Biochemical Society Transactions
Action of diphtheria toxin on Schizosaccharornyces pornbe John Davey School of Biochemistry, University of Birmingham, Birmingham B I 5 2TT, U.K. 728
Introduction Diphtheria toxin is a cytotoxic protein that inhibits eukaryotic protein synthesis by inactivating elongation factor-2 through ADP ribosylation. Inactivation requires the entry of the toxin, or at least an active fragment, into the cytoplasm and it is generally accepted that entry occurs via the endocytic pathway [reviewed in 11; the toxin binds to a specific receptor at the cell surface [2, 31, is concentrated in coated pits, and is then internalized by endocytosis. A conformational change in the toxin molecule takes place in an acidic compartment, probably the endosome [4],and results in the translocation of the enzymatically active A fragment into the cytosol where it exerts its toxicity. Yeast cells could be an ideal organism for studying these events, not only because of the ease with which biochemical analyses can be applied to these cells, but also because the biochemical approaches can be coupled to powerful genetic studies. Unfortunately, intact cells of the commonly used budding yeast Saccharomyces cereviszize are resistant to the toxin (although spheroplasts prepared from such cells are sensitive) [S] and so the interaction of diphtheria toxin with the fission yeast Schizosaccharomyces pombe has been investigated.
Materials and methods
containing the required concentration of diphtheria toxin. After aerated incubation at 29"C, ~ - [ 4 , 5 - ~ H ] leucine ( 120- 190 Ci/mmol, Amersham, Ayles bury, U.K.) was added to a final concentration of 100 nM. After further incubation, the cells were chilled to O"C, bovine serum albumin was added to 0.2 mg/ml and trichloroacetic acid was added to 10% (w/v). The precipitate was collected by filtration and radioactivity was determined as described previously 171. The effect of diphtheria toxin on cell viability was investigated in a similar fashion except that cells were not radiolabelled but were diluted and spread on agar plates. Spheroplasts were treated in the same way as intact cells exept that all media was supplemented with 1.2 M-sorbitol.
Results Toxicity of diptheria toxin t o intact cells and spheroplasts of S. pombe
Although S. cerevike spheroplasts are sensitive to diphtheria toxin, the intact cells are not [ S ] and this complicates the isolation of toxin-resistant mutants [S]. In contrast to these findings with the budding yeast, diphtheria toxin is toxic to both spheroplasts and intact cells of the fission yeast S. pombe (Fig. 1). Furthermore, the spheroplasts and cells are almost equally sensitive to the toxin. Using the data in Fig. 1, it is possible to estimate an IC,, for intact cells of
Yeast strains and culture conditions
All of the work used the wild-type haploid heterothallic M-type strain of S. pombe (L972). The cells were maintained on a defined minimal medium (Edinburgh minimal medium, EMM) which lacked leucine (EMM-Leu) [6]. Spheroplasts were prepared and maintained (EMM-Leu supplemented with Sorbitol) using standard techniques 161. Unless otherwise stated, all chemicals were reagent grade and were purchased from standard suppliers. Diphtheria toxin was obtained from ICN Riomedicals, High Wycombe, U.K.
Fig. I Effect of diphtheria toxin on cell viability Exponentially growing cells were resuspended at lo6celldml in EMM-Leu containing various concentrations of diphtheria toxin. After 4 h at 29"C, the cells were washed and spread on agar plates to determine viability which IS expressed as a percentage of a control not exposed to toxin. Spheroplasts were treated in the same way except that all media included I .2 M-Sorbitol.
Measurement of protein synthesis
Precise conditions (e.g. incubation times) varied from one experiment to another and are detailed in the text but, generally, exponentially growing cells were suspended at loh cells/ml of fresh EMM-Leu Abbreviations used: EMM-Leu, Edinburgh minimal medium lacking leucine; EF-2, elongation factor 2.
