Organelles isolated from castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation were assessed for purity by mixing isolated radiolabelled organelles with unlabelled homogenates and recentrifuging. The glyoxysomal fraction was found to be very pure but glyoxysomal membrane fragments contaminated the mitochondria and microsomes. Integral membrane proteins of the three major organelle fractions were isolated using the Triton X-114 method. SDS-PAGE analysis of Triton X-114 extracts revealed that the microsomes and glyoxysomes contained proteins of similar molecular weights. The slight glyoxysomal membrane contamination of the microsomes was dismissed as the cause of these similarities. The e.r.-derived microsomal fraction was shown to possess lipid glucosyltransferase activity. The microsomal fraction was subfractionated on flotation gradients. Two major subfractions were obtained and appeared to represent microsomes of rough- and smooth- e.r. origin, the lighter subfraction being enriched in N-acetyl glucosaminyl and mannosyl transferases while the heavier subfraction was slightly enriched in protein fucosyltransferase. Glycoproteins of the glyoxysomal membrane were shown by in vivo [3H] and [14c] sugar incorporation to possess oligosaccharide moieties containing glucosamine (GlcNAc), fucose and galactose residues. Mannose residues were detected by overlaying SDS-PAGE separated polypeptides with [125i] concanavalin A. The presence of GlcNAc and mannose indicated the presence of e.r. synthesized asparagine-linked oligosaccharide chains and this was confirmed by demonstrating (a) their susceptibility to endo-H and (b) inhibition of glycosylation by tunicamycin. Glyoxysomal membrane protein synthesis was studied in vivo and in vitro. Structural similarities between shared microsomal and glyoxysomal membrane polypeptides were shown using antiserum to glyoxysomal Triton X-114 extracts. Castor bean mRNA translation in vitro demonstrated that the major glyoxysomal membrane proteins did not undergo co-translational insertion into canine microsomes and were possibly synthesized on free, cytosolic ribosomes. The results are discussed with reference to recent models for microbody formation in other seeds and mammalian liver.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:354606 |
| Date | January 1984 |
| Creators | Conder, Michael John |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/113060/ |
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