Solubilization and properties of chloroplast lamellar protein.

An understanding of the detailed mechanisms of the chloroplast-mediated reactions of photosynthesis requires information on the components of the chloroplast as thorough as the information established for the mitochondrion.I Two procedures for solubilization of chloroplast lamellar protein have been reported.Weber2 extracted chloroplasts with methanol and ether and dissolved the remaining protein in anhydrous formic acid to obtain subunits of approximately 165,000 particle weight.Criddle and Park3 found that a combination of 0.1 per cent sodium dodecyl sulfate and 1 per cent urea yielded solubilized protein from an acetone powder of chloroplast fragments.In this communication, we report a method for solubilizing two thirds of the total chloroplast lamellar protein by butanol extraction and discuss some of the properties of this isolated protein.The protein can be obtained in aqueous solution without the presence of detergent or other surfactant material.Materials and Methods.-Swisschard (Beta.vulgaris), variety dark green, was grown in the greenhouse or in a plant growth chamber and nourished with Hoagland and Arnon's4 mineral medium.Light-grown field corn (Zea mays) was grown similarly!etiolated corn plants were grown in complete darkness, and the leaves were harvested 10-12 days after planting.C14labeled chard material was obtained from plants grown in a closed system in the presence of C 1402.5 Chloroplast fragments were prepared by a method similar to that of Park and Pon.6 Two hundred gm of leaves, separated from the petioles and mid-ribs, were homogenized in a Waring Blendor at 4°C with 200 ml of. 0.05 M Tris buffer (titrated to pH 8 with ascorbic acid) contain- ing 0.01 M EDTA and usually 0.5 M sucrose.Debris was removed by filtration through six layers of cheesecloth, and the green juice was centrifuged for 3 min at 1000 g.The precipitate was discarded, and the supernatant was centrifuged for 12 min at 4000 g to bring down the chloro- plasts.The chloroplasts were washed extensively with 10-3 M pH 7 phosphate buffer (this ruptured the chloroplasts) and then with distilled water to remove all soluble proteins.After the material from 200 gm of leaves was resuspended in 50 ml of distilled water, it was further dis- integrated in a Raytheon 10-kc sonic oscillator for 15-20 min, and the volume was adjusted to 100 ml with washings.Starch granules were separated from the lamellar fragments by centrifuging for 30 min at 9000 g. Preparation of micelles: One gm of asolectin, a commercial phospholipid preparation from soybeans (Associated Concentrates, Woodside, N.Y.), was sonicated for 30 min in 50 ml of a solution of 0.01 M Tris buffer, pH 8, 0.25 M sucrose, and 5 X 10-4 M EDTA7 to prepare micelles.The micelles could be prepared as well by disrupting in distilled water or dilute cacodylate buffer.Chard leaf lipid was obtained by extracting the leaves with alcohol-ether7 or chloroform-metha- nol,8 and leaf lipid micelles were obtained by sonication of the dried, extracted material in sucrose- Tris buffer-EDTA, distilled water, or dilute cacodylate buffer.Some chlorophyll, isolated from chard leaves by the procedure of Jacobs et al.9 or that of Anderson and Calvin'0 somewhat modified, could also be obtained in micellar suspension by sonication for 45 min.Assay procedures: Protein was assayed according to the Folin method of Lowry et al.11Total nitrogen was determined by semimicro Kjeldahl digestion, followed by quantitative steam dis- tillation and determination of ammonia with Nessler's reagent."2The method of Chen et al."3 was used to determine phosphorus.Chlorophyll was assayed according to the spectrophotometric method of Arnon.14The amount