Characterization of adrenodoxin precursor expressed in Escherichia coli.
The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.
Iwahashi
J
J
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Furuya
S
S
Mihara
K
K
Omura
T
T
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000302:Adrenal Cortex / Q000378:metabolism; D000325:Adrenodoxin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D007425:Intracellular Membranes / Q000502:physiology; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D010957:Plasmids; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D014508:Urea
D007506:Iron-Sulfur Proteins; D011498:Protein Precursors; D000325:Adrenodoxin; D000255:Adenosine Triphosphate; D014508:Urea; D004247:DNA