AUTOLYSIS OF HUMAN ERYTHROCYTE CALPAIN PRODUCES 2 ACTIVE ENZYME FORMSWITH DIFFERENT CELL LOCALIZATION

The 80 kDa human erythrocyte calpain, when exposed to Ca2+, undergoes autoproteolysis that generates a 75 kDa species, with an increase in C a2+ affinity, It is demonstrated here that this proteolytic modificati on proceeds through an initial step producing a 78 kDa form which is r apidly converted to the 75 kDa one, In the presence of the calpain inh ibitor 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 A2 3187, the native 80 kDa calpain subunit is extensively translocated an d retained at the plasma membrane, this process is accompanied by the appearance of only a small amount of the 75 kDa submit which is releas ed into the soluble fraction of the cells, Following exposure to mu M Ca2+, membrane-bound 80 kDa calpain is converted to the 78 kDa form, t his conversion being linearly correlated with the expression of the pr oteinase activity, Taken together, these results demonstrate that the initial step in calpain activation involves Ca2+-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 autoproteoly tic intermolecular digestion converts the 78 kDa to the 75 kDa form, n o longer being retained by the membrane, This process generates two ac tive forms of calpain, with different intracellular localisations.