Planta (Berl.) 95, 187--201 (1970) 9 by Springer-Verlag 1970
Aspects of D-Leucine and D-Lysine Metabolism in Maize and Ryegrass Seedlings* * * R. W . ALDAG a n d J. L. Y o u n G * * * Agricultural Research Service, U.S. Department of Agriculture in cooperation with Department of Soils, Oregon State University, Corvallis, Oregon Received June 1 / July 15, 1970
Summary. Maize and ryegrass seedlings (2.5 weeks old), the roots of which were dipped into 10-sM 14C-earboxyl-labeled D-leucine and 14C-e-labeled D-lysine, readyly absorbed and converted or conjugated within 34 hr some 75-90% of the labeled compound supplied. The metabolic intermediates and products were generally similar for both maize and ryegrass. Radioactive intermediates from the carboxyllabeled D-leueine were L-leueine, N-malonyl-n-leucine (provisionally identified), and ~-ketoisocaproic acid. Intermediates from e-labeled D-lysine were numerous, with greater amounts and numbers detected in roots than in tops. Pipecolic acid was a major intermediate particularly in shoot tissue. Pathways of conversion appeared analogous to those for the L-isomer, and conversion may be by the usual L-configuration machinery, since the labeled L-isomer of the originally supplied 14C-I)-amino acid was always found. How the 14C-D-amino acid gets to l~C-L-isomer is not known, but finding significant proportions of unlabeled n-alanine in plants treated with both the labeled D-leucine and n-lysine suggested that formation of the ~-keto-acid analog and subsequent reamination was possibly an important route. Introduction A l t h o u g h t h e n u t r i t i o n a l effects a n d t h e m e t a b o l i s m of D-amino acids in m i c r o o r g a n i s m s a n d in a n i m a l s h a v e been s t u d i e d to some e x t e n t (Meister, 1965, p. 221-4, 297-304, 357-8, 369-375; Corrigan, 1969) little * Approved for publication as Technical Paper No. 2,729 of the Oregon Agricultural Experiment Station. ** Trade, copyright or company names are for identification and reader benefit; their use does not constitute endorsement or preferential treatment by the U.S.D.A. *** Respectively, Research Associate, Department of Soils, Oregon State University; Research Chemist, Soil and Water Conservation Research Division, Agricultural Research Service, U.S. Department of Agriculture. Present address of senior author: Institut fiir Bodenkunde D-3400 G6ttingen, yon SieboldstraBe 4, West Germany. We thank the Oregon State University Research Council and the Bundesministerium ffir Ern~hrung, Landwirtschaft und Forsten, Bonn, Germany, for partial financial assistance to the senior author, and M. Yamamoto for technical assistance. I3 Plan~a(Berl.), Bd. 95
188
R. W. Aldag and J. L. Young:
is k n o w n a b o u t t h e presence or t r a n s f o r m a t i o n of these "non-protein", " u n n a t u r a l " a m i n o acids in p l a n t s or softs. Their possible i m p o r t a n c e in soft-plant s y s t e m s needs r e c o n s i d e r a t i o n in light of r e c e n t r e p o r t s (Kiek u t h a n d Aldag, 1967; R o s a a n d Neish, 1968; A l d a g et al., 1970). A c o m p a n i o n p a p e r b y A l d a g a n d Y o u n g (1970) d e a l t w i t h u p t a k e a n d gross d i s t r i b u t i o n of five 14C-labeled D-a~mino acids in shoots a n d roots of maize a n d r y e g r a s s seedlings, a n d r e p o r t e d for t h e first t i m e a few of t h e a p p a r e n t i n i t i a l m e t a b o l i c conversions of D-amino acids b y these plants. This p a p e r p r e s e n t s m o r e i n f o r m a t i o n a b o u t t h e m e t a bolism of two of these five D-amino acids, n a m e l y , D-leucine a n d D-lysine.
