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Inhibition of Coupling Factor Activity of Chloroplast Membranes by Diazonium Compounds

From the ¹ From the Purdue University, Department of Biological Sciences, Lafayette, Indiana 47907

1. Chemical modification of chloroplast membranes by diazonium benzenesulfonic acid (DABS) resulted in marked inhibition of noncyclic and cyclic photophosphorylation. Accompanying the inhibition of ATP synthesis there was an increased rate of basal electron flow and loss of the ability of ADP to stimulate the basal electron transport rate, both characteristic of uncoupling.

2. The calcium-dependent ATPase activity of the coupling factor was also severely inhibited after treatment with the diazonium compound. This, in conjunction with the binding of [³⁵S]DABS to the coupling factor subunits as revealed by sodium dodecyl sulfate disc gel electrophoresis, suggests that the inhibition of phosphorylation results, in part at least, from inactivation of the coupling factor complex by the chemical modifier.

3. Treatment of chloroplast membranes (from which coupling factor was largely removed by two EDTA washes) with [³⁵S]DABS, followed by digitonin fractionation of the membranes into Photosystem I and Photosystem II fractions revealed that the Photosystem I fraction incorporated 10-fold more [³⁵S]DABS than the Photosystem II fraction. This is consistent with previous findings (Dillley, R. A., Peters, G. A., and Shaw, E. R. (1972) J. Membrane Biol. 8, 163–180) using intact chloroplasts (with coupling factor present) that the intrinsic proteins associated with Photosystem I are localized at the external surface of the grana membrane.

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