scholarly journals Evidence that glutathione S-transferases B1B1 and B2B2 are the products of separate genes and that their expression in human liver is subject to inter-individual variation. Molecular relationships between the B1 and B2 subunits and other Alpha class glutathione S-transferases

1989 ◽  
Vol 264 (2) ◽  
pp. 437-445 ◽  
Author(s):  
J D Hayes ◽  
L A Kerr ◽  
A D Cronshaw
Keyword(s):  
Amino Acid ◽  
Human Liver ◽  
Sequence Data ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Amino Acid Sequence Analysis ◽  
Cdna Sequences ◽  
Glutathione S Transferases ◽  
Molecular Relationships ◽  
The Relationship

The Alpha class glutathione S-transferases (GSTs) in human liver are composed of polypeptides of Mr 25,900. These enzymes are dimeric, and two immunochemically distinct subunits, B1 and B2, have been described that combine to form GSTs B1B1, B1B2 and B2B2 [Stockman, Beckett & Hayes (1985) Biochem. J. 227, 457-465]. Gradient affinity elution from GSH-Sepharose has been used to resolve the three Alpha class GSTs, and this method has been applied to demonstrate marked inter-individual differences in the hepatic content of GSTs B1B1, B1B2 and B2B2. The B1 and B2 subunits can be resolved by reverse-phase h.p.l.c., and their elution positions suggest that they are equivalent to the alpha chi and alpha y h.p.l.c. peaks described by Ketterer and his colleagues [Ostlund Farrants, Meyer, Coles, Southan, Aitken, Johnson & Ketterer (1987) Biochem. J. 245, 423-428]. The B1 and B2 subunits have now been cleaved with CNBr and the fragments subjected to automated amino acid sequence analysis. The sequence data show that B1 and B2 subunits do not arise from post-translational modification, as had been previously believed for the hepatic Alpha class GSTs, but are instead the products of separate genes; B1 and B2 subunits were found to contain different amino acid residues at positions 88, 110, 111, 112, 116, 124 and 127. The relationship between the B1 and B2 subunits and the cloned GTH1 and GTH2 cDNA sequences [Rhoads, Zarlengo & Tu (1987) Biochem. Biophys. Res. Commun. 145, 474-481] is discussed.

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals Hepatic glutathione S-transferases in mice fed on a diet containing the anticarcinogenic antioxidant butylated hydroxyanisole. Isolation of mouse glutathione S-transferase heterodimers by gradient elution of the glutathione-Sepharose affinity matrix

1991 ◽  
Vol 277 (2) ◽  
pp. 501-512 ◽  
Author(s):  
J D Hayes ◽  
L A Kerr ◽  
S D Peacock ◽  
A D Cronshaw ◽  
L I McLellan
Keyword(s):  
Amino Acid ◽  
Mouse Liver ◽  
Sequence Data ◽  
Fibroblast Cell ◽  
Exchange Chromatography ◽  
Mouse Fibroblast ◽  
Cdna Libraries ◽  
Butylated Hydroxyanisole ◽  
N Terminus ◽  
Glutathione S Transferases

