Diamine oxidase (DAO) activities are elevated in boundaries separating quiescent and rapidly dividing cells where the transformation of molecular oxygen and biogenic amines to aminoaldehydes and hydrogen peroxide by these enzymes may play a significant role in the regulation of cell division in normal and cancerous tissues. Biochemical studies of this enzyme will help to elucidate the molecular mechanism of cellular response to changes in polyamine concentration in normal and disease states. Here we propose to 1) develop and optimize purification schemes for diamine oxidase from bovine liver and kidney and evaluate the substrate specificities of these isoforms 2) identify the role of manganese in ligand-binding and the catalytic mechanism, 3) characterize the heparin, and nucleic acid-binding interactions of DAO and the modulation of its catalytic activity associated with these biopolymers. The primary structure, glycosylation, cofactor content, specific activity, and substrate specificities of the purified isozymes will be compared. Catalytic reaction mechanisms of the purified enzymes will be studied through a combination of ligand binding, steady state, and pre-steady state kinetic measurements. DNA, RNA, heparin, and metal binding mechanisms, as well as the polynucleotide sequence binding specificity of these isozymes will be investigated by using a combination of titrametric, stopped-flow and continuous flow measurements. The role of polynucleotide, metal, and heparin-binding in catalysis will be established through the measurement of ligand-specific effects on steady-state kinetic parameters and reaction product distributions. The influence of nucleic acid binding on individual reaction steps will be established by observing changes in the kinetics of the oxidative and reductive half reactions by stopped-flow spectroscopy. The role of polyamines in the DAO-nucleic acid binding interaction and the kinetics of oxidation of DAO with polyamines will be investigated. Elevated concentrations of diamines and increased diamine oxidase activity generates significant concentrations of hydrogen peroxide and aminoaldehydes, which may lead to oxidative damage and the formation of inter- and intramolecular cross-links between reactive groups of susceptible proteins and nucleic acids. Products recovered from in vitro reactions of defined composition will be screened for DAO-induced molecular modifications by capillary electrophoresis and mass spectrometry.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM065158-02
Application #
6838246
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Ikeda, Richard A
Project Start
2003-06-01
Project End
2006-05-31
Budget Start
2004-06-01
Budget End
2006-05-31
Support Year
2
Fiscal Year
2004
Total Cost
$75,000
Indirect Cost
Name
San Francisco State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
942514985
City
San Francisco
State
CA
Country
United States
Zip Code
94132