Localization, purification, and characterization of dimethylallylpyrophosphate:Umbelliferone dimethylallyltransferase from Ruta graveolens

ARCHIVES

OF

BIOCHEMISTRY

AND

BIOPHYSICS

177,

74-83

(1976)

Localization, Purification, and Characterization of Dimethylallylpyrophosphate:Umbelliferone Dimethylallyltransferase from Ruta graveolensl D. S. DHILLONZ Department

of Chemistry,

Trent

STEWART

AND

University, Received

Peterborough, March

A. BROWN” Ontario,

Canada

K9J

7B8

23, 1976

Fractionation of Ruta gruveolens L. leaf extracts showed chloroplasts to be the major if not exclusive site of umbelliferone:dimethylallyl pyrophosphate dimethylaliyl transferase (EC 2.5.1.3 tentative), which mediates the synthesis of demethylsuberosin, the first committed step in the formation of linear furanocoumarins. Tritium from [G“Hlumbelliferone administered to leaves is concentrated in association with the chloroplasts, as demonstrated by autoradiography. Most of the enzyme associated with a particulate fraction sedimenting at 30,OOOg could be solubilized by washing in lowmolarity (5 mM) Tris-HCI buffer, suggesting attachment to the membrane by weak ionic bonds. The soluhilized enzyme from cultured cells of R. gruueolens was purified 330-fold and characterized. It exhibits a broad pH optimum between 7.4 and 7.6. The apparentKi, approximated 60 PM for umbelliferone and 20 FM for dimethylallyl pyrophosphate. Sulihydryl-binding reagents were not inhibitory, indicating no involvement of thiol groups at the active site. Even at high concentrations, strong inhibition was not observed in the presence of demethylsuberosin or several biosynthetically related coumarins. The significance of these findings in the biosynthesis of furanocoumarins is discussed.

pothesis that the benzpyrone nucleus of furanocoumarins derives from umbelliferone by the partial pathway shown in Fig. 1. It is now well established that the conversion of umbelliferone to linear furanocoumarins proceeds via demethylsuberosin (6-dimethylallylumbelliferone), and that the prenylation of umbelliferone is the first committed step in the biosynthesis of psoralen and substituted linear furanocoumarins. Evidence for the nature of the pathway beyond demethylsuberosin also exists and has been discussed elsewhere (5, 7, 10-13). Ellis and Brown (14) have described an enzyme, a dimethylal1ylpyrophosphate:umbelliferone dimethylallyltransferase, which we shall call a prenyl transferase hereafter, mediating the prenyl group transfer to umbelliferone in cell cultures and young shoots of Ruta graveolens L., a species that elaborates a

Furanocoumarins form a well-known group of naturally occurring substances, some of which (those falling into the linear furanocoumarin category) have strong skin photosensitizing properties (1). In addition, when irradiated at 365 nm, they exhibit other biological effects of which two examples are mutagenic action on Sarcina lutea (2) and the loss of the tumor transmitting capacity of Ehrlich ascites cells (3). Studies on their biosynthesis are of interest in this context and also for the connection these compounds probably represent between the shikimate and mevalonate pathways. Such studies in several laboratories (4-9) have supported the hy‘This research was supported by Operating Grant A2487 of the National Research Council of Canada. 2 Present address: Department of Biochemistry, University of Chicago, Chicago, Ill. 60637. n To whom requests for reprints should be sent. 74 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

UMBELLIFERONE:DIMETHYLALLYLTRANSFERASE

oimethylaliyl Pyropharphate

FIG.

1. Partial

DeMlethYiWbWOSl”

pathway

variety of linear furanocoumarins. This particulate enzyme catalyzes attachment of a &carbon, but not lo-carbon prenyl side chain at position-6 of umbelliferone, forming none of the g-isomer which is the precursor of the angular furanocoumarins that are formed together with the linear in some other species. In the present paper we describe procedures leading to the solubilization of this particle-bound prenyl transferase and its 330-fold purification. It has been more fully characterized, and its location within the cell has been established. A preliminary report of part of this work has been presented (151. EXPERIMENTAL

