Biol. Chem. Hoppe-Seyler Vol. 373, pp. 419-425, July 1992
Characterisation of the Activity and Stability of Single-chain Cathepsin L and of Proteolytically Active Cathepsin L/ Cystatin Complexes CLIVE DENNISON, ROBERT PIKE, THERESA COETZER AND KIRSTEN KIRK Department of Biochemistry, University of Natal, P. O. Box 375, Pietermaritzburg 3200, South Africa.
Summary The activity of single-chain cathepsin L was found to be markedly dependent on cysteine concentration, while a covalent, proteolytically active cathepsin L/ cystatin complex was less cysteine-dependent. Cysteine levels and ionic strength did not affect the stability of either enzyme form and both enzyme forms were found to be stable for significant periods of time at or near physiological pH.
Introduction We have previously reported [1,2] on the isolation of proteolytically active complexes of single-chain cathepsin L with a cystatin, thought to be stefin B, and on the formation of similar complexes in vitro, from purified constituents. These complexes can only be disrupted by boiling in sodium dodecyl sulfate in the presence of a reducing agent and in these proteolytically active complexes the enzyme and cystatin may, therefore, be covalently bound [2]. We report here on the characterisation of the activity and stability of the singlechain sheep liver enzyme and of a proteolytically active complex fraction. Twochain forms of cathepsin L [3] and single-chain chicken cathepsin L [4] have previously been characterised in this respect, while the activity and stability of cathepsin L/cystatin complexes have not previously been documented. Because cathepsin L has been implicated in cancer [5,6], the activity of this enzyme under extracellular conditions of pH, ionic strength and redox potential is of some interest, in the context of invasion. Abbreviations: NHMec, 7-(4-methyl)coumarylamide; Z-, benzyloxycarbonyl.
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420
C. Dennison, R. Pike, T. Coetzer and K. Kirk
Vol. 373 (1992)
Methods Single-chain sheep liver cathepsin L, and a proteolytically active fraction of sheep liver cathepsin L complexed with a sheep liver cystatin, were isolated as described previously [7], through the pH 5.5 step on S-Sepharose. The active peak was subsequently separated on Sephadex G-75, the first included peak being the complex fraction and the second being free cathepsin L. Acetate-MES-Tris buffer systems, of defined ionic strength (0.1, unless stated otherwise), described by Ellis & Morrison [8] were used in the assays for activity and stability. Enzyme activity was measured at 30°C according to Barrett & Kirschke [9]; unless stated otherwise, all other assays were done at 37°C. As a measure of stability, the half-life of the enzyme was determined by continuously monitoring the fluorogenic products produced, from Z-Phe-Arg-NHMec, by an enzyme sample incubated in a defined buffer containing a thiol activator. (Activity could also be estimated from the initial slope of the same progress curve.) For determination of the half-life, the period of measurement was usually 30 min. As a null hypothesis it was assumed that the loss of activity was a first-order process. In each case, therefore, the logarithm of the activity (i.e. the logarithm of the slope of the plot of fluorescence intensity vs time) was plotted against time, and the slopes of the regression lines were used to calculate Kobs and hence T^/2 values, where; K
obs =sl°Pe x ~2·3 = 0.693/Kobs.
Statistical measures of significant differences may be done at the level of the slopes of the semi-log plots. Error bars were determined from 95% confidence limit upper and lower values of the slope of the semi-log plot, calculated as slope ± (1.96 χ standard error of the slope). The program "Statgraphics" [10] was used to calculate the standard error of the slope. Upper and lower values of K obs aRd hence of Ί\/2 were calculated from upper and lower values of the slope. Note that the error bars are asymmetrically distributed, being larger on the "upper" side. For this measure of the half-life to be useable it is important that substrate is never limiting and that there is both measurable activity, initially, and a measurable loss of activity over the period of measurement. To meet these conditions, it may be necessary to change the enzyme concentration over a wide range, as the conditions are altered: the effect which this change in concentration itself might have on the half-life is accepted as a limitation of the method. If the enzyme is unusually stable, under the conditions used, the progress curve will tend to a straight line (which corresponds to infinite stability). Occasionally, the 95% confidence limit encompasses the possibility that the enzyme is infinitely stable - in this case, the upper limit of the slope of the semi-log plot will be >0, and the calculated value of the upper limit of T|/2 will be less than the value of Tj/2! Such a "nonsensical" result should, of course, be interpreted to indicate that the upper limit represents "infinite" stability.
