Steinemann D, Lill H, Junge W, Engelbrecht S
Fachbereich Biologie/Chemie, Universität Osnabrück, Germany.
Biochim Biophys Acta. 1994 Sep 27;1187(3):354-9. doi: 10.1016/0005-2728(94)90009-4.
We studied the functioning of chimeric F0F1-ATPases by replacing subunits delta and epsilon of spinach CF1 with their counterparts from Synechocystis sp. PCC 6803. The sequence identities between these subunits are 26 and 41%, respectively. For a systematic approach to such studies and later extension to genetically modified subunits recombinant proteins are required. The genes coding for spinach and Synechocystis delta and epsilon were cloned into pET3 expression vectors and expressed in Escherichia coli. Upon expression at 37 degrees C the recombinant subunits formed inclusion bodies within the host cells except for spinach delta, which was soluble. Synechocystis delta and epsilon could be obtained in soluble form upon expression at 20 degrees C. After purification (and refolding of spinach epsilon) both epsilon subunits inhibited the Ca(2+)-ATPase activity of soluble CF1(- epsilon). Subunits delta and epsilon from both species raised the rate of ATP synthesis in partially CF1-depleted spinach thylakoids when added together with CF1(- delta) or CF1(- delta, epsilon). This showed the functionality of recombinant Synechocystis and spinach delta and epsilon together with spinach alpha 3 beta 3 gamma. The molar excess of epsilon necessary for saturation was higher for Ca(2+)-ATPase inhibition than for reconstitution of photophosphorylation thus pointing to a direct interaction between epsilon and both CF1 and CF0.
我们通过用来自集胞藻属PCC 6803的对应亚基替换菠菜CF1的δ和ε亚基，研究了嵌合F0F1 - ATP酶的功能。这些亚基之间的序列同一性分别为26%和41%。为了系统地进行此类研究并随后扩展到转基因亚基，需要重组蛋白。编码菠菜和集胞藻属δ和ε的基因被克隆到pET3表达载体中，并在大肠杆菌中表达。在37℃表达时，除了可溶的菠菜δ亚基外，重组亚基在宿主细胞内形成包涵体。集胞藻属δ和ε在20℃表达时可以以可溶形式获得。纯化后（以及菠菜ε的重折叠），两种ε亚基均抑制了可溶性CF1（-ε）的Ca（2 +）-ATP酶活性。当与CF1（-δ）或CF1（-δ，ε）一起添加时，来自这两个物种的δ和ε亚基提高了部分CF1缺失的菠菜类囊体中ATP合成的速率。这表明重组集胞藻属和菠菜的δ和ε与菠菜α3β3γ一起具有功能。Ca（2 +）-ATP酶抑制所需的ε的摩尔过量比光磷酸化重建所需的更高，因此表明ε与CF1和CF0之间存在直接相互作用。
