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Specific radioactivities of carotenes synthesized from C14-labeled terpenol pyrophosphates by isolated tomato plastids
ARCHIVES
OF
Specific
BIOCHEMISTRY
AND
Radioactivities
Terpenol
the Radioisotope
167-170 (1963)
I@),
of Carotenes
Pyrophosphates DOKALD
From
BIOPHYSICS
Synthesized
by Isolated
A. BEELER
AND
JOHN
from
Tomato
Piastids’
W. PORTER
Unit, Veterans Administration Hospital; and the Department Chemistry, University of Wisconsin, Madison, Wisconsin Received
October
C14-Labeled
of Physiological
23, 1962
Specific radioactivities were determined for carotenes biosynthesized from Cialabeled terpenol pyrophosphates by isolated tomato plastids. A decline in specific radioactivities of the acyclic carotenes was obtained with an increase in unsaturation of the carotene (i.e., phytoene to lycopene), which is consistent with the proposal of Porter and Anderson of sequential desaturation of phytoene as the pathway for the biosynthesis of lycopene. The specific radioactivities obtained for the cyclic carotene @-carotene were higher in some samples than those for lycopene, thereby indicating that some of the o-carotene of tomatoes is synthesized through cyclization of a precursor other than lycopene. A suggested precursor for this cyclization is neurosporene. INTRODUCTION
Proof was presented in previous studies that mevalonic acid-Z-C’* is incorporated into the carotenes of ripening tomatoes (1) and that CMabeled terpenol pyrophosphates are incorporated into carotenes by isolated tomato plastids (2). In each of these studies radioactive contaminants were separated from the carotenes through a series of purification steps. The demonstration of the incorporation of (Ylabeled terpenol pyrophosphates into carotenes by isolated tomato plastids indicates that each of these compounds arises from a common source, probably geranyl geranyl pyrophosphate. Furthermore, this result, when coupled with a knowledge of the structures of the acyclic carotenes, suggests that these carotenes arise through sequential desaturation (3). In
the
present
study
V-labeled
terpenol
pyrophosphates of high specific radioactivity (5.5 X lo6 and 6.7 X lo6 counts/ min./pmole of farnesyl pyrophosphate)
were synthesized and then incubated with isolated tomato plastids. The carotenes of the plastids were isolated without the addition of carrier, and the quantity and the radioactivity of each carotene (phytoene, phytofluene, {-carotene, and neurosporene) were determined at each step of purification. The final step involved gas-liquid chromatography of the hydrogenated acyclic carotenes (lycopersane). Specific radioactivities were calculated from the quantity of carotene before hydrogenation and the quantity of radioactivity eluted with the lycopersane peak on gas-liquid chromatography. Lycopene and @carotene were crystallized to constant specific radioactivity. Aliquots of the crystals were catalytically hydrogenated at each re‘crystallization before determinations were made of radioactivity. The specific radioactivities obtained for the carotenes provide further evidence in support of the proposal of Porter and Anderson (3) on the pathway of the biosynthesis of carotenes. EXPERIMENTAL
1 This work a-as supported by a research grant -4.1383 from the National Institute of Arthritis and Metabolic Diseases of the NationaI Institutes of Health, U. S. Public Health Service.