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Vol. 373 (1992)
Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
421
Results and Discussion
The effect of cysteine concentration, on the activity of free and complexed cathepsin L, is shown in Figure 1. The results suggest that complexing of the enzyme with the cystatin has an effect similar to that of high cysteine concentrations. Also, the free single-chain enzyme itself apparently requires much higher levels of cysteine, for complete activation, than has previously been reported to be the case for single- and two-chain forms of cathepsin L [4,9]. Alternatively, this may indicate that the single-chain sheep enzyme might be able to tolerate higher levels of cysteine. The complex can be disrupted by reducing SDS-PAGE [2] which suggests the involvement of a cysteine residue (or residues) in the linkage between the enzyme and the cystatin. Perhaps the formation of this link requires the disruption of an intramolecular disulfide bond in the enzyme; an effect which can be mimicked by a high cysteine concentration. We can envisage that, in the two-chain version of the enzyme, disruption of this disulfide bond might lead to
50
100
150
200
mM Cysteine Figure 1. The effect of cysteine concentration on the activity of single-chain sheep liver cathepsin L. 4, free cathepsin L; Q, cystatin-complexed cathepsin L. A, pH 7.0; B, pH 5.5.
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422
C. Dennison, R. Pike,T. Coetzerand K. Kirk
Vol. 373 (1992)
loss of the light chain, with consequent loss of activity. A single-chain enzyme might, therefore, be a prerequisite for the formation of catalytically active enzyme/cystatin complexes. In other words, two-chain forms of cathepsin L might be susceptible to inactivation by high cysteine concentrations and/or covalent bonding to the cystatin. As shown in Figure 2, the single-chain form apparently suffers a slight decrease in stability with increasing cysteine concentration, but there is less of a differential in stability between the two forms, compared to the difference in activity. The results in Figure 1 suggest that 150 mM cysteine is required for full activation and this concentration was consequently used in determination of the pH activity curves shown in Figure 3. Qualitatively similar results were obtained using 50 mM cysteine, however, and the pH optima were the same (results not shown). Note that, at equimolar concentrations, the cystatincomplexed cathepsin L has approximately four-fold greater activity than the free 40
3020-
10 -
0
E^ CD
40
CO
30-
X
50
100
150
200
50
100
150
200
20100
mM Cysteine Figure 2. The effect of cysteine concentration, at pH 7.0, upon the stability of single-chain sheep liver cathepsin L. A, free cathepsin L; B, cystatin-complexed cathepsin L.
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Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
Vol. 373 (1992)
>»
500
ο
400 -
rt
300 -
ce
200 -
-
100 J 3.0
4.0
5.0
6.0
7.0
423
8.0
PH
Figure 3. The effect of pH on the activity of sheep liver cathepsin L. +, free cathepsin L; Q, cystatin-complexed cathepsin L. Ionic strength, 0.1. The results are normalised to equimolar concentrations of the two forms of cathepsin L and the activities are expressed as % of the maximal activity of free cathepsin L.
enzyme (Fig. 3), suggesting that, bound in this alternate manner, the cystatin is acting as an activator. Both the free and complexed enzyme have an apparent optimum at pH 6.0 and both have significant activity at pH 7.0 (ca. 60% of their respective maximal activities) (Fig. 3). Stability determinations suggest that both forms of the enzyme are maximally stable at ca. pH 5.5-6.0 (Fig. 4).
Figure 4. The effect of pH on the stability of single-chain sheep liver cathepsin L. Free cathepsin L, at (A) 50 mM cysteine and (B) 150 mM cysteine; Cystatin-complexed cathepsin L, at (C) 50 mM cysteine and (D) 150mM cysteine.
