The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.

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A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the -calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on -calpain independently from the source of the proteinase.The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca 2؉ -dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation.Calpains are a family of dimeric proteinases all characterized by an absolute dependence on Ca 2ϩ (1-7). In the absence of this metal ion, calpains are stabilized in an inactive conformational state, by inter-and intramolecular constraints (8, 9). Binding of Ca 2ϩ to the proteinase molecules produces both dissociation of the heterodimers (10) and conformational changes of the 80-kDa catalytic subunits, triggering the enzyme activation that is completed by an autoproteolytic event (11,12). The concentrations of Ca 2ϩ inducing the conformational changes required for the activation of both -and mcalpain are at least one order of magnitude higher than the actual concentrations of this metal ion in cells. Experiments designed to identify possible mechanisms effective in reducing the calcium requirement of calpains have demonstrated that association of the proteinase to phospholipid vesicles (12) or to nuclei (13) are effective in increasing its affinity for Ca 2ϩ . A more relevant physiological significance is represented by a calpain activator protein recently identified in human red blood cells (14) and in rat skeletal muscle (15). This protein factor, which is significantly effective in reducing the requirement of the proteinase for calcium ions, binds Ca 2ϩ with high affinity (10) and associates to the particulate fraction of the cells; in fact, it is recovered in the soluble fraction only when cell lysis is performed by a medium containing metal chelators.Furthermore, the activator-Ca 2ϩ complex interacts with calpain and thereby induces those conformational changes required to trigger the activation pro...

In the presence of micromolar concentrations of Ca2+, both protein kinase C and a cytosolic Ca2+-requiring neutral proteinase of human neutrophils become associated with the neutrophil membrane. Binding to the membrane results in activation of the proteinase, which then catalyzes limited proteolysis of the kinase to produce a form that is fully active in the absence of Ca2' and phospholipid. This irreversibly activated protein kinase is released from the membrane and may thus have access, in the intact cell, to intracellular protein substrates. In the absence of the proteinase, Ca2+ promotes the binding of protein kinase C, but conversion to the Ca2+/phospholipid-independent form does not occur and the kinase remains associated with the membrane fraction.Protein kinase C was originally described in rat brain as a soluble, cAMP-independent proenzyme (1) that was converted to the active kinase by the action of a cytosolic Ca2+-requiring proteinase (2, 3). The native "proenzyme" was later shown to require Ca2' and phospholipid (4,5) and to be further activated by diacylglycerol (6), which markedly increased its affinity for both Ca2' and phospholipid (for reviews, see refs. 7 and 8). Activation of protein kinase C in stimulated platelets has been attributed to the formation of diacylglycerol generated by phospholipase C from inositol phospholipids (7,9). An irreversible activation by limited proteolysis has also been described in platelets treated with phospholipase C (10) or phorbol 12-myristate 13-acetate (11) Isolation of Neutrophils. This was based on the procedure of Boyum (14). Freshly collected, heparinized human blood (100 ml) from healthy donors was treated with 1.6% (wt/vol) dextran (final concentration) and left at 25-28°C for -1 hr. The sedimented erythrocytes were removed and the supernatant solution (40 ml) was collected and layered onto 10 ml of 6% Ficoll 400 solution containing 0.17% (vol/vol) Urovison. The gradient was centrifuged at 800 x g for 20 min and the pellet obtained was resuspended in 10 ml of 0.2% NaCl to lyse the contaminating red cells. After 30 sec, 10 ml of 1.6% NaCl was added; the cells were recovered by centrifugation at 400 x g for 5 min and washed three times with 0.01 M sodium phosphate, pH 7.4/5 mM KCl/0.12 M NaCl/24 mM NaHCO3/5 mM glucose. Prior to use, the cells were maintained in an ice bath in the same medium at a concentration of 15-20 x 106 cells per ml. The cell population obtained consisted of 96% neutrophils, as evaluated by microscopic examination. The remaining 4% consisted of 3.5% eosinophils and 0.4% monocytes.Isolation of Human Platelets. Fresh human blood platelet concentrates were obtained from a blood bank and washed platelets were prepared as described by Baenziger and Majerus (15). The platelets were washed and suspended at a final concentration of 1010 cells per ml in the same buffer employed for the neutrophils.Isolation of the Soluble and Particulate Fractions from Neutrophils and Platelets. These were prepared from lysates obtained by sonicating ...

Calpains, the thiol proteinases of the calciumdependent proteolytic system, are regulated by a natural inhibitor, calpastatin, which is present in brain tissue in two forms. Although both calpastatins are highly active on human erythrocyte calpain, only one form shows a high inhibitory efficiency with both rat brain calpain isozymes. The second calpastatin form is almost completely inactive against homologous proteinases and can be converted into an active one by exposure to a phosphoprotein phosphatase, also isolated from rat brain. Phosphorylation of the active calpastatin by protein kinase C and protein kinase A promotes a decrease in its inhibitory efficiency. The interconversion between the two inhibitor forms seems involved in the adjustment of the level of intracellular calpastatin activity on specific cell requirements.