The biosynthesis of phytoene and other carotenes by enzymes of isolated higher plant plastids

aRCHIVES

OF

BIOCHEMISTRY

The Biosynthesis

AND

97,

!i%-519

of Phytoene and Isolated Higher

DAVID From

BIOPHYSICS

the Radioisotope of Physiological

G. ANDERSON2

(1962)

Other Plant AND

Carotenes Plastids’

JOHN

by Enzymes

of

W. PORTER

Unit, Veterans Administrution Hospital, and the Department Chemistry, Univexity of Tl’isconsin Nadison, Wisconsin Received

January

3, 1962

A soluble rat liver enzyme system was utilized to synthesize V-labeled terpenol pyrophosphates from radioactive mevalonic acid. After heat inactivation of the animal enzymes the components of the incubation mixture served as substrate for the biosynthesis of phytoene by carrot or tomato plastids during an incubation of l&l8 hr. Incorporation of C” int,o phptoene was proved through chromatography on alumina, catalytic hydrogenation and chromatography of the lycopersane on alumina, and gas chromatography followed by counting of trapped eluates for radioactivity. Experiments are reported which are consistent with the presumption that a Go terpenol pyrephosphat)e (presumably geranyl geranyl pyrophosphate) served as the substrate for the synthesis of phytoene. Evidence is also presented that either TPN or DPS, and not, the reduced nucleotides, serred as the cofactor for this reaction in plastids and in more purified enzyme systems. The incorporation of Cl4 of terpenol pyrophosphatcs into carotenes other than phytoene was also achieved with tomato plastids. Proof for the incorporation of C’” into these compounds was obtained in the same day as for phytoene. exceut in the case of &carotene and lycopene. These compounds were crystallized to constant specific radioactivity. I

_

Phytoene, the most saturated of the naturally occurring carotenes, is postulated to be the precursor of the more highly unsaturated carotenes (4). A study of its formation in isolated enzyme systems is, therefore, of importance to an understanding of the mechanism of the biosynthesis of carotenes. In the present paper we report upon studies of the formation of this compound, and upon results which lead us to suggest that phytoene, and not lycopersenc, is the first, hydrocarbon formed in the biosynthesis of carotenes from terpenol pyrophosphates.

Plastids derived from a number of higher plant sources form radioactive phytoene when supplied with acid-labile derivatives of radioactive mevalonic acid. In addition, tomato plastids incorporate c’” of these substrates into the sequence of carotenes of the proposed biosynthetic route (4). Detergent treatment of carrot plastids makes the phytoene synthetic syst,em nonsedimentable, and this system may, in turn, be dialyzed, precipitated with (NH,‘) &301 and dialyzed, and treated with charcoal to remore nucleotides. Throughout these treatments, the ability to synthesize phytoene is retained. Furthermore, TPN” enhances this

‘This work was supported by a research grant, A-1383, from the National Institute of Arthrit,is and Metabolic Diseases of t,he National Institutes of Health, U. S. Public Health Service. Preliminary reports of this work have been presented (l-3). ’ Present address: The Marine Laboratory, No. 1 Rickenbacker Causeway, Miami 49, Florida.

’ Thr following abhrerintions are used : TPN and TPNH, oxidized and reduced triphosphopyridine nurleotide ; DPT and Dl’iSH, oxidized and reduced diphosphopyridine nucleotide; ATP, adenosine triphosphatc; Pt,O, platinum oxide catalyst ; EDTrl. ethylenedinmine tetraacetat,e; MV-4, mevnlonic: arid; B:\L, 2,3-11imrrcaptopropanol.

INTRODUCTION

509

510

BNDERSON

synthesis while TPNH suppressed the activity, thus suggesting that TPK may be a cofactor in the synthesis of phytoene. EXPERIMENTAL Many of the materials and methods used in this study were reported in a previous publication (5). Other materials and methods are described in the following sections. MATERIALS Tomato fruits, red-ripe but not soft, carrot roots, and spinach were purchased locally. Tangerine variety tomatoes were the generous gift of Dr. Mark L. Tomes of the Department of Botany and Plant Pathology, Purdue University. Rat livers were the gift of members of the Department of Zoology, University of Wisconsin. These livers were obtained from animals sacrificed for the removal of their pituitary glands. Mevalonic acid-2-Cl4 was obtained as the lactone from Volk Radiochemical Co. A stock solution of this compound was prepared through treatment with excess KOH, followed by careful neutralization with acid to pH 8. ATP, TPN, TPNH, DPN, and DPNH were obtained from the Sigma Chemical Company. Phytoene for use in the preparation of carrier lycopersane was isolated from carrot oil obtained from Nutritional Research Associates, Inc. of South Whitley, Indiana. After the oil was saponified, nonsaponifiable compounds were extracted with petroleum ether, and the extract was chromatographed on alumina. Phytoene isolated by this procedure was hydrogenated catalytically over PtO in an isopropyl alcohol-petroleum ether solution. Complete reduction was assured through a second hydrogenation in glacial acetic acid. The resultant lycopersane was purified and characterized as reported in the following section. Squalane was prepared from squalene purchased from Distillation Products Industries, Rochester, N. Y. Squalene was purified through saponification, followed by chromatography of the nonsaponifiable fraction on alumina. Hydrogenation to squalane was accomplished in glacial acetic acid-petroleum ether with a PtO catalyst. Carot,enes. used as carrier in t,he isolation of the biosynthesized radioactive compounds, were isolated from tomato paste or carrot oil. Crystalline p-carotene was obtained from Nutritional Biochemicals Corporation. Lycopene and p-carotene were recrystallized before use as carrier in experiments on the crystallization of biosynthesized compounds to constant specific radioactivity (5).

