Isolation and Properties of $\beta$-N-Acetyl-D-glucosaminidase B from Rat Uterus

  • Jung, Jin-Ha (Department of Chemistry, College of Natural Sciences, Seoul National University) ;
  • Yang, Chul-Hak (Department of Chemistry, College of Natural Sciences, Seoul National University)
  • Published : 1983.06.20

Abstract

${\beta}$-N-Acetyl-D-glucosaminidase B was highly purified with the following sequence of steps; DEAE-cellulose, CM-cellulose, and Sephadex G-200 gel filtration chromatograpies. The specific activity of the purified ${\beta}$ -N-acetyl-D-glucosaminidase B was 2.2 units/mg protein with 12.9 % yield and 196.2 fold purity. The purified ${\beta}$-N-acetyl-D-glucosaminidase B showed single band on polyacrylamide gel electrophoresis. The final preparation of ${\beta}$ -N-acetyl-D-glucosaminidase B was completely free friom arylsulfatase and ${\beta}$-glucuronidase. ${\beta}$ -N-Acetyl-D-glucosaminidase B had pH optimum of 4.5 in 0.5 M sodium citrate buffer. The molecular weight of ${\beta}$-N-acetyl-D-glucosaminidase B was 133,000 by Sephadex G-200 gel filtration. The Km value of ${\beta}$-N-acetyl-D-glucosaminidase B using p-nitrophenyl-N-acetyl-${\beta}$-D-glucosaminide as substrate was 1.0 mM and $V_{max}$ was 0.014 ${\mu}$ mole/min. ${\beta}$-N-Acetyl-D-glucosaminidase B was stable at $55^{circ}C$ for 70 minutes. The crude ${\beta}$ -N-acetyl-D-glucosamiinidase in 70 % ammonium sulfate retained 93 % activity after 7 months storage at -$55^{circ}C$. Bovine serum albumin, sodium chloride, and phosphate activated ${\beta}$ -N-Acetyl-D-glucosaminidase B. N-Acetyl-D-glucosamine, ${\alpha}$-methyl-D-mannoside, and acetate inhibited ${\beta}$ -N-acetyl-D-glucosaminidase B.

Keywords

References

  1. J. Biochem. v.67 R. Heyworth;J. Borooah;D. H. Leaback
  2. Fed. Eur Biochem. Soc. Lett. v.4 K. Sandhoff
  3. Biol. Reprod. v.2 T. O. Abney;W. L. Williams
  4. Proc. Roy. Soc., B v.149 C. R. Austin;M. W. H. Bishop
  5. J. Reprod. Fert. v.14 W. R. Dukelow;H. N. Chernoff;W. L. Williams
  6. Methods Enzymol. v.XXVIII A. S. Tarentino;F. Maley
  7. EFBS Lett. v.29 R. G. Harris;J. J. M. Rowe;P. S. Stewart;D. C. Williams
  8. J. Biochem. v.159 C. H. Yang;P. N. Srivastava
  9. J. Biol. Chem. v.193 O. H. Lowry;N. J. Rosebrough;A. L. Farr;R. J. Randall
  10. J. Biochem. v.96 P. Andrews
  11. J. Chem. Education v.46 J. M. Brewer;R. B. Ashworth
  12. J. Biochem. v.69 J. Findlay;G. A. Levvy;C. A. Marsh
  13. J. Biochem. v.77 J. Findlay;G. A. Levvy
  14. Hoppe-Seyler's Z. Physiol. Chem. v.352 K. Sandhoff;W. Wassle
  15. Chem. Phys. Lipids v.13 J. F. Tallman
  16. J. Biochem. v.65 D. Pugh;D. H. Leaback;P. G. Walker
  17. Biochemistry v.13 J. A. Verpoorte
  18. Biochemistry v.15 B. Geiger;R. Arnon
  19. Int. Congr. Anim. Reprod. 5th v.2 L. Pico;A. Tyler
  20. Science v.177 G. L. Nicolson;R. Yanagimachi
  21. Biochim. Biophys. Acta. v.483 A. Kahr;S. R. Anand
  22. Int. J. Biochem. v.10 A. A. Farooqui;P. N. Srivastava
  23. J. Biochem. v.71 J. Conchie;J. Findlay;G. A. Levvy
  24. J. Biochem. v.107 D. Robinson;J. L. Stirling
  25. Science v.165 S. Okada;J.S.O' Brien
  26. Fed. Eur. Biochem. Soc. Lett. v.13 A. Goldstone;P. Konecny;H. Koenig
  27. J. Biochem. v.131 M. Carrol;D. Robinson