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Fine structure of protein bodies isolated from rice endosperm
ARCHIVES
OF
RIOCHEMISTRY
-13-D
RIOPHYSICS
103, 678-680 (1969)
COMMUNICATIONS Fine Structure
of
Protein
Bodies
Isolated
from
Rice Endosperm Most of the proteins in numerous plant seeds are localized in subcellular storage particles which are commonly called “protein bodies” or aleurone grain. Recently these protein bodies (l-6) have been morphologically well described by electronmicroscopy and isolated from original tissues. In previous papers (3,4), we have demonstrated the protein bodies in rice endosperm by electronmicroscopy and isolated fairly pure protein bodies in good yield and analyzed their proximate chemical composition. This paper reports fine structure of the isolated protein bodies as revealed by electron-microscopy. The protein bodies were prepared by modifying the previous procedure (3, 4) as follows: In 60 ml of isolation buffer’ was suspended 10 g of rice polish, together with 100 mg each of cellulase and macerating enzyme2 and the suspension was incubated for 3 hr at 30” with continuous shaking. The resultant macerate was homogenized slightly in Potter Elvehjem homogenizer and centrifuged at SOOOgfor 50 min. The precipitate was diluted to 75 ml with distilled water, placed on top of tube containing a density gradient medium which consisted of 45, 55, and 65% sucrose solution and then centrifuged for 15 min at 40,OOOg in a swing-out head rotor. After centrifugation, protein bodies were clearly separated into three fractions on the interfaces of the respective sucrose solution. Protein body fractions were gathered, respectively, and washed with distilled water. The respective fractions were freeze-dried and analyzed for protein, lipid, and carbohydrate by the method previously reported (3, 4). Protein content was fairly constant throughout the protein body fractions (about 60yo), however, contents of lipid and carbohydrate were variable among the fractions (from 10 to 28yo for lipid and from 12 to 29% for carbohydrate). The fraction of 1 0.5 M Sucrose containing 0.2 M phosphate at pH 5.8 and 0.1% KBr03. 2 A mixture of protopectinase, pectinase, and hemicellulase, purchased from Kinki Yakult Co. Ltd., Nishinomiya, Japan.
higher density contained less lipid and more carbohydrate. The respective freeze-dried fractions of the protein bodies were combined and submitted to the following chemical analysis of their composition. Over 50% of protein and lipid of rich polish were recovered as the protein bodies. A small amount of ash, RNA, phospholipid, phytic acid, nicotinic acid, and niacin were found in the protein bodies. The protein bodies contained most protein components detected in rice polish by the techniques of solubility fractionation, electrophoresis membrane and gel-filtration on on Cellogel Sephadex G-100 and G-150 column. No remarkable difference was found in amino acid composition between the protein bodies and rich polish. As observed under the light microscope, the protein bodies were round in shape, ranging in diameter from 1.5 to 4 EC. Some of them were clumped together forming an assembly of discrete bodies. The protein bodies were fixed with glutaraldehyde and successively with osmium t,etraoxide, dehydrated, embedded, sliced, and poststained with lead citrate and uranyl acetate for their electron-microscopic observation. Details of this procedure are described in legend to Fig. 1. Electron microscopy revealed that the specimen from the respective fractions consisted mostly of electron-dense bodies, more than half of which have a limiting membrane and distinct concentric strata structure as shown in Fig. 1. The strata structure which is especially clearly visible around the outer portion of the bodies consists of electron-dense and electron-thin layers arrayed alternatively and respective electron-dense layers appear to be composed of minute granules (about 15O d) of exceedingly high electron density. These granular structures became visible clearly by poststaining of the thin sections with uranyl acetate and lead citrate. The uniformity of these granules in size and their general presence in the protein bodies may suggest that they are a basic unit of structure of the protein bodies. Buttrose (6) has shown that protein deposits in developing wheat endosperm usually appear to 3 Purchased Italy. 678
from
Chemetron
Ltd.,
Milano,
COMMUXICATIONS have a granular structure with individual particles of approximately 100 i diameter. And very recently, St. Angels et al. (7) have also reported that almost all volume of protein bodies isolated from viable hempseeds is occupied by the crystalloids which are composed of repeating polygonalshaped macromolecules, even down to 80 A level. These ideas are compatible with the present ob-
679
servation concerned with fine granulation of the protein bodies of rice endosperm. The concentric strata structure observed in the protein bodies of rice endosperm is unique among various protein body structures reported up to this time. In developing wheat endosperm, however, Jennings et al. (5) have observed that protein bodies are often embedded in lamella structure of
FIG. 1. Electron-micrograph of thin section of protein body isolated from rice endosperm. Bar in figure represents 1 p. Photograph represents a body folmd in the fraction sedimented on 135% sucrose interface after the density gradient centrifugation. The protein body fraction was fixed with 57, glutaraldehyde for 1 hr at 0” and successively with l$&, osmium tetraoxide and 2y0 potassium dichromate buffer, pH 7.0 for another 2 hr. The fixed material was dehydrated by an ethanol series and embedded in epoxy resin. Ultra-thin sections of the embedded material were observed after staining with uranyl acetate and lead citrate.