Many of the materials and methods used in this study were reported in previous publications (1, 2). 167
168
BEELER
AND
MATERIALS The procurement of tomato fruits and rat livers was reported previously (2). Mevalonic acid-2-Cl4 was obtained as either the lactone or the DBED2 salt from the Volk Radiochemical Co. A stock solution of the lactone was prepared through treatment with excess KOH, followed by careful neutralization with acid to pH 8.0. The DBED salt, was dissolved in water and used without further treatment. ATP and TPN were obtained from the Sigma Chemical Co., and BAL was obtained from the Aldrich Chemical Co. Solvents and adsorbents and methods of purification of the solvents did not differ from those used previously (1, 2). SE-30 and Chromosorb W were obtained from Wilkens Instrument & Research, Inc. Lycopersane and squalane, used as reference standards for gas-liquid chromatogwere prepared as reported previously raphy,
(2). METHODS ENZYMIC
SYXTHESIS OF CAROTENES
Rat liver enzymes and tomato plastids were prepared by the methods reported earlier (2). Terpenol pyrophosphates were synthesized by a system containing ATP, 10 rmoles; BAL, 1.6 pmoles; MgCl2, 6 rmoles; potassium phosphate buffer, pH 7.0,50 pmoles; rat liver enzymes [40-60 fraction of Witting and Porter (4)], 10 mg. protein; and mevalonic acid-2-C14. In Expt. 1, 1 pmole substrate (2.23 X 106 counts/min./pmole) was added to each incubation mixture, and in Expt. 2, 0.8 rmole (1.84 X 10” counts/min./lmole) and 30 pmoles KF were added. All incubations were 1 ml. in volume. The enzymic reactions were stopped by heating at 70°C. for 2 min., after 90 min. of incubation at 37°C. One milliliter of a tomato plastid preparation (2) and 2 rmoles TPN were then added, and the incubation was continued under nitrogen at 25°C. for 16-18 hr. Eight (Expt. 1) and 10 (Expt. 2) individual incubations were combined for determinations of specific radioactivities of the carotenes.
SEPARATION AND PURIFICATION OF THE CAROTENES Enzyme activity in the incubation mixture was stopped by the addition of an equal volume of 10% alcoholic KOH containing 2 mg. hydro2 The following abbreviations are used: DBED salt, the dibenzylethylenediamine salt of mevaionic acid; ATP, adenosine triphosphate; TPN, triphosphopyridine nucleotide; BAL, 2,3-dimercaptopropanol; PPO, 2,5diphenyloxazole.
PORTER quinone/ml. Saponification was effected at 70°C. for 30 min. and, on cooling, carotenes were extracted with petroleum ether. The extract was then washed thoroughly with water, dried, and chromatographed on a 1.8 X 10 cm. column of alumina. Increasing percentages of ethyl ether (O-50’%) were used to elute phytoene, phytofluene, and p-carotene from the column. The remaining carotenes were removed from the column with 10% ethanol in petroleum ether. After the ethanol was washed from the petroleum ether, the solution was dried and then chromatographed on MgO-Super-Cel (1). The chromatogram was developed with increasing concentrations of acetone in petroleum ether, and <-carotene was elutedfrom thecolumn. r-carotene, neurosporene, and lycopene were sectioned from the column and then eluted from the MgO with 10% ethanol in petroleum ether. After the ethanol was washed from the petroleum ether, the solutions of r-carotene and neurosporene were dried and chromatographed on Ca(OH)n-Super-Cel. Each of the carotenes, except lycopene and p-carotene which were crystallized (1) to constant specific radioactivity, was rechromatographed until spectrally pure. Where possible, spectral analyses (Process and Instruments recording spectrophotometer) and determinations of radioactivity (Packard liquid-scintillation spectrometer) were made on successive eluates of a carotene. In other cases the carotenes were sectioned from the column and analyzed spectrally, and the Cl4 content was determined. All carotenes, except phytoene and phytofluene, were hydrogenated catalytically before determinations were made of radioactivity. Aliquots of a carotene with similar specific radioactivities were combined and hydrogenated catalytically (l), and the reduction product was chromatographed on alumina. The reduced compound was eluted from the column with petroleum ether and assayed for radioactivity, and then it was subjected to gas-liquid chromatography on a 6 ft. X 6 mm. I.D. column of 5% SE-30 on Chromosorb W. A temperature of 270°C. and an argon flow rate of 100 ml./min. was used. Eluates from the gas-liquid chromatograph were trapped on glass wool, eluted with toluene (containing 0.150/, PPO), and then assayed for radioactivity with a Packard liquidscintillation spectrometer. RESULTS
AND
DISCUSSION
The specific radioactivities of the acyclic carotenes (Table I) decline with an increase in unsaturation (i.e., phytoene to lycopene). This decline in specific radioactivities may be taken as evidence in
RADIOACTIVITIES TABLE
I
SPECIFIC RADIOACTIVITIES OF CAROTENES SYNTHESIZED BT~ ISOLATED TOMATO PLASTIDS FROMW-LABELED TERPENOLPYROPHOSPHATES 1
Experiment
1
coNnts/’ min.img. Phytoene Phytofluene I-Carotene Neurosporene Lycopene p-Carotene
mL?21s/ min./mg.
