Rudolf Bender

Tendamistat (HOE 467), a tight‐binding α‐amylase inhibitor from Streptomyces tendae 4158

Culture fluids of Streptomyces tendae 4158 (ATCC 31210) contain a new kind of polypeptide alpha-amylase inhibitor, tendamistat (HOE 467). Several methods of isolating this inhibitor are described, including two rapid crystallisation methods, which produce homogeneous material. A characteristic of tendamistat is its tight-binding, pH-independent inhibition kinetics and the specific inhibition of the mammalian alpha-amylase form a stoichiometric 1:1 complex, which cannot be separated into its individual components by sodium dodecyl sulphate or molecular sieve chromatography. Studies of the mode of action reveal that the alpha-amylase-inhibiting activity is linked to the intact disulphide bridges of the inhibitor. It is assumed that the multipoint protein-protein bond exists between the enzyme and tendamistat. It is shown that extracellular tendamistat inhibits amylase formed by streptomyces. We therefore assume a regulatory function in the microorganism. By-products of tendamistat, which possess similar enzyme-inhibiting properties, are also described.

[45] d-Gluconate dehydratase from Clostridium pasteurianum

Publisher Summary This chapter describes the assay, purification procedure, and properties of the enzyme D-gluconate dehydratase that catalyzes the irreversible dehydration of D-gluconate to form 2-keto-3-deoxy-D-gluconate (KDG). The enzyme from C. pasteurianum is inactivated by exposure to oxygen but the activity can be completely restored by incubation with sulfhydryl compounds and ferrous ions. The assay involves three steps: (1) the activation of D-gluconate dehydratase; (2) the incubation of enzyme with D-gluconate; and (3) the colorimetric determination of KDG. The purification procedure involves preparation of crude extract, cetyltrimethylammonium bromide (CTAB) treatment, heat treatment, ammonium sulfate fractionation, diethylaminoethyl (DEAE)-Sephadex chromatography, and Sephadex G-200 chromatography. The molecular weight of the enzyme is 130,000 and the enzyme consists of two subunits of identical size. Activation of the dehydratase requires the presence of both 2-mercaptoethanol (or dithiothreitol or other sulfhydryl compounds) and ferrous ions. D-gluconate dehydratase exhibits a high substrate specificity. The pH optimum of the dehydratase reaction is pH 7.3 to pH 7.8.