COMMUNICATIONS
680
lipoprotein membranes and some of the lipoprotein structure is an integral part of the protein bodies. Jennings’s finding on structure of the wheat protein bodies are somewhat similar, but not entirely, to the strata structure reported in this paper. These distinct strata structure including fine granulation may be reminiscent of the well-known layered structure of starch granules (8, 9). Starch granules contaminated in the protein bodies preparation, however, were electron-thin and were quite different from the feature of the protein bodies. The population of the stratified bodies in the respective fractions and chemical analysis of the protein bodies fractions also allow us to conclude that the stratified type of the bodies are neither starch granules or lipid bodies but the protein bodies. Beside the stratified bodies, however, there were a significant number of uniformly electrondense bodies or their fragments in the respective fractions which might be an unresolved form of the stratified bodies or may represent another type of the protein bodies. Taking in consideration together of chemical analyses data and electron micrographs of the isolated protein bodies, the authors are seeking for possible existence of different types of protein bodies in rice endosperm with respect to their composition, fine structure, and biological function. ACKNOWLEDGMENT This research has been financed in part by a grant from the United States Department of Agriculture under P. L. 480. REFERENCES 1. ALTSCHUL, A.M., YATSU, L. Y., ORY, R. L., AND ENQLEMAN, E. M., Ann. Rev. Plant. Physiol. 17, 113 (1966). 2. TOMBS, M. P., Plant Physiol. 42, 797 (1967). 3. MITSUDA, H., YASUMOTO, K., MURAKAMI, K., KUSANO, T., AND KISHIDA, H., Agr. Biol. Chem. 31, 293 (1967). 4. MITSUDA, H., Y.UUMOTO, K., MURAKAMI, K., KUSANO, T., AND KISHIDA, H., 2Mem. Coil. Agr., Kyoto Univ., No. 92, p. 17 (1967). 5. JENNINGS, A. C., MORTON, R. K., AND PALK, B. A., Australian J. Biol. Sci., 16,366 (1963). 6. BUTTROSE, M. S., Australian J. Biol. Sci. 16, 305 (1963). 7. ST. ANGELO, A. J., YATSU, L. Y., AND ALTSCHUL, A. M., Arch. Biochem. Biophys. 124, 199 (1968). 8. MUSSIJLMAN, W. C., AND WAGONER, J. A., Cereal Chem. 46, 99 (1968).
9. FREY-WYSSLING, A., AND MUHLETHALER, K., “Ultrastructural Plant Cytology,” p. 243. Elsevier, New York (1965). HISATERU MITSUDA KAZUO MURAKAMI TAKANORI KUSANO KYODEN YASUMOTO Laboratory of llrutritional Chemistry Department of Food Science and Technology Faculty of Agriculture Kyoto University Kyoto, Japan Received August 5’1, 1968; accepted December S, 1968.
An Intermediate
Density
Lipoprotein
of
Rat Serum Structural investigations of the serum lipoproteins have been concentrated on those isolated from humans, with the individual lipoprotein entities usually distinguished by flotation analysis in high density salt solutions. In contrast, most metabolic studies on circulating lipoproteins have employed experimental animals, commonly the rat, although the methods of separation of molecular species and the interpretation of results have relied heavily on the physical characteristics determined for the human serum lipoproteins (1). Several reports have appeared indicating that ratserum lipoproteins differ significantly in quantitative distribution and in chemical and physical properties when compared with the human analogs
(2-4). By centrifuging rat serum in the density range 1.05-1.075 a lipoprotein fraction can be isolated as an individual component with a hydrated density of about 1.065, intermediate between the hydrat,ed densities of fl, or low density lipoprotein (LDL), and 01, or high density lipoprotein (HDL), which are the principal lipoprotein components of rat serum (4, 5). The hydrated density of this intermediate density lipoprotein (IDL) falls very near the point at which LDL and HDL are separated by conventional procedures. Lipoproteins were isolated from the serum of fasted male albino rats by a preparative ultracentrifugation technique (5) based on the classical method of Have1 et al. (6). The desired densities were obtained using KBr and were measured by pycnometry. The density class of each lipoprotein fraction was defined by measuring the density of the second milliliter of solution in the preparative ultracentrifuge tubes, as suggested by Ewing et al. (7). A typical ultracentrifuge pattern of the lipoproteins (densit,y <1.21), isolated from the
OF
RIOCHEMISTRY
-13-D
RIOPHYSICS
103, 678-680 (1969)
COMMUNICATIONS Fine Structure
of
Protein
Bodies
Isolated
from
