α-Mannosidase activity from antibody raised against a glucal antigen

Bioorganic and Medicinal Chemistry Letters

Volumn 8, Issue 10, 1998, Pages 1145-1148

  Yu, Jaehoon ^a  

^a Korea Institute of Science and Technology   (South Korea)

Author keywords

[No Author keywords available]

Indexed keywords

ALPHA MANNOSIDASE; ANTIGEN; HAPTEN; MONOCLONAL ANTIBODY;

ANIMAL CELL; ANTIBODY COMBINING SITE; ANTIGEN ANTIBODY REACTION; ARTICLE; CATALYSIS; CELL LINE; CHEMICAL MODIFICATION; ENZYME ACTIVITY; ENZYME MECHANISM; HYBRIDOMA; NONHUMAN; PH;

ALPHA-MANNOSIDASE; ANIMALS; ANTIBODIES, MONOCLONAL; BINDING SITES, ANTIBODY; CALCIUM GLUCONATE; CATALYSIS; CELL LINE; HAPTENS; HYDROGEN-ION CONCENTRATION; KINETICS; MANNOSIDASES; MOLECULAR STRUCTURE;

ANIMALIA; FELIS CATUS;

EID: 0032546501     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(98)00191-7     Document Type: Article

References (19)

1. 1. Reymond J.-L.; Janda, K. D.; Lerner, R. A. Angew. Chem. Int. Ed. Engl. 1991, 30, 1711.
2. 2. Suga, H.; Tanimoto, N.; Sinskey, A. J.; Masamune, S. J. Am. Chem. Soc. 1994, 116, 11197.
3. 3. Yu, J.; Hsieh, L.; Kochersperger, L.; Yonchovich, S.; Stephans, J. C.; Gallop, M. A.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 1994, 33, 339.
4. 4. Janda, K. D.; Lo, L.-C.; Lo, C. L.; Sim, M-M.; Wang, R.; Wong, C.-H.; Lerner, R. A. Science, 1997, 275, 945.
5. 5. Yu, J.; Lee, S.; Choi, S. Y.; Youn, H. J.; Jeong, S.; Park, H.; Schultz, P. G. J. Chem. Soc. Chem. Comm. 1997, in press.
6. 6. (a) Feizi, T.; Childs, R. A. Trends Biochem. Sci. 1985, 70, 24.
7. (b) Quiocho, F. A. Ann. Rev. Biochem. 1986, 55, 287.
8. (c) Varki, A. Glycobiology 1993, 3, 97.
9. 7. (a) Lee, Y. C. Biochem. Biophys. Res. Commun. 1969, 35, 161.
10. (b) Walsh, C. Enzymatic Reaction Mechanisms; W. H. Freeman and Co.: New York, 1979.
11. 8. Wentworth, R; Wolfenden, R. Biochemistry 1974, 13, 4715.
12. 9. Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Press: New York, 1988.
13. 10. The SP-Separose® (HiLoad™ 16/10) column (Pharmacia) was used for the stationary phase. For the mobile phase, buffer "A" (50 mM MES at pH 5.5) and buffer "B" (50 mM MES, 1.0 M NaCl at pH 5.5) were co-eluted. Antibodies were usually eluted at 30-35% of buffer "B".
14. 11. To 90 μL of antibody solution (2-3 mg/mL, 10 mM of N-(2-morpholino)ethansulfonic acid (MES), 100 mM of NaCl, 0.02% of sodium azide at 37°C at pH 4.5.), 10 μL of 20 mM (same buffer) substrate solution was added. The resulting mixture was incubated at 37°C for 3 days. The activity was measured by injecting 10 μL of this incubated mixture into HPLC in every 24 h, and monitoring at 315 nm. A C18 column (Microsorb; 4.6 mm x 15 cm, 5 μm) with a guard module (4.6 mm x 2.5 cm) was used as a stationary phase. Isocratic run of 50% methanol with 0.8 mL/min gave 5.7 min retention time for p-nitrophenol. The amount of p-nitrophenol in the injected solution was determined both by peak height and integration unit. The rate was determined when less than 1 % of the substrate was converted to the product.
15. obs). The reaction temperature was 37°C for all reactions. The buffer salts used were HCl/acetate, acetate, MES, N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS), N,N-bis-(2-hydroxyethyl)glycine (BICINE) for pH 2-3, 3-4.5, 4.5-7, 7-8, 8-9, respectively. Ionic strength was adjusted to be 100 mM NaCl.
16. 13. A solution of antibodies (1-2 mg/mL) in the assaying buffer was add to the dry protein-A agarose gel. The resulting solution was gently stirred for 30 min and the gel was filtered out. The resulting filtrate was incubated with the substrate at 37°C for 24 h, showing activity decrease, which is the same amount of antibody decrease in solution. The filtered agarose gel was re suspended in the substrate solution and incubated at 37°C for 24 h, and showed glycosidase activities.
17. 14. The whole antibody (IgG) was digested with papain as was suggested in Parham, P. J. Immunol. 1983, 131, 2895. The digested Fab soluton was ion-exchange chromatographed as was refered in 10. The Fab was eluted at 20% of buffer "B".
18. 4, 20 mM boric acid, pH 8.0) was added and stirred for 8 h at room temperature. The resulting solution was dialyzed in the assaying buffer (10 mM MES, 100 mM NaCl; pH 4.5) and concentrated for the assay. Grossberg, A.L. Pressman, D. J. Am. Chem. Soc. 1960, 52, 5478.
19. 16. Miyashita, H.; Hara, T.; Tanimura, R.; Tanaka, F.; Kikuchi, M.; Fujii, I. Proc. Nad. Acad. Sci. USA 1994, 91, 6045.