- Email: [email protected]
Purification of thiogalactoside transacetylase by affinity chromatography
\NALYTICAL BIOCHEMISTRY 136,493-496
Purification
(1984)
of Thiogalactoside IRVING
Department
Transacetylase
ZABIN
of Biological Chemistry, University of California,
by Affinity Chromatography
AND AUDREE V. FOWLER School of Medicine, and Molecular Los Angeles, Los Angeles, California
Biology 90024
Institute,
Received August 16, 1983 Thiogalactoside transacetylase, the product of the 1acA gene of the lactose operon of Escherichia has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration. KEY WORDS: Cibacron blue F3GA chromatography; agarose-coenzyme A chromatography. co/i,
The lactose operon of Escherichia coli contains three structural genes. Extensive structure-function studies of the products of the 1acZ and lacy genes have been carried out ( l-4), and the 1acZ and lacy genes themselves
have been completely sequenced (5,6). In contrast, less is known about the lad gene and its product, the enzyme thiogalactoside transacetylase (EC 2.2.1.18) (7). The reaction catalyzed is
acetyl coenzyme A + isopropylthiogalactoside coenzyme A + 6-O-acetyl isopropylthiogalactoside The enzyme apparently functions as a detoxifier, since wild-type strains of E. coli containing the transacetylase grow faster on media containing lactose and a thiogalactoside than isogenic strains without the enzyme (8). In the presence of the transacetylase, acetyl thiogalactosides diffuse into the medium and have no effect on growth rate. In its absence, high concentrations of thiogalactosides remain within the cell and tend to inhibit growth. The molecular weight and subunit composition of thiogalactoside transacetylase are known and the protein has been crystallized (9). Its substrate specificity, kinetics, and mechanism of reaction also have been examined ( 10). Purification to homogeneity was first accomplished using both anion- and cation-exchange chromatography. This procedure with minor modifications was used re-
cently to compare the transacetylases in two different strains of E. coli (11). Although the yields of pure protein obtained by such methods are satisfactory, the procedure is somewhat tedious. We report here a simpler, more rapid method for the isolation of thiogalactoside transacetylase in excellent yield and in pure form. MATERIALS
AND
METHODS
Materials. Affi-Gel Blue (Cibacron Blue F3GA immobilized on agarose; 100-200 mesh) was obtained from Bio-Rad Laboratories. Agarose-Hexane-Coenzyme A, Type 5, (AgCoA)’ was a product of P-L Biochem’ Abbreviations used: AgCoA, agarose-hexane-coenzyme A; SDS, sodium dodecyl sulfate. 493
0003-2697184 $3.00 Copyright 0 1984 by Academic FVes, Inc. All rights of reproduction in any form reserved.
494
ZABIN
AND
icals. The CoA is bound to the agarose by a thiol ester linkage with a six-carbon spacer. Sephacryl S-200 was obtained from Pharmacia. Protein and enzyme assays. Protein was measured by absorbance at 280 nm or by the Lowry procedure. Thiogalactoside transacetylase was assayed with dithiobis(2-nitrobenzoic acid) as described by Alpers et al. (I 2) and modified by Fried (11). Bacterial growth conditions and preliminary fractionations. Cultures (150 liters) of E. coli
A324-5 were grown on succinic acid. This strain produces large amounts of &alactosidase and thiogalactoside transacetylase. Extracts were prepared by sonication and nucleic acids were removed by precipitation with streptomycin sulfate. &Galactosidase was precipitated below 40% saturation with ammonium sulfate and the transacetylase remained in the supernatant solution (13). /3-Galactosidase in the ammonium sulfate precipitate was readily purified on DEAE-BioGel and Bio-Gel A 1.5 (14). A_ffi-Gel blue chromatography. The supernatant solution obtained after addition of ammonium sulfate to 40% saturation was dialyzed thoroughly against 0.03 M potassium phosphate, pH 7.2, 1 mM EDTA, 0.5 mM mercaptoethanol in the cold. Solutions were clarified by centrifugation, and were then passed through a column ( 1.5 X 5 cm) of AffiGel Blue at room temperature. The column was washed with 100 ml of the same buffer followed by 100 ml of the buffer containing 0.1 M KCl. The transacetylase was then eluted at room temperature with 200 ml of the same buffer containing 2 M KCl. Agarose-coenzyme A chromatography. The 2 M KC1 eluate from the Affi-Gel Blue column was dialyzed against 0.03 M sodium phosphate, pH 7.2, 1 mM EDTA, 0.5 mM mercaptoethanol and was applied to a 1.5 X 15cm column of AgCoA in the same buffer at room temperature. After washing with 100 ml buffer, the column was eluted with a linear gradient of O-2 M NaCl in buffer in a total volume of 500 ml.
