Laboratoire de Biochimie Végétale, Institut de Botanique, 28, rue Goethe, 67083 Strasbourg, Cédex, France.
Plant Physiol. 1987 Sep;85(1):51-5. doi: 10.1104/pp.85.1.51.
Solubilization and partial purification of the microsomal UDP-glucose sterol glucosyl transferase activity from maize coleoptiles by chromatography on DEAE-cellulose resulted in a highly delipidated (>95%) and inactive enzymic preparation. Addition of sterols revealed part of the activity and subsequent addition of phospholipids further increased the activity. Negatively charged phospholipids were shown to be by far the best activators. The purification step also produced the elimination of two interfering microsomal enzymic activities: UDPase and steryl glucoside acyl transferase. The removal of these two enzymic activities was a prerequisite for kinetic studies including product-inhibition studies, since the substrates of these two latter enzymes are the products of UDPG-SGTase activity. The results of the kinetic studies strongly suggest an ordered bi-bi mechanism for the glucosylation of sterols. Finally the effect of different phospholipids on the kinetic parameters of the reaction was studied. Both phosphatidylcholine and phosphatidylglycerol significantly decrease K(m-sterol) (and not K(m-UDPglucose)) and increase the reaction V(max). The decrease of K(m-sterol) is similar with both phospholipids whereas the increase of V(max) is much greater with phosphatidylglycerol than with phosphatidylcholine.
通过 DEAE-纤维素层析，从小麦胚芽微粒体中分离出 UDP-葡萄糖甾醇葡萄糖基转移酶，并对其进行增溶和部分纯化，得到高度脱脂（>95%）和无活性的酶制剂。添加甾醇可显示部分活性，随后添加磷脂可进一步提高活性。带负电荷的磷脂是迄今为止最好的激活剂。该纯化步骤还消除了两种干扰的微粒体酶活性：UDP 酶和甾醇糖苷酰基转移酶。消除这两种酶活性是进行动力学研究（包括产物抑制研究）的前提条件，因为这两种酶的底物是 UDPG-SGTase 活性的产物。动力学研究的结果强烈表明，甾醇的葡萄糖基化遵循有序的双酶促机制。最后，研究了不同磷脂对反应动力学参数的影响。磷脂酰胆碱和磷脂酰甘油均显著降低 K(m-甾醇)（而不是 K(m-UDPglucose)）并增加反应 V(max)。两种磷脂均可降低 K(m-甾醇)，而与磷脂酰胆碱相比，磷脂酰甘油增加 V(max)的作用更大。
