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A new cold agglutinin from Achatina fulica snails
ARCHIVES
OF BIOCHEMISTRY
Vol. 233, No. 1, August
AND BIOPHYSICS
15, pp. 286-289, 1984
A New Cold Agglutinin MANJU
SARKAR,’ Indian
BIMAL
KUMAR
Institute
from Achatina fulica Snails BACHHAWAT,
of Chemical Biology,
Received December
Calcutta
AND
CHITRA
MANDAL
700 032, India
28, 1983, and in revised form April
5, 1984
An electrophoretically homogeneous agglutinin was purified from the albumin gland of Achatina fulica snails using asialofetunin-Sepharose 4B as an affinity column. The agglutinin was found to be temperature sensitive; it agglutinated rabbit and human umbilical cord erythrocytes only at low temperature. It was found to be specific for methyl-P-D-galactoside, and the best inhibitor was N-acetyllactosamine.
Cold agglutinins are a special class of monoclonal macroglobulins that show specificity towards blood group I, i, and Pr antigens (1, 2). These agglutinins agglutinate human erythrocytes at temperatures below 37°C (3). So far cold agglutinins have been found in human patients with Waldenstrom macroglobulinemia, autoimmunohemolytic anemia, and breast carcinoma (4-6). These cold agglutinins are autoantibodies mainly of the IgM2 type (4). In the present communication we report the isolation and characterization of a cold agglutinin from an invertebrate, Achatina fulica snail. To the best of our knowledge, this is the first report of a cold agglutinin in an animal other than the human. MATERIALS
AND
METHODS
Achutinafulicu snails were purchased from the local market. All the monosaccharides and the disaccharides were purchased from Sigma Chemical Company. The disaccharide N-acetyllactosamine was obtained from Dr. E. A. Kabat (Columbia University, N. Y.). Protein was estimated by the method of Lowry et aL (7) using crystalline bovine serum albumin as the standard. Asialofetuin was prepared from fetuin (Sigma) (8). The affinity column was prepared by coupling asialofetuin with CNBr-activated Sepharose 4B
r To whom correspondence should be addressed, at 4 Raja S. C. Mallick Road, Calcutta 700 032, India. * Abbreviations used: IgM, immunoglobulin M, PBS, phosphate-buffered saline. 0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
according to Cuatrecasas (9). All the eluates from the columns were checked by absorption at 280 nm, and also by assaying hemagglutinating activity using rabbit erythrocytes. The albumin glands (165 g) from 180 Achatinufulicu snails were dissected, homogenized in a blender, and extracted with 200 ml 0.01 M phosphate-buffered saline (PBS), pH 7.2, at 10°C. The extract was centrifuged at 12,OOOgfor 20 min at lO”C, and the precipitate was reextracted with PBS. The combined supernatants (360 ml) were centrifuged at 150,OOOgfor 2.5 h. The clear yellowish-green layer (150 ml), containing ‘70 mg/ml protein, was tested for agglutinin activity. An aliquot (10 ml) (sp act, 0.114 unit/mg) of the crude extract was directly applied onto a lo-ml asialofetuinSepharose 4B column (1.5 X 6 cm) at 10°C. Elution of specific agglutinin was accomplished by raising the temperature to 37°C with PBS or by eluting with 0.5 M D-gahCtOSe in 1 M NaCl at 10°C. The eluted material was dialyzed against 0.01 M phosphate buffer (pH 7.7), and then passed through a DEAE-cellulose colwith the umn (1.5 X 30 cm) previously equilibrated same buffer. The agglutinin was eluted with 0.05 M NaCl. The 0.05 M NaCl eluate was pooled and dialysed against PBS, and concentrated by ultrafiltration. Disc gel electrophoresis was performed as described by Davis (10). An alkaline buffer, pH 8.3, and 7.5 and 5% gels were employed. Hemagglutination was performed with a Takatsy microtitrator (Cooke Engineering Co., Alexandria, Va.) using 25-nl loops and 25 ~1 of a 2% suspension of rabbit erythrocytes (2 ml packed erythrocytes diluted to 100 ml). Equal volumes of the red blood cell suspension and the agglutinin solution or dilutions were mixed, incubated for 1 h, and scored. Temperature sensitivity of this agglutinin was evaluated at different temperatures, ranging from 4 to 30°C. The effects of metal ions were checked at 286