Material and Methods Growth conditions, exposure of plants to and uptake of labeled ])-amino acids, harvest, extraction, and methods to study enzymatic oxidation of the amino acids have been more fully described elsewhere (Aldag and Young, 1970; Aldag et al., 1970). Thus, only minimal detail is repeated below. Seeds. Ryegrass (Lolium spec.) and corn (Zea may~ L.) ev. "Pride No. 5". ])-Amino Acids. Labeled and unlabeled compounds were purchased from Calbiochem, Los Angeles, California. Specific activities were 4.1 mC millimole-1 for ])-leucine, and 3.0 mC millimole-1for D-lysine. D-leucine was labeled in the carboxyl position and D-lysine in the e-carbon position. Plant Culture. Seedlings were grown under sterile conditions in 2.5 • 19 cm Pyrex glass tubes on 80-90 ml of nutrient agar. Microbial contamination was readily detected by viewing tubes against a bright light. Colonies, agar liquefaction, discoloration or profuse fungal growth signalled inadequate seed sterilization or contamination, generally within 2-5 days. Tubes suspected of any contamination were discarded. After 18 days, when most of the nutrients and water had been used, the plants were removed from the tubes and the remaining adhering agar was carefully rinsed away with water. Then the seedlings were returned to the clean growing tube and 5 ml of sterile 10-aM labeled (approximately, 10 ~zC 1~C) ])-amino acid solution was supplied to the roots. Ryegrass seedlings absorbed essentially all of the labeled solution in about 6 hr; the corn took about 24 hr. Supplement unlabeled D-amino acid (to raise ninhydrin levels) or distilled H~O (to prevent desiccation) were added as needed at 6, 24, and 29 hr. At the end of the 34-hr exposure time, roots were separated from the shoot, each immediately frozen with dry ice, lyophilized and stored dry under vacuum until analyzed. Analytical Methods. Free amino acids and other organic acids were extracted with 80 % ethanol. Chlorophyll was removed with petroleum ether and the ethanol extract concentrated under vacuum at 35 ~. The final volume was divided into equal parts, and each taken to dryness. One half was picked up in H20 and the other half in lithium citrate buffer (pH 2.2, as used for sample application to the amino-acid analyzer). All preparations were immediately refrigerated (--10 ~ or -}-1-2 ~ until analyzed. Bound or hydrolyzable amino acids in the residual plant material (i.e. previously extracted with 80% ethanol) were hydrolyzed in 6N HC1 for 6 hr under reflux. The amino acids were separated by ion exchange chromatography with an automatic amino-acid analyzer (Phoenix, Model K-8,000A, modified to equivalent of current high-pressure, high flow-rate, high resolution, nanomole sensitivity units).
D-Leucine and D-Lysine Metabolism
189
Radioactivity of the separated amino acids was continuously monitored with a liquid-flow scintillation detector (Packard 317). Evidence for identification of a-keto-acid analogs of applied D-amino acids came from a combination of results including column chromatography (peak positions, relative 14Cactivity and ninhydrin reactivity), paper chromatography and IR spectra, the labeled organic acids having been pre-separated from the amino acids with Dowex 50 cation exchange-resin column. Enzymatic oxidation of the D-amino acids was studied using the following incubation mixture: Tris-HC1buffer (150 micromoles, pH 8.3), ca. 50-500 nanomoles of substrata, electrophoretically pure D-amino-acid oxidase (0.5 units), and catalase (6,000 units) in a total volume of 0.5-1.0 ml. Incubation in a 10 ml centrifuge tube at 37~ with a continuous shaking for 60 rain was terminated by adding 50 ~l of 10% trichloroacetic acid. The supernatant, cleared by centrifugation, was quantitatively transferred to and assayed in the automated amino-acid analyzer (see Aldag et al., 