ABSTRACT
Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris--HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 +/- 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.
Subject(s)
Glycine max/enzymology , Iron/chemistry , Lipoxygenase/chemistry , Apoenzymes/chemistry , Apoenzymes/metabolism , Catalysis , Chelating Agents/chemistry , Electron Spin Resonance Spectroscopy , Iron Chelating Agents/chemistry , Lipoxygenase/metabolism , Metalloproteins/chemistry , Metalloproteins/metabolism , Phenanthrolines/chemistry , Resins, SyntheticABSTRACT
The Smad family of intracellular proteins mediates signals generated by activin and other transforming growth factor beta-related proteins via specific heteromeric complexes of transmembrane receptor serine kinases (1, 2). xSmad2 has been implicated as an activin signal mediator that may participate in transcriptional regulation (3, 4). We have employed an interaction cloning strategy to identify xSmad2-binding proteins and found that calmodulin directly associated with Smads. xSmad2, generated either by in vitro translation or by overexpression in COS cells, specifically bound to calmodulin-agarose; the association was calcium-dependent and required xSmad2 N-terminal residues. In the same assay, xSmad1 and hSmads 2, 3, and 4 also bound to calmodulin-agarose. Furthermore, a calmodulin antagonist, W13, increased expression of the activin-inducible transcriptional reporter, 3TP-Lux, whereas overexpression of calmodulin suppressed this reporter. These observations demonstrate that Smad proteins interact with calmodulin in a calcium-dependent way through conserved N-terminal amino acids and suggest a role for calmodulin in regulating Smad function.
