Formation and properties of hybrid photosynthetic F1âATPases: Demonstration of different structural requirements for stimulation and inhibition by tentoxin
2001
Tucker, Ward C. | Du, Ziyun | GrometâElhanan, Zippora | Richter, Mark L.
A hybrid ATPase composed of cloned chloroplast ATP synthase β and γ subunits (βC and γC) and the cloned α subunit from the Rhodospirillum rubrum ATP synthase (αR)Â was assembled using solubilized inclusion bodies and a simple singleâstep folding procedure. The catalytic properties of the assembled were compared to those of the core complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R.ârubrumα and β subunits and the CF1γ subunit ( ). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast γ subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1e subunit. Thus the CF1γ subunit conferred full redox regulation and normal e binding to the two hybrid enzymes. Only the native CF1 complex was inhibited by tentoxin, confirming the requirement for both CF1α and β subunits for tentoxin inhibition. However, the complex, like the complex, was stimulated by tentoxin at concentrations in excess of 10âµm. In addition, replacement of the aspartate at position 83 in βC with leucine resulted in the loss of stimulation in the hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the β subunit, but differ in their requirements for α subunit structure.
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