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The relative permeability of lysosomes from Tetrahymena pyriformis to carbohydrates, lactate and the cryoprotective nonelectrolytes glycerol and dimethylsulphoxide
55 °
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
75494
T H E R E L A T I V E P E R M E A B I L I T Y OF LYSOSOMES FROM T E T R A H Y M E I \ L 4 PYRIFOR3IIS
TO CARBOHYDRATES, LACTATE AND T H E
CRYOPROTECTIVE N O N E L E C T R O L Y T E S GLYCEROL AND I)IMETHYI.SULPHOXIDE D()N:kLI) I,E I,; Division of I.o~, Tcmpcralttrc l~ioloL~7, Clinical Research Ccnl~e Laborah)ries, National lnstiluh' f,~ Medical Research, 3 Mill Hill, London N. W.7 (Great Britain)
(Received May 4th, ~07o)
SUMMARY Lysosomes in homogenates of T e t r a h y m e u a p y r i f o r m i s have been shown to be permeable to the cryoprotective nonelectrolvtes glycerol and dimethylsulphoxide, to the hexoses glucose, galactose and ~-methylglucose and to the hexitols mannitol, sorbitol and inositol. They are impermeable to the disaccharides sucrose, maltose and melibiose and to the trisaccharide raffinose. The presence of a charged group has a great influence on permeability, the lvsosomes being impermeable to the gluconate and glucuronate anions and only slightly permeable to the lactate anion and the glucosaminium cation.
INTRODU('TION
Lysosomes have now been described in many types of cells 1 and, although some of the earlier studies on rat liver lysosomes concerned their permeability 2 ~, it was only recently a that a systematic study was made of their permeability to carbohydrates. As nothing is known about the permeability characteristics of lysosomes from the ciliate protozoan T e t r a h y m e n a p3,riformis, it was of interest to study their response to carbohydrates and, in addition, to the cryoprotective agents glycerol and dimethylsulphoxide, as preliminary results G had shown dimethylsulphoxide to protect these lysosomes against damage during freezing. MATERIALSANI} METHODS Dinmthylsulphoxide (Laboratory Reagent Grade), glycerol (A.R.), glucose (A.R.) and litlfium lactate were from Hopkin and Williams, Chadwell Heath, Essex. Mannitol was the product of British Drug Houses, Poole, Dorset. fl-Glycerophosphate (Grade I), Triton X-Ioo and all other carbohydrates were supplied by Sigma (London), London S.W.6. Biochim. Biophys..4 eta, 2t i (r 97°) 55o-554
PERMEABILITY OF LYSOSOMES FROM T. pyriformis
551
Growth and harvesting of cells T.pyriformis, strain S, was grown in tile dark at room t e m p e r a t u r e L Cells were h a r v e s t e d between 5 a n d 7 days after inoculation, washed with 0.25 M sucrose in the m a n n e r described previously8 (except t h a t centrifugation was at 15oo x g for 2.5 rain) a n d resuspended in 0.25 M sucrose at a c o n c e n t r a t i o n between IO7 a n d 5" IO7 cells per ml before disruption at 4 ° b y exposure to p H I I for between 45 sec a n d i rain. Acid phosphatase (orthophosphoric monoester phosphoroilydrolase, EC 3.1.3.2) was assayed in triplicate in tile m a n n e r described previouslyS; all assays contained o.25 M sucrose in addition to tile sucrose which was present in the homogenates.
Assessment of permeability of the lysosomal membrane After preparation, homogenates were placed in ice a n d an aliquot diluted with 4 vol. of the solution u n d e r e x a m i n a t i o n . At t i m e d intervals thereafter, samples of the diluted homogenates were assayed for acid phosphatase activity. For the d e t e r m i n a tion of total activity, assay m i x t u r e s c o n t a i n e d 0.05 °o T r i t o n X - i o o a n d latency is defined as the percentage of tile total a c t i v i t y u n m a s k e d b y incorporation of T r i t o n X - i o o into the assay mixture. As there is no i n d i c a t i o n in tile literature t h a t a n y of the c o m p o u n d s u n d e r e x a m i n a t i o n has a direct lytie action on biological m e l n b r a n e s at tile c o n c e n t r a t i o n s studied, it was assumed t h a t a decrease in l a t e n c y of acid phosphatase was the result of p e r m e a t i o n of the substance through the lvsosomal m e m b r a n e . RESULTS
I n Fig. I it can be seen t h a t the l a t e n c y of acid phosphatase in an h o m o g e n a t e d i l u t e d with o.25 M sucrose decreased only slightly d u r i n g the s u b s e q u e n t 2-tl period, while the l a t e n c y in homogenates diluted with o.25 M glucose or o.25 M e - m e t h y l glucose decreased d r a m a t i c a l l y (although in a p p r o x i m a t e l y the same fashion). I n another e x p e r i m e n t tile l a t e n c y was well m a i n t a i n e d on dilution with the trisaccharide raffinose, although it decreased on dilution with the hexitols sorbitol a n d inositol.
