Chinese Hamster Rhodanese cDNA: Activity of the Expressed Protein Is Not Blocked by a C-Terminal Extension

Trevino, R. J.; Hunt, Jay D.; Horowitz, Paul M.; Chirgwin, John M.

doi:10.1006/prep.1995.1091

Cited by 5 publications

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“…Identity analysis of the entire amino acid sequence of rat liver MST compared with rat liver (deduced from cDNA) (16), mouse liver (deduced from cDNA) (18), avian liver (determined from purified enzyme) (14), hamster ovary (deduced from cDNA) (17), and bovine liver (determined from purified en-zyme) (12) rhodaneses shows 60, 60, 60, 59, and 59% identity, respectively (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…HUM RHOD, deduced primary structure of human liver rhodanese (15); RAT RHOD, deduced primary structure of rat liver rhodanese (16); HAM RHOD, deduced primary structure of hamster ovary rhodanese (17); MOU RHOD, deduced primary structure of mouse liver rhodanese (18); BOV RHOD, primary structure of purified bovine liver rhodanese (12); AVI RHOD, amino acid sequence from purified chicken liver rhodanese (14); SseA, deduced primary structure of E. coli sseA gene (43); shaded box, identical amino acid residues: arginine 187 (#1), arginine 196 (#2), cysteine 247 (#3), arginine 248 (#4), and lysine 249 (#5). AAQ, the order of these three amino acid residues was not determined (14); ‫,ށދއ‬ these three amino acids were not detected by protein sequencing of purified mature rhodanese from bovine liver (12) but were contained in the deduced primary structure of bovine adrenal rhodanese cDNA (13 could increase its MST activity.…”

Section: Figmentioning

confidence: 99%

“…Primary structures of the enzyme from various sources were determined from protein or deduced from cDNA, e.g. bovine liver (12) and adrenal (13), chicken liver (14), human liver (15), rat liver (16), hamster ovary (17), and mouse liver (18). Further, recombinant bovine liver (13) and adrenal (19), rat liver (20), hamster ovary (17), and mouse liver (18) rhodanese were overexpressed in Escherichia coli and characterized.…”

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Role of Amino Acid Residues in the Active Site of Rat Liver Mercaptopyruvate Sulfurtransferase

Nagahara

Nishino

1996

Journal of Biological Chemistry

107

134

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A complete amino acid structure of rat liver mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) was determined by sequence analysis of cDNA and purified enzyme. The enzyme consists of 296 amino acid residues with a calculated molecular mass of 32,808 Da. Sequence identity in cDNA and the deduced amino acid sequence are 65 and 60% respectively, between rat MST and rhodanese. By their entire sequence similarity MST and rhodanese are confirmed to be evolutionarily related enzymes (Nagahara, N., Okazaki, T., and Nishino, T. (1995) J. Biol. Chem. 270, 16230 -16235). The conversion of MST to rhodanese was attempted, and the role of amino acid residues was studied by site-directed mutagenesis with the isolated cDNA of rat liver MST. In the reaction the outer sulfur atom of thiosulfate is transferred to the Cys residue of the enzyme molecule to form a persulfide intermediate, which is subsequently attacked by cyanide anion to give thiocyanide. However, the enzyme has rather wide substrate specificity. Certain other sulfur compounds such as thiosulfonate (2) or persulfides (3, 4) may substitute for thiosulfate; and sulfite (5), sulfinates (5), or various thiol compounds (6) may substitute for cyanide. This enzyme was reported to be widely distributed in prokaryote and eukaryote mitochondria (7-9). Although the physiological role of this enzyme is not well understood, the enzyme is well characterized. The enzyme was first isolated from bovine liver (10) and subsequently from rat liver (11). Primary structures of the enzyme from various sources were determined from protein or deduced from cDNA, e.g. bovine liver (12) and adrenal (13), chicken liver (14), human liver (15), rat liver (16), hamster ovary (17), and mouse liver (18). Further, recombinant bovine liver (13) and adrenal (19), rat liver (20), hamster ovary (17), and mouse liver (18) rhodanese were overexpressed in Escherichia coli and characterized. The enzyme from bovine liver was crystallized (10, 21), and the three-dimensional structure was determined. (22)(23)(24). On the other hand, mercaptopyruvate sulfurtransferase (MST, 1 EC 2.8.1.2) catalyzes the following reaction.The enzyme was discovered in rat liver quite a long time ago (25-28), but compared with rhodanese the enzyme was not well characterized. Although the enzyme responsible for this reaction was at first assumed to be rhodanese, subsequent investigation with crystallized rhodanese showed that rhodanese did not catalyze this reaction and that another enzyme, mercaptopyruvate sulfurtransferase, was responsible for the reaction (28). Similar to rhodanese, compounds other than cyanide may function as sulfur acceptors in the reaction catalyzed by this enzyme (29 -32), and its physiological role is a matter of discussion (33, 34). This enzyme was reported to be distributed in both prokaryotes and eukaryotes and to be located in the cytosol of eukaryotic cells (26,27, 34,35), but the existence of this enzyme in mitochondria is also reported (36). Recently rat liver MST was purified to homogeneity, and th...