ms
0.764 215,35@ 12 lOWa * 0.058 15:650~~ bI 0.094 4,500” 0.032
489,600~ 1t%,700* 149,800h 57,800h 1,200c 2,480”
Experiment 2
1.020 0.098
4OC 140”
2.100 0.340
a Determined by spectraphotometric assay after elution from an aluminum oxide chromatogram and by determination of the radiochemical purity of the reduced compound on gas-liquid chromatography. b Determined by spectrophotometric assay after elution from an aluminum oxide chromatogram and/or a magnesium oxide-Super-Cel column and by a determination of the radioactivity coincident with lycopersane on reduction and gas-liquid chromatography. c Determined through crystallization to constant specific radioactivity. d Determined by spectrophotometric assay after elution from an aluminum oxide chromatogram and a Ca(OH)n-Super-Cel column and by a determination of radioactivity coincident with lycopersane after reduction and chromatography on alumina. The absolute value for the specific radioactivity of this compound may be lower than the value given since the quantity of radioactivity present was insufficient for an analysis by gasliquid chromatography.
169
OF CAROTENES
of the intermediate phytofluene remains closely associated with the enzyme and does not enter the pool of free phytofluene before conversion to [-carotene. The remainder would be lost from the enzyme surface, and it would enter the phytofluene pool. Whether this is a correct interpretation of the results must await additional experimentation on the enzymic conversion of phytoene to c-carotene. The specific radioactivities of neurosporene and lycopene are in the order expected on the basis of the proposed conversion: {-carotene -+ neurosporene -+ lycopene . The specific radioactivities of lycopene and p-carotene (Table I) indicate that some p-carotene must arise in some tomato selections from a precursor other than lycopene. However, other results reported from our laboratory (3), and Table II, indicate that ,&carotene does arise from lycopene in some tomato selections. Possibly the selection of a system for synthesizing the carotenes is of paramount importance in the formation of p-carotene. Other information which has a bearing on this question has been supplied by Decker and Uehleke (8) and by Davies (9). Decker and Uehleke (8) reported the conversion of lycopene to p-carotene by chloroplasts and the reverse reaction by parenchymatous tissue of red tomatoes. Davies (9) reported the synthesis of y-carotene by Rhixophlyctis rosea by a pathway which did not involve TABLE
support of the proposal of Porter and Lincoln (5) as modified by Porter and Anderson (3), that the biosynthesis of lycopene proceeds by sequential desaturation of phytoene. Further support for this conclusion has been provided recently by the finding that phytoene is converted to phytofluene by isolated tomato plastids (6) and to d-carotene by cell-free extracts of a mutant of Staphylococcus aureus (7). The similar specific radioactivities of and r-carotene (Table I) phytofluene might be interpreted as evidence that one enzyme is involved in the conversion of phytoene to [-carotene (3). If so, a portion
SPECIFIC
II
RADIOACTIVITIES OF LYCOPENE ~-CAROTENE BIOSYNTHESIZED BI-
AND
TOMATO PREPARATIONS system
LyCOpeIltT p-carotene ClJUfltS/?lliVZ./Mg. COl~lZlSjl~li~./I~Zg.
Tomato
Tomato
fruita
plastids*
2140 5530 2210 3110 1200 40
c Mevalonic acid-2-C14 was injected ing tomato fruits [see Ref. (l)]. * See Table I.
