H⁺转运ATP酶的碳二亚胺结合蛋白以及二环己基碳二亚胺对H⁺传导的抑制作用

Carbodiimide-binding protein of H+-translocating ATPase and inhibition of H+ conduction by dicyclohexylcarbodiimide.

作者信息

Sone N, Yoshida M, Hirata H, Kagawa Y

出版信息

J Biochem. 1979 Feb;85(2):503-9. doi: 10.1093/oxfordjournals.jbchem.a132357.

DOI:10.1093/oxfordjournals.jbchem.a132357

Abstract

H+-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F1) and a membrane moiety (F0). Using highly-purified F0 from a thermophilic bacterium PS3 (TF0), the following results were obtained. 1. Inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) of H+ conduction through TF0 followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5 times 10(3) M(-1)-min(-1). 2. H+ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF0. When only one-third of the band 8 protein was labeled with DCCD, TF0 hardly transported any H+. 3. By extracting TF0 with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6,000. 4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6,500. 5. The role of band 8 protein (DCCD-binding protein) in H+ conduction through TF0 is discussed on the basis of these results.

摘要

H⁺转运ATP酶催化生物膜中的ATP合成，由头部（F1）和膜部分（F0）组成。使用从嗜热细菌PS3（TF0）中高度纯化的F0，得到了以下结果。1. N，N'-二环己基碳二亚胺（DCCD）对通过TF0的H⁺传导的抑制遵循假一级动力学。抑制剂与酶相互作用的二级速率常数为5×10³ M⁻¹·min⁻¹。2. 被DCCD阻断的H⁺传导率与掺入TF0的8带蛋白中的DCCD量成正比。当只有三分之一的8带蛋白用DCCD标记时，TF0几乎不转运任何H⁺。3. 用氯仿 - 甲醇萃取TF0，得到作为蛋白脂质的8带蛋白。十二烷基硫酸钠和尿素聚丙烯酰胺凝胶电泳显示分子量约为6000。4. 8带蛋白的氨基酸组成表明该蛋白含有极高比例的疏水氨基酸（极性为0.29），并且不含组氨酸、色氨酸、半胱氨酸和赖氨酸。其最小分子量为6500。5. 根据这些结果讨论了8带蛋白（DCCD结合蛋白）在通过TF0的H⁺传导中的作用。