Purification of dihydroxyacetone phosphate acyltransferase from guinea pig liver peroxisomes.
Purification of dihydroxyacetone phosphate acyltransferase from guinea pig liver peroxisomes.
复制标题
从豚鼠肝过氧化物酶体中纯化二羟丙酮磷酸酰基转移酶。
DOI：
10.1006/abbi.1993.1013
复制
发表时间：
1993
影响因子：
3.9
通讯作者：
Hajra,AK
中科院分区：
文献类型：
--
作者：
Webber,KO;Hajra,AK
二羟丙酮磷酸酰基转移酶（EC 2.3.1.42）是一种在高等真核生物中启动甘油脂（特别是醚连接甘油脂）生物合成的过氧化物酶，已从豚鼠肝脏中纯化了超过3250倍。纯化的初始阶段需要通过微分和密度梯度离心的组合分离肝脏过氧化物酶体。用3-[3-胆酰胺丙基)二甲胺]-1-丙烷磺酸盐在中等离子强度（0.15 M NaCl）下从过氧化物酶体膜中溶解二羟丙酮磷酸酰基转移酶。溶解后的酶通过尺寸排除层析、阳离子交换层析和羟基磷灰石层析进一步纯化。在酶纯化过程中不同组分的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上，一个69-kDa的蛋白带与酶活性共纯化，表明该酶单体的amr0可能为69000。当SDS-PAGE上观察到一条69-kDa的条带时，进一步用色谱聚焦纯化酶证实了这一点。凝胶过滤法测定的二羟丙酮磷酸酰基转移酶分子量为90 kDa。在棕榈酰辅酶a饱和浓度下，酶的vmax为每分钟每毫克蛋白质生成4 pmol酰基二羟丙酮磷酸（acylDHAP）， km为70 μM。游离辅酶A抑制酰基转移酶反应的抑制常数（Ki）约为0.76 mM。迄今为止，这是哺乳动物源性中纯度最高的dhp酰基转移酶（>3200倍）。
Dihydroxyacetone phosphate acyltransferase (EC 2.3.1.42), a peroxisomal enzyme which initiates the biosynthesis of glycerolipids (especially the ether-linked glycerolipids) in higher eukaryotes, has been purified by over 3250-fold from guinea pig liver. Initial stages of purification entailed isolation of liver peroxisomes by a combination of differential and density-gradient centrifugation. Dihydroxyacetone phosphate acyltransferase was solubilized from peroxisomal membranes with 3-[3-cholamidopropyl)dimethylammonio]-1-propane sulfonate at moderate ionic strength (0.15 M NaCl). The solubilized enzyme was further purified by a regimen of size-exclusion chromatography, cation-exchange chromatography, and hydroxylapatite chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions during the purification of the enzyme, a 69-kDa protein band copurified with the enzyme activity, indicating that the monomeric enzyme may have aMrof 69,000. This was verified by further purifying the enzyme by chromatofocusing, when a single 69-kDa band was observed on SDS-PAGE. TheMrof dihydroxyacetone phosphate acyltransferase determined by gel filtration is 90 kDa. TheVmaxof the purified enzyme was 4 pmol acyldihydroxyacetone phosphate (acylDHAP) formed per minute per milligram protein and theKm(DHAP)is 70 μM when assayed at saturating concentrations of palmitoylCoA. Free coenzyme A inhibits the acyltransferase reaction with an inhibition constant (Ki) of approximately 0.76 mM. To date, this is the most highly purified DHAP acyltransferase(>3200-fold) of mammalian origin.