FOWLER
Gelfiltration. The pooled fraction containing active enzyme from the AgCoA column was dialyzed against 0.05 M ammonium bicarbonate and 0.5 mM @-mercaptoethanol. It was then lyophilized to a volume of 2-3 ml, and applied to a 1.5 X loo-cm column of Sephacryl S-200 equilibrated and eluted with the same buffer. Agarose A 0.5 (Bio-Rad) could also be used instead of Sephacryl S-200. This step was carried out in the cold. RESULTS
AI&Gel Blue (Cibacron Blue) adsorbent was found to be highly effective in removing thiogalactoside transacetylase from the other proteins. Elution with NaCl at various temperatures and in the presence of 0.1 mM CoA were ineffective in removing the enzyme from the column. However, when KC1 was used as eluant, the transacetylase was released in good yield. Both sodium and potassium phosphate buffers could be used for binding of the transacetylase to the column without effect on subsequent elution with KCl. The capacity of the Affi-Gel Blue was quite high so that only a small column was needed. Normally a 1.5 X 5cm column of Al&Gel Blue was used to adsorb the transacetylase from a fraction containing 20 g of protein. Batch rather than gradient elution was performed in order to obtain the material in a minimum volume. The yield in the experiment shown in Table 1 was 64% with a 68-fold purification. Yields have varied from 25 to 80% and were generally higher when a minimum amount of adsorbent was used. Previous kinetic studies on thiogalactoside transacetylase had indicated that CoA could be expected to be an excellent affinity ligand (lo), and chromatography on AgCoA of the enzyme obtained from the A&Gel Blue column gave yields of 95%. The transacetylase was eluted from the column at a salt concentration of less than 1 M (second peak in Fig. 1) and was pooled as indicated. Final purification to a satisfactory degree of purity was achieved by gel filtration through
THIOGALACTOSIDE
TRANSACETYLASE TABLE
CHROMATOGRAPHY
OF THI~GALMX~SIDE
495
PURIFICATION
I TRANSACETYLASE
ON AFFI-GEL
BLUE
(mol)
Protein (mg)
Enzyme activity (units)
Yield (%I
Specific activity (units/mg protein)
2640 2640 100 100 200
19000 18000 90 10 180
76,600 0 500 1500 49,oOil
(loo) 0 7 2 64
4 0 6 150 272
Vol
Ammonium sulfate fraction 40-75% saturation Unabsorbed solution Buffer wash Buffer + 0.1 M KCl eluate Buffer + 2 M KC1 eluate
CHROMATOGRAPHIC
Nate. The Affi-Gel Blue column (1.5 X 5 cm) was equilibrated in buffer (0.03 M potassium phosphate, pH 7.2, containing 1 mM EDTA and 0.5 mM /3-mercaptoethanol).
Sephacryl S-200. Pooled fractions containing active enzyme from the AgCoA column were dialyzed and lyophilized and applied to columns of Sephacryl S-200. Yields in this step were at least 85%. Only one band was seen on SDS-gel electrophoresis. An overall summary of the purification of thiogalactoside transacetylase is shown in Table 2. Specific activities ranged from 3000 to 3300 in different preparations. A specific activity of 3540 units/mg protein has been re-
ported for thiogalactoside transacetylase purified with DEAE and CM ion exchangers (11). DISCUSSION
The E. coli strain used here produces about 15 to 20 times more &galactosidase than the transacetylase by weight (9). For much of the work in this laboratory, relatively large amounts of the former enzyme have been required, and it has been useful to isolate both enzymes by the procedure shown above. It is also possible to obtain thiogalactoside transacetylase directly from crude extracts. In one experiment, 100 ml of an dialyzed sonicate was passed through a 1.5 X 2.5-cm column of A&Gel Blue. The enzyme was eluted in a yield of 55% and a 37-fold purification. pGalactosidase does not bind to Affi-Gel Blue and can be isolated from the unabsorbed fraction. Crude extracts and ammonium sulfate fractions can also be placed directly on AgCoA VOLUME [ml) columns. Their capacity is about 1 to 2 mg enzyme/ml adsorbent, considerably lower FIG. 1. The 2 M KCl eluate from At&Gel Blue was dialyzed in the cold against 0.03 M sodium phosphate than that of Affi-Gel Blue columns. More imbuffer, pH 7.2, containing 1 mM EDTA and 0.5 mM j3- portantly, such affinity columns have been efmercaptoethanol, and was applied at room temperature fective in our hands for no more than three to a column of AgCoA (1.5 X 15 cm). The column was or four preparations, possibly because of loss washed with 150 ml of buffer and was then eluted with a linear gradient (500 ml total volume) of O-2 M NaCl of CoA from the adsorbent by hydrolysis of in buffer. Fractions of 4 ml were collected. The enzyme the thiol ester catalyzed by thiolases in the E. was pooled as indicated. coli extracts.
