Plant Physiol. (1982) 69, 308-313 0032-0889/82/69/0308/06/$00.50/0

['5NJNMR Determination of Asparagine and Glutamine Nitrogen Utilization for Synthesis of Storage Protein in Developing Cotyledons of Soybean in Culture Received for publication May 26, 1981 and in revised form September 2, 1981 THOMAS A. SKOKUT', JOSEPH E. VARNER, JACOB SCHAEFER, EDWARD 0. STEJSKAL, AND ROBERT A. MCKAY Department ofBiology, Washington University, St. Louis, Missouri 63130 (T. A. S., J. E. V.) and Physical Sciences Center, Monsanto Co., St. Louis, Missouri 63166 (J. S., E. 0. S., R. A. McK.)

ABSTRACT Although asparagine and glutamine are similar in chemical structure, the metabolic pathways responsible for transfer of their Solid-state I'5NINMR was used to measure the use of the amide and nitrogen to other compounds are basically different. The amide amino nitrogens of glutamine and asparagine for synthesis of storage nitrogen of glutamine can be directly transferred to a-ketoglutar- protein in cotyledons of soybean (Glycine max L. cv. Elf) in culture. No ate to form glutamate by the action of glutamate synthase (13). If major discrimination in the incorporation of the amide or amino nitrogens the amide nitrogen of asparagine is to be transferred to other of glutamine into protein is apparent, but the same nitrogens of asparagine amino acids, the asparagine molecule first must be hydrolyzed to are used with a degree of specificity. During the first seven days in culture ammonium and aspartate by the enzyme asparaginase; an enzyme with asparagine as the sole nitrogen source, the amino nitrogen donates system similar to glutamate synthase that could transfer asparagine approximately twice as much nitrogen to protein as does the anmde amide nitrogen to a-ketoglutarate or glutamate has not been nitrogen. The use of the amide nitrogen increases with longer periods of detected in higher plants (7). The free ammonium is reassimilated culture. The reduced use of the amide nitrogen was confirmed by its early to form glutamine via glutamine synthetase (13). These two path- appearance as ammonium in the culture medium. The amide nitrogen of ways of utilization of amide nitrogen are believed to be operating asparagine was found at aDl times to be an essential precursor for protein during seed development because potassium-dependent asparagi- because of its appearance in protein in residues whose nitrogens were not nase activity has been detected in the developing seeds of a supplied by the amino nitrogen. In addition, methionine sulfoximine in- number of legumes (23), and glutamate synthase activity has been hibited growth completely on asparagine, indicating that some ammonium detected in developing cotyledons of soybean and pea (24, 25). assimilation is essential for storage protein synthesis. These results indi- The amino nitrogens of glutamine and asparagine can both be cate that in a developing cotyledon, a transaminase reaction is of major transferred to other amino acids by the action of transaminases importance in the utilization of asparagine for synthesis of storage protein (29) using the glutamate and aspartate formed as a result of the and that, at least in the early stages of cotyledon development, reduced reactions described above. However, the amino nitrogen of aspar- activities of ammonium-assimilating enzymes in the cotyledon tissue or in agine can also be transferred directly to pyruvate, glyoxylate, ox- other tissues of the seed or pod may be a limiting factor in the use of aloacetate, or a-ketoglutarate by the action of asparagine trans- asparagine-amide nitrogen. aminase (7). Asparagine transaminase activity has been found in leaves of lupin, soybean, and pea (8, 10, 27) and thus might be present in developing seeds. In this paper we report the use of '5N-labeled asparagine and glutamine, and solid-state magic-angle cross-polarization [15N]- NMR (19, 20) to study the metabolism of these compounds in developing cotyledons of soybean in culture. The culture method The production of storage protein in a developing embryo is we use, Thompson et al. (28), has been shown to be an appropriate dependent upon the flow of nitrogen compounds to the immature system