COLD
AGGLUTININ
FROM TABLE
PURIFICATION
Fractions Crude albumin gland extract AsialofetuinSepharose 4B affinity column eluate DEAE column eluate
OF AGGLUTININ
Total volume (ml)
150
22.5 9.4
Achatina
fulica
I
FROM THE ALBUMIN
GLANDS
OF Achatina
Total activity (units)
Specific activity (units/mg)
105,000
1200
0.114
10.1
720
Total protein (mg)
1.12
602
10°C by addition, in each case, of 25 mM CaCl,, MgCla. and MnClz after dialyzing the agglutinin against saline. The reciprocal of the highest dilution of the agglutinin that produced visible hemagglutination was taken as the titer. The specific activity was defined as the total hemagglutination titers or units per milligram protein. The effect of pH on hemagglutination was checked at 10°C by testing the purified agglutinin in 0.01 M citrate buffer (pH 5.0), 0.01 M phosphate buffer (pH 6.2), 0.01 M PBS (pH ‘7.2), or 0.01 M Tris-HCl buffer (pH 8.4). All buffers were made up in saline. Hemagglutination was also performed using a 2% suspension of adult human erythrocytes (irrespective of ABO blood group) of six mothers and their umbilical cord erythrocytes at 10°C as well as at 30°C. For the inhibition of hemagglutination at 10°C appropriate sugar solutions were serially diluted in saline. Equal volumes of a 2% suspension of rabbit erythrocytes and a solution containing 10 hemagglutinating units of agglutinin were added to each well. Total volume of each well was 0.1 ml.
287
SNAILS
fulica
SNAILS
Purification (fold)
Recovery (%I
100
1
60
625
50
4711
71.3 537
eluate is shown in Fig. 1. The purified agglutinin showed a single band on 7.5% as well as 5% polyacrylamide gel at pH 8.3 (Fig. 2). The hemagglutinating activity was found to be temperature dependent (Fig. 3). Agglutination was maximal near 13°C. Activity was not appreciably affected by the presence of metal ions, e.g., Ca’+, Mn2+, and Mga+. The agglutinin was stable in the pH range 6.2-7.2, and became labile at pH 5.0 and 8.4. With six samples of human umbilical cord erythrocytes, the agglutinin showed a high titer, approximately 64, at
RESULTS
The purification of the agglutinin was carried out in two steps, including affinity chromatography followed by ion-exchange chromatography. The specific activity of the purified agglutinin was increased approximately 5000-fold compared to the crude extract (Table I), with a recovery of 50% and a yield 1.12 mg of purified agglutinin from 165 g of snail albumin gland. The agglutinin was bound to the asialofetuin affinity column only in the cold (lO”C), and not at 30°C. Elution at 37°C with PBS, or with 0.5 M D-galactose in 1 M NaCl at 10°C yielded the same purified agglutinin. The elution profile of the PBS
4 256
TEST
TUBE
NO
FIG. 1. Elution profile of the affinity column. Elution at 37°C with PBS monitored for absorbance at 280 nm, l ; the reciprocal of hemagglutination titer of each fraction against rabbit erythrocytes, 0, the elution profile of the same affinity column at 37°C with PBS when the extract was incubated at 30 instead of 10% A.
288
SARKAR,
BACHHAWAT,
AND
MANDAL
The results of inhibition experiments with various sugars are presented in Table II. Among the monosaccharides, D-galactose was a strong inhibitor, while D-mannose, D-glucose, and N-acetylneuraminic acid were inactive. 2-Acetamido-2-deoxyD-galactose was about as potent as D-g% lactose, but 2-acetamido-2-deoxy-D-glucose was inactive. Methyl-P-D-galactoside was much more active than a-D-galactoside. Similarly, lactose was at least 12 times more active than melibiose. However, the most active oligosaccharide was N-acetyllactosamine, which was 4 times more active than methyl-P-D-galactoside and 110 times more active than lactose. DISCUSSION
FIG. 2. Polyacrylamide gel electrophoresis purified agglutinin at pH 8.3 (7.5% gel).
of the
lO”C, whereas with the maternal erythrocytes, three of the six samples showed low titers of about 4 while the others were negative. All the hemagglutination tests carried out with umbilical cord erythrocytes and maternal erythrocytes at 30°C were negative.