•
on
~- ~
"~
~c
%
•
....
o~
~c
4c ~2c o ~
"6
o o za
o &
Time(h)
Fig. I. Latency of acid phosphatase in two homogenates prepared with 0.25 M s u c r o s e . ( I ) After dilution with sucrose ([~), with glucose (&), with a-methylglucose (O). (2)After dilution with raffinose ( • ), sorbitol (~k), with inositol (O). The concentration of the diluting solutions was o.25 M. Fig. 2. Latency of acid phosphatase in two homogenates prepared with o.25 ~I sucrose. (i) After dilution with melibiose (El), with glycerol (A), with dimethylsulphoxide (O). (2) After dilution with maltose ( • ), with galactose (A), with mannitol (O). The concentration of the diluting solutions was 0.25 M.
Biochim. Biophys. Acta, 211 (197o) 55o-554
55 2
D. LEE
Fig. 2 shows the latency of acid phosphatase to remain high after dilution with maltose but to decrease on dilution with galactose and mannitol. Similarly, latencv was well maintained with the disaccharide melibiose, although dilution with glycerol or dinmthylsulphoxide resulted in a much more rapid decrease in latency than was seen with hexoses or hexitols. All of the compounds tested above are unchanged, and it was decided to study the effect of charged groups on permeability. Fig. 3 shows that dilution of an homogenate with o.25 M potassium gluconate or o.25 M potassium glucuronate resulted in only a slight decrease in latency of acid phosphatase over the subsequent 2-h period. This decrease probably does not differ significantly from that found in the control holnogenate diluted with sucrose and, in any' case, it is clearly seen that the presence of an anionic grouping had a profound effect on the permeability of these lnolecules as coinpared to glucose. o
i 6C
(,'i
.1(i
s 5
5
I
"
T r T e (q~
i 'irre
',n~
Fig. 3. L a t e n c v of acid p h o s p h a t a s e in an h o m o g e n a t e prepared with 0.2 5 M sucrose after dilution with sucrose ( • ), with p o t a s s i n m glucuronate ( A ), with p o t a s s i u m gluconate ( • ) . The concentration of the diluting solutions was 0.2 5 M. t:ig. 4. L a t e n c y of acid p h o s p h a t a s e in an h o m o g e n a t e prepared with o.2 5 M sucrose after dilution with sncrose ( ~ ) , with lithium lactate (A), with glucosamine. HC1 which had been adjusted to p H 7.o (o). The concentration of the diluting solutions was 0.25 M.
Dilution with litlfium lactate and glucosamine. HC1 resulted in decreases in latency (Fig. 4) which were greater than those found with the sucrose control although not as great as those found with hexoses and hexitols DISCUSSION
In Figs ~ and 2 it is shown that the latency of acid phosphatase decreased only, slightly when homogenates prepared in 0.25 M sucrose were diluted with identical concentrations of tile disaccharides maltose or melibiose or with tile trisaccharide raffinose. This is in agreement with the findings for rat liver lysosomes 5 where the latency of nitrocatechol sulphatase was always maintained when the lysosomes were resuspended in solutions of disaccharides. The results with monosaccharides are also in agreement with those for rat liver lysosomes as glucose, galactose and ~-inethylglucose all appear to permeate rapidly into both types of lysosome. There is, however, conBiochim. Biophys. Acla, 21] (t97 o) 550-554
PERMEABILITY OF LYSOSOMES FROM
Z.
