Rat liver alcohol dehydrogenase. Purification and properties

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Alcohol dehydrogenase (EC 1.1.1.1) plays a crucial role in alcohol metabolism.
• Understanding the properties of purified alcohol dehydrogenase is essential for metabolic research.

Purpose of the Study:

• To purify and partially characterize alcohol dehydrogenase from rat liver supernatant.
• To investigate the enzyme's structural and functional properties, including cofactor specificity and stability.

Main Methods:

• Enzyme purification using ammonium sulphate fractionation, DEAE-Sephadex chromatography, and gel filtration.
• Homogeneity assessment via cellulose acetate and polyacrylamide-gel disc electrophoresis.
• Characterization using sulphoethyl-Sephadex chromatography, immunoelectrophoresis, and molecular weight estimation.

Main Results:

• A 200-fold purification of rat liver alcohol dehydrogenase was achieved, yielding a preparation with a minor impurity.
• The enzyme contains approximately 4 mol of zinc per mole, has a molecular weight of 65,000, and comprises two subunits.
• The enzyme is NAD(+)-dependent, exhibits broad alcohol specificity, and is deactivated by heavy metals, thiol reagents, urea, low pH, and chelating agents, with irreversible deactivation.

Conclusions:

• Rat liver alcohol dehydrogenase is a zinc-containing enzyme with specific structural and substrate-binding characteristics.
• The enzyme's sensitivity to various deactivating agents highlights the importance of its thiol groups and metal ions for activity.
• The broad substrate specificity suggests a role in metabolizing various alcohol compounds.