Incorporation and metabolic conversion of cyanocobalamin by ehrlich ascites carcinoma cells in vitro and in vivo

348

Biochimica et Biophysica Acta, 381 (1975) 348--358 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27585

INCORPORATION AND METABOLIC CONVERSION OF CYANOCOBALAMIN BY EHRLICH ASCITES CARCINOMA CELLS IN VITRO AND IN VIVO

K A R E N PEIRCE, T S U K A S A ABE* and B E R N A R D A. COOPER**

Hematology Division, Royal Victoria Hospital, and Departments of Experimental Medicine and Physiology, McGill University, Montreal, Quebec (Canada) (Received September 5th, 1974)

Summary 1. Cyano[STCo]cobalamin bound to murine transcobalamin, associates with Ehrlich ascites carcinoma cells. Association was found to be dependent on temperature, and to require between 7.2 • 10 -s and 2 • 10 -4 M ionized calcium. 2. Association was blocked by vinblastine and colchicine, but not cytochalasin, suggesting that microtubules may be involved in this phenomenon. 3. Although irreversible association of radioactivity with cells was observed withirt minutes, appearance of significant radioactivity associated with the intracellular B 12 binder, and conversion of cyanocobalamin to methyl- and 5'deoxyadenosylcobalamin required more than 18 h of incubation. 4. A pool of free vitamin B12 was found in cell extracts. This was composed of metabolically-active cobalamins characteristic of the interior of the cell, and not of cyanocobalamin recently incorporated. 5. Incorporation of 5 VCo.labelle d vitamin B 12 by these cells involves two major processes: a rapid irreversible association of transcobalamin-B 12 complex following reaction with a presumably calcium
Introduction Incorporation of vitamin B~ 2 by some mammalian cells is facilitated by prior association of the binder with an extracellular protein. The vitamin-* Present address: T o k y o Medical and Dental College, Tokyo, Japan. ** Medical Research Associate, Medical Research Council of Canada. Requests for reprints should be directed to: Dr B.A. Cooper, Division of Hematology, Royal Victoria Hospital 687 Pine Ave. West, Montreal, Quebec, C a n a d a H 3 A 1A1.

349 protein complex then associates with the cell surface [1--7]. This transport p h e n o m e n o n may be similar to the intrinsic factor-mediated transport of vitamin B~2 across the mammalian intestine. Similarities between the intrinsic factor-mediated system, and that observed with non-intestinal cells include the specificity of the B~ 2 binder, and the calcium dependence of association of the complex with the cell [8,9]. Murine leukemia cells in culture, and h u m a n bone marrow cells in vivo probably utilize transcobalamin 2 to incorporate vitamin B~ 2 • Murine leukemia L5178Y develops deficiency of vitamin B~ z in the absence of this transcobalamin [7]. Two h u m a n subjects have been described with congenital absence of transcobalamin 2 [10]. In these children, megaloblastic anemia was observed when serum B~ z concentration was not extremely high. Because these observations suggest that this mechanism of B~ 2 utilization by cells may be important physiologically, we have studied the transport of vitamin B~ 2 into Ehrlich ascites carcinoma cells, utilizing the murine transcobalamin in the ascitic fluid of tumor-bearing mice as the binder responsible for incorporation of vitamin B~ 2 by the cells. Materials and Methods Swiss white mice were injected intraperitoneally with Ehrlich ascites carcinoma, and t u m o r cells were harvested from the peritoneal cavity on the 6th, 7th, or 8th day after infection [2]. Cells were washed free of suspending ascitic fluid with three washes of cold Krebs--Ringer--Henseleit at pH 7.4, and resuspended in the same buffer at a concentration of 20% (v/v). Such suspensions were found to contain 2.5--5.0 × 107 cells per ml, representing a mean of 11.2--22.4 mg protein. Ascitic fluid was pooled, and stored in aliquots at--20°C. Only a single B1 z binder was observed during filtration of this fluid through Sephadex G-200: a fraction which filtered with a Stokes radius of about 2.5 nm. The same binder was observed in the plasma of the mice, and the BI 2 binding capacity of plasma was similar to that of ascitic fluid. The binding capacity for added cyanocobalamin of four batches of ascitic fluid used in these experiments was 40.2, 39.7, 36.7, and 36.1 ng per ml. In vitro incubations were conducted in 6-ml volumes in 25-ml Erlenmeyer flasks which were stoppered after gassing with 5% CO2--95% 0 2 . Incubation solutions contained 10 ng of cyano[ s 7Co] cobalamin, ascitic fluid (approx. 0.25 ml) 0.2 ml of ascites cells, and other test materials in Krebs--Ringer Henseleit buffer at pH 7.4. During 2 h of incubation at 37 ° C in vitro, pH did n o t fall below 7.1. After incubation, the contents of each flask were decanted into 7.5 ml of ice-cold saline buffered with 0.1 M sodium phosphate at pH 7.4, and immediately centrifuged at 10 000 × g for 0.5 min. The supernatant solution was removed, and the cells washed twice with the same solution. Care was taken to gently resuspend the cells during washing, and to keep them cold. Before the last centrifugation, a sample was taken for cell c o u n t and/or protein determination. The final pellet was counted for radioactivity in a well-type of scintillation spectrometer. Results were expressed as ng of radioactive B~ 2 by compari-

