A rapid technique for isolating chloroplasts with high rates of endogenous photophosphorylation

Abstract

THE MAIN FEATURES OF THE PROCEDURE DEVELOPED FOR RAPID CHLOROPLAST ISOLATION ARE: 1) gentle grinding of the plant material in a special nylon bag which retains nearly all whole cells and large debris, 2) osmoticum concentration chosen on the basis of the measured endogenous photophosphorylation, 3) a single, brief, low-speed centrifugation, 4) pellet resuspension by means of a vortex mixer, and 5) a total elapsed time from harvesting the plants to the obtaining of a resuspended chloroplast pellet of only 2 minutes. The usual isolation medium consists of an osmoticum (0.2 m sucrose) and a buffer (0.02 m N-tris-(hydroxymethyl) methyl-2-aminoethanesulfonate-NaOH, pH 7.9). In addition to these, the incubation medium contains only 200 mum ADP and 200 mum phosphate. Photophosphorylation rates of 24 mumoles ATP formed per mg chlorophyll per hour are consistently obtained using chloroplasts isolated from peas (Pisum sativum var. Laxton's Superb). The rate of endogenous photophosphorylation is maximal when the isolation and incubation media have an osmolarity of about 0.19 made up either with sucrose or with NaCl. The high rates and ease of measurement of endogenous photophosphorylation may facilitate the study of certain soluble components of chloroplasts as well as the general state of the photosynthetic ability of the plant.