Distinction between Cytosol and Chloroplast Fructose-Bisphosphate Aldolases from Pea , Wheat , and Corn Leaves

A reinvestigation of cytosol and chloroplast fructose-1,6-bisphosphate (FBP) aldolases from pea (Pisum sativum L.), wheat (Triticum aestivum L.) and corn leaves (Zea mays L.) revealed that the two isoenzymes can be separated by chromatography on diethylaminoethyl (DEAE)-cellulose although the separation was often less clear-cut than for the two aldolases from spinach leaves. Definite distinction was achieved by immunoprecipitation of the two isoenzymes with antisera raised against the respective isoenzymes from spinach leaves. The proportion of cytosol aldolase as part of total aldolase activity was 8, 9, 14, and 4.5% in spinach (Spinacia oleracea L.), pea, wheat, and corn leaves, respectively. For corn leaves we also obtained values of up to 15%. The K. (FBP) values were about 5-fold lower for the cytosol (1.1-2.3 micromolar concentration) than for the chloroplast enzymes (8.0-10.5 micromolar concentration). The respective Km (fructose-i-phosphate, FIP) values were about equal for the cytosol (1.0-23 millimolar concentration) and for the chloroplast aldolase (0.6-1.7 millimolar concentration). The ratio V (FIP)/V (FBP) was 0.20 to 0.27 for the cytosol and 0.07 to 0.145 for the chloroplast aldolase. Thus, cytosol and chloroplast aldolases from spinach, pea, wheat, and corn leaves differ quite considerably in the elution pattern from DEAEcellulose, in immunoprecipitability with antisera against the respective isoenzymes from spinach leaves, and in the affinity to FBP. In green leaf cells, sugar phosphate metabolism takes place in the cytosol and in chloroplasts (e.g. 26). Thereby, the chloroplast envelope controls a limited exchange of sugar phosphates and other molecules either by diffusion or translocator mediated countercurrent exchange (9, 15). Since the first discovery of cytosol and chloroplast specific isoenzymes of sugar phosphate metabolism in the respective cell compartments (1), the view has developed that two isoenzymes exist for virtually all reactions of glycolysis, gluconeogenesis, and the oxidative pentose phosphate cycle, with one isoenzyme located in the cytosol and the other in chloroplasts (23). Just a few key enzymes of the Calvin cycle are present in the chloroplasts exclusively, e.g. ribulose bisphosphate carboxylase/oxygenase and ribulose-5-P kinase. While all pairs of isoenzymes of sugar phosphate metabolism differ in charge, many of them have very similar molecular sizes and catalytic properties. This may imply that they are different but very similar proteins. The ratter may apply particularly to the two aldolases of pea and corn which were reported to have virtually identical mol wt, substrate affinities, and terminal amino acid sequences (3, 4, 12). However, recent investigations ' Present address: Botanisches Institut, Technische Universitait Darmstadt, D-6 100 Darmstadt, Schnittspahnstraf3e, FRG. ofthe two spinach aldolases indicated rather profound differences between these two isoenzymes as deduced from their subunit mol wt, from the lack of extensive immunochemical crossreaction, from peptide analyses after tryptic digestion, and from terminal amino acid sequencing (17, 20). In leaves of wheat and corn, it was difficult to identify the cytosol aldolase by conventional methods (17, 22) so that even the existence of separate cytosol and chloroplast aldolases had to be questioned. For this reason we have applied an improved combination of ion-exchange chromatography and immunoprecipitation to identify the two pea, wheat, and corn aldolases. Quantitative estimates of the two aldolases in green leaves are reported. In addition we found large differences in the apparent affinity for FBP2 but not in the apparent affinity for FIP and in the V(FlP)/ V(FBP) ratio for the two spinach, pea, wheat, and corn aldolases. MATERIALS AND METHODS Materials. Spinach (Spinacia oleracea L., type Monopa), pea (Pisum sativum L., type Kleine Rheinlanderin), wheat (Triticum aestivum L., type Kolibri), and corn (Zea mays L., type Inrafruh) were grown from seeds in greenhouses. Leaves (or shoots in the case of pea) of well developed plants were harvested and used immediately for analyses. Separation of Cytosol and Chloroplast Aldolases. The two aldolases were separated by anion-exchange chromatography on DEAE-cellulose. About 20 g of tissue were homogenized for 1 min at 4C in 4to 5-fold ofgrinding buffer (10 mm K-phosphate, pH 8.6, and 10 mM f3-mercaptoethanol) using a Waring Blendor. Corn leaves were homogenized for an additional minute with a Virtis "45" homogenizer in order to break the bundle sheath cells. The homogenates were squeezed through two layers of Miracloth and centrifuged for 12 min at 48,000g. The supernatant was readjusted to pH 8.6 and diluted with 10 mm f3mercaptoethanol, pH 8.6, to a final conductivity of 3 mS or less. The solution was immediately applied to 50 ml of wet packed DE32-cellulose (Whatman Lab. Sales, Springfield Mill, Maidstone, Kent/England) and equilibrated with grinding buffer on a funnel under suction. The resin was packed into a 3 cm wide glass column and proteins were eluted by a linear 200 ml gradient of0 to 0.5 M KCI in grinding buffer overnight. Fractions of 3 ml were collected and assayed for aldolase activity (see below) and the KCI concentration measured by conductimetry. Assays and Immunoprecipitation of Aldolases. Fructose-bisphosphate aldolase (EC 4.1.2.13) was measured spectrophotometrically at 20°C in a standard assay by coupling the reaction to the glycerol-3-P dehydrogenase (NAD+) (EC 1.1.1.8) reaction according to Wu and Racker (29). The reaction mixture of 1 ml 2Abbreviations: FBP, fructose-1,6-bisphosphate; FIP, fructose-lphosphate. 301 https://plantphysiol.org Downloaded on December 31, 2020. Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved. SCHNARRENBERGER AND KRUGER contained 50 mm Tris-HCl, pH 7.5, 4.5 mM MgCl2; 1 mm EDTA; 1 unit each of triosephosphate isomerase and glycerol-3-P dehydrogenase; 250 gM NADH, and 1.0 mM FBP trisodium salt (Boehringer, Mannheim, FRG). Activity of FIP aldolase was measured accordingly except that Fl P disodium salt (Boehringer, Mannheim, FRG) was substituted for FBP. The enzyme activity of 1 unit is equivalent to the cleavage of 1 Mmol FBP (= 2 umol NADH) or F1P (= 1 Mmol NADH) per min. To distinguish between cytosol and chloroplast aldolases, one ofthe two aldolases was removed by immunoprecipitation before assaying enzyme activity. Antisera against the cytosol and chloroplast aldolase were prepared as described previously ( 17). For immunoprecipitation 20 Ml of an aldolase preparation and 20 Ml of antiserum were incubated for 1 min in an Eppendorf reaction vessel. Five ,l of a 12% (v/v) suspension of Staphylococcus aureus Cowan I cells (immunoprecipitin from Bethesda Research Laboratory, Bethesda, MD) were added to bind immune complexes to protein A on the surface of these cells. After centrifugation in a table top centrifuge for 1 min, the residual enzyme activity in the supernatant was determined in the standard assay.