Plant Physiol. (1984) 76, 814-8180032-0889/84/76/0814/05/$0 1.00/0

Conversion and Distribution of Cobalamin in Euglena gracilis z,with Special Reference to Its Location and Probable Functionwithin Chloroplasts

Received for publication May 4, 1984 and in revised form July 25, 1984

YUJI ISEGAWA, YOSHIHISA NAKANO, AND SHOZABURO KITAOKA*Department ofAgricultural Chemistry, University ofOsaka Prefecture, Sakai, Osaka 591, Japan

ABSTRACT

Cobalamin is essentially required for growth by Eugkna gracilis andshown to be converted to coenzyme forms promptly after feeding cyano-cobalamin. Concentrations of coenzymes, methylcobalamin, and 5'-de-oxyadenosylcobalamin, reached about 1 femtomole/lO' cells 2 hours afterfeeding cyanocobalamin to cobalamin-limited cells. Cobalamins all werebound to proteins in Eugkna cells and located in subcellular fractions ofchloroplasts, mitochondria, microsomes, and cytosol. Incorporated co-balamin into chloroplasts was localized in thylakoids. Methylcobalaminexisted in chloroplasts, mitochondria, and cytosol, while 5'-deoxyadeno-sylcobalamin was in mitochondria and the cytosol, 2 h after feedingcyanocobalamin to Euglena cells. Quantitative alterations of methylco-balamin and 5'-deoxyadenosylcobalamin in chloroplasts suggest theirimportant functions as coenzymes in this organelle. The occurrence offunctional cobalamins in chloroplasts has not been reported in otherphotosynthetic eukaryotes.

Euglena gracilis requires Cbl' for growth and rapidly accu-mulates Cbl in cells (1, 4). When grown on a CN-Cbl-limitedmedium (below 50 ng CN-Cbl/l), Euglena cells show an increaseof chloroplast number (3, 4), enlargement of cells (3, 29), andconcomitant increase of cell components such as Chl, protein,RNA, and DNA (1, 3, 4). When CN-Cbl is replenished to theCN-Cbl-limited cells, they return to the normal state in 24 h (1,4).Although plants and yeasts had not been thought to require

Cbl for growth, Poston (24) and Poston and Hemmings (25)reported occurrence of a coenzyme Cbl-dependent enzyme, leu-cine-2,3-aminomutase, in Phaseolus vulgaris, Solanum tuber-esum, and Candida utilis. The subcellular location ofthis enzymehas not been known. Nobody has recognized the existence andfunction ofCbl in the chloroplasts ofhigher plants. In the presentreport, we describe the transformation and distribution ofCbl inEuglena cells after feeding CN-Cbl, and demonstrate the presenceof functional forms of Cbls in chloroplasts. The probable func-tion of Cbl in Euglena chloroplasts is discussed.

MATERIALS AND METHODS

Chemicals. [G-3H]CN-Cbl (4,380 mCi/mmol) and NaH'4CO2(0.1 mCi/mmol) were purchased from Amersham International

'Abbreviations: Cbl, cobalamin; CN-Cbl, cyanocobalamin; Me-Cbl,methylcobalamin; HO-Cbl, hydroxocobalamin; Ado-Chl, 5'-deoxyaden-osylcobalamin.

Limited and authentic cold Cbls, Ado-, Me-, HO-, and CN-Cblfrom Sigma. All other reagents were of analytical grade.Orgnism and Culture. Euglena gracilis z was cultured pho-

toheterotrophically under CN-Cbl-limited conditions (50 ng/l)according to Tokunaga et al. (32).

Subcellular Fractionation and Purification of Chloroplasts.Partial trypsin digestion ofthe pellicle followed by mild mechan-ical disruption of Euglena cells and subcellular fractionation bydifferential centrifugation were conducted according to Shigeokaet al. (30).A suspension of crude chloroplasts obtained by the method

described above was layered on the top of discontinuous Percollgradients and centrifuged in a swing rotor (Hitachi RPS 6-2) for30 min at 2,300g with a Hitachi 2ORP-5 refrigerated centrifuge.The gradients were prepared by layering into centrifugal tubes1.5 ml each of 25 mM glycylglycine-KOH buffer (pH 7.4),containing 3% Ficoll, 0.33 M mannitol and in the shown order10, 20, 30, and 50% Percoll solutions. The isopycnically bandedchloroplasts were recovered from the gradients, washed, andresuspended in 0.33 M mannitol-glycylglycine-KOH buffer (pH7.4).

