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Xylose assimilation in higher plants
582
PRELIMINARY NOTES
VOL. 36 (1959)
Xylose assimilation in higher plants* The curious fact that L-arabinose is incorporated by higher plants into pentosans without substantial cleavage of the carbon chain whereas D-xylose experiences considerable rearrangement has strongly suggested that the primary metabolic reaction of these pentoses are different. Thus NEISHz has inferred from such studies that D-xylose passes through the pentose phosphate cycle, and is hence subject to extensive cleavage and recombination prior to its combination with uridine nucleotide while L-arabinose, following phosphorylation, is directly converted to the uridine derivative. The suggested pathways have been largely substantiated by HASSlD et al. who have isolated and identified many of the intermediate compounds and enzymic steps leading from free pentoses to pentosans. A recent summary of this work reports the discovery of L-arabinokinase in higher plants 2, the last link required to complete the pathway from L-arabinose to the pentosans. The primary step in the introduction of D-xylose into the metabolic stream of the plant, however, remains to be elucidated. We wish to note here the occurrence in a tissue of a higher plant, specifically corn pollen, an enzyme system which converts D-xylose to D-xylulose. In a typical experiment, IOO mg corn pollen was homogenized in 2.0 ml o.oi M phosphate buffer, pH 6.5, and incubated with 500,000 counts/min of D-[14C]xylose (2.35.IO e counts/min/mg, counted at 24 % efficiency) for 6 h at 25 °. The reaction was stopped by adding 4 vol. 99.5 % ethanol and heating for 2 min at 80 °. The clear supernatant was chromatographed on filter paper with ethyl a c e t a t e - p y r i d i n e - w a t e r (8:2:1) 8. Two radioactive areas were detected, by radioautography and by a recording scanner employing a windowless Geiger tube (Forro Radiochromatograph). The more active region corresponded to unreacted xylose, the other to xylulose. The suspected xylulose, following elution, appeared to be identical on co-chromatography with authentic xylulose but different from ribulose, all of the aldopentoses, the pentitols and the common hexoses in several different solvents. The eluted material also resembled authentic xylulose in the rate of color development in the DISCHE-BORENFREUND test 4. This substance on reduction with NaBH4 yieldeO a material which exhibited the chromatographic behavior of authentic xylitol. (Apparently no appreciable amount of the diastereisomer, D-arabitol was formed.) Under the conditions described above a 3.0--4. 3 % conversion of D-xylose to xylulose was obtained; the remainder of the D-xylose was recovered unchanged. Exclusion of air did not affect the reaction. When adenosine triphosphate (I.I. lO -3 M) was present in the reaction mixture, no radioactivity appeared on the chromatogram as xylulose but a corresponding number of counts were found close to the origin. In the solvent employed sugar phosphates move to this region. The isomerization, unlike that noted in mammalian tissues 5, did not involve the intermediate formation of free xylitol and was independent of the presence of di- and triphosphospyridine nucleotides. The reaction resembles therefore that noted in Lactobacillus pe~tosus 6. The isomerase activity can be recovered as a pellet after centrifuging the homogenate at IOO,OOO × g for 20 min but it is not sedimented at io,ooo × g in 15 min. Xylose isomerase in cooperation with xylulose kinase (whose presence may be inferred from the effect of adenosine triphosphate) can serve to convert xylose to * Journal Paper No. i489 of the Purdue University Agricultural Experiment Station.
VOL. 36 (1959)
PRELIMINARY NOTES
583
xylulose-5-phosphate, which can then be metabolized by the pentose phosphate enzymes, whose presence in higher plants is already well known.
Department o/ Biochemistry, Purdue University, La/ayette, Ind. (U.S.A.)
