Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 μkat mg(-1) protein. The enzyme exhibited a Mr of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of Mr 43,000. Monospecific antibodies were raised against the homogeneous protein.

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Several forms of microbodies have been characterized on the basis of their biochemical functions. We have investigated cucumber cotyledons which house two different microbody forms during their development. In these cells, a shift from organelles with the enzymes of beta-oxidation and glyoxylate cycle to peroxisomes with the enzymes of the photosynthetic C2-cycle can be induced by light. The transition state and the time course of changes was studied at different levels of gene expression during the first 2 days of illumination, by quantifying the rate of de novo protein synthesis in cotyledons and by measuring the mRNA activities in vitro. Synthesis and turnover of particular proteins were determined during the transition stage by immunoprecipitation of malate synthase, isocitrate lyase, catalase, multifunctional protein, and thiolase, and quantified by fluorography. From the mRNA activities and the rate of protein synthesis, gene expression for enzymes of the glyoxylate cycle and beta-oxidation started to decrease 24-36 h after onset of continuous light. At that time the rate of synthesis of glycolate oxidase, a leaf peroxisomal marker, is already maximal. By pulse-chase experiments 0-48 h after the onset of light the speed and intensity of protein turnover were measured. Rates of proteolytic degradation of individual enzymes indicated that the different enzymes were not lost simultaneously or all at once. This excludes a destruction of the whole organelle by the lytic compartment.

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Earlier work on microbody biosynthesis has shown that glyoxysomal and liver peroxisomal proteins synthesized in the cytosol are made as precursors which are then transferred into the organelles and processed. Here, it is demonstrated that the unprecessed precursor detected in the cytosol after protein synthesis in vivo for an enzyme at the transition stage between glyoxysomes and leaf peroxisomes is indistinguishable from the product of translation in vitro. It is assumed that the transfer of extraorganellarly made precursor across the glyoxysomal membranes is followed by processing of the precursor and oligomerization to the tetrameric or 16-meric form of the enzyme. Oligomerization was, however, also observed in a portion of the cytosolic form.