Subcellular Fractionation by Linear Sucrose Density GradientCentrifugation. Euglena cells were incubated in the Oda medium(22) with 10-9 M [3H]CN-Cbl for 30 min under illumination(2,500 lux) at 27C. The cell homogenate obtained by the abovemethod was subjected to 20 to 45% linear sucrose density gra-dient centrifugation at 100,000g for 2.5 h with a Hitachi RPS 25rotor as described by Shigeoka et al. (30).

Subfractionation of Chloroplasts. Preparation of chloroplastsfrom [3H]CN-Cbl-fed cells was performed as described above. Agentle osmotic shock of chloroplasts to detach the envelope wascarried out according to Douce et al. (7) with some modificationsas follows. Intact, purified chloroplasts were suspended in 0.5 mlof 0.8 M sucrose (about 5 mg of protein of chloroplasts per ml)and stirred gently for 5 min at OC after adding 6 ml of 25 mMglycylglycine-KOH buffer (pH 7.4). The suspension was loadedon the discontinuous sucrose gradients which were prepared bylayering 3 ml each of 25 mM glycylglycine-KOH buffer (pH 7.4)containing in the shown order, 0.6, 0.93, 1.2, and 1.8 M sucroseand centrifuged with the Hitachi RPS 25 rotor for 1 h at100,000g. Each interface of the sucrose layers was recoveredfrom the gradient, diluted four times with 25 mM glycylglycine-KOH buffer (pH 7.4), and centrifuged for 1 h at 100,000g withthe Hitachi RPS 25 rotor. The pellets were suspended in 25 mMglycylglycine-KOH buffer (pH 7.4) containing 0.33 M mannitol.Radioactivity of each fraction was counted with an Aloka LSC903 scintillation counter.

Determination of Chloroplast Integrity. Integrity of purifiedchloroplasts was assessed by the activities of photosynthetic CO2fixation and ferricyanide reduction of purified chloroplasts as

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LOCATION OF COBALAMIN IN CHLOROPLASTS OF EUGLENA

measured by the procedures of Forsee and Kahn (10) and Neu-mann and Drechsler (21), respectively.Enzyme Assays. Activity of ribulosebisphosphate carboxylase

(EC 4.1.1.39), a marker enzyme for stroma of chloroplasts, wasmeasured according to Rabinowitz et aL (26). Adenylate kinase(EC 2.7.4.3), a marker enzyme for chloroplast envelopes, wasassayed according to Murakami and Strotmann (20). Succinate-semialdehyde dehydrogenase (EC 1.2.1.16), a mitochondrialmarker enzyme, was assayed by the method of Tokunaga et aL(32). Glucose-6-phosphatase, (EC 3.1.3.9), a microsomal markerenzyme, was assayed according to de Duve et al. (6). The activityof glutamate dehydrogenase (EC 1.4.1.2), a cytosolic markerenzyme (30), was measured according to Corman and Inamonar(5).

Chl and protein were determined by the methods of Mackin-ney ( 17) and Lowry et al. (15), respectively.Assay of Cobalamin-Binding Activity. Subcellular fractions

and chloroplast subfractions in 0.1 ml of chilled 25 mM glycyl-glycine-KOH buffer (pH 7.4) were placed inside Visking dialysistube (6.4 mm diameter) and dialyzed against 100 ml of 10 mmK-phosphate buffer (pH 7.2), containing 30 nm KCN and 10-9M [3H]CN-Cbl at 4°C for 24 h in the dark. The Cbl-bindingactivity was determined from the increased radioactivity of thesample-containing sacks relative to the radioactivity of the con-trol sack to which only 25 mM glycylglycine-KOH buffer wasadded. Protein in the Visking tube was again determined at theend of each dialysis.Gel Filtration Chromatography. Chloroplasts purified from

