Purification and biosynthesis of cottonseed (Gossypium hirsutum L.) catalase.
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.
D002374:Catalase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D003368:Gossypium / Q000201:enzymology*; D005779:Immunodiffusion; D008715:Methionine / Q000378:metabolism; D008830:Microbodies / Q000201:enzymology; D014176:Protein Biosynthesis
D008715:Methionine; D002374:Catalase