- Email: [email protected]
An intermediate density lipoprotein of rat serum
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
COMMUNICATIONS
681
1 (A)
FIG. 1. Ult.rncent.rifugal flotation patterns of rat-serum lipoproteins concent.rat.ed 5.7.fold. Flotat,ion from right to left. Schlieren patterns 23 min after reaching 42,040 rpm, 2G”, 05” bar angle. KBr: ti = 1.19, L = LDL, I = IDL, H = IIDL. (A) normal rat, (13) hypothyroid rat. ser,,m of normal fasted rats, is shown in Fig. 1A. By measuring F rates of the mid-ordinate of each peak, corrected for viscosit,y, at various bnckground densities and extrapolating them to zero flotation, an estimate of the hydrated density of each component was obtained, (Fig. 2). Very low density lipoproteins (VLDL) moved too rapidly for accurate flotation measurement. The calculated values have been confirmed by measuring the minimum background density at which the components can be isolated by preparative Ikltracentrifugation, and the follr components can be isolated free of contamination by adjacettt fractions in the following density ranges: VLT)L (d < 1.01); LDL (1.02 < d < 1.05); IDL (1.05 < cl < 1.075); HDL (1.08 < d < 1.2). On electrophoresis in polyacrylamide gel at pII 9.1, IDL demonstrated a mobility int.ermediate between the@ (LDL) and thecu (HI)L) bands (Fig.
1.064
d FIG. 2.
FIG. 2. Flotation rate as a function of density. Each point represents a flotation rate (F) calculated from an ultracentrifugal rul, at the given density (d). Vallles for 7, the viscosity relative to were obtained from the International water, Critical Tables.
COMMUNICATIONS under different conditions. It has usually been considered as the highest density member of a heterogeneous LDL group, and has generally been ignored. IDL may be analogous to HDLr, a minor component of the human serum lipoprotein spectrum which has a reported hydrated density of 1.05 (9) and has never been fully characterized. Since nothing is known concerning the synthesis, metabolism, or functional role of this lipoprotein, it was of particular interest to find that when rats were made hypothyroid by radiosurgery with 1311 (lo), a several-fold increase in the concentration of IDL was observed along with slight increases in the concentrations of the other lipoprotein components (Fig. 1B). IDL from these TABLE AMINO
FIG. 3. Vertical polyacrylamide gel phoresis of rat serum lipoproteins. Four cyanogum; pH 9.1, Tris-EDTA-borate 106 V/20 cm; 7 hr; amido black stain. 1 2 = LDL, 3 = IDL, 4 = HDL, and 5 serum.
electroper cent buffer; = HDL, = whole
3). Immunochemical studies by double diffusion indicate that IDL contains the a peptide characteristic of HDL and does not react significantly with antiserum to LDL. Amino acid analyses of the isolated purified fractions (Table I) show that the protein moieties of IDL and HDL are not identical. The chemical compositions of IDL and HDL (Table II) are sufficiently different to serve as further evidence that IDL is not a higher numbered polymer of HDL subunits. The original procedure (6) for the separation of human lipoproteins specified a density of 1.063 as the dividing point between LDL and HDL. When this method is applied to rat serum it may result in LDL or HDL fractions that are contaminated by IDL, depending on the type of salt employed, and the subsequent redistribrition of salt during ultracentrifugation. IDL is probably the minor component isolated with the LDL fraction from rat serum by Camejo (3) and Barclay (8)
I
ACID COMPOSITION OF ISOLATED LIPOPROTEINS~
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
IDL
HDLb
LDL
39.5 5.4 60.7 79.4 47.8 47.8 147.6 25.0 48.0 65.7 46.0 -
57.9 13.9 49.4 85.4 43.2 36.3 132.6 22.7 39.2 52.5 41.2 20.5 11.3 80.0 18.1 23.2
46 12 29 88 48 67 110 31 41 45 47 4 38 92 14 34
24.2 87.6 11.3 23.1
a Performed on Beckman 120 C analyzer. All values given in moles of amino acid per 100,000 g protein. 1,From the data of Camejo (3). TABLE
II
COMPOSITION OF ISOLATED LIPOPROTEINS" componentb Protein Total cholesterol Triglyceride Phospholipid
IDL
HDLC
LDL
39 2G 3 23
40 19 2 35
20 46 10 23
a Percentage of dry weight. b The four components were assayed as in Refs. 11-14 respectively. c From t,he data of Camejo (3).