for study of legume seed storage protein synthesis under seed from other parts of the plant and the subsequent transfer of controlled conditions. The NMR analysis can be performed on nitrogen from these compounds to the amino acids required for intact cotyledons thereby avoiding elaborate digestion, separation, protein synthesis. Although ureides are found in substantial quan- derivitization, and purification procedures normally required in tities in the translocation stream of many legumes (12, 18), aspar- stable isotope studies. From our labeling and NMR experiments, agine and glutamine are usually the major amino acids present we have determined the extent to which the amide and amino (16, 17). In soybean, asparagine can represent as much as 60% of nitrogens of these amino acids contribute to the synthesis of the total amino acid nitrogen extracted from stem exudate (26), storage protein. We find that the amide and amino nitrogens of suggesting that it is an important nitrogen source for protein glutamine are used similarly in protein synthesis, whereas the synthesis in developing seeds. Glutamine plays a central role in corresponding nitrogens of are not. the assimilation of ammonium in plants (13) and has been found asparagine to support substantial growth of soybean cotyledons in culture (28), indicating that it, too, may play an important role in synthesis MATERIALS AND METHODS of storage protein. Growth of Plants. Glycine max (cv. Elf) were grown in chambers under conditions previously described (21). At the time of plant- 'Present address: Monsanto Agricultural Products Co., 800 N. Lind- ing, seeds were inoculated with Rhizobium japonicum. After the bergh Boulevard, St. Louis, MO 63166. appearance of the first trifoliolate, the plants were fertilized three 308 PROTEIN SYNTHESIS IN SOYBEAN COTYLEDONS 309 times a week with Hoagland solution. Between 70 and 90 days above reagents was subtracted from all values. The concentrations after planting, selected pods were removed and the immature of ammonium in the unknown samples were calculated from a seeds were used to initiate organ cultures of cotyledons. standard absorbance curve obtained with known concentrations Growth of Cotyledons in Culture. Immature cotyledons were of NH4Cl in 0.01 N HCI. grown in culture using a slightly modified procedure ofThompson Ammonium content of the cotyledon tissue was obtained from et al. (28). The excised cotyledons were rinsed with sterile water, the 80% ethanol extract. The extract was acidified with HCI to pH blotted dry, and transferred to a culture flask which contained 2.0 and evaporated to dryness. The dried residue was redissolved liquid medium. Each flask was weighed before and after adding in H20 and a portion was subjected to the above ammonium assay the cotyledon to obtain an initial fresh weight. The initial fresh procedure. weight of each cotyledon ranged between 5 and 20 mg. Stable Isotopes. '5N-labeled amino acids (95-98 atom % 15N) The culture medium used was that described by Thompson et were obtained from Merck (Montreal, Canada) and Stohler Iso- al. (28), with the only difference being the concentration of the tope Chemicals (Azusa, CA). The ['5N]amide asparagine used in nitrogen source. The medium was prepared without the amino these experiments was also labeled with 13C at the number 4 acid nitrogen source and adjusted to pH 6.0 with NaOH. Ten ml carbon only (90 atom % 13C). of medium, in 50-ml screw-top Erlenmeyer flasks, were sterilized [15NINMR. Magic-angle [15N]NMR spectra were obtained at by autoclaving. The amino acid nitrogen source was prepared as 9.12 MHz using matched spin-lock cross-polarization transfers a stock solution, adjusted to pH 6.0 with NaOH, and sterilized by with 1-ms single radio frequency contacts and 25 kHz His with filtration with an Acrodisc filter (0.2 iLm pore size; Gelman, Ann the dried samples contained in a Beams-Andrew 420-!L hollow Arbor, MI). Two ml of the sterile amino acid stock solution were rotor spinning at 1.5 kHz (19). Technical details of the spinning added to each culture flask containing sterile medium so that a and cross-polarization procedures have been reported elsewhere final concentration