Molecular and catalytic characterization of intact heterodimeric and derived monomeric calpains isolated under different conditions from pig polymorphonuclear leukocytes.
Evidence is presented of polymorphonuclear (PMN) cells derived from pig peripheral blood containing two molecular species of Ca2+-dependent cysteine endopeptidases, calpains I and II, which require low and high concentrations of Ca2+, respectively, for activation. Calpains I and II, purified from PMN homogenates, are heterodimers consisting of 83 plus 29 kDa and 80 plus 29 kDa subunits, respectively, which can be identified by using subunit-specific antibodies and which are identical with those of calpain species in other pig tissues and cells hitherto reported. However, a 70-kDa calpain can also be detected when pig PMN cells are disrupted by the nitrogen cavitation method under rather mild conditions, i.e., with minimal destruction of the lysosomes. Lines of evidence are presented showing that the 70-kDa species is devoid of the light subunit, that it is artificially derived from naturally occurring heterodimeric calpain I, and that the PMN cells before disruption contained no such monomeric form. The isolated 70-kDa calpain I, or monomeric artifact, requires only 1 microM Ca2+ for half-maximal activation, and it is less pH stable and much less heat stable than the parent heterodimeric calpain I. A possible mechanism for the production of this artifact is discussed.
Fukui
I
I
Department of Clinical Science and Laboratory Medicine, Kyoto University, Japan.
Toyohara
H
H
Ito
K
K
Hamakubo
T
T
Murachi
T
T
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002154:Calpain / Q000097:blood* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007122:Immunoelectrophoresis; D007074:Immunoglobulin G; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009504:Neutrophils / Q000201:enzymology*; D013552:Swine
D000596:Amino Acids; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D002154:Calpain