780 3325
1470 G-100 2480
140 int,o ripen-
170
BEELER
AND PORTER
the intermediate formation of lycopene. The simplest explanation for the formation of @-carotene, which is consistent with all the existing data, is that p-carotene may be formed from lycopene, and also from neurosporene via p-zeacarotene and y-carotene (3). Accurate determinations of the specific radioactivity of y-carotene were not made because of the small quantity of this compound, the presence of appreciable quantities of contaminating radioactivity, and the presence of small amounts of radioactivity in the -y-carotene. However, the specific radioactivity of y-carotene (determined after reduction and chromatography on alumina) was less than that of neurosporene. It was reported in an earlier publication (1) that radioactive contaminants accompany the carotenes during purification and that great care must be exercised to remove these contaminants. Some of these contaminants were also present in the isolated system used in these experiments. In Expt. 1, 78,500 counts/min. (124,500,OOO counts/min./mg.) was found in association with neurosporene after this compound was purified through chromatography on MgO-Super-Cel, Ca(OH)2-Super-Cel, and then catalytically hydrogenated. Chromatography of the reduced product on a 1.8 X 7 cm. column of alumina reduced the 14,000 counts/min. radioactivity to (22,200,OOO counts/min./mg.). Most of this radioactivity appeared in the solvent front on gas-liquid chromatography on SE-30
at 270°C. The remainder was coincident with the lycopersane eluate from t,he column. The latter was used as a true measure of the radioactivity present in neurosporene. These results indicate the extreme care that must be taken to insure proof of the incorporation of radioactivity into a carotene. Most of the radioactive contaminants of the carotenes have not been identified. One that has been identified (through gas-liquid chromatography) is farnesol. This compound chromatographs slightly before {-carotene on a MgO-Super-Cel column. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Marcia L. Hipke and Mr. William J. Schelble for their technical assistance. REFERENCES 1. ANDERSON, D. G., NORGARD, D. W., AND PORTER, J. W., Arch. Biochem. Biophys. 33,
68 (1960). 2. ANDERSON, D. G., AND PORTER, J. W., Biochem. Biophys. 97, 509 (1962). 3. PORTER, J. W., AND ANDERSON, D. G., Biochem. Biophys. 97, 520 (1962). 4. WITTING, L. A., AND PORTER, J. W., J. Chem. 234, 2841 (1959). 5. PORTER, J. W., AND LINCOLN, R. E.,
Arch. Arch. Biol. Arch.
Biochem. 27,390 (1950). 6. BEELER, D. A., AND PORTER, J. W. Biochem. Biophys. Research Communs. 8, 367 (1962). et Biophys. Acta 60, 7. SUZUE, G., Biochim. 593 (1961). 8. DECKER, K., AND UEHLEKE, H., 2. physiol. Chem. 323, 61 (1961). 9. DAVIES, B. H., Biochem. J. 80, 48P (1961).
OF
Specific
BIOCHEMISTRY
AND
Radioactivities
Terpenol
the Radioisotope
167-170 (1963)
I@),
of Carotenes
Pyrophosphates DOKALD
From
BIOPHYSICS
Synthesized
by Isolated
A. BEELER
AND
JOHN
from
Tomato
Piastids’
W. PORTER
Unit, Veterans Administration Hospital; and the Department Chemistry, University of Wisconsin, Madison, Wisconsin Received
October
C14-Labeled
of Physiological
23, 1962
Specific radioactivities were determined for carotenes biosynthesized from Cialabeled terpenol pyrophosphates by isolated tomato plastids. A decline in specific radioactivities of the acyclic carotenes was obtained with an increase in unsaturation of the carotene (i.e., phytoene to lycopene), which is consistent with the proposal of Porter and Anderson of sequential desaturation of phytoene as the pathway for the biosynthesis of lycopene. The specific radioactivities obtained for the cyclic carotene @-carotene were higher in some samples than those for lycopene, thereby indicating that some of the o-carotene of tomatoes is synthesized through cyclization of a precursor other than lycopene. A suggested precursor for this cyclization is neurosporene. INTRODUCTION
Proof was presented in previous studies that mevalonic acid-Z-C’* is incorporated into the carotenes of ripening tomatoes (1) and that CMabeled terpenol pyrophosphates are incorporated into carotenes by isolated tomato plastids (2). In each of these studies radioactive contaminants were separated from the carotenes through a series of purification steps. The demonstration of the incorporation of (Ylabeled terpenol pyrophosphates into carotenes by isolated tomato plastids indicates that each of these compounds arises from a common source, probably geranyl geranyl pyrophosphate. Furthermore, this result, when coupled with a knowledge of the structures of the acyclic carotenes, suggests that these carotenes arise through sequential desaturation (3). In
the
present
study
V-labeled
terpenol
pyrophosphates of high specific radioactivity (5.5 X lo6 and 6.7 X lo6 counts/ min./pmole of farnesyl pyrophosphate)
were synthesized and then incubated with isolated tomato plastids. The carotenes of the plastids were isolated without the addition of carrier, and the quantity and the radioactivity of each carotene (phytoene, phytofluene, {-carotene, and neurosporene) were determined at each step of purification. The final step involved gas-liquid chromatography of the hydrogenated acyclic carotenes (lycopersane). Specific radioactivities were calculated from the quantity of carotene before hydrogenation and the quantity of radioactivity eluted with the lycopersane peak on gas-liquid chromatography. Lycopene and @carotene were crystallized to constant specific radioactivity. Aliquots of the crystals were catalytically hydrogenated at each re‘crystallization before determinations were made of radioactivity. The specific radioactivities obtained for the carotenes provide further evidence in support of the proposal of Porter and Anderson (3) on the pathway of the biosynthesis of carotenes. EXPERIMENTAL
1 This work a-as supported by a research grant -4.1383 from the National Institute of Arthritis and Metabolic Diseases of the NationaI Institutes of Health, U. S. Public Health Service.