496
ZABIN AND FOWLER TABLE 2 PURIFICATION
Step Crude extract Ammonium sulfate traction, supematant after 40% saturation Affi-Gel Blue eluate AgCoA pooled fraction S-200 pooled fraction
OF
THICGALAC~OSIDE TRANSACETYLASE
Volume (ml)
Protein (mg)
Enzyme activity (units)
4460
162,000
486,000
3
(1W
5580 800 95 30
89,000 1136 91 60
360,008 210,080 200,000 180,000
4 185 2200 3000
74 43 41 37
Cibacron Blue F3GA has been shown to be useful in the purification of a large number of proteins (15). Apparently, binding occurs to any protein possessing a cluster of aromatic and other apolar groups (16). In this regard it is of interest that potassium ions but not sodium can release thiogalactoside transacetylase from the dye. The enzyme is active with both cations but is about 50% more active in potassium phosphate than in sodium phosphate buffers. Since catalytic sites are generally considered to be in hydrophobic pockets, this suggests that potassium is effective an an eluant because it competes for or changes a hydrophobic catalytic structure in the protein. ACKNOWLEDGMENTS We thank David Dupont and Steven Wong for expert technical assistance. This work was supported by NIH Grant AI-04 18 1 and NSF Grant PCM-8 118 112.
REFERENCES 1. Fowler, A. V., and Zabin, I. (1978) J. Biol. Chem. 253, 5521-5525. 2. Zabin, I. (1980) in The Evolution of Protein Structure and Function (Sigman, D. S., and Brazier,
3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15.
16.
Specific activity (units/mg)
Yield (%)
M. A. B., eds.), pp. 49-62, Academic Press, New York. Ehring, R., Beyreuther, K., Wright, J. K., and Gverath, P. (1980) Nature (London) 283, 537-540. Kaczorowski, G. J., Robertson, D. E., Garcia, M. L., Padan, E., Patel, L., LeBlanc, G., and Kaback, H. R. (1980) Ann. N. Y. Acad. Sci. 358,307-321. Kalnins, S. A., Otto, K., Ruther, V., and Muller-Hill, B. (1983) Eur. Mol. Biol. Org. J. 2, 593-597. Buchel, D. E., Gronenbom, B., and Muller-Hill, B. (1980) Nature (London) 283, 541-545. Zabin, I., Keyes, A., and Monod, J., (1959) B&hem. Biophys. Res. Commun. 1, 289-292. Andrews, K. S., and Lin, E. C. C. (1976) J. Bacterial. X28,510-513. Zabin, I. (1963) J. Biol. Chem. 238, 3300-3306. Musso, R. E. and Zabin, I. (1973) Biochemistry 12, 553-557. Fried, V. A. (1980) J. Bacterial. 143, 506-509. Alpers, D. H., Appel, S. H., and Tompkins, G. M. (1965) J. Biol. Chem. 240, 10-13. Fowler, A. V. (1972) J. Bacterial. 112, 856-860. Fowler, A. V., and Zabin, I. (1983) J. Biol. Chem., in press. Haff, L. A., and Easterday, R. L. (1978) in Theory and Practice in Affinity Techniques (Sum&am, P. V., and Eckstein, F., eds.), pp. 23-44, Academic Press, New York. Subramanian, S., and Kaufman, B. T. (1980) J. Biol. Chem. 255, 10587-10590.
