Purification, stabilization, and characterization of rat hepatic triglyceride lipase.
Purification, stabilization, and characterization of rat hepatic triglyceride lipase.
复制标题
大鼠肝甘油三酯脂肪酶的纯化、稳定和表征。
DOI：
10.1016/0003-2697(81)90073-7
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发表时间：
1981
影响因子：
2.9
通讯作者：
Bensadoun,A
中科院分区：
文献类型：
--
作者：
Jensen,GL;Bensadoun,A
肝甘油三酯脂肪酶（H-TGL）从含肝素的大鼠肝脏灌注液中纯化至接近均匀性，柱层析步骤：肝素- sepharose亲和层析，DEAE-Sephacel阴离子交换层析，Ultrogel AcA 34凝胶过滤。最终比活性为45,000 μmol脂肪酸/mg/h，总催化活性回收率为31%。肝素- sepharose步骤纯化了20倍，而DEAE和凝胶过滤步骤进一步纯化，完全恢复了活性。对各种洗涤剂作为H-TGL活性的潜在稳定剂的广泛调查导致选择Triton N-101用于DEAE和凝胶过滤步骤的柱缓冲液。相对于在不含洗涤剂的缓冲液中稀释后的初始H-TGL活性，含Triton n -101的缓冲液在20°C下孵育24小时后，回收率始终在90%至100%之间。相比之下，在20°C下孵育24小时后，缺乏洗涤剂的对照样品至少失活95%。高纯度的H-TGL经十二烷基硫酸钠电泳显示为单主带。使用DEAE层析和Triton N-101稳定H-TGL是纯化的改进，导致比先前报道的最高纯化大鼠肝灌注液的比活性提高了8倍。
Hepatic triglyceride lipase (H-TGL) was purified to near homogeneity from heparin-containing rat liver perfusates with the following column chromatography steps: heparin-Sepharose affinity chromatography, anion-exchange chromatography on DEAE-Sephacel, and gel filtration on Ultrogel AcA 34. A final specific activity of 45,000 μmol fatty acid/mg/h was obtained with an overall 31% recovery of catalytic activity. The heparin-Sepharose step resulted in a 20-fold purification, while the DEAE and gel filtration steps led to further purification with complete recovery of activity. An extensive survey of various detergents as potential stabilizers of H-TGL activity led to the selection of Triton N-101 for use in the column buffers of the DEAE and gel filtration steps. Relative to initial H-TGL activity upon dilution in buffer without detergent, recoveries between 90 and 100% were consistently obtained with Triton N-101-containing buffers following a 24-h incubation at 20°C. In contrast after a 24-h incubation at 20°C those control samples lacking detergent were at least 95% inactivated. The highly purified H-TGL exhibited a single major band by sodium dodecyl sulfate-electrophoresis. The use of DEAE chromatography and stabilization of H-TGL with Triton N-101 are the improvements in purification that resulted in an 8-fold enhancement in specific activity relative to the highest previous report of purification from rat liver perfusates.