Many of the materials and methods used in this study were reported in previous publications (1, 2). 167
168
BEELER
AND
MATERIALS The procurement of tomato fruits and rat livers was reported previously (2). Mevalonic acid-2-Cl4 was obtained as either the lactone or the DBED2 salt from the Volk Radiochemical Co. A stock solution of the lactone was prepared through treatment with excess KOH, followed by careful neutralization with acid to pH 8.0. The DBED salt, was dissolved in water and used without further treatment. ATP and TPN were obtained from the Sigma Chemical Co., and BAL was obtained from the Aldrich Chemical Co. Solvents and adsorbents and methods of purification of the solvents did not differ from those used previously (1, 2). SE-30 and Chromosorb W were obtained from Wilkens Instrument & Research, Inc. Lycopersane and squalane, used as reference standards for gas-liquid chromatogwere prepared as reported previously raphy,
(2). METHODS ENZYMIC
SYXTHESIS OF CAROTENES
Rat liver enzymes and tomato plastids were prepared by the methods reported earlier (2). Terpenol pyrophosphates were synthesized by a system containing ATP, 10 rmoles; BAL, 1.6 pmoles; MgCl2, 6 rmoles; potassium phosphate buffer, pH 7.0,50 pmoles; rat liver enzymes [40-60 fraction of Witting and Porter (4)], 10 mg. protein; and mevalonic acid-2-C14. In Expt. 1, 1 pmole substrate (2.23 X 106 counts/min./pmole) was added to each incubation mixture, and in Expt. 2, 0.8 rmole (1.84 X 10” counts/min./lmole) and 30 pmoles KF were added. All incubations were 1 ml. in volume. The enzymic reactions were stopped by heating at 70°C. for 2 min., after 90 min. of incubation at 37°C. One milliliter of a tomato plastid preparation (2) and 2 rmoles TPN were then added, and the incubation was continued under nitrogen at 25°C. for 16-18 hr. Eight (Expt. 1) and 10 (Expt. 2) individual incubations were combined for determinations of specific radioactivities of the carotenes.
SEPARATION AND PURIFICATION OF THE CAROTENES Enzyme activity in the incubation mixture was stopped by the addition of an equal volume of 10% alcoholic KOH containing 2 mg. hydro2 The following abbreviations are used: DBED salt, the dibenzylethylenediamine salt of mevaionic acid; ATP, adenosine triphosphate; TPN, triphosphopyridine nucleotide; BAL, 2,3-dimercaptopropanol; PPO, 2,5diphenyloxazole.
