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Leucine biosynthesis in higher plants
456
SHORT COMMUNICATIONS
Although the occurrence of exchange reactions independent of and in a d d ~ c m to the reactions that lead to net uptake of amino acids a n d peptides b y bacteria complicates measurement of the kinetics of accumulation with radioactive tracers, previous conclusions t h a t transport of these substances into the cell occurs b y a n active, energy-dependent process with structural specificity characteristic off enzymic processes remain unchanged. This research was sponsored in part b y grant E-I575 from the U.S. Public HeM~h Service. F . R . L . was a National Research Fellow in the Medical Science (I957--~9)-
Department of Biochemistry, University of California, Berkeley, Calif. (U.S.A.)
FRANKLIN R. LY-~'CH° ESMOND E. SNELL
F. R. LEACHAND E. E. SNELL, Bioohim. Biophys. Acta, 34 (I959) 292. ¢ F. R. LEACHAND E. E. SNELL,J. Biol. Chem., 235 (196o) 3523. 3 E. M. LEVXN~AND S. SIMMONDS,J. Biol. Chem., 235 (196o) 29o2. 4 G. N. COHENAND H. V. RICKENBERG,Compt. Rend., 24o (1955) 2o86. BG. N. COHENAND H. V. RICKENBERG, Ann. Inst. Pasteur, 91 (I956) 693. R. J. BRITTEN,R. B. ROBERTSAND E. F. FRENCH, Proc. Natl. Acad. Sci. U.S., 41 (1955) 805G. N. COHENAND J. MONOD,Bacteviol. Rev., 2I (1957) 169. 8 E. HEINZ,J. Biol. Chem,, 21I (1954) 781. o E. HEINZAND P. M. WALSH,J. Biol. Chem., 233 (1958) 1488. ]
lo j . MANDELSTAM, Biochem. J., 69 (1958) 1o3.
* Present address: Department of Biochemistry, Oklahoma Statc Univez~ity, Stillwater. ,Ot~L Received October 18+.h, 1962 Biochim. Biophys ,4eta, 7 x (1963) 4 ~ 4 - 4 ~
SC 2 2 1 6
Leucine biosynthesis in higher plants There is considerable evidence from labelling experiments t h a t in mieroorga~is~ns leucine arises from the condensation of 2-ketoisovaleric acid (ketovaline) a n d a ~ tate 1-3. Support for such a pathway, involving the intermediate formation of propylmalate, has recently been obtained at the enzyme level in S a / m o ~ lylff~murium and Torulopsis utilis4, 5 and ,,-isopropylmalate has been isolated from ~ t m ~ e s of several microorganismsn. I n the course of an investigation of the biosynthesis off linamarin in flax, some evidence for the presence of this p a t h w a y for l e u c ~ e synthesis was obtained incidentally and the results are briefly reported here. Flax seeds (Linum usitatissimum L. var. Imperial) were germinated a n d grotto for 72 h in the dark on gauze moistened with dilute n u t r i e n t solution. They next exposed to artificial light of i n t e n s i t y approx. 2ooo ft-candles from a n descent lamp for 16 h. Uniformly labelled L-[14C]valine was administered b y of the following methods: (a) 20 seedlings selected for uniformity were placed in a 5-ml beaker conlmiml~g 5 F moles L-[14C]valine (I ~C) in ~- ml water. This was administered for 48 h ~41h aeration a n d continuous illumination. (b) The stems of 20 seedlings were cut with a sharp razor blade 2 a m b e ~ w the cotyledon leaves a n d placed in a 1-ml beaker containing 5 Vanoles x-[a~C]~ (I FC) in o.2 ml water. Successive a m o u n t s of o.i rnl water were added as during a 7-h absorption period in continuous light.
