To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant.

As part of our research on peroxisome biogenesis, catalase was purified from cotyledons of dark-grown cotton (Gossypium hirsutum L.) seedlings and monospecific antibodies were raised in rabbits. Purified catalase appeared as three distinct electrophoretic forms in non-denaturing gels and as a single protein band (with a subunit Mr of 57,000) on silver-stained SDS/polyacrylamide gels. Western blots of crude extracts and isolated peroxisomes from cotton revealed one immunoreactive polypeptide with the same Mr (57,000) as the purified enzyme, indicating that catalase did not undergo any detectable change in Mr during purification. Synthesis in vitro, directed by polyadenylated RNA isolated from either maturing seeds or cotyledons of dark-grown cotton seedlings, revealed a predominant immunoreactive translation product with a subunit Mr of 57,000 and an additional minor immunoreactive product with a subunit Mr of 64000. Labelling studies in vivo revealed newly synthesized monomers of both the 64000- and 57,000-Mr proteins present in the cytosol and incorporation of both proteins into the peroxisome without proteolytic processing. Within the peroxisome, the 57,000-Mr catalase was found as an 11S tetramer; whereas the 64,000-Mr protein was found as a relatively long-lived 20S aggregate (native Mr approx. 600,000-800,000). The results strongly indicate that the 64,000-Mr protein (catalase?) is not a precursor to the 57,000-Mr catalase and that cotton catalase is translated on cytosolic ribosomes without a cleavable transit or signal sequence.

Morphometric procedures were used with light and electron microscopy to examine glyoxysome number, volume, shape and distribution as well as mesophyll cell volume, in cotyledons of mature (50 d postanthesis), imbibed (5h) and germinated (24 and 37 h) cotton (Gossypium hirsutum L.) seeds. Additionally, activities of five glyoxysomal marker enzymes in cotyledon extracts were assayed at each of the above ages. Cell volume was determined from photomicrographs of Epon-embedded sections by the point-counting procedure. Analysis of variance showed that cell volume was not different among the tissue segments studied. Glyoxysomes were cytochemically stained for catalase (EC 1.11.1.6) activity with the 3,3'-diaminobenzidine-tetrahydrochloride procedure. Analyses involving both phase and electron microscopy, and two separate sterologic calculations for determining the number of glyoxysomes per cell, indicate that glyoxysomes are numerous in mature seeds, persist through desiccation and imbibition, then increase dramatically in volume (seven fold) but not number (a maximum of 1.5-fold), when enzyme activities increase two to six times (depending on the enzyme). During the entire period of increase in glyoxysomal enzyme activities, no ultrastructural evidence was found for glyoxysome formation or destruction. Our data, in contrast to some proposals in the literature, indicate that cottonseed glyoxysomes form during seed maturation, then develop following seed imbibition into pleomorphic organelles by posttranslational accumulation of proteins from the cytosol and transfer of membrane components probably from the endoplasmic reticulum.

Malate synthase (EC 4.13.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE element for studying certain aspects of protein trafficking is the availability of monospecific antibodies to specific organellar proteins. As part of our ongoing efforts to elucidate mechanisms of glyoxysomal enzyme acquisition in cotton seeds, we have purified and raised antibodies to isocitrate lyase (4) and catalase (13) and now report results for MS.MS has been partially purified from cottonseeds (20), purified to apparent homogeneity from only three oilseed species (cucumber 10, 22; castor bean 2, 5; corn 24), and purified to varying degrees in bacteria, yeast, and Euglena (Ref. 20 for references). In all procedures described for oilseeds, the enzyme was purified from glyoxysome fractions. In the procedure presented here the enzyme was extracted from homogenized cotyledons and purified using the same buffer medium for all steps. The final product was free of contaminating proteins as judged from silver-stained gels; this criterion for purity had not been applied previously to other purified MS preparations. New information is also presented on the immunological characteristics and cross-reactivity of the polyclonal antibodies raised in rabbits relative to different forms of MS in germinated and maturing seeds.MATERIALS AND METHODS Chemicals. PMSF, DTT, aprotinin, benzamidine-HCl, iodoacetamide, Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose CL-4B, Protein-A Sepharose CL-4B, bovine thyroglobulin, BSA (98-99%), bovine heart L-lactic dehydrogenase, bovine liver catalase (C-I 0), glyoxylic acid (free acid and sodium salt), alkaline phosphatase conjugated goat anti-rabbit IgG, fast red violet LB salt, naphthol AS-BI phosphate (sodium salt), MOPS, Triton X-100, and PVP-10 were purchased from Sigma Chemical Co.

Biochemical, electrophoretic and immunological studies were made among peroxisomal enzymes in three organs of soybean [Glycine max (L.) Merr. cv. Centennial] to compare the enzyme distribution and characteristics of specialized peroxisomes in one species. Leaves, nodules and etiolated cotyledons were compared with regard to several enzymes localized solely in their peroxisomes: catalase (EC 1.11.1.6), malate synthase (EC 4.1.3.2), glycolate oxidase (EC 1.1.3.1), and urate oxidase (EC 1.7.3.3). Catalase activity was found in all tissue extracts. Electrophoresis on native polyacrylamide gels indicated that leaf catalase migrated more anodally than nodule or cotyledon catalase as shown by both activity staining and Western blotting. Malate synthase activity and immunologically detectable protein were present only in the cotyledon extracts. Western blots of denaturing (lithium dodecyl sulfate) gels probed with anti‐cotton malate synthase antiserum, reveal a single subunit of 63 kDa in both cotton and soybean cotyledons. Glycolic acid oxidase activity was present in all three organs, but ca 20‐fold lower (per mg protein) in both nodule and cotyledon extracts compared to leaf extracts. Electrophoresis followed by activity staining on native gels indicated one enzyme form with the same mobility in nodule, cotyledon and leaf preparations. Urate oxidase activity was found in nodule extracts only. Native gel electrophoresis showed a single band of activity. Novel electrophoretic systems had to be developed to resolve the urate oxidase and glycolate oxidase activities; both of these enzymes moved cathodally in the gel system employed while most other proteins moved anodally. This multifaceted study of enzymes located within three specialized types of peroxisomes in a single species has not been undertaken previously, and the results indicate that previous comparisons between the enzyme content of specialized peroxisomes from different organisms are mostly consistent with that for a single species, soybean.