Volume 20
0
1
0.0
0.5
1.0
1.5
20
2.5
3.0
3.5
40
45
50
Toxin (log[nM])
Endocytosis, Toxins, lmmunotoxins and Viruses
about 6 x lo-' M; the ICso is the concentration of toxin necessary to kill 50% of the cells. Toxicity of diphtheria toxin via inhibition of protein synthesis
The cytotoxicity of diphtheria toxin in higher eukaryotic cells is via an ADP ribosylation of elongation factor-2 (EF-2) and a consequent inhibition of protein synthesis. For this reason, the effect of toxin on protein synthesis in intact cells of S. pombe was examined. Fig. 2 shows that the incorporation of [jH]leucine into protein reduces following treatment with toxin; the simultaneous effect of toxin on cell viability is repeated for comparison. The inhibition of protein synthesis precedes cell death and suggests that death occurs as a consequence of the inhibition in protein synthesis. Effects of varying concentrations of toxin on the rate of protein synthesis
Diphtheria toxin clearly inhibits protein synthesis in
S. pombe (see Fig. 2) but the experimental design is relatively crude and simply monitors the amount of protein synthesized during a 2 h period, 2 h after the addition of toxin. To more closely investigate the effect of toxin on the rate of protein synthesis, a 30-min pulse of [3H]leucine at varying times after the addition of toxin was used (Fig. 3). In uninhibited cells the rate of synthesis increases during the time of the experiment and probably reflects the ongoing growth and division of the cells (under the Fig. 2
conditions of the experiment the cells divide once every 2.3 h; not shown). Exposure to a high level of toxin (3 p ~caused ) an almost immediate inhibition of protein synthesis which did not recover even after a further 6 h of incubation (not shown). This is consistent with the result using continuous labelling and confirms that this level of toxin is sufficient to completely inhibit protein synthesis. Addition of lower concentrations of toxin had a less drastic but perhaps more interesting effect. Although these caused an initial decrease in the rate of protein synthesis, the rate began to increase and, after a longer incubation (not shown), the rate of increase paralleled that in the untreated cells. The simplest explanation is that the toxin causes a transient inhibition of protein synthesis and that, after a lag period, the length of which increases with increased toxin, the rate of synthesis returns to that observed in the untreated cells. Exposure to a high level of toxin, however, causes a prolonged inhibition of protein synthesis from which the cells are unable to recover.
Discussion This study shows that diphtheria toxin is toxic to S. pombe and that this is probably through inhibition of protein synthesis. Furthermore, unlike the situation with S. cerevz'siae in which only spheroplasts are sensitive to the toxin [S], both spheroplasts and intact cells are equally sensitive to the toxin. Under the conditions used (i.e. a 4 h exposure to toxin prior to plating), the concentration of toxin required
Effect of diphtheria toxin on protein synthesis
Fig. 3
Exponentially growing cells were resuspended at lo6 cells/ml in EMM-Leu containing various concentrations of diphtheria toxin. After 2 h at 29"C, [3H]leucine was added t o a final concentration of 100 nM and incubation was continued for a further 2 h. The sample was then divided in t w o and, while one aliquot was used t o determine cell viability (see Fig. I), the other was used t o determine the incorporation of [3H]leucine into proteins (see Methods section). The results are expressed relative t o a sample not exposed t o toxin.
Effect of varying concentrations of toxin on the rate of protein synthesis Exponentially growing cells were resuspended at lo6 cells/ml in EMM-Leu and, after I h at 29°C diphtheria toxin was added at different concentrations. The rate of protein synthesis was measured at various time points by pulse-labelling for 30 min with [3H]leucine,the graph shows the times at which the labelling periods were started.
501
/" No toxin
\\
a\
0.0
0s
1.0
I.)
