scholarly journals Cloning and expression of a novel Mu class murine glutathione transferase isoenzyme

2002 ◽  
Vol 366 (3) ◽  
pp. 817-824 ◽  
Author(s):  
Jianxia GUO ◽  
Ludwika ZIMNIAK ◽  
Piotr ZIMNIAK ◽  
John L. ORCHARD ◽  
Shivendra V. SINGH
Keyword(s):  
Small Intestine ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutathione Transferase ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Amino Acid Residues ◽  
Reverse Transcription Pcr ◽  
Mouse Small Intestine

The present study describes the cDNA cloning, expression and characterization of a novel Mu class murine glutathione transferase (GST) isoenzyme. Screening of a cDNA library from the small intestine of a female A/J mouse using consensus probes derived from Mu class murine GST genes (mGSTM1—mGSTM5) resulted in the isolation of a full-length cDNA clone of a previously unknown Mu class GST gene (designated as mGSTM7). The choice of tissue was based on our previous identification in female A/J mouse small intestine of a potentially novel Mu class GST isoenzyme. The deduced amino acid sequence of mGSTM7, which comprises of 218 amino acid residues, exhibited about 67—78% identity with other Mu class murine GSTs. Recombinant mGSTM7-7 cross-reacted with anti-(GST Mu) antibodies, but not with anti-(GST Alpha) or anti-(GST Pi) antibodies. The pI and the reverse-phase-HPLC elution profile of recombinant mGSTM7-7 were different from those of other Mu class murine GSTs. The substrate specificity of mGSTM7-7 was also different compared with other Mu class murine GSTs. Interestingly, mGSTM7 had a higher identity with the human Mu class isoenzyme hGSTM4 (87% identity and 94% similarity in the amino acid sequence) than with any of the known mouse Mu class GSTs. Specific activities of recombinant mGSTM7-7 and human GSTM4-4 were comparable towards several substrates. For example, similar to hGSTM4-4, recombinant mGSTM7-7 was poorly active in catalysing the GSH conjugation of 1-chloro-2,4-dinitrobenzene and ethacrynic acid, and lacked activity towards 1,2-dichloro-4-nitrobenzene and 1,2-epoxy-3-(p-nitrophenoxy)propane. These results suggested that hGSTM4-4 might be the human counterpart of mouse GSTM7-7. Reverse transcription-PCR analysis using mGSTM7-specific primers revealed that mGSTM7 is widely expressed in tissues of female A/J mice, including liver, forestomach, lung, kidney, colon and spleen.

scholarly journals Cloning and Expression of ntnD, Encoding a Novel NAD(P)+-Independent 4-Nitrobenzyl Alcohol Dehydrogenase from Pseudomonas sp. Strain TW3

2000 ◽  
Vol 182 (11) ◽  
pp. 3136-3141 ◽  
Author(s):  
Keith D. James ◽  
Michelle A. Hughes ◽  
Peter A. Williams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Cloning And Expression ◽  
Significant Similarity ◽  
Reverse Transcription Pcr ◽  
Nitrobenzyl Alcohol ◽  
Dehydrogenase Gene

ABSTRACT Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)+ independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)+-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.

Characterization of the sulfated glycopeptide of chicken pepsinogen

1982 ◽  
Vol 47 (2) ◽  
pp. 709-718 ◽  
Author(s):  
Miroslav Baudyš ◽  
Vladimír Kostka ◽  
Karel Grüner ◽  
Jan Pohl
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Protein Molecule ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Sugar Moiety ◽  
Pepsinogen A ◽  
Identical Amino Acid Sequence ◽  
High Voltage Electrophoresis

S-sulfonated chicken pepsinogen was digested with TPCK-trypsin; large tryptic peptides, separated on Sephadex G-25 fine, were subjected to additional cleavage with α-chymotrypsin. The hold-up fraction of the chymotryptic digest from the Sephadex G-25 column, was resolved by high voltage electrophoresis. The three most acidic zones contained glycopeptides of identical amino acid sequence Val-Ser-Thr-Asn-Glu-Thr-Val-Tyr, yet differed in the composition of the sugar moiety. These glycopeptides, moreover, bear different numbers of sulfate groups which enabled the resolution of the peptides. The most acidic glycopeptide contains 7 glucosamine residues, 3 mannose residues and 5 sulfate groups, the second one 6 glucosamine residues, 3 mannose residues and 4 sulfate groups and the slowest, minority glycopeptide, 5 glucosamine residues, 2 mannose residues and 2 sulfate groups. The entire sugar moiety is attached to one of the chain viaasparagine. In other experiments the glycopeptides were also isolated from the thermolytic digest of chicken pepsin; their C-terminal sequence was shorter by two amino acid residues. The tentative assignment of the glycopeptides to the amino acid sequence of pepsinogen resulted from the analysis of the limited tryptic digest of the whole protein molecule. Chicken pepsinogen is glycosylated at the site of the chain occupied by a phosphoserine residue in hog pepsinogen A.

