Related Experiment Videos
Human micro-calpain: simple isolation from erythrocytes and characterization of autolysis fragments
D Gabrijelcic-Geiger1, R Mentele, B Meisel
1Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik-Innenstadt, Klinikum der Ludwig-Maximilians-Universität Munchen, Germany.
Abstract:
Heterodimeric p-calpain, consisting of the large (80 kDa) and the small (30 kDa) subunit, was isolated and purified from human erythrocytes by a highly reproducible four-step purification procedure. Obtained material is more than 95% pure and has a specific activity of 6-7 mU/mg. Presence of contaminating proteins could not be detected by HPLC and sequence analysis. During storage at -80 degrees C the enzyme remains fully activatable by Ca2+, although the small subunit is partially processed to a 22 kDa fragment. This novel autolysis product of the small subunit starts with the sequence 60RILG and is further processed to the known 18 kDa fragment. Active forms and typical transient and stable autolysis products of the large subunit were identified by protein sequencing. In casein-zymograms only the activatable forms 80 kDa+30 kDa, 80 kDa+22 kDa and 80 kDa+18 kDa displayed caseinolysis.
Insights
Human erythrocyte p-calpain, a heterodimer, was purified and characterized. The enzyme remains active after storage, with partial processing of its small subunit into novel autolysis products.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- p-Calpain is a heterodimeric protease composed of large (80 kDa) and small (30 kDa) subunits.
- Understanding p-calpain's structure and function is crucial for cellular processes.
Purpose of the Study:
- To isolate and purify human erythrocyte p-calpain.
- To characterize its stability and autolysis products during storage.
Main Methods:
- Four-step purification procedure for p-calpain isolation.
- High-Performance Liquid Chromatography (HPLC) and protein sequencing for purity assessment.
- Calcium activation and casein-zymography to analyze enzyme activity and autolysis.
Main Results:
- Highly pure ( >95%) heterodimeric p-calpain (80 kDa + 30 kDa) was obtained with specific activity of 6-7 mU/mg.
- The enzyme remained fully activatable by Ca2+ after storage at -80°C.
- The small subunit underwent partial autolysis to a 22 kDa fragment (starting with 60RILG) and further to an 18 kDa fragment.
- Active forms (80+30 kDa, 80+22 kDa, 80+18 kDa) displayed caseinolysis.
Conclusions:
- Human erythrocyte p-calpain can be purified to high homogeneity.
- The enzyme exhibits remarkable stability at -80°C, with novel autolysis products identified.
- These findings provide insights into p-calpain's post-translational modifications and enzymatic activity.