1970). The precision of the coupled enzymatic oxidation and chromatographic assay for 14C-labeled compounds is exemplified by the extremes of deviation from the mean of six replicate pairs (oxidized and unoxidized) as follows: ~4% for D-alanine, • for D-valine, and ~7% for D-leucine--all in the presence of larger amounts of their corresponding L-forms and all the other amino acids commonly occurring in plant extracts. Tests for N-Malonyl conjugates of the supplied D-amino acids included decarboxylation by heating and simultaneous conversion to the N-acetyl conjugate (Eschrich and tIartmarm, 1969) and acid hydrolysis (6N HC1) resulting in concurrent loss of labeled organic acid and corresponding reappearance of the original labeled amino acid. Results Table 1 summarizes the gross distribution and uptake of laC-labcled D-leucine and D-lysine in various extracts of corn and ryegrass seedlings at the end of a 34-hr treatment period. Uptake, given as percent of the total supplied laC-activity, varied between 92 and 99 % (column 2). Amounts of radioactivity extracted by 80% ethanol at the end of the 3 4 h r appear in columns 4 and 6. Columns 5 and 7 show the radioactivity of the bound amino acids, i.e., incorporated into 6N HC1 hydrolyzable linkages. Column 8 represents the total 14C-activity from all extracts, and column 9, the net 14C-loss. The net loss is about 15% higher in the corn plants than in the ryegrass seedlings for these two applied D-amino acids. The distribution of radioactivity within the 80 %-ethanol extracts of shoots and roots and the total D-amino acid metabolized are shown in Table 2. Also, the stereospecificity of the laC-amino acids in the extracts, as determined enzymatically with D-amino-acid oxidase, is given. These data show that not only organic acid 14C-compounds were formed but that significant amounts of the corresponding L-e-amino acid isomer, carrying the 14C label, also appeared. The amount of D-eompomld converted to the labeled L-isomer was generally higher in the root than in the shoot extracts (column 5b versus 2b). The total D-amino acid which 13"
(2)
98.9 99.7
97.7 92.2
(1)
I)-Leucine D-Lysine
D-Leueine I)-Lysine
0.6 0.4
0.5 0.7
(3)
33.4 16.6
11.4 31.5
(4)
nil nil
nil nfl
(5a) b 15.5 18.0 17.2 38.2
Ryegrass 11.1 1.8
(6)
13.3 1.8
Corn
5b) c
80%ethanol extract
~IC1 hydrolysate of residue after alcohol extraction
Ether extract (chlorophyll)
80% ethanol extract
R o o t tissue
Shoot tissue
laC found, % of t o t a l t a k e n u p
nil 3.2 (?)
nil 1.8 (?)
(7a) b
3.9 6.5
10.5 3.8
(7b) c
HC1 hydrolysate of residue after alcohol extraction
66.2 66.7
51.2 57.6
(8)
Total shoot a n d root (3, 4, 5, 6, 7)
31.5 25.5
47.7 42.1
(9)
N e t laC loss a (2)--(8)
a Assumed is released as laC02. Suggestions t h a t ~4C0~ was lost from ~-N-malonyl or similar derivatives during e x t r a c t i o n appear untenable. N-malonyl conjugates are acid- a n d heat-labile b u t COs evolved is from t h e malonyl-COOH r a t h e r t h a n t h e amino acid where the xdC label was located. b Identified as originally supplied 1*C-D-amino acid. While a p p r o x i m a t e l y one t h i r d of t h e hydrolysis-released labeled lysine appeared as residual ])-form, this is n o t evidence for incorporation of D-isomer into peptide-type linkages since incomplete alcohol extraction, for example, could be a n explanation. The emphasis a t this time is on t h e presence of t h e 14C-labeled L-isomer. c Identified as L-amino acid, formed b y t h e p l a n t from the supplied D-amino acid.
Uptake (% of supplied)
Amino acid supplied
Table 1. Uptake ot 14C-labeled D-amino acids and their gross distribution i n extracts /rom corn and ryegrass seedlings at the end o] a 34-hr treatment period Absolute a m o u n t of laO label supplied was 2.83 • 107 cpm for D-leucine a n d 2.81 • 10 ~ for D-lysine in 5 ml 10-sM solution.