Biochim. Biophys. Aaa, ?x (i965) 4:V6.--.t$$
SHORT COMMUNICATIONS
457
The plant material was harvested, frozen in liquid nitrogen, ground to a fine powder with a mortar and pestle and extracted with IOO vol. boiling 8o o,3 (v/v) aq. ethanol for 5 rain. The solvent was evaporated in -,aczto and the residue extracted with 2 ml io % (v/v) aq. isopropanol. The extract was clarified by centrifugation if necessary and suitable aliquots were used for paper chromatography. Similar experiments were also carried out with seedlings of Sorghum vulgare var. Honey Drip. In some experiments the plant residue remaining after aq. ethanol extraction was refluxed with zo vol. 6 N HC1 for I6 h and the acid removed by repeated evaporation to d~'ness. The residue was extracted with 2 ml Io % (v/v) aq. isopropanol and suitable aliquots taken for paper chromatography. Paper chromatography in one dimension on Whatman No. I paper was carried out in each of the following solvent systems: n - b u t a n o l - p y r i d i n e - w a t e r (6o:4o:3o, v/v), methylethyl k e t o n e - a c e t o n e - w a t e r (30: lO:6, v/v), butanol-acetic a c i d - w a t e r (12o :3o: 5 o, v/v) and phenol-water (80: 20, v/v). The distribution of radioactivity was determined using a chromatogram strip scanner (Nuclear Chicago Model R IOOO). With ethanol extracts from flax seedlings the major radioactive peaks corresponded with linamarin and L-valine markers in all solvents; a smaller peak was also seen corresponding with L-leucine marker. With ethanol extracts from sorghum seedlings two peaks were seen corresponding to L-valine and L-leucine markers. Chromatograms of the acid hydrolysates from both flax and sorghum seedlings showed the presence of two radioactive peaks corresponding to L-valine and L-leucine markers in all solvent systems. The identity of the suspected [l*C!leucine in flax seedlings was definitely established by the preparation of the naphthalene-fl-sulphonyl (nasyl) derivativeL Partial purification was first accomplished by successive paper chromatography and elution using b u t a n o l - p y r i d i n e - w a t e r and methylethyl ketone-acetone-water. L-Leucine (5° nag) was added and the nasvl derivative prepared and dried. A suitable sample was dissolved in a known wflume of ethanol and aliquots were evaporated on planchettes and counted at infinite thinness in a gas-flow counter (NuclearChicago Model x8IB). The counting procedure was repeated through 3 recrystallisations with the same specific activity (29o counts/rain/rag derivative) being observed throughout. Table I shows the percentages of 14C found in free and bound leucine in an experiment with sorghum seedlings. The low ratio of 2.7 for the 14C in bound valine and leucine shows that leucine biosynthesis by a pathway of the type proposed represents an important source of leucine in this tissue. Attempts to demonstrate the participation of ~,-isopropylmalate as an intermediate were inconclusive. [lac~Isopropylmalate could not be detected in aq. ethanol extracts of seedlings administered L-[t4C]valine. A competition experiment was carried out with tops of green flax seedlings in which the amount of label incorporated into free and bound leucine from L-[t4C]valine was compared in the presence and absence of synthetic m.-e-isopropylmalate. It will be seen front Table I I that a competitive or inhibitory effect of isopropylmalate was evident from the specific activity of bound leucine, but not in the case of free leucine. It is of interest in this connexion that STmaSS.~IA.xaXI) CECI5 found s3qathetic DL-**-isopropylmaiate was not converted *o e-ketoisocaproic acid by yeast extracts whereas the natural e-isopropylmalate was converted. Biochim. Biophys. Acta, 7I {I963) 456--458
PERCENTAGE INCORPORATION O~" I~C ff~ ~"$~.H~I~ z~g'D~LEUCINE OF ETHANOL EXTRACTS AND HYDROLYSATES OF SORGHTu~I~~SIE~im~..II~ ~Ib"EEl~ L-~14CIVALINE ADMIN:STRATION L-rt~C Valine administered by m e t h o d i(~).. ~ i ~ r determined b y planimetric m e a s u r e m e n t of the areas of peaks from c h r o m a * o g r ~ ~¢~.=m~r~ ~md comparison with s t a n d a r d a m o u n t s of
~J~mmala:ttr~ Ami,oacid
a~c
"ml~ITi'a~a~
(°,v}
~]~lfiiiina
Leucine
"C ('!o)
L3
3.5
~.3
9. 8
Hydrolysate ["C.lValme .fiIC]Letw,ine
2.7 Valine
]'.ABLE H SPECIFIC ACTIVITIES OF FREE /kl~D ~O~7!~'~ *~:K.E'~CE~.4"E AFTER L-rI4C!VALINE ADMINISTRATION TO FLAX TOI~S ~qIW~ ~k.~k1~~ll'll~O~ DL-~X-ISOPROPYLMALATE L-[I~C~Valine administered b y ~ /~)).. Lcm~im~ separated b y paper c h r o m a t o g r a p h y s a n d determined by a n i n h y d r i n m,vtilae~P. ~ ~ t i v i t y of a d m i n i s t e r e d L-[x~C]valine w a s
~_s #=/~ote. Spe~i~ activity (m~C/~molt)
Treatment
L- ~.14C!Valine, (.o.2a , m ~ ) ) L- [14C2Valine, (o.2a//~a~)) ~ d DL-Or-isop'ro ~ y 3 " ~ ffH~ ~I~llO~tL~)
Free leucine
Boundlet~ine
33
26
33
XO
W e t h a n k D r . M. STP,ASSm~x f f ~ a g i f t O{ s y n t h e t i c D L - ~ - i s o p r o p y l m a l a t e a n d D r . E . E . Cox.,," t o r h e i p h f i , d i ~ . ~ work was supported in part by U.S. Public Health Service Grant R 5~x-
Depurtment of Biochemistry and B~p~.'~,. Urdversity o f C a l i f o r n i a , D a v i s , C ~ / ~ ((U..~_4.)) 1 p. H. ABELSON AND H. J. VO~EL, J . B ~ . C ~ . , ¢ M. STRASSMAN, L. A. Locm~, 2k. J . ~ ~ 1599. 30. 4 C. 6 M. e W.
