Solubilization and partial purification of a thylakoidal enzyme of spinach involved in the processing of D1 protein

Abstract

The enzyme involved in the processing of DI precursor protein was solubilized from spinach thylakoids by Triton X-100 treatment and then partially purified in the presence of the detergent by Sephadex G-75 gel-filtration chromatography. The apparent molecular mass of the enzyme was estimated via this procedure to be about 34 kDa. The D1 precursor protein translated from the extracted spinach chloroplast RNA by a wheat germ cell-free system was used here as a substrate in measurements of the activity.

Cleavage of a synthetic COOH‐terminal oligopeptide of D1 precursor protein by a purified processing enzyme

“…The activity depended linearly on the amount of enzyme solution added to the reaction mixture under the experimental conditions used. The activity was completely abolished by heat treatment (70°C, 3 min) of the enzyme solution, in a manner similar to that of the processing activity for the native precursor of D1 protein [14]. The enzyme activity was relatively stable at room temperature as shown by the results in fig.…”

“…The possibility exists that the additional cleavage at the C-side of Arg-344 is due to a contaminating trypsin-type protease in the enzyme solution. However, this does not appear to be the case, since when in vitro translated D1 precursor protein was incubated with our enzyme solution, the product we were able to detect was only the mature-sized protein [14].…”

“…The enzyme involved in the processing has recently been solubilized from a wild-type strain of Scenedesmus obliquus [13] and spinach [14]. The spinach enzyme, which has a molecular mass of about 34 kDa [14], has recently been highly purified and characterized [15].…”

“…X-100 extracts o f spinach thylakoids as in [ 14,15]. An oligopeptide corresponding to the C-terminal 29-amino-acid sequence deduced from the spinach psbA gene was synthesized on an Applied Biosystems model 430 A peptide synthesizer and purified by preparative HPLC using a reverse-phase column (Applied Biochem, Aquapore RP-300).…”

Transcript Levels and Synthesis of Photosystem II Components in Cyanobacterial Mutants with Inactivated Photosystem II Genes.

“…PCC 6803, there is no evidence for a "33.5-kD" D1 precursor as observed in plants (Reisfeld et al, 1982;Marder et al, 1984;Minami and Watanabe, 1985;Inagaki et al, 1989). However, this may be due to a decreased life time of the precursor compared with the mature protein in cyanobacteria and does not imply that there would be a fundamental difference between D1s from plants and cyanobacteria in this respect.…”