Jeannine Lincoln A, Donat Nathaniel, Palmer Gary, Prochaska Lawrence J
Department of Biochemistry and Molecular Biology, School of Medicine and College of Science and Mathematics, Wright State University, Dayton, OH 45435, USA.
Arch Biochem Biophys. 2003 Aug 1;416(1):81-91. doi: 10.1016/s0003-9861(03)00202-9.
Antibodies were raised against conserved amino acid sequences in four extramembranous portions of subunit III (sIII) from beef cytochrome c oxidase (COX) and the role of these domains in the functional activities of the enzyme was investigated. The binding of one antipeptide antibody corresponding to an externally exposed (facing the intermembrane space) domain of COX sIII (amino acids 180-189 in the primary sequence) exhibited a 30-50% stimulation of electron transfer activity in both detergent-dispersed COX and COX incorporated into phospholipid vesicles (COV). Antibody binding to two different matrix-faced domains (amino acids 57-66 and 148-159 in the sequence) resulted in small stimulations (10-25%) of COX electron transfer activity. The remaining antipeptide antibody (against amino acids 119-128) had no effect on electron transfer activity of COX in detergent solution, but exhibited a slight inhibition of activity (15%) in COV. The mechanism of antibody-induced stimulation of COX electron transfer activity was determined to be an increase in the maximum velocity of the enzyme and not due to a change in the apparent K(m) of cytochrome c interaction with COX as determined by steady state kinetic assays. Antibody binding to COX in COV increased the respiratory control ratio (an indicator of endogenous proton permeability) of COV, but had no effect on the vectorial proton pumping activity of COV. These results suggest that these conserved, hydrophilic domains of COX sIII are conformationally linked to the electron transfer function of the enzyme in subunits I and II and that sIII may serve as a regulatory subunit for COX electron transfer and proton pumping activities.
制备了针对牛细胞色素c氧化酶（COX）亚基III（sIII）四个膜外部分保守氨基酸序列的抗体，并研究了这些结构域在该酶功能活性中的作用。一种对应于COX sIII外部暴露（面向膜间隙）结构域（一级序列中的氨基酸180 - 189）的抗肽抗体，在去污剂分散的COX和掺入磷脂囊泡（COV）的COX中，均表现出对电子传递活性30 - 50%的刺激作用。抗体与两个不同的面向基质的结构域（序列中的氨基酸57 - 66和148 - 159）结合，导致COX电子传递活性有小幅度的刺激（10 - 25%）。其余抗肽抗体（针对氨基酸119 - 128）在去污剂溶液中对COX的电子传递活性没有影响，但在COV中表现出轻微的活性抑制（15%）。通过稳态动力学分析确定，抗体诱导COX电子传递活性刺激的机制是酶的最大反应速度增加，而非细胞色素c与COX相互作用的表观米氏常数（K(m)）发生变化。抗体与COV中的COX结合增加了COV的呼吸控制率（内源性质子通透性的指标），但对COV的矢量质子泵活性没有影响。这些结果表明，COX sIII的这些保守亲水区在构象上与亚基I和II中酶的电子传递功能相关联，并且sIII可能作为COX电子传递和质子泵活性的调节亚基。
