A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells.

A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Research Abstract Details 

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A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Abstract Text:

J Fujii,Y Ikeda,T Watanabe,Y Kawasaki,K Suzuki,C Fujii,M Takahashi,N Taniguchi,

Wild-type and several mutant human manganese superoxide dismutases (Mn-SODs) were produced in a baculovirus/insect cell system and characterized. The enzymatic activity of a homogenate of Sf21 cells, infected with baculovirus carrying wild-type Mn-SOD and grown in the conventional medium, was indistinguishable from that of control cells, but was augmented by supplementation with Mn2+. The protein produced was largely imported into the mitochondria, as judged from the enrichment in the mitochondrial fraction, the mobility of the protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results of N-terminal processing, which was confirmed by sequencing of the purified enzyme. However, a significant amount of precursor was also detected by an antibody raised against the human Mn-SOD signal peptide. While both Mn2+ and Fe3+ stimulated Mn-SOD accumulation within mitochondria, the active form was produced in the presence of submillimolar Mn2+ only. Amino acid substitutions at a signal peptide-cleavage site, His-Ser-Leu4 to Pro-Met-Va14, in the mature Mn-SOD prevented the processing of the precursor protein, and thus resulted in the accumulation of the precursor protein within mitochondria, as judged on immunostaining with an anti-Mn-SOD antibody. Mutant Mn-SODs with a truncated signal peptide or carboxyl region (8, 13, and 42 amino acid residues in the mature form) were barely solubilized, even with a nonionic detergent, and exhibited no activity, suggesting inappropriate folding of these mutant SODs. They were also susceptible to proteolytic degradation, while the wild-type and precursor forms were resistant. Thus, the baculovirus/insect cell expression system appears to be adequate for the analysis of mitochondrial import using intact cells as well as for the large scale production of active Mn-SOD.

A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Publishing Authors By Initials

J Fujii,Y Ikeda,T Watanabe,Y Kawasaki,K Suzuki,C Fujii,M Takahashi,N Taniguchi,

For similar enzymes and coenzymes: enzymes: oxidoreductases: superoxide dismutase research abstracts see: enzymes and coenzymes: enzymes: oxidoreductases: superoxide dismutase research

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A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of biochemistry

VOLUME: 124

Page Numbers: 340-6

Journal Abbreviation: J. Biochem.

ISSN: 0021-924X

DAY: 19

MONTH: Aug

YEAR: 1998

A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 376600

A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Keywords Mesh Terms:

KEYWORDS: Superoxide Dismutase

MESH TERMS: metabolism

Chemical & Substance for Abstract: A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells. Information

Substance Name: Superoxide Dismutase

Registry Number: EC 1.15.1.1

Grant and Affiliation Information for A defect in the mitochondrial import of mutant Mn-superoxide dismutase produced in Sf21 cells.

AFFILIATION: Department of Biochemistry, Osaka University Medical School, Suita, Osaka, 565-0871, Japan.

Country: JAPAN

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MEDLINETA: J Biochem

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