Plant Physiol. (1976) 57, 589­593 Development of Ribulose 1,5­Diphosphate Carboxylase in Nonphotosynthetic Endosperms of Germinating Castor Beans 1 Received for publication October 27, 1975 and in revised form December 22, 1975 LEE ANN FRY GILLEN, JOSHUA H. H. WONG, AND C. ROY BENEDICT Texas A&M University, College Station, Texas 77843 ABSTRACT Ribulose 1,5­diphosphate (RuDP) carboxylase has been partially purified from dark­grown nonphotosynthetic endosperms of germinating castor beans (Ricinus communis var. Hale). The Km values for RuDP, HC03­, and Mg 2+ are 0.51, 33, and 1.78 mM, respectively. The pH optimum for the carboxylation reaction is pH 7.5. Germination is required for the development of the carboxylase in the endosperms. The enzyme reaches a maximal activity in 4­ to 5­day­old dark­grown seedlings (which have an endosperm weight of approximately 0.75 g fresh weight/bean) and then declines. Total endosperm carboxylase activity is 1230 nmoles/min­g fresh weight, which is 25 and 50% of the total activity developed in soybean and maize leaves, respectively. Specific activity of the carboxylase in crude soluble endosperm preparations (which contain enzymic and storage protein) is 0.05 mmole/min mg protein. This is 5 times greater than the specific activity of RuDP carboxylase in soluble preparations from etiolated leaves. During germination the Vmax of the endosperm carboxylase for RuDP increases 10­fold. Development of the enzyme is inhibited 90% by the exposure of the endosperm to 2 mg/ml cycloheximide or 50
mg/ml chloramphenicol. Light (or phytochrome Pfr) is not required for the synthesis of the enzyme. Electron photomicrograpbs of dark­grown endosperm cells (with peak RuDP carboxylase activity) show proplastids with several invaginations of the inner membrane but no prolamellar­like structures. Ribulose­1,5­diphosphate carboxylase is responsible for fixing the CO2 that is used by plants for photosynthesis. It comprises a large percentage of the total soluble protein in green leaves and may well be the most abundant protein in the natural world (Ellis, 1973). The reaction catalyzed by RuDP carboxylase: CO2 + RuDP + H2O 2 3­PGA has an absolute divalent cation requirement. Magnesium ion appears to be the best candidate for the activator with a Km of 1.1 – 1.4 x 10 ­3 M (Paulsen and Lane, 1966). Unlike many other carboxylating enzymes, RuDP carboxylase uses molecular CO2 as the active species rather than HCO3 ­ . However, since CO2 is a 1 Support for this research was provided by the Robert A. Welch Foundation under Grant A­482 and was part of a thesis presented by L.A.F.G.
gas, most experimentation with this enzyme is done using HCO3 ­ as the source of “CO2”. The activity of the enzyme in higher plants is associated with the large subunit and can exhibit catalytic activity by itself (Nishimura, et.al. 1973). The role of the small subunit is not yet clear. It is possibly related to the binding of the Mg +2 and appears to function as a regulator of the activity of the larger subunit (Takabe and Akazawa. 1973). Not only are the subunits different in composition, they are also different in their site of synthesis within the cell and their rate of synthesis (Smillie, et.al. 1967). The large subunit is synthesized on the chloroplastic ribosomes (70S) and is inhibited by chloramphenicol, while the small subunit is synthesized on cytoplasmic ribosomes (80S) and is inhibited by cycloheximide (Criddle, et.al.1970). In the lower plants, sites of synthesis have been examined only in the green algae, Chlamydomonas reinhardi and Chlorella ellipsoidea, with the enzyme inhibited by both chloramphenicol and cycloheximide (Given and Criddle, 1972; Sugiyama,et.al, 1971). Previous reports have described the presence and properties of RuDP 2 carboxylase in castor bean endosperms (4Benedict, 1973; Osmond, et.al., 1975). These are the first and only reports of the occurrence of this enzyme in nonphotosynthetic higher plant tissue. RuDP carboxylase has been localized in the proplastids of germinating castor beans (Osmond, et.al., 1975) and the enzyme reaches a peak activity in the dark in 4 days (Benedict, 1973). Dark­ grown etiolated leaves contain low amounts of RuDP carboxylase activity which increases substantially only after exposure of the leaves to light (Chollet and Ogren, 1972;,Kleinkopf, et.al., 1970; Margulies, 1964. The purpose of this research is to further investigate the unique development of RuDP carboxylase and proplastids in the endosperms of germinating castor beans and to determine some of its enzymatic properties in relation to photosynthetic RuDP carboxylase. This was accomplished by studying the increase in activity of the enzyme with growth, its location and synthesis within the endosperm and some of its biochemical properties. RESULTS The results of the purification procedures for RuDP carboxylase from castor bean endosperms are shown in Table 1. The enzyme has been shown to be inhibited by S04 2­ (Paulsen and Lane, 1966) which accounts for the low carboxylase activity in the 35 to 55% (NH4)2SO4 precipitate and the reappearance of activity in the 35­55% (NH4)2SO4 dialyzate. The specific activity of RuDP carboxylase eluted from Sephadex G­200 columns was 0.200 mmole CO2 fixed/min­mg protein. This specific activity is 10 times greater than reported 2 Abbreviations: RuDP: ribulose­1,5­diP; PGA: 3­phosphoglyceric acid; PEP: phosphoenolpyruvate: CHI: cycloheximide; CAP: chloramphenicol.
