Moreno de la Garza M, Schultz-Borchard U, Crabb J W, Kunau W H
Eur J Biochem. 1985 Apr 15;148(2):285-91. doi: 10.1111/j.1432-1033.1985.tb08837.x.
A multifunctional protein from oleate-grown cells of Candida tropicalis has been purified and partially characterized. A simple two-step purification has been developed involving ion-exchange chromatography followed by dye-ligand chromatography on blue Sepharose CL-6B. Homogeneous enzyme with a subunit Mr of 102 000 is obtained in 60% yield. The native relative molecular mass, determined by three different methods, yielded values which suggest that the enzyme is dimeric. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the purified protein revealed a single polypeptide band and reverse-phase high-performance liquid chromatography indicated a single component suggesting that this protein may consist either of two identical or very similar subunits. Three beta-oxidation activities, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-hydroxyacyl-CoA epimerase, co-purified with this protein. The ratio of the three beta-oxidation enzyme activities remained constant during purification and was unchanged by additional chromatographic methods (adsorption and affinity chromatography), thus indicating the multifunctional nature of this protein. Enzymatic staining of the purified protein for 3-hydroxyacyl-CoA dehydrogenase and epimerase, following electrophoresis in a polyacrylamide density gradient, further supported the multifunctionality of this protein. After isopycnic centrifugation of a particulate fraction from oleate-grown cells in a linear sucrose gradient the activities of all individual beta-oxidation enzymes cosedimented with catalase and with the glyoxylate bypass enzymes. This result demonstrated the peroxisomal localization of the multifunctional enzyme. The relationship of this multifunctional protein to the two bifunctional beta-oxidation enzymes isolated from peroxisomes of rat liver and from glyoxysomes of cucumber seeds is discussed.
已对热带假丝酵母油酸生长细胞中的一种多功能蛋白进行了纯化和部分特性鉴定。已开发出一种简单的两步纯化方法，包括离子交换色谱，随后在蓝色琼脂糖凝胶CL-6B上进行染料配体色谱。以60%的产率获得了亚基Mr为102 000的均一酶。通过三种不同方法测定的天然相对分子质量得出的值表明该酶是二聚体。纯化蛋白的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示出一条单一的多肽带，反相高效液相色谱表明是单一成分，这表明该蛋白可能由两个相同或非常相似的亚基组成。三种β-氧化活性，即烯酰辅酶A水合酶、3-羟酰基辅酶A脱氢酶和3-羟酰基辅酶A差向异构酶，与该蛋白共纯化。三种β-氧化酶活性的比例在纯化过程中保持恒定，并且通过额外的色谱方法（吸附和亲和色谱）没有变化，因此表明该蛋白具有多功能性质。在聚丙烯酰胺密度梯度中进行电泳后，对纯化蛋白进行3-羟酰基辅酶A脱氢酶和差向异构酶的酶促染色，进一步支持了该蛋白的多功能性。在用线性蔗糖梯度对油酸生长细胞的颗粒部分进行等密度离心后，所有单个β-氧化酶的活性与过氧化氢酶和乙醛酸循环旁路酶一起沉降。这一结果证明了该多功能酶的过氧化物酶体定位。讨论了这种多功能蛋白与从大鼠肝脏过氧化物酶体和黄瓜种子乙醛酸循环体中分离出的两种双功能β-氧化酶的关系。
