The relation of the alteration of Photosystem II. Activity to the inhibition of energy transfer and the proton pump in tris-washed chloroplasts

Abstract

Washing of spinach chloroplasts with high concentrations of Tris 3 3 Abbreviations used in this paper include: ADP, adenosine diphosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)amino methane; Tricine, tris (hydroxymethyl) methyl-glycine; DCMU, dichlorophenyl-1,1-dimethyl-urea; EDTA, ethylenediaminetetraacetic acid; DTT, dithiothreitol; PSII, Photosystem 2; PMS, N-methylphenazonium methosulfate. induces pH-dependent changes in chloroplast reactions. At high pH (8.4) Tris washing causes the inhibition of Photosystem 2 activity which can be prevented by the maintenance of reducing conditions during washing. Washing at low pH (7.2) causes an enhancement of oxygen evolution and increased rate of ferricyanide photoreduction which is not influenced by the presence of reducing conditions. The increased rate of electron flow is accompanied by the inhibition of light mediated phosphorylating activity, acid-induced ATP synthesis, light-induced proton uptake and light triggered Mg 2+ ATPase activity. Tris treatment at low pH also causes a sensitization of Photosystem 2 activity such that oxygen evolution is inhibited by low concentrations of tris at high pH. This inhibition of the stimulated electron flow is not accompanied by a reconstitution of the photophosphorylation activity. A detailed analysis of the effect of tris treatment on Photosystem II activity and membrane dependent energy conversion shows that the treatment of chloroplasts causes an inhibition of the energy conversion process which is independent of the effect on oxygen evolution. Determination of the presence of coupling factor (as determined by ATPase activity) and membrane osmotic properties reveal normal levels of enzyme activity and osmotic response in treated chloroplasts. The inhibition of the energy conversion process is accompanied by reduced capacity to maintain a proton gradient. Kinetic analysis of the proton uptake reaction reveals that Tris treatment renders the grana membranes more permeable to protons.