zn
2.s
3.0
3.5
4.0
4.s
5.0
Toxin (log[nM])
0 -2
toxin
0 4 5 Time (h) '
-I
0
1
2
3
I992
729
Biochemical Society Transactions
7 30
to kill SO% of the cells (ICSO) is approximately 6 x lo-' M. This compares favourably with spheroplasts from S. cerevkzize (ICSO - 3 x lo-' hi, estimated from [S]) but is at least 1000-fold greater than the level required to kill sensitive mammalian cells [9]. There could be many explanations for the low sensitivity of yeast cells to the toxin. One of the principle determinants of a cell's sensitivity to diphtheria toxin is the number of toxin-specific receptors on the cell surface: the more receptors a cell possesses the more sensitive it is to the toxin [ 10-121. If yeast cells possess only a few receptors, then they would be relatively insensitive to the toxin, but while ligand binding experiments (using '2'I-labelled toxin) might reveal the number of receptors per cell, preliminary experiments have failed to show a specific interaction between the toxin and the yeast (not shown). Although this is a negative result, the lack of a specific interaction is consistent with there being only a few receptors per cell. T o be effective diphtheria toxin not only needs to bind to a cell but also must enter the cytoplasm, and the cytosolic concentration of the toxin plays an important role in determining cell death [ 131. It is possible that translocation of the toxin across the yeast cell membrane is inefficient and this could contribute to the low sensitivity of the yeast. A further problem could be inactivation of the toxin in the yeast cytoplasm. There is evidence to suggest that diphtheria toxin, and toxin-related products such as CRh4176 (a mutant toxin with reduced enzymic activity), are continuously destroyed in the cell cytoplasm [ 131. In sensitive mammalian cells, the inhibition of protein synthesis by diphtheria toxin is mediated via an ADP ribosylation of EF-2 and although this is also true for S. pombe (J. Davey and A. Brown, unpublished work) we have no insights into the parameters of this conversion. It could be, for example, that the yeast EF-2 is more resistant to ribosylation than mammalian EF-2 or that the yeast cells contain a higher amount of EF-2. Alternatively, the rate of turnover of EF-2 in yeast cells could be very high while the absolute rate of synthesis could also be significantly higher than in mammalian cells (see below). A final observation that is likely to have an effect on the interaction between the toxin and its target cell is the rate at which the cell grows and divides. The fact that S. pombe divides approximately every 2 to 3 h, whereas cultured mammalian cells have a much longer division time, could contribute to a higher lethal dose for the yeast. Not only would the toxin be continually diluted during
Volume 20
growth but also such a rapid division cycle requires a high rate of synthesis of essential macromolecules such as EF-2. Such synthesis would therefore replenish the cell's reserves of EF-2 and alleviate the effects of the toxin. It is difficult to determine which, if any, of the above possibilities is responsible for the low sensitivity of yeast cells to diphtheria toxin but the lack of an explanation does not necessarily mean that the mechanism of cytotoxicity in the yeast is significantly different from the mechanism in sensitive mammalian cells. In fact the results would strongly indicate that the action of diphteria toxin on yeast cells is the same as on mammalian cells. Studies are currently in progress to further characterize the interaction of diphtheria toxin with S. pombe and to develop methods of isolating mutants that are resistant to toxin. These studies will benefit from the fact that both spheroplasts and intact cells are sensitive to the toxin. I am grateful to A. M. Pappenheimer Jr. and J. W. Hodley for helpful comments and to Alison Brown for technical assistance. This work was supported by the Cancer Kesearch Campaign. 1. Olsnes, S., Moskaug, J. O., Stenmark, H. & Sandvig, K. (1988) Trends Hiol. Sci. 13, 348-35 1 2. Mekada, E., Okada, Y. & Uchida, T. (1988) J. Cell Hiol. 107, 511-519 3. Mekada, E., Senoh. H.. Iwamoto, K., Okada, Y. & Uchida, T. (1991) J. Hiol. Chem. 266,20457-20462 4. Morris, R. E., Gerstein, A. S., Honventre, 1'. F. & Saelinger, C. €3.( 1985) Infect. Immun. 50.72 1-727 5. Murakami, S., Bodley, J. W. & Livingston, D. M. (1982) Mol. Cell. Biol. 2. 588-592 6. Moreno, S., Klar, A. & Nurse, 1'. (199 1) in Guide to Yeast Genetics and Molecular Hiology (Guthrie, C. & Fink, G. R., eds.), pp. 795-823, Academic Press, New York 7. Hartwell, I,. H. (1967) J. Hacteriol. 93, 1602-1670 8. Chen, J.-Y. C., Hodley, J. W. & Livingston, 1). M. (1985) Mol. Cell. Biol. 5, 3357-3360 9. Middlebrook, J. I,. & Dorland, R. H. (1977) Can. J. Microbiol. 23, 183- 189 10. Middlebrook, J. L., Dorland, K. €3. & Leppla, S. H. (1978) J. Biol. Chem. 253,7325-7330 11. Mekada. E.. Kohno, K., Ishiura, M.. Uchida, T. & Okada, Y. (1982) Biochem. Hiophys. Kes. Commun. 109,792-799 12. Kohno, K., Uchida, T., Mekada, E. & Ikada, Y. (1985) Somatic Cell Genet. 11,421-43 1 13. Uchida, T., Pappenheimer. A. J.. Jr. & Harper. A. (1973) J. Biol. Chem. 248,3845-3850 Received 10 July 1002
Action of diphtheria toxin on Schizosaccharornyces pornbe John Davey School of Biochemistry, University of Birmingham, Birmingham B I 5 2TT, U.K. 728
Introduction Diphtheria toxin is a cytotoxic protein that inhibits eukaryotic protein synthesis by inactivating elongation factor-2 through ADP ribosylation. Inactivation requires the entry of the toxin, or at least an active fragment, into the cytoplasm and it is generally accepted that entry occurs via the endocytic pathway [reviewed in 11; the toxin binds to a specific receptor at the cell surface [2, 31, is concentrated in coated pits, and is then internalized by endocytosis. A conformational change in the toxin molecule takes place in an acidic compartment, probably the endosome [4],and results in the translocation of the enzymatically active A fragment into the cytosol where it exerts its toxicity. Yeast cells could be an ideal organism for studying these events, not only because of the ease with which biochemical analyses can be applied to these cells, but also because the biochemical approaches can be coupled to powerful genetic studies. Unfortunately, intact cells of the commonly used budding yeast Saccharomyces cereviszize are resistant to the toxin (although spheroplasts prepared from such cells are sensitive) [S] and so the interaction of diphtheria toxin with the fission yeast Schizosaccharomyces pombe has been investigated.