pyriformis
553
siderable difference between the two types in their permeability to hexitols. As shown in Figs. I and 2 mannitol, sorbitol and the cyclic hexitol inositol all permeate the lysosomes of T.pyriformis, while the 5, permeate very slowly, if at all, into rat liver lysosomes, even at 25 °. This permeability to mannitol and sorbitol was also found with homogenates prepared by passing a suspension of cells through a nylon Millipore filter (mean pore diameter 14 #) so it would appear that the permeability to hexitols is, in fact, a property of the lysosonIes of T.pyriformis and not an artifact of the isolation procedure which exposes the lvsosomes to high pH. It m a y be that the intracellular digestive system of T.pyriformis is adapted to the utilisation of hexitols found predominantly in the vegetable matter of their natural habitat 9,a°. The permeability to glucose of the lvsosomes from T.pymformis is not affected by the incorporation of o.2 mM CN- into the diluted homogenate (D. LEE, unpublished results), and it seems that their permeability to hexoses is not mediated by' an active transport system. It appears, therefore, that they will be permeable to all uncharged molecules with molecular dimensions similar to (and less than) that of glucose. Rat liver lysosomes have been shown 2 to be permeable to sinIple salts such as NaC1, KC1, Na2SO 4 and CaCI~ and, as it seems probable that the lysosomes from T.pyriformis will also be permeable to such simple salts, the influence of the organic ion only will be considered in the following discussion. The lysosomes from T.pyriformis are impermeable or only very slightly permeable to gluconate and glucuronate in spite of the fact that their molecular weight is closer to that of glucose than to a disaccharide and the presence of an anionic grouping nmst be having considerable influence on the permeability of the molecule. This is also found with lactate which appears to penetrate the lysosomes nmch more slowly than glucose in spite of its lower molecular weight. Another example of the influence of a charged group in a small molecule is shown by the fact that lvsosomes from T.flyrz/ormis and from rat liver2, a are permeable to glycerol although impermeable, or only slightly permeable, to fi-glycerophosphate. Nevertheless, if the cell has the ability to maintain a suffteientlv low intralysosomal pH n - ' a it seems possible that organic acids could pass from the lysosomes into the cell sap in the uncharged form. The presence of a cationic grouping in the molecule also has a considerable effect on the permeability as can be seen by comparing the decrease in latency found with glucose (Fig. I) with that found with glucosamine (Fig. 4). However, the fact that the lysosomal membrane shows some permeability towards glucosamine does not preclude the possibility that an initially low intralysosomal p H which allows the passage of uncharged organic acids could not be followed by a progressively higher p H which subsequently enhances the passage of organic basesn, ~2. The precipitous decrease in latency of acid phosphatase found when homogenates were diluted with glycerol and dimethylsulphoxide, shows that these compounds traverse the lvsosomal membrane with great ease and suggests that the protective effect of dimethylsulphoxide during the freezing of lysosomes 6 might be produced by the same mechanism as operates with whole cellsl4, is. ACKNOWLEDGEMENT
It is a pleasure to thank Miss Jacqueline Osborne for her excellent technical assistance.
Biochim. Biophys. Acta, 2ii (197o) 550-554
554
D. LEE
REFERENCES [ A. L. TAPPEL, P. L. SA\VANT AND S. SHIBKO, in A. V. S. DE[{EUCK AND hi. P. CAMERON, Ciba Foundation Syrup. 'I.ysosomes', Churchill, London, 1963, p. 78. 2 J. BERTHET, L. BERTHET, F. APPELMANS AND C. DE I)UVE, Biochem. J., 5 ° (I952) I82. 3 F, APPELMANS AND C. DE DUVE, Biochem. J., 59 (19551 426. 4 R. GIANETTO AXD C. DE DVvE, Biochem..]., 50 (19551 433. 5 J. B. LLOYD, Biochem. J., 115 (1969) 7o3 • 0 D. LEE, Cryobiology, 6 (~969) 269. 7 D. LEE, .[. Celhdar Comp. Physiol., 74 (r0691 205. 8 D. LEE, J. Cellular Comp. Physiol., in the press. 9 R. LOH~AR AND R. M. C-AEpH, ,4dvan. Carbohydrate Chem., 4 (~949) 2 I I . to L. F. \VIe;GINS, Hdva*L Carbohydrate Chem., 5 (195 °) I91. i i P. R o v s , J. Exptl. ,'lied., 41 (I925) 370. I2 P. R o u s , J. Exptl. 3led., 4 t (1925) 39913 M. G. SPRICK,AD1. ReV. Tuberc. l~uhnoJ~m3~ Diseases, 74 (I956) 552. 14 J. E. LOVELOCK, Biochim. Biophys. Acta, I~ (I953) 28. 15 J. [~. LOVELOCK AND ,~l. X,\7. H. BISHOP, Nature, 183 (19591 1394.