Purification
(1984)
of Thiogalactoside IRVING
Department
Transacetylase
ZABIN
of Biological Chemistry, University of California,
by Affinity Chromatography
AND AUDREE V. FOWLER School of Medicine, and Molecular Los Angeles, Los Angeles, California
Biology 90024
Institute,
Received August 16, 1983 Thiogalactoside transacetylase, the product of the 1acA gene of the lactose operon of Escherichia has been purified by an improved procedure. The enzyme binds tightly to immobilized Cibacron Blue F3GA columns and can be eluted by potassium chloride in high concentrations. Final purification was obtained by affinity chromatography on an agarose-coenzyme A column followed by gel filtration. KEY WORDS: Cibacron blue F3GA chromatography; agarose-coenzyme A chromatography. co/i,
The lactose operon of Escherichia coli contains three structural genes. Extensive structure-function studies of the products of the 1acZ and lacy genes have been carried out ( l-4), and the 1acZ and lacy genes themselves
have been completely sequenced (5,6). In contrast, less is known about the lad gene and its product, the enzyme thiogalactoside transacetylase (EC 2.2.1.18) (7). The reaction catalyzed is
acetyl coenzyme A + isopropylthiogalactoside coenzyme A + 6-O-acetyl isopropylthiogalactoside The enzyme apparently functions as a detoxifier, since wild-type strains of E. coli containing the transacetylase grow faster on media containing lactose and a thiogalactoside than isogenic strains without the enzyme (8). In the presence of the transacetylase, acetyl thiogalactosides diffuse into the medium and have no effect on growth rate. In its absence, high concentrations of thiogalactosides remain within the cell and tend to inhibit growth. The molecular weight and subunit composition of thiogalactoside transacetylase are known and the protein has been crystallized (9). Its substrate specificity, kinetics, and mechanism of reaction also have been examined ( 10). Purification to homogeneity was first accomplished using both anion- and cation-exchange chromatography. This procedure with minor modifications was used re-
cently to compare the transacetylases in two different strains of E. coli (11). Although the yields of pure protein obtained by such methods are satisfactory, the procedure is somewhat tedious. We report here a simpler, more rapid method for the isolation of thiogalactoside transacetylase in excellent yield and in pure form. MATERIALS
AND
METHODS
Materials. Affi-Gel Blue (Cibacron Blue F3GA immobilized on agarose; 100-200 mesh) was obtained from Bio-Rad Laboratories. Agarose-Hexane-Coenzyme A, Type 5, (AgCoA)’ was a product of P-L Biochem’ Abbreviations used: AgCoA, agarose-hexane-coenzyme A; SDS, sodium dodecyl sulfate. 493
0003-2697184 $3.00 Copyright 0 1984 by Academic FVes, Inc. All rights of reproduction in any form reserved.
494
ZABIN
AND
icals. The CoA is bound to the agarose by a thiol ester linkage with a six-carbon spacer. Sephacryl S-200 was obtained from Pharmacia. Protein and enzyme assays. Protein was measured by absorbance at 280 nm or by the Lowry procedure. Thiogalactoside transacetylase was assayed with dithiobis(2-nitrobenzoic acid) as described by Alpers et al. (I 2) and modified by Fried (11). Bacterial growth conditions and preliminary fractionations. Cultures (150 liters) of E. coli
A324-5 were grown on succinic acid. This strain produces large amounts of &alactosidase and thiogalactoside transacetylase. Extracts were prepared by sonication and nucleic acids were removed by precipitation with streptomycin sulfate. &Galactosidase was precipitated below 40% saturation with ammonium sulfate and the transacetylase remained in the supernatant solution (13). /3-Galactosidase in the ammonium sulfate precipitate was readily purified on DEAE-BioGel and Bio-Gel A 1.5 (14). A_ffi-Gel blue chromatography. The supernatant solution obtained after addition of ammonium sulfate to 40% saturation was dialyzed thoroughly against 0.03 M potassium phosphate, pH 7.2, 1 mM EDTA, 0.5 mM mercaptoethanol in the cold. Solutions were clarified by centrifugation, and were then passed through a column ( 1.5 X 5 cm) of AffiGel Blue at room temperature. The column was washed with 100 ml of the same buffer followed by 100 ml of the buffer containing 0.1 M KCl. The transacetylase was then eluted at room temperature with 200 ml of the same buffer containing 2 M KCl. Agarose-coenzyme A chromatography. The 2 M KC1 eluate from the Affi-Gel Blue column was dialyzed against 0.03 M sodium phosphate, pH 7.2, 1 mM EDTA, 0.5 mM mercaptoethanol and was applied to a 1.5 X 15cm column of AgCoA in the same buffer at room temperature. After washing with 100 ml buffer, the column was eluted with a linear gradient of O-2 M NaCl in buffer in a total volume of 500 ml.
