Gabrijelcic-Geiger, Dusica; Mentele, Reinhard; Meisel, Barbara; Hinz, Heide; Assfalg-Machleidt, Irmgard; Machleidt, Werner; Möller, Achim; Auerswald, Ennes A.
Human μ-calpain: Simple isolation from erythrocytes and characterization of autolysis fragments.
In: Biological Chemistry, Vol. 382, No. 12: pp. 1733-1737
Heterodimeric μ-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 °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 (60)RILG 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.