Related Experiment Videos
Acid sphingomyelinase from human urine: purification and characterization
L E Quintern1, G Weitz, H Nehrkorn
1Institut für Organische Chemie und Biochemie der Universität Bonn, F.R.G.
Biochimica Et Biophysica Acta
|December 14, 1987
Summary
Human urine contains acid sphingomyelinase, an enzyme that breaks down sphingomyelin and other phospholipids. A detergent-free assay using tripalmitoylglycerol enhances enzyme activity and detection.
Area of Science:
- Biochemistry
- Enzymology
- Human Physiology
Background:
- Acid sphingomyelinase (sphingomyelin phosphodiesterase) is an enzyme involved in lipid metabolism.
- Understanding its properties and activity is crucial for various biological processes.
- Human urine is a potential source for enzyme purification.
Purpose of the Study:
- To purify acid sphingomyelinase from human urine.
- To characterize its enzymatic activity and substrate specificity.
- To develop an efficient assay system for the enzyme.
Main Methods:
- Enzyme purification using sequential chromatography (octyl-Sepharose, concanavalin A-Sepharose, blue Sepharose, DEAE-cellulose).
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular mass determination.
- Enzyme activity assays with and without detergents and specific inhibitors.
Main Results:
- Acid sphingomyelinase was purified 23,000-fold from human urine, yielding a 70 kDa enzyme.
- The purified enzyme hydrolyzed sphingomyelin, phosphatidylcholine, and phosphatidylglycerol.
- A detergent-free assay using tripalmitoylglycerol stimulated sphingomyelinase activity 20-fold.
- Specific inhibitors like phosphatidylinositol 4',5'-bisphosphate significantly inhibited enzyme activity.
Conclusions:
- Human urine is a viable source for purifying acid sphingomyelinase.
- The enzyme exhibits broader phosphodiesterase activity than previously recognized.
- A novel, sensitive, and detergent-free assay system for acid sphingomyelinase was developed.