A New Purification Procedure for Fumarase Based of Affinity Chromatography. Isolation and Characterization of Pig-Liver Fumarase

Abstract

A new procedure has been developed for the isolation of fumarase (EC 4.2.1.2). It is described for the purification of pig heart and liver enzyme. Pyromellitic acid has been covalently coupled to Sepharose-4B with diaminopropanol as spacer arm. When a dialysed 0.55 saturated ammonium sulphate precipitate is applied to the column, in Tris-acetate buffer, pH 7.3, fumarase remains quantitatively bound. It is eluted by competition, together with a few other proteins, by the natural product L-malate. Malate is removed from the eluate by dialysis. After this highly efficient purification step the enzyme is very easily crystallized. The final yield is 67 % for both pig heart and liver preparations. The specific activity of fumarase purified from both tissues is found to be the same.Polyacrylamide gel electrophoresis in dodecylsulphate shows one single band corresponding with a subunit molecular weight of 48 500. A single band is also obtained by electrophoresis in acid urea. This new procedure based on biospecific affinity chromatography allows a fast and easy preparation of gram quantities of fumarase.The enzyme fumarase catalyses the reversible hydration of fumaric acid to L-malic acid. It is involved in the mitochondria1 citric acid cycle as well as in many metabolic processes which occur in the cytoplasm.An easy purification method has been reported earlier for pig heart fumarase by Kanarek and Hill [l]. The critical step in this preparation is the ability to crystallize this enzyme from a rather impure solution (about 1 %), i.e. immediately after the ammonium sulphate steps (0.35 -0.55 saturation). However, when other organisms are considered this procedure usually fails and even with extracts from pig liver, crystallization does not occur.We now report a simple and fast purification technique based on affinity chromatography, using a column with pyromellitic acid as affinity ligand, coupled to the classic Sepharose-4B matrix. The method is described here for pig heart and liver preparations. Purification of the enzyme from other organisms has been successful and will be reported elsewhere. EXPERIMENTAL PROCEDURE MatevialsPig hearts and livers were immediately chilled in ice or frozen at the slaughterhouse. Sepharose-4B EHZJWX. Fumarase, fumarate hydratase (EC 4.2 1.2) was obtained from Pharmacia Fine Chemicals. Pyromellitic acid was from Merck and was used without further purification. l-Cyclohexyl-3-(2-morpholinoethy1)carbodiimide metho-p-toluenesulfonate was from Aldrich. N,N-Dimethylformamide (Merck) and 2-mercaptoethanol (Fluka AG) were freshly distilled before use. Urea solutions were freed from cyanates by passing them through an Amberlite MB-3 column (Rohm-Haas Comp.) ; final concentrations were calculated by way of refractive index. Acrylamide and N,N '-methylene-bisacrylamide were purchased from Fluka AG ; the latter was recrystallized from chloroform. L-Malate and Trizma base (Tris) were from Sigma Chemical Co. All other reagents were 'pro analysis' grade and used without further purification.All pH me...

The visualization by affinity electrophoresis of a specific association between the consecutive citric acid cycle enzymes fumarase and malate dehydrogenase

“…Interactions between previously purified cycle enzymes were found to become apparent exclusively in conditions which are considered to reduce the water concentration or to alter the physical properties of water in the vicinity of the enzymes, thereby imitating the actual in situ environment. Two experimental [38,441. These techniques allowed the detection of associations, which in all instances were proven to be specific, between fumarase and mitochondrial malate dehydrogenase [38,441, mitochondrial malate dehydrogenase and citrate synthase [34,35,38,39,631, fumarase and citrate synthase [38], citrate synthase and mitochondrial aconitase [62], between NAD-dependent isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex [64], as well as the 2-oxoglutarate dehydrogenase complex and succinate thiokinase [40].…”

“…Two experimental [38,441. These techniques allowed the detection of associations, which in all instances were proven to be specific, between fumarase and mitochondrial malate dehydrogenase [38,441, mitochondrial malate dehydrogenase and citrate synthase [34,35,38,39,631, fumarase and citrate synthase [38], citrate synthase and mitochondrial aconitase [62], between NAD-dependent isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex [64], as well as the 2-oxoglutarate dehydrogenase complex and succinate thiokinase [40]. The latter two associations, both involving the bulky 2-oxoglutarate dehydrogenase complex, could however also be assessed by simple gel filtration or cosedimentation experiments.…”

“…Purity of these enzymes was confirmed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate according to Laemmli [49]. Activity measurements were performed as described before [38].…”

“…Both mitochondrial and cytoplasmic malate dehydrogenase were covalently immobilized on Sepharose 4B (Pharmacia) as previously described [38]. Wet Sepharose (2.5 g) was activated with 1 g cyanogen bromide.…”

“…This approach offers several advantages over our previously described affinity chromatographic technique [Beeckmans, S. & Kanarek, L. (1981) Eur. J .…”