Biochim. Biophys..4cta, 2 [ i (197 o) 55 ° 554
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
75494
T H E R E L A T I V E P E R M E A B I L I T Y OF LYSOSOMES FROM T E T R A H Y M E I \ L 4 PYRIFOR3IIS
TO CARBOHYDRATES, LACTATE AND T H E
CRYOPROTECTIVE N O N E L E C T R O L Y T E S GLYCEROL AND I)IMETHYI.SULPHOXIDE D()N:kLI) I,E I,; Division of I.o~, Tcmpcralttrc l~ioloL~7, Clinical Research Ccnl~e Laborah)ries, National lnstiluh' f,~ Medical Research, 3 Mill Hill, London N. W.7 (Great Britain)
(Received May 4th, ~07o)
SUMMARY Lysosomes in homogenates of T e t r a h y m e u a p y r i f o r m i s have been shown to be permeable to the cryoprotective nonelectrolvtes glycerol and dimethylsulphoxide, to the hexoses glucose, galactose and ~-methylglucose and to the hexitols mannitol, sorbitol and inositol. They are impermeable to the disaccharides sucrose, maltose and melibiose and to the trisaccharide raffinose. The presence of a charged group has a great influence on permeability, the lvsosomes being impermeable to the gluconate and glucuronate anions and only slightly permeable to the lactate anion and the glucosaminium cation.
INTRODU('TION
Lysosomes have now been described in many types of cells 1 and, although some of the earlier studies on rat liver lysosomes concerned their permeability 2 ~, it was only recently a that a systematic study was made of their permeability to carbohydrates. As nothing is known about the permeability characteristics of lysosomes from the ciliate protozoan T e t r a h y m e n a p3,riformis, it was of interest to study their response to carbohydrates and, in addition, to the cryoprotective agents glycerol and dimethylsulphoxide, as preliminary results G had shown dimethylsulphoxide to protect these lysosomes against damage during freezing. MATERIALSANI} METHODS Dinmthylsulphoxide (Laboratory Reagent Grade), glycerol (A.R.), glucose (A.R.) and litlfium lactate were from Hopkin and Williams, Chadwell Heath, Essex. Mannitol was the product of British Drug Houses, Poole, Dorset. fl-Glycerophosphate (Grade I), Triton X-Ioo and all other carbohydrates were supplied by Sigma (London), London S.W.6. Biochim. Biophys..4 eta, 2t i (r 97°) 55o-554
PERMEABILITY OF LYSOSOMES FROM T. pyriformis
551
Growth and harvesting of cells T.pyriformis, strain S, was grown in tile dark at room t e m p e r a t u r e L Cells were h a r v e s t e d between 5 a n d 7 days after inoculation, washed with 0.25 M sucrose in the m a n n e r described previously8 (except t h a t centrifugation was at 15oo x g for 2.5 rain) a n d resuspended in 0.25 M sucrose at a c o n c e n t r a t i o n between IO7 a n d 5" IO7 cells per ml before disruption at 4 ° b y exposure to p H I I for between 45 sec a n d i rain. Acid phosphatase (orthophosphoric monoester phosphoroilydrolase, EC 3.1.3.2) was assayed in triplicate in tile m a n n e r described previouslyS; all assays contained o.25 M sucrose in addition to tile sucrose which was present in the homogenates.
Assessment of permeability of the lysosomal membrane After preparation, homogenates were placed in ice a n d an aliquot diluted with 4 vol. of the solution u n d e r e x a m i n a t i o n . At t i m e d intervals thereafter, samples of the diluted homogenates were assayed for acid phosphatase activity. For the d e t e r m i n a tion of total activity, assay m i x t u r e s c o n t a i n e d 0.05 °o T r i t o n X - i o o a n d latency is defined as the percentage of tile total a c t i v i t y u n m a s k e d b y incorporation of T r i t o n X - i o o into the assay mixture. As there is no i n d i c a t i o n in tile literature t h a t a n y of the c o m p o u n d s u n d e r e x a m i n a t i o n has a direct lytie action on biological m e l n b r a n e s at tile c o n c e n t r a t i o n s studied, it was assumed t h a t a decrease in l a t e n c y of acid phosphatase was the result of p e r m e a t i o n of the substance through the lvsosomal m e m b r a n e . RESULTS
I n Fig. I it can be seen t h a t the l a t e n c y of acid phosphatase in an h o m o g e n a t e d i l u t e d with o.25 M sucrose decreased only slightly d u r i n g the s u b s e q u e n t 2-tl period, while the l a t e n c y in homogenates diluted with o.25 M glucose or o.25 M e - m e t h y l glucose decreased d r a m a t i c a l l y (although in a p p r o x i m a t e l y the same fashion). I n another e x p e r i m e n t tile l a t e n c y was well m a i n t a i n e d on dilution with the trisaccharide raffinose, although it decreased on dilution with the hexitols sorbitol a n d inositol.
