Takeda K, Hirano M, Kanazawa H, Nukiwa N, Kagawa Y, Futai M
J Biochem. 1982 Feb;91(2):695-701. doi: 10.1093/oxfordjournals.jbchem.a133742.
ATPase complexes were reconstituted from homologous and heterologous combinations of alpha, beta, and gamma subunits of coupling factor ATPase TF1 of thermophilic bacterium PS3 and EF1 of Escherichia coli. TF1 and alpha beta gamma complex reconstituted from TF1 subunits were thermostable and activated by methanol, sodium dodecyl sulfate and anions and they were halophilic, whereas EF1 and its three-subunit complex did not show these properties. The hybrid ATPase alpha T beta T gamma E (complex of the alpha and beta subunits of TF1 and the gamma subunit of EF1) showed closely similar properties to TF1 except for thermostability, while alpha E beta E gamma T (alpha and beta from EF1 and gamma from TF1) had similar properties to EF1. These results suggest that alpha and/or beta is required for the properties of F1. The complex alpha E beta T gamma E showed similar properties to EF1 except for its optimum pH: this complex had a broad pH optimum at about pH 7, whereas EF1 had an optimum at pH 8.5. No hybrid complexes were thermostable, suggesting that all three subunits of TF1 are required for heat stability. These hybrids showed higher halophilicity than EF1, although they were less halophilic than TF1. The hybrid enzymes studied here are the first thermophilic-mesophilic hybrid enzymes obtained.
ATP酶复合物由嗜热细菌PS3的偶联因子ATP酶TF1和大肠杆菌的EF1的α、β和γ亚基的同源和异源组合重构而成。从TF1亚基重构的TF1和αβγ复合物具有热稳定性，可被甲醇、十二烷基硫酸钠和阴离子激活，且具有嗜盐性，而EF1及其三亚基复合物则不具备这些特性。杂合ATP酶αTβTγE（TF1的α和β亚基与EF1的γ亚基的复合物）除热稳定性外，表现出与TF1极为相似的特性，而αEβEγT（EF1的α和β以及TF1的γ）具有与EF1相似的特性。这些结果表明，F1的特性需要α和/或β。复合物αEβTγE除最适pH外，表现出与EF1相似的特性：该复合物在约pH 7时有较宽的最适pH范围，而EF1的最适pH为8.5。没有杂合复合物具有热稳定性，这表明TF1的所有三个亚基对于热稳定性都是必需的。这些杂合体表现出比EF1更高的嗜盐性，尽管它们的嗜盐性低于TF1。这里研究的杂合酶是首次获得的嗜热-嗜温杂合酶。