[3H]CN-Cbl-fed cells were completely solubilized with 0.1% Tri-ton X-100 by stirring at 4°C for 15 min and then applied onSephadex G-25 column (1 x 30 cm) equilibrated with 10 mM K-phosphate buffer (pH 7.2) at 40C. Elution was carried out withthe above buffer and the eluant collected with a fraction collectorto count the radioactivity. The column was standardized withblue dextran 2,000 and CN-Cbl.HPLC of Cbl Analogs. Separation of CN-Cbl analogs was

performed according to Frenkel et al. ( 11) with some modifica-tions.The sample, whole cell, or each organelle, to which [3H]CN-

Cbl was fed for a definite period, was mixed with 10 ug of eachunlabeled Cbl analog (CN-, HO-, Me-, and Ado-Cbl), and to thissolution was added ethanol to the final concentration of 80%.The mixture was stirred for 30 min at room temperature andthen centrifuged for 10 min at 2,500g. The clear supernatant wasevaporated, and dissolved in 0.5 ml of 0.04 M tataric acid-NaH2PO4 buffer (pH 3.0), containing 28% methanol. The solu-tion was centrifuged for 5 min at 15,000g. The supernatant wasfiltered through membrane filter TM-2P (pore size 0.45 jsm,Toyo Filter, Inc.) in a stainless steel swinney adaptor attached toa 2-ml syringe. A 100-,ul aliquot of this extract was then applieddirectly onto a HPLC which was equipped with a stainless steelcolumn (0.4 x 30 cm) packed with Unisil QC 18, 5 ,m particlesize (Gasukuro Kogyo Inc.). Elution was carried out with a lineargradient of methanol concentration of 28 to 70% in 0.04 Mtartaric acid-NaH2PO4 buffer (pH 3.0) in 22 min followed byeluting with 70% methanol buffer for 1 min. The flow rate wasset at 1.0 ml/min and column oven at 40°C. The column eluatewas monitored at 350 nm with an absorbance detector andcollected sequentially, and radioactivity was counted. AuthenticHO-, CN-, Ado-, and Me-Cbls had the retention times of 6, 10.5,22, and 25 min, respectively.

RESULTSConversion of 3HICN-Cbl to I3HJCbl Coenzymes in E. gracilis.

The conversion of [3H]CN-Cbl to its analogs in Euglena cells,after feeding [3H]CN- Cbl to Cbl-limited cells, is shown in Figure1. Total Cbl accumulated in Euglena rapidly reached plateau

(a00

E

.0

-

U

8 I I 1

6

4-

2

00 1 2 3

Time (h)4 5

FIG. 1. The conversion of [3H]CN-Cbl to the analogs by Euglenacells. Accumulated total 3H-labeled Cbl (0), CN-Cbl (A), Me-Cbl (0),Ado-Cbl (0), HO-Cbl (A).

within 30 min. The decreasing percentage ofradioactivity in CN-Cbl with incubation time (from 100% to 10% in 2 h) apparentlyaccounted for increases of labeled HO-Cbl, Me-Cbl, and Ado-Cbl. Accumulation ofHO-Cbl was initially more rapid than thatof either coenzyme; accumulation of labeled Ado-Cbl in 1 happeared to be more rapid than that of labeled Me-Cbl. After 2h, 32% of total [3H]Cbl was present as HO-Cbl, while Me-Cblwas 21% and Ado-Cbl, 16%. After 4 h, [3H]HO-Cbl represented35% of the incorporated total radioactivity; Me-Cbl, 17%; andAdo-Cbl, 23%. That radioactivity was found to be accumulatedpromptly in HO-Cbl indicates the reaction is operative, in whichremoval of the cyanide group from CN-Cbl takes place. On theother hand, concentrations ofMe-Cbl and Ado-Cbl were maximaafter 2 and 4 h, respectively.