E > IO3 c 2 s LL hi +!!L % IO20
Early reports have suggested that invertebrate agglutinins, mainly from Gastropoda (Mollusca), agglutinate human erythrocytes of specific blood group types (11). The first report of the agglutinin was found to be an anti-A agglutinin isolated from the albumin gland of the snail Helix pomatia (12). The presence of agglutinins in A. fulica and A. granulata has been reported, but these have not been studied extensively (13-15). The present study on the agglutinin from the albumin gland of TABLE
INHIBITION BY VARIOUS SUGARS OF THE HEMAGGLUTINATING ACTIVITY OF PURIFIED AGGLUTININ USING 2% RABBIT ERYTHROCYTES AT 10°C
Sugars
IO TEMPERATURE
20
30 1°C)
FIG. 3. Effect of temperature on the hemagglutination. Incubations were carried out at the indicated temperatures for a period of 1 h. Conditions for the experiment were described under Materials and Methods. Specific activity (units/mg protein) was plotted against temperature.
II
D-Galactose D-Mannose D-Glucose N-acetylneuraminic acid 2-Acetamido-Z-deoxy-Dgalactose 2-Acetamido-Z-deoxy-Dglucose Methyl-a-D-galactoside Methyl-@-D-galaetoside Melibiose (~Gakl + 6~Gk) Lactose (~Gal@l -+ 4DGlc) N-acetyllactosamine (~Gal@l - 4~GlcNAc)
Minimum concentration required for complete inhibition of agglutination (in 0.1 ml) (pmol) 3.5 >222 >222 >60 5 >&I >103 0.4 >160 13.3 0.121
COLD
AGGLUTININ
FROM
A. fulica shows that it is clearly different from agglutinins from plants or from animals because it agglutinates at low temperature. A report
x4chatin.a fulica
289
SNAILS
Department of Microbiology, New York, for his generous amine.
Columbia University, gift of N-acetyllactos-
REFERENCES 1. ROELCKE, D. (1974) Clin. ImmunoL Immuwpathol 2,266-280. 2. FEIZI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARSH, W. L. (1971) J. ImmuneL 106,15781592. 3. WALDENSTR~~M, J. G. (1968) in Monoclonal and Polyclonal Hypergammaglobulinemia, Vanderbilt Univ. Press, Nashville. 4. TSAI, C. M., ZOPF, D. A., Yu, R. K., WISTER, R., JR., AND GINSBURG, V. (1977) Proc. Natl. Acad. Sci. USA 74, 4591-4594. 10, 127-156. 5. Feizi, T. (1981) Immunol Cmtmun 6. DUBE, V., IWAKI, Y., ANDERSON, B., AND TERASAKI, P. (1982) Voz Sang. 43, 113-121. LOWRY, 0. H., ROSEBROUGH, J. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. LUNNEY, J., AND ASHWELL, G. (1976) Proc. Natl. Acad Sci. USA 73, 341-343. 9. CUATRECASAS, P., AND ANFINSEN, C. B. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 345-378, Academic Press, New York. 10. DAVIS, B. J. (1964) Ann. N. lT Acad Sci. 121,404427. 11. PROKOP, 0.. UHLENBRUCK, G., AND KOHLER, W. (1968) Vex. Sang. 14, 321-333. 12. HAMMARSTR~M, S., AND KABAT, E. A. (1971) Bicchemistry 10, 1684-1692. 13. GOLD, E. R., CANN, G. B., AND THOMPSON, T. E. (1967) VOX. Sung. 12, 461-464. 14. BRAIN, P., AND GRACE, H. J. (1968) Voz. Sang. 15, 297-299. 15. KHALAP, S., THOMPSON, J. E., AND GOLD, E. R. (1971) Voz. Sang. 20, 150-173. 16. OKOT~RE, R. O., KLEIN, P. J., ORTMANN, M., AND UHLENBRUCK, G. (1981) Camp Biochem PhysioL B 70, 469-475. 17. DE WAARD, A., HICKMAN, S., AND KORNFELD, S. (1976) J. Biol. Chem. 25, 7581-7587. 18. CHILDS, R. A., AND FEIZI, T. (1979) FEBS Lett. 99, 175-179. 19 IGLESIAS, J. L., LIS, H., AND SHARON, N. (1982) Eur. J. B&hem 123,247-252. 20. KEMLER, R., BANINET, C., EISEN, H., AND JACOB, F. (1977) Proc. NatL Acad Sci. USA 74,44494452. 21. NIEMANN, H., WATANABE, K., HAKOMORI, S., CHILDS, R., AND FEIZI, T. (1978) B&hem. Biophys. Res. Commun 81, 1286-1293. 22. WATANABE, K., HAKOMORI, S., CHILDS, R. A., AND FEIZI, T. (1979) J. BioL Chem. 254, 3221-3228.
