Heavy membrane-associated caspase 3. Identification, isolation, and characterization

Heavy membrane preparations from 697 lymphoblastoid cells contain a tightly bound caspase zymogen. This heavy membrane-bound procaspase can be efficie ntly liberated from membrane preparations using detergents. Alternatively, the procaspase can be rapidly processed and activated from membrane prepara tions by caspase-1 without detergents. The activated caspase-3 was purified using affinity chromatography and characterized by amino acid sequencing a nd inhibitor specificity analysis. The sequence indicates that this heavy m embrane bound caspase is caspase-3. The kinetic properties and inhibitor bi nding specificity also show that this purified caspase is enzymologically i ndistinguishable from cytoplasmic or recombinant caspase-3. However, the N- termini of activated heavy membrane-bound and cytoplasmic caspase-3 are sli ghtly different; peptide sequencing data indicate that the heavy membrane c aspase-3 begins at Lys 14, whereas the cytoplasmic enzyme begins at Ser 10. Implications of this structural difference are discussed.