~
O
t
9~ (}q
>
21.9 1.4
D-Leueine D-Lysine
4.3 1.1
0.9 0.1
5.3 13.4 b
4.7 25.6 b
(3)
1.9 0.7
0.3 2.3
(4)
(5a)
1.3 12.5
3.1 6.7
D-
(5b)
2.4 10.7
1.6 3.5
L-
13.0 8.9 b
t~yegrass
8.8 3.2 b
Corn
(6)
0.5 6.1
2.0 4.6
(7)
Other and unidentiffed
23.2 13.9
8.6 10.2
D-
(8a)
6.7 11.8
2.5 3.6
L-
(8b)
Total " f r e e " amino acid (shoot a n d root) (2) + (5)
18,3 22.3 b
13.5 28.8 b
(9)
74.5 78.3
90.3 87.7
(10) a
Total deriv- Total ative organic D.amino a n d keto acid acid (shoot metaba n d root) olized (3) + (6)
a Col. 10 values axe t h e sum of cols. 4, 7, 8b, 9 of this table plus cols. 3, 5b, 7b, a n d 9 of Table 1. b Is largely t h e piperidine-ring amino compound, pipecolic acid. This a n d d a t a in Table 3 emphasize t h e extensiveness of metabolism beyond first-reaction or " d e t o x i f y i n g " or " i m m o b i l i z i n g " waste products.
5.5 3.5
(2b)
L-
(2a)
D-
Derivative organic a n d o~keto acid
Supplied 14Camino acid present as D- or Lcompound
Other and unidentiffed
Supplied laCamino acid present as D - or Lcompound
Derivafire organic a n d aketo acid
I n ethanol extract from root
D-amino acid metabolized
I n ethanol extract from shoot
D-Leucine D-Lysine
(1)
Amino acid supplied
Values are percent of total laC t a k e n up.
Table 2. Distribution o/ radioactivity within the 80 % ethanol extracts o/shoots and roots, and minimum total
R. W. Aldag and J. L. Young:
192
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Aspects of D-Leucine and D-Lysine Metabolism in Maize and Ryegrass Seedlings* * * R. W . ALDAG a n d J. L. Y o u n G * * * Agricultural Research Service, U.S. Department of Agriculture in cooperation with Department of Soils, Oregon State University, Corvallis, Oregon Received June 1 / July 15, 1970
Summary. Maize and ryegrass seedlings (2.5 weeks old), the roots of which were dipped into 10-sM 14C-earboxyl-labeled D-leucine and 14C-e-labeled D-lysine, readyly absorbed and converted or conjugated within 34 hr some 75-90% of the labeled compound supplied. The metabolic intermediates and products were generally similar for both maize and ryegrass. Radioactive intermediates from the carboxyllabeled D-leueine were L-leueine, N-malonyl-n-leucine (provisionally identified), and ~-ketoisocaproic acid. Intermediates from e-labeled D-lysine were numerous, with greater amounts and numbers detected in roots than in tops. Pipecolic acid was a major intermediate particularly in shoot tissue. Pathways of conversion appeared analogous to those for the L-isomer, and conversion may be by the usual L-configuration machinery, since the labeled L-isomer of the originally supplied 14C-I)-amino acid was always found. How the 14C-D-amino acid gets to l~C-L-isomer is not known, but finding significant proportions of unlabeled n-alanine in plants treated with both the labeled D-leucine and n-lysine suggested that formation of the ~-keto-acid analog and subsequent reamination was possibly an important route. Introduction A l t h o u g h t h e n u t r i t i o n a l effects a n d t h e m e t a b o l i s m of D-amino acids in m i c r o o r g a n i s m s a n d in a n i m a l s h a v e been s t u d i e d to some e x t e n t (Meister, 1965, p. 221-4, 297-304, 357-8, 369-375; Corrigan, 1969) little * Approved for publication as Technical Paper No. 2,729 of the Oregon Agricultural Experiment Station. ** Trade, copyright or company names are for identification and reader benefit; their use does not constitute endorsement or preferential treatment by the U.S.D.A. *** Respectively, Research Associate, Department of Soils, Oregon State University; Research Chemist, Soil and Water Conservation Research Division, Agricultural Research Service, U.S. Department of Agriculture. Present address of senior author: Institut fiir Bodenkunde D-3400 G6ttingen, yon SieboldstraBe 4, West Germany. We thank the Oregon State University Research Council and the Bundesministerium ffir Ern~hrung, Landwirtschaft und Forsten, Bonn, Germany, for partial financial assistance to the senior author, and M. Yamamoto for technical assistance. I3 Plan~a(Berl.), Bd. 95
