In vitro assembly of the core catalytic complex of the chloroplast ATP synthase

The regulatory subunit and an αβ complex were isolated from the catalytic F 1 portion of the chloroplast ATP synthase. The isolated subunit was devoid of catalytic activity, whereas the αβ complex exhibited a very low ATPase activity ( 200 nmol/min/mg of protein). The αβ complex migrated as a hexameric α 3 β 3 complex during ultracentrifugation and gel filtration but reversibly dissociated into α and β monomers after freezing and thawing in the presence of ethylenediamine tetraacetic acid and in the absence of nucleotides. Conditions are described in which the and αβ preparations were combined to rapidly and efficiently reconstitute a fully functional catalytic core enzyme complex. The reconstituted enzyme exhibited normal tight binding and sensitivity to the inhibitory subunit and to the allosteric inhibitor tentoxin. However, neither the αβ complex nor the isolated subunit alone could bind the subunit or tentoxin with high affinity. Similarly, high affinity binding sites for ATP and ADP, which are characteristic of the core α 3 β 3 enzyme, were absent from the αβ complex. The results indicate that when the subunit binds to the αβ complex, it induces a three-dimensional conformation in the enzyme, which is necessary for tight binding of the inhibitors and for high-affinity, asymmetric nucleotide binding.