Subcellular Location of Cbl. After incubation of the Euglenacells with [3H]CN-Cbl, distribution of 3H-labeled Cbl in Euglenasubcellular fractions was estimated and is shown in Figure 2A.Location ofCN-Cbl-binding activities and marker enzyme activ-ities are given in Figure 2, B and C, respectively. Succinate-semialdehyde dehydrogenase, a marker enzyme for mitochon-dria, gave a sharp peak ofthe activity in fraction 5, correspondingto an equilibrium density of 1.198 g/cm3. A peak of the activityof glucose-6-phosphatase, a microsomal marker enzyme, wasseen with an equilibrium density of 1.130 g/cm3 in fraction 24.Glutamate dehydrogenase, which is located solely in the cytosolin Euglena (30), showed the activity in fractions 28 to 38. Amajor peak of the activity of ribulosebisphosphate carboxylasewas present with an equilibrium density of 1.165 g/cm3 in thesame fractions as those of Chl. The radioactivity ofincorporated[3H]CN-Cbl was distributed to all subcellular fractions, indicatingthat the labeled Cbl and Cbl-binding activities are present in allsubcellular fractions. Incorporated Cbl amounted to 0.43, 0.70,1.38, and 1.56 pmol/mg protein in mitochondria, chloroplasts,microsomes, and cytosol, respectively, and Cbl-binding activitieswere 0.58, 0.91, 0.78, and 1.55 pmol/mg protein, respectively.Subcellular distribution, after correction for the leakage ofmarker enzymes, of incorporated Cbl in mitochondria, chloro-plasts, microsomes, and cytosol was 8, 13, 22, and 57%, respec-tively, and that of Cbl-binding activity was 11, 21, 9, and 59%,respectively.

Location and Chemical Forms of Cbls in Organelles. Table Ishows distribution and chemical forms of Cbl in the subcellularfractions from differential centrifugation of a homogenate of E.gracilis cells fed 3H-labeled CN-Cbl for 2 h. Incorporated Cblwas located 21.5% in chloroplasts, 12.6% in mitochondria,18.2% in microsomes, and 47.6% in cytosol. In chloroplasts,Me-Cbl comprised 54% of the incorporated Cbl; in mitochon-dria, 18%; and Ado-Cbl, 25%. Microsomes were largely occupiedby HO-Cbl and CN-Cbl, being 53 and 35%, respectively. Cytosol

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Plant Physiol. Vol. 76, 1984

3 15 1 .20=

0"30 0.6 60~~~~~~~~~~~~~~-1.10

2i[ .E,0.-. J2

2 JO-~~2 s'E2 E0 10.lNL -U

0 10 20 30 40Fraction number

FIG. 2. Subcellular distribution of incorporated Cbl (A) and Cbl-

binding activity (B). See the text for details. Activities of marker enzymes

(C) were assayed as described in "Materials and Methods". Succinate-

semialdehyde dehydrogenase (SSADH) (0), ribulosebisphosphate car-

boxylase (RuBPC) (0), glucose-6-phosphatase (G-6-Pase) (A), glutamate

dehydrogenase (GDH) (0), protein (.s), Chl (A), sucrose density

( -)-

had the rates of 15% Me-Cbl, 23% Ado-Cbl, 29% HO-Cbl, and

33% CN-Cbl. Me-Cbl in chloroplasts corresponded to 54% of

total cellular Me-Cbl; 33% was present in cytosol, 10% in mito-

chondria, and it was hardly found in microsomes.

Locato of Cbl in Chioroplast Subfaions. The purified

chloroplasts from the Euglena cells fed the labeled CN-Cbl were

solubilized with 0.1% Triton X-100 and applied on Sephadex G-

25 column. The radioactivity was eluted only in the fraction of

a void volume (Fig. 3) but not in free, authentic Cbl fractions,

suggesting that Cbl incorporated in chloroplasts is completely

bound to macromolecular substances.

