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix α3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V max. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

scholarly journals Mutation of Key Residues in β-Glycosidase LXYL-P1-2 for Improved Activity

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1042
Author(s):  
Jing-Jing Chen ◽  
Xiao Liang ◽  
Tian-Jiao Chen ◽  
Jin-Ling Yang ◽  
Ping Zhu
Keyword(s):  
Amino Acid ◽  
Lentinula Edodes ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Recent Success ◽  
Key Residues ◽  
Positive Effect ◽  
Candidate Strain

The β-glycosidase LXYL-P1-2 identified from Lentinula edodes can be used to hydrolyze 7-β-xylosyl-10-deacetyltaxol (XDT) into 10-deacetyltaxol (DT) for the semi-synthesis of Taxol. Recent success in obtaining the high-resolution X-ray crystal of LXYL-P1-2 and resolving its three-dimensional structure has enabled us to perform molecular docking of LXYL-P1-2 with substrate XDT and investigate the roles of the three noncatalytic amino acid residues located around the active cavity in LXYL-P1-2. Site-directed mutagenesis results demonstrated that Tyr268 and Ser466 were essential for maintaining the β-glycosidase activity, and the L220G mutation exhibited a positive effect on increasing activity by enlarging the channel that facilitates the entrance of the substrate XDT into the active cavity. Moreover, introducing L220G mutation into the other LXYL-P1-2 mutant further increased the enzyme activity, and the β-d-xylosidase activity of the mutant EP2-L220G was nearly two times higher than that of LXYL-P1-2. Thus, the recombinant yeast GS115-EP2-L220G can be used for efficiently biocatalyzing XDT to DT for the semi-synthesis of Taxol. Our study provides not only the prospective candidate strain for industrial production, but also a theoretical basis for exploring the key amino acid residues in LXYL-P1-2.

scholarly journals Structure of Looped Regions in β-α- and α-β-Arches in Abcd-Units of Globular Proteins

2016 ◽  
Vol 11 (2) ◽  
pp. 159-169
Author(s):  
Е.В. Бражников ◽  
E.V. Brazhnikov
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Protein Structures ◽  
Three Dimensional ◽  
Structural Motif ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Homologous Proteins ◽  
Reverse Turn ◽  
Structure Of Proteins

Conformations of about 600 looped regions (loops) in β-α- and α-β-arches of a structural motif occurring in the abCd-unit of proteins were analyzed. On the whole, 258 abCd-units with a reverse turn of the polypeptide chain (236 PDB files) and 69 abCd-units with a direct turn (65 PDB files) were selected in non-homologous proteins. Four types of arches were studied: β-α- and α-β-ones at a direct turn of the chain; β-α- and α-β-ones at a reverse turn of the chain. For each type of arches, frequencies of loops occurrence of different lengths were determined and corresponding histograms were plotted. It was found that abCd-units with loops up to three amino acid residues long occur most frequently (57 %). In β-α-arches with a direct turn of the chain, loops consisting of two amino acid residues occur most often (44 %) and in 86% cases they have the βmαβαn - conformation. They have no Gly and Pro residues, and in position β there is an Asn residue. In such type of arches, the loops of one residue (βmεαn- or βmαLαn- conformation) contain the Gly residue most frequently. α-β-Arches with a direct turn of the chain have most commonly (18 %) loops of four amino acid residues. In this case, there is no predominant conformation of the loops. In β-α-arches with a reverse turn of the chain, most common are loops of seven amino acid residues (17%), and most part of them (88 %) have the βmαLββααββαn - conformation. α-β-Arches with a reverse turn of the chain contain most frequently (32%) loops of one amino acid residue (all Gly ones) with arch conformations αmεβn or αmαLβn. The above structural analysis of the abCd-unit has useful information for prediction of the three-dimensional structure of proteins and for molecular simulation of the de novo design of protein structures.

Sign in / Sign up

Export Citation Format

Share Document