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A membrane bound substance P degrading endopeptidase from rat brain
1Institut für Biochemie und Molekularbiologie, Berlin, F.R.G.
NIDA Research Monograph
|January 1, 1986
Summary
Researchers purified a substance P-degrading endopeptidase (SPE) from rat brain. This enzyme, crucial for substance P breakdown, shows specific inhibition patterns and hydrolysis sites.
Area of Science:
- Neuroscience
- Biochemistry
- Enzymology
Background:
- Substance P is a neuropeptide involved in various physiological processes.
- Understanding the enzymes that degrade Substance P is crucial for neuroscience research.
- A specific endopeptidase responsible for Substance P degradation was previously uncharacterized.
Purpose of the Study:
- To purify and characterize a novel membrane-bound endopeptidase from rat brain responsible for Substance P degradation.
- To determine the enzyme's biochemical properties, including optimal conditions, molecular weight, and sensitivity to inhibitors.
- To identify the specific peptide bonds within Substance P that are hydrolyzed by the purified enzyme.
Main Methods:
- Purification of the enzyme using a multi-step chromatographic approach: CHAPS extraction, DEAE-cellulose ion exchange, hydroxyapatite adsorption, Ultrogel AcA 44 gel filtration, and FPLC on Mono Q.
- Determination of enzyme activity at different pH levels.
- Assay of enzyme inhibition using various metal chelators, sulfhydryl modifying reagents, serine-protease inhibitors, and peptide-based inhibitors.
- Analysis of Substance P degradation products to identify hydrolysis sites.
Main Results:
- A membrane-bound substance P-degrading endopeptidase (SPE) was purified 1580-fold to near homogeneity from rat brain.
- The purified enzyme has a molecular weight of 70,000 and is optimally active at pH 7.5.
- The enzyme is inhibited by metal chelators (EDTA, EGTA), sulfhydryl reagents, and bacitracin, but not by diisopropyl-fluorophosphate, phosphoramidon, or captopril.
- Degradation of Substance P was inhibited by neurotensin, somatostatin, ACTH 1-39, and LHRH.
- Substance P was preferentially hydrolyzed at the Gln6-Phe7 bond, with additional fragmentation observed at other peptide bonds.
Conclusions:
- A novel, membrane-bound endopeptidase (SPE) with specific activity against Substance P has been successfully purified and characterized from rat brain.
- The enzyme's properties suggest it plays a significant role in regulating Substance P levels in the brain.
- Further investigation into SPE's physiological function and its potential as a therapeutic target is warranted.