350 son with standards counted simultaneously. Cell counts were determined manually. Incorporation of [ 3H] sucrose by the cells was measured as described by Wagner et al. [11] in a total volume of 3.0 ml. After incubation, cells were centrifuged, washed thrice in 5 ml of 80 mM sucrose, dissolved in Beckman Biosolv BBS-2, mixed with 10 ml of toluene containing PPO and POPOP and counted in a liquid scintillation spectrometer. Counts were compared with those of cells mixed with sucrose immediately before washing. Homogenates of these cells did not hydrolyse sucrose, and none of the 3H.label in the preparation filtered through Sephadex G-10 with monosaccharides. Association of ferritin with the cells was determined by incubating cells in solutions containing 95 mg of ferritin per 6 ml of incubation solution. Experiments with ferritin were done simultaneously with studies of B, 2 -uptake using the same inhibitors for both. After incubation, 2 ml of incubation solution was added to 2 ml of ice-cold 3% glutaraldehyde in cocodylate buffer for 5 min, supernatant was removed by centrifugation and replaced with 2 ml of glutaraldehyde-cocodylate for 150 min, the cells were washed thrice in cocodylate buffer, concentrated by centrifugation at 1200 X g, aspirated into capillary tubes and centrifuged. The pellets were expressed into 0.33% osmium tetraoxide for 1 h, dehydrated in increasing concentrations of ethanol, embedded in epon and sectioned. Samples were examined at 45 000 X, and 10 smooth areas of cell surface were selected for photography. Final magnification of photographs was 90 000 X. Incorporation of radioactive BI 2 by Ehrlich cells in vivo was studied by injecting 10 ng of cyano[ s 7Co ] cobalamin into the peritoneal cavity of mice 6 days after infection with tumor. The mice were returned to cages, and the cells collected after the selected interval. Cells were washed as described above. B 12 forms also were fractionated in liver, kidney, plasma, and ascitic fluid of mice so studied. 48 h after injection of cyano[ s 7Co]cobalamin ' the proportion of s 7Co.activity identified as cyanocobalamin was 33, 62, 63, and 88%, respectively, and as 5'deoxyadenosylcobalamin as 65, 38, 24, and 8%, respectively. Fractionation of B, 2 forms in cells and fluid utilized the technique of Linnell et al. [12] as follows: Cells were washed thrice in ice cold Krebs-Ringer Henseleit, sonified in ice at full power for 2 min in a Branson sonifier, centrifuged at 100 000 X g for 1 h and the cobalamins in the supernatant extracted. The solution was desalted in a Torbal desalting apparatus, and chromatographed on Eastman 6061 thin-layer silica gel plates in secondary butanol: ammonia: water, 95 : 2 : 25. All f~actionation was conducted in the dark, with no illumination except a red safe-light. For determination of radioactivity, plates were cut into 1-cm strips; for determination of biologically-active cobalamins, the thin-layer plates were dried in air and applied to agar containing Bacto-EC medium, Escherichia coli ATCC 10799, and 2,3,5-triphenyl-2,4 tetrazolium chloride, and incubated for 18 h. Red spots indicated areas of bacterial growth. Spots were quantitated by comparison with standards of purified cobalamins kindly provided by Dr Kiyoshi Kwabe, Esai Co. Ltd., Tokyo, Japan. The use of Eastman silica gel thin-layer plates allowed separation of hydroxocobalamin from 5'-deoxyadenosylcobalamin. The former remained at the origin, whereas the latter migrated 1--2 cm during chromatography.

