Reactivation by chloride of hill activity in heat- and tris-treated thylakoid membranes from Beta vulgaris

Abstract

Hill activity (photoreduction of 2,6,dichlorophenol indophenol) of heat inactivated (40°C, 3 min) and Tris-washed (0.8M, pH 8.3) thylakoids of Beta vulgaris (beet-spinach) was partially restored if they were incubated with 150 mM MgCl2 prior to the assay. Mg(NO3)2 or MgSO4 were unable to restore this activity. The extent of this reactivation was dependent upon the degree of inactivation by heating and upon the composition of the isolation and the resuspension buffer used during the heat treatment. Washing of heat-treated thylakoids with phosphate-EDTA buffer prior to incubation with MgCl2 did not affect the extent of this reactivation. Chloride ions seem to be required for the reactivation of Hill activity damaged either by heat or by Tris.Most commonly used chloroplast isolation and resuspension media, except for Tris-HCl as resuspension medium, were suitable for restoration of Hill activity in heat-damaged thylakoids by preincubation with 150 mM MgCl2 prior to the assay. Pretreatment with MgCl2 stimulated Hill activity in Tris-treated and heat-damage thylakoids if phosphate buffer was used for their resuspension. However, the same pretreatment inhibited Hill activity in unheated thylakoids isolated in Tris medium and resuspended in the same medium. On the other hand, MgCl2 pretreatment induced restoration of the Hill activity of the heated thylakoids when Tricine or Hepes was used as the resuspension medium. It appears that the presence of Tris somehow hampers the Cl(-) induced reactivation. The stimulation of Hill activity by MgCl2 treatment in unheated (control) thylakoids is possibly induced by Mg(2+) ions and not by Cl(-) ions.