Related Experiment Videos
Purification and characterization of a dynorphin-processing endopeptidase
Y L Berman1, L Juliano, L A Devi
1Department of Pharmacology, New York University Medical Center, New York 10016, USA.
The Journal of Biological Chemistry
|October 6, 1995
Summary
Researchers purified the dynorphin-converting enzyme (DCE), a metallopeptidase from bovine pituitary. This enzyme processes dynorphin B at monobasic sites, crucial for neuropeptide biosynthesis.
Area of Science:
- Biochemistry
- Neuroendocrinology
- Proteomics
Background:
- Dynorphin B (Dyn B-13) is derived from Dyn B-29 (leumorphin) via cleavage at a single arginine residue.
- Enzymatic activity for this monobasic processing exists in neurosecretory vesicles of the bovine pituitary.
Purpose of the Study:
- To purify and characterize the enzyme responsible for processing Dyn B-29 into Dyn B-13.
- To determine the enzyme's properties and its role in neuropeptide biosynthesis.
Main Methods:
- Purification of the dynorphin-converting enzyme (DCE) using hydrophobic chromatography, preparative isoelectrofocusing, and non-denaturing polyacrylamide gel electrophoresis.
- Characterization of DCE's pI, molecular mass, pH optimum, and inhibition profile.
- Mass spectrometry (MALDI-TOF) for cleavage site determination.
Main Results:
- DCE was purified to homogeneity from the bovine pituitary neurointermediate lobe.
- DCE is a metallopeptidase with a neutral pH optimum, pI of ~5.1, and molecular mass of ~54 kDa.
- DCE specifically cleaves Dyn B-29 at Arg14, generating Dyn B-13 and Dyn B-(14-29), and recognizes a consensus motif for monobasic processing.
Conclusions:
- The purified enzyme, DCE, is distinct from known metallopeptidases like enkephalinase.
- DCE plays a significant role in the biosynthesis of peptide hormones and neuropeptides by processing at monobasic sites.