G.W. LAURA
BUTLER* SHEN**
~ 3 (t955) 355S. W i n . aOUSE, J. A m . Chem. Sot., 78 (x956}
REISS A N D K. BLOCH, J. B ~ . C ~ m . , 2~6 ~r%55) 703 • J U N G W I R T H A N D H. E. U ~ F ~ P~oc. zi (i962) IO. STRASSMAN A N D L. !~. < ~ , ~ ~ . , , 2,~ (~962) IO. R. i~[ARTIN, W . H. C O L ~ D ~ , ~. ~ ~ W R G , R. CANTRELL, M. JACKSON A N D F. W .
DENISON, Biochira. Bio'phys. A a ~ , ~ ({a~a,'~))x ~ 7 M. BERGMANN AND W. Sama~. ~ . ~ ~,. ~2~ (~939~ 6o9. s G. H6GSTROM, A a a Chem. Sr~md.~ ~a ({a~F)); ~ 9 E. W. YEIdM AND E. C. C O C ~ , . ~ y d # , ~ ~I~5~5) 209. Received November
6th, x~o2
" U.S. Public H e a l t h Service ~ Print-Doctoral Research Fellow (FF-246). P r e s e n t address: P l a n t C h e m i s t r y I M v i ~ m , D . ~ L R _ ° l ~ m e t ~ o n North (New Zealand). "* U.S. Public H e a l t h Seawioe ~ - ~ T r ~ l ~ (2Gr x9).
Biochim. Biopha, s. Acta, 7z (x963) 456-458
SHORT COMMUNICATIONS
Although the occurrence of exchange reactions independent of and in a d d ~ c m to the reactions that lead to net uptake of amino acids a n d peptides b y bacteria complicates measurement of the kinetics of accumulation with radioactive tracers, previous conclusions t h a t transport of these substances into the cell occurs b y a n active, energy-dependent process with structural specificity characteristic off enzymic processes remain unchanged. This research was sponsored in part b y grant E-I575 from the U.S. Public HeM~h Service. F . R . L . was a National Research Fellow in the Medical Science (I957--~9)-
Department of Biochemistry, University of California, Berkeley, Calif. (U.S.A.)
FRANKLIN R. LY-~'CH° ESMOND E. SNELL
F. R. LEACHAND E. E. SNELL, Bioohim. Biophys. Acta, 34 (I959) 292. ¢ F. R. LEACHAND E. E. SNELL,J. Biol. Chem., 235 (196o) 3523. 3 E. M. LEVXN~AND S. SIMMONDS,J. Biol. Chem., 235 (196o) 29o2. 4 G. N. COHENAND H. V. RICKENBERG,Compt. Rend., 24o (1955) 2o86. BG. N. COHENAND H. V. RICKENBERG, Ann. Inst. Pasteur, 91 (I956) 693. R. J. BRITTEN,R. B. ROBERTSAND E. F. FRENCH, Proc. Natl. Acad. Sci. U.S., 41 (1955) 805G. N. COHENAND J. MONOD,Bacteviol. Rev., 2I (1957) 169. 8 E. HEINZ,J. Biol. Chem,, 21I (1954) 781. o E. HEINZAND P. M. WALSH,J. Biol. Chem., 233 (1958) 1488. ]
lo j . MANDELSTAM, Biochem. J., 69 (1958) 1o3.