OF RUTA

PROCEDURES

Cell culture. Cells from cultures ofR. graveolens, maintained as previously described (13), were harvested lo-14 days after transfer, during which period the prenyl transferase activity is near its maximum (14). Reagents. The dimethylallyl pyrophosphate used was the same as in a previous study (14). Umbelliferone, tritiated by catalytic exchange (Amershami Searle) was purified chromatographically before use on a column of silica gel H developed with chloroform containing 5% methanol. Glutaraldehyde was a 2.5% aqueous solution (Fisher Scientific Co.). Hydroxylapatite was prepared according to Levin (16). Farnesyl pyrophosphate, the generous gift of Professor H. C. Rilling, was tested for purity before use by paper chromatography (Whatman No. 1) in propanol-concentrated NH,OH-water, 36:9:15. Farnesyl pyrophosphate CR,, 0.8) was made visible by spraying, first with 0.1% FeCl,% and then with 0.5% sulfbsalicylic acid, both in 80%~ ethanol. All other reagents were of the highest commercial purity a,lailable. Analyses. Protein was estimated by the method of Lowry et al. (17) with a crystalline bovine serum albumin (fraction V, Sigma) as standard. Effluent from columns was monitored by the procedure of Warburg and Christian (18). Tritium was analyzed by scintillation spectrometry. Enzyme assay. The assay system contained the

of psoralen

75

Psordlen

biosynthesis.

following in a total volume of 1.0 ml: Tris-HCI (pH 7.5), 100 pmol; [G-:‘H]umbelliferone (2.1 x lo6 dpm), 10 nmol; dimethylallyl pyrophosphate, 100 nmol; MnSO,, 5 pmol; enzyme protein, lo-28 pg. Incubation was for 60 min at 30°C. Isolation and measurement of the labeled demethylsuberosin were as previously described (14). Studies of enzyme activity in chloroplasts. Chloroplasts containing active prenyl transferase were prepared as described in Table I. Chloroplasts prepared by this procedure were suspended in 50 ml of 5 mM Tris-HCI buffer, 1 rnM in EDTA (pH 7.6) and ruptured by treatment with a Bronwill Biosonik ultrasonic oscillator for 30 s at maximum output. The broken chloroplast suspension was then stirred for 30 min, and particulate matter was separated by centrifugation at 20,OOOg for 30 min. Autoradiographic studies. [G-:‘HlUmbelliferone (70 pg, 5.8 mCi/mmol) in 0.5 ml of water was fed as the sodium salt to 1.0 g of young, garden-grown leaves of R. graveolens under continuous illumination for 26 h, the leaves being placed in water after uptake of the initial solution. After the feeding period the leaves were chilled for 10 min and homogenized in a mortar at 4°C with buffered sucroseglutaraldehyde solution as described by Sabatini et al. (19). The solution contained 10.5 ml of 0.1 M phosphate buffer (pH 7.41, 10.5 ml of 0.4 M sucrose, and 6.5 ml of glutaraldehyde solution. The addition of glutaraldehyde lowered the pH to 6.9. The leaf debris was removed by filtration through four layers of cheesecloth and the resulting filtrate was centrifuged at 200g for 2 min at 4°C to remove cell debris. The supernatant was then centrifuged 7 min at 700g. The resulting pellet was suspended in 5 ml of the above buffer and recentrifuged for 7 min at 700g to remove any radioactivity adsorbed on the chloroplast surfaces. This second pellet was resuspended in the buffer and used for autoradiographic studies. Acid-cleaned microscope slides were immersed in a solution composed of 5.0 g of gelatin and 0.5 g of chrome alum per liter (20) and dried in air. A drop of chloroplast suspension was then spread on a treated slide; after drying, the mounted chloroplasts were placed in contact with Kodak autoradiographic stripping emulsion, AR 10, for 8-10 days. Procedures used in the preparation and processing of the stripping emulsion were essentially those given in the

76

DHILLON TABLE

I

DISTRIBUTION OF PRENYL TRANSFERASE FROM Ruta gravedens LEAVES

Fraction”

Chloroplast Supernatant

ACTIVITY

Specific activity” (Xl@“)