In an attempt to determine the location of binding site of Cbl

in chloroplasts, the organelles prepared from the Euglena cells

preincubated with [3HJCN-Cbl were purified with a Percoll gra-

dient and then subfractionated. Chloroplasts, recovered at theinterface between 20 and 30% Percoll solutions, were capable offixing C02 at the rate of 28.4 ;&mol of C02/mg Chl-h. Thisphotosynthetic activity corresponded to 50% of that of the orig-inal Euglena cells. The 1.5-fold increased specific activity ofphotosynthesis against crude chloroplasts and was seen after thepurification of the crude chloroplasts with Percoll. Reducingactivity of ferricyanide by the purified chloroplasts was notdetected at all. These results indicate that the envelopes of thepurified chloroplasts were intact (28). In addition, intactness ofthe purified chloroplasts was also supported from a phase contrastmicroscopic observation. Subfractionation of the chloroplastswas performed by an osmotic treatment followed by sucrose-stepwise gradient centrifugation into 4 fractions of the interfaceof the sucrose layers (0.6, 0.93, 1.2, and 1.8 M). There was noband at the first interface (fraction 1), a yellow band at the secondinterface (fraction 2), a green band at the third interface (fraction3), and a dark green band at the fourth interface on 1.8 M sucrose(fraction 4).

Table II shows subchloroplast distribution of 3H-labeled CN-Cbl, activities of adenylate kinase and ribulosebisphosphate car-boxylase, and contents of Chl and protein in each fraction.Fraction 1 corresponds to the stroma in which ribulosebisphos-phate carboxylase is located. Fraction 2 shows the highest specificactivity ofadenylate kinase which is the markerenzyme ofplastidenvelopes. However, fraction 1 shows a higher total activity ofadenylate kinase than that in fraction 2, probably due to releaseof adenylate kinase located in stroma into the soluble fraction(13). Consequently, plastid envelopes were recovered in fraction2. Fraction 3 contains thylakoid membranes in which Chl wasrecovered, and it hardly contaminated other fractions. Fraction4 apparently contained partially broken chloroplasts containingplastid envelopes and thylakoids with no ribulosebisphosphatecarboxylase activity; the fraction showed about 20% of totalactivity of adenylate kinase as the membrane-bound enzyme ofplastid envelopes. These results manifest that stroma, plastidenvelopes, and thylakoids ofEuglena chloroplasts were separatedwell from each other.

Distribution of [3HJCN-Cbl incorporated into Euglena chlo-roplasts was about 80, 15, and 5% in thylakoids, envelopes, andstroma, respectively.The Chemical Forms of Cbl in Chloroplasts. When E. gracilis

was fed [3H]CN-Cbl for 2 h, about 54% of the labeled Cbl inchloroplasts was recovered in the Me-Cbl fraction after HPLC;the rates of HO-, CN-, and Ado-Cbls were 29, 13, and 3%,respectvely (Fig. 4). Four h after feeding Cbl to the cells, theamount of Me-Cbl immediately decreased. About 10% of totalCbls in chloroplasts existed as Me-Cbl 2 h after Cbl feedingwhich, however, accounted for about 50% oftotal Me-Cbl in thecells.

DISCUSSIONConcomitant with Cbl depletion, cellular enlargement takes

place in E. gracilis (29), because both nuclear and cellular

Table I. Contents ofCbl Analogs in Subcellular Fractions ofEuglenaCells were grown for 5 d under illumination (2500 lux) and Cbl-limited conditons. Two h after feeding 10-9

M [3H]CN-Cbl, the cells were fractionated and then Cbls were extracted with 80% ethanol from each organelle.The Cbl analogs were separated by HPLC as described in "Materials and Methods". Distribution ofCbl analogsinto each organelle is given at pmol/109 cells and per cent distribution given in parentheses, after correctionfor the leakages of marker enzymes.