•
on
~- ~
"~
~c
%
•
....
o~
~c
4c ~2c o ~
"6
o o za
o &
Time(h)
Fig. I. Latency of acid phosphatase in two homogenates prepared with 0.25 M s u c r o s e . ( I ) After dilution with sucrose ([~), with glucose (&), with a-methylglucose (O). (2)After dilution with raffinose ( • ), sorbitol (~k), with inositol (O). The concentration of the diluting solutions was o.25 M. Fig. 2. Latency of acid phosphatase in two homogenates prepared with o.25 ~I sucrose. (i) After dilution with melibiose (El), with glycerol (A), with dimethylsulphoxide (O). (2) After dilution with maltose ( • ), with galactose (A), with mannitol (O). The concentration of the diluting solutions was 0.25 M.
Biochim. Biophys. Acta, 211 (197o) 55o-554
55 2
D. LEE
Fig. 2 shows the latency of acid phosphatase to remain high after dilution with maltose but to decrease on dilution with galactose and mannitol. Similarly, latencv was well maintained with the disaccharide melibiose, although dilution with glycerol or dinmthylsulphoxide resulted in a much more rapid decrease in latency than was seen with hexoses or hexitols. All of the compounds tested above are unchanged, and it was decided to study the effect of charged groups on permeability. Fig. 3 shows that dilution of an homogenate with o.25 M potassium gluconate or o.25 M potassium glucuronate resulted in only a slight decrease in latency of acid phosphatase over the subsequent 2-h period. This decrease probably does not differ significantly from that found in the control holnogenate diluted with sucrose and, in any' case, it is clearly seen that the presence of an anionic grouping had a profound effect on the permeability of these lnolecules as coinpared to glucose. o
i 6C
(,'i
.1(i
s 5
5
I
"
T r T e (q~
i 'irre
',n~
Fig. 3. L a t e n c v of acid p h o s p h a t a s e in an h o m o g e n a t e prepared with 0.2 5 M sucrose after dilution with sucrose ( • ), with p o t a s s i n m glucuronate ( A ), with p o t a s s i u m gluconate ( • ) . The concentration of the diluting solutions was 0.2 5 M. t:ig. 4. L a t e n c y of acid p h o s p h a t a s e in an h o m o g e n a t e prepared with o.2 5 M sucrose after dilution with sncrose ( ~ ) , with lithium lactate (A), with glucosamine. HC1 which had been adjusted to p H 7.o (o). The concentration of the diluting solutions was 0.25 M.
Dilution with litlfium lactate and glucosamine. HC1 resulted in decreases in latency (Fig. 4) which were greater than those found with the sucrose control although not as great as those found with hexoses and hexitols DISCUSSION
In Figs ~ and 2 it is shown that the latency of acid phosphatase decreased only, slightly when homogenates prepared in 0.25 M sucrose were diluted with identical concentrations of tile disaccharides maltose or melibiose or with tile trisaccharide raffinose. This is in agreement with the findings for rat liver lysosomes 5 where the latency of nitrocatechol sulphatase was always maintained when the lysosomes were resuspended in solutions of disaccharides. The results with monosaccharides are also in agreement with those for rat liver lysosomes as glucose, galactose and ~-inethylglucose all appear to permeate rapidly into both types of lysosome. There is, however, conBiochim. Biophys. Acla, 21] (t97 o) 550-554
PERMEABILITY OF LYSOSOMES FROM
Z.
