1987
DOI: 10.1042/bj2440801
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Intermediate forms of human β-N-acetylhexosaminidase lack activity towards 4-methylumbelliferyl β-N-acetylglucosaminide 6-sulphate

Abstract: 4-Methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate was purified from a mixture containing its unsulphated precursor. The substrate was used to test for the presence of functional alpha-subunits in 'intermediate' forms of human beta-N-acetylhexosaminidase in samples of normal and pregnancy serum and in extracts of placenta and lymphocytes from a patient with common acute lymphoblastic leukaemia. Intermediate forms in these samples had no activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6… Show more

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Cited by 50 publications
(16 citation statements)
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“…and other reagents from Merck.Leukocytes were prepared from heparinized blood(21) and frozen at -70°C until used. Total homogenates obtained after sonication in distilled water were used for enzyme analysis.Total protein was determined according toLowry et al(13) using bovine serum albumin (BSA) as standard.4-MU-GlcNAc-6-S was purified from the purchased preparation as described by Beccari et al(1). An aqueous solution of impure 4-MU-GlcNAc-6-S (28 g/l) was loaded onto a DEAE-cellulose column (10 ml) equilibrated with destilled water.…”
mentioning
confidence: 99%
“…and other reagents from Merck.Leukocytes were prepared from heparinized blood(21) and frozen at -70°C until used. Total homogenates obtained after sonication in distilled water were used for enzyme analysis.Total protein was determined according toLowry et al(13) using bovine serum albumin (BSA) as standard.4-MU-GlcNAc-6-S was purified from the purchased preparation as described by Beccari et al(1). An aqueous solution of impure 4-MU-GlcNAc-6-S (28 g/l) was loaded onto a DEAE-cellulose column (10 ml) equilibrated with destilled water.…”
mentioning
confidence: 99%
“…Eluates were assayed for MUG versus MUGS hydrolysis to assign the isoenzyme peaks. As shown in Figure 2a, b-hexosaminidase from normal brain extract resolved in three peaks of activity, eluting in positions corresponding to Hex B (bb structure, as revealed by its inability to hydrolyze the MUGS substrate), a Hex intermediate form which displays a Hex B-like structure 14 and Hex A (ab), respectively. In the brain extract from a Hexb À/À mouse (Figure 2b), only the Hex S isoenzyme (aa structure) was detected, eluting within the NaCl gradient just after the elution point of Hex A.…”
Section: Resultsmentioning
confidence: 99%
“…The assay with MUGS was performed as previously described. 30 Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin Elmer LS-3 fluorometer (PerkinElmer, Branchburg, NJ, USA), with 360 nm excitation and 446 nm emission. The fluorometer was calibrated with 4-methylumbelliferone solution in 0.2 mol/L glycine buffer (pH 10.6).…”
Section: Enzyme Activity Determinationmentioning
confidence: 99%