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Section: Resultsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

mentioning

confidence: 99%

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Role of Amino Acid Residues in the Active Site of Rat Liver Mercaptopyruvate Sulfurtransferase

Nagahara

Nishino

1996

Journal of Biological Chemistry

107

134

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“…Rhodanese, a 33 kDa sulfur transferase, was synthesized from plasmid DNA in a cell-free transcription/translation system derived from E. coli (Kudlicki et al, 1992). Cterminal extensions of the cloned wild-type gene were produced in three different ways: (a) A synthetic tRNAAl4 which functions as a suppressor tRNA (Ma et al, 1993) was used to insert alanine at the UGA stop codon of the wildtype protein so that a naturally occurring in-frame UAG stop codon would be used after translation of an additional 13 amino acids, (b) A coding sequence had been engineered that lacked the codons for the five C-terminal amino acids of native hamster rhodanese but coded for an extension of this rhodanese species by 28 other amino acids (Trevino et al, 1995). (c) A + 160-amino acid fusion protein was generated containing bovine rhodanese linked at its Cterminus to mouse dihydrofolate reductase (DHFR).…”

Section: Resultsmentioning

confidence: 99%

“…The chaperones DnaJ, DnaK, GrpE, GroES, and GroEL were bought from Epicentre Technologies. The following coding sequences inserted in the indicated plasmids were used: bovine rhodanese in pSP65 (Kudlicki et al, 1994a); hamster rhodanese coding sequence minus its five terminal amino acids, plus 28 unrelated amino acids in pETl Id (Trevino et al, 1995); bovine rhodanese minus its stop codon fused to mouse DHFR1 in pETlld (J.C., unpublished results).…”

Section: Methodsmentioning

confidence: 99%

Folding of an Enzyme into an Active Conformation While Bound as Peptidyl-tRNA to the Ribosome

et al. 1995

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Rhodanese bound to bacterial ribosomes as peptidyl-tRNA can be folded into an enzymatically active conformation by generating C-terminal extensions of the wild-type enzyme. Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system from plasmids containing the coding sequences for the wild-type enzyme or its C-terminally extended mutants. Two proteins with extensions of 23 amino acids or longer were enzymatically active while bound to the ribosomes whereas wild-type protein and a 13-amino acid extension were not. All forms of the enzyme were active after termination and release of the full-length protein from the ribosomes. All five of the bacterial chaperones were required to substantially increase the specific enzymatic activity of the extended rhodanese while the nascent protein was bound to ribosomes. The results provide direct support for the hypothesis that proteins acquire tertiary structure as they are formed in ribosomes.

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Cloning and Expression of Human Liver Rhodanese cDNA

Aita

Ishii

Akamatsu

et al. 1997

Biochemical and Biophysical Research Communications

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Chinese Hamster Rhodanese cDNA: Activity of the Expressed Protein Is Not Blocked by a C-Terminal Extension

Cited by 5 publications

References 0 publications

Role of Amino Acid Residues in the Active Site of Rat Liver Mercaptopyruvate Sulfurtransferase

Role of Amino Acid Residues in the Active Site of Rat Liver Mercaptopyruvate Sulfurtransferase

Folding of an Enzyme into an Active Conformation While Bound as Peptidyl-tRNA to the Ribosome

Cloning and Expression of Human Liver Rhodanese cDNA

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