Reversible inactivation of calpain isoforms by nitric oxide.
S-nitrosylation by sodium nitroprusside, a nitric oxide-generating agent, inactivates, almost completely at neutral pH, the proteolytic activity of the high Ca2+ requiring calpain form (m-calpain) from skeletal muscle. This inhibition is reversed by treating the inactivated proteinase with dithiothreitol. When exposed to sodium nitroprusside, the single m-calpain-like isoform from human neutrophils is inactivated too. On the contrary, the activities of muscle mu-calpain isoform and the human erythrocyte single mu-calpain-like isoform are poorly affected by nitric oxide treatment at neutral pH; however, inactivation is progressively enhanced if the pH of incubation mixtures is shifted to acidic values, a condition which conversely reduces NO-mediated inactivation of m-calpain. On the basis of these results, it is conceivable to postulate that nitric oxide may exert a regulatory role of muscle calpain activity by modulation of either one or the other proteinase isoform, also in concomitance with fluctuations of hydrogen ions in contracting cells occurring in physiological or pathological conditions. The regulatory role of nitric oxide is also supported by the observation that S-nitrosylation induces inactivation of calpain also in intact human neutrophils. Furthermore, the reversibility of the inactivation of calpain by nitric oxide may be exploited to study the relationship between the molecular structure and the catalytic and regulatory mechanisms of this neutral proteinase.
Michetti
M
M
Institute of Biological Chemistry, Genoa, Italy.
Salamino
F
F
Melloni
E
E
Pontremoli
S
S
D002154:Calpain / Q000037:antagonists & inhibitors*; D004229:Dithiothreitol / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors*; D009504:Neutrophils / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009599:Nitroprusside / Q000378:metabolism / Q000494:pharmacology
D007527:Isoenzymes; D009599:Nitroprusside; D009569:Nitric Oxide; D002154:Calpain; D004229:Dithiothreitol