Chloroplast and Cytoplasmic Enzymes

Abstract

Several peaks of aldolase activity are found in the isoelectric focusing pattern of pea (Pisum sativum) leaf chloroplast extracts. One peak, separated by 0.5 pH unit from the major chloroplast aldolase peak, is found when cytoplasmic extracts are focused. The chloroplast and cytoplasmic enzymes have a pH 7.4 optimum with fructose 1,6-diphosphate. In mammalian systems fructose-1,6-diP aldolases from different tissues have kinetic properties related to the function (synthetic or degradative) of the enzyme. Differences in the ratio V.a. fructose-1,6-diP cleavage to Vma. fructose-1,6-diP synthesis led Rutter and coworkers (15) to conclude that muscle aldolase is "more effective in cleaving fructose-1 ,6-diP whereas liver aldolase is relatively more effective in synthesis." Since in green plants chloroplast and cytoplasmic aldolases might also be expected to have opposing functions, it seemed of interest to compare the properties of the plant aldolases. Although spinach whole leaf (8, 14) and chloroplast (7) aldolases and a pea seed aldolase (10,19,20) had been studied prior to the present investigation, no higher plant aldolase known to be cytoplasmic had been characterized. The purpose of the experiments reported here was to characterize the pea leaf chloroplast and cytoplasmic aldolases and to compare the physical and kinetic properties of the isoenzymes.The results indicate that the chloroplast and cytoplasmic aldolases are exceedingly similar enzymes, the pH optima and Michaelis constants for fructose-1 ,6-diP, fructose-1-P, and sedoheptulose-1,7-diP being almost identical. Isoelectric Focusing. Chloroplast and cytoplasmic extracts, prepared as described previously (5) (except that the chloroplast extract was centrifuged 45 min at 500,000g), and containing 10 to 15 mg of protein, were subjected to electrophoresis for 1.5 days in pH 3 to 6 ampholytes at 450 v or pH 4 to 6 ampholytes at 500 v, in a 100-ml LKB isoelectric focusing column at 10 C. Ampholyte and electrode solutions were made up with 5 mm 2-mercaptoethanol. The anode solution (made with 0.5 N H2SO4) was at the bottom of the column. The cathode solution was 0.25 N NaOH. The density gradient was usually made with sucrose. Sorbitol was used in place of sucrose in experiments where aldolase activity was measured as fructose-1,6-diP formation. Fractions (9 drops, 0.7 ml) were collected and analyzed for enzyme activity. The pH of the individual fractions was measured at 0 C using a Radiometer pH meter 26.Purification of Chloroplast and Cytoplasmic Aldolases. Chloroplast and cytoplasmic extracts were prepared as described previously (5) except that the procedure was scaled up for 1 kg of tissue, and the entire shoot of the plant was used. Chloroplasts were usually prepared beforehand and stored at -20 C. The method of Fluri et al. (8) for purification of spinach aldolase was followed, with some modifications. The chloroplast extract, in 10 mm potassium phosphate buffer, pH 7.5, was brought to pH 5.5 with 3.5 M acetic acid, and the supernatant fractio...

Studies of protein-protein interaction using countercurrent distribution in aqueous two-phase systems. Partition behaviour of six Calvin-cycle enzymes from a crude spinach (Spinacia oleracea) chloroplast extract

“…The observed heterogeneity is not caused by entrapment of the enzymes by membrane vesicles or their adhesion to membrane fragments. The presence of a membrane form of RuBPC in spinach chloroplasts is well established [35][36][37][38][39][40], and activities associated with membranes have also been reported for PGK, GAPDH and aldolase from pea (Pisum sativum) chloroplasts [41][42][43] and for FBPase from soybean (Glycine max) chloroplasts [44]. However, the chloroplast extracts used in our experiments ( Fig.…”

Chloroplast and Cytoplasmic Enzymes

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“…This may restrict the GAP to DHAP back conversions. Furthermore, numerous common metabolites inhibit TPI competitively (Anderson, 1971; Grüning et al ., 2014; Flügel et al ., 2017; Li et al ., 2019). In addition, the activity of cytosolic TPI decreases significantly upon treatment with reactive oxygen species, especially H2O2 (Lopez-Castillo et al ., 2016).…”