Purification and characterization of arylamidase from monkey brain

Area of Science:

• Biochemistry
• Enzymology
• Neuroscience

Background:

• Arylamidases are enzymes that cleave amino acids from the N-terminus of peptides.
• Understanding the properties of arylamidase in brain tissue is crucial for neurological research.

Purpose of the Study:

• To isolate and purify arylamidase from monkey brain extract.
• To characterize the biochemical properties of the purified enzyme.

Main Methods:

• Six-step purification procedure including chromatography and gel filtration.
• Polyacrylamide disc electrophoresis and SDS-PAGE for purity assessment.
• Enzyme inhibition studies and substrate specificity assays.

Main Results:

• Arylamidase was purified ~2100-fold with 11% yield.
• The enzyme is a single polypeptide chain with a molecular weight of 92,000 Da.
• It is inhibited by PCMB, TPCK, and puromycin, with competitive inhibition by puromycin.
• Enzyme activity is dependent on divalent metal ions (Zn2+, Co2+, Mn2+) and is lost upon EDTA treatment.
• L-alanine beta-naphthylamide was the preferred substrate.

Conclusions:

• Monkey brain arylamidase is a metalloenzyme with specific substrate preferences.
• The purified enzyme provides a valuable tool for studying aminopeptidase activity in the brain.