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 efficiently liberated from membrane preparations using detergents. Alternatively, the procaspase can be rapidly processed and activated from membrane preparations by caspase-1 without detergents. The activated caspase-3 was purified using affinity chromatography and characterized by amino acid sequencing and inhibitor specificity analysis. The sequence indicates that this heavy membrane bound caspase is caspase-3. The kinetic properties and inhibitor binding specificity also show that this purified caspase is enzymologically indistinguishable from cytoplasmic or recombinant caspase-3. However, the N-termini of activated heavy membrane-bound and cytoplasmic caspase-3 are slightly different; peptide sequencing data indicate that the heavy membrane caspase-3 begins at Lys 14, whereas the cytoplasmic enzyme begins at Ser 10. Implications of this structural difference are discussed.
Krebs
J F
JF
IDUN Pharmaceuticals, 11085 North Torrey Pines Road, Suite 300, La Jolla, California 92037, USA.
Srinivasan
A
A
Wong
A M
AM
Tomaselli
K J
KJ
Fritz
L C
LC
Wu
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D020170:Caspase 1 / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D003374:Coumarins / Q000378:metabolism; D003902:Detergents; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D005960:Glucosides; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017830:Octoxynol; D009842:Oligopeptides / Q000378:metabolism; D013379:Substrate Specificity
D061945:Caspase Inhibitors; D003374:Coumarins; D003902:Detergents; D004792:Enzyme Precursors; D005960:Glucosides; D008565:Membrane Proteins; D009842:Oligopeptides; C102580:acetyl-aspartyl-glutamyl-valyl-aspartyl-amino-4-methylcoumarin; C018619:octyl-beta-D-glucoside; D017830:Octoxynol; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1