Dans les chemins metaboliques d'Embden-Meyerhof et du phosphate pentose, les enzyrnes suivantes sont presentes: la dehydrogenase glucose-6-phosphate ...
Carbohydrate metabolism in Acanthamoeba castellanii. 1. The activity of key enzymes and [14C]-glucosemetabolism1
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LANSING M. PRESCOTT? HAROLD E. HOYME, DARLENE CROCKETT, AND ELENA HUI Department of Biology, Augustana College, Sioux Falls, South Dakota 57102 Accepted May 28, 1973 PRESCOTT, L. M., H. E. HOYME, D. CROCKETT, and E. HUI. 1973. Carbohydrate metabolism in Acanthamoeba castellanii. 1. The activity of key enzyrnes and ['4C]-glucose metabolism. Can. J. Microbiol. 19: 1131-1136. The specific activities of a number of the key enzyrnes involved in carbohydrate metabolism in Acantha1noeba castellanii (Neff clone 1-12) have been determined. The following Embden-Meyerhof and pentose phosphate pathway enzymes were present: glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, hexokinase, phosphofructokinase, hexose diphosphatase, aldolase, glyceraldehydephosphate dehydrogenase, pyruvate kinase, and pyruvate-phosphate dikinase. The following tricarboxylic acid cycle enzyrnes were also found: citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, and malate dehydrogenase. The degradation of glu~ose-U-'~C to 14C02was was 3.4-fold greater than anaerobic producexamined. Aerobic 14C02production from glu~ose-U-'~C tion. The data provide further evidence that the Embden-Meyerhof, pentose phosphate, and tricarboxylic acid cycle pathways are probably functional in A. castellanii. PRESCOTT, L. M., H. E. HOYME, D. CROCKETT et E. HUI. 1973. Carbohydrate metabolism in Acanthamoeba castellatzii. 1. The activity of key enzyrnes and [14C]-glucosemetabolism. Can. J. Microbiol. 19: 1131-1136. Nous avons determine les activitks spkcifiques de plusieurs enzyrnes clees impliquies dans le metabolisme des carbohydrates chez Acanthatnoeba castellanii (Neff clone 1-12). Dans les chemins metaboliques d'Embden-Meyerhof et du phosphate pentose, les enzyrnes suivantes sont presentes: la dehydrogenase glucose-6-phosphate, la dehydrogenase 6-phosphogluconate, l'hexokinase, la phosphofructokinase, la diphosphatase hexose, l'aldolase, la dehydrogenase glyceraldehyde phosphate, la kinase pyruvate, et la dikinase pyruvate-phosphate. Dans le cycle de l'acide tricarboxylique, les enzyrnes suivantes sont aussi trouvees: la synthase citrate, l'aconitase, la dehydrogenase isocitrate, la dehydrogenase succinate, l'hydratase fumarate et la dehydrogenase malate. La degradation du g l ~ c o s e - U - ~en ~ C14C02 fut examinee. La production aerobique du 14C02 a partir du glucose-U-14C est 3.4 fois superieure que la production anaerobique. Les donnees fournissent d'autres evidences que les chemins metaboliques d'Embden-Meyerhof, du phosphate pentose et du cycle de l'acide tricarboxylique sont probablement praduit par le journal] fonctionnels chez A. castellanii.
Introduction The free-living soil amoeba Acantlzamoeba castellanii has recently been the focus of a number of investigations of metabolic changes during encystment and has proved to be an excellent system for the study of molecular changes during differentiation (6, 23, 27, 30, 36). The general nature of carbohydrate metabolism in A. castellanii has been partially elucidated (1, 7, 14, 18, 24, 32, 34). However, relatively few detailed enzymological and radioisotopic studies of carbohydrate metabolism in this protozoan have been published. Such studies are of importance both in a comparative biochemical sense and to provide a firm foundation for the biochemical study of encystment. Therefore we have exmined some of the key enzymes in carbohydrate metabolism and the catabolism of [14C]-glucose to l4co,. 'Received March 19, 1973. 2Author to whom all reprint requests should be sent.
