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Alternate Catalytic States of Bovine Kidney Diamine Oxidase Resolved by Hydroxyapatite Chromatography
Author(s) -
Elgart David M.,
Guzman Creobelle,
Silva Blanca,
Gassner George T.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a52-d
Subject(s) - diamine oxidase , chemistry , enzyme , allosteric regulation , substrate (aquarium) , biochemistry , compartmentalization (fire protection) , biology , ecology
Diamine oxidase (DAO, EC 1.4.3.6) has a crucial role in the down‐regulation of biogenic amines known to be involved in cellular proliferation and differentiation. Through the oxidative diamination of polyamines and production of their corresponding aminoaldehydes, DAO acts as the principal mechanism for down regulating the immediate effects of polyamine synthesis. Various isoforms of copper and TPQ‐dependent diamine oxidases are prevalent in various tissues, where the compartmentalization of these enzymes further defines their physiological functions. We have developed a purification scheme for the isolation of the bovine kidney isoform of diamine oxidase (BKDAO), and find that the purified enzyme can be resolved into two distinct populations by calcium phosphate chromatography. Each form is quite stable in solution, but the interconversion between these two peaks is possible by passage through the column a second time. This salt‐dependence of DAO was further examined by the measurement of steady‐state parameters in presence of various salts (KCl, KPi, NaCl, and NaPi). Global fitting of steady‐state kinetic data recorded over a broad range of substrate and salt concentrations suggests that this enzyme exists in solution in interchangeable allosteric states each having unique sensitivity to substrate inhibition. The close structural homology of BKDAO and other closely related enzymes suggests that this may represent a general catalytic mechanism for this group of enzymes. FUNDING RIMI Program NIH GM0651158