Comp. Biochem. Physiol. Vol. 94B, No. 4, pp. 797-800, 1989 0305-0491/89 $3.00 + 0.00 Printed in Great Britain © 1989 Pergamon Press pie

TRANSFER SYSTEM OF COBALAMIN FROM PELLICLE TO CYTOSOLIC BINDING PROTEINS IN

EUGLENA GRACILIS

FUMIO WATANABE,*~" YOSHIHISA NAKANO,~ YOSHIYUKI TAMURA* and SHOZABURO KITAOKAJ~ *Laboratory of Nutrition and Food Science, College of Hagoromo-gakuen, Sakai, Osaka 592, Japan; and

~:Department of Agricultural Chemistry, University of Osaka Prefecture, Sakai, Osaka 591, Japan

(Received 2 May 1989)

Abstract--1. A cobalamin (Cbl)-transfer system from the pellicle to cytosolic Cbl-binding proteins occurs in Euglena gracilis.

2. The Cbl-transfer activity showed thermal dependency. The optimum temperature was 50°C. The Cbl-transfer activity was increased significantly above pH 7.0.

3. ATPase, thiol-groups and metal ions were not involved in the Cbl transfer.

INTRODUCTION

The intestinal absorption of Cbl, the subsequent plasma transport, and the related Cbl-binding

, proteins and their receptors have been extensively studied in mammals (Seetharam and Alpers, 1982). The mechanisms and kinetics of Cbl uptake have also been reported in some prokaryotic and eukary- otic cells (Bradbeer, 1971; Bradbeer and Woodrow, 1976; Youngdahl-Turner et al., 1979). However, little information on the intracellular Cbl-transport system has been available yet because of the lack of studies on intracellular Cbl-carrier protein(s).

Euglena gracilis z, which requires Cbl for growth (Ross, 1952), has well-developed organelles and also is suitable for preparation of Cbl-limited ceils. E. gracilis z takes up Cbl by a biphasic process, consisting of energy-independent and -dependent phases (Sarhan et al., 1980). Our previous studies (Isegawa et al., 1984; Watanabe et al., 1987a,b, 1988) indicated that all the Cbl taken up by Euglena cells were bound to some specific binding proteins, which were distributed in some organelles (cytosol, mito- chondira, chloroplasts, and microsomes) and the pellicle, cell membrane complex, and reported that some Cbl-binding proteins have been purified to homogeneity and characterized. We have further suggested that Euglena cytosolic Cbl-binding proteins function as intracellular Cbl carriers (Watanabe et al., 1987a). These observations indicate that E. gra- cilis z is a useful organism for elucidation of the intracellular Cbl-transport system. In this paper we describe the occurrence of the Cbl-transfer system from the pellicle to cytosolic binding proteins.

tAuthor to whom correspondence should be addressed. Abbreviations used: Cbl, cobalamin; Mops, 3-(N-mor-

pholino)propane-sulfonic acid; CCCP, carbonylcya- nide-m-chlorophenyl hydrazone; DTNB, 5,5'-dithio- bis(2-nitrobenzoic acid); EDTA, ethylenediaminete- traacetic acid; EGTA, ethyleneglycol-bis(fl-aminoethyl ether)tetraacetic acid; pI, isoelectric point.

MATERIALS AND METHODS

Organisms and culture Euglena gracilis SM-ZK, a streptomycin-bleached mutant

of E. gracilis z, was cultured for 5 days at 27°C at 2000 lux in a Cbl-limited (0.05/~g/1) Koren-Hutner medium (Koren and Hutner, 1967).

Preparation of E. gracilis pellicle E. gracilis pellicle was prepared as described previously

(Watanabe et al., 1988).

Preparation of pellicle-[3H]CN-Cbl complex

All procedures of the preparation of pellicle-Cbl complex were done at 0-4°C unless otherwise specified. [3H]CN-Cbl (5.1 Ci/mmol) was added to the pellicle at 10 -7 M and the mixture was incubated at 20°C for 10 min to bind [3H]CN- Cbl to the pellicular Cbl-binding proteins and was then centrifuged at 10,000g for 5rain. The precipitate, pelli- cle-[3H]CN-Cbl complex, was washed several times with 10mM 3-(N-morpholino)propanesulfonic acid (Mops)- KOH buffer, pH 7.5, to remove any excess unbound [3H]CN-Cbl. The washed pellicle-[3H]CN-Cbl complex was suspended in 2 ml of the washing buffer and the suspension was used as pellicle-[3H]CN-Cbl complex. The pellicle-[3H]CN-Cbl complex was freshly prepared for experiments.

