Purification and Characterization of Pea Chloroplastic Phosphoriboisomerase

Abstract

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.Phosphoriboisomerase (EC 5.3.1.6) participates in the interconversion ofribose-5-phosphate and ribulose-5-phosphate in both the oxidative and reductive pentose phosphate pathways. We have now developed a rapid procedure for the isolation of the chloroplastic phosphoriboisomerase from pea (Pisum sativum) shoots. Although the enzyme has been purified from other sources (24) and spinach ( 18), this is the first time that the enzyme has been purified to apparent homogeneity from the chloroplasts of a higher plant.We have characterized the enzyme with respect to kinetic parameters and inhibitor constants, and we have determined the N-terminal sequence through 34 amino acids. Phase partitioning experiments indicate that the chloroplast phospho-'In memory of Professor Mordhay Avron.

Enzyme-enzyme interaction in the chloroplast: Glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase and aldolase

“…When sedoheptulose-l,7-bisphosphate was substrate the concentration was 0.1 mM. Fructosebisphosphatase was assayed as described by Marques et al (1987) but at pH 8.8, triose-P isomerase as described by Anderson and Advani (1970), phosphoglycerate kinase as described by Macioszek et al (1990), ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) as described by Marques et al (1987), and phosphoribulokinase as described by Skrukrud et al (1991). Change in A340 was followed on a Cary 210 or a Cary 219 recording spectrophotometer.…”

“…Chloroplasts for the isoeleetric-focusing experiments were isolated as in (Skrukrud et al 1991) from pea shoots, except that 0.1% glycine-KOH (pH 7), 5 mM mercaptoethanol, 1/3 M sorbitol replaced the resuspension buffer used in the final washing step. The pelleted chloroplasts were stored at -20~ Extract was prepared by adding 0.1% glycine-KOH (pH 7), 5 mM mercaptoethanol to the thawed pellet.…”

“…Pea (Pisum sativum L.) plants were grown as described in (Skrukrud et al 1991) from seed (cv. Little Marvel; J. MoUema and Son, Grand Rapids, Mich., USA), except that the shoots used for the preparation of aldolase for the fluorescence experiments were an unnamed local variety obtained from the Hongqiaoxiang Farm near Shanghai.…”

“…Change in A340 was followed on a Cary 210 or a Cary 219 recording spectrophotometer. Phosphoriboisomerase activity was assayed as increase in A280 with ribose-5-phosphate as substrate as described by Skrukrud et al (1991).…”

“…For purification of triose-P isomerase, chloroplasts were isolated from about 0.9 kg of 10-to 13-d-old pea seedling shoots as described previously (Skrukrud et al 1991) except that the chloroplasts were washed twice by resuspension in the original grinding media and collected by centrifugation. The pelleted chloroplasts were lysed by suspension in 10 mM potassium phosphate (pH 7.0), 10 mM mercaptoethanol, and membranes and debris were removed by centrifugation (27000.g, 15min).…”