AND

PORTER METHODS

Plastid

and Enzyme Preparations

Rat liver enzymes were prepared according to Witting and Porter (6). The protein fraction precipitated between 40 and 60% of saturation with (NH&SO, was employed in the present studies. A preparation of soluble protein of carrot root was obtained following pulverization of frozen carrot slices with a pestle in a chilled mortar. The thawed powder was extracted with an equal volume of 0.1 M phosphate buffer, pH 7.4, and the solubles were then pressed through cheesecloth. Protein of the supernatant solution, obtained after centrifugation at 105,000 X g for 15 min., was precipitated with solid (NH,)&lO, between 65 and 90% of saturation. After centrifugation at 5000 X g, the protein pellet was redissolved in 0.01 M phosphate buffer, pH 7.0, and the protein solution was dialyzed against the same buffer. Carrot plastids were prepared from shredded carrots by homogenizing for 1 min. in a Waring blendor at 70% of maximum speed in a medium of 2 vol. of 0.4 M sucrose and 0.1 iM phosphate buffer, pH 7.4. After the homogenate was pressed through cheesecloth the suspension was centrifuged at 700 X g to remove cell debris. Plastids were then obtained on centrifugation at 4000 X g. Tomato plastids were prepared from tomato parenchyma tissue. The tissue was homogenized for 5 sec. at full speed in a Waring blendor in an equal volume of 0.2 M Tris, pH 8.2, and 0.001 M EDTA4. EDTA, which probably acts by sequestering calcium, was added to protect against gel formation. After the homogenate was squeezed through a cheesecloth-glass wool mat to remove cell debris, plastids were removed by centrifugation at 4000 X g. Spinach plastids were prepared by the method of Avron and Jagendorf (7). Each of the above plastid preparations was resuspended in 0.1 M phosphate buffer, pH 7.0, at a concentration equivalent, to 30-40 g. of original tissue/ml. An equal volume of 0.1% sodium lauryl sulfate was added to the final plastid suspension in the preparation of plastid extracts. After 15 min. of stirring, the suspension was centrifuged at 105,000 X g for 15 min. The supernatant solution was then dialyzed against 0.05 M phosphate buffer, pH 7.0, or treated with solid (NH,)?SO, between the limits of O-90% of saturation. The protein pellet obtained after centrifugation was dissolved in 0.05 M phosphate buffer, pH 7.0, dialyzed against the same buffer, and in some instances treated with acidwashed Nuchar C-190-N (5 mg. charcoal/mg. pro-

BIOSYNTHESIS OF PHYTOENE ter enzymes were chest.

Substru te Form tion and Pwifica tion

RIGIN

1

-

SOLVENT

stable on storage in a Dry Ice

FRONT

-

FIG. 1. Diagram of paper chromstographic separation of terpenol pyrophosphates. The strips are 6 X 22 in. (the width is denoted by the line marked solvent front, and the length by the lines marked “strip for counting.“) The strip for counting serves to locate the radioactive peaks of the chromatogram, and the border area on the right serves as a wick for the elution of the radioactive terpenol pyrophosphates. tein) for 30 min. Charcoal was removed through centrifugation and filtration. The protein content of each extract was determined by the biuret method of Gornall et al. (8). All of the above enzymes and plastids were prepared in the cold, and each, with the exception of the carrot-soluble enzymes and the rat liver enzymes, was prepared fresh before use. The lat-

