Abstract
Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, α-globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (Mrs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH2-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with Mrs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the α-globulin and one of the new low-molecular-weight proteins of Mr 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH2-terminal amino acids.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- HMW:
-
high molecular weight
- IgE:
-
immunoglobulin E
- LMW:
-
low molecular weight
- Mr :
-
relative molecular mass
- PAGE:
-
polyacrylamide gel electrophoresis
- RP-HPLC:
-
reversed-phase high-performance liquid chromatography
- SDS:
-
sodium dodecyl sulphate
References
Asano, K., Svensson, B., Poulsen, F.M., Nygard, O., Nilsson, L. (1986a) Influence of a protein synthesis inhibitor from barley seeds upon different steps of animal cell-free protein synthesis. Carlsberg Res. Commun. 51, 75–81
Asano, K., Svensson, B., Svendsen, L, Poulsen, F.M., Roepstorff, P. (1986b) The complete primary structure of protein synthesis inhibitor II from barley seeds. Carlsberg Res. Commun. 51, 129–141
Barber, D., Limas, G.G., Gavilanes, J.G., Méndez, E. (1988) Isolation and characterization of thirteen new salt-soluble proteins from barley by reversed-phase high-performance liquid chromatography. Planta 176, 221–229
Bietz, J.A. (1985) High performance liquid chromatography: How proteins look in cereals. Cereal Chem. 62, 201–212
Bradford, M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Cagampang, G.B., Perdon, A.A., Juliano, B.O. (1976) Changes in salt-soluble proteins of rice during grain development. Phytochemistry 15, 1425–1429
Calés, C., Fando, J.L., Azuara, C., Salinas, M (1986) Developmental studies of the first step of the initiation of brain protein synthesis role for initiation factor 2. Mech. Ageing Dev. 33, 147–156
Carrasco, L., Vázquez, D., Hernández-Lucas, C., Carbonero, P., Garcia-Olmedo, F. (1981) Thionins: plant peptides that modify permeability in cultured mammalian cells. Eur. J. Biochem. 116, 185–189
Coleman, H.W., Roberts, W.K. (1982) Inhibitors of animal cellfree protein synthesis from grains. Biochim. Biophys. Acta 696, 239–244
Cosgrove, J.W., Brown, I.R. (1981) Characterization of an initiating cell-free protein synthesis from rabbit brain. J. Neurochem. 36, 1026–1036
Fando, J.L., Alaba, I., Escarmis, C., Fernández-Luna, J.L., Méndez, E., Saunas, M. (1985) The mode of action of restrictocin and mitogillin on eukaryotic ribosomes. Inhibition of brain protein synthesis cleavage and sequence of the ribosomal RNA fragment. Eur. J. Biochem. 149, 29–34
Fischer, S., Reimann, F., Wittmann-Liebold, B. (1989) A new modular sequencer. In: Methods in protein sequence analysis, pp. 98–107, Witmann-Liebold, B., ed. Springer, Berlin Heidelberg New York
Fischer, S., Wittmann-Liebold, B. (1987) Microsequencing with a new module sequencer, p. 107, Abstracts 18th Int. FEBS Congress, Ljubljana, Abstr., p. 107
Golenkov, V.F. (1983) Comparative amino acid composition of proteins of wheat, rye and triticale grain. In: Amino acid composition and biological value of cereal proteins, pp. 349–357, Lasztity, R., Hidvegi, M., eds. Reidel, Dordrecht
Guo, Y.J., Bishop, R., Ferhnström, H., Yu, G.Z., Lian, Y.N., Hua, S.D. (1986) Classification of Chinese rice varieties by electrofocusing. Cereal Chem. 63, 1–3
Houston, D.F., Mohammad, A. (1970) Purification and partial characterization of a major globulin from rice endosperm. Cereal Chem. 47, 5–9
Houston, D.F., Mohammad, A., Hernández, N.E.P. (1964) Highsulfur seed proteins. Cereal Chem. 41, 427–430
Jones, B.L., Lookhart, G.L. (1985) High performance liquid Chromatographic separation of peptides for sequencing studies. Cereal Chem. 62, 89–96