351 Cobalamins associated with Ehrlich ascites cells were also fractionated by determining the size of the materials with which they were associated. After washing, sonification, and centrifugation at 100 000 × g, the supernatant solution was filtered through Sephadex G-200 at 4--6°C in the dark. Tubes composing each fraction were pooled after determination of radioactivity of aliquots, and the distribution of cobalamins in them was determined: Cytochalasin B was purchased from Imperial Chemical Industries, Cheshire, England. Its p o t e n c y was verified by observing inhibition of phagocytosis of bacteria by human neutrophils incubated in 10 pg per ml of this material. Vinblastine and vincristine were purchased from Eli Lilly, Toronto, colchicine from Laboratories Houde, Paris, as 1 mg per ml in saline, and ethyleneglycol-bis-(~-aminoethylether)-N,N'-tetraacetic acid (EGTA) from Eastman Organic Chemicals, New York. Results

Incorporation of radioactive cyanocobalamin by cells Ehrlich ascites cells incubated in vitro in solutions containing cyanoIS 7Co] cobalamin b o u n d to murine transcobalamin incorporated radioactivity. Cells washed after 1 h of incubation and transferred to fresh medium containing radioactive cyanocobalamin b o u n d to transcobalamin continued to incorporate radioactivity (Fig. 1). Those transferred to medium containing nonradioactive cyanocobalamin b o u n d to transcobalamin exchanged some of the radioactivity previously incorporated, b u t the remainder of the radioactivity remained associated with the cells.

of

uploke

STCoB~z

by

Ehrlich

cells /.

90"

control

/-"//" o'.///,,"* 240 ¢n

---- 2 0 0 ~C)

160'

"--

120

o

_~

wo/h

~

~

6o-

40-

~.~

,//"

,/

Q)~,,"

,,/ °

,//'"

cE ,.;

SO'

30-

.-. SgCOB ~2

20,,

40

3

,,7t/"

STCoB2, ~50"

CL

n'l

70-

b

,......... .......

E G T A

i0 £

•

0

_.:.

;

i

~ hours

;,

'[

o

5'0 incubotion

6'0 time

9'0

I{0

(min.)

Fig. 1. U p t a k e of c y a n o [ 57Co ] c o b a l a m i n b y E h r l i c h ascites cells in v i t r o . Cells w e r e i n c u b a t e d in m e d i u m c o n t a i n i n g r a d i o a c t i v e B 12 a n d ascitic fluid for 1 h, w a s h e d , a n d t h e n r e i n c u b a t e d in i d e n t i c a l s o l u t i o n s (o o), or in s o l u t i o n s c o n t a i n i n g n o n r a d i o a c t i v e B 12 ( e . . . . . . e). Fig. 2. E f f e c t o f c a l c i u m c h e l a t i o n of u p t a k e o f c y a n o [ s 7Co] c o b a l a m i n b y E h r l i c h ascites cells in v i t r o . Cells w e r e i n c u b a t e d in c y a n o [ S ? C o ] c o b a l a m i n w i t h ascitic fluid at 3 7 ° C . × . . . . . . X, E G T A p r e s e n t in t h e i n c u b a t i o n vessel a t t h e s t a r t of t h e e x p e r i m e n t , a n d at the t i m e s i n d i c a t e d , e x c e s s c a l c i u m w a s a d d e d ; • -•, c a l c i u m p r e s e n t at t h e start, a n d a t t h e t i m e s i n d i c a t e d , e x c e s s E G T A w a s a d d e d .