of 30 mm ~lutamine or 60 mm asparagine ('5N- (20). Fast cross-polarization rates for protonated nitrogens, long labeled or natural abundance 5N depending upon the experiment) proton rotating-frame lifetimes, and high concentrations of pro- was obtained. tons in these biological samples ensure representative NMR inten- The culture flasks containing one cotyledon each were incu- sities for all nitrogens (19, 20). The one exception is nitrogen in bated for various times at 28 ± 1 °C in an incubator shaker (Model the form of ammonia or ammonium ion, where internal molecular G-25R; New Brunswick Scientific, Edison, NJ). The flasks were motion decreases the cross-polarization transfer rates, resulting in shaken with a rotary motion describing a 1-inch circle at 100 rpm signal intensities which underestimate the ammonium nitrogen and were illuminated from above with a 20-w cool white fluores- present. The degree of the underestimate can be evaluated by a cent bulb (GE) at a distance of 30 cm. measurement of cross-polarization transfer from protons to nitro- Preparation of Cotyledons for NMR Analysis. After incubation, gens as a function of the time during which the two spin systems the cotyledons were rinsed with distilled H20, blotted, weighed, are in radio frequency contact (20). frozen in liquid N2, and lyophilized. After lyophilization, the The amount of 1 N present in the protein of the ethanol- cotyledons were subjected to [ 5N]NMR analysis. When only the extracted cotyledons was calculated from the NMR spectra, using nitrogen present in protein was to be observed, the cotyledons a natural abundance cross-polarization ['5N]NMR spectrum of a were extracted with 80% ethanol to remove free amino acids. The soybean seed as a standard. The amount of 15N represented by the ethanol extraction was performed by first grinding 150 to 300 mg soybean-seed [15N]NMR spectrum was calculated by multiplying dry weight of cotyledon tissue to a fine paste using a few ml 80% the natural abundance of 15N (0.00365) times the amount of ethanol in a mortar and pestle. Additional 80% ethanol was added protein nitrogen present in the sample as determined by protein to obtain a total of 20 ml. The mixture was allowed to stand for and total nitrogen assays. This analytical procedure was confirmed 10 min with intermittent mixing, after which it was centrifuged at by comparisons with integrated intensities from the spectra of 1,000 rpm for 10 min. This extraction procedure was repeated on accurately known quantities of 4-['3C-'5N](amide) asparagine. the resultant pellet until the supernatant was ninhydrin negative (usually three extractions were sufficient). The final pellet was RESULTS dried over a stream of N2 gas and subsequently observed by NMR. Determination of Protein Content. Protein content of the coty- Growth of Cotyledons in Culture. When 30 mm glutamine was ledons was determined by two methods. For the growth experi- given as the nitrogen source, immature cotyledons in culture grew ments, protein was determined by the method of Bradford (3). rapidly and exhibited increases in fresh weight, dry weight, and Ten mg of the dried cotyledon tissue were extracted with 10 ml 35 protein ranging between 10- and 15-fold in 7 days. For example, mm K-phosphate (pH 7.6), containing 0.4 M NaCl and 10 mm a cotyledon having an initial fresh weight of 15 mg reached a fresh dithioerythritol (5). A portion (100 ,il) of this extract was subjected weight of 190 mg, a dry weight of 47 mg, and a protein content of to the Bradford assay. BSA dissolved in the extraction buffer was 11 mg after 7 days. The cotyledons did not grow as well on used as a standard. Protein content of the cotyledons subjected to asparagine as they did on glutamine (Table I); increases in fresh NMR analysis was determined by the method of Lowry et al. (1 1). weight, dry weight, and protein were usually about 70 to 80% of The dried cotyledons were extracted with ethanol as described that observed with glutamine. The cotyledons grew better on 60 above. A portion of the dried, ethanol-extracted material was mM asparagine than on 30 mm asparagine; 60 mM asparagine was used throughout these experiments. When no nitrogen was