OF BIOCHEMISTRY
Vol. 233, No. 1, August
AND BIOPHYSICS
15, pp. 286-289, 1984
A New Cold Agglutinin MANJU
SARKAR,’ Indian
BIMAL
KUMAR
Institute
from Achatina fulica Snails BACHHAWAT,
of Chemical Biology,
Received December
Calcutta
AND
CHITRA
MANDAL
700 032, India
28, 1983, and in revised form April
5, 1984
An electrophoretically homogeneous agglutinin was purified from the albumin gland of Achatina fulica snails using asialofetunin-Sepharose 4B as an affinity column. The agglutinin was found to be temperature sensitive; it agglutinated rabbit and human umbilical cord erythrocytes only at low temperature. It was found to be specific for methyl-P-D-galactoside, and the best inhibitor was N-acetyllactosamine.
Cold agglutinins are a special class of monoclonal macroglobulins that show specificity towards blood group I, i, and Pr antigens (1, 2). These agglutinins agglutinate human erythrocytes at temperatures below 37°C (3). So far cold agglutinins have been found in human patients with Waldenstrom macroglobulinemia, autoimmunohemolytic anemia, and breast carcinoma (4-6). These cold agglutinins are autoantibodies mainly of the IgM2 type (4). In the present communication we report the isolation and characterization of a cold agglutinin from an invertebrate, Achatina fulica snail. To the best of our knowledge, this is the first report of a cold agglutinin in an animal other than the human. MATERIALS
AND
METHODS
Achutinafulicu snails were purchased from the local market. All the monosaccharides and the disaccharides were purchased from Sigma Chemical Company. The disaccharide N-acetyllactosamine was obtained from Dr. E. A. Kabat (Columbia University, N. Y.). Protein was estimated by the method of Lowry et aL (7) using crystalline bovine serum albumin as the standard. Asialofetuin was prepared from fetuin (Sigma) (8). The affinity column was prepared by coupling asialofetuin with CNBr-activated Sepharose 4B
r To whom correspondence should be addressed, at 4 Raja S. C. Mallick Road, Calcutta 700 032, India. * Abbreviations used: IgM, immunoglobulin M, PBS, phosphate-buffered saline. 0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
according to Cuatrecasas (9). All the eluates from the columns were checked by absorption at 280 nm, and also by assaying hemagglutinating activity using rabbit erythrocytes. The albumin glands (165 g) from 180 Achatinufulicu snails were dissected, homogenized in a blender, and extracted with 200 ml 0.01 M phosphate-buffered saline (PBS), pH 7.2, at 10°C. The extract was centrifuged at 12,OOOgfor 20 min at lO”C, and the precipitate was reextracted with PBS. The combined supernatants (360 ml) were centrifuged at 150,OOOgfor 2.5 h. The clear yellowish-green layer (150 ml), containing ‘70 mg/ml protein, was tested for agglutinin activity. An aliquot (10 ml) (sp act, 0.114 unit/mg) of the crude extract was directly applied onto a lo-ml asialofetuinSepharose 4B column (1.5 X 6 cm) at 10°C. Elution of specific agglutinin was accomplished by raising the temperature to 37°C with PBS or by eluting with 0.5 M D-gahCtOSe in 1 M NaCl at 10°C. The eluted material was dialyzed against 0.01 M phosphate buffer (pH 7.7), and then passed through a DEAE-cellulose colwith the umn (1.5 X 30 cm) previously equilibrated same buffer. The agglutinin was eluted with 0.05 M NaCl. The 0.05 M NaCl eluate was pooled and dialysed against PBS, and concentrated by ultrafiltration. Disc gel electrophoresis was performed as described by Davis (10). An alkaline buffer, pH 8.3, and 7.5 and 5% gels were employed. Hemagglutination was performed with a Takatsy microtitrator (Cooke Engineering Co., Alexandria, Va.) using 25-nl loops and 25 ~1 of a 2% suspension of rabbit erythrocytes (2 ml packed erythrocytes diluted to 100 ml). Equal volumes of the red blood cell suspension and the agglutinin solution or dilutions were mixed, incubated for 1 h, and scored. Temperature sensitivity of this agglutinin was evaluated at different temperatures, ranging from 4 to 30°C. The effects of metal ions were checked at 286
COLD
AGGLUTININ
FROM TABLE
PURIFICATION
Fractions Crude albumin gland extract AsialofetuinSepharose 4B affinity column eluate DEAE column eluate
OF AGGLUTININ
Total volume (ml)
150
22.5 9.4
Achatina
fulica
I
FROM THE ALBUMIN
GLANDS
OF Achatina
Total activity (units)
Specific activity (units/mg)
105,000
1200
0.114
10.1
720
Total protein (mg)
1.12
602
10°C by addition, in each case, of 25 mM CaCl,, MgCla. and MnClz after dialyzing the agglutinin against saline. The reciprocal of the highest dilution of the agglutinin that produced visible hemagglutination was taken as the titer. The specific activity was defined as the total hemagglutination titers or units per milligram protein. The effect of pH on hemagglutination was checked at 10°C by testing the purified agglutinin in 0.01 M citrate buffer (pH 5.0), 0.01 M phosphate buffer (pH 6.2), 0.01 M PBS (pH ‘7.2), or 0.01 M Tris-HCl buffer (pH 8.4). All buffers were made up in saline. Hemagglutination was also performed using a 2% suspension of adult human erythrocytes (irrespective of ABO blood group) of six mothers and their umbilical cord erythrocytes at 10°C as well as at 30°C. For the inhibition of hemagglutination at 10°C appropriate sugar solutions were serially diluted in saline. Equal volumes of a 2% suspension of rabbit erythrocytes and a solution containing 10 hemagglutinating units of agglutinin were added to each well. Total volume of each well was 0.1 ml.