683
COMMUNICATIONS rats could be readily isolated by ultracentrifugation and had a hydrated density similar to that of the corresponding component of normal rat serum. Marked increases in IDL concentration were also observed in the hypothyroid states induced by feeding rats propylthiouracil, aminotriazole, or an iodine-deficient diet. IDL from these rats was found to be similar in immunochemical behavior and amino acid composition to IDL isolated from normal rats. These findings describe IDL, a class of lipoprotein in the ultracentrifugal pattern of ratserum lipoproteins, with flotation and other characteristics intermediate between LDL and HDL. This component can be isolated in the predicted density range, and its chemical and physical properties are all consistent with the proposition that IDL is a distinct, albeit minor, class of lipoprotein. The specific increase of IDL observed in experimental hypothyroidism in the rat is further evidence that IDL is a distinct, and perhaps metabolically important, class of lipoprotein. That increase finds no direct parallel in human myxedema, since in the human, hypothyroidism results in specific increases in the concentration of serum p lipoproteins (1). ACKNOWLEDGMENTS This work was supported by Grant 6%AG-32 from the Florida Heart Association and Grant AM 02457 from the National Institutes of Health. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9.
10.
11.
12.
SCANU, A., Advan. Lipid Res. 3, 64 (1965). NAR~YAN, K. A., Anal. Biochem. 20,582 (1967). CAMEJO, G., Biochemistry 6, 3228 (1967). WINDMUELLER, H. G., AND LEVY, R. I., J. Biol. Chem. 242, 2246 (1967). FALOONA, G. R., STEW.~RT, B. N., AND FRIED, M., Biochemistry 7,720 (1968). HAVEL, R. J., EDER, H. A., AND BRAGDON, J. H., J. Clin. Invest., 34, 1345 (1955). EWIXG, A. M., FREEMAN, N. K., AND LINDGREN, F. T., Advan. Lipid Res. 3, 25 (1965). BARCLAY, M., TEREBUS-KEKISH, O., SKIPSKI, V. P., AND BSRCLAY, R. K., Clin. Chim. Acta 11, 339 (1965). LINDGREN, F. T., AND NICHOLS, A. V., in “The Plasma Proteins” (F. W. Putnam, ed.) Vol. 2. Academic Press, New York (1960). GOLDBERG, R. C., CHAIKOFF, I. L., LINDSAY, S., AND FELLER, D. D., Endocrinology 46, 72 (1950). LOWRY, 0. H., ROSEBROUGH, N. J., FSRR, A. L., GNU RBNDALL, R. J., J. Biol. Chem. 193, 265 (1951). ZAK, B., DICKENI~AN, R. C., WHITE, E. G.,
BURNETT, H., AND CHERNEY, P. J., Am. J. Clin. Path. 24, 1307 (1954). 13. VAN HANDEL, E., Clin. Chem. 7, 249 (1961). 14. BARLETT, G. R., J. Biol. Chem. 234,466 (1959). GERALD R. FALOONA~ BRUCE N. STEWART MELVIX FRIED Department of Biochemistry College of Medicine Unizlersity of Florida Gainesville, Florida %?601 Received September 3,1968; accepted 16, 1968
December
1 Present address: Research Division, Hospital, 4500 S. Lancaster Road. Dallas, 75216.
Biosynthesis
of Riboflavin-a-glucoside Plant
V. A. Texas
by
Grains
Riboflavin-a-glucoside (5’-D-riboflavin-Lu-D-glucopyranoside) has been synthesized by rat liver preparations (1) and by cultures and enzyme preparations from different microorganisms (210). The present paper deals with the biosynthesis of riboflavin-or-glucoside by enzyme preparations from plant grains. The crude enzyme solution was prepared as follows: The grains of glutinous millet were soaked in 0.5% NazSzOk solution for 20 min, washed with tap water, ground in a mortar, suspended in 0.1 &r acetate buffer (pH 5.3) at 4”, strained through two layers of cloth, and centrifuged. (NH4)#04 was added to the supernatant solution to 0.95 saturation and the precipitate was collected, dissolved in acetate buffer, and centrifuged. The supernatant solution was used as the crude enzyme solution. A reaction mixture containing 90 g of maltose, 900 mg of riboflavin, 900 ml of 0.1 M acetate buffer (pH 5.3) and 900 ml of crude enzyme solution prepared from 23 kg of grain powders of glutinous millet was incubated in darkness at 25” for 6 hr. Six batches were incubated in this way. After incubation, the reaction mixture was treated with an equal volume of ethanol, cooled rapidly to O”, and filtered to remove the precipitate. The filtrate was concentrated under reduced pressure, treated with (NH&S01 to 0.66 saturation, and the resulting precipitate removed by centrifugation. The yellow solution was shaken vigorously with phenol in a separatory funnel, and the phenol layers combined. A sample of this phenolic solution was analyzed by paper chromatography (Fig. I), and the yield of riboflavin-cy-
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
COMMUNICATIONS
681
1 (A)
FIG. 1. Ult.rncent.rifugal flotation patterns of rat-serum lipoproteins concent.rat.ed 5.7.fold. Flotat,ion from right to left. Schlieren patterns 23 min after reaching 42,040 rpm, 2G”, 05” bar angle. KBr: ti = 1.19, L = LDL, I = IDL, H = IIDL. (A) normal rat, (13) hypothyroid rat. ser,,m of normal fasted rats, is shown in Fig. 1A. By measuring F rates of the mid-ordinate of each peak, corrected for viscosit,y, at various bnckground densities and extrapolating them to zero flotation, an estimate of the hydrated density of each component was obtained, (Fig. 2). Very low density lipoproteins (VLDL) moved too rapidly for accurate flotation measurement. The calculated values have been confirmed by measuring the minimum background density at which the components can be isolated by preparative Ikltracentrifugation, and the follr components can be isolated free of contamination by adjacettt fractions in the following density ranges: VLT)L (d < 1.01); LDL (1.02 < d < 1.05); IDL (1.05 < cl < 1.075); HDL (1.08 < d < 1.2). On electrophoresis in polyacrylamide gel at pII 9.1, IDL demonstrated a mobility int.ermediate between the@ (LDL) and thecu (HI)L) bands (Fig.