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.

scholarly journals Uncovering the Protocatechuate 2,3-Cleavage Pathway Genes

2009 ◽  
Vol 191 (21) ◽  
pp. 6758-6768 ◽  
Author(s):  
Daisuke Kasai ◽  
Toshihiro Fujinami ◽  
Tomokuni Abe ◽  
Kohei Mase ◽  
Yoshihiro Katayama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Coenzyme A ◽  
Sole Source ◽  
Pcr Analysis ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
Reverse Transcription Pcr ◽  
Semialdehyde Dehydrogenase ◽  
Cell Extracts

ABSTRACT Paenibacillus sp. (formerly Bacillus macerans) strain JJ-1b is able to grow on 4-hydroxybenzoate (4HB) as a sole source of carbon and energy and is known to degrade 4HB via the protocatechuate (PCA) 2,3-cleavage pathway. However, none of the genes involved in this pathway have been identified. In this study, we identified and characterized the JJ-1b genes for the 4HB catabolic pathway via the PCA 2,3-cleavage pathway, which consisted of praR and praABEGFDCHI. Based on the enzyme activities of cell extracts of Escherichia coli carrying praI, praA, praH, praB, praC, and praD, these genes were found to code for 4HB 3-hydroxylase, PCA 2,3-dioxygenase, 5-carboxy-2-hydroxymuconate-6-semialdehyde decarboxylase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate (OCA) tautomerase, and OCA decarboxylase, respectively, which are involved in the conversion of 4HB into 2-hydroxypenta-2,4-dienoate (HPD). The praE, praF, and praG gene products exhibited 45 to 61% amino acid sequence identity to the corresponding enzymes responsible for the catabolism of HPD to pyruvate and acetyl coenzyme A. The deduced amino acid sequence of praR showed similarity with those of IclR-type transcriptional regulators. Reverse transcription-PCR analysis revealed that praABEGFDCHI constitute an operon, and these genes were expressed during the growth of JJ-1b on 4HB and PCA. praR-praABEGFDCHI conferred the ability to grow on 4HB to E. coli, suggesting that praEGF were functional for the conversion of HPD to pyruvate and acetyl coenzyme A. A promoter analysis suggested that praR encodes a repressor of the pra operon.

scholarly journals The amino acid sequence of ferredoxin from Brassica napus (rape)

1980 ◽  
Vol 185 (1) ◽  
pp. 239-243 ◽  
Author(s):  
I Takruri ◽  
D Boulter
Keyword(s):  
Amino Acid ◽  
Brassica Napus ◽  
Amino Acid Sequence ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Plant Type ◽  
Single Polypeptide Chain ◽  
N Terminus ◽  
Single Polypeptide

The amino acid sequence of the ferredoxin of Brassica napus was determined by using a Beckman 890C sequencer in combination with the characterization of peptides obtained by tryptic and chymotryptic digestion of the protein; some peptides were subdigested with thermolysin. The molecule consists of a single polypeptide chain of 96 amino acid residues and has an unblocked N-terminus. The primary structure shows considerable similarity with other plant-type ferredoxins.

Identification and characterization of phenol hydroxylase from phenol-degrading Candida tropicalis strain JH8

2014 ◽  
Vol 60 (9) ◽  
pp. 585-591 ◽  
Author(s):  
Yan Long ◽  
Sheng Yang ◽  
Zhixiong Xie ◽  
Li Cheng
Keyword(s):  
Amino Acid ◽  
Candida Tropicalis ◽  
Amino Acid Identity ◽  
Phenol Hydroxylase ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Native Molecular Mass ◽  
Its Sequence Analysis

The gene phhY encoding phenol hydroxylase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The gene phhY contained an open reading frame of 2130 bp encoding a polypeptide of 709 amino acid residues. From its sequence analysis, it is a member of a family of flavin-containing aromatic hydroxylases and shares 41% amino acid identity with phenol hydroxylase from Trichosporon cutaneum. The recombinant phenol hydroxylase exists as a homotetramer structure with a native molecular mass of 320 kDa. Recombinant phenol hydroxylase was insensitive to pH treatment; its optimum pH was at 7.6. The optimum temperature for the enzyme was 30 °C, and its activity was rapidly lost at temperatures above 60 °C. Under the optimal conditions with phenol as substrate, the Km and Vmax of recombinant phenol hydroxylase were 0.21 mmol·L–1 and 0.077 μmol·L–1·min−1, respectively. This is the first paper presenting the cloning and expression in E. coli of the phenol hydroxylase gene from C. tropicalis and the characterization of the recombinant phenol hydroxylase.