352 6O --~ 50 % L)

6050-

%

40: ~

1 min.

30 -~ 2O

~

40

-

50

co-

20

o_

~0 ~ ' : 0

rain. "0005 0010 M CaCI2

"0015

O

C O CBICB2CB5 0

C D

incubation

CB2CB5 30 time

C D CBICB2CB3 60 (rain.)

Fig. 3. T i t r a t i o n o f c a l c i u m and E G T A e f f e c t o n u p t a k e o f c y a n o [ 5 ?Co ] c o b a l a m i n b y E h r l i c h cells. Cells w e r e i n c u b a t e d in vitro at 3 7 ° C in m e d i u m c o n t a i n i n g 0 . 0 0 1 M EGTA a n d t h e c a l c i u m c o n c e n t r a t i o n indicated above. Uptake over the periods of incubation indicated was determined. Fig. 4. E f f e c t o f c y t o c h a l a s i n B o n u p t a k e o f c y a n o [ 57Co ] c o b a l a m i n . Cells w e r e i n c u b a t e d in m e d i u m a l o n e (C), m e d i u m w i t h dL,n e t h y l s u l f o x i d e (DMSO) (D), or in m e d i u m w i t h c y t o c h a l a s i n B in DMSO (CB), f o r t h e p e r i o d s i n d i c a t e d . C B 1 r e p r e s e n t s 1, CB 2 r e p r e s e n t s 1 0 , a n d CB 3 r e p r e s e n t s 50/~g per ml o f t h e i n h i b i t o r . S h a d e d areas r e p r e s e n t u p t a k e o f O°C, o p e n areas t h e d i f f e r e n c e b e t w e e n u p t a k e at 37°C a n d t h a t at 0 ° C.

Uptake in vitro was calcium-dependent, as described previously [ 1 ] . Incubation of cells in 1.5 "10-~M EGTA stopped uptake of cyano[57Co] cobalamin b o u n d to transcobalamin; uptake of radioactivity resumed when calcium (1.8 • 10 -3 M) was added to the incubation medium (Fig. 2). The concentration of ionized calcium required for the p h e n o m e n o n was calculated from data in Fig. 3, in which increasing concentrations of calcium were added to incubation medium containing 0.001 M EGTA, c y a n o [ s 7Co]cobalamin b o u n d to transcobalamin, and ascites cells. Based on data presented by Portzehl et al. [ 1 3 ] , the free calcium required for half-maximum uptake at 0 and 90 min at 37°C was between 7.2 • 10 -~ and 2 • 10 -4 M. Uptake in vitro was n o t affected by cytochalasin B 1--50 pg per ml (Fig. 4), but was reduced when cells were incubated in iodoacetate (data not shown), vinblastine or colchicine (Fig. 5). Cells were preincubated in inhibitor I00

control

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~

~

c°lchicineZ

~ ' _ - _ : : ' - : ~ . ..... ,,j ...............

3'0 incubation

do time

do

t2b

(rain.)

Fig. 5. E f f e c t o f c o l e h i e i n e o n u p t a k e o f c y a n o [ 5 7 C o ] c o b a l a m i n in vitro. S o l i d l i n e s i n d i c a t e i n c u b a t i o n at 3 7 ° C ; b r o k e n l i n e s at 0 °. L i n e s m a r k e d w i t h X ' s ( c o l c h i c i n e 1) r e p r e s e n t e x p e r i m e n t s w i t h 2 . 5 • 1 0 -4 M c o l e h i c i n e ; t h o s e w i t h O's ( c o l c h i c i n e 2 ) , w i t h 5 • 1 0 - 4 M.