PORTER quinone/ml. Saponification was effected at 70°C. for 30 min. and, on cooling, carotenes were extracted with petroleum ether. The extract was then washed thoroughly with water, dried, and chromatographed on a 1.8 X 10 cm. column of alumina. Increasing percentages of ethyl ether (O-50’%) were used to elute phytoene, phytofluene, and p-carotene from the column. The remaining carotenes were removed from the column with 10% ethanol in petroleum ether. After the ethanol was washed from the petroleum ether, the solution was dried and then chromatographed on MgO-Super-Cel (1). The chromatogram was developed with increasing concentrations of acetone in petroleum ether, and <-carotene was elutedfrom thecolumn. r-carotene, neurosporene, and lycopene were sectioned from the column and then eluted from the MgO with 10% ethanol in petroleum ether. After the ethanol was washed from the petroleum ether, the solutions of r-carotene and neurosporene were dried and chromatographed on Ca(OH)n-Super-Cel. Each of the carotenes, except lycopene and p-carotene which were crystallized (1) to constant specific radioactivity, was rechromatographed until spectrally pure. Where possible, spectral analyses (Process and Instruments recording spectrophotometer) and determinations of radioactivity (Packard liquid-scintillation spectrometer) were made on successive eluates of a carotene. In other cases the carotenes were sectioned from the column and analyzed spectrally, and the Cl4 content was determined. All carotenes, except phytoene and phytofluene, were hydrogenated catalytically before determinations were made of radioactivity. Aliquots of a carotene with similar specific radioactivities were combined and hydrogenated catalytically (l), and the reduction product was chromatographed on alumina. The reduced compound was eluted from the column with petroleum ether and assayed for radioactivity, and then it was subjected to gas-liquid chromatography on a 6 ft. X 6 mm. I.D. column of 5% SE-30 on Chromosorb W. A temperature of 270°C. and an argon flow rate of 100 ml./min. was used. Eluates from the gas-liquid chromatograph were trapped on glass wool, eluted with toluene (containing 0.150/, PPO), and then assayed for radioactivity with a Packard liquidscintillation spectrometer. RESULTS
AND
DISCUSSION
The specific radioactivities of the acyclic carotenes (Table I) decline with an increase in unsaturation (i.e., phytoene to lycopene). This decline in specific radioactivities may be taken as evidence in
RADIOACTIVITIES TABLE
I
SPECIFIC RADIOACTIVITIES OF CAROTENES SYNTHESIZED BT~ ISOLATED TOMATO PLASTIDS FROMW-LABELED TERPENOLPYROPHOSPHATES 1
Experiment
1
coNnts/’ min.img. Phytoene Phytofluene I-Carotene Neurosporene Lycopene p-Carotene
mL?21s/ min./mg.
ms
0.764 215,35@ 12 lOWa * 0.058 15:650~~ bI 0.094 4,500” 0.032
489,600~ 1t%,700* 149,800h 57,800h 1,200c 2,480”
Experiment 2
1.020 0.098
4OC 140”
2.100 0.340
a Determined by spectraphotometric assay after elution from an aluminum oxide chromatogram and by determination of the radiochemical purity of the reduced compound on gas-liquid chromatography. b Determined by spectrophotometric assay after elution from an aluminum oxide chromatogram and/or a magnesium oxide-Super-Cel column and by a determination of the radioactivity coincident with lycopersane on reduction and gas-liquid chromatography. c Determined through crystallization to constant specific radioactivity. d Determined by spectrophotometric assay after elution from an aluminum oxide chromatogram and a Ca(OH)n-Super-Cel column and by a determination of radioactivity coincident with lycopersane after reduction and chromatography on alumina. The absolute value for the specific radioactivity of this compound may be lower than the value given since the quantity of radioactivity present was insufficient for an analysis by gasliquid chromatography.