Rice Endosperm Most of the proteins in numerous plant seeds are localized in subcellular storage particles which are commonly called “protein bodies” or aleurone grain. Recently these protein bodies (l-6) have been morphologically well described by electronmicroscopy and isolated from original tissues. In previous papers (3,4), we have demonstrated the protein bodies in rice endosperm by electronmicroscopy and isolated fairly pure protein bodies in good yield and analyzed their proximate chemical composition. This paper reports fine structure of the isolated protein bodies as revealed by electron-microscopy. The protein bodies were prepared by modifying the previous procedure (3, 4) as follows: In 60 ml of isolation buffer’ was suspended 10 g of rice polish, together with 100 mg each of cellulase and macerating enzyme2 and the suspension was incubated for 3 hr at 30” with continuous shaking. The resultant macerate was homogenized slightly in Potter Elvehjem homogenizer and centrifuged at SOOOgfor 50 min. The precipitate was diluted to 75 ml with distilled water, placed on top of tube containing a density gradient medium which consisted of 45, 55, and 65% sucrose solution and then centrifuged for 15 min at 40,OOOg in a swing-out head rotor. After centrifugation, protein bodies were clearly separated into three fractions on the interfaces of the respective sucrose solution. Protein body fractions were gathered, respectively, and washed with distilled water. The respective fractions were freeze-dried and analyzed for protein, lipid, and carbohydrate by the method previously reported (3, 4). Protein content was fairly constant throughout the protein body fractions (about 60yo), however, contents of lipid and carbohydrate were variable among the fractions (from 10 to 28yo for lipid and from 12 to 29% for carbohydrate). The fraction of 1 0.5 M Sucrose containing 0.2 M phosphate at pH 5.8 and 0.1% KBr03. 2 A mixture of protopectinase, pectinase, and hemicellulase, purchased from Kinki Yakult Co. Ltd., Nishinomiya, Japan.
higher density contained less lipid and more carbohydrate. The respective freeze-dried fractions of the protein bodies were combined and submitted to the following chemical analysis of their composition. Over 50% of protein and lipid of rich polish were recovered as the protein bodies. A small amount of ash, RNA, phospholipid, phytic acid, nicotinic acid, and niacin were found in the protein bodies. The protein bodies contained most protein components detected in rice polish by the techniques of solubility fractionation, electrophoresis membrane and gel-filtration on on Cellogel Sephadex G-100 and G-150 column. No remarkable difference was found in amino acid composition between the protein bodies and rich polish. As observed under the light microscope, the protein bodies were round in shape, ranging in diameter from 1.5 to 4 EC. Some of them were clumped together forming an assembly of discrete bodies. The protein bodies were fixed with glutaraldehyde and successively with osmium t,etraoxide, dehydrated, embedded, sliced, and poststained with lead citrate and uranyl acetate for their electron-microscopic observation. Details of this procedure are described in legend to Fig. 1. Electron microscopy revealed that the specimen from the respective fractions consisted mostly of electron-dense bodies, more than half of which have a limiting membrane and distinct concentric strata structure as shown in Fig. 1. The strata structure which is especially clearly visible around the outer portion of the bodies consists of electron-dense and electron-thin layers arrayed alternatively and respective electron-dense layers appear to be composed of minute granules (about 15O d) of exceedingly high electron density. These granular structures became visible clearly by poststaining of the thin sections with uranyl acetate and lead citrate. The uniformity of these granules in size and their general presence in the protein bodies may suggest that they are a basic unit of structure of the protein bodies. Buttrose (6) has shown that protein deposits in developing wheat endosperm usually appear to 3 Purchased Italy. 678
from
Chemetron
Ltd.,
Milano,
COMMUXICATIONS have a granular structure with individual particles of approximately 100 i diameter. And very recently, St. Angels et al. (7) have also reported that almost all volume of protein bodies isolated from viable hempseeds is occupied by the crystalloids which are composed of repeating polygonalshaped macromolecules, even down to 80 A level. These ideas are compatible with the present ob-
679
servation concerned with fine granulation of the protein bodies of rice endosperm. The concentric strata structure observed in the protein bodies of rice endosperm is unique among various protein body structures reported up to this time. In developing wheat endosperm, however, Jennings et al. (5) have observed that protein bodies are often embedded in lamella structure of
FIG. 1. Electron-micrograph of thin section of protein body isolated from rice endosperm. Bar in figure represents 1 p. Photograph represents a body folmd in the fraction sedimented on 135% sucrose interface after the density gradient centrifugation. The protein body fraction was fixed with 57, glutaraldehyde for 1 hr at 0” and successively with l$&, osmium tetraoxide and 2y0 potassium dichromate buffer, pH 7.0 for another 2 hr. The fixed material was dehydrated by an ethanol series and embedded in epoxy resin. Ultra-thin sections of the embedded material were observed after staining with uranyl acetate and lead citrate.