353 150-

1201

~

control

50

u~

901 % 801 701 Q3 601 so i 405o I

57° controI

r

.-x

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vinblostine

50-

° control

o ~oIo-

30 60 90 120 incubation lime (min.)

0

~

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iodoacetate

5b io 9'o ,~o incubation time (min.)

Fig. 6. R e v e r s i b i l i t y of i n h i b i t i o n c a u s e d b y v i n b l a s t i n e . Ceils w e r e i n c u b a t e d in v i t r o at 3 7 ° C . T h e line j o i n i n g p o i n t s r e p r e s e n t s c o n t r o l ; t h o s e j o i n i n g × 's r e p r e s e n t flasks c o n t a i n i n g 1.2 • 1 0 -4 M v i n b l a s t i n e . A t i n t e r v a l s , cells in v i n b l a s t i n e w e r e w a s h e d a n d r e s u s p e n d e d in fresh, v i n b l a s t i n e - f r e e m e d i u m (X . . . . . . × ): ( A ) a f t e r p r e i n c u b a t i o n w i t h v i n b l a s t i n e , b u t s i m u l t a n e o u s w i t h i n c u b a t i o n in c y a n o [ 57Co ] c o b a l a m i n a n d aseitic fluid, (B) a f t e r 30 rain o f i n c u b a t i o n in STCo-labelled v i t a m i n B12 a n d ascitic fluid, a n d (C) a f t e r 6 0 rain. Cells in (D) (× X ) r e m a i n e d in v i n b l a s t i n e t h r o u g h o u t t h e e x p e r i m e n t . Fig. 7. E f f e c t of i n h i b i t o r s on i n c o r p o r a t i o n o f s u r c r o s e b y cells in v i t r o . Cells w e r e i n c u b a t e d in 0.08 M s u c r o s e , 0 . 0 1 4 M glucose, 0.2 m l of m o u s e aseitic fluid in 2 m l v o l u m e s . C y t o c h a l a s i n B w a s p r e s e n t at 2 0 #g p e r ml; i o d o a c e t a t e at 0.01 M.

for 60 min before each experiment. The effect of vinblastine was only partly reversible; recovery being inversely proportional to the duration of incubation with the inhibitor (Fig. 6). Inhibition of uptake was less complete with these agents than when calcium was removed from the medium and these inhibitors did not significantly affect the small association of cyano[STCo] cobalamin bound to transcobalamin, which was observed at 0 ° C, or after a few seconds of incubation at 37°C, whereas calcium chelation reduced this. The lower Y intercept of the vinblastine curve in Fig. 6 was not typical of all experiments. The effect of inhibitors on pinocytosis was measured using [ 3H] sucrose and ferritin. Sucrose uptake was temperature dependent, inhibited by iodoacetate, and only slightly altered by cytochalasin B (Fig. 7). Sucrose uptake TABLE

I

FERRITIN

UPTAKE

BY EHRLICH

ASCITES

CELLS

IN V I T R O

N u m b e r s represent m e a n per 16 × 16 c m photograph, final magnification 9 0 0 0 0 X.

Control

Surface L e n g t h (era) Ferritin Vesicles Number Ferritin * t = 2.13,

Vinblastine 60 rain

Cytochalasin 60 min

1 rain

60 rain

9.7

12.3

9.6

10.2

6.8

9.9

6.7

6.0

2.4 0.2*

3.0 0.8*

2.8 0.5

2.2 1.1

v = 17, P < 0.05.

354 was unaffected by the presence of ascitic fluid. Ferritin uptake was quantitated by measuring the ferritin bound per cm of surface on photomicrographs of standard dimension, and counting ferritin particles observed in vesicles (Table I). It was apparent that whereas more ferritin was observed in vesicles after 60 min of incubation than after 1 min of incubation Co < 0.05), there was no significant difference in any of these parameters in cells incubated with ferritin with or w i t h o u t vinblastine or cytochalasin. Plaques of microtubules were observed in photographs of cells incubated with vinblastine.