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424
Vol. 373 (1992)
C. Dennison, R. Pike,T. Coetzer and K. Kirk
In general, the activity of both the free and complexed forms of the enzyme declined with an increase in ionic strength of the buffer, an effect which was more pronounced at pH 7.0, than at pH 5.5 (Fig. 5). Nevertheless, significant activity remains at physiological ionic strengths (/ = ca. 0.15). Ionic strength does not appear to have a significant effect upon the stability of either form of the enzyme (result not shown). Previous measures of the activity and stability of cathepsin L have suggested that this enzyme is insufficiently active and insufficiently stable, at physiological pH values, to have significant activity in extracellular locations. These previous measurements have been made on two-chain enzymes, which, as we have previously argued [7], may be artifacts of the isolation methods used. The singlechain enzyme, studied here, appears to have significant activity at pH 7.0 and, very significantly, appears to be activated by its interaction with a cystatin (thought to be stefin B [2]), which it might only encounter as a result of tissue destruction. This suggests that cathepsin L may, in fact, have significant activity in the extracellular milieu, and might be further activated by tissue destruction. 100-
100 -
8060-
80 60 40 -
(
E 'χ 03 Ε
20
0.0
0.1
0.0
0.2
100806040-
0.1
0.2
0.1
0.2
100 8060 40 20
0.0
0.1
0.0
0.2
Ionic strength Figure 5.
The effect of ionic strength on the activity of single-chain sheep liver cathepsin L. Free cathepsin L at (A) pH 5.5 and (B) pH 7.0; Cystatin-complexed cathepsin L at (C) pH 5.5 and (D) pH 7.0. All at 50 mM cysteine.
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Vol. 373 (1992)
Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
425
Methods used previously for the measurement of the pH stability of cathepsin L have involved incubation of the enzyme at different pH values for Ih, before measurement of the residual activity at pH 5.5 [3], or at the pH of incubation [11]. While arguments can be made for both of these assays, they do tend to underestimate the stability under circumstances where the half-life is significantly less than the incubation period. The measure of stability employed in this study does not suffer from this particular limitation and its use has suggested that single-chain sheep cathepsin L might be more stable at physiological pH than previously surmised. The activity and stability of the free single-chain form of the enzyme and of the enzyme/cystatin complexes, therefore, appear to be such that they could have significant activity in the extracellular milieu. Such properties are not inconsistent with a possible role of similar enzymes and enzyme complexes in tumor invasion, in which cathepsin L has been implicated. References 1. Pike, R. N., Coetzer, T. H. T. and Dennison, C. (1991) South African Biochemical Society 10th Congress. Pietermaritzburg, January 1991. 2. Pike, R. N., Coetzer, T. H. T. and Dennison, C. (1992) Arch. Biochem. Biophvs., (in press) 3. Mason, R. W. (1986) Biochem. T.. 240, 285-288. 4. Dufour, E., Obled, A., Valin, C. and Bechet, D. (1987) Biochemistry. 26, 5689-5695. 5. Denhardt, D. T., Greenberg, A. H., Egan, S. E., Hamilton, R. T. and Wright, J. A. (1987) Qncogene, 2, 55-59. 6. Rozhin, J., Wade, R. L., Honn, K. V. and Sloane, B. F. (1989) Biochem. Biophys. Res. Commun.. 164, 556-561. 7. Pike, R. N. and Dennison, C. (1989) Prep. Biochem.. 19, 231-245. 8. Ellis, K. J. and Morrison, T. F. (1982) in: Methods of Enzymology (Purich, D. L., ed.), Vol. 87, pp.405-426, Academic Press, San Diego. 9. Barrett, A. J. and Kirschke, H. (1981) in: Methods of Enzymology (Lorand, L., ed.), Vol. 80, pp.535-561, Academic Press, San Diego. 10. "Statgraphics", STSC Software Products Inc., 2115 E. Jefferson St. Rockville, MD, USA. 11. Kirschke, H., Langner, J., Wiederanders, B., Ansorge, S. and Bohley, P. (1977) Eur. T. Biochem.. 74, 293-301.