Terpenol pyrophosphates were formed by incubating the following mixture or multiples thereof: ATP, 10 pmoles; BAL, 1.6 pmoles: MgCh, 6.0 pmoles; potassium phosphate buffer, pH 7.0, 50 pmoles; MVA-2-C?, 3.0 rmolee, (6.8 X loj counts/min./pmole) ; and rat liver enzyme [4&60 fraction of Witting and Porter (611, 10 mg.; all in 1 ml. The enzymic reactions were stopped b) heating at 7O’C. for 2 min., after 90 min. of incubation. Most frequently this heat-innct,ivated mixture was used directly as the source of terpenol pyrophosphates. In other designated experiments the radioactive acid-labile pyrophosphntes were isolated and reintroduced into incubation mixtures. In the isolation of pyrophosphates the protein of the incubation mixture was heat denatured, as reported above, and ethanol was added to 90% (v/v) to aid in the precipitation of protein and other contaminants. After ccntrifugation, the supernatant solution was made basic with 0.3’6 NHhOH, reduced to a small \-olurne in a flash evaporator, and finally transferred to a small flask with 0.3% NH&OH washes in preparat,ion for lyophilization. The dry powder obtained on lyophilization was taken utj in a minimal volume of 0.3% NHIOH and streaked on specially prepared \Vhatman No. 3 paper. The chromatogram was developed with a system of isopropyl alcohol-isobutyl alcohol-NH,OH-water (40:20:1:39) (9-11) by descending techniclue. A diagram of the rhromatogram (6 X 22 in.) is shown in Fig. 1. A l-cm. strip was cut from the left side and assayed for radioactivity in a strip counter while the rcmnindei of the chromatogram was held in the chromatographic chambei~ to keep it moist and basic. The position of the terpenol pyrophosl~hnte~, IZI 0.75-0.80, was marked on the original chromatogram, and this strip was cut out and cluted with 0.3% NH&OH, the blank area on the right-hand side of the chromatogram serving as a wick. The eluate was stored frozen or lyophilized. Analyses of t.hc solution (9) generally indicated that 90-100% of the radioactivity present was in acid-labile terpenol pyrophosphates. Splitting of the terpenol pyrophoaphates was effected by incubating for 10 min. at pH 1.0 and 37°C. Whenever material of lr~ than 90% radiochemical lmritq wras ol)tained, it was rec.hromatopraphed. The paper used for the chromatography of tcrpenol pyrophosphatea was washed with methanol-formic acid-water (80:15:5), then with 0.1 Jf di-Na EDT;\-NH&OH-water (3:3:94), and 6nally with water. Deionized distilled water was

512

ANDERSON

used in each case. A final wash of the paper with the developing system (see above) served to remove a slight brown color which appeared during chromatography and moved close to the front. Isopropyl alcohol and isobutyl alcohol were purified prior to use with 10 g. potassium borohydride/l. This solution was shaken for 2 days in the cold and then distilled, and the purified alcohols were stored in dark bottles in the cold.

Incubation

System for the Biosynthesis of Carotenes

One milliliter of a heat-inactivated terpenol pyrophosphate mixture was supplemented with either 1 ml. of a plastid suspension, 1 ml. of solubilized carrot plastids (2 mg. prot,ein/ml.), or 0.5 ml. of an (?;Hd)?SOa-precipitated soluble carrot plastid preparation (5 mg. protein/ml.). Pyridine nucleotide cofactors were added at a concentration of 1 rmole/ml., and carrot-soluble protein, (0.5 ml. and 6.0-8.0 mg./ml.) was added as indicated. Each incubation mixture was kept at 25°C. for 18 hr.

Isolation

of Phy toene

Enzyme activity in the incubation mixture was stopped by the addition of an equal volume of 10% alcoholic KOH. Saponification was then effected at 70°C. for 30 min. and, on cooling, nonsaponifiable compounds were extracted with petroleum ether. The addition of small amounts of acetone aided in the complete extraction of carotenes. Carrier phytoene was added to the petroleum ether extract to minimize destruction of plastid phytoene by light and oxygen in subsequent operations. The extract was then washed thoroughly with water and chromatographed on a 1.8 X 10 cm. column of alumina. Development of the chromatogram was effected with successive additions of 50 ml. of petroleum ether, 50 ml. of 1% ethyl ether in petroleum ether, and 70 ml each of 2 and 4% ethyl ether in petroleum ether. Ten-milliliter fractions were collected, except for 5-ml. quantities collected at the end of the squalene and the start of the phytoene elution from the column. The smaller fractions were collected to insure a complete separation between squalene and phytoene. Squalene begins to appear in the eluate at the change over to 2% ethyl ether, whereas phytoene appears at t,he change over to 4% ethyl ether. An occasional lot of alumina showed a decreased adsorptive affinity for squalene and phytoene. To obtain adequate separation on these lots of alumina, it was necessary to change the per cent of ethyl ether in petroleum ether to 0.75, 1.5, and 4%. A single chromatographic separation on alumina was adequate for phytoene purification,

AND

PORTER

according to the criteria of purity that we previously established (5). Eluate fractions closely agreeing in specific radioactivities were then combined and catalytically hydrogenated, and the lycopersane formed was isolated by chromatography on alumina. The recovery of radioactivity in the isolated lycopersane was taken as a measure of the purity of the phytoene separated on alumina. The total radioactivity of the phytoene was then calculated from the recovery of radioactivity (generally 90-100%) per unit weight of phytoene and the quantity of phytoene present in the whole chromatogram, as determined spectrophotometrically, before reduction. Duplicate incubations gave values which agreed within 25%.