287
SNAILS
fulica
SNAILS
Purification (fold)
Recovery (%I
100
1
60
625
50
4711
71.3 537
eluate is shown in Fig. 1. The purified agglutinin showed a single band on 7.5% as well as 5% polyacrylamide gel at pH 8.3 (Fig. 2). The hemagglutinating activity was found to be temperature dependent (Fig. 3). Agglutination was maximal near 13°C. Activity was not appreciably affected by the presence of metal ions, e.g., Ca’+, Mn2+, and Mga+. The agglutinin was stable in the pH range 6.2-7.2, and became labile at pH 5.0 and 8.4. With six samples of human umbilical cord erythrocytes, the agglutinin showed a high titer, approximately 64, at
RESULTS
The purification of the agglutinin was carried out in two steps, including affinity chromatography followed by ion-exchange chromatography. The specific activity of the purified agglutinin was increased approximately 5000-fold compared to the crude extract (Table I), with a recovery of 50% and a yield 1.12 mg of purified agglutinin from 165 g of snail albumin gland. The agglutinin was bound to the asialofetuin affinity column only in the cold (lO”C), and not at 30°C. Elution at 37°C with PBS, or with 0.5 M D-galactose in 1 M NaCl at 10°C yielded the same purified agglutinin. The elution profile of the PBS
4 256
TEST
TUBE
NO
FIG. 1. Elution profile of the affinity column. Elution at 37°C with PBS monitored for absorbance at 280 nm, l ; the reciprocal of hemagglutination titer of each fraction against rabbit erythrocytes, 0, the elution profile of the same affinity column at 37°C with PBS when the extract was incubated at 30 instead of 10% A.
288
SARKAR,
BACHHAWAT,
AND
MANDAL
The results of inhibition experiments with various sugars are presented in Table II. Among the monosaccharides, D-galactose was a strong inhibitor, while D-mannose, D-glucose, and N-acetylneuraminic acid were inactive. 2-Acetamido-2-deoxyD-galactose was about as potent as D-g% lactose, but 2-acetamido-2-deoxy-D-glucose was inactive. Methyl-P-D-galactoside was much more active than a-D-galactoside. Similarly, lactose was at least 12 times more active than melibiose. However, the most active oligosaccharide was N-acetyllactosamine, which was 4 times more active than methyl-P-D-galactoside and 110 times more active than lactose. DISCUSSION
FIG. 2. Polyacrylamide gel electrophoresis purified agglutinin at pH 8.3 (7.5% gel).
of the
lO”C, whereas with the maternal erythrocytes, three of the six samples showed low titers of about 4 while the others were negative. All the hemagglutination tests carried out with umbilical cord erythrocytes and maternal erythrocytes at 30°C were negative.