FOWLER
Gelfiltration. The pooled fraction containing active enzyme from the AgCoA column was dialyzed against 0.05 M ammonium bicarbonate and 0.5 mM @-mercaptoethanol. It was then lyophilized to a volume of 2-3 ml, and applied to a 1.5 X loo-cm column of Sephacryl S-200 equilibrated and eluted with the same buffer. Agarose A 0.5 (Bio-Rad) could also be used instead of Sephacryl S-200. This step was carried out in the cold. RESULTS
AI&Gel Blue (Cibacron Blue) adsorbent was found to be highly effective in removing thiogalactoside transacetylase from the other proteins. Elution with NaCl at various temperatures and in the presence of 0.1 mM CoA were ineffective in removing the enzyme from the column. However, when KC1 was used as eluant, the transacetylase was released in good yield. Both sodium and potassium phosphate buffers could be used for binding of the transacetylase to the column without effect on subsequent elution with KCl. The capacity of the Affi-Gel Blue was quite high so that only a small column was needed. Normally a 1.5 X 5cm column of Al&Gel Blue was used to adsorb the transacetylase from a fraction containing 20 g of protein. Batch rather than gradient elution was performed in order to obtain the material in a minimum volume. The yield in the experiment shown in Table 1 was 64% with a 68-fold purification. Yields have varied from 25 to 80% and were generally higher when a minimum amount of adsorbent was used. Previous kinetic studies on thiogalactoside transacetylase had indicated that CoA could be expected to be an excellent affinity ligand (lo), and chromatography on AgCoA of the enzyme obtained from the A&Gel Blue column gave yields of 95%. The transacetylase was eluted from the column at a salt concentration of less than 1 M (second peak in Fig. 1) and was pooled as indicated. Final purification to a satisfactory degree of purity was achieved by gel filtration through
THIOGALACTOSIDE
TRANSACETYLASE TABLE
CHROMATOGRAPHY
OF THI~GALMX~SIDE
495
PURIFICATION
I TRANSACETYLASE
ON AFFI-GEL
BLUE
(mol)
Protein (mg)
Enzyme activity (units)
Yield (%I
Specific activity (units/mg protein)
2640 2640 100 100 200
19000 18000 90 10 180
76,600 0 500 1500 49,oOil
(loo) 0 7 2 64
4 0 6 150 272
Vol
Ammonium sulfate fraction 40-75% saturation Unabsorbed solution Buffer wash Buffer + 0.1 M KCl eluate Buffer + 2 M KC1 eluate
CHROMATOGRAPHIC
Nate. The Affi-Gel Blue column (1.5 X 5 cm) was equilibrated in buffer (0.03 M potassium phosphate, pH 7.2, containing 1 mM EDTA and 0.5 mM /3-mercaptoethanol).
Sephacryl S-200. Pooled fractions containing active enzyme from the AgCoA column were dialyzed and lyophilized and applied to columns of Sephacryl S-200. Yields in this step were at least 85%. Only one band was seen on SDS-gel electrophoresis. An overall summary of the purification of thiogalactoside transacetylase is shown in Table 2. Specific activities ranged from 3000 to 3300 in different preparations. A specific activity of 3540 units/mg protein has been re-
ported for thiogalactoside transacetylase purified with DEAE and CM ion exchangers (11). DISCUSSION
The E. coli strain used here produces about 15 to 20 times more &galactosidase than the transacetylase by weight (9). For much of the work in this laboratory, relatively large amounts of the former enzyme have been required, and it has been useful to isolate both enzymes by the procedure shown above. It is also possible to obtain thiogalactoside transacetylase directly from crude extracts. In one experiment, 100 ml of an dialyzed sonicate was passed through a 1.5 X 2.5-cm column of A&Gel Blue. The enzyme was eluted in a yield of 55% and a 37-fold purification. pGalactosidase does not bind to Affi-Gel Blue and can be isolated from the unabsorbed fraction. Crude extracts and ammonium sulfate fractions can also be placed directly on AgCoA VOLUME [ml) columns. Their capacity is about 1 to 2 mg enzyme/ml adsorbent, considerably lower FIG. 1. The 2 M KCl eluate from At&Gel Blue was dialyzed in the cold against 0.03 M sodium phosphate than that of Affi-Gel Blue columns. More imbuffer, pH 7.2, containing 1 mM EDTA and 0.5 mM j3- portantly, such affinity columns have been efmercaptoethanol, and was applied at room temperature fective in our hands for no more than three to a column of AgCoA (1.5 X 15 cm). The column was or four preparations, possibly because of loss washed with 150 ml of buffer and was then eluted with a linear gradient (500 ml total volume) of O-2 M NaCl of CoA from the adsorbent by hydrolysis of in buffer. Fractions of 4 ml were collected. The enzyme the thiol ester catalyzed by thiolases in the E. was pooled as indicated. coli extracts.