by Osmondet.al., 1975 for a similar preparation of RuDP carboxylase from castor beans, but 10 times lower than more purified preparations from green leaves (Ellis, 1973). The elution profile for the enzyme from two, four, and six day old endosperm was the same. The only difference between the three days was the height of the void volume peak. During this same time period, the total soluble protein in the endosperm steadily decreased. The reaction velocity varied linearly with change in the enzyme concentration, indicating that any effect on the initial velocity of the reaction was not due to limited substrate concentration (Fig. 1). The reaction velocity was also approximately linear with time for at least fifteen minutes. Therefore, to insure that the initial velocity was being measured, a time period of fifteen minutes was employed for all assays. The specific activity of RuDP carboxylase in the soluble protein fraction from castor bean endosperms was 0.05 mmole CO2 fixed/min­mg protein. The specific activity of RuDP carboxylase in comparable soluble protein fractions from 6­day­ old darkgrown leaves was 0.011 mmole CO2 fixed/min­mg protein (Margulies, 1964). The enzyme activity was 5 times greater in endosperms compared to etiolated leaves. The activity of the enzyme increased with development of the endosperm, resulting in a maximum at the sixth day after germination. Before day two, no activity was measurable and after the seventh day, activity dropped to virtually zero (Fig. 2). The pH optimum was 7.5 in tris buffer. Another amine buffer, bis­tris, was inhibitory to the reaction above pH 6.5. Both phosphate and imidazole buffers were quite inhibitory to the reaction (Fig. 3). The Km value for HCO 3­ was 33 mM. The Km value for Mg 2+ was 1.78 mM. The reaction was inhibited at concentrations of Mg 2+ greater than 20 mM and was completely inactive in the absence of Mg 2+ . The development of RuDP carboxylase activity in endosperms of germinating castor beans in the dark is shown in Fig. 4. The enzyme activity peaked 4 days after germination and decreased to a low or negligible value 7 days after germination. There was no significant enzyme activity in dry or imbibed seeds and germination was required for the appearance of RuDP carboxylase activity in the endosperms. At three different times during the development of RuDP carboxylase activity in the endosperms, the Vmax and Km values for RuDP were determined on the enzyme fraction eluted from Sephadex G­200 columns (Fig. 5). For enzyme preparations isolated from 2­, 4­, and 6­day­old seedlings, the Vmax at saturating concentrations of RuDP increased 10­fold. The Km values for 2­ and 6­day­old endosperm carboxylase were 0.27 and 0.51 mM, respectively. The effect of light on the development of RuDP carboxylase in the castor bean endosperms is shown in Fig. 6. In these experiments the imbibed seeds, without seedcoats, were exposed to continuous illumination. During the growth of the castor bean seedlings, light had no promotive effect on the rate of development or on the total level of RuDP carboxylase activity in the endosperms. Apparently, phytochrome (Pfr) is not involved in the synthesis of the carboxylase.