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Characterisation of the Activity and Stability of Single-chain Cathepsin L and of Proteolytically Active Cathepsin L/ Cystatin Complexes CLIVE DENNISON, ROBERT PIKE, THERESA COETZER AND KIRSTEN KIRK Department of Biochemistry, University of Natal, P. O. Box 375, Pietermaritzburg 3200, South Africa.
Summary The activity of single-chain cathepsin L was found to be markedly dependent on cysteine concentration, while a covalent, proteolytically active cathepsin L/ cystatin complex was less cysteine-dependent. Cysteine levels and ionic strength did not affect the stability of either enzyme form and both enzyme forms were found to be stable for significant periods of time at or near physiological pH.
Introduction We have previously reported [1,2] on the isolation of proteolytically active complexes of single-chain cathepsin L with a cystatin, thought to be stefin B, and on the formation of similar complexes in vitro, from purified constituents. These complexes can only be disrupted by boiling in sodium dodecyl sulfate in the presence of a reducing agent and in these proteolytically active complexes the enzyme and cystatin may, therefore, be covalently bound [2]. We report here on the characterisation of the activity and stability of the singlechain sheep liver enzyme and of a proteolytically active complex fraction. Twochain forms of cathepsin L [3] and single-chain chicken cathepsin L [4] have previously been characterised in this respect, while the activity and stability of cathepsin L/cystatin complexes have not previously been documented. Because cathepsin L has been implicated in cancer [5,6], the activity of this enzyme under extracellular conditions of pH, ionic strength and redox potential is of some interest, in the context of invasion. Abbreviations: NHMec, 7-(4-methyl)coumarylamide; Z-, benzyloxycarbonyl.
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420
C. Dennison, R. Pike, T. Coetzer and K. Kirk
Vol. 373 (1992)
Methods Single-chain sheep liver cathepsin L, and a proteolytically active fraction of sheep liver cathepsin L complexed with a sheep liver cystatin, were isolated as described previously [7], through the pH 5.5 step on S-Sepharose. The active peak was subsequently separated on Sephadex G-75, the first included peak being the complex fraction and the second being free cathepsin L. Acetate-MES-Tris buffer systems, of defined ionic strength (0.1, unless stated otherwise), described by Ellis & Morrison [8] were used in the assays for activity and stability. Enzyme activity was measured at 30°C according to Barrett & Kirschke [9]; unless stated otherwise, all other assays were done at 37°C. As a measure of stability, the half-life of the enzyme was determined by continuously monitoring the fluorogenic products produced, from Z-Phe-Arg-NHMec, by an enzyme sample incubated in a defined buffer containing a thiol activator. (Activity could also be estimated from the initial slope of the same progress curve.) For determination of the half-life, the period of measurement was usually 30 min. As a null hypothesis it was assumed that the loss of activity was a first-order process. In each case, therefore, the logarithm of the activity (i.e. the logarithm of the slope of the plot of fluorescence intensity vs time) was plotted against time, and the slopes of the regression lines were used to calculate Kobs and hence T^/2 values, where; K
obs =sl°Pe x ~2·3 = 0.693/Kobs.
Statistical measures of significant differences may be done at the level of the slopes of the semi-log plots. Error bars were determined from 95% confidence limit upper and lower values of the slope of the semi-log plot, calculated as slope ± (1.96 χ standard error of the slope). The program "Statgraphics" [10] was used to calculate the standard error of the slope. Upper and lower values of K obs aRd hence of Ί\/2 were calculated from upper and lower values of the slope. Note that the error bars are asymmetrically distributed, being larger on the "upper" side. For this measure of the half-life to be useable it is important that substrate is never limiting and that there is both measurable activity, initially, and a measurable loss of activity over the period of measurement. To meet these conditions, it may be necessary to change the enzyme concentration over a wide range, as the conditions are altered: the effect which this change in concentration itself might have on the half-life is accepted as a limitation of the method. If the enzyme is unusually stable, under the conditions used, the progress curve will tend to a straight line (which corresponds to infinite stability). Occasionally, the 95% confidence limit encompasses the possibility that the enzyme is infinitely stable - in this case, the upper limit of the slope of the semi-log plot will be >0, and the calculated value of the upper limit of T|/2 will be less than the value of Tj/2! Such a "nonsensical" result should, of course, be interpreted to indicate that the upper limit represents "infinite" stability.