given dissolved in 1 N NaOH and assayed for protein. BSA dissolved in (Table I), growth was considerably less than that on glutamine or 1 N NaOH was used as a standard. Total nitrogen was determined by pyrolysis and gas chromatography. asparagine. Determination of Ammonium. Ammonium ion concentration of The soybean cotyledons would not grow on asparagine in the presence of 1 MSX,2 whereas MSX had no effect when the the medium was determined by a modified method of Kaplan (6). mM Fifty Al medium were mixed with 50 1A 0.01 N HCI. The reaction cotyledons were grown on glutamine (Table II). was initiated by adding successively to this 100,l sample, 1 ml of I15NINMR of Cotyledons. An [15N]NMR spectrum of five cot- yledons grown on 30 ['5N]amide glutamine (98.2 atom % '5N) 0.2 mm sodium nitroprusside in 1% w/v phenol and 1 ml of 0.125 mm is shown in Figure 1 (left). After extraction of the sample with N NaOH in 0.05% v/v NaOCl. After mixing, the solutions were 80% ethanol, a spectrum was obtained that represents only the incubated at room temperature for 30 min, during which time a blue color developed. A at 625 nm was then determined. The absorbance of a blank which consisted of 0.01 N HCI plus the 2Abbreviation: MSX, methionine sulfoximine. 310 SKOKUT ET AL. Plant Physiol. Vol. 69, 1982

Table 1. Growth of Soybean Cotvledons on Glutamine and Asparagine cotykdom Immature seeds of equal weight were cut in two so that each half don, " nWtmmtu consisted of one intact cotyledon. One cotyledon was grown on 30 mM glutamine while the other was grown on 60 mm asparagine or no nitrogen. After 7 days, the fresh weight, dry weight, and protein of each cotyledon was determined, averaged and expressed as the percentage (± SE) of that observed with growth on 30 mM glutamine. The cotyledons grown on 30 mM glutamine had an average initial fresh weight of 10 mg and an average, final fresh weight, dry weight, and protein content of 152, 33, and 5 mg, respectively. Growth on 60 mm glutamine and 30 mm asparagine was 80 ± 5% and 59 ± 6% of the 30 mm glutamine control. Nitrogen Source Fresh Weight Dry Weight Protein x4 % of control 30 mM Glutamine 100 ± 10 100 ± 12 100 ± 12 60 mM Asparagine 82 ± 7 73 ± 6 69 ± 5 No nitrogen 29 ± 2 36 ± 2 17 ± I inract ethAnoL uisotubt fracton- FIG. 1. Magic angle cross-polarization 9.12 MHz ['5N]NMR spectra of Table II. Effect ofMethionine Sulfoximine on Growth of Cotyledons in lyophilized cotyledons cultured 8 days on a medium containing 30 mM Culture I'5Njamide glutamine. The spectrum on the left is of five intact cotyledons Five cotyledons were growt on 30 mM glutamine ± I mM MSX or 60 and that on the right is of the same five cotyledons following an extraction mM asparagine ± I mm MSX. After 7 days, the cotyledons were harvested with 80%o ethanol. The large central peak of the spectrum on the left and their fresh weight, dry weight, and protein determined and averaged. represents '5N present as amide nitrogen (peptide nitrogen of protein plus The growth in the presence of MSX is presented as the percentage of that amide nitrogen of glutamine and asparagine). This peak appears about observed without MSX. 100 ppm downfield from that of solid ammonium sulfate as an external reference (19). (Downfield is measured from right to left.) The small peak Nitrogen Source Weight Dry Weight Protein at the left of the amide peak arises from nitrogen of histidine rings. The double peak immediately to the right of the amide peak represents the % ofcontrol guanidino nitrogens of arginine. The two peaks immediately to the right, Glutamine 100 100 100 or high-field side of the arginine peak, represent primarily the amino Glutamine + MSX 103 105 118 nitrogen of free amino acids and the e nitrogen oflysine, respectively. The Asparagine 100 100 100 remaining two peaks at the high-field and low-field extremes of the Asparagine + MSX 8 6 6 spectrum appear as a result of the mechanical spinning procedure and are called spinning sidebands (SSB). amino acids present in protein (Fig. 1, right). That is, the central peak represents only amide nitrogen of protein with no contribu- Table III. Incorporation of '5N into Cotyledon Protein tion from the amide nitrogen of