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The crystal structure of the Man/Glc-specific seed lectin from Pterocarpus angolensis was determined in complex with methyl-␣-D-glucose, sucrose, and turanose. The carbohydrate binding site contains a classic Man/ Glc type specificity loop. Its metal binding loop on the other hand is of the long type, different from what is observed in other Man/Glc-specific legume lectins. Glucose binding in the primary binding site is reminiscent of the glucose complexes of concanavalin A and lentil lectin. Sucrose is found to be bound in a conformation similar as seen in the binding site of lentil lectin. A direct hydrogen bond between Ser-137(OG) to Fru(O2) in Pterocarpus angolensis lectin replaces a water-mediated interaction in the equivalent complex of lentil lectin. In the turanose complex, the binding site of the first molecule in the asymmetric unit contains the ␣Glc1-3␤Fruf form of furanose while the second molecule contains the ␣Glc1-3␤Frup form in its binding site.Lectins are carbohydrate-binding proteins other than immunoglobulins that display no enzymatic activity toward the recognized sugars. Lectins are found in all kingdoms of life ranging from micro-organisms (1-4) to plants (5-8) and animals (9 -15). The biological functions associated with their carbohydrate binding activities are diverse. Many different, evolutionary unrelated, lectin families have been identified.The legume lectin family has proven to be a very useful model system to study protein carbohydrate interactions. Their highly variable carbohydrate specificity makes them ideal to study the structural basis of carbohydrate specificity. Because they are often expressed in high yields in legume seeds, they can easily be purified in amounts suitable for experimental approaches that require large amounts of protein such as microcalorimetry and x-ray crystallography. Indeed, the crystal structures of 22 legume lectins and three other family members without lectin activity have been determined by x-ray crystallography. For most of these, complexes with one or more carbohydrates have been studied (8 -9, 16 -24).The seed lectin from the tropical legume Pterocarpus angolensis (bloodwood tree) belongs to the mannose/glucose specificity group that contains several well studied members such as concanavalin A and the lectins from Lathyrus ochrus, Lens culinaris, Pisum sativum, and Dioclea grandiflora as well as several other Canavalia and Dioclea species. Although they share a common monosaccharide specificity, all these lectins differ in the details of their specificity for oligosaccharides. In the current paper we present the crystal structures of the complexes of Pterocarpus angolensis lectin (PAL) 1 with glucose, sucrose, and turanose, which were shown to inhibit the hemagglutination activity of this lectin.

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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...