Isolation

of Car0 tenes

Six individual incubations were combined to determine whether C” of the terpenol pyrophosphates is incorporated into other carotenes by tomato or carrot plastids. After saponification, nonsaponifiable compounds were extracted with petroleum ether. Carrier carotenes were then added to this extract at a concentration of at least fivefold that present in the incubation mixture. The addition of known levels of carrier carotenes protected the plastid carotenes against extensive destruction during the various isolation procedures. The addit.ion of carrier also enabled us to correct for losses inherent in the purification procedure. Chromatography of the carotenes was carried out as described previously (5). Individual chromatographic eluates of a carotene were analyzed spectrophotometrically for spectral purity and quantity, combined, and catalytically hydrogenated, and the product (lycopersane or perhydron/-carotene) was isolated by chromatography on alumina. The total radioactivity in each carotene was calculated from the radioactivity found in the reduction product isolated on chromatography, and the recovery of added carrier carotene measured spectrophotometrically previous to reduction. Lycopene and B-carotene were purified, after the original chromatographic separation on MgOSuper-Cel, through crystallization to constant specific radioactivity. Aliquots of the crystals from each cryst,allization were decolorized by hydrogenation for 30 min. in the presence of PtO, and then counted in a Packard Tri-Carb liquid scintillation spectrometer.

Gas Chromatography Reduced squalene (squalane) and carotene samples (lycopersane or perhydro-y-carotene) were chromatographed in a Barber-Colman model 10 gas chromatograph. Authentic squalane and lyco-

BIOSYNTHESIS persane were added to unknown samples when adequate quantities were not already present. Squalane and lycopersane were separated on a 6-ft. X 6 mm. column of 5% Se-30 on Chromosorb W at 270-C., with an argon flow rate of 100 ml./ min. (Fig. 2). The flash heater was operated at 325”C., and the ionization detector was kept at 295°C. Effluent peaks and intervening areas were trapped separately on silicone-coated anthracene crystals in a Packard model 830 fraction collector. Trapping tubes were modified to contain glass wool plugs and reduced amounts of anthracene crystals (approximately 400 mg./tube). Comparisons were made of the count of aliquots placed directly on the anthracene crytals, aliquots added to the toluene-phosphor solution, and the summation of count of the fractions trapped on anthracene crystals on gas chromatography. Recoveries of radioactivity were nearly quantitative.

Determination Measurements a Packard Liquid

of Radioactivity

513

OF PHTTOENE

12UCOPERSANE

IO 6SQUAL ANE \

642-

0

5

IO MINUTES

15

20

FIG. 2. The gas chromatographic separation of squalane and lycopersane. Details of the conditions of seljaration are given in the text.

of radioactivity were made with Scintillation Spectrometer. RESULTS

CRITERIA OF PHPTOEKE SYNTHESIS

The operations previously used (5) to assay for the formation of radioactive phytoene were shortened to a single chromatographic separation on alumina, instead of successive separations on MgO-Super-Cel and alumina. A comparison of the radiochemical purity of the phytoene obtained with that of previous studies (5) showed that virtually all of the radioactive contaminants of phytoene that were present on the MgO chromatogram (5) were removed by this procedure. A major portion of the eluate fractions of phytocne from the alumina chromatogram showed nearly equal specific radioactivities (Fig. 3). When t’he fractions of essentially equal specific radioactivity were combined, catalytically reduced, chromatographed, and counted for radioactivity, 9776 of the radioactivity attributed to phytoene was present in the lycopersane fraction. Gas chromatographic separation of this fraction, followed by trapping of effluent peaks and intermediate areas and counting of the t,rapped samples (see Table IV) showed that 90% or more of the radioactivity was associated with the lycopersane peak. Thus the synthesis of phytoene by carrot and tomato plastids was est,ablished.

FIG. 3. The chromatographic separation of pbytoenc on alumina. Specific radioactivities for each fraction are given at, the top of each bar graph.

Samples of phytoene obt’ained by t’he procedures outlined above were routinely reduced and then assayed by gas chromatography to make certain that modifications in incubation mixtures did not yield radioactive impurities which chromatographecl with phytoene. In addition, the squalene (3) area, which immediately precedes phytoene on the chromatogram, was reduced and tested for the presence of lyropersane. Any lycopersene synthesized in the incubation mixture would be expected t’o chromatograph with squalene on an alumina chromatogrnm. No evidence of lysopersene was obtained.

514

ANDERSON TABLE

THE

AND

I

OF CARROT-SOLUBLE ENZYME ON THE FORMATION OF PHYTOENE BY HIGHER PLANT PLASTIDS EFFECT

All samples contained the rat liver incubation system (see text), except where noted, plus the additions noted below. Incubation conditions are reported in the text. Total radioactivity in phytoene

Incubation system Rat liver enzyme system minus plastids Minus rat liver enzyme + carrot plastids Minus rat liver enzyme + tomato plastids Carrot plastids + TPN Repeat of above Carrot plastids + TPN + carrotsoluble enzyme Repeat of above Tomato plastids + TPN Tomato plastids + TPN + carrotsoluble enzyme Spinach plastids + TPN Spinach plastids + TPN + carrotsoluble enzyme

15 10 10 1500 1560 2050 2140 810 2160

PORTER

phate as a buffer (Table II) did not increase the incorporation of radioactivity into phytoene. Neither did the presence of KF, a phosphatase inhibitor, increase the synthesis of phytoene. Addition of the carrot-soluble system was based upon the possibility that it might catalyze the formation of an increased amount of geranyl geranyl pyrophosphate from farnesyl and isopentenyl pyrophosphates of the incubation mixture. The effect of the carrot solubles was small (Table II), but the further addition of biosynthetic isopentenyl pyrophosphate greatly enhanced the incorporation of radioactivity into phytoene. This result suggested that t’he carrotsoluble enzyme is capable of catalyzing the condensation of C, and Cl5 pyrophosphate esters with the formation of a C& pyrophosphate ester. In a few experiments, isolated terpenol pyrophosphates (50,000-100,000 counts/ TABLE THE EFFECT

