Causeret C, Bentejac M, Albet S, Teubner B, Bugaut M
Laboratoire de Biologie Moléculaire et Cellulaire, Faculté des Sciences Mirande, Université de Bourgogne, Dijon, France.
Biochimie. 1997 Jul;79(7):423-33. doi: 10.1016/s0300-9084(97)86152-4.
Dihydroxyacetone-phosphate acyl-transferase (DHAP-AT), a peroxisomal membrane-bound enzyme that catalyzes the first step of ether-glycerolipid synthesis, was purified from liver of rats treated with fenofibrate, a peroxisome proliferator. The protocol first included isolation of peroxisomes, their purification through a discontinuous gradient and solubilization of membranes in CHAPS. DHAP-AT was further purified by four chromatographic steps, namely low-pressure size-exclusion, cation-exchange, hydroxylapatite and chromatofocusing. The chromatofocusing step led to a 4000-fold increase in the specific activity of DHAP-AT with respect to the liver homogenate with a yield of about 0.2%. Trypsin digestion of a 64-kDa protein band upon SDS-PAGE resulted in a peptide sequence unknown in databases. A corresponding degenerated oligonucleotide was used as a probe in Northern blotting, and a transcript of 3.3 kb was detected in some rat tissues. Moreover, the overall procedure allowed co-purification of four major peroxisomal enzymes: urate-oxidase, catalase, multifunctional enzyme and palmitoyl-CoA oxidase, respectively.
磷酸二羟丙酮酰基转移酶（DHAP - AT）是一种过氧化物酶体膜结合酶，催化醚甘油脂质合成的第一步，它是从用氯贝丁酯（一种过氧化物酶体增殖剂）处理过的大鼠肝脏中纯化得到的。该方案首先包括过氧化物酶体的分离，通过不连续梯度进行纯化，以及在CHAPS中溶解膜。DHAP - AT通过四个色谱步骤进一步纯化，即低压尺寸排阻、阳离子交换、羟基磷灰石和色谱聚焦。色谱聚焦步骤使DHAP - AT相对于肝脏匀浆的比活性增加了4000倍，产率约为0.2%。SDS - PAGE上对一条64 kDa蛋白带进行胰蛋白酶消化后得到一个数据库中未知的肽序列。相应的简并寡核苷酸用作Northern印迹的探针，在一些大鼠组织中检测到一个3.3 kb的转录本。此外，整个过程还分别共纯化了四种主要的过氧化物酶体酶：尿酸氧化酶、过氧化氢酶、多功能酶和棕榈酰辅酶A氧化酶。
