从淡水贻贝（Lamellidens corrianus）中纯化两种己糖胺酶并进行生化/生物物理特性分析。

Purification and biochemical/biophysical characterization of two hexosaminidases from the fresh water mussel, Lamellidens corrianus.

机构信息

School of Chemistry, University of Hyderabad, Hyderabad 500046, India.

Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India.

出版信息

Int J Biol Macromol. 2020 Apr 15;149:754-766. doi: 10.1016/j.ijbiomac.2020.01.241. Epub 2020 Jan 24.

DOI:10.1016/j.ijbiomac.2020.01.241

PMID:31987953

Abstract

Two thermostable isoforms of a hexosaminidase were purified to homogeneity from the soluble extract of fresh water mussel Lamellidens corrianus, employing a variety of chromatographic techniques. Hexosaminidase A (HexA) is a heterodimer with subunit masses of ~80 and 55 kDa. Hexosaminidase B (HexB) is a homodimer with a subunit mass of 55-60 kDa. Circular dichroism spectroscopic studies indicated that both HexA and HexB contain β-sheet as the major secondary structural component with considerably lower content of α-helix. The temperature and pH optima of both the isoforms were found to be 60 °C and 4.0, respectively. The IC values for HexA with N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-galactosamine, d-glucosamine, methyl α-d-mannopyranoside and d-mannose are 3.7, 72.8, 307, 216, 244 and 128 mM, respectively, whereas the corresponding IC values for HexB were estimated as 5.1, 61, 68, 190, 92 and 133 mM, respectively. Kinetic parameters K and V for HexA and B with p-nitrophenyl N-acetyl-β-d-glucosaminide are 4 mM, 0.23 μmol·min·mL and 2.86 mM, 0.29 μmol·min·mL, respectively, and with p-nitrophenyl N-acetyl-β-d-galactosaminide are 4.5 mM, 0.054 μmol·min·mL and 1.4 mM, 0.14 μmol·min·mL, respectively. GalNAc inhibited both isoforms in a non-competitive manner, whereas a mixed mode of inhibition was observed with GlcNAc with both forms.

摘要

从淡水贻贝（Lamellidens corrianus）的可溶性提取物中，通过多种色谱技术，纯化得到两种热稳定的己糖胺酶同工酶，达到均一性。己糖胺酶 A（HexA）是一种异二聚体，亚基分子量约为 80 和 55 kDa。己糖胺酶 B（HexB）是一种同二聚体，亚基分子量为 55-60 kDa。圆二色性光谱研究表明，HexA 和 HexB 均含有β-折叠作为主要的二级结构成分，α-螺旋含量相当低。两种同工酶的最适温度和 pH 值分别为 60°C 和 4.0。HexA 与 N-乙酰-d-半乳糖胺、N-乙酰-d-葡萄糖胺、半乳糖胺、葡萄糖胺、甲基-α-d-甘露吡喃糖苷和 D-甘露糖的 IC 值分别为 3.7、72.8、307、216、244 和 128 mM，而 HexB 的相应 IC 值分别估计为 5.1、61、68、190、92 和 133 mM。HexA 和 B 与对硝基苯基 N-乙酰-β-d-葡糖胺的动力学参数 K 和 V 分别为 4 mM、0.23 μmol·min·mL 和 2.86 mM、0.29 μmol·min·mL，与对硝基苯基 N-乙酰-β-d-半乳糖胺的动力学参数 K 和 V 分别为 4.5 mM、0.054 μmol·min·mL 和 1.4 mM、0.14 μmol·min·mL。GalNAc 以非竞争性方式抑制两种同工酶，而 GlcNAc 对两种形式均表现出混合抑制模式。