496
ZABIN AND FOWLER TABLE 2 PURIFICATION
Step Crude extract Ammonium sulfate traction, supematant after 40% saturation Affi-Gel Blue eluate AgCoA pooled fraction S-200 pooled fraction
OF
THICGALAC~OSIDE TRANSACETYLASE
Volume (ml)
Protein (mg)
Enzyme activity (units)
4460
162,000
486,000
3
(1W
5580 800 95 30
89,000 1136 91 60
360,008 210,080 200,000 180,000
4 185 2200 3000
74 43 41 37
Cibacron Blue F3GA has been shown to be useful in the purification of a large number of proteins (15). Apparently, binding occurs to any protein possessing a cluster of aromatic and other apolar groups (16). In this regard it is of interest that potassium ions but not sodium can release thiogalactoside transacetylase from the dye. The enzyme is active with both cations but is about 50% more active in potassium phosphate than in sodium phosphate buffers. Since catalytic sites are generally considered to be in hydrophobic pockets, this suggests that potassium is effective an an eluant because it competes for or changes a hydrophobic catalytic structure in the protein. ACKNOWLEDGMENTS We thank David Dupont and Steven Wong for expert technical assistance. This work was supported by NIH Grant AI-04 18 1 and NSF Grant PCM-8 118 112.
REFERENCES 1. Fowler, A. V., and Zabin, I. (1978) J. Biol. Chem. 253, 5521-5525. 2. Zabin, I. (1980) in The Evolution of Protein Structure and Function (Sigman, D. S., and Brazier,
3. 4. 5. 6. I. 8. 9. 10. 11. 12. 13. 14. 15.
16.
Specific activity (units/mg)
Yield (%)
M. A. B., eds.), pp. 49-62, Academic Press, New York. Ehring, R., Beyreuther, K., Wright, J. K., and Gverath, P. (1980) Nature (London) 283, 537-540. Kaczorowski, G. J., Robertson, D. E., Garcia, M. L., Padan, E., Patel, L., LeBlanc, G., and Kaback, H. R. (1980) Ann. N. Y. Acad. Sci. 358,307-321. Kalnins, S. A., Otto, K., Ruther, V., and Muller-Hill, B. (1983) Eur. Mol. Biol. Org. J. 2, 593-597. Buchel, D. E., Gronenbom, B., and Muller-Hill, B. (1980) Nature (London) 283, 541-545. Zabin, I., Keyes, A., and Monod, J., (1959) B&hem. Biophys. Res. Commun. 1, 289-292. Andrews, K. S., and Lin, E. C. C. (1976) J. Bacterial. X28,510-513. Zabin, I. (1963) J. Biol. Chem. 238, 3300-3306. Musso, R. E. and Zabin, I. (1973) Biochemistry 12, 553-557. Fried, V. A. (1980) J. Bacterial. 143, 506-509. Alpers, D. H., Appel, S. H., and Tompkins, G. M. (1965) J. Biol. Chem. 240, 10-13. Fowler, A. V. (1972) J. Bacterial. 112, 856-860. Fowler, A. V., and Zabin, I. (1983) J. Biol. Chem., in press. Haff, L. A., and Easterday, R. L. (1978) in Theory and Practice in Affinity Techniques (Sum&am, P. V., and Eckstein, F., eds.), pp. 23-44, Academic Press, New York. Subramanian, S., and Kaufman, B. T. (1980) J. Biol. Chem. 255, 10587-10590.