The presence of 2 mg/ml CHI or 50 mg/ml CAP severely inhibits the development of the carboxylase in the endosperms. Chloramphenicol inhibited the synthesis 56% while cycloheximide inhibited the synthesis 90%. RuDP carboxylase has been localized in the endosperm proplastid fraction (Osmond, et.al., 1975). The equilibrium density of these proplastids from 5­day­old endosperms is 1.22 g cm 3 (Miflin and Beevers, 1974). The fine structure of endosperm proplastids in 5­day­old seedlings is shown in the electron photomicrographs in Fig. 7. The proplastids appear as double membrane structures with invaginations of the inner membrane. In these structures there was no crystalline center or prolamellar body formation indicative of the development of proplastids into etioplasts. In electron photomicrographs of dry castor bean seeds, we were able to detect only a few small plastids between extensively developed aleurone grains. DISCUSSION Developmental RuDP Carboxylase Activated In the Absence of Light. The endosperm of castor bean is very high in fat, and this fat is greater than 60% of the dry seed. During germination, there is a precipitous decline in this fat content and a concomitant increase in sugars, primarily sucrose. This conversion of fat to sucrose, involves the glyoxylate cycle and requires two specialized enzymes, malate synthetase and isocitrate lyase. These enzymes are only found together in germinating seeds that are high in stored fat. These two enzymes and the other enzymes associated with gluconeogenesis, citrate synthetase, aconitase, malate dehydrogenase, succinic dehydrogenase and fumarase rise from virtually zero activity in the ungerminated seed to a peak at the time of most rapid fat breakdown, usually four or five days after germination. They then disappear when the process is completed (Beevers, 1969; Breidenbach, 1969). In addition, gluconeogenesis appears intimately associated with a particular cell organelle, the glyoxysome, which also appears at the onset of germination and disappears after the conversion of fats to sucrose is completed (Beevers, 1969; Breidenbach, 1969). The glyoxylate pathway and its related organelle is just one of the "mini" life cycles which occurs in the castor bean endosperm. Ribulose diphosphate carboxylase and all the enzymes of the reductive pentose phosphate cycle (Calvin cycle), except triose phosphate dehydrogenase (NADP), are present in the endosperm and exhibit this "life and death" pattern during germination. It seems significant that in green leaves, these enzymes are repressed until exposed to light (Smillie, et.al. 1967) while in the castor bean endosperm, the enzymes are de­repressed and activated during germination in the absence of light.
RuDP Carboxylase Associated with the Proplastid. While ribulose­1,5­diphosphate carboxylase, the first enzyme of the Calvin cycle, is associated with the chloroplasts in green leaves, this same enzyme in the endosperm appears to be associated with the proplastid, which is the progenitor of the chloroplast (Osmond, et.al.,1975). The equilibrium density of these proplastids from 5­day­old endosperms is 1.22 g cm 3 (Miflin and Beevers, 1974). However, the proplastids, by definition, are small undifferentiated cell organelles enclosed in a double membrane. This inner membrane is lost through differentiation to the lamellar system of the chloroplasts or the prolamellar body of the etioplast. (Fig. 8). The proplastids of the castor bean endosperm, from a six day old seed, are larger than the normal proplastid; and although the matrix of the proplastid is still undifferentiated, the inner membrane exhibits some invaginations (Fig. 7). However, these invaginations never develop into a lamellar system or prolamellar body and the proplastids gradually disappear along with the endosperm protein during germination. Our investigation has shown that in the dry ungerminated seed, virtually no proplastids are detectable (Fig. 9), while at the same time, there is also no measurable enzyme active. In fact, the enzyme exhibits no activity until the second day of germination. This apparent lag of two days in the appearance of the enzyme may be explained on the basis of the synthesis of the proplastid organelle which is needed to house the enzyme. The proplastid is composed of two membranes and the formation of these membranes requires the synthesis of at least three phospholipids: phosphatidycholine, phosphatidylinositol, and phosphatidylserine which are known to be major components of membranes. (Kagawa, et.al.,1973) The synthesis of these phospholipids was shown by Moore, et.al, 1973 to occur primarily in the endoplasmic reticulum and on the cytoplasmic ribosomes. The endosperm of castor beans however, are very