* Present address: Department of Biochemistry, Oklahoma Statc Univez~ity, Stillwater. ,Ot~L Received October 18+.h, 1962 Biochim. Biophys ,4eta, 7 x (1963) 4 ~ 4 - 4 ~
SC 2 2 1 6
Leucine biosynthesis in higher plants There is considerable evidence from labelling experiments t h a t in mieroorga~is~ns leucine arises from the condensation of 2-ketoisovaleric acid (ketovaline) a n d a ~ tate 1-3. Support for such a pathway, involving the intermediate formation of propylmalate, has recently been obtained at the enzyme level in S a / m o ~ lylff~murium and Torulopsis utilis4, 5 and ,,-isopropylmalate has been isolated from ~ t m ~ e s of several microorganismsn. I n the course of an investigation of the biosynthesis off linamarin in flax, some evidence for the presence of this p a t h w a y for l e u c ~ e synthesis was obtained incidentally and the results are briefly reported here. Flax seeds (Linum usitatissimum L. var. Imperial) were germinated a n d grotto for 72 h in the dark on gauze moistened with dilute n u t r i e n t solution. They next exposed to artificial light of i n t e n s i t y approx. 2ooo ft-candles from a n descent lamp for 16 h. Uniformly labelled L-[14C]valine was administered b y of the following methods: (a) 20 seedlings selected for uniformity were placed in a 5-ml beaker conlmiml~g 5 F moles L-[14C]valine (I ~C) in ~- ml water. This was administered for 48 h ~41h aeration a n d continuous illumination. (b) The stems of 20 seedlings were cut with a sharp razor blade 2 a m b e ~ w the cotyledon leaves a n d placed in a 1-ml beaker containing 5 Vanoles x-[a~C]~ (I FC) in o.2 ml water. Successive a m o u n t s of o.i rnl water were added as during a 7-h absorption period in continuous light.
Biochim. Biophys. Aaa, ?x (i965) 4:V6.--.t$$
SHORT COMMUNICATIONS
457
The plant material was harvested, frozen in liquid nitrogen, ground to a fine powder with a mortar and pestle and extracted with IOO vol. boiling 8o o,3 (v/v) aq. ethanol for 5 rain. The solvent was evaporated in -,aczto and the residue extracted with 2 ml io % (v/v) aq. isopropanol. The extract was clarified by centrifugation if necessary and suitable aliquots were used for paper chromatography. Similar experiments were also carried out with seedlings of Sorghum vulgare var. Honey Drip. In some experiments the plant residue remaining after aq. ethanol extraction was refluxed with zo vol. 6 N HC1 for I6 h and the acid removed by repeated evaporation to d~'ness. The residue was extracted with 2 ml Io % (v/v) aq. isopropanol and suitable aliquots taken for paper chromatography. Paper chromatography in one dimension on Whatman No. I paper was carried out in each of the following solvent systems: n - b u t a n o l - p y r i d i n e - w a t e r (6o:4o:3o, v/v), methylethyl k e t o n e - a c e t o n e - w a t e r (30: lO:6, v/v), butanol-acetic a c i d - w a t e r (12o :3o: 5 o, v/v) and phenol-water (80: 20, v/v). The distribution of radioactivity was determined using a chromatogram strip scanner (Nuclear Chicago Model R IOOO). With ethanol extracts from flax seedlings the major radioactive peaks corresponded with linamarin and L-valine markers in all solvents; a smaller peak was also seen corresponding with L-leucine marker. With ethanol extracts from sorghum seedlings two peaks were seen corresponding to L-valine and L-leucine markers. Chromatograms of the acid hydrolysates from both flax and sorghum seedlings showed the presence of two radioactive peaks corresponding to L-valine and L-leucine markers in all solvent systems. The identity of the suspected [l*C!leucine in flax seedlings was definitely established by the preparation of the naphthalene-fl-sulphonyl (nasyl) derivativeL Partial purification was first accomplished by successive paper chromatography and elution using b u t a n o l - p y r i d i n e - w a t e r and methylethyl ketone-acetone-water. L-Leucine (5° nag) was added and the nasvl derivative prepared and dried. A