From Chinese Cabbage and Photosynthetic Microorganisms

Ribulose-1,5-diphosphate carboxylase (carboxydismutase) was prepared from Chinese Cabbage [Brassica petsai (Parl)] and the Km values and molecular weight were determined. These parameters were found to be in good agreement with values reported for this enzyme from other higher plants. Investigation of carboxydismutase activity from the photosynthetic micro-organisms Chlamydomonas reinhardi (IU 89+), Plectonema boryanum (IU 594), and Chromatium strain D showed striking similarity to the higher plant enzyme, when the sedimentation coeffioients were compared. It is now well established that carboxydismutase (ribulose-1,5-diphosphate [RUD'P] carboxylase) is the enzyme primarily respon-sible for photosynthetic "CO.," fixation in many species of plants [(3); for notable exceptions see Slack and Hatch (20) and Evans et al. (6)]. The unique features of the higher plant enzyme which distinguish it from other chloroplast soluble proteins are its high sedimentation coefficient of approximately 18 S and its molecular weight of approximately 500,00. Fraction I protein (a soluble component of higher plant cytoplasm) exhibits these same properties (5,19), and recent evidence strongly supports the conclusion that Fraction I protein and carboxydisinutase are identical (23, 24). The experiments of a number of investigators (7, 9, 17, 21 ) have establis,hed the existence of carboxydismutase in a number of photosynthetic microorganisms, including blue-green algae and photosynthetic bacteria. No information relating to the molecular nature of this enzyme was reported in these studies and only recently have physical properties of carboxydismutase froml such souirces been reported (1, 11, 14). It was our purpose, therefore, to characterize carboxydismutase from 3 photosynthetic microorganisms '(blue-green alga, green alga, and photosyn1 This investigation was supported by USPHIS grants No. Al 04448 and AI 06729 to R. Haselkorn and USPHS Predoctoral Traineeship to F. J. Kieras. R. Haselkorn is the recipient of a Research Career Development Award from USPHS. This material is taken from a thesis presented 'by F. J. Kieras to the Division of Biological Sciences, Department of Biochemistry at the University of 'Chicago in partial fulfillment of the requirements for the Ph.D. degree. thetic bacterium) and to compare these properties with those of the higher plant enzyme. In the work described below striking similarities in S values (and in 1 case in Km values) to the higher plant enzyme were found with all of the photosynthetic ilmicroorganisms tested. Further characterization of carboxydismutase from Chinese cabbage, which was the subject of an earlier report (10), is described. The Km values, S, and molecular weight are in good agreement with values for carboxydismutase from other plants (23, 24, 26). Materials and Methods Carboxydismutase was prepared and purified from Chinese cabbage [Brassica petsai (Parl)] using a procedure similar to Trown's (24) except that whole leaves rather than chloroplasts were used as starting material. All operations were carried out at 0 to 50. The leaves were harvested at various stages of growth, were washed with distilled water, and the midribs were removed. They were then ground in a meat grindler with 2 times their weight of a pH 7.5 extraction buffer (0.2 M tris-SO4, 1 mM GSH, and 1 mM EDTA). This slurry was then transferred to a Waring Blendor and blended for 3 minutes at top speed. The homogenate was then filtered through 4 to 8 layers of cheesecloth and the filtrate brought to 35 % saturation by addition of solid (NH4)9S04; the pH was adjusted to approximately 7 with 2 N NH4OH. After centrifugation of this suspension for 20 minutes at 8000 rpm in the Servall GSA rotor, the resulting pellet was discarded; the supernatant was brought to 50 % saturation by addition of solid (NH4)2SQ4 and the pH adjusted as described. This was cen1264 www.plantphysiol.org on October 30, 2017 Published by Downloaded from Copyright © 1968 American Society of Plant Biologists. All rights reserved. KIERAS AND HASELKORN-CARBOXYDISMUTASE FROM VARIOUS SOURCES trifuged as described above, and the supernatant discarded. The 35 to 50 % (NH4)2SO4 pellet is crude carboxydismutase. After being dissolved in 0.05 M tris-S04, 1 mm GSH (pH 17.5) the crude enzyme was reprecipitated 3 times by bringing the solution to 50 % saturation of (NH4)2S04, redissolved in 0.05 M tris-SO4, 1 mm GSH (pH 7.5) and dialyzed against the same buffer. This preparation was then purified by chromatography on Sephadex G-200. Generally, a 3 to 5 ml zone containing 200 to 300 mg of protein was layered on a column of dimension 25 cm X 100 cm and eluted with a buffer (0.05 M tris-SO pIT 7.4; 0.2 M (NH4)2S0A; 1 mM EDTA; and 1 mMi 2-mercaptoethanol) used by Trown (24). Five ml fractions were collected and assayed for carboxydismutase activity and the absorbancy at 280 mIX was determined. On some fractions phosphoriboisomerase activity was also assayed using the procedure of Axelrod and Jang (2). Carboxydismutase activity wvas assayed according to a modification of the procedure of Trown (24) in a system of volume 0.20 ml containing the following components: tris-Cl, pH 8.0, 0.1 m; RULDP, 0.5 mm; NaH14CO3 50 mM (0.054 ,uc/,umole); GSH, 1 mM; EDTA, 50 /M; M,gCl.2, 10 mM and varying amounts of protein, usually approximately 10 ug. After incubation at 250 the reaction was terminated by addition of 0.05 ml of 6 N CH3COOH. The reaction mixture was plated on a planchet. dried in a 1000 oven, and counted in a Beckman "Loow Beta" gas flow counter at an efficiency of approximately 20 %. All results are uncorrected for counting efficiency, except where otherwise noted. Appropriate controls lacking the substrate RUDP were also performed. Protein concentrations were determined by the Lowry method (12) using bovine serum albumin (BSA) or lysozyme as standards. In the case of purified preparations, the concentration was determined from the absorbancy at 280 mj.c assuiming the extinction coefficient to be the same as that determined by Trown (24) for spinaclh carboxydismutase. i.e., at a concentration of 1cm