Materials and Methods Materials Glucose-U-14C, hyamine hydroxide, and PCS solubilizer counting solution were purchased from the Amersham/Searle Corporation. Enzymes used in enzyme activity determinations were obtained from the Sigma Chemical Company and the Boehringer Mannheim Corporation. Culture Methods A. castellunii (Neff clone 1-12) was cultured axenically at 30°C in 200 ml of NefF's optimal growth medium (25) contained in siliconized 500-ml Erlenmeyer culture flasks. The flasks were agitated at 100 rpm by a New Brunswick gyrotory shaker. The media was prepared using the modified procedure described by Byers et al. (6). Usually 6- to 7-day-old cultures were used in experiments because more cysts were formed during longer incubation periods. Cell-fLee Extract Preparation and Mitochondria1 Isolation Extracts were prepared in two ways. When glycolytic or pentose phosphate pathway enzyrnes were to be studied the following procedure was used. The cells from one or two culture flasks were collected by centrifugation at O°C and 650 g for 10 min in a Sorvall refrigerated centrifuge. The pellet was resuspended gently in about 20 ml of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-HC1
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M mercaptoethanol buffer pH 7.2, containing 2 x and the cells were collected by centrifugation in the Sorvall for 5 min at 755 g. The washed cells were resuspended in 8 to 10 rnl of the above buffer and homogenized for 30 s at about 0°C with an aluminum cold-shoulders cooling cell and a Branson sonifier cell disruptor, model W185, equipped with a 3 in. probe (power setting number 7, about 75-80 W power). The homogenate was then clarified by centrifugation at 25 000g for 10 min at P C in a Sorvall refrigerated centrifuge. The supernatant was dialyzed for a total of 4 to 6 h at 4°C against two, 500-ml changes of the same buffer used for extraction. When tricarboxylic acid cycle enzymes were to be studied, the mitochondrial fraction was prepared using a buffer containing 0.3 M mannitol essentially as described by Lloyd and Griffiths (18). Sintered glass funnels (150 ml volume) with a pore size of 10-15 pm were used for cell disruption. After a preliminary 1000 g centrifugation, the mitochondria were collected by centrifugation at IOOOOg for 15 min and then washed once (18). The washed mitochondrial pellet was resuspended in 6.0 ml of 0.02 M potassium phosphate buffer, pH 7.2, containing M ethylenediaminetetraacetic acid (EDTA). 1 x The mitochondria were then disrupted by treatment with a Branson sonifier cell disruptor at 0°C and about 75-80 W power for 2 min after the addition of 1 ml of 25 pm diameter glass beads. The homogenate was clarified by centrifugation at 650g for 5 min at 0°C. The disrupted mitochondrial fraction was then dialyzed against two, 2-liter changes of the previously mentioned phosphate buffer for a total of 4 h and immediately assayed to minimize activity losses. Enzyme Assuys The activities of some of the enzymes in cell-free extracts were determined according to the procedures given in the following references: pyruvate-phosphate dikinase (28); citrate synthase (E.C. 4.1.3.7), assay using 5,5'dithiobis-(2-nitrobenzoic acid) (10); nicotinamide adenine dinucleotide phosphate (NADP)-dependent isocitrate dehydrogenase (E.C.1.1.1.42) (15); nicotinamide adenine dinucleotide (NAD)-dependent isocitrate dehydrogenase (E.C. 1.1.1.41) (16); succinate dehydrogenase (E.C. 1.3.99.1) (17); fumarate hydratase (E.C.4.2.1.2) (13); malate dehydrogenase (E.C.1.1.1.37), with pH at 7.4 instead of 8.8 (37); and hexose diphosphatase (E.C. 3.1.3.11), with pH 8.5 instead of 8.9 (8). Aconitate hydratase (E.C. 4.2.1.3) activity was determined by following the increase in absorbance at 240 nm uDon the conversion of isocitrate to cis-aconitate in a Aodification of the procedure of Fansler and Lowenstein (9). The reaction mixture (3.0 ml volume) contained TrisHCI buffer, pH 7.4, 60 pmol; NaCI, 30 pmol; D,L-isocitrate, I pmol; and enzyme. Glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) and phosphogluconate dehydrogenase (E.C.1.1.1.44) were assayed essentially as described by Eidels and Preiss (8). The reaction mixture (3.0 ml volume) contained the following components: Tris-HC1 buffer, pH 7.5, 100 pmol; MgCI2, I0 pmol; reduced glutathione, 5 pmol; NADP, 1 pmol; either glucose-6-phosphate or 6-phosphogluconate, 5 pmol; and enzyme. The reduction of NADP was followed spectrophotometrically at 340 nm. Hexokinase (E.C.2.7.1.1) activity was determined by
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measuring NADP reduction in the presence of glucose-6phosphate dehydrogenase (8). The reaction mixture contained in 2.0 ml the following: Tris-HC1 buffer, pH 7.5, 200 pmol ; mercaptoethanol, 10 pmol ; MgCI,, I0 pmol ; glucose, 5 pmol; ATP, 6 pmol; NADP, 2 pmol; excess glucose-6-phosphate dehydrogenase; and enzyrne extract. The control consisted of the total mixture minus adenosine 5'-triphosphate (ATP). Phosphofructokinase (E.C.2.7.1.11) activity was followed spectrophotometrically with a coupled enzyme system (2). The reaction mixture (2.0 ml) contained TrisHCI buffer, pH 7.5, 200 pmol; MgCI,, IOpmol; mercaptoethanol, I0 pmol; fructose-6-phosphate, 4 pmol; NADH, 0.3 pmol; ATP, 2 pmol; an excess of aldolase, triosephosphate isomerase, and a-glycerophosphate dehydrogenase; and enzyrne extract. The activity of fructosediphosphate aldolase (E.C. 4.1.2.13) was determined by modification of the coupled systems of Rutter et nl. (31). The reaction mixture (1.0 ml volume) contained either 50 prnol of Tris-HC1 buffer, pH 7.5 (class I assay) or 50 pmol of Tris-HC1 buffer, pH 7.5, containing 50 pmol KC1 and 0.1 pmol mercaptoethanol (class I1 assay); fructose-l,6-diphosphate, 2 pmol; NADH, 0.2 pmol; an excess of triosephosphate isomerase and cr-glycerophosphate dehydrogenase; and enzyme extract. In some class I1 aldolase assays, 2 prnol of EDTA were included. The oxidation of reduced NAD (NADH) was followed spectrophotometrically at 340 nm. The control assay mixtures contained all components except fructose-l,6-diphosphate. Glyceraldehydephosphatedehydrogenase(E.C.l.2.1.12) was determined svectrovhotometricalIy by following NAD reduction (5). he reaction mixture