1.064
d FIG. 2.
FIG. 2. Flotation rate as a function of density. Each point represents a flotation rate (F) calculated from an ultracentrifugal rul, at the given density (d). Vallles for 7, the viscosity relative to were obtained from the International water, Critical Tables.
COMMUNICATIONS under different conditions. It has usually been considered as the highest density member of a heterogeneous LDL group, and has generally been ignored. IDL may be analogous to HDLr, a minor component of the human serum lipoprotein spectrum which has a reported hydrated density of 1.05 (9) and has never been fully characterized. Since nothing is known concerning the synthesis, metabolism, or functional role of this lipoprotein, it was of particular interest to find that when rats were made hypothyroid by radiosurgery with 1311 (lo), a several-fold increase in the concentration of IDL was observed along with slight increases in the concentrations of the other lipoprotein components (Fig. 1B). IDL from these TABLE AMINO
FIG. 3. Vertical polyacrylamide gel phoresis of rat serum lipoproteins. Four cyanogum; pH 9.1, Tris-EDTA-borate 106 V/20 cm; 7 hr; amido black stain. 1 2 = LDL, 3 = IDL, 4 = HDL, and 5 serum.
electroper cent buffer; = HDL, = whole
3). Immunochemical studies by double diffusion indicate that IDL contains the a peptide characteristic of HDL and does not react significantly with antiserum to LDL. Amino acid analyses of the isolated purified fractions (Table I) show that the protein moieties of IDL and HDL are not identical. The chemical compositions of IDL and HDL (Table II) are sufficiently different to serve as further evidence that IDL is not a higher numbered polymer of HDL subunits. The original procedure (6) for the separation of human lipoproteins specified a density of 1.063 as the dividing point between LDL and HDL. When this method is applied to rat serum it may result in LDL or HDL fractions that are contaminated by IDL, depending on the type of salt employed, and the subsequent redistribrition of salt during ultracentrifugation. IDL is probably the minor component isolated with the LDL fraction from rat serum by Camejo (3) and Barclay (8)
I
ACID COMPOSITION OF ISOLATED LIPOPROTEINS~
Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine
IDL
HDLb
LDL
39.5 5.4 60.7 79.4 47.8 47.8 147.6 25.0 48.0 65.7 46.0 -
57.9 13.9 49.4 85.4 43.2 36.3 132.6 22.7 39.2 52.5 41.2 20.5 11.3 80.0 18.1 23.2
46 12 29 88 48 67 110 31 41 45 47 4 38 92 14 34
24.2 87.6 11.3 23.1
a Performed on Beckman 120 C analyzer. All values given in moles of amino acid per 100,000 g protein. 1,From the data of Camejo (3). TABLE
II
COMPOSITION OF ISOLATED LIPOPROTEINS" componentb Protein Total cholesterol Triglyceride Phospholipid
IDL
HDLC
LDL
39 2G 3 23
40 19 2 35
20 46 10 23
a Percentage of dry weight. b The four components were assayed as in Refs. 11-14 respectively. c From t,he data of Camejo (3).
