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Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization
B S Slusher1, M B Robinson, G Tsai
1Department of Neuroscience and Pharmacology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
The Journal of Biological Chemistry
|December 5, 1990
Summary
Researchers purified N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), an enzyme crucial for neuropeptide processing. The purified enzyme, identified at 94 kDa, showed specific activity and immunoreactivity in brain and kidney tissues.
Area of Science:
- Neuroscience
- Biochemistry
- Enzymology
Background:
- N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase.
- It cleaves glutamate from the neuropeptide N-acetyl-L-aspartyl-L-glutamate.
Purpose of the Study:
- To solubilize and purify NAALA dipeptidase from synaptosomal membranes.
- To characterize the purified enzyme and generate specific antibodies.
Main Methods:
- Solubilization using Triton X-100.
- Purification via sequential chromatography (DEAE-Sepharose, CM-Sepharose, lentil lectin-Sepharose).
- SDS-PAGE, enzymatic gel staining, antibody generation, and immunocytochemistry.
Main Results:
- Achieved 720-fold purification with 1.6% yield.
- Identified a single 94 kDa band as NAALA dipeptidase.
- Demonstrated pharmacological similarity to previously described activity and high immunoreactivity in cerebellar and renal cortices.
Conclusions:
- NAALA dipeptidase was successfully purified to homogeneity.
- The purified enzyme is pharmacologically characterized and immunologically specific.
- Intense immunoreactivity suggests a significant role in cerebellar and renal tissues.