title: Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization.
abstract: The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in Ca2+ affinity. It is demonstrated here that this proteolytic modification proceeds through an initial step producing a 78 kDa form which is rapidly converted to the 75 kDa one. In the presence of the calpain inhibitor E-64, the 78 kDa form accumulates and only small amounts of the 75 kDa polypeptide are formed. Following loading of erythrocytes with micromolar concentration of Ca2+, in the presence of the ionophore A23187, the native 80 kDa calpain subunit is extensively translocated and retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa subunit which is released into the soluble fraction of the cells. Following exposure to microM Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, this conversion being linearly correlated with the expression of the proteinase activity. Taken together, these results demonstrate that the initial step in calpain activation involves Ca(2+)-induced translocation to the inner surface of plasma membranes. In the membrane-bound form the native inactive 80 kDa subunit is converted through intramolecular autoproteolysis to a locally active 78 kDa form. Further autoproteolytic intermolecular digestion converts the 78 kDa to the 75 kDa form, no longer being retained by the membrane. This process generates two active forms of calpain, with different intracellular localisations.
authors.affiliation: Institute of Biological Chemistry, University of Genoa, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Michetti
authors.forename: F
authors.initials: F
authors.lastname: Salamino
authors.forename: I
authors.initials: I
authors.lastname: Tedesco
authors.forename: M
authors.initials: M
authors.lastname: Averna
authors.forename: R
authors.initials: R
authors.lastname: Minafra
authors.forename: E
authors.initials: E
authors.lastname: Melloni
authors.forename: S
authors.initials: S
authors.lastname: Pontremoli
mesh_terms: D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D005914:Globins / Q000378:metabolism; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D015853:Cysteine Proteinase Inhibitors; D005914:Globins; D002154:Calpain; D007930:Leucine; C024974:E 64; D002118:Calcium