Preparation of E. gracilis cytosolic Cbl-binding proteins Purification of the cytosolic Cbl-binding proteins was

done as described previously (Watanabe et al., 1987a). A partially purified preparation, which contained the cytosolic Cbl-binding proteins with pls of 3.8 and 4.7, was used unless otherwise specified. Euglena cytosol prepared by the tryptic digestion method (Tokunaga et al., 1979) was put on a DEAE-cellulose column equilibrated with 10mM potas- sium phosphate buffer, pH 7.2. The column was washed with the same buffer and the cytosolic Cbl-binding proteins were eluted with a linear KC1 (0-0.4 M) gradient in the same buffer. The active fractions were combined, concentrated by the use of polyethylene glycol 20,000 and then dialyzed against 10 mM Mops-KOH buffer, pH 7.5. The dialyzed preparation was used as the partially purified preparation.

797

798 FUMIO WATANABE et al.

Preparation of the antibody against Euglena cytosolic Cbl- binding protein

The antibody against the cytosolic Cbl-binding protein with a pI of 3.8 was prepared as described previously (Watanabe et al., 1987a).

Assay of transfer of Cbl from pellicle-Cbl complex to cytosolic Cbl-binding protein

The Cbl-transfer activity was assayed by a sucrose density gradient centrifugation method. The reaction mixture (0.2 ml) contained 50 mM Mops-KOH buffer, pH 7.5, pelli- cle-[3H]CN-Cbl complex (13.4 pmol/mg protein), 30-40 pg of protein, and cytosolic Cbl-binding protein (binding ca- pacity of 23.4 pmol/mg protein), 40-50 pg of protein. The reaction mixture was put on the top of 0.1 ml of 1 M sucrose laid on the bottom on microcentrifuge tube. The reaction was started by the addition of the cytosolic Cbl-binding protein, allowed to proceed for 40 min at 25°C in the dark and stopped by centrifuging rapidly at 10,000g for 1 min, separating the cytosolic Cbl-binding protein from the pelli- cle complex. The upper fraction (0.2 ml), which contained the cytosolic Cbl-binding protein, on the sucrose layer was collected and analyzed with a liquid scintillation counter (Aloka LSC 903). The Cbl-transfer activity was determined by subtracting the radioactivity in the absence of cytosolic Cbl-binding protein from that in its presence.

Protein determination Protein was measured according to Bradford (1976) using

bovine serum albumin as a standard.

Chemicals [3H]CN-Cbl (5.1 Ci/mmol, 188.7GBq/mmol) was ob-

tained from New England Nuclear. Sephadex G-25 (fine grade) was obtained from Pharmacia Fine Chemicals. Cen- tricon-30 was obtained from Am±con Corporation. All other chemicals used were reagent grade.

RESULTS AND DISCUSSION

Time and iemperature dependence of the Cbl transfer activity

The Cbl- t ransfer activity was not detected at 0°C, while at 25°C there was a rapid initial Cbl t ransfer with the rate decreasing as equi l ibr ium was reached. Twenty-one per cent of Cbl b o u n d to the pellicle was t ransferred to the cytosolic b inding prote in after 40 min of react ion (Fig. 1). When only the pellicle- [3H]CN-Cbl complex was incubated in the absence of the cytosolic Cbl -b inding prote in at 25°C and then centrifuged, all the radioactivi ty was recovered in the pellicle fraction. The results suggest tha t the system

3.0

2.0

i' I 0 10 20 30 40 50 60

Incubation time (min)

Fig. 1. Incubation time and thermal dependencies of the Cbl-transfer activity. The reaction was started by the addition of the cytosolic Cbl-binding protein, allowed to proceed for the indicated time at 0 and 25°C, and stopped by centrifugation at 10,000g for 1 min at 0-4°C. (C)) 0°C and (O) 25°C. The data represent the mean + SD from five

experiments.

for Cbl t r anspor t f rom the pellicle to the cytosolic Cbl-b inding prote in occurs in E. gracilis.

