Large-scale purification of porcine calpain I and calpain II and comparison of proteolytic fragments of their subunits.
Large-scale purification of calpain [Ca2+-dependent cysteine proteinase; EC 3.4.22.17] from porcine tissues is described. The methods used included chromatographies on DEAE-cellulose, Ultrogel AcA 34, Blue Sepharose CL-6B, and DEAE Bio-Gel A which yielded homogeneous enzyme proteins: 27.0 mg of calpain I (low Ca2+-requiring form) from 5 liters 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 5,800-fold purification. Porcine calpains I and II are half-maximally activated at 2.8 microM and 150 microM Ca2+, respectively. They are composed of large and small subunits: Mr 83,000 and 29,000 for calpain I and Mr 80,000 and 29,000 for calpain II. Gel-electrophoretic analysis of the digest with a-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. Mono-specific 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. several porcine tissues of calpain I but also that of calpain II.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000032:analysis; D002154:Calpain; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D013552:Swine
D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium