The polyglutamate nature of plant folates

Phyrochemisfry. Vol. 31, No. 7, pp. 2277 2282, 1992 Printed in Grcac Britain.

THE POLYGLUTAMATE Lr-LI ZHENG,* MNG

003 1 9422p2 $5.00 + 0.00 Q 1992 Pergamon Press Ltd

NATURE LIN, SONG LIN

OF PLANT

FOLATES

and EDWIN A. COSSINS

Department of Botany, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada (Receioed in reuisedjorm 25 September 1991) Key Word Index-Higher

plants; folylpolyglutamatw; HPLC; folate hydrolase.

Abstract-Folylpolyglutamates were extracted from a variety of higher plant tissues and cleaved to p-aminobenzoyl polyglutamates by acid-base and Zn-HCI treatments. After purification as azo dyes and reconversion to p-aminobenzoylpolyglutamates these derivatives were separated according to their glutamyl chain lengths by HPLC on Partisil SAX. Polyglutamate concentrations were calculated by reference to standard calibration curves. Tomato leaf extracts contained relatively high folate contents (7.55 nmol/g fr. wt) and these were mainly hexa- (23%) and heptaglutamates (30%). Carrot root extracts had low folate contents (0.22 nmol/g fr. wt) with diglutamates accounting for 69% of the folate recovered after chromatography. Carboxypeptidase treatments of broccoli floret extracts indicated that the folylpolyglutamates principally contained y-glutamyl linkages. C3H]Glutamate and [14C]p-aminolxnzoate were incorporated into the polyglutamates of broccoli seedlings but equilibrium of the label with the endogenous pools was not attained after a 48 hr pulse feeding. Differential, oxidative cleavage of broccoli leaf folates into three polyglutamate pools showed that the formyl- and methylfolates were mainly (80%) diglutamyl derivatives. The methylene and unsubstituted folates of this tissue were principally (78%) hexaglutamates.

INTRODUCTION

One-carbon substituted derivatives of tetrahydrofolate (H,PteGlu) are important metabolic precursors of purines, thymidylate, formylmethionyltRNA, serine and methionine [2]. In their roles as donors of one-carbon units these folates function most effectively as y-glutamyl conjugates [3]. As a result, mutant cells that lack the ability to generate these folylpolyglutamates commonly display auxotrophies for methionine and other products of one-carbon metabolism [4]. In addition, folatedependent enzymes usually display much greater affinities for the polyglutamate forms of their folate substrates than for the corresponding monoglutamyl folates [3, S]. Folylpolyglutamates are also preferentially retained by living cells [3, S] and appear to have variable glutamyl chain lengths [3, 63 in different species. There is also good

evidence that these folates have roles in metabolic regulation [S, 7, 81. A number of microbiological assays have confirmed that plant folates,.like those of other organisms, occur principally as highly conjugated derivatives [2,6]. Plant folates are also compartmented [4] and are produced from H,PteGlu by folylpolyglutamate synthetase activity [9, lo]. There is, however, relatively little information on *Present address: Xiongyue Agricultural College, Xiongyue, Liaoning Province, People’s Republic of China. Abbreviations:The abbreviations for folate derivatives are those suggested by the IUPAC-IUB Commission aa summarized by Blakley and Benkovic [I]: e.g. H,PteGlu = 5,6,7,8tetrahydropteroylmonoglutamate; S,lO-CH,-H+PteGlu = 5,10methylenetetrahydropteroylmonoglutamate; H,PteGly,=polyy-glutamyl derivatives, where n= the number of ~-glutamate moieties. p-ABA and pABAGlu,=paminobenzoate and its polyglutamates where II= the number of r-glutamate moieties.

the polyglutamate chain lengths of native higher plant folates. Column chromatography, combined with differential microbiological assays, provided evidence for 5-CH,-H,PteGlu, and 5-CH,-H,PteGlu, in cabbage [I l] and for a range of mono- to pentaglutamyl folates in lettuce [12]. In more recent work [ 133, we used the folate cleavage and HPLC procedures of Shane [ 141 to analyse a pool of pentaglutamyl folates in pea seedlings. As this latter method allows the concentration of p-ABAGlu, derived from folate polyglutamate cleavage, it appears to have considerable potential for use in the analysis of folates from a range of plant species. In the present work, we have examined extracts of nine different plant tissues by HPLC to determine their polyglutamate chain length distributions.

RESULTS HPLC analysis of plant folates and eflects of carboxypeptidase treatments

Extracts of several plant species (Table 1) were examined for polyglutamyl folates using HPLC [ 14, 151. This involved reductive cleavage of the folates lo p-ABAGlu, followed by separation according to chain length on Partisil 10 SAX [13, 153. For quantitative determinations, calibration curves were constructed using standard p-ABAGlu, (n = l-6) solutions. The relationship between peak area and the quantity of pABAGlu, injected was linear in the 0.1-10 nmol range. However, the line for each of these polyglutamates had a significantly different slope, even after correction for non-folate impurities present in the standard solutions. Recoveries of standard PteGlu, averaged 42.4% when samples containing 36 nmol were taken through the complete acid-base and

2277

2218

L.-L.

ZHENC

et d.

Table I. Polyglutamate chain lengths of some plant folates

Species

Tissue

Cauliflower Broccoli

Florets Florets Florets Florets 3-week-old leaves 12-week-old leaves Leaves Leaves

Tomato Lettuce Cabbage

LGiVCS

Leaves IO-day-old leaves 4-week-old leaves Roots

Pea Carrot

Total folate recovered*

-. -. 2

0.8 1 1.37 1.00 1.20 7.55 2.29 1.87 1.50 0.26 0.2 1 4.65 1.20 0.22

22.1 41.9 40.9

I x.0 8.4 19.4 27.6 49.6 45.5 45.7 53.2 69.3

.-

---- ---3 7.8 19.5 22.6 1.3 8.5 29.9 26.9 11.5 15.5 18.6

Polyglutamate distributlont .4 5 6

5.2 3.1 10.2 12.7 16.5 23.4 24.8 32.5 9.2

4.5 4.4 5.4 14.8 9.6 15.2 37.1 72.1 27.3 29.7 10.5 22.1 I21

7

8

22.2 7.2 3.2 11.8 23.2 33.7 --

36.0 13.2 10.3 30.0 30.4 --. --

6.8 7.7 12.3 38.5

-. ..-

-

_.

*Data are expressed in nmol/g fr. wt and are average values of three separate extractions of each tissue sample. tData are expressed as percentages of total polyglutamates recovered after Zn-HCI cleavage and HPLC as p-ABAGlu, derivatives.

Zn-HCI cleavage procedure [14]. This value rose to 67.3% if the acid-base steps were omitted. None of the extracts examined contained detectable amounts of p-ABAGlu, (Table 1). However, peaks of more highly conjugated derivatives were resolved for each of the analysed tissues (Table 1). In leaf extracts of lettuce, cabbage and pea (Table I), these polyglutamates had chain lengths up to pentaglutamate. In cauliflower, broccoli and tomato the pool contained significant amounts of more highly conjugated folates. In broccoli, a small absorbance peak, corresponding to the pentaglutamate (average R, 45.3 min) was followed by a distinct and slightly larger peak with an average R, of 47.9 min. This latter peak did not co-chromatograph with either p-ABAGIu, or p-ABAGlu, standards. The polyglutamate nature of the folates in broccoli floret extracts was also examined after carboxypeptidase treatments (Table 2). The peptidases of chicken pancreas and pea cotyledons hydrolyse folylpolyglutamates to short-chain products by cleavage of y-glutamyl bonds [3]. In contrast, yeast carboxypeptidase Y specifically cleaves cr-carboxyl-linked glutamates in naturally occurring peptides but not the ;I-glutamyl bonds found in most folates [ 163. As expected, incubation of broccoli extracts with the avian and plant enzymes caused a redistribution of polyglutamate chain lengths that favoured less highly conjugated derivatives (Table 2). Incubation of the extracts with the yeast enzyme resulted in loss of the unidentified peak (R, 47.9 min) and this was accompanied by an increase in the recovery of p-ABAGlu, (Table 2). Incorporation

qf

[‘4C]p-ABA

into broccoli

polyglutamates The folate pool of 14-day-old broccoli seedlings and cotyledon disks excised from these seedlings was examined after feeding [“C]p-ABA (Fig. 1) and C3H]glutamate (data not shown). These precursors were incorporated into a number of folylpolyglutamates as

indicated by the labelling of pABAGlu, peaks. However, the distribution of 14C did not reflect the pool sizes of the various polyglutamates detected by absorbance measurements (Fig. 1). For example, radioactivity was readily detected in p-ABAGlu, but this derivative did not give a measurable peak area during HPLC analyses. Furthermore, the hexa- and heptaglutamyl derivatives, which had larger pool sizes, contained smaller percentages of incorporated “% (Fig. I). Differential cleavage of broccoli leaffolates The polyglutamate nature of methylene, methyl and formyl folates was determined after these pools were converted to p-ABAGI, derivatives by acid treatment and subsequent oxidation [17-193. Although this method has been used successfully in assays of liver folates [l7, 193, there have be-en no reports of analyses involving plant extracts. Data for the analysis of broccoli extracts, prepared from leaves of 90-day-old seedlings, are given in Table 3. These tissues were found to contain greater total folate contents than younger seedlings but they also lacked detectable pools of mono- and triglutamates. The three pools recovered by the differential cleavage procedure had quite different polyglutamate chain lengths (Table 3). In this regard, the derivatives of Pool 1 were mainly penta- and hexaglutamates. On the other hand, the methyl and formyl folates of broccoli leaves were mainly diglutamates accompanied by smaller amounts of tetraand heptaglutamates. Polyglutamate hydrolysis by broccoli leaf extracts Extracts of broccoli leaves had the ability to hydrolyse PteGlu, and PteGlu, in vitro. Optimal rates of hydrolysis occurred at pH 5.0 and 50% of maximal rates were observed at pH 4.3 and 5.8. This enzyme activity was recovered with protein precipitating in the 40-70% range of saturation with (NH,),SO,, a treatment that was

Plant folylpolyglutamates Table

Source

2. Polyglutamate

of

distributions

1

None (control) Chicken pancreas Pea cotyledons Yeast enzyme Y

extracts

Polyglutamate -------------.----__-_-

--.---

carboxypeptidase

in broccoli

2219 after carboxypeptidase distribution

treatments

(%)*

2

3

4

5

6

7

8

II.7

12.9

4.6

10.5

15.4

II.1 n.d. nd. 10.0

n.d.

33.8

n.d.

81.7

1.0

1.6

5.0

5.1

5.0

9.1

72.6

4.5

4.9

n.d.

53.6

2.1

7.8

8.8 n.d.

n.d. 13.1

n.d. 13.1

*Expressed as percentages of total folylpolyglutamates recovered as pABAGlu, derivatives after HPLC. Broccoli floret extracts, prepared from 10 g fr. wt and containing an average of 16.3 nmol total folate, were incubated with the various carboxypeptidases for 24 hr (see Experimental). The pentaglutamate data include a peak of absorbance at 280 nm (R, 47.9 min) that did not co-chromatograph with authentic pABAGlu, (R, 45.3 min).

the enzymic hydrolysis of PteGlu, was accompanied by formation of several, shorter chained polyglutamate derivatives (Fig. 3). DISCUSSION

”

(0 33) (1.19)

Ill

0L-L

Clu*

Glu3

Clq

Polyglutamate

Glug

GlU6

Chain

Lengths

(1 30)

Ill Glu7

Fig. 1. Incorporation of [‘4C]p-aminobenzoate into the folylpolyglutamates of broccoli seedlings. The labelled substrate (10 pCi, 0.17 pmol) was supplied to duplicate samples (0, n ) of excised 14-day-old seedlings (A) and 5 mm cotyledon disks (B). Tissues were maintained in a regime of I6 hr light: 8 hr dark at 20” for 48 hr (A) and 24 hr (B). Values m brackets are the average pool sizes (nmol/g fr. wt) of individual folylpolyglutamates recovered from each tissue sample after cleavage to p-ABAGlu, and analysis by HPLC.

accompanied by an increase in specific enzyme activity of cc five-fold. Analysis of the non-folate reaction products (Fig. 2) showed that [‘4C]PteGlu3 was converted into diglutamyl peptide and glutamate, with the latter being the major labelled product after 1 hr incubations. HPLC analyses of the folate products indicated that PteGlu accumulated during this reaction period without significant production of PteGlu, (data not shown). In contrast,

In agreement with earlier microbiological assays [l, 2,63, the total folate contents (Table 1) of young leaf tissues such as pea and tomato were relatively high when compared to those recovered from carrot root tissue. Older leaves of pea and tomato seedlings and leaf samples derived from market-purchased lettuce and cabbage had lower folate contents. Extracts of cauliflower and broccoli florets contained ca 1 nmol of total folate/g fr. wt. Assuming a recovery similar to that observed for PteGlu,, this value would be ca 2.5 nmol/g fr. wt. Other plants of dietary importance contain between 0.63 and 6.93 nmol of total folate/g fr. wt [6]. Despite good resolution of p-ABAGlu, by HPLC, we failed to detect this monoglutamate in the present study (Table 1). It therefore follows that these plant species, like other eukaryotes [3, 43, probably contain very low concentrations of monoglutamyl folates. On the other hand, all of the tissues examined contained significant amounts of polyglutamyl folates. The distribution of these derivatives varied in the plants examined and this has been noted in earlier studies of mammalian and avian tissues [3, 63. The carboxypeptidase experiments (Table 2) imply that the folylpolyglutamates of broccoli florets are mostly y-glutamyl derivatives. These enzymes, however, failed to reduce the area of the peak that was eluted just after authentic p-ABAGIu,. Although further analyses are necessary to establish the nature of this unidentified derivative the present work suggests that it may be a folate containing a-carboxyl-linked glutamates. Compounds of this nature have recently been isolated from E. coli extracts [16]. It is interesting to note that Zn-HCI cleavage of these folyl-sc-polyglutamates produces pABAGlu, derivatives with HPLC R,s that are slightly greater than those of the corresponding y-linked pABAGlu, standards [ 163. The data for [“C]p-ABA incorporation (Fig. l), like those reported for folate labelling in various animal tissues [6], suggest that the major polyglutamate pools in these plant tissues are not in equilibrium with the radioactive precursor, even after incubation for 48 hr. This could be partly due to the relatively slow glutamylation of

L.-L. ZHEF~G et al.

2280

Table

3. Distribution

of polyglutamate

chain lengths after differential

cleavage of broccoli leaf folates

Polyglutamate Folate pools subjected to oxidative 1. Methylene-H,PteGlu,,

cleavage

H,PteGlu,

H,PteGlu,

Pool size*

GIU,

GIU,

1.06

Trace

n.d.

4.0

2. Methyl-H,PteGlu, 3. Formyl-H,PteGlu,

I

*Data

analysis

are expressed as in Table

1. Pools I, 2 and 3 represented

detected. Trace, minor peak area not recorded by instrument

Note: Mono- and triglutamate derivatives were not parentheses are percentages of each folate pool occurring

E 0

z

6000

Glu, n.d.

0.23

0.83

(21.7)

(78.3)

0.19

n.d.

n.d.

(4.7) 0.02

nd.

n.d.

(5.7) 0.2 I

0.23

0.83

3.51

5.63

Glu,

3.23

(80.0) Total folate recovered after HPLC

Glu,

(X0.5) 0.28

0.35

methyl-H,PteGlu,

content (nmol/g fr. wt)

19.69’0, 74.0%

0.59 (14.7) 0.05 (14.3)

and 6.5%

0.64

of total folate, respectively. n.d.. not

integrator.

detected during

differential

as polyglutamates

cleavage of broccoli

leaf extracts.

Data

in

of the specified cham length

.

c3

s-

4000

2 d

D@ksnyl

0 0

10

20

30

40

50

Fraction

Fig. 2. Formation

of glutamate

60

with (NH&SO,

a0

90

peptide during

Broccoli leaf extracts

(see Experimental) and

Non-folate

by passage through

exchange

were recovered

cellulose and then separated

x 1.5 cm column of Dowex

by elution

respectively. ion-

from a 20

1-X8 (acetate form). Glutamate

eluted with 0.5 N acetic acid. Diglutamyl

were

and incubated

I hr (0)

with the folate substrate for 30 min (0) products

100 Polyglutemate

and diglutamyl

enzymic hydrolysis of [‘4C]PteGluJ. fractionated

70

number

was

peptide was recovered

by washing the column, after collection of fraction 57, with 5 N acetic acid. Fraction

size, 1.5 ml.

H,PteGlu in the folylpolyglutamate synthetase reaction [3, lo]. In addition, these plant polyglutamates are probably compartmented [2-41 into pools with variable rates of turnover. As a consequence, the distribution of label after pulse feeding will not reflect the endogenous folate patterns [ 17, 201. When the folates of broccoli leaves were subjected to differential cleavage (Table 3) it was clear that the various pools of C-l substituted derivatives had different polyglutamate chain distributions. For example, Pool 1 contained only trace amounts of diglutamyl folates whereas Pools 2 and 3 were largely of this chain length. Diglutamates were not detected in similar analyses of rat liver polyglutamates where Pools 1 and 2 are mainly pentaglutamates and Pool 3 is predominantly hexaglutamate in nature [17]. It Seems unlikely that the diglutamates we detected in Pools 2 and 3 (Table 3) arose by chemical modification of longer chain polyglutamates during the

Cham

Lengths

Fig. 3. Formation of polyglutamate products during enzymic hydrolysis of PteGlu,. Broccoli leaf extracts (see Experimental) were incubated wrth 30 nmol of PteGlu, in acetate buffer (pH 5.0) at 37.’ for 30 min

(?)

and

1 hr

(m). Folatc products were

recovered by elution from ron-exchange ABAGlu,

and analysed

duplicate

reaction

30 min and

1 hr

Polyglutamatcs

by HPLC.

systems. Total

cellulose, cleaved IO p

Data folate

are the averages of recovered

after

the

incubations was 33.6 and 27.0 nmol respectively. other

than

PteGlu,

were

not

recovered

in

systems lacking leaf extract.

cleavage procedure. If this had occurred diglutamates should have been detected in Ito and Krumdieck’s study [17]. We also rule out the possibility that diglutamates arose by enzymic hydrolysis during tissue extraction because we found only traces of p-ABAGlu, when the initial extracts were analysed for Pool 1 derivatives (Table 3). The presence of different polyglutamate pools in broccoli (Table 3) raises questions about their physiological roles and their intracellular localization. Work on various mammalian tissues [5] supports the contention that these conjugated folates are the preferred substrates for the pathways of one-carbon metabolism. If this conclusion has validity in higher plants it follows that S-CH,-H,PteGlu, may be a methionine precursor in broccoli as reported earlier for fungi [2]. Similarly, the more highly conjugated folates of Pool 1 (Table 3) should have importance in the serine hydroxymethyltransferase

Plant folylpolyglutamates

reaction, which in leaf tissues will be associated with the mitochondrial oxidation of glycine [2, 43. These conclusions, although feasible, may be too simplistic since work on liver folates has shown that polyglutamate chain lengths are affected by conditions that alter the steady state of one-carbon metabolism [21, 223. As a result, the concentration of various folate substrates and their degree of glutamyl conjugation affects the flux of one-carbon units through the major folate-dependent pathways [4,5]. The factors which alter polyglutamate distributions in plants are still largely unknown. Recent work [13] suggests that folate concentration may determine the degree of H,PteGlu conjugation by pea cotyledon folylpolyglutamate synthetase, a property shared with this enzyme from other eukaryotes [3]. The importance of folate hydrolases in shortening the polyglutamate chain of dietary folate is well documented [33. Recent inhibitor studies imply that endogenous folates may also be shortened by these enzymes [23]. It remains to be determined whether this activity in leaf extracts (Figs 2 and 3) has a physiological role in modifying folate pools and if such a change affects the pathways of one-carbon metabolism. EXPERIMENTAL

Chemicals. PteGlu, (n = 2-6). p-ABAGIu, (n = l-5) and y-glutamyl dipeptide were obtained from Dr B. Schircks Laboratories, Jona, Switzerland. Purity of polyglutamates was determined by calorimetric analysis [24]. [U-3H]Glutamate was supplied by Amersham-Searle and diluted with carrier Lglutamate to give 2.5 pCi/l.S pmol/O.l ml. Carboxyl-labelled [‘*C]pABA from Research Products International was dissolved in a minimal vol. of 95% EtOH and diluted with Ha0 to give 10 pCi/O.17 pmol/O.l ml of soln. [‘*C]PteGlu,, labelled in the terminal glutamate moiety, supplied by Dr Carlos Krumdieck, Department of Biochemistry, University of Alabama, U.S.A., was diluted with carrier PteGlu, to give 0.033 pCi/O.l pmol/ml of soln. Yeast carboxypeptidase Y was from Sigma and y-glutamyl carboxypeptidase was isolated from Me&O powders of chicken pancreas (Difco Laboratories). Plant material. Seeds of broccoli (Brassica oieracea var. it&u, Green Valiant) and pea (Pisum sariuum L. cv. Homesteader) were purchased from commercial sources. Seedlings were produced under controlled growth conditions [ 131.Tomato (Lycopersicon esculenrum cv. Bounty) seedlings were grown under greenhouse conditions. Samples of broccoli, cabbage (B. oleracea var. capita@, cauliflower (B. oleracea var. borryris) lettuce (Loctuca soriua L.) and carrot (Doucus carom) were obtained from local market sources. Folore extraction and cleavage. Tissue samples (co 10 g fr. wt) were rapidly sliced and heated at loo” in 20 mM KH,PO, buffer containing 2-mercaptoethanol [13]. After cooling and amtrifugation [13], folates in the supernatants were cleaved to pABAGlu, derivatives by a series of acid-base and Zn-HCI treatments [14]. These products were then converted to azo dyes, purified on columns of Bio-Gel P2, and reconverted to p-ABAGlu, by a second Zn-HCI treatment [14]. HPLC of pABAGlu, deriuariues. After drying in uacuo, samples were dissolved in co 1.5 ml of H,O, adjusted to pH 6.5 and centrifuged to remove Zn(OH), [14]. These solns were stored in darkness at -20”. Immediately before HPLC, samples were filtered through 0.45 pm nylon filters. Sepn of individual p ABAGlu, derivatives by HPLC was achieved on Whatman Partisil 10 SAX (250 mm x 4.6 mm columns). The chromatographic programme was that of ref. [IS]. The quantity of each

2281

polyglutamate was determined by reference to a standard calibration curve [I33 for the corresponding pABAGlu, species. All HPLC analyses were carried out in triplicate. For isolation of labelled polyglutamates the effluent from the HPLC column was collected in I ml fractions and assessed for radioactivity by scintillation counting [13]. Carboxypepridase treatment offolure exrracrs. Enzyme treatments were applied to 10 ml samples of the initial folate extracts. Incubation conditions for the enzymes of pea cotyledons [25], chicken pancreas [26] and yeast carboxypeptidase Y [27] were as previously described. Hydrolysis was allowed to proceed for 16 hr followed by folate cleavage to pABAGlu, and HPLC analysis (see above). Incorporation of [‘*C]p-ABA into folares. Fourteen-day-old broccoli seedlings were excised under H,O at the hypocotyl base. Samples of 20 excised seedlings were placed in 100 ml beakers containing 10 ml of 50% strength Hoagland’s soln and 10 PCi of [i4CJpABA. In other expts, 5 mm disks were excised from the cotyledons of 14day-old broccoli seedlings and 1 g fr. wt samples were placed in 25 ml flasks containing 4 ml of 50% strength Hoagland’s soln and 10 pCi of [i4C]pABA. Tissues were exposed to a light/dark regime [ 131 for 24 and 48 hr for disks and excised seedlings respectively. Folates were extd and cleaved as described above. Differential clearage of broccoli leuffolares. Leaves (25 g fr. wt) of 90-day-old plants were placed in 100 ml of Ar-satd 0.1 N HCI and homogenized under a stream of Ar to maintain anaerobic conditions. The homogenate was stirred for I5 min under Ar, centrifuged and divided into three equal portions for oxidative folate cleavage [19]. The resulting pABAGlu, derivatives were converted to azo dyes of naphthylethylenediamine [19], purified on 4x0.8 cm columns of Bio-Gel P2 and reconverted to pABAGlu, [I43 prior to HPLC analysis [IS]. Hydrolysis of PreGlu, by broccoli leaf extracts. Leaf samples (40 g) excised from 60-9Oday-old broccoli plants were homogenized in 80 ml of 10 mM Pi buffer (pH 6.0) containing 50 mM 2-mercaptoethanol. After passage through cheesecloth the homogenate was centrifuged (10 000 g for 10 min). Nucleic acids were removed by addition of streptomycin sulphate (final concn 1%) and protein was fractionated with (NH&SO,. Protein ppting in the 40-70% range of satn with (NH&SO, was dissolved in co 1.5 ml of extn buffer and dialysed overnight against this buffer. Enzymatic hydrolysis of [i4CJPteGlul and PteGlu, was examined in 0.8 ml reaction systems containing 50 pmol acetate buffer (pH 5), 0.1 pm01 L-glutamate, 10 nmol folate substrate and co 0.1 mg of leaf protein. The reaction was allowed to proceed at 37” for periods up to 1 hr. When release of [‘*C]glutamate was followed, the reaction was terminated with TCA and excess substrate was removed using activated charcoal [28]. When folate products were examined, the reaction was terminated by addition of 30 mM 2-mercaptoethanol(3 ml) and polyglutamates were recovered using 4 x 0.8 cm Bio-Rad Econo-columns containing Whatman DE-52 cellulose [29]. These folate products were then cleaved to p-ABAGlu, [I43 and characterized by HPLC [IS]. Reaction products not absorbed to DE-52 cellulose were characterized by ion-exchange chromatography using Dowex (acetate) as summarized in Fig 2. The identity of diglutamyl peptide was also confirmed by HPLC analysis using a Whatman Partisil SAX column (250 mm x 4.6 mm) and isocratic elution in 50 mM ammonium phosphate (pH 3.0) at 34”. Product detection was at 215 nm. Typical R,s for glutamate, di- and triglutamyl peptide were 3.9, 4.7 and 7.0 min respectively. Acknowledgements-This

work was supported by a research

grant awardedto E.A.C. by the Natural Sciences and Engineering Research Council of Canada. These investigations were

2282

L.-L. ZnF.PU’Get al.

carried out while Li-Li Zheng was a Visiting Scholar from Xiong-yue Agricultural College, The People’s Republic ofchina. The authors thank Professor Carlos Krumdieck, University of Alabama, for advice regarding the assay of folate hydrolase activity.

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1. Blakley,

2. 3.

4. 5.

R. L. and Benkovic, S. J. (1984) in Folates and Prerins (Blakley, R. L. and Benkovic, S. J., cds), p. xi. Wiley, New York. Cossins, E. A. (1980) in The Biochemisrry o/Plants (Davies, D. D., ed.), Vol. 2, p. 365. Academic Press, New York. McGuire, J. J. and Coward, J. K. (1984) in Folafes and Pterins (Blakley, R. L. and Benkovic, S. J.. eds). p. 135. Wiley, New York. Cossins, E. A. (1987) in The Biochemistry o/P/ants (Davies, D.D., cd.), Vol. 11, p. 316. Academic Press, New York. Schirch, V. and Strong, W. B. (1989) Arch. Biochem. Biophys. 269, 371.

6. Cossins,

E. A. (1984) in Folates and Pterins (Blakley. R. L. and Benkovic, S. J.. eds). p. 1. Wiley, New York. 7. Kisliuk, R. L. (1981) Molec. Cell Biochem. 39, 331. 8. Selhub, J., Savm, M. A., Sakami, W. and Flavm, M. (1971) Proc. Narn.

9. Imeson,

Acad. Sci. U.S.A. 68, 312.

H. C. and Cossins,

E. A. (1991) J. PIam

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10. Imeson, H. C. and Cossins, E. A. (1991) J. Planr Physiol. 1% 483. 11. Chan, C.. Shin, Y. S. and Stokstad, E. L. R. (1973) Can. J. Biochem. 51, 1617.

12. Batra. K. K., Wagner,

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