free glutamine and asparagine. Five cotyledons were grown on medium containing [15NJamide gluta- After ethanol extraction, the intensities of the histidine, amide, mine (8 days), [15NJamide asparagine (7 days) or ['5N]amino asparagine and arginine peaks decrease slightly, and the double peak repre- (7 days). After the incubation period, they were lyophilized, pooled senting free amino acids disappears, leaving behind a small peak together as one sample, extracted with 80%o ethanol, dried, and observed representing the nitrogen of lysine side chains in protein. Virtually with NMR. The amount of "5N present in protein was quantitatively no protein was lost on extraction as determined by protein assay. determined as described in the text and is presented as the percentage of The amount of '5N present in the protein of the ethanol-extracted total protein nitrogen. cotyledons was calculated from this spectrum, using ['5N]NMR Percentage of Total Protein the natural abundance ['5N]NMR spectrum of a soybean seed as a standard; this value is presented in Table III as the percentage Nitrogen of total nitrogen in protein. Slightly more than one-half of the Composed nitrogen incorporated into protein was '5N. Nitrogen Source The spectra (before and after 80%1o ethanol extrac- Present at of '4N (in- 115NJNMR startstroff Composed tion) of five cotyledons grown on 60 mm [15Njamide asparagine of 15N corporated (98 atom % 15N) for 7 days were similar to those of the cotyledons expt during fed ['5NJamide glutamine. However, the '5N present in the protein expt) of the 80%o ethanol extracted tissue represented only 32% of the ['6NJamide glutaminea 10 48 42 total protein nitrogen (Table III) and accounted for about one- I'5Nlamide asparaginea 11 32 57 third of the nitrogen incorporated into protein during the growth ['5Nlamino asparagine 13 71 16 period. Similar calculations were made for cotyledons grown on a Average of three replicate experiments. ['5N]amide asparagine for 3, 7, 14, and 21 days. Between 3 and 21 days the ratio of 14N to 15N incorporated decreased from 6.1 to 0.8 medium. Seven days later, these cotyledons were harvested. Con- (Table IV). sistent with the results of Table IV, NMR analysis of these two To confirm the increased use of asparagine amide with time in samples showed that 38% of the total nitrogen incorporated into culture, the following experiment was performed. Cotyledons of protein in the first 7 days was 1"N, while 67% of the total nitrogen five immature seeds were prepared for culture. One cotyledon incorporated into protein during the second 7 days was 15N. from each seed was cultured on [15Njamide asparagine and the When 60 mm [' NJamino asparagine (99 atom % '5N) was used other cotyledon on natural abundance asparagine. After 7 days in as the nitrogen source, the ['5N]NMR spectra of the cotyledons is culture, the cotyledons growing on the labeled asparagine were qualitatively dissimilar to that of cotyledons fed amide-labeled harvested, while the sister cotyledons growing on the natural asparagine (Fig. 2). On a relative basis, little of the nitrogen from abundance medium were transferred to the ['5N]amide asparagine the 1 5NJamino asparagine appears in NH4', or in arginine and PROTEIN SYNTHESIS IN SOYBEAN COTYLEDONS 311

Table IV. Time Course ofIncorporation of 15N into Cotyledon Protein Bo,idculzureI mdim from ['5NJAmide Asparagine (97AW) Five cotyledons were grown on medium containing I'5Nlamide aspar- agine for various time periods. At the end of incubation, they were lyophilized, pooled together as one sample, extracted with 80%o ethanol, dried, and observed with NMR. The amount of "5N present in protein was XMAI 9fN(amidt)gt quantitatively determined as described in the text, and is presented as the percentage of total protein nitrogen. Percentage of Total Protein Nitrogen '4N Incor- Composed porated/ at Present Composed of '4N (in- "5N Incor- start of of '5N corporated porated expt during expt) 6mM 4- «C, 0N(azmide4t) d ratio FIG. 3. Magic angle cross-polarization 9.12 MHz NMR spectra of the 3 43 8 49 6.1 culture media containing ['5Njamide glutamine (top) and ['5N]amide 7a 11 32 57 1.8 asparagine (bottom) used to grow soybean