suitable sample was dissolved in a known wflume of ethanol and aliquots were evaporated on planchettes and counted at infinite thinness in a gas-flow counter (NuclearChicago Model x8IB). The counting procedure was repeated through 3 recrystallisations with the same specific activity (29o counts/rain/rag derivative) being observed throughout. Table I shows the percentages of 14C found in free and bound leucine in an experiment with sorghum seedlings. The low ratio of 2.7 for the 14C in bound valine and leucine shows that leucine biosynthesis by a pathway of the type proposed represents an important source of leucine in this tissue. Attempts to demonstrate the participation of ~,-isopropylmalate as an intermediate were inconclusive. [lac~Isopropylmalate could not be detected in aq. ethanol extracts of seedlings administered L-[t4C]valine. A competition experiment was carried out with tops of green flax seedlings in which the amount of label incorporated into free and bound leucine from L-[t4C]valine was compared in the presence and absence of synthetic m.-e-isopropylmalate. It will be seen front Table I I that a competitive or inhibitory effect of isopropylmalate was evident from the specific activity of bound leucine, but not in the case of free leucine. It is of interest in this connexion that STmaSS.~IA.xaXI) CECI5 found s3qathetic DL-**-isopropylmaiate was not converted *o e-ketoisocaproic acid by yeast extracts whereas the natural e-isopropylmalate was converted. Biochim. Biophys. Acta, 7I {I963) 456--458
PERCENTAGE INCORPORATION O~" I~C ff~ ~"$~.H~I~ z~g'D~LEUCINE OF ETHANOL EXTRACTS AND HYDROLYSATES OF SORGHTu~I~~SIE~im~..II~ ~Ib"EEl~ L-~14CIVALINE ADMIN:STRATION L-rt~C Valine administered by m e t h o d i(~).. ~ i ~ r determined b y planimetric m e a s u r e m e n t of the areas of peaks from c h r o m a * o g r ~ ~¢~.=m~r~ ~md comparison with s t a n d a r d a m o u n t s of
~J~mmala:ttr~ Ami,oacid
a~c
"ml~ITi'a~a~
(°,v}
~]~lfiiiina
Leucine
"C ('!o)
L3
3.5
~.3
9. 8
Hydrolysate ["C.lValme .fiIC]Letw,ine
2.7 Valine
]'.ABLE H SPECIFIC ACTIVITIES OF FREE /kl~D ~O~7!~'~ *~:K.E'~CE~.4"E AFTER L-rI4C!VALINE ADMINISTRATION TO FLAX TOI~S ~qIW~ ~k.~k1~~ll'll~O~ DL-~X-ISOPROPYLMALATE L-[I~C~Valine administered b y ~ /~)).. Lcm~im~ separated b y paper c h r o m a t o g r a p h y s a n d determined by a n i n h y d r i n m,vtilae~P. ~ ~ t i v i t y of a d m i n i s t e r e d L-[x~C]valine w a s
~_s #=/~ote. Spe~i~ activity (m~C/~molt)
Treatment
L- ~.14C!Valine, (.o.2a , m ~ ) ) L- [14C2Valine, (o.2a//~a~)) ~ d DL-Or-isop'ro ~ y 3 " ~ ffH~ ~I~llO~tL~)
Free leucine
Boundlet~ine
33
26
33
XO
W e t h a n k D r . M. STP,ASSm~x f f ~ a g i f t O{ s y n t h e t i c D L - ~ - i s o p r o p y l m a l a t e a n d D r . E . E . Cox.,," t o r h e i p h f i , d i ~ . ~ work was supported in part by U.S. Public Health Service Grant R 5~x-
Depurtment of Biochemistry and B~p~.'~,. Urdversity o f C a l i f o r n i a , D a v i s , C ~ / ~ ((U..~_4.)) 1 p. H. ABELSON AND H. J. VO~EL, J . B ~ . C ~ . , ¢ M. STRASSMAN, L. A. Locm~, 2k. J . ~ ~ 1599. 30. 4 C. 6 M. e W.
G.W. LAURA
BUTLER* SHEN**
~ 3 (t955) 355S. W i n . aOUSE, J. A m . Chem. Sot., 78 (x956}
REISS A N D K. BLOCH, J. B ~ . C ~ m . , 2~6 ~r%55) 703 • J U N G W I R T H A N D H. E. U ~ F ~ P~oc. zi (i962) IO. STRASSMAN A N D L. !~. < ~ , ~ ~ . , , 2,~ (~962) IO. R. i~[ARTIN, W . H. C O L ~ D ~ , ~. ~ ~ W R G , R. CANTRELL, M. JACKSON A N D F. W .
DENISON, Biochira. Bio'phys. A a ~ , ~ ({a~a,'~))x ~ 7 M. BERGMANN AND W. Sama~. ~ . ~ ~,. ~2~ (~939~ 6o9. s G. H6GSTROM, A a a Chem. Sr~md.~ ~a ({a~F)); ~ 9 E. W. YEIdM AND E. C. C O C ~ , . ~ y d # , ~ ~I~5~5) 209. Received November
6th, x~o2
" U.S. Public H e a l t h Service ~ Print-Doctoral Research Fellow (FF-246). P r e s e n t address: P l a n t C h e m i s t r y I M v i ~ m , D . ~ L R _ ° l ~ m e t ~ o n North (New Zealand). "* U.S. Public H e a l t h Seawioe ~ - ~ T r ~ l ~ (2Gr x9).
Biochim. Biopha, s. Acta, 7z (x963) 456-458