M. H. PUBOLS B. AXELROD
A. C. NEISH, Can. J. Biochem. and Physiol., 36 (1958) 187. 2 W. Z. HASSID, E. F. NEUFELD AND D. S. FEINGOLD, Proc. Natl. Acad. Sci,. U.S., 45 (1959) 905. 3 K. T. WILLIAMS AND A. BEVENUE, Cereal Chem., 28 (1951) 416. 4 Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., 192 (I95 I) 583 . 5 S. HOLLMAN AND O. TOUSTER, J. Biol. Chem., 225 (1957) 87. 6 S. MITSUHASHI AND J. O. LAMPEN, J. Biol. Chem., 204 (1953) i O l i . z
Received September 4th, 1959
Intracerebral incorporation of [2-14C] mevalonic acid into adult rat brain squalene and cholesterol When sodium [2-14C]acetate was injected intracerebrally into rats the non-saponifiable fractions of the brains contained 14C in a non-polar form, in addition to that incorporated into cholesterol1. Subsequent investigation showed the non-polar material from adult rats contained 14C-labeled squalene since: (I) on dilution with squalene and preparation of squalene hexahydrochloride the 14(: could not be removed by repeated crystallization, and (2) re-injection of the labeled non-polar fraction into a young rat produced 14C-labeled cholesterol. The metabolism of squalene and cholesterol in the adult rat brain was studied b y injecting intracerebrally 0.048 tzmole sodium [2-z4C]mevalonate* (in o.I ml of aqueous solution) into each of a number of female rats weighing 290-350 g by a modification of the method previously described 2. After sacrifice by decapitation the whole brain was quickly weighed and digested in alcoholic KOH under N v A nonsaponifiable fraction was obtained in petroleum ether S. This was chromatographed on alumina, the eluate yielding a crude "non-polar" fraction, after which the columns were eluted with acetone-ethyl ether ( I : I ) to obtain a crude cholesterol fraction. Each non-polar fraction was diluted with IOO.Omg shark squalene and converted to the hexahydrochlorides3. The precipitated hydrochlorides were washed with acetone then recrystallized from acetone to constant specific activity. The crude cholesterol fractions were converted to the dibromides 4 either directly or after conversion to free sterol via the digitonide (14C values the same by either method). Radioactive determinations were performed on infinitely thin samples with a windowless gas-flow counter. The relationship between the crude squalene and crude cholesterol fractions is shown in Fig. I. The purified squalene hexahydrochlorides and cholesterol dibromides follow analogous curves (Fig. 2). The squalene and cholesterol curves bear the same relationship to liver squalene and cholesterol shown in another type of experiment 5, * T h e s o d i u m [2-14C]mevalonate w a s p r e p a r e d f r o m t h e N , N - d i b e n z y l e t h y l e n e d i a m i n e salt p u r c h a s e d f r o m I s o t o p e s Specialities Inc., B u r b a n k , Calif., a n d h a d a specific a c t i v i t y of 2.26 m C ] m m o l e as t h e t o t a l salt. T h e N a salt w a s diluted w i t h o.067 M p h o s p h a t e buffer, p H 7.4, so t h a t o.I m l c o n t a i n e d 0 . o 4 8 / , m o l e m e v a l o n i c acid.
PRELIMINARY NOTES
VOL. 36 (1959)
Xylose assimilation in higher plants* The curious fact that L-arabinose is incorporated by higher plants into pentosans without substantial cleavage of the carbon chain whereas D-xylose experiences considerable rearrangement has strongly suggested that the primary metabolic reaction of these pentoses are different. Thus NEISHz has inferred from such studies that D-xylose passes through the pentose phosphate cycle, and is hence subject to extensive cleavage and recombination prior to its combination with uridine nucleotide while L-arabinose, following phosphorylation, is directly converted to the uridine derivative. The suggested pathways have been largely substantiated by HASSlD et al. who have isolated and identified many of the intermediate compounds and enzymic steps leading from free pentoses to pentosans. A recent summary of this work reports the discovery of L-arabinokinase in higher plants 2, the last link required to complete the pathway from L-arabinose to the pentosans. The primary step in the introduction of D-xylose into the metabolic stream of the plant, however, remains to be elucidated. We wish to note here the occurrence in a tissue of a higher plant, specifically corn pollen, an enzyme system which converts D-xylose to D-xylulose. In a typical experiment, IOO mg corn pollen was homogenized in 2.0 ml o.oi M phosphate buffer, pH 6.5, and incubated with 500,000 counts/min of D-[14C]xylose (2.35.IO e counts/min/mg, counted at 24 % efficiency) for 6 h at 25 °. The reaction was stopped by adding 4 vol. 99.5 % ethanol and heating for 2 min at 80 °. The clear supernatant was chromatographed on filter paper with ethyl a c e t a t e - p y r i d i n e - w a t e r (8:2:1) 8. Two radioactive areas were detected, by radioautography and by a recording scanner employing a windowless Geiger tube (Forro Radiochromatograph). The more active region corresponded to unreacted xylose, the other to xylulose. The suspected xylulose, following elution, appeared to be identical on co-chromatography with authentic xylulose but different from ribulose, all of the aldopentoses, the pentitols and the common hexoses in several different solvents. The eluted material also resembled authentic xylulose in the rate of color development in the DISCHE-BORENFREUND test 4. This substance on reduction with NaBH4 yieldeO a material which exhibited the chromatographic behavior of authentic xylitol. (Apparently no appreciable amount of the diastereisomer, D-arabitol was formed.) Under the conditions described above a 3.0--4. 3 % conversion of D-xylose to xylulose was obtained; the remainder of the D-xylose was recovered unchanged. Exclusion of air did not affect the reaction. When adenosine triphosphate (I.I. lO -3 M) was present in the reaction mixture, no radioactivity appeared on the chromatogram as xylulose but a corresponding number of counts were found close to the origin. In the solvent employed sugar phosphates move to this region. The isomerization, unlike that noted in mammalian tissues 5, did not involve the intermediate formation of free xylitol and was independent of the presence of di- and triphosphospyridine nucleotides. The reaction resembles therefore that noted in Lactobacillus pe~tosus 6. The isomerase activity can be recovered as a pellet after centrifuging the homogenate at IOO,OOO × g for 20 min but it is not sedimented at io,ooo × g in 15 min. Xylose isomerase in cooperation with xylulose kinase (whose presence may be inferred from the effect of adenosine triphosphate) can serve to convert xylose to * Journal Paper No. i489 of the Purdue University Agricultural Experiment Station.