Sephadex G-25 gel filtration

After react ion at 25°C for 40 min, the Cbl- t rans- ferred fraction, which conta ined the cytosolic Cbl- b inding protein, was put on a Sephadex G-25 column (0.5 x 4 0 c m ) equi l ibrated with 100 m M potass ium phospha te buffer, pH 7.2, and eluted with the same buffer at 20°C. The radioactivi ty in the fract ion was recovered as a single peak in void fractions. W h e n the void fraction, after being combined and concentra ted with a Centr icon-30 microconcent ra tor , were incu- ba ted with the an t ibody against Euglena cytosolic Cbl-b inding prote in or an un immunized rabb i t serum at 25°C for 1 hr and then centrifuged at 10,000g for 10 min to remove immunoprecipi ta te , all the radioac- tivity of the fract ion t reated with the an t ibody against the cytosolic b inding prote in was recovered in the precipitate but tha t of the fract ion treated with the un immunized serum was recovered in the supernatant .

Table 1. Effects of some compounds on the Cbl-transfer activity. The Cbl-transfer activity was assayed as described in the text. To correct non-specific release of Cbl from the pellicle-Cbl complex by these reagents, the activity in the addition of these reagents was determined by subtracting the radioactivity

in the absence of cytosolic Cbl-binding protein from that in its presence Relative activity Relative activity

Addition (%) Addition (%) None 100 CCCP (0.1) 85.9 + 7.1 ATP (5) 111.8 ± 7.3 Thymol (0.1) 93.9 ± 5.3 ATP (5), Mg 2÷ (5) 97.9 + 9.7 Phlorizin (1) 121.5 ± 3.6 ATP (5), Ca 2÷ (5) 96.0 ± 8.4 Quercetin (1) 103.8 ± 7.3 ATP (5), Na + (5), K + (5) 88.9 ± 7.5 DTNB (1) 115.8±4.3 Mg 2-" (5) 119.7 + 6.4 Mersaly (I) 113.4 ± 8.3 Ca 2+ (5) 78.6 ± 3.4 N-Ethylmaleimide (1) 121.0 ± 7.5 Na ÷ (5), K ÷ (5) 106.6 ± 9.3 EDTA (10) 99.7 + 6.2

EGTA (10) 111.4 ± 5.3

Final concentrations (mM) of each reagent added to the reaction mixture are given in parenthesis. The radioactivity in the control (none) was 300 DPM. The data represent the mean ± SD from three experiments.

Transfer of cobalamin within Euglena 799

The results indicate that Cbl is evidently transferred from the pellicle~Cbl complex to the cytosolic protein and that the cytosolic protein-Cbl complex is formed.

Effects of some compounds and metal ions on the Cbl-transfer activity

The Cbl-transfer activity was hardly affected by ATP and ATP plus some metal ions (Mg 2÷, Ca 2÷, Na ÷ and K ÷ ) (Table 1). Such ATPase inhibitors as thymol, phlorizin, and quercetin, did not inhibit the activity. The Cbl-transfer activity was also not inhib- ited by some thiol-group inhibitors and chelaters e.g. 5,5'-dithio-bis(2-nitorobenzoic acid), mersalyl, N- ethylmaleimide, EDTA and EGTA. The results sug- gest that ATPase and thiol-groups are not involved in the Cbl transfer to the cytosolic binding protein from the pellicle. In mammals, the attachment of the intrinsic factor-Cbl complex and the transcobalamin II-Cbl complex to their receptors have been reported to require some divalent cations, especially Ca 2÷, and to be reduced by chelating reagents like EDTA (Khadarkar and Vakil, 1982), while in E. gracilis, Ca 2÷ and EDTA did not affect the Cbl-transfer activity.

pH dependence of the Cbl-transfer activity Cbl in the pellicle-Cbl complex was not released

between pH 5.0 and 11.0 in the absence of the cytosolic Cbl-binding protein, while the Cbl-transfer activity was increased significantly above pH 7.0 and showed a maximum activity between pH 9.0 and 11.0 (Fig. 2). This activation of the Cbl transfer could be due to a conformational change of the Cbl-binding site located in the pellicle under alkaline conditions.