Materials and methods
containing the required concentration of diphtheria toxin. After aerated incubation at 29"C, ~ - [ 4 , 5 - ~ H ] leucine ( 120- 190 Ci/mmol, Amersham, Ayles bury, U.K.) was added to a final concentration of 100 nM. After further incubation, the cells were chilled to O"C, bovine serum albumin was added to 0.2 mg/ml and trichloroacetic acid was added to 10% (w/v). The precipitate was collected by filtration and radioactivity was determined as described previously 171. The effect of diphtheria toxin on cell viability was investigated in a similar fashion except that cells were not radiolabelled but were diluted and spread on agar plates. Spheroplasts were treated in the same way as intact cells exept that all media was supplemented with 1.2 M-sorbitol.
Results Toxicity of diptheria toxin t o intact cells and spheroplasts of S. pombe
Although S. cerevike spheroplasts are sensitive to diphtheria toxin, the intact cells are not [ S ] and this complicates the isolation of toxin-resistant mutants [S]. In contrast to these findings with the budding yeast, diphtheria toxin is toxic to both spheroplasts and intact cells of the fission yeast S. pombe (Fig. 1). Furthermore, the spheroplasts and cells are almost equally sensitive to the toxin. Using the data in Fig. 1, it is possible to estimate an IC,, for intact cells of
Yeast strains and culture conditions
All of the work used the wild-type haploid heterothallic M-type strain of S. pombe (L972). The cells were maintained on a defined minimal medium (Edinburgh minimal medium, EMM) which lacked leucine (EMM-Leu) [6]. Spheroplasts were prepared and maintained (EMM-Leu supplemented with Sorbitol) using standard techniques 161. Unless otherwise stated, all chemicals were reagent grade and were purchased from standard suppliers. Diphtheria toxin was obtained from ICN Riomedicals, High Wycombe, U.K.
Fig. I Effect of diphtheria toxin on cell viability Exponentially growing cells were resuspended at lo6celldml in EMM-Leu containing various concentrations of diphtheria toxin. After 4 h at 29"C, the cells were washed and spread on agar plates to determine viability which IS expressed as a percentage of a control not exposed to toxin. Spheroplasts were treated in the same way except that all media included I .2 M-Sorbitol.
Measurement of protein synthesis
Precise conditions (e.g. incubation times) varied from one experiment to another and are detailed in the text but, generally, exponentially growing cells were suspended at loh cells/ml of fresh EMM-Leu Abbreviations used: EMM-Leu, Edinburgh minimal medium lacking leucine; EF-2, elongation factor 2.