Juliano, B.O. (1985) Polysaccharides, proteins and lipids of rice. In: Chemistry and technology, 2nd edn., page 59, Juliano, B.O., ed. Am. Assoc. Cereal Chem., St. Paul, Minn
Katzenstein, G.E., Vrona, S.A., Wechsler, R.J., Steadmen, B.L., Lewis, R.V., Middaugh, H.R. (1986) Role of conformational changes in the elution of proteins from reversed-phase HPLC columns. Proc. Natl. Acad. Sci. USA 83, 4268–4272
Larkins, B.A. (1980) Seed storage proteins: characterization and biosynthesis. In: The biochemistry of plants, vol. 6, pp. 449–467, Marcus, A. ed. Academic Press], New York
Lázaro, A., Barber, D., Salcedo, G., Méndez, E., Garcia-Olmedo, F. (1985) Differential effects of high-lysine mutations on the accumulation of individual members of a group of proteins encoded by a disperse multigene family in the endosperm of barley (Hordeum vulgare L.). Eur. J. Biochem. 149, 617–623
Matsuda, T., Sugiyama, M., Nakamura, R., Torii, S. (1988) Purification and properties of an allergenic protein in rice grain. Agric. Biol. Chem. 52, 1465–1470
Moore, S. (1963) On the determination of cystine as cysteic acid. J. Biol. Chem. 238, 235–237
Morita, Y., Yoshida, C. (1968) Studies on γ-globulin of rice embryo. Agric. Biol. Chem. 32, 664–670
Ozaki, Y., Wada, K., Hase, T., Matsubara, H., Nakanishi, T., Yoshizumi, H. (1980) Amino acid sequence of a purothionin homolog from barley flour. J. Biochem. (Tokyo) 87, 549–555
Padhye, V.W., Salunkhe, D.K. (1979) Extraction and characterization of rice proteins. Cereal Chem. 56, 389–393
Pan, S.J., Reeck, G.R. (1988) Isolation and characterization of rice α-globulin. Cereal Chem. 65, 316–319
Pascual, C.G., Juliano, B.O., Tanaka, Y. (1981) Fractionation of globulins of milled rice. Phytochemistry 20, 2471–2475
Perdon, A.A., Juliano, B. (1978) Properties of a major α-globulin of rice endosperm. Phytochemistry 17, 351–353
Rahman, S., Shewry, P.R., Miflin, B.J. (1982) Differential protein accumulation during barley grain development. J. Exp. Bot. 33, 717–728
Reinbolt, J., Hounwanou, N., Boulanger, Y., Wittman-Liebold, B., Bosserhoff, A. (1983) Reversed-phase liquid chromatography of peptides for direct micro-sequencing. J. Chromatogr. 259, 121–130
Sadler, A.J., Micanovic, R., Katzenstein, G.E., Lewis, R.V., Mid-daugh, C.R. (1984) Protein conformation and reversed-phase high-performance liquid chromatography. J. Chromatogr. 317, 93–101
Stirpe, F., Barbieri, L. (1986) Ribosome-inactivating proteins up to date. FEBS Lett. 195, 1–8
Takahashi, T., Yamada, N., Iwamoto, K., Shimabayashi, Y., Izut-su, K. (1973) Some properties and characterization of rice seed hemagglutinin. Agric. Biol. Chem. 37, 29–36
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Walsh, M., McDougall, J., Wittmann-Liebold, B. (1988) Extended N-terminal sequencing of proteins of archaebacterial ribosomes blotted from two-dimensional gels onto glass fiber and poly(vinylidene difluoride) membrane. Biochemistry 27, 6867–6876
Wittmann-Liebold, B. (1988) A new approach for liquid-phase sequencing of minute polypeptide amounts on filters in the Berlin sequencers. J. Prot. Chem. 7, 304–306
Yamagata, H., Sugimoto, T., Tanaka, K., Kasai, Z. (1982) Biosynthesis of storage proteins in developing rice seeds. Plant Physiol. 70, 1094–1100
Author information
Authors and Affiliations
Additional information
We thank F. Soriano and F. Colillia for technical assistance, and Shirley McGrath for secretarial work. We also appreciate the cheerful assistance of the members of Instituto Nacional de Semillas, specially Mr. L. Solaices, who provided samples of rice. This work was supported by a grant from Comision Asesora de Investigación Ciéntifica y Técnica.
Rights and permissions
About this article
Cite this article
Limas, G.G., Salinas, M., Moneo, I. et al. Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins. Planta 181, 1–9 (1990). https://doi.org/10.1007/BF00202318
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00202318