Induction of glutathione S-transferases (GSTs) is believed to represent an important mechanism whereby butylated hydroxyanisole inhibits chemical carcinogenesis. The soluble hepatic GSTs expressed by mice fed on normal diets are all homodimers comprising Ya3 (Mr 25,800), Yb1 (Mr 26,400) and Yf (Mr 24,800) subunits. In addition to these constitutively expressed GSTs, we have identified enzymes containing Ya1 (Mr 25,600), Ya2 (Mr 25,600), Yb2 (Mr 26,200) and Yb5 (Mr 26,500) subunits from the livers of Balb/c mice fed on diets containing butylated hydroxyanisole (BHA). Gradient affinity elution of GSH-Sepharose has been used to resolve the mouse liver enzymes into several discrete pools of activity from which GSTs were purified by cation-exchange chromatography. The inducible Mu-class Yb2 and Yb5 subunits were separately isolated as the heterodimers GST Yb1Yb2 and GST Yb1Yb5 and their catalytic properties are described; this showed that 1,2-dichloro-4-nitrobenzene and trans-4-phenylbut-3-en-2-one are marker substrates for the mouse Yb1 and Yb2 subunits respectively, but no discriminating model substrate was found that allows the identification of the Yb5 subunit. Individual GST subunits were resolved by reverse-phase h.p.l.c. and their amino acid compositions were determined. Certain subunits (Yb1, Yb2, Yb5 and Yf) were also subjected to automated amino acid sequence analysis, and this demonstrated that the Yb5 subunit has a blocked N-terminus. The mouse Yb1, Yb2 and Yb5 subunits from the major inducible Mu-class heterodimers were cleaved with CNBr and purified peptides from the Yb2 and Yb5 subunits were sequenced. These data show that the Yb2 subunit is distinct from the GSTs that are encoded by the cDNAs that have been cloned from mouse liver cDNA libraries but possesses identity with the protein that is encoded by pmGT2, a cDNA isolated from a mouse fibroblast cell line by Townsend, Goldsmith, Pickett & Cowan [(1989) J. Biol. Chem. 264. 21582-21590]. The sequence data also show that the cDNA encoding the mouse Yb5 subunit has not, to date, been cloned, and the relationship between this subunit and Mu-class GSTs in other species that possess a blocked N-terminus (e.g. rat GST YoYo) is discussed.

scholarly journals cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 291-299 ◽  
Author(s):  
Christine A. Swann ◽  
Rory M. Hope ◽  
William G. Breed
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Amino Acid Sequence Identity ◽  
Sequence Data ◽  
Coding Region ◽  
Cdna Sequences ◽  
Sequence Identity ◽  
Murid Rodents

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.

scholarly journals Localization of Ca2+-dependent conformational changes of calretinin by limited tryptic proteolysis

1995 ◽  
Vol 308 (2) ◽  
pp. 607-612 ◽  
Author(s):  
J Kuźnicki ◽  
T L Wang ◽  
B M Martin ◽  
L Winsky ◽  
D M Jacobowitz
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Conformational Changes ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Amino Acid Sequence Analysis ◽  
Sds Page ◽  
Ef Hand ◽  
Digestion Pattern

Calretinin is an EF-hand Ca(2+)-binding protein expressed predominantly in some neurons. We have found that the tryptic digestion pattern of rat recombinant calretinin depends on Ca2+ concentration as determined by SDS/PAGE, amino-acid-sequence analysis and electrospray-ionization MS. Ca(2+)-saturated calretinin was cleaved between amino acids 60 and 61 to yield two fragments, which accumulated during cleavage. Small amounts of the larger fragment (amino acid residues 61-271) were further cleaved from the C-terminal end. Ca(2+)-free calretinin was also cleaved between residues 60 and 61; however, under the latter conditions the fragment 61-271 was further cleaved from the N-terminal end. Native rat calretinin was cleaved by trypsin in a similar Ca(2+)-dependent fashion. All identified fragments of recombinant calretinin bound 45Ca2+ on nitrocellulose filters, although to a different extent. The 61-271 fragment was released by EGTA from an octyl-agarose column in a manner similar to intact calretinin, while fragment 61-233 was not eluted by EGTA. These observations show that there are trypsin cleavage sites in calretinin that are available regardless of Ca2+ binding, other sites that are completely protected against trypsin on Ca(2+)-binding and sites which become partially available on Ca(2+)-binding. Together these data show that calretinin changes its conformation on Ca2+ binding and identify the regions which are exposed in apo and Ca(2+)-bound form.

