Datta S C, Ghosh M K, Hajra A K
Department of Biological Chemistry, University of Michigan, Ann Arbor 48104-1687.
J Biol Chem. 1990 May 15;265(14):8268-74.
The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.
过氧化物酶体酰基/烷基二羟基丙酮磷酸还原酶（EC 1.1.1.101）从豚鼠肝脏中溶解并纯化了5500倍。该酶仅在高离子强度且存在共底物NADPH的情况下才能被去污剂溶解。通过Nycodenz阶梯密度梯度离心从肝脏中分离得到的过氧化物酶体，首先用0.2% Triton X-100处理以去除可溶性蛋白和大部分膜结合蛋白。通过在pH 7.5的Tris-HCl缓冲液（10 mM）中加入0.05% Triton X-100、1 M KCl、0.3 mM NADPH和2 mM二硫苏糖醇，从所得残渣中溶解该酶。通过透析去除KCl使酶沉淀，然后用1 M KCl中的0.8%辛基葡糖苷（加NADPH和二硫苏糖醇）重新溶解，对该酶进行进一步纯化。通过在Sepharose CL-6B柱上进行凝胶过滤，然后在NADPH-琼脂糖凝胶基质上进行亲和层析，将第二次溶解的酶纯化至同质（相对于过氧化物酶体纯化了370倍）。NADPH-琼脂糖是通过将高碘酸盐氧化的NADP⁺与己二酸二酰肼-琼脂糖反应，然后用NaBH₄还原固定化的NADP⁺制备而成。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上，纯化后的酶显示出一条单一的同质条带，表观分子量为60,000。通过尺寸排阻色谱法估计天然酶的分子量为75,000。对纯化蛋白的氨基酸分析表明，疏水氨基酸占分子的27%。纯化后的酶对十六烷基二羟基丙酮磷酸（DHAP）的Km值为21 μM，在存在0.07 mM NADPH时的Vmax值为67 μmol/min/mg。在校正共底物NADP³H的同位素效应后，假设该酶分子量为60,000，计算其转换数（Kcat）为6,000 mol/min/mol酶。纯化后的酶也使用棕榈酰二羟基丙酮磷酸作为底物（Km = 15.4 μM，Vmax = 75 μmol/min/mg）。棕榈酰-DHAP竞争性抑制十六烷基-DHAP的还原，表明同一酶催化酰基-DHAP和烷基-DHAP的还原。NADH可以替代NADPH，但该酶对NADH的Km值（1.7 mM）远高于对NADPH的Km值（20 μM）。纯化后的酶受到NADP⁺和棕榈酰辅酶A的竞争性抑制（针对NADPH）。该酶在存在NADPH和二硫苏糖醇的情况下于4℃储存时稳定。