cotyledons for 7 to 8 days. The 14b 3 46 51 1.1 residual labeled amide nitrogen in the glutamine medium is less than half 21 1 56 43 0.8 that in the asparagine medium due to increased incorporation of '5N during protein synthesis and because only one-halfas much glutamine was Average of three replicate experiments. present initially in the medium. b Average of two replicate experiments. ('5NH4)2SO4 as a standard. The 15NH4' in the asparagine medium yOptzc&d itract represents about 75 ,umol 15NH4+. This quantity is enough to cuLurcdvcotykdor4s account for the amount ofasparagine-amide nitrogen released but not utilized by the cotyledons for synthesis of protein. About one-half of the 15NH4' present in the ['5Njamide gluta- mine medium was found to be present as a contaminant of the [I5N]amide glutamine. Correcting for this amount, 12 umol '5NH4' were present in the glutamine medium as a result of the growth of H$+ one cotyledon. Cotyledons grown on 60 mm asparagine contained about 1 ,imol ammonium per cotyledon, whereas cotyledons grown on 30 mM x4 glutamine contained 0.5 ,imol ammonium.

DISCUSSION [I6N]Amide Nitrogen ofGlutamine. When ['5N]amide glutamine '5N ami&)Asn mcd(um. IN(aniin)asnmmediuW was fed to soybean cotyledons in culture, approximately equal FIG. 2. Magic angle cross-polarization 9.12 MHz ["NJNMR spectra of amounts of 14N and '5N were incorporated into storage protein intact lyophilized cotyledons cultured 7 days on a medium containing 60 (Table III). This result can be explained by assuming that the mM asparagine in which either the amide (left) or amine (right) nitrogens [15N]amide glutamine is first deamidated by the action of gluta- were "N-labeled. The relative intensities of the two spectra have been mate synthase. The presence of glutamate synthase activity in adjusted so that the intense peptide-nitrogen peaks appear equal. various plant tissues, including developing cotyledons, has been well documented (13, 24, 25). The glutamate thus formed, half of histidine residues of protein, as evidenced by the diminishedI which was "5N-labeled, results in an indiscriminate use of label intensities of those resonances, while more appears in peptide for the synthesis of protein. The excretion of ammonium into the nitrogen. "N incorporation into protein was 71% of the total medium could have occurred as a result ofthe action of glutamate protein nitrogen for the cotyledons grown on ["5N]amino aspara- dehydrogenase in the cotyledon, if there were a substantial accu- gine (Table III). mulation of glutamate in this tissue. An [15N]NMR spectrum of I'5NINMR of Media. The ['5N]NMR spectra of lyophilizedI the medium (Fig. 3) has a sizable peak at the position where ["5Niamide glutamine medium, and an equal weight of [f5N]amide [5N]amino nitrogen appears, suggesting that glutamate might also asparagine medium, on which the growth of single cotyledons hadL have been extracted into the medium. been supported for 8 and 7 days, respectively, are shown in Figure I15NIAmide Nitrogen of Asparagine. The results obtained when 3. The asparagine medium (bottom spectrum) has an approxi- ['5N]amide asparagine was fed to the cotyledons indicate that the mately 3-fold more intense '5NH4' resonance than that of the metabolism of the amide nitrogen of asparagine is different from glutamine medium, while the glutamine medium has the more that of the amide nitrogen of glutamine. After 7 days in culture intense ["5N]amino nitrogen resonance (peak directly to the left of on ['5N]amide asparagine medium, onlyr about one-third of the the ammonium peak). No "5NH4' was detected in the ["5N]amino nitrogen incorporated into protein was 5N (Table III). We con- asparamine medium used to support the growth of one cotyledon. clude that the amide nitrogen of asparagine is not immediately The 'NH4' present in the asparagine medium was the result of transferred to a compound such as glutamate. The amide and growth on [15N]amide asparagine. No "5NH4' was detected in the amino nitrogens do not, therefore, enter a common pool. starting ["5N]amide asparagine. The amount of "5NH4' present in The NMR experiments also show that incorporation of 15N the medium was estimated from the spectrum of Figure 3 com- (32% of total protein nitrogen after 7 days) was more than can be pared to a spectrum obtained from a known amount of attributed solely to incorporation of ['5N]amide asparagine. Ifonly 312 SKOKUT ET AL. Plant Physiol. Vol. 69, 1982 the asparagine residues were labeled with '5N, the percentage of percentage of the nitrogen incorporated into protein as 15N in- total protein nitrogen composed of '5N would be between 2 and creased from 38 to 67%. 