Wang Mei Mei, Olsher Michelle, Sugár István P, Chong Parkson Lee-Gau
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Biochemistry. 2004 Mar 2;43(8):2159-66. doi: 10.1021/bi035982+.
Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model. Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 degrees C using cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, approximately 800 nm in diameter). In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol %. Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C(r)) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation. The activity variation with cholesterol content was correlated well with the area of regular distribution (A(reg)) in the plane of the membrane as determined by nystatin fluorescence. A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (approximately 168 nm in diameter). These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (approximately 168-800 nm). The present work on COD and the previous study on phospholipase A(2) (sPLA(2)) [Liu and Chong (1999) Biochemistry 38, 3867-3873] suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner. When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C(r). When phospholipid is the substrate, the minimum activity is at C(r), as is the case with sPLA(2). Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.
在此，以土壤细菌胆固醇氧化酶（COD）为模型，研究了膜胆固醇侧向组织与膜表面活性酶活性之间的相互作用。具体而言，在37℃下，使用胆固醇/1-棕榈酰-2-油酰基-l-α-磷脂酰胆碱（POPC）大单层囊泡（LUVs，直径约800nm），研究了膜胆固醇摩尔分数对COD催化胆固醇氧化初始速率的影响。在所研究的三个浓度范围内（18.8 - 21.2、23.6 - 26.3和32.2 - 34.5mol%胆固醇），COD的初始活性随胆固醇摩尔分数呈双相变化，在19.7、25.0和33.4mol%处出现局部最大值。在实验误差范围内，这些摩尔分数与理论预测的最大超晶格形成的临界胆固醇摩尔分数（C(r)）（20.0、25.0和33.3）一致。COD活性随胆固醇含量的变化与制霉菌素荧光测定的膜平面内规则分布面积（A(reg)）密切相关。在脱氢麦角固醇（DHE）/POPC LUVs（直径约168nm）中，在临界甾醇摩尔分数20mol%处也检测到了类似的COD活性双相变化。这些结果表明，对于两种甾醇（DHE和胆固醇）以及广泛的囊泡尺寸范围（约168 - 800nm），COD的活性受甾醇超晶格程度的调节。目前关于COD的研究以及之前关于磷脂酶A(2)（sPLA(2)）的研究[Liu和Chong（1999年）《生物化学》38卷，3867 - 3873页]表明，一些表面活性酶的活性可能以底物依赖的方式受膜中甾醇超晶格程度的调节。当底物是甾醇时，如COD的情况，酶活性在C(r)处达到局部最大值。当磷脂是底物时，最小活性在C(r)处，如sPLA(2)的情况。这两种现象均符合甾醇超晶格模型，体现了膜胆固醇含量的功能重要性。