Molecular characterisation of some species of Mylonchulus (Nematoda: Mononchida) from Japan and comments on the status of Paramylonchulus and Pakmylonchulus

Nematology ◽  
2009 ◽  
Vol 11 (3) ◽  
pp. 337-342 ◽  
Author(s):  
Masaaki Araki ◽  
Wasim Ahmad ◽  
Majid Olia ◽  
Nobuhiro Minaka
Keyword(s):  
Phylogenetic Trees ◽  
Sequence Data ◽  
Preliminary Investigation ◽  
Sister Group ◽  
Small Subunit ◽  
Ssu Rdna ◽  
Rdna Sequences ◽  
The Status ◽  
Molecular Relationships ◽  
The Relationship

AbstractComparative analyses of different regions of ribosomal DNA have become a popular tool in understanding the relationship among different species and genera and nematodes are no exception to this. In this study, molecular relationships were inferred from a nearly complete small subunit (SSU) of total 16 OTUs for five species of Mylonchulus, Paramylonchulus and Pakmylonchulus collected from various parts of Japan with two out-group taxa (Mononchus aquaticus and Clarkus papillatus) to examine the relationship among these species. Out of 1685 bp SSU rDNA sequences, phylogenetic trees using distance (NJ), parsimony and likelihood algorithms were performed. Obtained tree topologies were stable across algorithms and sequence data show that populations of the same species clustered together and four out of five species (M. brachyuris, M. hawaiiensis, M. oceanicus, M. sigmaturus) formed a monophyletic assemblage while M. mulveyi formed a sister group. Populations of species lacking subventral teeth but with a double gonad (M. oceanicus) stand with other Mylonchulus species, thereby confirming the synonymy of Pakmylonchulus, while populations with a narrow buccal cavity with few rows of denticles, no subventral teeth and a single prodelphic gonad (M. mulveyi = Paramylonchulus mulveyi) support to some extent the validity of the genus Paramylonchulus. Though a preliminary investigation, it is the first report on molecular relationships in Mylonchulus, probably a paraphyletic genus. Our results suggest that SSU rDNA sequence data are useful in understanding the relationship between genera and species.

Prokaryotic Expression and Functional Characterization of the 19 kDa Protein in Balanus albicostatus Cement

2013 ◽  
Vol 461 ◽  
pp. 445-450
Author(s):  
Chao Liang ◽  
Yun Qiu Li ◽  
Bi Ru Hu ◽  
Wen Jian Wu
Keyword(s):  
Amino Acid ◽  
Molecular Design ◽  
Functional Characterization ◽  
Blue Staining ◽  
Amino Acid Residues ◽  
Adhesive Ability ◽  
Solid Liquid ◽  
Dental Applications ◽  
The Relationship

Barnacle is a unique sessile crustacean, which produces a multi-protein complex historically called barnacle cement to attach to diverse immersed materials permanently. The proteinaceous cement exhibits powerful adhesive property and special waterproof capability to cure at solid-liquid boundaries, which makes it ideal biomaterial for technical, medical and dental applications. It has been proved that a 19 kDa protein component, termed cp-19k in the cement plays a key role in surface coupling during underwater attachment. To verify whether the bacterial recombinant 19 kDa protein retains the adhesive ability, we cloned and sequenced the Bacp-19k gene in Balanus albicostatus. It encodes 173 amino acid residues, with seven biased ones, Thr, Lys, Gly, Ala, Val, Ser and Leu, comprising about 80% of the total. Two amino acid substitutions (F69L, I106L) were discovered in Bacp-19k due to the polymorphisms in barnacle cp-19ks, compared with the submitted one (GenBank: AB242295.1). Recombinant Bacp-19k was highly expressed in host strain Escherichia coli BL21 (DE3) and purified by affinity chromatography. Adsorption of recombinant Bacp-19k to glass substrata was examined by Coomassie brilliant blue staining. Future study will reveal the relationship between specific structures and functions for molecular design of novel biomimetic underwater adhesives.

scholarly journals An Activated Glutamate Residue Identified in Photosystem II at the Interface between the Manganese-stabilizing Subunit and the D2 Polypeptide