Percentage of total activity

9.93 2.13

67.7 32.3

(1 Young leaves (20 g) were freed of petioles, washed twice with deionized water, blotted dry, and frozen at -20°C to facilitate homogenization. Subsequent manipulations were at 0-4°C. The material was homogenized in a Waring Blender for 2 min at low speed with 2 vol (w/v) of Honda’s (211 medium (2.5% Ficol, 5% dextran T-40, 0.25 M sucrose, 0.1 M MgCl,, 0.025 M Tricine, 1% sodium ascorbate, final pH 7.9) and then for 2 min at high speed. The suspension was filtered through six layers of cheesecloth and centrifuged for 1.5 min at 4000g. The resulting green pellet constituted the chloroplast fraction. Chloroplasts were ruptured by sonication before assay (cf. Experimental Procedures). b Expressed as nanomoles of demethylsuberosin formed per milligram of protein per hour at 30°C under standard assay conditions. c Calculated from specific activity and total amount of protein in the fraction. Kodak data sheet SC-lo. The emulsion, with the chloroplast specimen attached, was developed and dried in a stream of cold air and the slides were mounted in glycerol jelly. Photomicrographs of the developed film were taken at a magnification of 500 with a green filter. Enzyme purification. R. gruueolens culture cells (100 g) were harvested, washed three times with deionized water, and drained for 5 min. All further manipulations were at 0-4°C. The cell clumps were homogenized with twice their weight (w/v) of 0.5 M Tris-HCl buffer (pH 7.51, 1 mM in P-mercaptoethanol, in a blender at top speed for 5 min. The resulting suspension was squeezed through eight layers of cheesecloth and centrifuged at 10,OOOg for 10 min. After suspension in 5 mM Tris buffer, the particulate fraction was stirred for 1 h to dissolve the enzyme. The supernatant from centrifugation at 20,OOOg for 30 min was subjected to ammonium sulfate fractionation, the precipitated protein being collected by centrifugation (lO,OOOg, 10 min). The active fraction was dissolved in the minimal volume of buffer and dialyzed overnight against 5 mM Tris buffer (pH 7.51, 1 mM in P-mercaptoethanol, and the dialyzate was concentrated against sucrose for 5 h. Further purification on columns of DEAE-cellulose and hydroxylapatite is described in Figs. 3 and 4. RESULTS

Localization

of the enzyme

in the cell.

AND

BROWN

The distribution of prenyl transferase in the cell was examined by isolation of the chloroplasts from R . graveolens leaves. The results in Table I clearly indicate that the activity resides predominantly in the “low-speed” chlorophyll-containing fraction - the chloroplasts. Approximately 68% was recovered in the pellet (4OOOg), which also contained 80% of the chlorophyll. The intracellular location of the enzyme was next studied by rupture of the chloroplasts by sonication in hypotonic buffer and assay of the stroma and lamellar membranes for enzyme activity. Sixty-two percent was soluble and was found in the stroma remaining in the supernatant after centrifugation at ZO,OOOg, but 38% remained bound to the membranes, from which it could be liberated by further washings in low-molarity buffer (see next section). Supporting evidence for the involvement of the chloroplasts in the prenylation reaction was obtained from autoradiographic studies. Tritiated umbelliferone was administered to Ruta leaves, and chloroplasts were isolated and submitted to autoradiography on stripping emulsion. Development after 8 days of exposure showed a relatively heavy distribution of silver grains in the emulsion in contact with the chloroplasts, while very few grains were detectable in the background areas (Fig. 2). As the figure shows, photomicrographs of the chloroplasts and the superimposed silver grains indicated that most of the radioactivity was associated with the chloroplasts, either within these organelles or residing on their surfaces. Solubilization of the enzyme. When the “low-speed” homogenate from culture cells of R. graveolens was centrifuged at 30,OOOg for 30 min, the enzyme activity was found to be quantitatively associated with the pellet (14). As shown in Table II, it could be released by washing of this particulate fraction with Tris-HCI buffer solutions (pH 7.6) of decreasing molarity, upon which increasing amounts of activity appeared in the supernatant. Three such washings for 30 min at 4°C were sufficient to release over 92% of the enzyme originally associated with the particulate frac-

UMBELLIFERONE:DIMETHYLALLYLTRANSFERASE

FOG. 2. Photomicrograph isolated from R. g~uueolens

(X 500) of autoradiographic leaves after administration

tion. Much less activity was released in this way from the particulate fraction of leaves. To determine whether the solubilized enzyme could be resedimented by prolonged centrifugation, previously solubi-

OF

emulsion exposed of [G-‘lH1umbelliferone.

