Nicotinamide riboside phosphorylase from beef liver: purification and characterization

Area of Science:

• Biochemistry
• Enzymology

Background:

• Nicotinamide riboside phosphorylase (NR phosphorylase) is an enzyme involved in nucleotide metabolism.
• Understanding the properties of NR phosphorylase is crucial for metabolic pathway research.

Purpose of the Study:

• To purify and characterize NR phosphorylase from beef liver.
• To investigate the substrate specificity and kinetic properties of the purified enzyme.

Main Methods:

• Purification of NR phosphorylase from beef liver using standard biochemical techniques.
• Enzyme kinetics assays to determine kinetic constants (Km, Vmax).
• Molecular weight determination (native and subunit) and amino acid composition analysis.

Main Results:

• NR phosphorylase was purified 300-fold with 35% yield.
• Kinetic constants for nicotinamide riboside and inorganic phosphate were determined.
• The enzyme is tetrameric with identical subunits (MW 131,000 native, 32,000 subunit).
• The enzyme exhibits phosphorolytic activity towards nicotinamide riboside, inosine, and guanosine with constant activity ratios throughout purification.
• Inosine and guanosine competitively inhibit nicotinamide riboside phosphorolysis.
• Thermal denaturation studies indicate a single enzyme catalyzes all observed reactions.

Conclusions:

• Beef liver NR phosphorylase is a tetrameric enzyme with identical subunits.
• The enzyme catalyzes the phosphorolysis of nicotinamide riboside, inosine, and guanosine, suggesting a single active site.
• The characterized enzyme provides insights into purine and pyrimidine salvage pathways.