6%, assuming that the asparagine content of the soybean protein Direct incorporation of aspartate into protein is apparently not is 25 to 75% of the aspartyl residues present (2). Qf course, the affected by the increased use of asparagine amide. Since the general appearance of the [15N]NMR spectrum (Fig. 2, left) con- [15NJamide asparagine used in these experiments was labeled at firms that 5N from the asparagine amide nitrogen appears in a the number 4 carbon with '3C, we also followed the fate of the wide variety of functional groups. ['5N-'3C]amide bond of asparagine using double cross-polarization MSX is an inhibitor of glutamine synthetase (15) which is the NMR as described in a concurrent paper (22). Although different major enzyme responsible for ammonium assimilation (13). MSX amounts of asparagine amide are incorporated into protein, de- completely inhibited protein production in cotyledons given as- pending on the time in culture, the percentage of the 15N which paragine as the sole nitrogen source (Table II). This suggests that remains as ['5N-'3C]amide, i.e. the amount of asparagine that is for protein to be produced, there is an absolute requirement for directly incorporated into protein, is relatively constant for the the ammonium released from the asparagine by the action of different times (22). asparaginase. Recently, Murray and Kennedy (14) have reported decreasing The importance of asparaginasein the transfer of nitrogen from activities of asparaginase and aspartate: a-ketoglutarate amino- asparagine has been stressed by other workers. Asparagine amide transferase in the seed coats of pea seeds in an early stage of nitrogen from ['5N]amide asparagine applied to fruiting shoots of development with concomitant increasing activities in the devel- lupin was found to appear in various amino acids in the lupin oping cotyledons. Although the results we report here indicate endosperm and seed (1). In addition, activities of asparaginase that a developing soybean cotyledon differs in its ability to utilize detected in developing cotyledons of various species were clearly the amide nitrogen of asparagine, depending upon its age or stage sufficient to account for the synthesis of all the protein of the of development, this is not necessarily due to the lack of asparag- in cotyledon (23). Finally, MSX and azaserine are known to inhibit inase young cotyledons. The excess ['5N]ammonium detected asparagine-dependent protein synthesis in developing cotyledons in the [1 N]amide asparagine culture medium strongly suggests of three legumes (other than soybean) in culture (9). that asparaginase is active all of the time. The presence or avail- Use of Amide Versus Amino Nitrogen of Asparagine. Although ability of an ammonium-assimilating enzyme, glutamine synthe- asparaginase appears to be functioning in the soybean cotyledons, tase, or possibly glutamate dehydrogenase, may therefore be an our results indicate that in the early stages of cotyledon develop- important limiting factor in the utilization of asparagine nitrogen ment, the amide nitrogen of asparagine is used to a lesser extent during the early stages of cotyledon development. than the amino nitrogen of asparagine (Table III). At this time, a Future Experiments. We have demonstrated the use of magic- transaminase reaction appears to be more important than the angle cross-polarization ['5N]NMR for the quantitative study of asparaginase reaction in making available the nitrogen required nitrogen metabolism in developing cotyledons of soybean in cul- for synthesis of protein. Because considerable amounts of 1 NH4' ture. The directness and simplicity of the NMR method permit an were detected in the ['5N]amide asparagine medium (Fig. 