E > IO3 c 2 s LL hi +!!L % IO20
Early reports have suggested that invertebrate agglutinins, mainly from Gastropoda (Mollusca), agglutinate human erythrocytes of specific blood group types (11). The first report of the agglutinin was found to be an anti-A agglutinin isolated from the albumin gland of the snail Helix pomatia (12). The presence of agglutinins in A. fulica and A. granulata has been reported, but these have not been studied extensively (13-15). The present study on the agglutinin from the albumin gland of TABLE
INHIBITION BY VARIOUS SUGARS OF THE HEMAGGLUTINATING ACTIVITY OF PURIFIED AGGLUTININ USING 2% RABBIT ERYTHROCYTES AT 10°C
Sugars
IO TEMPERATURE
20
30 1°C)
FIG. 3. Effect of temperature on the hemagglutination. Incubations were carried out at the indicated temperatures for a period of 1 h. Conditions for the experiment were described under Materials and Methods. Specific activity (units/mg protein) was plotted against temperature.
II
D-Galactose D-Mannose D-Glucose N-acetylneuraminic acid 2-Acetamido-Z-deoxy-Dgalactose 2-Acetamido-Z-deoxy-Dglucose Methyl-a-D-galactoside Methyl-@-D-galaetoside Melibiose (~Gakl + 6~Gk) Lactose (~Gal@l -+ 4DGlc) N-acetyllactosamine (~Gal@l - 4~GlcNAc)
Minimum concentration required for complete inhibition of agglutination (in 0.1 ml) (pmol) 3.5 >222 >222 >60 5 >&I >103 0.4 >160 13.3 0.121
COLD
AGGLUTININ
FROM
A. fulica shows that it is clearly different from agglutinins from plants or from animals because it agglutinates at low temperature. A report
x4chatin.a fulica
289
SNAILS
Department of Microbiology, New York, for his generous amine.
Columbia University, gift of N-acetyllactos-
REFERENCES 1. ROELCKE, D. (1974) Clin. ImmunoL Immuwpathol 2,266-280. 2. FEIZI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARSH, W. L. (1971) J. ImmuneL 106,15781592. 3. WALDENSTR~~M, J. G. (1968) in Monoclonal and Polyclonal Hypergammaglobulinemia, Vanderbilt Univ. Press, Nashville. 4. TSAI, C. M., ZOPF, D. A., Yu, R. K., WISTER, R., JR., AND GINSBURG, V. (1977) Proc. Natl. Acad. Sci. USA 74, 4591-4594. 10, 127-156. 5. Feizi, T. (1981) Immunol Cmtmun 6. DUBE, V., IWAKI, Y., ANDERSON, B., AND TERASAKI, P. (1982) Voz Sang. 43, 113-121. LOWRY, 0. H., ROSEBROUGH, J. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 8. LUNNEY, J., AND ASHWELL, G. (1976) Proc. Natl. Acad Sci. USA 73, 341-343. 9. CUATRECASAS, P., AND ANFINSEN, C. B. (1971) in Methods in Enzymology (Jakoby, W. B., ed.), Vol. 22, pp. 345-378, Academic Press, New York. 10. DAVIS, B. J. (1964) Ann. N. lT Acad Sci. 121,404427. 11. PROKOP, 0.. UHLENBRUCK, G., AND KOHLER, W. (1968) Vex. Sang. 14, 321-333. 12. HAMMARSTR~M, S., AND KABAT, E. A. (1971) Bicchemistry 10, 1684-1692. 13. GOLD, E. R., CANN, G. B., AND THOMPSON, T. E. (1967) VOX. Sung. 12, 461-464. 14. BRAIN, P., AND GRACE, H. J. (1968) Voz. Sang. 15, 297-299. 15. KHALAP, S., THOMPSON, J. E., AND GOLD, E. R. (1971) Voz. Sang. 20, 150-173. 16. OKOT~RE, R. O., KLEIN, P. J., ORTMANN, M., AND UHLENBRUCK, G. (1981) Camp Biochem PhysioL B 70, 469-475. 17. DE WAARD, A., HICKMAN, S., AND KORNFELD, S. (1976) J. Biol. Chem. 25, 7581-7587. 18. CHILDS, R. A., AND FEIZI, T. (1979) FEBS Lett. 99, 175-179. 19 IGLESIAS, J. L., LIS, H., AND SHARON, N. (1982) Eur. J. B&hem 123,247-252. 20. KEMLER, R., BANINET, C., EISEN, H., AND JACOB, F. (1977) Proc. NatL Acad Sci. USA 74,44494452. 21. NIEMANN, H., WATANABE, K., HAKOMORI, S., CHILDS, R., AND FEIZI, T. (1978) B&hem. Biophys. Res. Commun 81, 1286-1293. 22. WATANABE, K., HAKOMORI, S., CHILDS, R. A., AND FEIZI, T. (1979) J. BioL Chem. 254, 3221-3228.