low in cytoplasmic ribosomes and endoplasmic reticulum in the dry, unhydrated seed (Sturani, 1968) due to ribosomal degradation during ripening of the seed (Sturani, 1965). Therefore, it takes twenty­four hours after hydration for single ribosomes to appear, and an additional forty­eight hours for these ribosomes to join together to form the polysomes of the endoplasmic reticulum. When this delay is added together, it equals approximately seventy­two hours and can easily account for the two­day delay in the appearance of the viable enzyme. RuDP Carboxylase Synthesized De Novo RuDP carboxylase itself is probably synthesized de novo. This observation is based not only on the needed synthesis of the ribosomes within the endosperm, but also because the enzyme requires O2 during its synthesis, indicating an active respiratory process occurring (Bahr and Jensen, 1974). Rutner and Lane, 1967 have shown that the enzyme is composed of two subunits, and Criddle, et. al., have shown that the larger subunit is synthesized on chloroplast ribosomes while the smaller is synthesized on the cytoplasmic ribosomes. These data are also consistent with the data of Margulies, 1964 and Smillie, et.al., 1967 showing
the sensitivity of the synthesis of RuDP carboxylase to CHI and CAP, and the data of Kleinkopf, et.al., 1970 showing the sensitivity of the synthesis of the large subunit and small subunit of RuDP carboxylase to CAP and CHI, respectively. The inhibitory effects of CHI and CAP seen in the present study could be consistent with the conclusion that protein synthesis on 80S cytoplasmic ribosomes and on 70S proplastid ribosomes is required for the synthesis of small and large polypeptides of endosperm RuDP carboxylase. Therefore, any inhibition in the activity of the enzyme caused by exposure to antibiotics which prevent protein synthesis on chloroplastic or cytoplasmic ribosomes could indicate active de novo synthesis of the enzyme to be occurring. (Table II). Further evidence for de novo synthesis is suggested by the fact that while the total soluble protein in the endosperm is decreasing, the void volume protein from the G­200 column, which is predominantly RuDP carboxylase, is increasing. (Fig. 10). RuDP Carboxylase Activity by Gram Fresh Weight of Tissue The development data shown in Fig. 2, are similar to the RuDP carboxylase developmental curve previously published (Benedict, 1973) with the important exception that total units of RuDP carboxylase/g fresh weight have now been determined. This makes possible a comparison of carboxylase units from castor bean endosperms and green leaves. The RuDP carboxylase activity in endosperms is 1230 nmole /min­g fresh weight in comparison to about 2500 nmole / min­g fresh weight in green maize leaves, and 5000 nmole / min­g fresh weight in green soybean leaves (Chollet and Ogren, 1972). The amount of RuDP carboxylase activity in castor bean endosperms is 25% of soybean leaves and 50% of maize leaves. The developmental curve for RuDP carboxylase is similar to the developmental curves for malate synthetase and isocitric lyase in endosperms of germinating castor beans (Beevers, 1969). Biochemical Buffers May Inhibit Mg 2+ Binding Sites The biochemical properties were investigated and compared to the same properties of RuDP carboxylase from other species. In general, the pH and kinetic properties are quire similar. The pH optimum for castor bean endosperm RuDP carboxylase of 7.5, agrees with the value reported by Osmond et.al.,1975 but is shifted 0.5 pH unit down from the optimum found for the enzyme from green leaves (Bassham, et.al., 1968). Bis­tris buffer is a neutral buffer above pH 6.5 while tris buffer is protonated below 8.1. Despite both these buffers being amine buffers, the difference in their pK values may lead to an enhancement or an inhibition of the enzyme activity. In addition, it is possible that bis­tris may be binding Mg 2+ ions. Both Sugiyama et al., 1968 and Bassham et.al.,1968 have shown that the pH optimum of RuDP carboxylase is sensitive to the concentration of Mg 2+ ions. In agreement with the observations made by
