Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

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Abstract
Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.