Optimum temperature of Cbl-transfer activity Because of release of Cbl from the pellicle-Cbl

complex by heat denaturation of the pellicular Cbl-

t3.. a

%

O "O

4.0

3.0

2.0

1.0

0

6 7 8 10 11

(pH) Fig. 2. pH dependency on the Cbl-transfer activity. The pH dependency was determined by using Tris-acetate buffer for pH 5.(~9.0, Tris-HC1 buffer for pH 9.0 and 10.0, and glycine NaOH buffer for pH 10.0 and 11.0, all at I00 mM as the reaction buffer. The Cbl-transfer activity was assayed by the method described in the text. The activity with (O) and without (C)) cytosolic Cbl-binding protein. The data

represent the mean __+ SD from three experiments.

2.0

¢ o

:E 1.o

e~

0

, , 210 ' , ; 0 10 30 4O 5 60

Temperature (°C)

Fig. 3. Optimum temperature of the Cbl-transfer activity. The optimum temperature was determined by measurement of the activity at the reaction temperature between 0 and 60°C with (0) and without (©) the cytosolic Cbl-binding protein. True Cbl-transfer activity (11) was calculated by subtracting the radioactivity without the cytosolic Cbl-bind- ing protein from that with the protein. The data represent

the mean + SD from three experiments.

binding proteins, true Cbl-transfer activity at each temperature was calculated by subtracting radioactiv- ity in the absence of the cytosolic Cbl-binding protein from that in its presence. The optimum temperature of the Cbl-transfer activity was 50°C (Fig. 3). Using Arrhenius plots, the activation energy of the Cbl transfer was calculated to be 13.3kcal/mol. The considerably high activation energy seems to be required for conformational change of the cytosolic Cbl-binding protein so that the cytosolic binding protein can take up Cbl bound tightly to the pellicu- lar Cbl-binding proteins (Ks values: 0.19-0.22nM) which have a higher affinity for Cbl than the cyto- solic Cbl-binding proteins (Ks values: 1.0-2.0 nM) (Watanabe et al., 1987a, 1988).

Cbl transfer from pellicle-Cbl complex to purified cytosolic Cbl-binding protein

We have purified two Cbl-binding proteins with different pI values (pI 3.9 and 4.7) from E. gracilis cytosol and further reported that the cytosolic binding proteins are immunologically identical with each other (Watanabe et al., 1987a). We then determined which of the cytosolic binding proteins was involved in the Cbl transfer. Both cytosolic Cbl-binding proteins showed the same degree of the Cbi-transfer activity (Table 2). The Cbl-transfer ac- tivity of the cytosolic Cbl-binding proteins was not replaced by bovine serum albumin, ovalbumin or any protein fractions other than the fraction of the cyto- solic binding proteins from the DEAE-cellulose

800 FUMIO WATANABE et al.

Table 2. The Cbl-transfer activity of two cytosolic Cbl-binding proteins. The reaction mixture of the transfer activity contained 50raM Mops-KOH buffer, pH 7.5, pellicle-[3H]CN-Cbl complex (13,4pmol/mg protein), 2.25mg protein and purified cytosolic Cbl-binding proteins (each 20#g of protein). The reaction was

allowed to proceed for 40 min at 25°C in the dark

Cbl-transfer activity (pmol/mg protein)

Cytosolic Cbl-binding protein with pI 3.8 9.2 _+ 0.3 Cytosolic Cbl-binding protein with pI 4.7 8.4 + 0.4 The data represent the mean -2_ SD from three experiments.

chromatography of Euglena cytosol (each added to 50-100 # g of protein). Our previous studies indicated that the cytosolic fraction contained about half of the total intracellular Cbl-binding activity in both E. gracilis z and SM-ZK, and that in in vivo experiments of Euglena labeling with radioactive CN-Cbl for 2 hr, some 47.6% of the Cbl taken up by the cells was found in the cytosol (Watanabe et al., 1987a; Isegawa et al., 1984). These observations and the results obtained here suggest that both cytosolic Cbl-binding proteins function as intracellular Cbl accumulators or carriers.

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Youngdahl-Turner P., Mellman I. S., Allen R. H. and Rosenberg L. E. (1979) Protein mediated vitamin uptake: Adsorptive endocytosis of the transcobalamin II-cobal- amin complex by cultured human fibroblasts. Expl Cell Res. 118, 127-134.