Weissbach et.al.,1956; Kuehn and McFadden, 1969, and Paulsen and Lane,1966, phosphate buffer is inhibitory to the RuDP carboxylase reaction. Imidazole buffer also inhibits the reaction in the same manner as the phosphate buffer. There is no report in the literature of this inhibitory effect, even though other investigators have employed imidazole and a buffer similar to it, histidine, in their studies (Kuehn and McFadden, 1969; Sugiyama et al., 1968). It is possible that the Mg 2+ ions, needed for the reaction, complexed with the buffer instead of the enzyme. RuDP Carboxylase ‘s Specific Activity Is Comparable to Green Leaves The specific activity of RuDP carboxylase from green spinach leaves range from 0.23 to 0.35 mmole CO2 fixed/min­mg protein (Paulsen and Lane, 1966; Wishnick and Lane, 1971). RuDP carboxylase is a major protein in leaves and is 50% of the total soluble protein in leaf extracts (Kawashima and Wildman, 1970). However, there is no comparable soluble protein fraction (Fraction I) in germinating seeds. Nonetheless, RuDP carboxylase from castor bean endosperms has albumin type properties (Haurowitz, 1963). It is also soluble in H20, precipitated at 55% of saturation with (NH4)2SO4 and remains in solution following an extensive dilution by dialysis. In addition, the following composition has been reported for the protein of castor bean meal: 60% globulins, 16% albumins, 4% proteoses, and 20% glutelins (Bolley and Holmes, 1958). If the specific activity of endosperm carboxylase is corrected for the globulin content in the soluble preparation, it becomes 0.23 mmole CO2 fixed/min.mg protein. This level of specific activity is comparable to the lower range of RuDP carboxylase reported for green leaves (Paulsen and Lane, 1966). Kinetic Measurements Support Other Investigations The Km for HCO3 ­ is quite similar in all species studies with the exception of soybean (Bowes and Ogren, 1972), Hydrogenomonas facilis (Kuehn and McFadden, 1969), and Chlorella fusca (Lord and Brown, 1975). High concentrations, greater than 50 mM, of HCO 3­ inhibit the reaction but there are no reports of this inhibition in the literature. The Km for RuDP is also quite similar to the values reported by other investigators. The present investigation revealed inhibition of the enzyme by RuDP concentrations greater than 7 mM. Two previous reports of inhibition were found at concentrations greater than 50 mM (Weissbach, et.al., 1956) and greater than 70 mM (Paulsen and Lane, 1966). RuDP carboxylase isolated from castor bean endosperm is similar to the high Km carboxylase described by Bahr and Jensen, 1974. MATERIALS AND METHODS Plants
Castor beans (Ricinus communis var. Hale) were obtained from McNair Seed Company, Plainview, Texas. The seeds were dusted with Arasan. The seeds were imbibed in H20 for 24 hr and germinated in moist vermiculite at 35° C in the dark. For developmental studies, the seedlings were harvested from 0 to 8 days. For antibiotic studies, the seedcoats were removed, and the seeds were imbibed in H20, 2 mg/ml CHI, or 50 mg/ml CAP. The imbibed seeds were germinated at 35° C in the dark in Petri dishes in H20 or antibiotic solution. To study the effect of light on the development of RuDP carboxylase, the imbibed seeds, without seedcoats, were germinated in the light or dark at 35° C. The light source was General Electric cool­white 20­w fluorescent bulbs. The light intensity at the seed surface was 30 meinsteins m ­2 sec ­1 . Previously (Benedict, 1973), it has been shown that RuDP carboxylase activity peaks in 4 day old germinating castor beans. We found that both the rate of growth of the castor bean seedlings and the rate of development of RuDP carboxylase in the seedlings are influenced by (a) removal of the hard seedcoat prior to germination and (b) the watering regime during the growth of the seedlings. Removing the seedcoats, prior to imbibing and germinating the seeds, increases the rate of seedling growth. In these seedlings growing in Petri dishes, RuDP carboxylase activity peaks 48 hr after germination. Depending on the watering regime, the peak carboxylase activity can be extended 1 to 2 days in seeds germinating in vermiculite. Because of these variables, in most experiments we have recorded the chronological age of the seedlings and the fresh weight of the endosperms. In most experiments, maximal RuDP carboxylase activity developed in seedlings with an endosperm weight between 0.60 and 0.84 g fresh weight/seed. Enzyme Isolation and Purification RuDP carboxylase was isolated and partially purified from endosperms of 5­day­old seedlings. (0.75 g endosperm/seed). All steps were carried out at 4° C. The endosperms were separated from the testa, hypocotyl, epicotyl, and cotyledons and rinsed with deionized H20. The endosperms were homogenized for 60 sec in a prechilled Waring Blendor in 0.1 M tris buffer, pH 7.5, containing 0.1 mM GSH. The homogenizing medium contained 1 ml buffer/g fresh weight of endosperm. The homogenate was filtered through two layers of cheesecloth and centrifuged 15 min at 1OOOg in a RC­5 Sorvall refrigerated centrifuge. The crude supernatant fraction was centrifuged 30 min at 17,000g. The soluble supernatant was fractionated with (NH4)2SO4. The protein which precipitated between 35 and 55% of saturation with (NH4)2SO4 was collected by centrifugation and dissolved in 3 ml of 0.1 M