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Vol. 373 (1992)
Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
421
Results and Discussion
The effect of cysteine concentration, on the activity of free and complexed cathepsin L, is shown in Figure 1. The results suggest that complexing of the enzyme with the cystatin has an effect similar to that of high cysteine concentrations. Also, the free single-chain enzyme itself apparently requires much higher levels of cysteine, for complete activation, than has previously been reported to be the case for single- and two-chain forms of cathepsin L [4,9]. Alternatively, this may indicate that the single-chain sheep enzyme might be able to tolerate higher levels of cysteine. The complex can be disrupted by reducing SDS-PAGE [2] which suggests the involvement of a cysteine residue (or residues) in the linkage between the enzyme and the cystatin. Perhaps the formation of this link requires the disruption of an intramolecular disulfide bond in the enzyme; an effect which can be mimicked by a high cysteine concentration. We can envisage that, in the two-chain version of the enzyme, disruption of this disulfide bond might lead to
50
100
150
200
mM Cysteine Figure 1. The effect of cysteine concentration on the activity of single-chain sheep liver cathepsin L. 4, free cathepsin L; Q, cystatin-complexed cathepsin L. A, pH 7.0; B, pH 5.5.
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422
C. Dennison, R. Pike,T. Coetzerand K. Kirk
Vol. 373 (1992)
loss of the light chain, with consequent loss of activity. A single-chain enzyme might, therefore, be a prerequisite for the formation of catalytically active enzyme/cystatin complexes. In other words, two-chain forms of cathepsin L might be susceptible to inactivation by high cysteine concentrations and/or covalent bonding to the cystatin. As shown in Figure 2, the single-chain form apparently suffers a slight decrease in stability with increasing cysteine concentration, but there is less of a differential in stability between the two forms, compared to the difference in activity. The results in Figure 1 suggest that 150 mM cysteine is required for full activation and this concentration was consequently used in determination of the pH activity curves shown in Figure 3. Qualitatively similar results were obtained using 50 mM cysteine, however, and the pH optima were the same (results not shown). Note that, at equimolar concentrations, the cystatincomplexed cathepsin L has approximately four-fold greater activity than the free 40
3020-
10 -
0
E^ CD
40
CO
30-
X
50
100
150
200
50
100
150
200
20100
mM Cysteine Figure 2. The effect of cysteine concentration, at pH 7.0, upon the stability of single-chain sheep liver cathepsin L. A, free cathepsin L; B, cystatin-complexed cathepsin L.
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Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
Vol. 373 (1992)
>»
500
ο
400 -
rt
300 -
ce
200 -
-
100 J 3.0
4.0
5.0
6.0
7.0
423
8.0
PH
Figure 3. The effect of pH on the activity of sheep liver cathepsin L. +, free cathepsin L; Q, cystatin-complexed cathepsin L. Ionic strength, 0.1. The results are normalised to equimolar concentrations of the two forms of cathepsin L and the activities are expressed as % of the maximal activity of free cathepsin L.
enzyme (Fig. 3), suggesting that, bound in this alternate manner, the cystatin is acting as an activator. Both the free and complexed enzyme have an apparent optimum at pH 6.0 and both have significant activity at pH 7.0 (ca. 60% of their respective maximal activities) (Fig. 3). Stability determinations suggest that both forms of the enzyme are maximally stable at ca. pH 5.5-6.0 (Fig. 4).
Figure 4. The effect of pH on the stability of single-chain sheep liver cathepsin L. Free cathepsin L, at (A) 50 mM cysteine and (B) 150 mM cysteine; Cystatin-complexed cathepsin L, at (C) 50 mM cysteine and (D) 150mM cysteine.