2007 ◽  
Vol 282 (38) ◽  
pp. 27802-27809 ◽  
Author(s):  
Sascha Rexroth ◽  
Catherine C. L. Wong ◽  
Jessica H. Park ◽  
John R. Yates ◽  
Bridgette A. Barry
Keyword(s):  
Amino Acid ◽  
Photosystem Ii ◽  
Oxygenic Photosynthesis ◽  
Photosynthetic Oxygen Evolution ◽  
Amino Acid Residues ◽  
Post Translational Modification ◽  
Steady State Rate ◽  
Reactive Groups ◽  
Psii Subunits

Photosystem II (PSII) catalyzes the oxidation of water during oxygenic photosynthesis. PSII is composed both of intrinsic subunits, such as D1, D2, and CP47, and extrinsic subunits, such as the manganese-stabilizing subunit (MSP). Previous work has shown that amines covalently bind to amino acid residues in the CP47, D1, and D2 subunits of plant and cyanobacterial PSII, and that these covalent reactions are prevented by the addition of chloride in plant preparations depleted of the 18- and 24-kDa extrinsic subunits. It has been proposed that these reactive groups are carbonyl-containing, post-translationally modified amino acid side chains (Ouellette, A. J. A., Anderson, L. B., and Barry, B. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2204–2209 and Anderson, L. B., Ouellette, A. J. A., and Barry, B. A. (2000) J. Biol. Chem. 275, 4920–4927). To identify the amino acid binding site in the spinach D2 subunit, we have employed a biotin-amine labeling reagent, which can be used in conjunction with avidin affinity chromatography to purify biotinylated peptides from the PSII complex. Multidimensional chromato-graphic separation and multistage mass spectrometry localizes a novel post-translational modification in the D2 subunit to glutamate 303. We propose that this glutamate is activated for amine reaction by post-translational modification. Because the modified glutamate is located at a contact site between the D2 and manganese-stabilizing subunits, we suggest that the modification is important in vivo in stabilizing the interaction between these two PSII subunits. Consistent with this conclusion, mutations at the modified glutamate alter the steady-state rate of photosynthetic oxygen evolution.

scholarly journals Completion of the amino acid sequences of the A and B chains of subcomponent C1q of the first component of human complement

1982 ◽  
Vol 203 (3) ◽  
pp. 559-569 ◽  
Author(s):  
K B M Reid ◽  
J Gagnon ◽  
J Frampton
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Sequence Data ◽  
Helical Structure ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Human Complement ◽  
Major Site ◽  
A Chain ◽  
Asparagine Residue

The sequences of amino acid residues 109-224 of the A chain, and residues 109-22 of the B chain, of human subcomponent C1q are given. These results, along with previously published sequence data on the N-terminal, collagen-like, regions of the A and B chains [Reid (1979) Biochem. J. 179, 367-371] yield the complete amino acid sequences of the A and B chains of subcomponent C1q. The asparagine residue at position A-124 has been identified as the major site of asparagine-linked carbohydrate in subcomponent C1q. When the sequences of the C-terminal, 135-residue-long, ‘globular’ regions of A and B chains are compared they show 40% homology. The degree of homology over certain stretches of 15-20 residues, within the C-terminal regions, rises up to values of 73%, indicating the presence of strongly conserved structures. Structure prediction studies indicate that both the A and B chain C-terminal regions may adopt a predominantly beta-type structure with apparently little alpha-helical structure.

scholarly journals Action of human liver cathepsin B on the oxidized insulin B chain

1983 ◽  
Vol 213 (2) ◽  
pp. 467-471 ◽  
Author(s):  
M J McKay ◽  
M K Offermann ◽  
A J Barrett ◽  
J S Bond
Keyword(s):  
Amino Acid ◽  
Cathepsin B ◽  
Human Liver ◽  
Degradation Products ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Amino Acid Content ◽  
Peptide Bond ◽  
Cleavage Sites ◽  
Amino Acid Residues

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.

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