10 130

CHARACTERISTICS OF THE PHYTOENE BIOSYNTHETIC SYSTEM

Plastids of carrot root, tomato fruit and spinach leaves synthesized phytoene from the terpenol pyrophosphates formed by the rat liver enzyme system. Table I presents evidence that carrot and tomato plastids catalyzed this reaction in the absence of other plant enzymes. However, synthesis of phytoene was increased when a soluble carrot enzyme system was added. Spinach plastids, in contrast, synthesized a significant amount of phytoene only in the presence of this soluble preparation. The carrot-soluble enzyme preparation was unable to synthesize phytoene in the absence of plastids. [The rationale for including TPN in the incubation mixture will be reported in a subsequent section, as will experiments to elucidate the role of the carrot-soluble enzyme system.] The substitution of imidazole for phos-

BUFFER,

II KF

AND ISOPENTENYL PYROPHOSPHATE ON THE BIOSYNTHESIS OF PHYTOENE OF

The incubation system included the rat liver enzyme system, as described in the text, carrot plastids, 1.0 ml.; TPN, 1.0 pmole/ml.; KF, when present, 5 /*mole/ml. ; and carrot-soluble enzyme, 0.5 ml. Radioactivity

in

phytoene

counts/min. Standard incubation system minus plastids Standard incubation system Incubation system in imidazolea Incubation system in imidazolea + KF Incubation system + carrot-soluble enzyme Incubation system + carrot-soluble enzyme + KF Incubation system f carrot-soluble enzyme + KF + isopentenyl pyrophosphateb (0.05 crmole)

15 2410 2270 1670 2670 2400 3650

0 The rat liver incubation system and the carrot plastid suspension were present in 0.04 M imidazole, pH 7.0. * Isopentenyl pyrophosphate was the gift of David Norgard.

BIOSYIVTHESIS TABLE

515

OF PHTTOESE

III

REQUIREMENTS FOR THE BIOSPNTHESIS OF PHYTOENE Each sample cont,ained the rat liver incubation system (see text) and additions as noted. Pyridine nucleotides were present at a concentration of 1.0 pmole/ml. Total radioactivity in phytoene

Incubation system Minus rat liver eneyme + plastids No plastids Heated plastids 12 min. at 1OO’C.) Plastids (1 ml.) Plastids (0.2 ml.) Plastids + N, Plastids + TPX Plastids + TPX + Nz Plastids f TPNH Plastids + TPNH + NZ

10 50 35 1300 840 1320 2000 2520 1140 1080

min.) were used as substrate for the biosynthesis of phytoene in incubation mixtures containing carrot plast,ids, TPN, and MgCl, . il maximum incorporation of 1010 counts/min. into phytoene was obt’ained. Gas chromatographic analysis of the terpenols of the acid-labile terpenol pyrophosphates showed the presence of small amounts of radioactivity associated with the peak of geranyl linalool,4 but most of the radioactivity was associat’ed with nerolidol. All of the data presented above support the proposal of CSOterpenol pyrophosphate (geranyl geranyl pyrophosphatrj as t,he probable substrate in the synthesis of phytoene by carrot plastids. In early experiments, a number of possible cofactors were added to carrot plastids to effect the synthesis of phytoene. However, it soon became evident that plastids alone were capable of this activity (Table III, sample 4), providing they were supplied with the terpenol pyrophosphates formed during the incubation with the rat liver enzyme system. The addition of cofactors other than pyridine nucleotides had ’ Authent.ic all-trnna-geranyl linalool generous gift, of Dr. 0. I&r of Hoffman b Co., Bask, Switzerland.

was the LaRoche

3 ‘““‘t/

, 5

IO HOURS

, 15

, 20

of phytoene as a funcFIG. 4. The biosynthesis tion of time. The conditions of incubation are detailed in the text.

only negligible effects on phytoene synthesis. In the absence of terptnol pyrophosphatcs, plastids alone were incapable of converting appreciable amounts of mevaionic acid-Cl4 to phytoenc. The rat liver enzyme system was also incapable of t’he synthesis of phytoene when incubated alone, and no synthesis of phytoene occurred when the plastids were heated. A comparison of samples 4 and 5, Table III, shows that phytoene synthesis was also dependent upon the amount of plastids used, and an examination of Fig. 4 shows that the synthesis of phytocne was a linear function of time. Since the plastids per se were capable of phytoene synthesis, the effect of any cofactors on the reaction could only be supplementary. The effects of t’he addition of pyridine nucleotides were not large, but they were consistent. The addition of TPK stimulated phytocne synthesis, while the presence of TPKH inhibited the reaction slightly. 1,ikewise the addition of DPNH had a negligible effect, whereas the addition of DPN slightly stimulated the reaction. Incubation in an atmosphere of nitrogen had no effect on phytoene formation by plastids with or without TPKH supplementation. An atmosphere of nitrogen did, however, increase the stimulation observed with TPN. The plast,id system is more rigorously drfined than the whole organ or a simple homogenate, but it is still quite crude for en-

