Purification and characterization of beta-N-acetylhexosaminidase I2 from human liver.
beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.
N N
NN
Dewji
D R
DR
De-Keyzer
J L
JL
Stirling
D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D006596:Hexosaminidases / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D001619:beta-N-Acetylhexosaminidases
D000939:Epitopes; D007527:Isoenzymes; D006596:Hexosaminidases; D001619:beta-N-Acetylhexosaminidases