title: Assay optimization and kinetic profile of the human and the rabbit isoforms of 11beta-HSD1.
abstract: Assay conditions for the 11beta-hydroxysteroid dehydrogenase have been optimized by adding phospholipids in the media buffer to increase and stabilize the enzymatic activity. The presence of phospholipids greatly facilitates the study of the binding of cortisone and NADPH at the enzyme catalytic site. Kinetic analyses conducted with the human and rabbit enzyme isoforms suggest that both enzymes behave according to an ordered sequential bi-bi mechanism where the NADPH is the first to bind at the active site followed by cortisone. The equilibrium dissociation constant, K(i)a as well as the apparent Michaelis-Menten constants K(m)a, K(m)b, k(cat)a, and k(cat)b for NADPH and cortisone, have been determined to be 147.5 microM, 14.4 microM, 43.8 nM, 0.21 min(-1), and 0.27 min(-1), respectively, for the human enzyme and 41.1 microM, 3.1 microM, 161.7 nM, 0.49 min(-1), and 0.52min(-1), respectively, for the rabbit enzyme.
authors: Pfizer Global Research and Development, La Jolla Laboratories, 10628 Science Center Drive, San Diego, CA 92121, USA.
authors: Arturo
authors: A
authors: Castro
authors: Jeff X
authors: JX
authors: Zhu
authors: Gordon R
authors: GR
authors: Alton
authors: Paul
authors: P
authors: Rejto
authors: Jacques
authors: J
authors: Ermolieff
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000737:chemistry*; D000818:Animals; D004789:Enzyme Activation; D004795:Enzyme Stability; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D011817:Rabbits; D013045:Species Specificity
chemical_list: D007527:Isoenzymes; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1