| Autor: |
Murata, Takeomi, Hattori, Takeshi, Amarume, Satoshi, Koichi, Akiko, Usui, Taichi |
| Předmět: |
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| Zdroj: |
European Journal of Biochemistry; Sep2003, Vol. 270 Issue 18, p3709, 13p |
| Abstrakt: |
Novel chromogenic substrates for endo-β-galactosidase were designed on the basis of the structural features of keratan sulfate. Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-p NP (2 ), which consists of two repeating units of N -acetyllactosamine, was synthesized enzymatically by consecutive additions of GlcNAc and Gal residues to p -nitrophenyl β-N -acetyllactosaminide. In a similar manner, GlcNAcβ1-3Galβ1-4GlcNAcβ-p NP (1 ), GlcNAcβ1-3Galβ1-4Glcβ-p NP (3 ), Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ-p NP (4 ), Galβ1-3GlcNAcβ1-3Galβ1-4Glcβ-p NP (5 ), and Galβ1-6GlcNAcβ1-3Galβ1-4Glcβ-p NP (6 ) were synthesized as analogues of 2 . Endo-β-galactosidases released GlcNAcβ-p NP or Glcβ-p NP in an endo-manner from each substrate. A colorimetric assay for endo-β-galactosidase was developed using the synthetic substrates on the basis of the determination of p -nitrophenol liberated from GlcNAcβ-p NP or Glcβ-p NP formed by the enzyme through a coupled reaction involving β-N -acetylhexosaminidase (β-NAHase) or β-d-glucosidase. Kinetic analysis by this method showed that the value of V [sub max] /K [sub m] of 2 for Escherichia freundii endo-β-galactosidase was 1.7-times higher than that for keratan sulfate, indicating that 2 is very suitable as a sensitive substrate for analytical use in an endo-β-galactosidase assay. Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position. In addition, the hydrolytic action of the enzyme toward 2 was shown to be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p -nitrophenyl group. This was the same in the case of a comparison of 1 and 3 . Furthermore, the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-p NP (9 ) and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-p NP (10 ) as the major products, which have N -acetyllactosamine repeating units. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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