Characteristics of hexosaminidase A in homogenates of white blood cells using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as substrate.
Using methylumbelliferyl-N-acetyl-beta-D-glucosaminide-6-sulphate as a substrate hexosaminidase A activity was measured in leukocyte homogenates with a one-step method without the need of thermoinactivation. According to the buffer system used--acetate or citrate--the enzyme has pH optima of 3.5 and 4, respectively, and is linear during at least 1 h of incubation. The enzyme is resistant to mechanical inactivation by homogenization or to freezing and thawing and is thermostable between 0 and 37 degrees C but it is rapidly inactivated at 50 degrees C. This thermoinactivation is retarded by acidification of the homogenate. The latency of the enzyme is in agreement with its known lysosomal localization. Like most lysosomal enzymes, hexosaminidase A binds to Concanavalin A. After homogenization of leukocytes and repeated freezing and thawing, about 60% of the enzyme is present in the supernatant. Hexosaminidase A activity was stimulated by taurocholate between 0.05 and 0.1 M and inhibited by dimethylsulfoxide, dimethylformamide, N-acetylglucosamine and N-acetylgalactosamine. No pronounced difference was noted in specific activity of a homogenate of mononuclear cells, granulocytes or total leukocytes.
Den Tandt
W R
WR
University of Antwerp (UIA), Faculty of Medicine, Antwerp-Wilrijk, Belgium.
Scharpe
S
S
D001665:Binding Sites; D002021:Buffers; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D054818:Hexosaminidase A; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000097:blood* / Q000737:chemistry
D002021:Buffers; C068972:4-methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulfoglucosamine; D006923:Hymecromone; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases; D005944:Glucosamine