Characteristics o f vitamin B12 associated with cells Radioactivity was associated with four fractions when supernatants of cell homogenates were filtered through Sephadex G-200 (Fig. 8). These corresponded to spherical proteins with molecular weights in excess of 300 000, about 120 000, about 40 000, and about 1000 [14]. The 40 000 fraction filtered in the same position as cyano[ s 7Co]cobalamin bound to the transcobalamin in murine ascitic fluid, and the 1000 fraction co-filtered with free cyanocobalamin. Antiserum prepared in rabbits to murine ascitic fluid binding cyanocobalamin reacted with the 40 000 fraction, and caused it to migrate with the excluded portion of the gel. This antiserum also combined with human transcobalamin 2. This suggests that the 40 000 fraction was identical with the murine transcobalamin present in the ascitic fluid. Little radioactivity was associated with the 120 000 fraction for the first 6 h of incubation, but as the time between injection of cyano[ s 7Co]cobalamin and collection of cells increased beyond this, an increasing proportion of radioactivity was associated with this fraction. These studies were done in vivo, but mice were fed on feed containing cyanocobalamin to provide some competition for later utilization of cyano[ S 7Co]cobalamin. The specific activity of cyanocobalamin assayed in ascitic fluid of mice after intraperitoneal injection of cyano[S 7Co] cobalamin decreased progressively over the first 6 h after injection, and then remained stable. B,2 distribution

in

sephodex G-2OO fractions

4000] •~

~ 0

48 hr

3000-] 2000

m 0

I00 0

I0 20 30 40 50 60 70 80 tu be

no.

Fig. 8. F i l t r a t i o n t h r o u g h S e p h a d e x G - 2 0 0 o f e x t r a c t s o f cells i n c u b a t e d in vivo w i t h c y a n o [ 5 7 C o ] cobalamin. C y a n o [ 57Co] c o b a l a m i n was injected into t u m o r - b e a r i n g mice, and cells harvested after the i n t e r v a l s i n d i c a t e d a b o v e . Cells w e r e h o m o g e n i z e d , a n d p a r t i c l e - f r e e s u p e r n a t a n t f i l t e r e d t h r o u g h S e p h a d e x . T h e p a t t e r n o f r a d i o a c t i v i t y f o r e a c h e x t r a c t is i n d i c a t e d . V o i n t h i s c o l u m n w a s i n t u b e 2 2 ; V t in tubes 68--69.

355

Fractionation of B, 2-forms in cells When the radioactivity and the microbiologically-active cobalamins were fractionated in the Sephadex fractions obtained from Fig. 8, different patterns of cobalamin distribution were found in each Sephadex fraction (Table II). Cyanocobalamin was present in significant quantities only associated with the 40 000 fraction presumed identical with the transcobalamin in ascitic fluid. The proportion of cyano[ s 7Co]cobalamin associated with this fraction decreased progressively as 5'-deoxyadenosylcobalamin increased. The distribution of cobalamins in the other fractions remained constant throughout the experiment, and similar to the distribution of microbiologically-active cobalamins within the cell. The proportion of the total [ s 7Co ] cobalamin associated with the cells which chromatographed as 5'-deoxyadenosylcobalamin increased progressively during the experiment, and corresponded roughly to the proportion of s 7Co_activity associated with the 120 000 fraction. Since conversion of cyanocobalamin to 5'-deoxyadenosylcobalamin required penetration of the vitamin into the cytoplasm for reduction and reaction with ATP, this suggests that association of radioactivity with the 120 000 fraction also represents penetration of the B, 2 into the cytoplasm. T A B L E II E F F E C T O F D U R A T I O N O F I N C U B A T I O N I N V I V O ON D I S T R I B U T I O N OF F O R M S O F V I T A M I N B12 IN C E L L S (%) T h e B12 ( e i t h e r 57Co-labeUed or m i c r o b i o l o g i c a l l y - a c t i v e ) in e a c h e x p e r i m e n t is e x p r e s s e d as the p e r c e n t o f t h e t o t a l B12 r e c o v e r e d f r o m t h e s u p e r n a t a n t of the cell e x t r a c t . S e p h a d e x f r a c t i o n s I, II, III, I V corres p o n d t o s p h e r i c a l p r o t e i n s w i t h m o l e c u l a r w e i g h t s of a b o u t o v e r 3 0 0 0 0 0 , 1 2 0 0 0 0 , 4 0 0 0 0 , a n d 1000, respectively.