188
R. W. Aldag and J. L. Young:
is k n o w n a b o u t t h e presence or t r a n s f o r m a t i o n of these "non-protein", " u n n a t u r a l " a m i n o acids in p l a n t s or softs. Their possible i m p o r t a n c e in soft-plant s y s t e m s needs r e c o n s i d e r a t i o n in light of r e c e n t r e p o r t s (Kiek u t h a n d Aldag, 1967; R o s a a n d Neish, 1968; A l d a g et al., 1970). A c o m p a n i o n p a p e r b y A l d a g a n d Y o u n g (1970) d e a l t w i t h u p t a k e a n d gross d i s t r i b u t i o n of five 14C-labeled D-a~mino acids in shoots a n d roots of maize a n d r y e g r a s s seedlings, a n d r e p o r t e d for t h e first t i m e a few of t h e a p p a r e n t i n i t i a l m e t a b o l i c conversions of D-amino acids b y these plants. This p a p e r p r e s e n t s m o r e i n f o r m a t i o n a b o u t t h e m e t a bolism of two of these five D-amino acids, n a m e l y , D-leucine a n d D-lysine.
Material and Methods Growth conditions, exposure of plants to and uptake of labeled ])-amino acids, harvest, extraction, and methods to study enzymatic oxidation of the amino acids have been more fully described elsewhere (Aldag and Young, 1970; Aldag et al., 1970). Thus, only minimal detail is repeated below. Seeds. Ryegrass (Lolium spec.) and corn (Zea may~ L.) ev. "Pride No. 5". ])-Amino Acids. Labeled and unlabeled compounds were purchased from Calbiochem, Los Angeles, California. Specific activities were 4.1 mC millimole-1 for ])-leucine, and 3.0 mC millimole-1for D-lysine. D-leucine was labeled in the carboxyl position and D-lysine in the e-carbon position. Plant Culture. Seedlings were grown under sterile conditions in 2.5 • 19 cm Pyrex glass tubes on 80-90 ml of nutrient agar. Microbial contamination was readily detected by viewing tubes against a bright light. Colonies, agar liquefaction, discoloration or profuse fungal growth signalled inadequate seed sterilization or contamination, generally within 2-5 days. Tubes suspected of any contamination were discarded. After 18 days, when most of the nutrients and water had been used, the plants were removed from the tubes and the remaining adhering agar was carefully rinsed away with water. Then the seedlings were returned to the clean growing tube and 5 ml of sterile 10-aM labeled (approximately, 10 ~zC 1~C) ])-amino acid solution was supplied to the roots. Ryegrass seedlings absorbed essentially all of the labeled solution in about 6 hr; the corn took about 24 hr. Supplement unlabeled D-amino acid (to raise ninhydrin levels) or distilled H~O (to prevent desiccation) were added as needed at 6, 24, and 29 hr. At the end of the 34-hr exposure time, roots were separated from the shoot, each immediately frozen with dry ice, lyophilized and stored dry under vacuum until analyzed. Analytical Methods. Free amino acids and other organic acids were extracted with 80 % ethanol. Chlorophyll was removed with petroleum ether and the ethanol extract concentrated under vacuum at 35 ~. The final volume was divided into equal parts, and each taken to dryness. One half was picked up in H20 and the other half in lithium citrate buffer (pH 2.2, as used for sample application to the amino-acid analyzer). All preparations were immediately refrigerated (--10 ~ or -}-1-2 ~ until analyzed. Bound or hydrolyzable amino acids in the residual plant material (i.e. previously extracted with 80% ethanol) were hydrolyzed in 6N HC1 for 6 hr under reflux. The amino acids were separated by ion exchange chromatography with an automatic amino-acid analyzer (Phoenix, Model K-8,000A, modified to equivalent of current high-pressure, high flow-rate, high resolution, nanomole sensitivity units).