COMMUNICATIONS
680
lipoprotein membranes and some of the lipoprotein structure is an integral part of the protein bodies. Jennings’s finding on structure of the wheat protein bodies are somewhat similar, but not entirely, to the strata structure reported in this paper. These distinct strata structure including fine granulation may be reminiscent of the well-known layered structure of starch granules (8, 9). Starch granules contaminated in the protein bodies preparation, however, were electron-thin and were quite different from the feature of the protein bodies. The population of the stratified bodies in the respective fractions and chemical analysis of the protein bodies fractions also allow us to conclude that the stratified type of the bodies are neither starch granules or lipid bodies but the protein bodies. Beside the stratified bodies, however, there were a significant number of uniformly electrondense bodies or their fragments in the respective fractions which might be an unresolved form of the stratified bodies or may represent another type of the protein bodies. Taking in consideration together of chemical analyses data and electron micrographs of the isolated protein bodies, the authors are seeking for possible existence of different types of protein bodies in rice endosperm with respect to their composition, fine structure, and biological function. ACKNOWLEDGMENT This research has been financed in part by a grant from the United States Department of Agriculture under P. L. 480. REFERENCES 1. ALTSCHUL, A.M., YATSU, L. Y., ORY, R. L., AND ENQLEMAN, E. M., Ann. Rev. Plant. Physiol. 17, 113 (1966). 2. TOMBS, M. P., Plant Physiol. 42, 797 (1967). 3. MITSUDA, H., YASUMOTO, K., MURAKAMI, K., KUSANO, T., AND KISHIDA, H., Agr. Biol. Chem. 31, 293 (1967). 4. MITSUDA, H., Y.UUMOTO, K., MURAKAMI, K., KUSANO, T., AND KISHIDA, H., 2Mem. Coil. Agr., Kyoto Univ., No. 92, p. 17 (1967). 5. JENNINGS, A. C., MORTON, R. K., AND PALK, B. A., Australian J. Biol. Sci., 16,366 (1963). 6. BUTTROSE, M. S., Australian J. Biol. Sci. 16, 305 (1963). 7. ST. ANGELO, A. J., YATSU, L. Y., AND ALTSCHUL, A. M., Arch. Biochem. Biophys. 124, 199 (1968). 8. MUSSIJLMAN, W. C., AND WAGONER, J. A., Cereal Chem. 46, 99 (1968).
9. FREY-WYSSLING, A., AND MUHLETHALER, K., “Ultrastructural Plant Cytology,” p. 243. Elsevier, New York (1965). HISATERU MITSUDA KAZUO MURAKAMI TAKANORI KUSANO KYODEN YASUMOTO Laboratory of llrutritional Chemistry Department of Food Science and Technology Faculty of Agriculture Kyoto University Kyoto, Japan Received August 5’1, 1968; accepted December S, 1968.
An Intermediate
Density
Lipoprotein
of
Rat Serum Structural investigations of the serum lipoproteins have been concentrated on those isolated from humans, with the individual lipoprotein entities usually distinguished by flotation analysis in high density salt solutions. In contrast, most metabolic studies on circulating lipoproteins have employed experimental animals, commonly the rat, although the methods of separation of molecular species and the interpretation of results have relied heavily on the physical characteristics determined for the human serum lipoproteins (1). Several reports have appeared indicating that ratserum lipoproteins differ significantly in quantitative distribution and in chemical and physical properties when compared with the human analogs
(2-4). By centrifuging rat serum in the density range 1.05-1.075 a lipoprotein fraction can be isolated as an individual component with a hydrated density of about 1.065, intermediate between the hydrat,ed densities of fl, or low density lipoprotein (LDL), and 01, or high density lipoprotein (HDL), which are the principal lipoprotein components of rat serum (4, 5). The hydrated density of this intermediate density lipoprotein (IDL) falls very near the point at which LDL and HDL are separated by conventional procedures. Lipoproteins were isolated from the serum of fasted male albino rats by a preparative ultracentrifugation technique (5) based on the classical method of Have1 et al. (6). The desired densities were obtained using KBr and were measured by pycnometry. The density class of each lipoprotein fraction was defined by measuring the density of the second milliliter of solution in the preparative ultracentrifuge tubes, as suggested by Ewing et al. (7). A typical ultracentrifuge pattern of the lipoproteins (densit,y <1.21), isolated from the