I

II

nI

IV

6h

I

II

III

IV

methylcyano5'-deoxyOH

0 2 5 2

0 1 8 2

4 37 5 6

2 5 17 2

24h methylcyano5'-deoxyOH-

1 2 5 ]

0 4 25 I0

0 10 10 0

2 8 18 1

12 h methylcyano5'-deoxyOH-

1 1 6 2

1 2 10 3

2 31 11 4

2 6 17 2

36 h methylcyano5'-deoxyOH-

1 1 4 1

3 2 40 1

2 9 8 1

2 6 17 2

18 h methylcyano5'-deoxyOH-

1 2 6 2

1 1 13 3

3 23 14 3

2 5 18 2

48 h methylcyano5'-deoxyOH-

1 1 4 1

2 4 33 8

0 12 7 4

1 4 14 3

Controls S7Co-cyano plus cells

methyleyano5'-deoxyOH-

1 7 2 2

0 0 0 0

0 39 11 3

0 30 3 2

M i c r o b i o l o gically-ac tive BI2 methylcyano5'-deoxyOH-

2 0 7 0

11 i 41 6

3 2 9 0

2 I 11 2

356 Discussion The demonstration that lymphoid cells in culture and human bone marrow in vivo probably require transcobalamins for effective utilization of vitamin BI : suggests that this transport system has physiological relevance. It differs from the transport of iron into immature erythroid cells [15] in the long delay observed in our experiments between irreversible association of a considerable proportion of the B12 with the cells, and its appearance in the pool of vitamin BI 2 converted to metabolically-active forms. As reported previously, association of transcobalamin-bound cyanocobalamin with Ehrlich ascites cells is temperature dependent, and requires ionized calcium [1,2]. Based on the free calcium calculated to be present in a mixture of EGTA and calcium [13], we have calculated that half-maximum association of transcobalamin-bound cyano[ s 7Co] cobalamin with the cells requires between 7.2 • 10 -~ M and 2 " 10 -4 M of free calcium ions. If the calcium effect is to activate the system by combining with anionic groups involved in the B ~2-uptake reaction, then the concentration of free calcium ions required for half-maximum activity should approximate the dissociation constant of calcium ions for the ligand required for the reaction. Salts of calcium with monocarboxylic amino acids, monocarboxylic organic acid, phosphate, and sulphate have much higher dissociation constants than 10 -4 M [16], whereas chelates of calcium have dissociation constants in this range. This suggests that calcium does not form a bridge between transcobalamin and the receptor on the surface of the cell, but chelates to either the cell surface or the transcobalamin. Data are not available to differentiate between these, but it may be that calcium is required to maintain the configuration of the surface receptor, as vitamin B~ 2 is required to maintain the configuration of transcobalamin. The data c f Finkler and Hall [17] indicate that the configuration of the transcobalamin 2--B~ 2 receptor on HeLa and Sarcoma 180 cells requires disulphide bridges. Iodoacetate, colchicine, and vinblastine did not decrease the radioactivity associated with vitamin BI .~ after a few seconds of incubation at 37°C, or prolonged incubation at 0 ° C, suggesting that association with the cell surface is not affected by these materials or by low temperature. Although the effect of vinca alkaloids and colchicine is not restricted to microtubule assembly, the similarity of effect of these on the B~ 2 uptake p h e n o m e n o n suggests that intact microtubules may be required for this phenomenon. Cytochalasin B did not affect B~ 2 uptake, but it appeared not to affect pinocytosis measured with sucrose and ferritin. Although much of the B~ 2 associated with the cells did not exchange with the medium after 60 rain of incubation in vitro, none of the B~ 2 at that time was associated with the large-radius intracellular binder of the native B~ 2 of the cell, nor was any significant quantity converted to 5'deoxyadenosylcobalamin, suggesting that the non-exchangeable B~ 2 had not entered the cytoplasm of the cells. It might have been associated with surface, and have become unexchangeable because of trapping in corrugations of the surface membrane, suggesting that the uptake p h e n o m e n o n observed in vitro is restricted to association with surface receptors, and its progression with time represents exposure of fresh