CN-Cbl HO-Cbl Ado-Cbl Me-Cbl Total

Mitochondria 0.32 0.14 0.20 0.14 0.80 (12.6)Chloroplasts 0.19 0.40 0.04 0.74 1.37 (21.5)Microsomes 0.42 0.61 0.09 0.04 1.16 (18.2)Cytosol 1.00 0.89 0.69 0.04 3.03 (47.6)Total 1.93 (30.3) 2.04 (32.1) 1.02 (16.0) 1.37 (21.5) 6.36 (100)

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LOCATION OF COBALAMIN IN CHLOROPLASTS OF EUGLENA

o

In

~0.25[-

0 5 10 15 20Fraction number

FIG. 3. Elution pattem ofCN-Cbl-binding protein complex and CN-Cbl from Sephadex G-25 column. Fractions of I ml were collected.Radioactivity ([3H]Cbl) (A), A at 360 nm (authentic CN-Cbl) (0), A at590 nm (blue dextran) (0).

divisions are halted (2). When CN-Cbl was fed to E. gracilisgrown in CN-Cbl-limited medium (50 ng/l) for 5 d, Cbl wasrapidly incorporated to reach a constant cellular content. How-ever, the taken-up CN-Cbl decreased rapidly 2 h after feeding tothe Cbl-limited cells and converted to HO-Cbl, Ado-Cbl, andMe-Cbl. HO-Cbl was predominant in the cells 2 h after the CN-Cbl feeding, suggesting that HO-Cbl is a reservoir of Cbl inEuglena cells. Ado- and Me-Cbls, which function as coenzymes,reached to about 1 fmol/106 cells within 2 h after CN-Cbl feedingand was almost kept at constant thereafter. The intracellularconcentration of both Cbl coenzymes was 1 nm each for anestimated cell volume of 1 ;L/106 cells, which was determinedwith hematocrit. Figure 1 shows a parallel behavior ofthe changesof Ado- and Me-Cbl contents, when CN-Cbl was fed to the Cbl-limited cells. The results suggest that stoppage of nuclear andcellular divisions is caused not only by ceasing ofDNA synthesisby decrease of Ado-Cbl-dependent deoxyribonucleotide synthe-sis, but also by blockage of protein synthesis due to decrease ofMe-Cbl-dependent methionine synthesis. In Cbl-limited Euglenacells, ceasing of DNA synthesis has been observed by Carell (4),which is reported 2 to 3 h after Cbl refeeding. On the other hand,Bertaux and Valencia (1) have proposed that methylation isrelated to the ability ofcoenzyme-Cbl to transfer or create methylgroups in Euglena. Shehata and Kempner (29) have proposedthat Euglena cells can respond in two different ways to Cblstarvation: one, by deoxyribonucleotide synthesis and another,by methylation. Ochromonas malhamensis, another protozoan,also has a growth requirement for Cbl (9) and Cbl can be replacedby methionine (12). In Ochromonas, methionine is synthesizedby a Me-Cbl-dependent enzyme (16). Me-Cbl-dependent methi-onine synthetase has been found in some bacteria and higheranimals (8) but, to date, has not been found in plants andEuglena.

Cbl, incorporated rapidly into Euglena, was distributed incytosol, chloroplasts, mitochondria, and microsomes (Fig. 2).This distribution is different from the one reported for E. gracilis(33) and 0. malhamensis (27), in which Cbl was mainly incor-porated in the particulate fractions. The cause ofthis discrepancyis not clear at this time. In Euglena all ofthe uptaken Cbl existedin bound form, but not in free form, unlike in rabbit liver whereCbl was present partly in free form (14).Two hours after feeding CN-Cbl to Euglena cells, 60% of Cbl