D-Leucine and D-Lysine Metabolism
189
Radioactivity of the separated amino acids was continuously monitored with a liquid-flow scintillation detector (Packard 317). Evidence for identification of a-keto-acid analogs of applied D-amino acids came from a combination of results including column chromatography (peak positions, relative 14Cactivity and ninhydrin reactivity), paper chromatography and IR spectra, the labeled organic acids having been pre-separated from the amino acids with Dowex 50 cation exchange-resin column. Enzymatic oxidation of the D-amino acids was studied using the following incubation mixture: Tris-HC1buffer (150 micromoles, pH 8.3), ca. 50-500 nanomoles of substrata, electrophoretically pure D-amino-acid oxidase (0.5 units), and catalase (6,000 units) in a total volume of 0.5-1.0 ml. Incubation in a 10 ml centrifuge tube at 37~ with a continuous shaking for 60 rain was terminated by adding 50 ~l of 10% trichloroacetic acid. The supernatant, cleared by centrifugation, was quantitatively transferred to and assayed in the automated amino-acid analyzer (see Aldag et al., 1970). The precision of the coupled enzymatic oxidation and chromatographic assay for 14C-labeled compounds is exemplified by the extremes of deviation from the mean of six replicate pairs (oxidized and unoxidized) as follows: ~4% for D-alanine, • for D-valine, and ~7% for D-leucine--all in the presence of larger amounts of their corresponding L-forms and all the other amino acids commonly occurring in plant extracts. Tests for N-Malonyl conjugates of the supplied D-amino acids included decarboxylation by heating and simultaneous conversion to the N-acetyl conjugate (Eschrich and tIartmarm, 1969) and acid hydrolysis (6N HC1) resulting in concurrent loss of labeled organic acid and corresponding reappearance of the original labeled amino acid. Results Table 1 summarizes the gross distribution and uptake of laC-labcled D-leucine and D-lysine in various extracts of corn and ryegrass seedlings at the end of a 34-hr treatment period. Uptake, given as percent of the total supplied laC-activity, varied between 92 and 99 % (column 2). Amounts of radioactivity extracted by 80% ethanol at the end of the 3 4 h r appear in columns 4 and 6. Columns 5 and 7 show the radioactivity of the bound amino acids, i.e., incorporated into 6N HC1 hydrolyzable linkages. Column 8 represents the total 14C-activity from all extracts, and column 9, the net 14C-loss. The net loss is about 15% higher in the corn plants than in the ryegrass seedlings for these two applied D-amino acids. The distribution of radioactivity within the 80 %-ethanol extracts of shoots and roots and the total D-amino acid metabolized are shown in Table 2. Also, the stereospecificity of the laC-amino acids in the extracts, as determined enzymatically with D-amino-acid oxidase, is given. These data show that not only organic acid 14C-compounds were formed but that significant amounts of the corresponding L-e-amino acid isomer, carrying the 14C label, also appeared. The amount of D-eompomld converted to the labeled L-isomer was generally higher in the root than in the shoot extracts (column 5b versus 2b). The total D-amino acid which 13"
(2)
98.9 99.7
97.7 92.2
(1)
I)-Leucine D-Lysine
D-Leueine I)-Lysine
0.6 0.4
0.5 0.7
(3)
33.4 16.6
11.4 31.5
(4)
nil nil
nil nfl
(5a) b 15.5 18.0 17.2 38.2
Ryegrass 11.1 1.8
(6)
13.3 1.8
Corn
5b) c
80%ethanol extract
~IC1 hydrolysate of residue after alcohol extraction
Ether extract (chlorophyll)
80% ethanol extract
R o o t tissue
Shoot tissue
laC found, % of t o t a l t a k e n u p
nil 3.2 (?)
nil 1.8 (?)
