Pearce R D, Callahan J W, Novak A, Little P B, Clarke J T
Department of Paediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Br Vet J. 1990 May-Jun;146(3):270-80. doi: 10.1016/s0007-1935(11)80014-x.
Lysosomal beta-D-mannosidase (EC 3.2.1.25) was purified 6900-fold from normal goat kidney by serial Concanavalin A-Sepharose 4B and Red A dye ligand affinity chromatography, followed by anion exchange and molecular sieve high performance liquid chromatography. The relative molecular mass of the enzyme was estimated by molecular sieving to be 79,000 +/- 3000. The apparent Km for the synthetic substrate, 4-methylumbelliferyl-beta-D-mannopyranoside, was 2.3-2.8 mM and the sharp, unimodal pH optimum was 5.5. Enzyme activity was inhibited by Hg2+, Zn2+, Ag+, Co2+ and the thiol reactive agent N-ethylmaleimide. The mannose derivatives p-nitrophenyl-beta-D- thiomannopyranoside and p-aminophenyl-beta-D-thiomannopyranoside inhibited enzyme activity and may be of use as immobilized ligands in future attempts to purify beta-D-mannosidase by specific affinity chromatography.
通过刀豆球蛋白A-琼脂糖4B和红色A染料配体亲和层析，随后进行阴离子交换和分子筛高效液相色谱，从正常山羊肾脏中纯化出溶酶体β-D-甘露糖苷酶（EC 3.2.1.25），纯化倍数达6900倍。通过分子筛估计该酶的相对分子质量为79,000±3000。合成底物4-甲基伞形酮基-β-D-甘露吡喃糖苷的表观Km为2.3 - 2.8 mM，尖锐的单峰pH最适值为5.5。酶活性受到Hg2+、Zn2+、Ag+、Co2+和硫醇反应剂N-乙基马来酰亚胺的抑制。甘露糖衍生物对硝基苯基-β-D-硫代甘露吡喃糖苷和对氨基苯基-β-D-硫代甘露吡喃糖苷抑制酶活性，并且在未来通过特异性亲和层析纯化β-D-甘露糖苷酶的尝试中可能用作固定化配体。
