A radiochemical assay for aminopeptidase N

Area of Science:

• Biochemistry
• Enzymology
• Assay Development

Background:

• Aminopeptidase N (AmN) plays a crucial role in peptide hormone degradation in vivo.
• Physiological conditions involve low substrate concentrations relative to enzyme concentration.
• Existing assays may be limited by interfering substances or non-physiological conditions.

Purpose of the Study:

• To develop a novel assay for aminopeptidase N (AmN) that accurately reflects in vivo conditions.
• To enable precise measurement of AmN activity and kinetics under physiological substrate concentrations.
• To facilitate the characterization of AmN inhibitors and substrates.

Main Methods:

• Developed a new assay using Arg-Phe-[3H]anilide as a substrate for AmN.
• Employed substrate concentrations (1-200 nM) significantly below Km (12 microM) and at or below enzyme concentration ([E]).
• Utilized enzyme sources including human serum, seminal plasma, guinea pig serum, and purified porcine kidney AmN.

Main Results:

• The Arg-Phe-[3H]anilide:AmN reaction demonstrated first-order enzyme kinetics across various enzyme sources and substrate concentrations.
• Determined kinetic parameters for porcine AmN (kcat/Km = 1.47 x 10^9 M^-1 min^-1, kcat = 17,640 min^-1).
• Confirmed high affinities of AmN for known substrates like angiotensin III and bradykinin analogs (Km values 5.7-14.3 microM).

Conclusions:

• The developed assay accurately measures AmN activity under physiologically relevant conditions.
• First-order enzyme kinetics simplifies Vmax/Km determination and allows for enzyme concentration ([E]) calculation.
• This assay is robust in the presence of chromophores/fluorophores and provides accurate IC50 values for inhibitors and alternative substrates.