scholarly journals Cloning and Expression of the Gene for the Na+-Coupled Serine Transporter from Escherichia coli and Characteristics of the Transporter

1998 ◽  
Vol 180 (24) ◽  
pp. 6749-6752 ◽  
Author(s):  
Wakano Ogawa ◽  
Young-Mog Kim ◽  
Tohru Mizushima ◽  
Tomofusa Tsuchiya
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Copy Number ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Low Copy Number ◽  
Hydropathy Analysis

ABSTRACT We cloned a gene (sstT) for the Na+/serine symporter from the chromosome of Escherichia coli by using a low-copy-number vector and sequenced it. According to the deduced amino acid sequence, the transporter (SstT) consists of 414 amino acid residues. Hydropathy analysis suggested that the SstT protein possesses 9, instead of 12, hydrophobic domains.

scholarly journals Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus

1997 ◽  
Vol 325 (2) ◽  
pp. 527-531 ◽  
Author(s):  
Naoki OHKURA ◽  
Hiroaki OKUHARA ◽  
Seiji INOUE ◽  
Kiyoshi IKEDA ◽  
Kyozo HAYASHI
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Phospholipase A2 ◽  
Blood Plasma ◽  
Amino Acid Residues ◽  
Group Iii ◽  
Group I ◽  
Binding Surface ◽  
Group Ii

Three distinct types of phospholipase A2 (PLA2) inhibitory proteins (PLIα, PLIβ, and PLIγ) were isolated from the blood plasma of the Chinese mamushi, Agkistrodonblomhoffiisiniticus. PLIα is an inhibitor that we have already purified and whose amino acid sequence we have already determined [Ohkura, Inoue, Ikeda and Hayashi (1993) J. Biochem. (Tokyo) 113, 413–419]. It inhibited selectively the group-II acidic PLA2s from Crotalidae venom. PLIβ was a 160-kDa glycoprotein having a trimeric structure composed of 50-kDa subunits. The amino acid sequence of the first 30 amino acids of the N-terminal part of the 50-kDa subunit was determined and found to have no significant homology to that of known proteins. PLIβ was a selective inhibitor against the group-II basic PLA2s from Crotalidae venom. Some amino acid residues located in or close to the interfacial binding surface of the group-II basic PLA2s were suggested to be involved in selective binding to PLIβ. PLIγ was a 100-kDa glycoprotein containing 25-kDa and 20-kDa subunits and inhibited all of the PLA2s investigated equally, including Elapidae venom PLA2s (group I), Crotalidae and Viperidae venom PLA2s (group II) and honey-bee PLA2 (group III). From the N-terminal sequences of the two subunits, PLIγ was found to be the same type of PLI that had been purified from Thailand cobra plasma.

Amino acid sequence of cyanogen bromide fragment CB5 of human hemopexin

1989 ◽  
Vol 54 (3) ◽  
pp. 803-810 ◽  
Author(s):  
Ivan Kluh ◽  
Ladislav Morávek ◽  
Manfred Pavlík
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Cyanogen Bromide ◽  
Polypeptide Chain ◽  
Amino Acid Residues ◽  
Mild Acid Hydrolysis ◽  
Methionine Residue ◽  
Cyanogen Bromide Fragment ◽  
Mild Acid

Cyanogen bromide fragment CB5 represents the region of the polypeptide chain of hemopexin between the fourth and fifth methionine residue (residues 232-352). It contains 120 amino acid residues in the following sequence: Arg-Cys-Ser-Pro-His-Leu-Val-Leu-Ser-Ala-Leu-Thr-Ser-Asp-Asn-His-Gly-Ala-Thr-Tyr-Ala-Phe-Ser-Gly-Thr-His-Tyr-Trp-Arg-Leu-Asp-Thr-Ser-Arg-Asp-Gly-Trp-His-Ser-Trp-Pro-Ile-Ala-His-Gln-Trp-Pro-Gln-Gly-Pro-Ser-Ala-Val-Asp-Ala-Ala-Phe-Ser-Trp-Glu-Glu-Lys-Leu-Tyr-Leu-Val-Gln-Gly-Thr-Gln-Val-Tyr-Val-Phe-Leu-Thr-Lys-Gly-Gly-Tyr-Thr-Leu-Val-Ser-Gly-Tyr-Pro-Lys-Arg-Leu-Glu-Lys-Glu-Val-Gly-Thr-Pro-His-Gly-Ile-Ile-Leu-Asp-Ser-Val-Asp-Ala-Ala-Phe-Ile-Cys-Pro-Gly-Ser-Ser-Arg-Leu-His-Ile-Met. The sequence was derived from the data on peptides prepared by cleavage of fragment CB5 by mild acid hydrolysis, by trypsin and chymotrypsin.

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