CYTOCHEMICAL AND BIOCHEMICAL STUDIES OF CATALASE IN COTTON SEEDS (PEROXISOME, GLYOXYSOME, COTYLEDONS)

Experiments were designed to test the hypothesis that peroxisomes of cotton accumulate enzymes such as catalase (H(,2)O(,2):H(,2)O(,2) oxidoreductase; EC 1.11.1.6) post-translationally. The first chapter represents an ultrastructural and morphometric analysis of cottonseed peroxisomes cytochemically stained for catalase. It provides the first microscopic evidence for peroxisomes in ungerminated seeds. The data indicate that peroxisomes are numerous in mature seeds, persist through desiccation and imbibition relatively unchanged, then increase dramatically in volume (7-fold), but not number, when glyoxysomal enzyme activity increases 2- to 6-fold (depending on the enzyme). No evidence was found for organelle formation or destruction.;The third chapter describes purification (single protein on silver-stained SDS gel) of catalase from germinated cotton seeds and generation of monospecific antibodies. Western blots of SDS gels containing purified catalase, crude extracts and isolated peroxisomes revealed only one catalase subunit (Mr of 55 kDa) indicating (i) that the enzyme did not undergo any detectable change in Mr during purification, and (ii) the absence of a detectable, larger putative precursor. In vitro catalase, directed by mRNA isolated from germinated cotton seeds, also possessed a subunit of Mr of 55 kDa. In vivo pulse labelling and immunoprecipitation revealed a cytosolic form of catalase (subunit Mr of 55 kDa) and a minor, peroxisomal protein (subunit Mr of 63 kDa; native Mr of 600-800 kDa) labelled prior to peroxisomal catalase. The identity and significance of the 63 kDa protein remains unknown.;Overall, the data strongly indicate that cotton catalase is translated on cystolic ribosomes, without a cleavable transit or signal peptide, and accumulates in peroxisomes by some posttranslational uptake mechanism.;The second chapter represents an investigation of possible charge and size heterogeneity of cotton catalase. Multiple forms of peroxisomal catalase and their differential expression at different developmental ages are demonstrated. The forms of catalase characteristic of ungerminated seeds are not replaced by different forms following germination, but are supplemented by additional forms. Results from nondenaturing IEF and PAGE (Ferguson plots) indicate the various forms of catalase are charge isomers with a approximately Mr and 230 kDa. Western blots of SDS gels revealed only one catalase subunit (55 kDa) in cotton at all developmental ages.