Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca 2؉ ] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of -calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca 2؉ -dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.How the calcium-dependent proteolytic system is activated is an intriguing question in terms of understanding its general properties, physiological functions, and involvement in the occurrence of tissue damage (1-4). Calpains, the enzymatic components of the system, are primarily regulated by calcium ions. The binding of Ca 2ϩ to calmodulin-like domains of the proteinases induces a conformational change that constitutes the initial limiting step of the overall sequential activation process (5). In this new conformation calpains undergo limited autoproteolysis that irreversibly removes the molecular constraints responsible for stabilizing the native inactive form (1-4). In their autoproteolyzed active forms, calpains acquire the highest affinity for calpastatin, which becomes the only negative modulator of calpain activity (6). In rat brain we have recently identified a protein factor, called UK114, which specifically interacts with -calpains and accelerates their activation steps, increasing the affinity for calcium ions (7). The reduced time of calpain activation is consistent with the proposed involvement of the Ca 2ϩ -dependent proteolytic system in specific signal transduction pathways, resulting, for instance, in granule secretion in various cell types (8) or in the conversion of protein kinase C into an autoproteolyzed form produced during the differentiation of murine erythroleukemia cells (9).The rat brain -calpain activator shows a strict specificity for all -calpain isoforms, whereas it has no effect on the classic m-calpains (10), the activation of which requires a calcium concentration at least two orders of magnitude higher than that required by -calpain. Despite reports indicating that free calcium ions can be accumulated in specific cell regions at high micromolar concentrations (11), the calcium requirement for m-calpain activation can apparently be satisfied only during tissue necrosis, a situation far removed from physiological conditions (12). We now report that a highly conserved protein, called acylCoA-binding protein (ACBP) 1 (13-15), shows a potent m-calpain-activating pr...