(7a) b
3.9 6.5
10.5 3.8
(7b) c
HC1 hydrolysate of residue after alcohol extraction
66.2 66.7
51.2 57.6
(8)
Total shoot a n d root (3, 4, 5, 6, 7)
31.5 25.5
47.7 42.1
(9)
N e t laC loss a (2)--(8)
a Assumed is released as laC02. Suggestions t h a t ~4C0~ was lost from ~-N-malonyl or similar derivatives during e x t r a c t i o n appear untenable. N-malonyl conjugates are acid- a n d heat-labile b u t COs evolved is from t h e malonyl-COOH r a t h e r t h a n t h e amino acid where the xdC label was located. b Identified as originally supplied 1*C-D-amino acid. While a p p r o x i m a t e l y one t h i r d of t h e hydrolysis-released labeled lysine appeared as residual ])-form, this is n o t evidence for incorporation of D-isomer into peptide-type linkages since incomplete alcohol extraction, for example, could be a n explanation. The emphasis a t this time is on t h e presence of t h e 14C-labeled L-isomer. c Identified as L-amino acid, formed b y t h e p l a n t from the supplied D-amino acid.
Uptake (% of supplied)
Amino acid supplied
Table 1. Uptake ot 14C-labeled D-amino acids and their gross distribution i n extracts /rom corn and ryegrass seedlings at the end o] a 34-hr treatment period Absolute a m o u n t of laO label supplied was 2.83 • 107 cpm for D-leucine a n d 2.81 • 10 ~ for D-lysine in 5 ml 10-sM solution.
~
O
t
9~ (}q
>
21.9 1.4
D-Leueine D-Lysine
4.3 1.1
0.9 0.1
5.3 13.4 b
4.7 25.6 b
(3)
1.9 0.7
0.3 2.3
(4)
(5a)
1.3 12.5
3.1 6.7
D-
(5b)
2.4 10.7
1.6 3.5
L-
13.0 8.9 b
t~yegrass
8.8 3.2 b
Corn
(6)
0.5 6.1
2.0 4.6
(7)
Other and unidentiffed
23.2 13.9
8.6 10.2
D-
(8a)
6.7 11.8
2.5 3.6
L-
(8b)
Total " f r e e " amino acid (shoot a n d root) (2) + (5)
18,3 22.3 b
13.5 28.8 b
(9)
74.5 78.3
90.3 87.7
(10) a
Total deriv- Total ative organic D.amino a n d keto acid acid (shoot metaba n d root) olized (3) + (6)
a Col. 10 values axe t h e sum of cols. 4, 7, 8b, 9 of this table plus cols. 3, 5b, 7b, a n d 9 of Table 1. b Is largely t h e piperidine-ring amino compound, pipecolic acid. This a n d d a t a in Table 3 emphasize t h e extensiveness of metabolism beyond first-reaction or " d e t o x i f y i n g " or " i m m o b i l i z i n g " waste products.
5.5 3.5
(2b)
L-
(2a)
D-
Derivative organic a n d o~keto acid
Supplied 14Camino acid present as D- or Lcompound
Other and unidentiffed
Supplied laCamino acid present as D - or Lcompound
Derivafire organic a n d aketo acid
I n ethanol extract from root
D-amino acid metabolized
I n ethanol extract from shoot
D-Leucine D-Lysine
(1)
Amino acid supplied
Values are percent of total laC t a k e n up.
Table 2. Distribution o/ radioactivity within the 80 % ethanol extracts o/shoots and roots, and minimum total
R. W. Aldag and J. L. Young:
192
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::;~:_ -
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'1" ~ Zu
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=:
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