incorported into each organelle was comprised of HO- and CN-Cbls. Subcellular distributions of Ado- and Me-Cbls were spe-cific, compared with those of HO- and CN-Cbls, which werewidely scattered in all fractions. Ado-Cbl was mainly located incytosol and mitochondria; metabolic conversion of HO-Cbl toAdo-Cbl was shown in mitochondria of rat liver (18) and also inEuglena mitochondria (Y. Isegawa et al., unpublished data).Ado-Cbl in mitochondria may act as the cofactor of methyl-malonyl-CoA mutase like in human fibroblasts (19), and Ado-Cbl in cytosol as the cofactor of ribonucleotide reductase. Sincemicrosomes predominantly contained HO-Cbl and CN-Cbl,metabolic conversion of CN-Cbl to HO-Cbl may occur in thisorganelle which most probably contain Cyt P-450, active in drugmetabolism in other organisms (8). About half of Me-Cbl waslocalized in chloroplasts and considerable amounts in cytosoland mitochondria, but not in microsomes. Me-Cbl is a cofactorof methionine synthetase (N5-methyltetrahydrofolate homocys-teine methyltransferase) in many organisms (31) and recently wehave found occurrence of the methyltransferase in Euglena inpreliminary experiments (Y. Isegawa et al., unpublished data).Occurrence of Me-Cbl in three subcellular fractions suggests thatmethionine synthetase is located in these fractions and that Me-Cbl functions as a cofactor. Accumulation ofCbl and occurrenceof Me-Cbl in chloroplasts are demonstrated in the present workfor the first time. Cbl has not been found at all so far inchloroplasts of photosynthetic organisms. It has been reportedthat an extract of potato tubers contains two Cbl-dependentenzymes, leucine-2,3,-aminomutase and methylmalonyl-CoAmutase (24), and that an eukaryotic alga, Emiliania huxleyi, hasbeen reported to synthesize Cbl de novo (23); Ochromonas mal-hamensis is reported to contain Cbl-dependent methionine syn-thetase (16). Subcellular location of Cbl or Cbl-dependent en-zyme in these organisms has not been known.

Subfractionation of purified and intact chlorpplasts from Eu-glena cells revealed that 80% of Cbl was located in thylakoidmembranes and 15% of Cbl in chloroplast envelopes, suggestingthat Cbl functions on the membranes. When CN-Cbl was fed toEuglena cells, the form in chloroplasts was predominantly Me-Cbl after 2 h, and Ado-Cbl after 4 h. The fact may suggest that

Table II. Distribution of3H-Label ofCN-Cbl in Chloroplast SubfractionsCells were grown for 5 d under illumination (2500 lux) and Cbl-limited conditions. Two h after feeding

109 M [3H]CN-Cbl, the cells were fractionated and then chloroplasts were purified by centrifugation of Percollgradient. The purified chloroplasts were subfractionated by centrifugation on a sucrose gradient after osmoticdisruption as described in "Materials and Methods". Specific activities of marker enzymes are shown in Amol/mg protein/min, except for CN-Cbl incorporated whose amount is expressed in pmol/mg protein and proteinand Chl are shown in mg. Total activities are given at per cent in parentheses.

CN-Cbl Adenylate Ribulose-Fraction Protein Icprt kinase bisphosphate Chl

carboxylasePurified chloroplasts 6.73 (100) 10.5 (100) 1.3 (100) 1.23 (100) 0.59 (100)I (Stroma) 2.12 (31.5) 0.4 (1.0) 1.3 (30.4) 3.51 (90.2) 0 (0)2 (Chloroplast envelopes) 0.17 (2.5) 24.5 (5.9) 12.5 (24.5) 0.12 (0.2) 0 (0)3 (Thylakoid mem-

branes) 0.78 (11.5) 33.6 (37.0) 0.1 (0.9) 0.14 (1.3) 0.13 (21.7)4 (Broken chloroplasts) 2.63 (39.1) 12.8 (47.4) 0.6 (19.5) 0.11 (3.5) 0.25 (39.1)

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818 ISEGAWA ET AL.

c

.I;1.0_

., 0. 8 _E

W1 o 0.6 -

0.2

0

0 2 4 6Incubation time (h)

FIG. 4. Contents of Cbl analogs in chloroplasts. Euglena cells grownfor 5 d in Cbl-limited medium were incubated with [3H]CN-Cbl fordesired period. Me-Cbl (0), Ado-Cbl (0), HO-Cbl (A).

Me-Cbl act in chloroplasts chiefly soon after cellular uptake ofCbl. The results heretofore reported suggest that Me-Cbl func-tions as a cofactor of methionine synthetase in Euglena chloro-plasts. The details of this enzyme and the use of synthesizedmethionine in chloroplasts will be described elsewhere.

Acknowledgment-The authors thank Mr. T. Ishikawa for his technical assist-ance for HPLC.

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Plant Physiol. Vol. 76, 1984

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