Cell-free synthesis of precursor forms of mitochondrial steroid hydroxylase enzymes of the bovine adrenal cortex

Robert E. Kramer, Raymond N. Du Bois, Evan R. Simpson, Christen M. Anderson, Kiyoko Kashiwagi, J. David Lambeth, Colin R. Jefcoate, Michael R. Waterman

    Research output: Contribution to journalArticlepeer-review

    54 Scopus citations

    Abstract

    Newly synthesized, [35S]methionine-labeled cholesterol side-chain cleavage cytochrome P-450, 11β-hydroxylase cytochrome P-450, adrenodoxin, and adrenodoxin reductase were immunoisolated from radiolabeled bovine adrenocortical cells and from rabbit reticulocyte lysate translation systems programmed with bovine adrenocortical RNA. Cholesterol side-chain cleavage cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight of 54,500 whereas this cytochrome P-450 immunoisolated from radiolabeled bovine adrenocortical cells had an apparent molecular weight of 49,000, an apparent molecular weight identical to that of the purified protein. Similarly, newly synthesized, [35S]methionine-labeled 11β-hydroxylase cytochrome P-450 immunoisolated from a reticulocyte lysate translation system had an apparent molecular weight 5500 daltons larger than that immunoisolated from radiolabeled adrenocortical cells (48,000) and the authentic cytochrome (48,000). The cell-free translation products of adrenodoxin and adrenodoxin reductase were also several thousand daltons larger than the corresponding mitochondrial proteins. The apparent molecular weight of adrenodoxin immunoisolated from a reticulocyte lysate translation system was 19,000, while that of the authentic protein was 12,000. Adrenodoxin reductase immunoisolated from a lysate translation system had an apparent molecular weight of 53,400; an apparent molecular weight 2300 daltons larger than that of adrenodoxin reductase immunoisolated from radiolabeled adrenocortical cells or purified by conventional techniques. These results demonstrate that all of the components of the mitochondrial steroid hydroxylase systems of the bovine adrenal cortex are synthesized as precursor molecules of higher molecular weight. Presumably, the precursor proteins are post-translationally converted to the mature enzymes upon insertion into the mitochondrion by a process which includes the proteolytic cleavage of the precursor segments.

    Original languageEnglish (US)
    Pages (from-to)478-485
    Number of pages8
    JournalArchives of Biochemistry and Biophysics
    Volume215
    Issue number2
    DOIs
    StatePublished - May 1982

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology

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