Expression of cloned bovine adrenal rhodanese

David M. Miller, Ruby Delgado, John Chirgwin, Stephen C. Hardies, Paul M. Horowitz

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70% sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.

Original languageEnglish (US)
Pages (from-to)4686-4691
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number8
StatePublished - Mar 15 1991
Externally publishedYes

Fingerprint

Thiosulfate Sulfurtransferase
Liver
Amino Acids
Escherichia coli
glycyllysine
Proteins
Plasmids
Enzymes
Amino Acid Sequence
Gels
Cells
Carboxypeptidase B
DNA sequences
Initiator Codon
Escherichia coli Proteins
Protein Sequence Analysis
Recombinant Proteins
Methionine
Post Translational Protein Processing
Sequence Homology

ASJC Scopus subject areas

  • Biochemistry

Cite this

Miller, D. M., Delgado, R., Chirgwin, J., Hardies, S. C., & Horowitz, P. M. (1991). Expression of cloned bovine adrenal rhodanese. Journal of Biological Chemistry, 266(8), 4686-4691.

Expression of cloned bovine adrenal rhodanese. / Miller, David M.; Delgado, Ruby; Chirgwin, John; Hardies, Stephen C.; Horowitz, Paul M.

In: Journal of Biological Chemistry, Vol. 266, No. 8, 15.03.1991, p. 4686-4691.

Research output: Contribution to journalArticle

Miller, DM, Delgado, R, Chirgwin, J, Hardies, SC & Horowitz, PM 1991, 'Expression of cloned bovine adrenal rhodanese', Journal of Biological Chemistry, vol. 266, no. 8, pp. 4686-4691.
Miller DM, Delgado R, Chirgwin J, Hardies SC, Horowitz PM. Expression of cloned bovine adrenal rhodanese. Journal of Biological Chemistry. 1991 Mar 15;266(8):4686-4691.
Miller, David M. ; Delgado, Ruby ; Chirgwin, John ; Hardies, Stephen C. ; Horowitz, Paul M. / Expression of cloned bovine adrenal rhodanese. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 8. pp. 4686-4691.
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abstract = "A cDNA for the enzyme rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) has been cloned from a bovine adrenal library. An initiator methionine codon precedes the amino-terminal amino acid found in the isolated protein. Rhodanese is synthesized in the cytoplasm and transferred to the mitochondrial matrix. Thus, any amino-terminal sequence required for organelle import is retained in the mature protein. Furthermore, the DNA sequence shows that there are three additional amino acids, Gly-Lys-Ala, at the carboxyl terminus that are not found by protein sequencing. Additionally, comparison of the published amino acid sequence with that encoded by the open reading frame revealed three differences in the amino acid sequence. Comparison of the bovine and chicken liver sequences shows an overall level of 70{\%} sequence homology, but there is complete identity of all residues that have been implicated in the function of the enzyme. When two mammalian cells, cos-7 and 293 cells, were transiently transfected with a plasmid containing the rhodanese coding region, rhodanese activity in lysates increased approximately 20-fold. Fluorograms of denaturing polyacrylamide gels detected a large increase in a polypeptide that co-migrated with the native protein and reacted with anti-rhodanese antibodies. Nondenaturing gels showed two active species that co-migrated with the two major electrophoretic forms purified by current procedures. Escherichia coli, transformed with a plasmid containing the rhodanese coding region, showed a 15-fold increase in rhodanese activity over baseline values. When the E. coli recombinant protein was analyzed on a nondenaturing gel, only one species was observed that co-electrophoresed with the more electropositive variant seen in purified bovine liver rhodanese. This single variant could be converted by carboxypeptidase B digestion to a form of the enzyme that co-migrated with the more electronegative species isolated from bovine liver. Thus, two major, enzymatically active electrophoretic variants, commonly observed in mammalian cells, can be accounted for by carboxyl-terminal processing without recourse to other post-translational modifications.",
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