Purification and characterization of galactocerebrosidase from human lymphocytes.
Galactocerebrosidase was purified about 22,600-fold using several hydrophobic column and gel filtration steps with a 4.8% recovery, from human lymphocytes. Its specific activity was 1.54 x 10(5) nmol/h/mg with tritium-labeled galactocerebroside as the substrate in the taurocholate system. The optimal pH for galactocerebroside was 4.2 in the taurocholate system and 4.6 in the cholate system. The Km values for galactocerebroside were 5 microM in the taurocholate system and 25 microM in the cholate system. The molecular weight of the purified enzyme was estimated to be 90 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and gel filtration. However, 70, 50, 40, and 30 kDa bands were also recognized on SDS-PAGE. The N-terminal amino acid sequences of the 70 kDa molecule and the three 50 kDa molecules were the same as that of the 90 kDa molecule. The N-terminal amino acid sequences of the 40 and 30 kDa molecules were unique. A monoclonal antibody raised against the purified enzyme effectively immunoprecipitated galactocerebrosidase activity, and an affinity column prepared with this monoclonal antibody bound the 90 and 50 kDa proteins. These results suggest that this enzyme is probably processed from the 90 kDa protein.
Sakai
N
N
Department of Pediatrics, Faculty of Medicine, Osaka University.
Inui
K
K
Midorikawa
M
M
Okuno
Y
Y
Ueda
S
S
Iwamatsu
A
A
Okada
S
S
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005698:Galactosylceramidase / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight
D000911:Antibodies, Monoclonal; D005698:Galactosylceramidase