Reconstitution of calpain I and calpain II from their subunits: interchangeability of the light subunits

Abstract

Calpain (Ca2 +-dependent cysteine proteinase) is known to be a heterodimer, composed of one heavy (called 80K) and one light (called 30K) subunit. Calpain I, a low-Ca2 +-requiring form from porcine and human erythrocytes, and calpain II, a high-Ca2 +-requiring form from porcine kidney, were separated into their respective 80K and 30K subunits by high-performance liquid chromatography on a TSK-Gel G3000SW column in 1 M NaSCN. The isolated 80K subunits from porcine calpains I and II showed Ca2 +-dependent proteolysis, though much depressed and requiring higher Ca2 + concentrations compared with the respective parent calpains. The optimal conditions were set for the reconstitution of a heterodimeric molecule from the once denatured and separated 80K and 30K subunits. In such reconstitution, the 30K subunits of calpains I and II were found to be functionally identical and interchangeable; either a homologous or heterologous 30K subunit lowered the Ca2 + requirement of an 80K subunit significantly and enhanced the proteolytic activity. Subunit interchange could also be demonstrated between porcine and human calpains.