IN-VITRO ASSEMBLY OF THE CORE CATALYTIC COMPLEX OF THE CHLOROPLAST ATP SYNTHASE

The regulatory gamma subunit and an alpha beta complex were isolated f rom the catalytic F-1 portion of the chloroplast ATP synthase, The iso lated gamma subunit was devoid of catalytic activity, whereas the alph a beta complex exhibited a very low ATPase activity (similar to 200 nm ol/min/mg of protein), The alpha beta complex migrated as a hexameric alpha(3) beta(3) complex during ultracentrifugation and gel filtration but reversibly dissociated into alpha and beta monomers after freezin g and thawing in the presence of ethylenediamine tetraacetic acid and in the absence of nucleotides, Conditions are described in which the g amma and alpha beta preparations were combined to rapidly and efficien tly reconstitute a fully functional catalytic core enzyme complex, The reconstituted enzyme exhibited normal tight binding and sensitivity t o the inhibitory epsilon subunit and to the allosteric inhibitor tento xin, However, neither the alpha beta complex nor the isolated gamma su bunit alone could bind the epsilon subunit or tentoxin with high affin ity, Similarly, high affinity binding sites for ATP and ADP, which are characteristic of the core alpha(3) beta(3) gamma enzyme, were absent from the alpha beta complex, The results indicate that when the gamma subunit binds to the alpha beta complex, it induces a three-dimension al conformation in the enzyme, which is necessary for tight binding of the inhibitors and for high-affinity, asymmetric nucleotide binding.