TY - JOUR
T1 - Human serum α-l-fucosidase
AU - Alhadeff, Jack A.
AU - Janowsky, Aaron J.
N1 - Funding Information:
This work was supported in part by the National Foundation March of Dimes grant No. l-395 and USPHS Grant lRO1 AM 20409 (to J.A. Alhadeff). The authors gratefully acknowledge Dr. John S. O’Brien for providing laboratory space and equipment for carrying out much of this work.
PY - 1978/1/2
Y1 - 1978/1/2
N2 - Human serum α-l-fucosidase has been purified 241 200-fold with 35% yield by an affinity Chromatographie procedure utilizing agarose-ε{lunate}-aminocaproyl-fucosamine. Isoelectric focusing of the purified enzyme indicated the presence of several forms, with the form at pI 5.0 comprising the majority of the activity. Assay of the purified α-l-fucosidase showed only trace amounts of contaminating glycosidases present, with β-galactosidase being the largest contamnant (0.5% by activity). Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the presence of two subunits with very similar molecular weights (56500 and 54000). Using the p-nitrophenyl substrate, the purified serum α-l-fucosidase had an apparent Michaelis constant of 0.52 mM and a broad pH optimum centered around pH 4.8 with a second, minor optimum at pH 6.1. Gel filtration on Sepharose 6-B indicated an apparent molecular weight of 296 000 ± 30 000. Preincubation with antibodies made previously against purified liver α-l-fucosidase led to quantitative immuno precipitation of the purified serum α-l-fucosidase. Assay of the purified serum α-l-fucosidase for sialic acid indicated the presence of 1.7 μg sialic acid per 100 μg enzyme, about twice that previously found for the purified liver enzyme.
AB - Human serum α-l-fucosidase has been purified 241 200-fold with 35% yield by an affinity Chromatographie procedure utilizing agarose-ε{lunate}-aminocaproyl-fucosamine. Isoelectric focusing of the purified enzyme indicated the presence of several forms, with the form at pI 5.0 comprising the majority of the activity. Assay of the purified α-l-fucosidase showed only trace amounts of contaminating glycosidases present, with β-galactosidase being the largest contamnant (0.5% by activity). Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated the presence of two subunits with very similar molecular weights (56500 and 54000). Using the p-nitrophenyl substrate, the purified serum α-l-fucosidase had an apparent Michaelis constant of 0.52 mM and a broad pH optimum centered around pH 4.8 with a second, minor optimum at pH 6.1. Gel filtration on Sepharose 6-B indicated an apparent molecular weight of 296 000 ± 30 000. Preincubation with antibodies made previously against purified liver α-l-fucosidase led to quantitative immuno precipitation of the purified serum α-l-fucosidase. Assay of the purified serum α-l-fucosidase for sialic acid indicated the presence of 1.7 μg sialic acid per 100 μg enzyme, about twice that previously found for the purified liver enzyme.
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U2 - 10.1016/0009-8981(78)90036-0
DO - 10.1016/0009-8981(78)90036-0
M3 - Article
C2 - 618676
AN - SCOPUS:0017918327
SN - 0009-8981
VL - 82
SP - 133
EP - 140
JO - Clinica Chimica Acta
JF - Clinica Chimica Acta
IS - 1-2
ER -