RUZ’A

to a chloroplast

preparation

lized enzyme from cell cultures, frozen for 4 days, was centrifuged for 5 h at 3O,OOOg, and the supernatant and particulate fractions were assayed. The bulk of the activity remained in the supernatant (920/c), and the small loss observed can be attrib-

78

DHILLON

AND

uted to the effect of freezing and thawing. Enzyme purification. Results of the purifkation of the cell culture transferase are summarized in Table III. In the ammonium sulfate fractionation the activity was

BROWN

concentrated in the protein precipitating between 35 and 90% saturation. Further purification was achieved on a DEAE-cellulose column developed with Tris buffer (Fig. 3); prenyl transferase activity was

TABLE

II

SOLUBILIZATION OF R. graveolens PARTICLE-BOUND PRENYL TRANSFERASE Source

Culture

cells

Leaves

Fraction

Treatment”

Protein

Enzyme

activity

(mg)

(%I*

with cen-

Particles Supernatant

150 119

55.8 44.2

(l), incumM Tris

Supernatant

35

13.0

700

10.4

(21, incumM Tris

Supernatant

5

1.9

990

14.7

(31, incumM Tris

Particles Supernatant

24 72

8.8 26.8

450 1,330

6.7 19.7

(1) Extraction of leaves with Tris buffer, then centrifugation (2) Particles from cl), incubated in 30 mM Tris buffer (3) Particles from (21, incubated in 5 mM Tris buffer

Particles Supernatant

27.8 11.4

70.9 29.1

20,000 2,250

89.9 10.1

Supernatant

16.9

43.1

4,890

22.0

Particles Supernatant

0.9 3.3

2.3 8.4

530 4,970

2.4 22.3

(1) Extraction of Tris buffer, trifugation (2) Particles from bated in 30 buffer (3) Particles from bated in 5 buffer (4) Particles from bated in 3 buffer

cells then

(totaUc

(%I

3,520 3,220

52.2 47.8

-

a Leaves or 14-day-old cultured cells of Ruta graueolens (15 g) were harvested (14) and homogenized with 10 ml of 0.5 M Tris-HCl buffer (pH 7.61, 1 mM in EDTA, with sand, in a mortar for 10 min. The resulting suspension was filtered through six layers of Miracloth and the filtrate was centrifuged for 30 min at 20,OOOg. The resulting particles were suspended in 10 ml of 30 mM Tris-HCl buffer, 1 mM in EDTA. The green particulate suspension was stirred for 30 min at 0-4°C and then centrifuged at 20,OOOg. The particles were subjected to repetitions of this treatment in the buffer solutions shown. The final pellet was suspended in 5 ml of 5 mM buffer. Protein and enzyme activity were assayed at each stage as described under Experimental Procedures. b In each case the total of particles and supernatant from Treatment 1 is taken as 100%. ’ Expressed as nanomoles of demethylsuberosin formed x lo3 under standard assay conditions. TABLE

III

PURIFICATION SCHEME FOR PRENYL TKANSFERASE OF R.graveolens CELL CULTURES Volume Protein Total proSpecific activYield Step yy$ 0 tein ity” (ml) (mgiml) (%I bid 20,000 g supernatant 35-90% ammonium sulfate DEAE-cellulose Hydroxylapatite a Expressed as nanomoles standard assay conditions.

(x103)

186.0

29.5

5506

31

1

100

47.0

62.3

2927

855

28

1470

21.0 8.0

38.0 90.0

798 720

2910 10,200

94 330

1360 4300

of demthylsuberosin

formed

per milligram

of protein

per hour

at 30°C under

UMBELLIFERONE:DIMETHYLALLYLTRANSFERASE

OF

79

RUTA

phosphate, like the geranyl pyrophosphate previously examined (14), was inactive as a substrate. The K, for umbelliferone was calculated to be 57 PM -t 180/o, and for dimethylallyl pyrophosphate, 18 PM f 15%. DISCUSSION

FIG. 3. Elution pattern of protein and prenyl transferase activity from DEAE-cellulose column. Solid line represents changesin absorbance at 280 nm; broken line, changes in prenyl transferase activity as nanomoles of demethylsuberosin formed by the fraction in I h at 30°C. A concentrated 35-90s ammonium sulfate fraction was applied to the column (2.5 x 30 cm, Whatman DE521 previously equilibrated with 5 mM Tris buffer. The column was washed with the starting buffer (about 1 liter) until the absorbance at 260 nm had dropped to 0.05 and then developed with a linear gradient of 200 ml each of 0.005 M and 0.5 M Tris buffer (pH 7.5). Fractions of 5 ml were collected.

