从猪脾脏中纯化的GM1-β-半乳糖苷酶的pH依赖性缔合-解离

pH-dependent association-dissociation of GM1-beta-galactosidase purified from porcine spleen.

作者信息

Yamamoto Y, Nishimura K

出版信息

J Biochem. 1980 Sep;88(3):705-13. doi: 10.1093/oxfordjournals.jbchem.a133023.

DOI:10.1093/oxfordjournals.jbchem.a133023

Abstract

A beta-galactosidase [EC 3.1.23] catalyzing the hydrolysis of GM1-ganglioside was purified from porcine spleen to a homogeneous form. By applying a hydrophobic chromatography procedure as the first purification step, the enzyme could be purified through subsequent purification steps as a dissociated form. The purified enzyme was a monomer with an apparent molecular weight of 70,000-74,000 at neutral pH and associated to a dimer with an apparent molecular weight of 158,000-160,000 at acidic pH, near optimal for its activity (pH 4.6). It had specific activities of 1,820 mumol/mg/h towards GM1 with an apparent Km of 3.18 X 10(-5) M, 1,880 mumol/mg/h towards lactosylceramide with an apparent Km of 1.99 X 10(-4) M, and 1,340 mumol/mg/h towards p-nitrophenyl-beta-galactopyranoside (pNp-beta galactoside) with an apparent Km of 2,14 X 10(-4) M. Kinetic studies suggested that common catalytic site(s) cleaved the two natural substrates mentioned above.

摘要

一种催化GM1神经节苷脂水解的β-半乳糖苷酶[EC 3.1.23]从猪脾脏中纯化至均一形式。通过将疏水层析程序作为第一步纯化步骤，该酶可以以解离形式通过后续纯化步骤进行纯化。纯化后的酶在中性pH下为表观分子量70,000 - 74,000的单体，在酸性pH（接近其活性最佳pH 4.6）下缔合为表观分子量158,000 - 160,000的二聚体。它对GM1的比活性为1,820 μmol/mg/h，表观Km为3.18×10⁻⁵ M；对乳糖基神经酰胺的比活性为1,880 μmol/mg/h，表观Km为1.99×10⁻⁴ M；对对硝基苯基-β-吡喃半乳糖苷（pNp-β半乳糖苷）的比活性为1,340 μmol/mg/h，表观Km为2.14×10⁻⁴ M。动力学研究表明，共同的催化位点切割上述两种天然底物。