VOL. 36 (1959)
PRELIMINARY NOTES
583
xylulose-5-phosphate, which can then be metabolized by the pentose phosphate enzymes, whose presence in higher plants is already well known.
Department o/ Biochemistry, Purdue University, La/ayette, Ind. (U.S.A.)
M. H. PUBOLS B. AXELROD
A. C. NEISH, Can. J. Biochem. and Physiol., 36 (1958) 187. 2 W. Z. HASSID, E. F. NEUFELD AND D. S. FEINGOLD, Proc. Natl. Acad. Sci,. U.S., 45 (1959) 905. 3 K. T. WILLIAMS AND A. BEVENUE, Cereal Chem., 28 (1951) 416. 4 Z. DlSCHE AND E. BORENFREUND, J. Biol. Chem., 192 (I95 I) 583 . 5 S. HOLLMAN AND O. TOUSTER, J. Biol. Chem., 225 (1957) 87. 6 S. MITSUHASHI AND J. O. LAMPEN, J. Biol. Chem., 204 (1953) i O l i . z
Received September 4th, 1959
Intracerebral incorporation of [2-14C] mevalonic acid into adult rat brain squalene and cholesterol When sodium [2-14C]acetate was injected intracerebrally into rats the non-saponifiable fractions of the brains contained 14C in a non-polar form, in addition to that incorporated into cholesterol1. Subsequent investigation showed the non-polar material from adult rats contained 14C-labeled squalene since: (I) on dilution with squalene and preparation of squalene hexahydrochloride the 14(: could not be removed by repeated crystallization, and (2) re-injection of the labeled non-polar fraction into a young rat produced 14C-labeled cholesterol. The metabolism of squalene and cholesterol in the adult rat brain was studied b y injecting intracerebrally 0.048 tzmole sodium [2-z4C]mevalonate* (in o.I ml of aqueous solution) into each of a number of female rats weighing 290-350 g by a modification of the method previously described 2. After sacrifice by decapitation the whole brain was quickly weighed and digested in alcoholic KOH under N v A nonsaponifiable fraction was obtained in petroleum ether S. This was chromatographed on alumina, the eluate yielding a crude "non-polar" fraction, after which the columns were eluted with acetone-ethyl ether ( I : I ) to obtain a crude cholesterol fraction. Each non-polar fraction was diluted with IOO.Omg shark squalene and converted to the hexahydrochlorides3. The precipitated hydrochlorides were washed with acetone then recrystallized from acetone to constant specific activity. The crude cholesterol fractions were converted to the dibromides 4 either directly or after conversion to free sterol via the digitonide (14C values the same by either method). Radioactive determinations were performed on infinitely thin samples with a windowless gas-flow counter. The relationship between the crude squalene and crude cholesterol fractions is shown in Fig. I. The purified squalene hexahydrochlorides and cholesterol dibromides follow analogous curves (Fig. 2). The squalene and cholesterol curves bear the same relationship to liver squalene and cholesterol shown in another type of experiment 5, * T h e s o d i u m [2-14C]mevalonate w a s p r e p a r e d f r o m t h e N , N - d i b e n z y l e t h y l e n e d i a m i n e salt p u r c h a s e d f r o m I s o t o p e s Specialities Inc., B u r b a n k , Calif., a n d h a d a specific a c t i v i t y of 2.26 m C ] m m o l e as t h e t o t a l salt. T h e N a salt w a s diluted w i t h o.067 M p h o s p h a t e buffer, p H 7.4, so t h a t o.I m l c o n t a i n e d 0 . o 4 8 / , m o l e m e v a l o n i c acid.