ANDERSON

516

THE

EFFECT

OF

TABLE IV TPN ON THE

BIOSYNTHESIS

OF PHYTOENE

The conditions of incubation Table I and in the text.

are described in

Total radioactivity TPN-

TPNHa

counts/min None (intact carrot plastids) Detergent and dialysis Detergent, (NH4)804 pptn, dialysis Deterient, (NH,)&04 pptn, dialysis, charcoal treatment 4 Incubation

mixture,

1300

2018

1010

256 237

552 390

152 210

179

376

242

0.1 pmole/ml.

zyme studies. Consequently attempts were made to solubilize the system. Table IV shows the results that were obtained. A portion of t,he enzyme activity capable of synthesizing phytoene was released from the plastids by treatment with sodium lauryl sulfate. This activity, in turn, survived dialysis and (NHJ2S04 precipitation. Treatment with charcoal to remove possible coenzymes from the protein also did not destroy activity. The possibility that the activity of the solubilized preparation for phytoene formation was low because of a loss of cofactors cannot be rigorously excluded, but supplementation with a boiled carrot plastid extract did not increase the act’ivity. The effects of TPN and TPNH on phytoene synthesis by each of the above enzyme preparations (Table IV) were similar to that found with the intact plastids. In each case TPN enhanced the activity while TPNH either inhibited or was but slightly stimulatory. However, in no case did the treatment of the enzyme reduce the synthesis of phytoene to zero. Thus, it has not been possible to demonstrate an absolute requirement for TPN in this reaction. However, stimulation by TPN has been thoroughly consistent, which suggests that it, and not TPNH, is a participant in the reaction in which CZOterpenol pyrophosphate molecules are condensed to form phytoene.

AND PORTER

Grob, Kirschner, and Lynen (12) have reported the synthesis of lycopersene from geranyl geranyl pyrophosphate by an enzyme obtained from ATeurospora. Evidence was also presented for the participation of TPNH in this reaction. Therefore, we have considered the possibility that the demonstrated inhibition of phytoene synthesis by TPNH would be reflected in lycopersene formation. Gas chromatographic separations were made of the catalytically hydrogenated squalene fraction obtained from tomato and carrot plastids incubated with labeled terpenol pyrophosphates and TPNH. Lycopersene and squalene are structural homologs, and thus have similar, if not identical, adsorption properties when chromatographed on alumina. In no case were we able to find evidence for a labeled CqOisoprenoid compound on gas chromatography. Either lycopersene is not formed in these systems, or it is formed in undetectable amounts. Therefore it is concluded that TPNH inhibition of phytoene formation does not result in the formation of lycopersene. BIOSYNTHESIS OF CAROTENES TOMATO PLASTIDS

BY

In a number of experiments in which carrot, plastids were incubated with radioactive terpenol pyrophosphates to form phytoene, examination was also made of phytofluene, [-carotene, and /?-carotene for radioactivity. Although radioactivity was present in eluate fractions separated on chromatography on MgO, further purification of these carotenes reduced the radioactivity to negligible levels. When tomato plastids were used, however, it was possible to demonstrate the incorporation of radioactivitv into the less saturated carotenes. Six individual incubations were made of tomato plastids and terpenol pyrophosphates formed by the rat liver enzyme system and then combined. After saponification of the incubation mixture and ext,raction of the nonsaponifiable compounds with petroleum ether, the carotenes were separated through chromatography on MgO. The pattern of labeling found on chromat.ography was similar to that found when MVA-2-C!14 was injected into tomato

BIOSYNTHESIS

fruit (5). However, most of t.he label was found in squalene [previously termed “unknown colorless polyene” (5)] which precedes phytoene on chromatography. Each carotene was again grossly contaminated with non-carotene radioactivity, although not at, as high a level as that found in the intact fruit (5). The contaminating radioactivity was separated from the carotenes on rechromatography of each on either alumina or Ca (OH) &Auper-Cel (5). Determinations of specific radioactivities, particularly of the less abundant carotenes such as y-carotene and neurosporene, were subject to considerable error because of the difficulties of cleanly separating small quantities of these substances, with relatively IOK specific radioactivities, from other carotenes without large losses. We attempted to avoid this dilhculty by the addit,ion of known quantities of pure carotenes to the carotenes extracted from the incubation mixture. We then determined the total radioactivity present in each carotene after purification. Carrier carotenes were added in a quantity at least five times that present in the incubation mixture, and the per cent recovery of carotene and the radioactivity of this carotene after purification were utilized to calculate the total radioactivity incorporated into each carotene. The results of four experiments on the incorporation of Cl4 into the carotenes of tomato plast,ids are presented in Table V. All of the carotenes of the proposed biosynthetic route (13) incorporated radioactivity from the terpenol pyrophosphates of the rat liver incubation system. In addition there was, in general, a stepwise decrease in total radioactivity of the carotenes with progression along the proposed biosynthetic route. This is particularly evident in t#hc radioactivity values of phytoene, phytofluenc, and t-carotene in the red tomato as well as the yellow tomato, where these carotenes predominate. The stepwise decrease was also observed with the sequence lycopene -+ y-carotene + p-carotene (see Expt. III). Considering the difficulties inherent in the experiments we have done, and the unknown dynamic relationships within the plastid, we may conclude that these results are consistent lvith, but do not prove, t,he

