Isolation and Characterization of Protein Bodies in Lupinus angustifolius

Abstract

Using Nycodenz, a novel density gradient medium, we isolated intact protein bodies from developing seeds of Lupinus angustifolius L. (cultivar Unicrop) and achieved excellent separation from the endoplasmic reticulum, mitochondria, and other organelles.The distribution of the storage protein conglutin-,# was taken as evidence that up to 96% of the protein bodies remained intact on the gradients and banded at 1.25 grams per milliliter. The protein bodies also contained the three other abundant proteins present in L. angustifolius seeds: conglutins-a, -y, and -a. Pulse labeling experiments were carried out to determine the site of proteolytic processing of conglutin-a, a legumin-like 1 lSvedberg unit storage protein. Cotyledons aged either 33 or 40 days after flowering were pulsed with [3H]leucine. Protein bodies obtained from the cotyledons aged 33 days after flowering contained only the labeled precursors of conglutin-a with molecular weights 85,000, 72,000, and 64,000, even after a 4 hour chase of the radioactivity. Protein bodies obtained from the cotyledons aged 40 days after flowering contained the same radioactive precursors if the tissue had been pulsed for 2 hours, and the processing products of these precursors when the tissue had been chased for 4 hours. These studies confirm that the subcellular location of proteolytic cleavage of this legumin-like protein is the protein body, that this activity is detected only in protein bodies from lupin seeds aged between 33 and 40 days of seed development after flowering and that protein bodies from seeds younger than this contain only unprocessed conglutin-a.Seeds of the grain legume Lupinus angustifolius contain three predominant storage proteins and one relatively minor storage protein. The predominant proteins, conglutins-a, -3, and -6, are members of the widespread lIS, 7S, and 2S families of storage proteins, respectively. The other protein in seeds of lupins, conglutin-y, appears to be an example of a fourth type of storage protein (3). Conglutin-y is a methionine and cysteine-rich protein and has physical and chemical properties quite distinct from the other classes of storage proteins (5, 25). Conglutin-y in lupins and the homologous relative in soybeans, "basic 7S globulin" (17) are both present only in relatively small amounts in seeds of the respective species.

Biosynthesis, cDNA and amino acid sequences of a precursor of conglutin ?, a sulphur-rich protein from Lupinus angustifolius

“…Unicrop was grown as described previously [13]. Pods to be used in labelling studies were removed from the plants approximately 33 days after flowering (DAF).…”

“…The suspension was stirred for 2 h at room temperature and insoluble material removed by centrifugation. Chromatography on C18 reversed-phase Sep-pak cartridges (WatersMillipore, USA) was carried out as previously described [13]. NEM-blocked conglutin i5 was selectively bound to the Sep-pak cartridge which had been activated with acetonitrile and equilibrated with water.…”

“…Cotyledons were therefore incubated with a short pulse of [35S]-cysteine to preferentially label conglutin b-related proteins. Cotyledons were placed on 20 #1 drops containing 0.37 MBq of L-[35S]cysteine and incubated as previously described [13,38]. In pulse-chase experiments, cotyledons were rinsed with sterile 881 water and the incubation continued on 20/A of sterile water or 1 mM unlabelled cysteine for the duration of the chase.…”

“…In this paper we report the results of a study of the biosynthesis and secretion of radiolabelled conglutin b in intact cotyledons, using a chemically based method to overcome the difficulties inherent in attempts to detect 2S storage proteins with conventional immunological procedures [ 13,19]. The results show that conglutin b is synthesized initially as a family of closely related precursors which form a major sink for [ 35S ]-cysteine in developing cotyledons and are sequestered into the endoplasmic reticulum.…”

Biosynthèse des protéines de réserve et formation des corps protéiques dans la graine de lupin jaune (Lupinus luteus L, Légumineuses)

“…Chez le lupin à feuilles étroites, seuls les précurseurs de la conglutine y ont été mis en évidence au stade correspondant (Johnson et al, 1985). La (Duranti et al, 1991;Gayler et al, 1984;Blagrove et al, 1984;Johnson et al, 1985;Gayler et al, 1989 (Gayler et al, 1990). La perte d'une séquence de 13 acides aminés conduit ensuite à la formation des sous-unités de la protéine mature (9 558 et 4 520 Da).…”