357

receptors as fresh membrane is exposed to cell movement. Iodoacetate, vinblastine, colchicine, and low temperature might then affect the p h e n o m e n o n by decreasing membrane m o v e m e n t and preventing exposure of fresh receptors. The observations also are consistent with incorporation of materials b o u n d to the surface receptor into the cell in micropinosomes, or similar sequestra. The incomplete inhibition of uptake of transcobalamin-bound cyanocobalamin by vinblastine and colchicine might then be due to dependence of such vesicles on microtubules for further processing. Electron microscopic studies did not clarify this possibility. The means by which vitamin BI 2 leaves the transcobalamin and enters the cytoplasm is unknown. The apparent del'ay in penetration into the cytoplasm might be caused by the time required to synthesize fresh intracellular binder. The long delay in presentation of the cyanocobalamin to the pool of vitamin B12 available for conversion to metabolically-active coenzyme forms makes difficult studies utilizing metabolic inhibitors. Radioactive cyanocobalamin added to fresh Ehrlich cells immediately before homogenization became associated with fraction 3 and 4 (the transcobalamin found in ascitic fluid, and free B j ~ ), slightly with fraction 1 (material excluded from Sephadex G-200), and not at all with fraction 2 (the presumed intracellular binder). This indicates that vitamin B~ 2 associated with all fractions except No. 2 exchanged somewhat in vitro, and that free B~ 2 from fraction 4 could have modified the distribution of cobalamins associated with fractions 1 and 3. Whether fraction 4 (free B~2) exists as free cobalamins in vivo, or represents endogenous cobalamins released during preparation, is unknown. The predominance of metabolically-active cobalamin coenTymes in this fraction, however, indicates that it does not represent cyanocobalamin released from the uptake complex. The function of the excluded material is unknown. Although it represented little of the radioactive material, microbiological assay revealed that a considerable quantity of endogenous cobalamins filtered through sephadex in this position. It may represent membrane-associated cobalamins and endogenous transcobalamins. It probably does not represent receptor--transcobalamin complex because of the types of cobalamins found in it at a time when the majority of cobalamin in ascitic fluid in the animal was cyanocobalamin. Although these data do not allow delineation of the mechanism of penetration of cyanocobalamin b o u n d to mouse transcobalamin into the cytoplasm of Ehrlich ascites cells, they do n o t support classical endocytosis as the mechanism of transcobalamin-Bl 2 b o u n d to surface receptor with release of free B i 2 by lysozomal enzymes, and subsequent binding to endogenous binder, before or after slow penetration of the free vitamin into the cytoplasm. They are consistent with several models, including a long period of membrane association or formation of micropinosomes w i t h o u t lysozomal fusion, followed by slow penetration of the cytoplasm by the vitamin as it becomes associated with an intracellular transcobalamin and is converted to hydroxocobalamin, 5'-deoxyadenosylcobalamin and methylcobalamin.

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Acknowledgments We are grateful to Mrs Barbara Mudd for technical assistance, and to the Medical Research Council of Canada for Grant MT-802 which supported these studies. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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