Cell-free translation and regulation of Candida tropicalis catalase messenger RNA.

Abstract

To gain information on metabolic control and peroxisome biogenesis in Candida tropicalis growing on n-alkanes, cell-free translation of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6), a general marker enzyme of peroxisomes, was performed. The level of catalase activity in alkane-grown cells was approximately 9-fold and 27-fold higher than that in ethanol-grown and glucose-grown cells, respectively. Immunochemical titration experiments with rabbit antiserum against the purified peroxisomal catalase from alkane-grown C. tropicalis indicated that the remarkable variation in the enzyme activity level on different carbon sources was ascribable to a corresponding change in the amount of the enzyme protein. When cell-free translation was carried out with the mRNA-dependent reticulocyte lysate system, total RNA prepared from alkane-grown cells was shown to direct the synthesis of catalase subunit in vitro. The identity of the cell-free translation product was ascertained by the following evidence: (a) the translation product was immuno-reactive with specific antibody to catalase and competed effectively with the authentic enzyme for immunoprecipitation; (b) it possessed a molecular weight indistinguishable from that of authentic catalase subunit (Mr 54000); (c) its peptide fragments formed by partial digestion with Staphylococcus aureus V8 protease were identical with those from the authentic enzyme. With the use of the cell-free translation system, it was indicated that the significant change in the amount of catalase protein on different carbon sources nearly paralleled that in the activity of the mRNA encoding the enzyme.