pyriformis
553
siderable difference between the two types in their permeability to hexitols. As shown in Figs. I and 2 mannitol, sorbitol and the cyclic hexitol inositol all permeate the lysosomes of T.pyriformis, while the 5, permeate very slowly, if at all, into rat liver lysosomes, even at 25 °. This permeability to mannitol and sorbitol was also found with homogenates prepared by passing a suspension of cells through a nylon Millipore filter (mean pore diameter 14 #) so it would appear that the permeability to hexitols is, in fact, a property of the lysosonIes of T.pyriformis and not an artifact of the isolation procedure which exposes the lvsosomes to high pH. It m a y be that the intracellular digestive system of T.pyriformis is adapted to the utilisation of hexitols found predominantly in the vegetable matter of their natural habitat 9,a°. The permeability to glucose of the lvsosomes from T.pymformis is not affected by the incorporation of o.2 mM CN- into the diluted homogenate (D. LEE, unpublished results), and it seems that their permeability to hexoses is not mediated by' an active transport system. It appears, therefore, that they will be permeable to all uncharged molecules with molecular dimensions similar to (and less than) that of glucose. Rat liver lysosomes have been shown 2 to be permeable to sinIple salts such as NaC1, KC1, Na2SO 4 and CaCI~ and, as it seems probable that the lysosomes from T.pyriformis will also be permeable to such simple salts, the influence of the organic ion only will be considered in the following discussion. The lysosomes from T.pyriformis are impermeable or only very slightly permeable to gluconate and glucuronate in spite of the fact that their molecular weight is closer to that of glucose than to a disaccharide and the presence of an anionic grouping nmst be having considerable influence on the permeability of the molecule. This is also found with lactate which appears to penetrate the lysosomes nmch more slowly than glucose in spite of its lower molecular weight. Another example of the influence of a charged group in a small molecule is shown by the fact that lvsosomes from T.flyrz/ormis and from rat liver2, a are permeable to glycerol although impermeable, or only slightly permeable, to fi-glycerophosphate. Nevertheless, if the cell has the ability to maintain a suffteientlv low intralysosomal pH n - ' a it seems possible that organic acids could pass from the lysosomes into the cell sap in the uncharged form. The presence of a cationic grouping in the molecule also has a considerable effect on the permeability as can be seen by comparing the decrease in latency found with glucose (Fig. I) with that found with glucosamine (Fig. 4). However, the fact that the lysosomal membrane shows some permeability towards glucosamine does not preclude the possibility that an initially low intralysosomal p H which allows the passage of uncharged organic acids could not be followed by a progressively higher p H which subsequently enhances the passage of organic basesn, ~2. The precipitous decrease in latency of acid phosphatase found when homogenates were diluted with glycerol and dimethylsulphoxide, shows that these compounds traverse the lvsosomal membrane with great ease and suggests that the protective effect of dimethylsulphoxide during the freezing of lysosomes 6 might be produced by the same mechanism as operates with whole cellsl4, is. ACKNOWLEDGEMENT
It is a pleasure to thank Miss Jacqueline Osborne for her excellent technical assistance.
Biochim. Biophys. Acta, 2ii (197o) 550-554
554
D. LEE
REFERENCES [ A. L. TAPPEL, P. L. SA\VANT AND S. SHIBKO, in A. V. S. DE[{EUCK AND hi. P. CAMERON, Ciba Foundation Syrup. 'I.ysosomes', Churchill, London, 1963, p. 78. 2 J. BERTHET, L. BERTHET, F. APPELMANS AND C. DE I)UVE, Biochem. J., 5 ° (I952) I82. 3 F, APPELMANS AND C. DE DUVE, Biochem. J., 59 (19551 426. 4 R. GIANETTO AXD C. DE DVvE, Biochem..]., 50 (19551 433. 5 J. B. LLOYD, Biochem. J., 115 (1969) 7o3 • 0 D. LEE, Cryobiology, 6 (~969) 269. 7 D. LEE, .[. Celhdar Comp. Physiol., 74 (r0691 205. 8 D. LEE, J. Cellular Comp. Physiol., in the press. 9 R. LOH~AR AND R. M. C-AEpH, ,4dvan. Carbohydrate Chem., 4 (~949) 2 I I . to L. F. \VIe;GINS, Hdva*L Carbohydrate Chem., 5 (195 °) I91. i i P. R o v s , J. Exptl. ,'lied., 41 (I925) 370. I2 P. R o u s , J. Exptl. 3led., 4 t (1925) 39913 M. G. SPRICK,AD1. ReV. Tuberc. l~uhnoJ~m3~ Diseases, 74 (I956) 552. 14 J. E. LOVELOCK, Biochim. Biophys. Acta, I~ (I953) 28. 15 J. [~. LOVELOCK AND ,~l. X,\7. H. BISHOP, Nature, 183 (19591 1394.
Biochim. Biophys..4cta, 2 [ i (197 o) 55 ° 554