Albumin Storage Proteins in the Protein Bodies of Castor Bean 1

Of the total protein in the protein bodies of castor bean (Ricinus communis L.), approximately 40% is represented by a group of dosely related albumins localized in the matrix of the organefle. This group of albumins has a sedimentation value of 2S and is resolved into several proteins of molecular weight around 12,000 daltons by sodium dodecyl sulfate-acrylamide gel electrophoresis. It has a high content of glutamate/glutamine and undergoes rapid degradation during the early stage of germination. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins, in addition to globulins, glutelins, and prolamines, are important storage proteins in seeds. Since the classic studies of seed proteins by Osborne (19) and Danielsson (6), the storage proteins of legumes and oil seeds have been considered to be globulins that are insoluble in water but soluble in salt solution. Water-soluble proteins, albumins, also exist in these seeds. Albumins have not been shown to be storage proteins, but are generally thought to be enzymes and other metabolic proteins (1, 2, 4, 7, 17). However, such consideration cannot explain the high content of albumins in many seeds (3, 5, 10). In this paper, we report the characterization of a major group of albumins isolated from the protein bodies of castor bean. We provide ample evidence to demonstrate its physiological role as a storage protein. In view of the abundance and ubiquitous occurrence of albumins in various seeds, we suggest that albumins are important storage proteins in seeds. We also establish the occurrence of two major but diverse classes of storage proteins having distinct suborganelle localization in the protein bodies of a seed. MATERIALS AND METHODS Preparation of Protein Fractions. Three g of dry deshelled castor bean (Ricinus communis L. var. Hale) were ground in 100 ml of 1 M NaCl, 0.035 M Na-phosphate buffer (pH 7.5) for 20 min in a VirTis 45 homogenizer. The crude extract was centrifuged at 10,000g for 30 min and the supernatant fraction beneath the fat layer was used as the total protein extract. Protein bodies from dry castor bean were isolated and purified by the nonaqueous technique reported previously (25). The purified protein bodies were lysed and subfractionated on a sucrose gradient to yield the matrix fraction and the crystalloid fraction (25). The 2S albumin proteins were purified from the matrix fraction of protein bodies isolated by the following method ' This work was supported by National Science Foundation Grant BMS-75-02320 and by National Institutes of Health Biochemical Research Support Grant 1S07 RR 07160. (23). Dry castor beans were ground in glycerol and the protein bodies were pelleted by centrifugation. The protein bodies were lysed by the addition of water and the matrix fraction was separated from the other organelle components by centrifugation on a sucrose gradient (25). The matrix proteins were applied to a Sephadex G-50 column (2.6 x 90 cm) equilibrated with 0.05 M imidazole-HCl buffer (pH 7) and eluated with the same buffer. Fractions were collected and assayed for protein composition by 15% gel electrophoresis. The 4-6S proteins were eluated in the void volume and the 2S proteins were eluated afterward. The 2S protein fractions were slightly contaminated by some of the 4-6S proteins (the lectins) which were presumably retained by sugar-binding interaction with dextran gel (18, 23). All fractions containing the 2S proteins were pooled, concentrated by lyophilization, and rechromatographed on the same column of Sephadex G-50. All of the eluated fractions with the 2S proteins were again pooled and the combined eluate showed no contamination on 15% gel electrophoresis. Determination of Sedimentation Values by Ultracentrifugation. Protein fractions were prepared for ultracentrifugation by dissolving in or dialyzing against 35 mm Na-phosphate buffer (pH 7.5) and 1 M NaCl. Purified protein bodies in hexane and CC14 were mixed with the buffer, and all of the organic solvents were allowed to evaporate under N2. After this treatment which solubilized the crystalloid and matrix proteins, the globoids and membrane were removed by centrifugation. Since catalase dissociated into its subunits in 1 M NaCl, the enzyme was dissolved in the buffer without NaCl. Sedimentation analyses were performed by sucrose gradient centrifugation according to Martin and Ames (15) with modification (10). Each 1-ml protein sample was applied on top of a linear density gradient composed of 34 ml of 5 to 30% (w/w) sucrose. The gradient solution also contained 35 mm Na-phosphate buffer (pH 7.5) and 1 M NaCl. In the centrifugation of catalase, NaCl was omitted in the gradient solution. The gradient was centrifuged at 25,000 rpm for 24 hr in a Beckman L2-65B ultracentrifuge using a SW 27 rotor. The gradient was fractionated into 1-ml fractions and the protein was assayed according to Lowry et al. (13) with BSA as the standard. Myoglobin (type II, Sigma) and bovine liver catalase (type C10, Sigma) were used as markers for sedimentation values. Catalase was assayed as described (14) and myoglobin was assayed by the Lowry method. Gel Electrophoresis. Disc and slab gel electrophoresis was performed in 7.5% or 15% polyacrylamide gel with SDS and a discontinuous buffer system (24, 25). Protein samples were incubated at room temperature for 30 min or boiled for 2 min in 0.01 M tris-HCI buffer (pH 6.8), 1% SDS, and as appropriate, 1% f-mercaptoethanol. Molecular wt standards were used as described previously (25). Protein Hydrolysis and Amino Acid Analysis. After dialysis against water and lyophilization, the protein samples were hydrolyzed to amino acids with 4 N methanesulfonic acid in vacuo. The procedure that preserved tryptophan was followed (20). The protein samples were reduced with DTT for half13 www.pl ntphysiol.org on October 31, 2017 Publis d by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 61, 1978 cystine analysis. Amino acid analyses were performed on a Beckman 120-C instrument. Germination. Castor beans were soaked overnight in running tap water and germinated in moist vermiculite in 30 C in darkness. The endosperm at various stages was ground with a mortar and pestle in grinding medium (11). All of the extracts were made to the same volume (2.66 ml/endosperm pair) with grinding medium. The extracts were centrifuged at 5,000g for 10 min and the supematant fractions beneath the fat layer were used for gel electrophoresis.