Volume 20
0
1
0.0
0.5
1.0
1.5
20
2.5
3.0
3.5
40
45
50
Toxin (log[nM])
Endocytosis, Toxins, lmmunotoxins and Viruses
about 6 x lo-' M; the ICso is the concentration of toxin necessary to kill 50% of the cells. Toxicity of diphtheria toxin via inhibition of protein synthesis
The cytotoxicity of diphtheria toxin in higher eukaryotic cells is via an ADP ribosylation of elongation factor-2 (EF-2) and a consequent inhibition of protein synthesis. For this reason, the effect of toxin on protein synthesis in intact cells of S. pombe was examined. Fig. 2 shows that the incorporation of [jH]leucine into protein reduces following treatment with toxin; the simultaneous effect of toxin on cell viability is repeated for comparison. The inhibition of protein synthesis precedes cell death and suggests that death occurs as a consequence of the inhibition in protein synthesis. Effects of varying concentrations of toxin on the rate of protein synthesis
Diphtheria toxin clearly inhibits protein synthesis in
S. pombe (see Fig. 2) but the experimental design is relatively crude and simply monitors the amount of protein synthesized during a 2 h period, 2 h after the addition of toxin. To more closely investigate the effect of toxin on the rate of protein synthesis, a 30-min pulse of [3H]leucine at varying times after the addition of toxin was used (Fig. 3). In uninhibited cells the rate of synthesis increases during the time of the experiment and probably reflects the ongoing growth and division of the cells (under the Fig. 2
conditions of the experiment the cells divide once every 2.3 h; not shown). Exposure to a high level of toxin (3 p ~caused ) an almost immediate inhibition of protein synthesis which did not recover even after a further 6 h of incubation (not shown). This is consistent with the result using continuous labelling and confirms that this level of toxin is sufficient to completely inhibit protein synthesis. Addition of lower concentrations of toxin had a less drastic but perhaps more interesting effect. Although these caused an initial decrease in the rate of protein synthesis, the rate began to increase and, after a longer incubation (not shown), the rate of increase paralleled that in the untreated cells. The simplest explanation is that the toxin causes a transient inhibition of protein synthesis and that, after a lag period, the length of which increases with increased toxin, the rate of synthesis returns to that observed in the untreated cells. Exposure to a high level of toxin, however, causes a prolonged inhibition of protein synthesis from which the cells are unable to recover.
Discussion This study shows that diphtheria toxin is toxic to S. pombe and that this is probably through inhibition of protein synthesis. Furthermore, unlike the situation with S. cerevz'siae in which only spheroplasts are sensitive to the toxin [S], both spheroplasts and intact cells are equally sensitive to the toxin. Under the conditions used (i.e. a 4 h exposure to toxin prior to plating), the concentration of toxin required
Effect of diphtheria toxin on protein synthesis
Fig. 3
Exponentially growing cells were resuspended at lo6 cells/ml in EMM-Leu containing various concentrations of diphtheria toxin. After 2 h at 29"C, [3H]leucine was added t o a final concentration of 100 nM and incubation was continued for a further 2 h. The sample was then divided in t w o and, while one aliquot was used t o determine cell viability (see Fig. I), the other was used t o determine the incorporation of [3H]leucine into proteins (see Methods section). The results are expressed relative t o a sample not exposed t o toxin.
Effect of varying concentrations of toxin on the rate of protein synthesis Exponentially growing cells were resuspended at lo6 cells/ml in EMM-Leu and, after I h at 29°C diphtheria toxin was added at different concentrations. The rate of protein synthesis was measured at various time points by pulse-labelling for 30 min with [3H]leucine,the graph shows the times at which the labelling periods were started.
501
/" No toxin
\\
a\
0.0
0s
1.0
I.)