169
OF CAROTENES
of the intermediate phytofluene remains closely associated with the enzyme and does not enter the pool of free phytofluene before conversion to [-carotene. The remainder would be lost from the enzyme surface, and it would enter the phytofluene pool. Whether this is a correct interpretation of the results must await additional experimentation on the enzymic conversion of phytoene to c-carotene. The specific radioactivities of neurosporene and lycopene are in the order expected on the basis of the proposed conversion: {-carotene -+ neurosporene -+ lycopene . The specific radioactivities of lycopene and p-carotene (Table I) indicate that some p-carotene must arise in some tomato selections from a precursor other than lycopene. However, other results reported from our laboratory (3), and Table II, indicate that ,&carotene does arise from lycopene in some tomato selections. Possibly the selection of a system for synthesizing the carotenes is of paramount importance in the formation of p-carotene. Other information which has a bearing on this question has been supplied by Decker and Uehleke (8) and by Davies (9). Decker and Uehleke (8) reported the conversion of lycopene to p-carotene by chloroplasts and the reverse reaction by parenchymatous tissue of red tomatoes. Davies (9) reported the synthesis of y-carotene by Rhixophlyctis rosea by a pathway which did not involve TABLE
support of the proposal of Porter and Lincoln (5) as modified by Porter and Anderson (3), that the biosynthesis of lycopene proceeds by sequential desaturation of phytoene. Further support for this conclusion has been provided recently by the finding that phytoene is converted to phytofluene by isolated tomato plastids (6) and to d-carotene by cell-free extracts of a mutant of Staphylococcus aureus (7). The similar specific radioactivities of and r-carotene (Table I) phytofluene might be interpreted as evidence that one enzyme is involved in the conversion of phytoene to [-carotene (3). If so, a portion
SPECIFIC
II
RADIOACTIVITIES OF LYCOPENE ~-CAROTENE BIOSYNTHESIZED BI-
AND
TOMATO PREPARATIONS system
LyCOpeIltT p-carotene ClJUfltS/?lliVZ./Mg. COl~lZlSjl~li~./I~Zg.
Tomato
Tomato
fruita
plastids*
2140 5530 2210 3110 1200 40
c Mevalonic acid-2-C14 was injected ing tomato fruits [see Ref. (l)]. * See Table I.
780 3325
1470 G-100 2480
140 int,o ripen-
170
BEELER
AND PORTER
the intermediate formation of lycopene. The simplest explanation for the formation of @-carotene, which is consistent with all the existing data, is that p-carotene may be formed from lycopene, and also from neurosporene via p-zeacarotene and y-carotene (3). Accurate determinations of the specific radioactivity of y-carotene were not made because of the small quantity of this compound, the presence of appreciable quantities of contaminating radioactivity, and the presence of small amounts of radioactivity in the -y-carotene. However, the specific radioactivity of y-carotene (determined after reduction and chromatography on alumina) was less than that of neurosporene. It was reported in an earlier publication (1) that radioactive contaminants accompany the carotenes during purification and that great care must be exercised to remove these contaminants. Some of these contaminants were also present in the isolated system used in these experiments. In Expt. 1, 78,500 counts/min. (124,500,OOO counts/min./mg.) was found in association with neurosporene after this compound was purified through chromatography on MgO-Super-Cel, Ca(OH)2-Super-Cel, and then catalytically hydrogenated. Chromatography of the reduced product on a 1.8 X 7 cm. column of alumina reduced the 14,000 counts/min. radioactivity to (22,200,OOO counts/min./mg.). Most of this radioactivity appeared in the solvent front on gas-liquid chromatography on SE-30
at 270°C. The remainder was coincident with the lycopersane eluate from t,he column. The latter was used as a true measure of the radioactivity present in neurosporene. These results indicate the extreme care that must be taken to insure proof of the incorporation of radioactivity into a carotene. Most of the radioactive contaminants of the carotenes have not been identified. One that has been identified (through gas-liquid chromatography) is farnesol. This compound chromatographs slightly before {-carotene on a MgO-Super-Cel column. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Marcia L. Hipke and Mr. William J. Schelble for their technical assistance. REFERENCES 1. ANDERSON, D. G., NORGARD, D. W., AND PORTER, J. W., Arch. Biochem. Biophys. 33,
68 (1960). 2. ANDERSON, D. G., AND PORTER, J. W., Biochem. Biophys. 97, 509 (1962). 3. PORTER, J. W., AND ANDERSON, D. G., Biochem. Biophys. 97, 520 (1962). 4. WITTING, L. A., AND PORTER, J. W., J. Chem. 234, 2841 (1959). 5. PORTER, J. W., AND LINCOLN, R. E.,
Arch. Arch. Biol. Arch.
Biochem. 27,390 (1950). 6. BEELER, D. A., AND PORTER, J. W. Biochem. Biophys. Research Communs. 8, 367 (1962). et Biophys. Acta 60, 7. SUZUE, G., Biochim. 593 (1961). 8. DECKER, K., AND UEHLEKE, H., 2. physiol. Chem. 323, 61 (1961). 9. DAVIES, B. H., Biochem. J. 80, 48P (1961).