1021-4437/01/4802- $25.00 © 2001 MAIK “Nauka /Interperiodica” 0171 Russian Journal of Plant Physiology, Vol. 48, No. 2, 2001, pp. 171–175. From Fiziologiya Rastenii, Vol. 48, No. 2, 2001, pp. 203–207. Original English Text Copyright © 2001 by Zervoudakis, Georgiou, Angelopoulos. INTRODUCTION Pyruvate kinase (PK; ATP: pyruvate phosphotrans- ferase, EC 2.7.1.40) catalyses the irreversible conver- sion of phosphoenolpyruvate (PEP) to pyruvate in the presence of Mg 2+ and K + , with concomitant synthesis of ATP according to the following reaction: PEP + ADP pyruvate + ATP. The enzyme has been isolated from a wide variety of tissues [1]. The kinetics and regulatory properties of the plant enzyme have been studied mainly in germi- nating or developing castor bean endosperm [2–4], in green algae [5, 6] and less in leaves of higher plants [3, 7]. It is considered to be a key regulatory enzyme in the final steps of aerobic glycolysis and its activity depends on the rates of respiration [8], photorespiration [9], and ammonia assimilation [10, 11]. The existence of two isoenzymes with different immunological, physical, and kinetic properties has also been reported [5, 6]. One isoenzyme was found in the cytosol (PK c ; cytosolic pyruvate kinase) and the other (PK p ; plastid pyruvate kinase) was found in the plastids of seed endosperms as well as in the chloroplasts of green algae and leaves from C 3 plants [5, 7, 12]. Research to this point has been focused mainly on the PK from tissues of C 3 plants and green algae, and, for studying PK, the lactate dehydrogenase-coupled assay [13, 14] has been used. PEPC interferes with this assay, although it does not present a major problem in C 3 plant extracts, where PEPC is low as compared to PK, and can be inactivated by the relatively effective inhibitor maleate [15]. In C 4 plants, however, PEPC activity is much higher as compared to PK, and its interference was eliminated by thermal inactivation of crude extracts of Zea mays [16]. Based on our findings that the purified enzyme has the optimum pH 6.2 [17] and PEPC is inactivated by acidic pH [18], we devel- oped simple extraction and LDH-coupled PK assay methods that least modified PK quaternary structure to provide a way of assessing its physiological properties in crude leaf extracts from C 4 plants. MATERIALS AND METHODS Reagents. Mes, Mops, PVP (10 kD), DTT, β- NADH, ADP, phosphoenolpyruvate, lactate dehydro- genase (from rabbit muscle, salt free form), EDTA were from Sigma (United States), Tris and sea sand from Merck (Germany). All other reagents used were of the highest purity. Plant material. Cynodon dactylon (L.) Pers. plants were grown from rhizomes in soil pots kept outside during summer months or kept in a greenhouse during winter months. Fully expanded mature leaves were taken during daytime hours for the preparation of crude extracts. Mature leaves from C 4 species Setaria verti- cillata (L.) Beauv., Sorghum halepense (L.) Pers., Dig- itaria sanguinalis (L.) Scop., and Amaranthus sp. were collected in fields. Extraction, assay conditions, and temperature effect on PK activity. For enzyme extraction, 1 g of leaf tissue from C. dactylon and from other C 4 species was ground at room temperature in a porcelain mortar with purified Pyruvate Kinase Activity in Crude Extracts of Leaves of Cynodon dactylon and Other C 4 Plants 1 G. Zervoudakis*, C. D. Georgiou**, and K. Angelopoulos* * Department of Biology, Laboratory of Plant Physiology, University of Patras, Patra, 26100 Greece fax +30(61)997228; e-mail: angelop@upatras.gr ** Department of Biology, Section of Genetics, Cell and Developmental Biology, University of Patras, Patra, 26100 Greece e-mail: c.georgiou@upatras.gr Received May 17, 2000 Abstract—A new extraction procedure and an LDH-coupled assay method are presented for the study of pyru- vate kinase (PK) in leaf crude extracts from Cynodon dactylon (L.) Pers and other C 4 plants. Extraction at pH 6.8 and assay at pH 6.2 facilitated the measuring of PK activity by eliminating phosphoenolpyruvate carboxylase interference more effectively than the thermal inactivation or chemical inhibition previously used. The method suggested did not affect the kinetic properties of PK as compared to the purified enzyme from C. dactylon. Key words: Amaranthus sp. - Cynodon dactylon - Digitaria sanguinalis - Setaria verticillata - Sorghum halepense - C 4 plants - pyruvate kinase 1 This article was submitted by the authors in English. Abbreviations: BSA—bovine serum albumin; DTT—dithiothrei- tol; LDH—lactate dehydrogenase; Mops—3-(N-morpholino)pro- panesulfonic acid; PEPC—phosphoenolpyruvate carboxylase; PK—pyruvate kinase; PVP—polyvinylpyrrolidone.