517

OF PHYTOEXE TABLE

V

OF Cl4 OF TERPENOL

INCORPORATION

PYROPHOSPHATE INTO CAROTENES BY TOMATO PLASTIDS

Plastids derived from red tomatoes were used t,anin Expts. I-III; plastids from the variety gerine were used in Expt. IV. Total

radioactivity

I

II

49,800 870 350

16,600 1,125” 455’” 28 340 27 43,’

III

IV

counts/n&

Phytoenc Phytofluene <-Carotene Neurosporene Lycopene Y-Carotene &Carotene a Correct’ed

-

2940 80” 620 12oa Q635a

for losses of added carrier TABLE

5750 200 170 ~ -

carotene.

VI

GAS CHROMATOGRAPHIC

CHARACTERIZ.4TION HYDROGENATED LABELED CAROTENES L~COPERSAXE

OF AS

Trap No. 2 contains the syualane peak. Trap No. 4 contains the lycopersane peak. Figure 2 and the text give the details of gas chromatography. The values of this table have not been corrected for background which normally ranges between 2.5 and 3.0 counts/mm. Radioactivity

1

2

of effluent ___--. 3

fraction 4

3

198.0 9.0 20.6 10.7

8.3 3.0 4.0 2.1

counts,‘,,iin

Phytoene Phytofluene I-Carot.ene Neurosporene

2.3 2.3 3.2 2.9

2.7 2.0 2.6 2.6

11.1 4.0 2.7 3.1

Porter-Lincoln hypothesis of sequential desaturation. The crystallization of lycopene and pcarotene to constant specific radioactivity WB considered to be completely adequate proof of the presence of radioactivity in these compounds. The other carotenes were chromatographed on two different adsorbents, hydrogenated, and chromatographed again upon alumina. It is conceivable, but unlikely, that some radioactive contaminant might accompany a carotene through all of these purification steps. Therefore we

AXDERSON

518

resorted to gas chromatography of the purified hydrogenated carotenes (lycopersane) , and we trapped effluent peaks for a determination of radioactivity. Gas chromatography provided a criterion in which separation was dependent mainly on molecular weight. The results obtained by this procedure are shown in Table VI. In each case, virtually all of the radioactivity of the reduced carotene was present in the lycopersane peak. DISCUSSIOS

Evidence that the enzymic synthesis of phytoene by carrot plastids, and extracts thereof, proceeds via the condensat,ion of two CZOterpenol pyrophosphates with TPN as a cofactor has been presented. Full proof of the nature of the substrate in this reaction must await further experimentation, but the participation of TPN in the reaction appears to be firmly based. The activation by TPN occurs in the original plastid preparation and persists through a variety of treatments, i.e., solubilization, dialysis, (NH&SO4 precipitation, and adsorption on charcoal. However, our inability to demonstrate an absolute requirement for TPN in phytoene synthesis requires that a certain caution be used in attributing a full role to TPN. Possily another hydrogen acceptor is intermediate between the substrate and TPN. Furthermore, the exact nature of the function of TPN must await elucidation of the substrate (s) of the condensation reaction. The enzyme or enzyme system which catalyzes the reaction (s) involved in the synthesis of phytoene is not identical with the enzyme (s) which catalyzes the formation of squalene in the same plastid system (14). The pyridine nucleotide requirements are different, and the treatments mentioned above result in markedly different inactivations of the enzyme activities. One would expect that the condensation of two C20 terpenol pyrophosphates to form the carotene skeleton would be analogous to the condensation of two farnesyl pyrophosphates to form squalene. The latter reaction has been studied intensively in rat liver microsomal preparat,ions by Popjak et al. (15, 16’1, and the results of their

