Properties of Pyruvate Kinase from Soybean Nodule

The properties ofpyruvate kinase from soybean ( Glycinemax L.) nodule cytosol were examind to determine what influence the N2 fixation process might have on this supposed key control enzyme. A crude enzyme preparation was prepared by chromatography of cytosol extract on a diethylaminoethyl-cellulose column. ATP and citrate at 5 mm concentrations inhibited pyruvate kinase 27 and 34%, respectively. Enzyme activation was hyperbolic with respect to both K+ and NH4' concentrations. In the presence of physiological concentrations of K+ and high phosphoenolpyruvate (PEP) concentrations, NH4' inhibited enzyme activity. Comparisons of kinetic parameters ( V. and apparent Ka) for NH4' and K' with inhibition curves indicated that inhibition was very likely a result of competition of the ions for activation site(s) on the pyruvate kinase. In addition, apparent Ka (monovalent cation) and Km (PEP) were influenced by PEP and monovalent cation concentrations, respectively. This effect may reflect a fundamental difference between plant and animal pyruvate kiaases. It is concluded that control of cytosol pyruvate kinase may be closely related to reactions involved in the assimilation of NH+. Pyruvate kinase occupies a key position in the regulation of carbohydrate metabolism (4, 6, 29). Control of pyruvate kinase activity is often exerted through ADP levels and activators like AMP and fructose 1,6-diP and inhibitors such as organic acids and ATP. Posttranslational modification (phosphorylation) may also be important (14). In gluconeogenic animal tissues, the enzyme is thought to be critical in regulating the flow of carbohydrates between the degradative glycolytic and synthetic gluconeogenic pathways (4, 29). The same may be true for the plant enzyme (4). Regardless of whether gluconeogenesis is operative in the nodule, the availability of carbon skeletons and cofactors for ammonium assimilation could be influenced by pyruvate kinase activity. In addition, pyruvate kinases from the majority of plant and animal sources examined have been shown to have an absolute requirement for a monovalent cation for activity. Ammonium ion is a strong activator (10). As ammonia is the primary product of N2 fixation, and because of the presumed role of pyruvate kinase in regulation ofcarbohydrate metabolism, regulatory properties of the soybean nodule cytosol enzyme were examined. MATERIALS AND METHODS Materials. Soybean (Glycine max L. var. Chippewa) root nodules were obtained from 25-day-old plants grown as described by Evans et al. (5). Commercial rhizobial inoculum was purchased from Nitragin (Milwaukee). Commercial enzymes, nucleotides, and phosphorylated sugars were obtained from Sigma Chemical 'This work was supported by a National Science Foundation Postdoctoral Energy-Related Fellowship 76-17954 to J. P. and by the OSU Agricultural Experiment Station. The contents ofthis paper were presented at the 1977 meetings of the ASPP in Madison, Wisconsin. Co. (St. Louis, Mo.). Tetramethylammonium hydroxide was purchased from Matheson, Coleman and Bell (Norwood, Ohio). Imidazole (Sigma) was recrystallized from ethyl acetate. PVP (Polyclar AT) was obtained from GAF Corp. (New York) and washed with acid (15). All chemicals were reagent grade. Enzyme Assays. Unless otherwise stated, pyruvate kinase assays contained 100 mm imidazole (pH 7.5, maleic acid), 50 mm TMAH2-maleate, 100 mM KCI, 10 mM MgCl2, 1 mm cyclohexylamine PEP, and 2 mM Tris ADP (prepared from Ba2ADP [21]). Total maleate concentration was 60 mm. Assays coupled to lactate dehydrogenase also contained 0.16 mm NADH and 8 units of rabbit muscle lactate dehydrogenase. In some studies the 2,4dinitrophenyihydrazine colorimetric assay (8, 21) was employed and reactions were terminated after 10 min. ADP and PEP concentrations were raised to 2.5 and 1.5 mm, respectively, for these assays. In both assays the reaction velocity was proportional to enzyme concentration over the range of concentrations used. Reactions were run at 30 C. In all cases, reaction mixtures without ADP were included in order to correct for PEP carboxylase activity. PEP carboxylase was assayed using a malate dehydrogenase enzyme couple. Assays contained 100 mm imidazole HCI (pH 7.5), 2 mm cyclohexylamine PEP, 5 mm MgCl2, 0; 16 mm NADH, 10 mm KHCO3, and 20 units of beef heart malate dehydrogenase. Coupled assays were performed with a Cary 11 or Carya 118 spectrophotometer. The Cary 118 was used for colorimetric assays and spectral studies. One unit of enzyme activity is defined as I ,umol of product/min at 30 C. Plant Extracts. All isolation steps were performed at 0 to 4 C. In a typical extraction, 18 g of nodules were extracted by grinding with 3 volumes of grinding medium and 6 g of insoluble PVP in a mortar and pestle. Grinding medium consisted of a 3:1 (v/v) mixture of buffer (100 mm imidazole-phosphate [pH 7.51 and 1.33 mM DTT)-glycerol. The extracts were filtered through four layers of cheesecloth and centrifuged 15 min at 37,000g. The supernatant was removed and 30 ml applied to a column (3 x 4 cm) ofDEAEcellulose. The DEAE column was prepared by equilibrating with a 3:1 (v/v) mixture of buffer (10 mm imidazole-phosphate [pH 7.51 and 0.13 mm DTT)-glycerol (equilibrating medium). After addition of the enzyme, the column was washed with 2 column volumes of the equilibration medium, 0.075 M with respect to KCI. One column volume of equilibration medium, 0.175 M with respect to KCI, removed the single peak ofpyruvate kinase activity (7.5-10 units) from the column. Column flow rate during the entire procedure was 60 ml/hr. The specific activity of pyruvate kinase was increased 3.5-fold (5.2-fold purification) over the crude extract when protein was estimated by the method of Lowry et al. (16) after trichloroacetic acid precipitation. The enzyme preparation was frozen at -79 C and retained full activity for at least 2 months. The preparation did not lose activity when thawed and kept at ice bath temperature for 1.5 hr. Carboxylase activity under standard pyruvate kinase assay conditions (with maleate) was 8 to 2 Abbreviations: TMAH-maleate: maleic acid titrated to pH 7.5 with tetramethylammonium hydroxide; OAA: oxaloacetate; PEP: phosphoenolpyruvate; cytosol: plant cell cytoplasm. 909 www.plantphysiol.org on January 14, 2018 Published by Downloaded from Copyright © 1978 American Society of Plant Biologists. All rights reserved.