zn
2.s
3.0
3.5
4.0
4.s
5.0
Toxin (log[nM])
0 -2
toxin
0 4 5 Time (h) '
-I
0
1
2
3
I992
729
Biochemical Society Transactions
7 30
to kill SO% of the cells (ICSO) is approximately 6 x lo-' M. This compares favourably with spheroplasts from S. cerevkzize (ICSO - 3 x lo-' hi, estimated from [S]) but is at least 1000-fold greater than the level required to kill sensitive mammalian cells [9]. There could be many explanations for the low sensitivity of yeast cells to the toxin. One of the principle determinants of a cell's sensitivity to diphtheria toxin is the number of toxin-specific receptors on the cell surface: the more receptors a cell possesses the more sensitive it is to the toxin [ 10-121. If yeast cells possess only a few receptors, then they would be relatively insensitive to the toxin, but while ligand binding experiments (using '2'I-labelled toxin) might reveal the number of receptors per cell, preliminary experiments have failed to show a specific interaction between the toxin and the yeast (not shown). Although this is a negative result, the lack of a specific interaction is consistent with there being only a few receptors per cell. T o be effective diphtheria toxin not only needs to bind to a cell but also must enter the cytoplasm, and the cytosolic concentration of the toxin plays an important role in determining cell death [ 131. It is possible that translocation of the toxin across the yeast cell membrane is inefficient and this could contribute to the low sensitivity of the yeast. A further problem could be inactivation of the toxin in the yeast cytoplasm. There is evidence to suggest that diphtheria toxin, and toxin-related products such as CRh4176 (a mutant toxin with reduced enzymic activity), are continuously destroyed in the cell cytoplasm [ 131. In sensitive mammalian cells, the inhibition of protein synthesis by diphtheria toxin is mediated via an ADP ribosylation of EF-2 and although this is also true for S. pombe (J. Davey and A. Brown, unpublished work) we have no insights into the parameters of this conversion. It could be, for example, that the yeast EF-2 is more resistant to ribosylation than mammalian EF-2 or that the yeast cells contain a higher amount of EF-2. Alternatively, the rate of turnover of EF-2 in yeast cells could be very high while the absolute rate of synthesis could also be significantly higher than in mammalian cells (see below). A final observation that is likely to have an effect on the interaction between the toxin and its target cell is the rate at which the cell grows and divides. The fact that S. pombe divides approximately every 2 to 3 h, whereas cultured mammalian cells have a much longer division time, could contribute to a higher lethal dose for the yeast. Not only would the toxin be continually diluted during
Volume 20
growth but also such a rapid division cycle requires a high rate of synthesis of essential macromolecules such as EF-2. Such synthesis would therefore replenish the cell's reserves of EF-2 and alleviate the effects of the toxin. It is difficult to determine which, if any, of the above possibilities is responsible for the low sensitivity of yeast cells to diphtheria toxin but the lack of an explanation does not necessarily mean that the mechanism of cytotoxicity in the yeast is significantly different from the mechanism in sensitive mammalian cells. In fact the results would strongly indicate that the action of diphteria toxin on yeast cells is the same as on mammalian cells. Studies are currently in progress to further characterize the interaction of diphtheria toxin with S. pombe and to develop methods of isolating mutants that are resistant to toxin. These studies will benefit from the fact that both spheroplasts and intact cells are sensitive to the toxin. I am grateful to A. M. Pappenheimer Jr. and J. W. Hodley for helpful comments and to Alison Brown for technical assistance. This work was supported by the Cancer Kesearch Campaign. 1. Olsnes, S., Moskaug, J. O., Stenmark, H. & Sandvig, K. (1988) Trends Hiol. Sci. 13, 348-35 1 2. Mekada, E., Okada, Y. & Uchida, T. (1988) J. Cell Hiol. 107, 511-519 3. Mekada, E., Senoh. H.. Iwamoto, K., Okada, Y. & Uchida, T. (1991) J. Hiol. Chem. 266,20457-20462 4. Morris, R. E., Gerstein, A. S., Honventre, 1'. F. & Saelinger, C. €3.( 1985) Infect. Immun. 50.72 1-727 5. Murakami, S., Bodley, J. W. & Livingston, D. M. (1982) Mol. Cell. Biol. 2. 588-592 6. Moreno, S., Klar, A. & Nurse, 1'. (199 1) in Guide to Yeast Genetics and Molecular Hiology (Guthrie, C. & Fink, G. R., eds.), pp. 795-823, Academic Press, New York 7. Hartwell, I,. H. (1967) J. Hacteriol. 93, 1602-1670 8. Chen, J.-Y. C., Hodley, J. W. & Livingston, 1). M. (1985) Mol. Cell. Biol. 5, 3357-3360 9. Middlebrook, J. I,. & Dorland, R. H. (1977) Can. J. Microbiol. 23, 183- 189 10. Middlebrook, J. L., Dorland, K. €3. & Leppla, S. H. (1978) J. Biol. Chem. 253,7325-7330 11. Mekada. E.. Kohno, K., Ishiura, M.. Uchida, T. & Okada, Y. (1982) Biochem. Hiophys. Kes. Commun. 109,792-799 12. Kohno, K., Uchida, T., Mekada, E. & Ikada, Y. (1985) Somatic Cell Genet. 11,421-43 1 13. Uchida, T., Pappenheimer. A. J.. Jr. & Harper. A. (1973) J. Biol. Chem. 248,3845-3850 Received 10 July 1002
