LARGE-SCALE PURIFICATION OF PORCINE CALPAIN-I AND CALPAIN-II AND COMPARISON OF PROTEOLYTIC FRAGMENTS OF THEIR SUBUNITS

Abstract

Large-scale purification of calpain from porcine tissues is described. The methods used included chromatographies on DEAE-cellulose, Ultrogel AcA 34, Blue Sepharose CL-6B and DEAD Bio-Gel A which yielded homogeneous enzyme proteins: 27.0 mg of calpain I (low Ca2+-requiring form) from 5 l of blood with 17,900-fold purification and 57.6 mg of calpain II (high Ca2+-requiring form) from 1.5 kg of kidneys with 5800-fold purification. Porcine calpains I and II are half-maximally activated at 2.8 .mu.M and 150 .mu.M Ca2+, respectively. They are composed of large and small subunits: MW 83,000 and 29,000 for calpain I and MW 80,000 and 29,000 for calpain II. Gel-electrophoretic anaysis of the digest with .alpha.-chymotrypsin or Staphylococcus aureus V8 protease revealed that the large subunits of calpains I and II are markedly different in structure whereas the small subunits are most likely identical. Monospecific antibodies directed toward the respective large and small subunits were used for immunoblotting experiments which established not only the identity among several porcine tissues of calpain I but also that of calpain II.