683
COMMUNICATIONS rats could be readily isolated by ultracentrifugation and had a hydrated density similar to that of the corresponding component of normal rat serum. Marked increases in IDL concentration were also observed in the hypothyroid states induced by feeding rats propylthiouracil, aminotriazole, or an iodine-deficient diet. IDL from these rats was found to be similar in immunochemical behavior and amino acid composition to IDL isolated from normal rats. These findings describe IDL, a class of lipoprotein in the ultracentrifugal pattern of ratserum lipoproteins, with flotation and other characteristics intermediate between LDL and HDL. This component can be isolated in the predicted density range, and its chemical and physical properties are all consistent with the proposition that IDL is a distinct, albeit minor, class of lipoprotein. The specific increase of IDL observed in experimental hypothyroidism in the rat is further evidence that IDL is a distinct, and perhaps metabolically important, class of lipoprotein. That increase finds no direct parallel in human myxedema, since in the human, hypothyroidism results in specific increases in the concentration of serum p lipoproteins (1). ACKNOWLEDGMENTS This work was supported by Grant 6%AG-32 from the Florida Heart Association and Grant AM 02457 from the National Institutes of Health. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9.
10.
11.
12.
SCANU, A., Advan. Lipid Res. 3, 64 (1965). NAR~YAN, K. A., Anal. Biochem. 20,582 (1967). CAMEJO, G., Biochemistry 6, 3228 (1967). WINDMUELLER, H. G., AND LEVY, R. I., J. Biol. Chem. 242, 2246 (1967). FALOONA, G. R., STEW.~RT, B. N., AND FRIED, M., Biochemistry 7,720 (1968). HAVEL, R. J., EDER, H. A., AND BRAGDON, J. H., J. Clin. Invest., 34, 1345 (1955). EWIXG, A. M., FREEMAN, N. K., AND LINDGREN, F. T., Advan. Lipid Res. 3, 25 (1965). BARCLAY, M., TEREBUS-KEKISH, O., SKIPSKI, V. P., AND BSRCLAY, R. K., Clin. Chim. Acta 11, 339 (1965). LINDGREN, F. T., AND NICHOLS, A. V., in “The Plasma Proteins” (F. W. Putnam, ed.) Vol. 2. Academic Press, New York (1960). GOLDBERG, R. C., CHAIKOFF, I. L., LINDSAY, S., AND FELLER, D. D., Endocrinology 46, 72 (1950). LOWRY, 0. H., ROSEBROUGH, N. J., FSRR, A. L., GNU RBNDALL, R. J., J. Biol. Chem. 193, 265 (1951). ZAK, B., DICKENI~AN, R. C., WHITE, E. G.,
BURNETT, H., AND CHERNEY, P. J., Am. J. Clin. Path. 24, 1307 (1954). 13. VAN HANDEL, E., Clin. Chem. 7, 249 (1961). 14. BARLETT, G. R., J. Biol. Chem. 234,466 (1959). GERALD R. FALOONA~ BRUCE N. STEWART MELVIX FRIED Department of Biochemistry College of Medicine Unizlersity of Florida Gainesville, Florida %?601 Received September 3,1968; accepted 16, 1968
December
1 Present address: Research Division, Hospital, 4500 S. Lancaster Road. Dallas, 75216.
Biosynthesis
of Riboflavin-a-glucoside Plant
V. A. Texas
by
Grains
Riboflavin-a-glucoside (5’-D-riboflavin-Lu-D-glucopyranoside) has been synthesized by rat liver preparations (1) and by cultures and enzyme preparations from different microorganisms (210). The present paper deals with the biosynthesis of riboflavin-or-glucoside by enzyme preparations from plant grains. The crude enzyme solution was prepared as follows: The grains of glutinous millet were soaked in 0.5% NazSzOk solution for 20 min, washed with tap water, ground in a mortar, suspended in 0.1 &r acetate buffer (pH 5.3) at 4”, strained through two layers of cloth, and centrifuged. (NH4)#04 was added to the supernatant solution to 0.95 saturation and the precipitate was collected, dissolved in acetate buffer, and centrifuged. The supernatant solution was used as the crude enzyme solution. A reaction mixture containing 90 g of maltose, 900 mg of riboflavin, 900 ml of 0.1 M acetate buffer (pH 5.3) and 900 ml of crude enzyme solution prepared from 23 kg of grain powders of glutinous millet was incubated in darkness at 25” for 6 hr. Six batches were incubated in this way. After incubation, the reaction mixture was treated with an equal volume of ethanol, cooled rapidly to O”, and filtered to remove the precipitate. The filtrate was concentrated under reduced pressure, treated with (NH&S01 to 0.66 saturation, and the resulting precipitate removed by centrifugation. The yellow solution was shaken vigorously with phenol in a separatory funnel, and the phenol layers combined. A sample of this phenolic solution was analyzed by paper chromatography (Fig. I), and the yield of riboflavin-cy-