3), we unambiguous identification of the differences in utilization of the suggest that asparagine is first hydrolyzed to ammonium and amide and amine nitrogens of asparagine and glutamine in storage aspartate. A large portion of the ammonium is then excreted into protein synthesis. the medium, but some is reassimilated and used for the synthesis Nevertheless, we recognize characterization of the cotyledon ofamino acids destined for protein synthesis. However, to account culture system is only one step towards an understanding of for the labeling patterns, the aspartate nitrogen must be used to nitrogen metabolism in soybeans. In particular, metabolism in make a larger portion of the protein amino acids via an aspartate more meaningful organ cultures (such as, for example, seed and transaminase. pod cultures) as well as in the intact plant, has yet to be explored. High levels of activity of aspartate aminotransferase have been, In addition, monitoring the metabolism of nitrogen labels in the in fact, reported at the time of maximum protein synthesis in presence of a multiplicity of amino acid and ureide sources is developing seeds of Lolium (4, 29) and recently in developing clearly important. We expect the procedures illustrated above will be applicable. Indeed, encouraging preliminary experiments seeds of pea (14). Nevertheless, we cannot rule out the possibility in- volving observation of the incorporation into protein of'3C-'5N- that some of the asparagine amino nitrogen is directly transferred double label from to alanine or glycine by the action of asparagine transaminase (7), 4-[ 3C-'5NJ(amide)-asparagine, in the presence with the a-ketosuccinamate formed (10) subsequently deamidated, of a mixture of eight unlabeled amino acids and allantoin, have thereby generating the15NH4' detected in the medium. However, indicated no qualitative differenc. s from those results reported here. The only inherent limitation on such experiments is the the similarity of thelineshapes of the major amide-nitrogen reso- sensitivity of the NMR spectrometer, which is a matter under nance for cotyledons fed either or [15NJamino ['5N]amide-labeled continuing technological development. asparagine indicates there is no large incorporation into protein of [1lNJalanine and[15N]glycine. We have observed that the amide- nitrogen signals for polyalanine and polyglycine are at opposite LITERATURE CITED extremes of the peptide nitrogen resonance bandwidth with that 1. ATKINS CA, JS PATE, PJ SHARKEY 1975 Asparagine metabolism-key to the from polyglycine occurring at higher field. Thus, high concentra- nitrogen nutrition of developing legume seeds. Plant Physiol 56: 807-812 tions of these two residues in the protein of tissue growing on 2. BOULTER D, E DERBYSHIRE 1971 Taxonomic aspects of the structure of legume asparagine would produce a broadening of the amide- proteins. In JB Harborne, D Boulter, BL Turner, eds, Chemotaxonomy of the ['5Njamino Leguminosae. Academic Press, New York, pp 285-308 nitrogen resonance relative to that for tissue growing on[15NJ- 3. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of amide asparagine. This is not observed (Fig. 2). microgram quantities of protein utilizing the principle of protein-dye binding. The ratio of14N incorporated to'5N incorporated into protein Anal Biochem 72: 248-254 decreases with the length of time that cotyledons are grown on 4. HEDLEY CL, JL STODDART 1972 Patterns of protein synthesis in Loliumtemulen- tum (L.)II. During seed development. J Exp Bot 23: 502-510 ['5N]amide asparagine medium (Table IV). This indicates that the 5. HILL JE, RW BREIDENBACH 1974 Proteins of soybean seeds. I. Isolation and longer the cotyledons are exposed to asparagine in culture, the characterization of the major components. Plant Physiol 53: 742-746 more amide nitrogen of asparagine they can utilize. This obser- 6. KAPLAN A 1965 Urea nitrogen and urinary ammonia. In S Meites, ed, Standard vation was confirmed by the experiment described above which Methods ofClinical Chemistry, Vol 5. Academic Press, London, pp 245-256 7. LEA PJ, L FOWDEN 1975 Asparagine metabolism in higher plants. 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