tris buffer, pH 7.5, containing 0.1 mM GSH. The protein solution was dialyzed overnight against 0.1 m tris buffer, pH 7.5, containing 0.1 mM GSH. The dialyzate was centrifuged at 17,000g to remove any insoluble material. The solution was layered on a Sephadex G­ 200 column (84 x 2.5 cm) which had been previously equilibrated with 0.1 M tris, pH 7.5, containing 0.1 mM GSH. The protein was eluted from the column with the same buffer and collected in 2­ml fractions. A plot of absorbance against fraction number shows 2 major peaks at 280 nm (Fig. 11). RuDP carboxylase was eluted from the column in the void volume. This void volume protein increases between 2­day­ and 6­day­old endosperms. In these experiments a seed with maximal carboxylase activity had a fresh weight of endosperm of 0.70 to 0.80 g and was called a 6­day­old bean. The 2­day­ and 4­day­old beans had endosperm fresh weights of 0.30 to 0.38 and 0.42 to 0.50 g/ bean, respectively. The void volume was pooled and concentrated to 3 A units/ml in an Amicon Diaflo containing a UM­2 membrane. This concentrated protein solution was used as the source of RuDP carboxylase for the kinetic analysis. RuDP carboxylase from Sephadex G­200 columns was too highly adsorbed onto DEAE­ cellulose columns for displacement with 0.5 M tris buffer containing 0.5 M NaCl. Protein Determinations Protein was determined by the Lowry method. Bovine Serum Albumin was used as the standard. Enzyme Assay
RuDP carboxylase was assayed by a modification of the method of Benedict, 1973. The reaction mixture contained in mmoles: 50 tris buffer (pH 7.5); 1.25, GSH; 5, MgCl2; 1.25, RuDP 14 tetrasodium salt; 25, KH CO3 which contained 5 mCi of radioactivity; 0.1 ml of enzyme and H20 to a final volume of 0.5 ml. The reaction blank contained all of the reagents except RuDP. The reaction tubes were incubated at 35° C for 15 min. After this incubation, the tubes were placed on a N2­bubbling apparatus and the reaction stopped with 0.1 ml of concentrated HCl (Fig. 12). The volume in the tubes was brought to 10 ml with H2O. The tubes were bubbled with N2 for 15 min 14 and the unreacted CO2 was collected in a saturated solution of Ba(OH)2. The reaction mixture 14 was assayed for C­PGA by placing a 0.1­ml aliquot in 15 ml of scintillation fluid containing 5 g of PPO, 100 g of naphthalene, 10 ml of H20 and dioxane to 1 liter and counting to a ±0.2% error in a Beckman LS­200 B scintillation spectrometer system. Electron Microscopy The endosperm tissue from germinating castor beans was fixed in 1.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. The tissue was then exposed to a phosphate­buffered osmium tetroxide fixation, dehydrated with ethanol, and embedded in Epon. The sections were stained with uranyl acetate and lead citrate. The sections were photographed with a Hitachi HU­11 E electron microscope. The endosperm tissue from the dry castor bean seed were fixed in a solution of 50% aqueous gluteraldehyde, 40% aqueous osmium tetroxide and 0.1 M collidine buffer in a ratio of 1:2:5. They were dehydrated with ethanol and embedded in Spurr’s low viscosity embedding media. The sections were stained with Reynold’s lead citrate and uranyl acetate and photographed with a Hitachi HU­11E electron microscope. LITERATURE CITED Bahr, J. T. and R. Jensen. 1974. Ribulose diphosphate carbbxylase from­freshly ruptured spinach chloroplasts having an in vivo Km [CO2]. Plant Physiol. 53: 39­44. Bassham, J. A., P. Sharp, and I. Morris. 1968. The effect of Mg 2+ concentrations of the pH optimum and Michaelis constants of the spinach chloroplast ribulose diphosphate carboxylase (carboxydismutase). Biochim. Biophys. Acta 153: 898­900. Beevers, H. 1969. Glyoxysomes of castor bean endosperm and their relation to gluconeogenesis. Ann. N. Y. Acad. Sci. 168: 313­324. Benedict, C. R. 1973. The presence of ribulose­1,5­diphosphate carboxylase in the nonphotosynthetic endosperm of germinating castor beans. Plant Physiol. 51: 755­759. Bolley, D. S. and R. L. Holmes. 1958. Inedible oilseed meals. In: A. M. Altschul, ed., Processed Plant Protein Foodstuffs. Academic Press, New York, pp. 829­857. Bowes, G. and W. L. Ogren. 1972. Oxygen inhibition and other properties of soybean ribulose 1,5­diphosphate carboxylase. J. Biol. Chem. 247: 2171­2176. Breidenbach, R. W. 1969. Characterization of some glyoxysomal proteins. Ann. N.Y. Acad. Sci. 168: 342­343. Chollet, R. and W. L. Ogren. 1972. Greening in a virescent mutant of maize. II. Enzyme studies. Z. Pflanzenphysiol. Bd. 68. S45­54. Criddle, R.S., B. Dau, G. E. Kleinkopf, and R. C. Huffaker. 1970. Differential synthesis of ribulose diphosphate carboxylase subunits. Biochem. Biophys. Res. Commun. 41: 621­627.
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