An ultracentrifugation-chloroplast membrane preparatory method for the fine-structural localization of polyphenol oxidase

Abstract

Abstract Polyphenol oxidase (PPO) has been localized at the fine structural level in whole plant chloroplasts (Czaninski and Catesson, 1972; Henry, 1976). It is assumed that the PPO of chloroplast thylakoids and lamellae can be either latent or active depending upon specific cellular metabolism, plant treatment, and exposure conditions. Detergents, such as digitonin, accelerate tthe activation of spinach thylakoid membrane-bound PPO (Lieberei and Biehl, 1976). As any PPO activity localized by ultrastructural procedures, would be expected to possibly exist on the outer surfaces of the thylakoids and lamellae, a comprehensive methodology was defined for the isolation of sorbitol disrupted spinach chloroplast membranes, with subsequent ult ultrastructural localization of PPO (Henry et al., 1981). Spinach ( Spinacia oleracea L.) leaves were homogenized in 0.33M sorbitol-Hepes buffer, pH 7.6, followed by filtration through cheesecloth. 1) The filtrate was centrifuged at 300xg for 5 min. 2) The chloroplast pellet was then recentrifuged at 1,200xg for 10 min. The combined filtrates were layered on a discontinuous sucrose gradient and centrifuged at 20,000xg for 6 . Thirty separate 1-ml fractions were recentrifuged at 60,000xg for 20 min, giving individual pellets, each of which were prepared for ultastructural PPO enzyme localization (Henry et al., 1981). PPO is localized in the grana lamellae membranes of the sorbitol disrupted chloroplasts (Fig. 1), using 3,4-dihydroxyphenylalanine (L-DOPA) as the substrate. In the presence of the L-DOPA substrate, plus the PPO inhibitor, sodium diethyldithiocarbamate (DDC), the grana lamellae do not show a positive staining reaction for PPO (Fig. 2). There is also an absence of positive PPO staining in control tissue, lacking pretreatment with either L-DOPA or DDC (Fig. 3). To summarize, sorbitol-disrupted chlorolast membranes have been isolated via discontinuous sucrose density gradient centrifugaion and subsequently stained for the presence of PPO. The grana lamellae show positive PPO reaction product (Figs. 1–2). It is suggested that PPO is an integral part of the grana lamellae, even after sorbitol treatment; however, there is a release of a small amount of PPO into the soluble fractions (Henry et al., 1981). It is suggested that the grana lamellae may represent the possible sites of origin of PPO biosynthesis.