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424
Vol. 373 (1992)
C. Dennison, R. Pike,T. Coetzer and K. Kirk
In general, the activity of both the free and complexed forms of the enzyme declined with an increase in ionic strength of the buffer, an effect which was more pronounced at pH 7.0, than at pH 5.5 (Fig. 5). Nevertheless, significant activity remains at physiological ionic strengths (/ = ca. 0.15). Ionic strength does not appear to have a significant effect upon the stability of either form of the enzyme (result not shown). Previous measures of the activity and stability of cathepsin L have suggested that this enzyme is insufficiently active and insufficiently stable, at physiological pH values, to have significant activity in extracellular locations. These previous measurements have been made on two-chain enzymes, which, as we have previously argued [7], may be artifacts of the isolation methods used. The singlechain enzyme, studied here, appears to have significant activity at pH 7.0 and, very significantly, appears to be activated by its interaction with a cystatin (thought to be stefin B [2]), which it might only encounter as a result of tissue destruction. This suggests that cathepsin L may, in fact, have significant activity in the extracellular milieu, and might be further activated by tissue destruction. 100-
100 -
8060-
80 60 40 -
(
E 'χ 03 Ε
20
0.0
0.1
0.0
0.2
100806040-
0.1
0.2
0.1
0.2
100 8060 40 20
0.0
0.1
0.0
0.2
Ionic strength Figure 5.
The effect of ionic strength on the activity of single-chain sheep liver cathepsin L. Free cathepsin L at (A) pH 5.5 and (B) pH 7.0; Cystatin-complexed cathepsin L at (C) pH 5.5 and (D) pH 7.0. All at 50 mM cysteine.
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Vol. 373 (1992)
Single-chain Cathepsin L and Active Cathepsin L / Cystatin Complexes
425
Methods used previously for the measurement of the pH stability of cathepsin L have involved incubation of the enzyme at different pH values for Ih, before measurement of the residual activity at pH 5.5 [3], or at the pH of incubation [11]. While arguments can be made for both of these assays, they do tend to underestimate the stability under circumstances where the half-life is significantly less than the incubation period. The measure of stability employed in this study does not suffer from this particular limitation and its use has suggested that single-chain sheep cathepsin L might be more stable at physiological pH than previously surmised. The activity and stability of the free single-chain form of the enzyme and of the enzyme/cystatin complexes, therefore, appear to be such that they could have significant activity in the extracellular milieu. Such properties are not inconsistent with a possible role of similar enzymes and enzyme complexes in tumor invasion, in which cathepsin L has been implicated. References 1. Pike, R. N., Coetzer, T. H. T. and Dennison, C. (1991) South African Biochemical Society 10th Congress. Pietermaritzburg, January 1991. 2. Pike, R. N., Coetzer, T. H. T. and Dennison, C. (1992) Arch. Biochem. Biophvs., (in press) 3. Mason, R. W. (1986) Biochem. T.. 240, 285-288. 4. Dufour, E., Obled, A., Valin, C. and Bechet, D. (1987) Biochemistry. 26, 5689-5695. 5. Denhardt, D. T., Greenberg, A. H., Egan, S. E., Hamilton, R. T. and Wright, J. A. (1987) Qncogene, 2, 55-59. 6. Rozhin, J., Wade, R. L., Honn, K. V. and Sloane, B. F. (1989) Biochem. Biophys. Res. Commun.. 164, 556-561. 7. Pike, R. N. and Dennison, C. (1989) Prep. Biochem.. 19, 231-245. 8. Ellis, K. J. and Morrison, T. F. (1982) in: Methods of Enzymology (Purich, D. L., ed.), Vol. 87, pp.405-426, Academic Press, San Diego. 9. Barrett, A. J. and Kirschke, H. (1981) in: Methods of Enzymology (Lorand, L., ed.), Vol. 80, pp.535-561, Academic Press, San Diego. 10. "Statgraphics", STSC Software Products Inc., 2115 E. Jefferson St. Rockville, MD, USA. 11. Kirschke, H., Langner, J., Wiederanders, B., Ansorge, S. and Bohley, P. (1977) Eur. T. Biochem.. 74, 293-301.
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