associated with two peaks eluted in fractions 18-30. When these combined fractions were chromatographed on a column of’ hydroxylapatite developed in phosphate buffer (Fig. 41, activity coincided with the major protein peak, appearing in fractions 3-8. These combined fractions were used as the source of the enzyme. The half-life of the enzyme, stored frozen, was about 2 weeks. Other methods, such as storing samples in glycerol solutions of various concentrations, or with bovine serum albumin, failed to stabilize the enzyme. Properties of the enzyme. The pH-activity curve exhibited a broad maximum between 7.4 and 7.8 in Tris-maleate buffer. The effects of sulfhydryl-binding reagents are shown in Table IV. Iodoacetate andpchloromercuribenzoate did not inhibit the enzyme and, in fact, augmented the activity, the latter almost twofold. The effect of several coumarins on the activity of the prenyl transferase was tested at concentrations approximating 0.5 mM. Marmesin, psoralen, isopimpinellin, and Chydroxybergapten had no clear effect on the activity. Bergapten and demethylsuberosin reduced the activity to 69 and 45% of the control, respectively. Farnesyl pyro-

Activation during purification. The final column of Table III reveals a striking activation of the enzyme in two stages of the purification process. In the ammonium sulfate precipitation the removal of just under half the total protein was accompanied by more than a 14-fold increase in 1.0

r

FIG. 4. Elution pattern of protein and prenyl transferase activity from hydroxylapatite column. Combined active fractions from a DEAE cellulose column were dialyzed overnight against three changes of deionized water, concentrated about eight-fold against sucrose and applied to the column (2 x 10 cm) of hydroxylapatite previously equilibrated with 1 mM phosphate buffer (pH 6.8). The column was washed with ca. 100 ml of the 1 mM buffer and elution of the enzyme was effected with a linear gradient of 100 ml each of 0.01-0.3 M phosphate buffer (pH 6.8). Fractions of 5 ml were collected. Solid and broken lines are interpreted as in Fig. 3.

TABLE EFFECT

OF SULFHYDRYL-BINDING PRENYL TRANSFERASE

Compound None Arsenite Iodoacetate p-Chloromercuribenzoate

added”

IV REAGENTS ACTIVITY

Percentage activity

ON

of

100 93 121 196

a Added as sodium salts. Normal order of addition was buffer, Mn’+, umbelliferone, dimethylallyl pyrophosphate, sulfhydryl-binding reagent, enzyme (28 pg). Assay conditions were otherwise standard.

80

DHILLON

AND

yield. Similarly, chromatography on hydroxylapatite resulted in a threefold increase in yield accompanied by only a 10% loss of protein. We have not pursued the reason for this phenomenon, but prima facie the most probable explanation would appear to be the removal of inhibitors during the two steps in question. Properties of the enzyme. The data presented in Table II show that, at 0°C in the presence of EDTA, successive incubations of the broken Ruta culture cells with TrisHCl buffers of decreasing molarity, down to 3 mM, solubilized about 92% of the enzyme. Much less of the leaf enzyme could be released in this way. Comparable behavior has been described for other enzymes. By washing the membranes prepared from various microbial cells with low-molarity Tris buffer, Abrams’ group (22, 23) and Munoz et al. (24) were able to solubilize much of the associated ATPase activity. Solubilization was accompanied by a rise in specific activity up to 50-fold. Similarly, the solubilization of particulate dehydrogenases from Clostridium kluyveri has been reported by Hillmer and Gottschalk (25). Nakagawa et al. (26) have found an analogous phenomenon with plant preparations; the p-fructofuranosidase from tomato cell walls could be solubilized by incubation in 10 mM phosphate buffer at alkaline pH, but under acidic conditions remained firmly attached to the cell wall. This effect has been attributed by Hillmer and Gottschalk (2.5) to a loosening of weak ionic bonds, which may involve divalent cations (231, connecting the enzyme protein to its matrix. It would appear that the present observation represents yet another example of the same effect. Failure of the transferase to be inhibited by sulfhydryl-binding reagents suggests that the sulfhydryl groups in this enzyme are relatively exposed, and not part of the active site. Some other enzymes as, for example, DNA polymerase (271, are likewise insensitive to inhibition by sulfhydryl-binding reagents. We can offer no explanation for the increased activity observed in the presence of iodoacetate and, especially, p-chloromercuribenzoate, but

