Isolation and properties of chloroplast particles of Scenedesmus obliquus D 3 with high photochemical activity

Abstract

An improved and standardized procedure for isolation of chloroplast particles from the unicellular green alga, Scenedesmus obliquus, D 3, is described. The method is generally applicable to heterotrophically- and autotrophically-grown cells of Scenedesmus as well as to Chlamydomonas reinhardti and Chlorella sorokiniana (7-11-05) cultures. Chloroplast particles with high NADP + photoreducing capacity are obtained from heterotrophic cultures only when the cell types are random and the culture is in the logarithmic growth phase; maximal rates of 240–260 μmoles NADP + reduced/h per mg chlorophyll are achieved. Optimal conditions for separation of such chloroplast particles require the use of Tricine buffer (20 mM, pH 7.5), 50 nM EDTA, 10 mM KCl and 0.5 mM dithiothreitol in the breaking medium; for the maintenance of high photochemical activity it is necessary to store particles in a solution consisting of 0.4 M sucrose, 30 mM KCl and 1% bovine serum albumin. Optimum reaction conditions were developed and the properties of the isolated particles investigated. Maximal activities are obtained when the sucrose concentration is maintained below 0.4 M; the pH optimum with Tricine buffer is between 7.8–8.1; and at least 30 mM Cl − is required. Red actinic light (wavelength >620 nm) with an intensity of 10 6 ergs/cm 2 per s is required for saturation. Ferrodoxin and ferredoxin-NADP + oxidoreductase are lost from the particles during the preparatory procedures and maximum photochemical activity is attained only when they are added back in balanced amounts. Stimulatory effects of added plastocyanin and cytochrome c-553 are noted only with particles having an initially low photochemical activity.