AND PORTER

studies are consistent only with the proposal that dehydrosqualene (the CsO homolog of phytoene) is not formed as an intermediate to squalene. TPNH or DPNH were also reported to be part,icipants in the reaction (9, 17). However, the presumably analogous reaction to form phytoene in plant plastids is stimulated by TPN and DPN, and inhibited or uninfluenced by TPNH and DPNH. In addition, attempts to demonstrate lycopersene, the homolog of squalene, as a possible precursor of phytoene were consistently unsuccessful. Davies et al. (18) have also reported that lycopersene is not involved in carotene biosynthesis. [Lycopersene synthesis in fungal extracts has been reported (12)) however.] This anomalous situation would appear to suggest that plant and animal enzymes catalyze essentially similar reactions by different mechanisms. In a future paper (14) we will report on the formation of squalene by plant plastids and we will also present the results of studies to determine whether dehydrosqualene is an intermediate in the biosynthesis of squalene. Possibly in higher plant systems, CZOand Cl5 condensations produce phytoene and dehydrosqualene as the first products. Phytoene would then be dehydrogenated to yield the various carotenes, and dehydrosqualene would be reduced in the presence of TPNH to give squalene. The latter would serve as substrate in the biosynthesis of plant sterols. Suggestive evidence for sequential desaturation in the biosynthesis of carotenes is supplied from experiments in which the carotenes of intact tomatoes are labeled after injection of MVA-2-C14 (5), and the carotenes of tomato plast.ids are labeled after incubation with radioactive terpenol pyrophosphates. Conclusive evidence of labeling in the latter experiments was obtained on gas chromatographic separation of the hydrogenated purified carotenes. The specific radioactivities (5) and stepwise decreases in the total radioactivities of the carotenes along the proposed biosynthetic route, are also evidence for this route, but they are not unequivocal proof. A major reason for this stat,ement lies in the unknown dynamic state of the carotenes

BIOSYNTHESIS

within the plastid. It is quite possible that the pools are not in a state of rapid equilibration; hence specific radioactivities may

not give evidence of the precursor-product relationship. Similar arguments may be used to explain, or discount, the precursorproduct relationships which might be deduced from the total radioactivity found in each carotene. Consequently, we wish to place major emphasis on the proof of radioactivity in each of the carotenes of the proposed scheme. Full proof of the sequential desaturation route of carotene biosynthesis must, await evidence of direct conversion of each carotene to its postulated product. Some proof which may meet this requirement, has already been published. Decker and Uehleke (19) have demonstrated the conversion of labeled lycopene t,o p-carotene by green leaf plastids, and the reverse reaction in tomato plastids. Suzue has reported some evidence that radioactive phyt,oene is converted to labeled a-carotene by extracts of wild type Staphylococcus aureus (20). All of these results, taken together with studies of carotene structure, physiological experiments, and labeling experiments, reported from many laboratories, have led us to incorporate these results into, and somewhat modify, the original proposal of Porter and Lincoln (4). A full discussion of the modified scheme will be made in the paper immediately following (13).

OF PHYTOENE

2. PORTER, J. W,, ASD AXDERSOX, D. G., Abstr., Intern. Cvngr. Riochem. 5th Congr., 1961, p. 451.

3.

and

5. 6. 7. 8.

6x11) LIXCOLS, R. E.. Arch. Riothem. 27,390 (1950). ANDERSON, D. G., KORGARD, D. W., ASD PORTER, J. W., Arch. Biochem. Biophys. 88,68 (1960). WITTING, L. A., .411~ PORTER, J. Vi’., J. Bid. Chem. 234,2841 (1959). AVRON (ABRA~ISRY), M., AND JAGESDORF, A. T., Arch. Biochem. Biophys. 65,475 (1956). GORNALL, A. G., BARDAWILL, C. J., AXD DAVID, M. N., J. Bid. Chena. 177,751 (1949).

9. ANDERSOX,

10.

11. 12. 13. 14. 15.

16.

17. 18. 19.

REFERENCES 1. AsDER~os, D. G., ASD PORTER, J. W., Federation Proc. 20. 350 (1961).

D. G., AND PORTER, J. %‘., Plant Physiol. 36, Suppl. xlv (1961).

ANDERSON,

4. PORTER, J. W.,

ACKNOWLEDGMENTS The technical assistance of Mr. Jack Hipke Miss Marcia Morse is gratefully acknowledged.

519

20.

D.

G.,

RICE, M.

S., .~KD PORTER,

J. W.,

Biochem. Biophys. Research Communs. 3, 591 (1960). PORTER, J. W., Ptvc. Symposium “Drugs Affecting Lipid Metabolism,” p. 30. Elsevier Publ. Co., Amsterdam, 1961. EBEI,, J. P., ASD T’OLSTAR. T., Compt. rend. 233, 415 (1951). GROB, E. C., KIRSCHNER, K., .~SD LYNEN, F., Chimia 15,308 (1961). PORTER, J. TT., .~KD ANDERSON, D. G., Arch Biothem. Biophys. 97,520 (1962). ANDERSOS, D. G. .~XD PORTER. J. W., unpuhlished. POPJAK, G., GOODXAS, DEW., CORSFORTH, J. W., CORP~FORTH, R. H., AKD Rr~r.sc~, R. Biothem. Biophyx. Research Commons. 4, 138 (1961). POPJAK, G., GOODX~S, DEW. S., CORZIFORTH, J. W.. CORSFORTH, R. H., AND Rr-~.4oe, R.. J. Bid. Chem. 236, 1934 (1961). GOODMAR-, DEW. S., ~30 POPJAK, G., J. Lipirl Research 1, 286 (1960). DAVIES, B. H., GOOD~IY, T. W., ASD MERCER, E. I,., Biochem. J. 81, 40~ (1961). DECKER, Ii. ASD UEHLEKE. H., Z. physiol. Chem. 323, 61 (1961). SUZUE, G., Biochim. it Biophys. Acta 50, 59:< 1961).