BROWN

we note that stimulation in the presence of sulfhydryl-binding reagents has been reported (see, e.g., 26). The K, values for dimethylallyl pyrophosphate and umbelliferone were calculated to be 57 FM 5 18% and 18 FM k 15%, respectively. The relatively high standard errors derive from the techniques employed for measurement of the reaction. In the absence of a continuous assay we were compelled to rely on the assay of tritium in the reaction product - demethylsuberosin - isolated chromatographically after brief incubations with tritiated umbelliferone (14), as a measure of initial reaction rates; such a procedure is obviously less accurate than, for example, a continuous spectrophotometric assay. Similar dificulties were encountered by Holloway and Popjak in their study of a prenyl transferase from pig liver mediating the synthesis of farnesyl pyrophosphate (28). Even at the high concentrations used, furanocoumarins and biosynthetically related coumarins did not strongly inhibit prenyl transferase activity. It is therefore clear that neither product inhibition by demethylsuberosin nor feedback inhibition by end-products of the biosynthetic pathway are important factors. Previous data (14) had indicated that sidechains longer than five carbons are not attached to umbelliferone by this transferase, in that geranyl pyrophosphate did not serve as a substrate. The finding in the present study that farnesyl pyrophosphate is similarly inactive reinforces the conclusion reached earlier that the enzyme is highly specific with respect to substrates. Significance for furanocoumarin biosynthesis. The involvement of demethylsuberosin in the biosynthetic pathway to linear furanocoumarins was earlier suggested by radiotracer experiments on both cell cultures and organized plants (6, 9, 13), which strongly indicated that this compound is an intermediate in the formation of psoralen and its substitution products. The identification by Ellis and Brown (14) of the prenyl transferase mediating the formation of demethylsuberosin from umbelliferone, and the characterization and purification described here, confirm

UMBELLIFERONE:DIMETHYLALLYLTRANSFERASE

its role as the first committed intermediate in this pathway. The reaction in question is one of a number found in nature in which an isoprenoid substituent is introduced into an aromatic nucleus. A few of the many examples known are the isoprenylation of p-hydroxybenzoic acid during the biosynthesis of novobiocin (29) and of the indole nucleus in the formation of cyclopiazonic acid (301, echinulin (311, and dimethylallyltryptophan (32). Enzymes mediating the attachment of the prenyl group of the latter two compounds, both microbial metabolites, have been described (31, 32). In the accompanying paper, Lee, Floss, and Heinstein (33) report purification of dimethylallylpyrophosphate: tryptophan dimethylallyl transferase to apparent homogeneity, and its extensive characterization. In view of the wide occurrence of these prenylation reactions, the studies reported in the present paper have yielded results which not only represent an important step in the understanding of linear furanocoumarin biosynthesis, but have wider implications as well. The investigations we have done on the intracellular site of the enzyme show that by far the greatest part, if not all of the activity in R . graveolens leaf cells resides in the chloroplast-rich fractions sedimentable by low-speed centrifugation. Since only a small part of the activity was found in the supernatant after removal of the ch loroplast fraction, it is unlikely that the enzyme is associated with lighter particles such as mitochondria or microsomes trapped in the pellet, because most of these would remain in the supernatant under the conditions employed. Phase contrast microscopy showed the presence of some broken chloroplasts in the preparation, and it appears much more likely that the act,ivity in the supernatant is attributable, at least for the most part, to leaching of the enzyme from these broken chloroplasts after rupture of the envelope. Autoradiographic studies showed a concentration of tritium associated with the chloroplasts after administration of tritiated umbelliferone to leaves. No inference can be drawn from this observation alone

OF

RUTA

81

about the site of the prenyl transferase in the cell, but it does reinforce the conclusion drawn from the assays of the enzyme in isolated chloroplasts and is consistent with localization of the enzyme in that organelle. It was also, of course, impossible to determine whether all of the tritium was present as umbelliferone, or if not, how much was in the form of its metabolic products. The autoradiographic studies and the data obtained following rupture of the chloroplasts do not provide an unambiguous answer to the question of where the enzyme is located within the chloroplast, revealing only that the major part of it is not readily sedimented. About twothirds was easily solubilized, but the remainder stayed with the lamellar membranes, and a more detailed examination of these two readily obtained fractions would be necessary to resolve this question. Evidence has accumulated in recent years suggesting that the chloroplast may be a major site of reactions leading to the simple coumarins (those lacking additional rings fused to the benzpyrone nucleus). Thus, in 1967, Sato (34) showed that esculetin (6,7-dihydroxycoumarin) can be formed from cis-caffeic acid in Saxifraga stolonifera chloroplasts, probably by a phenolase, with the ortho-quinone as intermediate. Although this is not a typical coumarin biosynthetic reaction, it is of interest in the light of later work by Kind1 (35) and by Gestetner and Conn (36) which has identified the chloroplast as the site of what can be regarded as the most fundamental reaction in the biosynthesis of all coumarins, the introduction of a hydroxyl function ortho to the sidechain of a phenylpropanoid precursor, in this case cinnamic acid. The present findings strongly suggest that chloroplast involvement in the formation of coumarins is not confined to simple members of this class, but must be considered in the biosynthesis of furanocoumarins as well. The extent to which subsequent reactions in the pathway may be mediated by chloroplast enzymes is unknown, but the fact that O-methyltransferase activity found in Ruta extracts, mediating the final step in the elaboration of

82

DHILLON

AND

methoxylated psoralens, does not appear to be associated with chloroplasts (H. J. Thompson and S. A. Brown, unpublished work), leaves open the possibility that other cellular sites may be involved in later steps of the pathway. If, in the species that have been tested, the early reactions in the conversion of mevalonate carbons 4 and 5 to furan ring carbons of furanocoumarins are confined to the chloroplast, the known low permeability of the chloroplast envelope membrane to mevalonate (37) provides an adequate explanation of the very low in uiuo incorporations of mevalonate into furanocoumarins which have been observed (8, 38). When the specificity of incorporation of 14C from labeled mevalonate into the expected carbon of the furan ring (4, 8) is also considered, there is no longer reasonable doubt that, at least for coumarins, the mevalonate-dimethylallyl pyrophosphate pathway is the major, if not exclusive, biosynthetic route to the furan structure. ACKNOWLEDGMENTS We are grateful to department for several methylallyl pyrophosphate by Professor B. E. Ellis Ontario, and farnesyl H. C. Rilling of the Center, Salt Lake City.

Prof. R. G. Annett of this very helpful discussions. Diwas generously provided of the University of Guelph, pyrophosphate by Professor University of Utah Medical

L., AND RODIGHIERO, G. (1970) PhotoPhotobiol. 11, 27-35. 2. MATHEWS, M. M. (1963) J. Bacterial. 85, 322-

AND 336.

L., RAZZI,

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(1966)

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23, 335Phyto-

S. A., EL-DAKHAKHNY, M., AND STECK, W. (1970) Canad. J. Biochem. 48, 863-871. GAMES, D. E., AND JAMES, D. H. (1972) Phytochemistry 11, 868-869. DALL’ACQUA, F., CAPOZZI, A., MARCIANI, S., AND CAPORALE, G. (1972) 2. Naturforsch. 27b, BROWN,

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18. WARBURG, O., AND CHRISTIAN, W. (1942) Biothem. 2. 310, 384-421. 19. SABATINI, D. D., BENSCH, K., AND BARRNETT, R. J. (1963) J. Cell Biol. 17, 19-58. 20. GUDE, W. D. (1968) Autoradiographic Techniques, p. 23, Prentice-Hall, New York. 21. HONDA, S. I., HONGLADAROM, T., AND LATIES, G. G. (1966) J. Exptl. Botany 17, 460-472. 22. ABRAMS, A. (1965) J. Biol. Chem. 240, 3675 3681. 23.

813-817. 8. KUTNEY, J. P., VERMA, A. K., AND YOUNG, R. N. (1973) Tetrahedron 29, 2645-2660. 9. BROWN, S. A., AND STECK, W. (1973) Phytochem-

ABRAMS, A., AND BARON, 7, 501-507. Murjoz, E., SALTON, M. SCHOR, 501. HILLMER,

M. T. (1969)

C. (1968) Biochemistry

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