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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 ...

Key words: Proteolysis; Calcium; Calpain; activity and thus it can be considered as an active species of Activation process calpain. Materials and methods I. Introduction Purification of human erythrocyte calpainThe Ca2+-dependent proteinase, calpain, is normally locaHuman erythrocyte calpain was purified as previously described lised in the cytosol of the cells [1][2][3][4][5], together with its natural[15], modified as follows: packed red cells 50 ml were lysed in inhibitor calpastatin. In this cell localisation, calpain is pos-5 vols. of water containing 1 mM EGTA; the membranes were discarded by centrifugation at 25 000 ×g for 10 min and the supernatant tulated to be inactive, due to the high requirement for Ca 2+ was treated with 125 g (wet powder) of DE 32 previously extensively [6][7][8], in the absence of which the enzyme retains a native washed with 50 mM sodium acetate, pH 6.7, containing 0.1 mM conformation with an inaccessible active site. The transition EGTA (buffer A) and stirred for 10 min at 5°C. The resin was colfrom a high to low calcium requiring form results from aulected on a Buckner funnel, washed with 1 1 of buffer A and transferred on a glass column (2.5x 15 cm). The absorbed proteins were toproteolysis, which causes the removal of fragments at the Neluted in a single step with 0.2 M NaC1 dissolved in buffer A. The terminal region of both 80 kDa catalytic and 30 kDa subunits fractions containing calpain activity were collected, precipitated in [9][10][11][12]. The native catalytic subunit is then converted into a 75 50% saturated ammonium sulfate and centrifuged at 25000×g for kDa species, and the small subunit into a 18 kDa fragment.10 min. The pellet was suspended in buffer A and dialyzed for 4 h This new enzyme form expresses full catalytic activity at conin the same buffer solution. The dialyzed material was then loaded onto a column (2.5×3 cm) of Source 15Q (Pharmacia) previously centrations of calcium 50-100-times lower than that required equilibrated in buffer A. The proteins were eluted with a linear graby the native form, and has also been identified in red cells dient of 0~).3 M sodium chloride. The fractions containing calpain enriched with Ca 2+ following exposure of cells to a ionophore, activity were collected, dialyzed for 4 h against sodium borate (50 It seems therefore that autoproteolysis is an essential step that mM, pH 7.5) containing 0.1 mM EGTA (buffer B). To the dialyzed material was added sodium chloride at a final concentration of 0.3 M and the solution loaded onto a column (1 × 10 cm) containing PhenylSepharose CL 4B (Pharmacia) equilibrated with buffer B, containing *Corresponding author. Fax: (39) (10) 354415.0.3 M NaC1. The resin was washed and the bound proteins were 0014-57931961512.00

Hexamethylenebisacetamide (HMBA) is a potent inducer of murine erythroleukemia (MEL) cell differentiation. The mechanism of action of HMBA is not known. In this study we provide evidence that protein kinase C has a role in inducer-mediated MEL cell differentiation: (i) HMBA induces the formation of a soluble, proteolytically activated form of protein kinase C that is catalytically active in the absence of Ca2' and phospholipid; (it) the protease inhibitor leupeptin blocks formation of this activated form of the kinase and inhibits HMBA-induced MEL cell hemoglobin accumulation; (iiW) phorbol 12-myristate 13-acetate (PMA) inhibits HMBAinduced MEL differentiation and causes depletion of total protein kinase C activity; (iv) MEL cells depleted in protein kinase C activity by culture with PMA are resistant to induction by HMBA; (v) upon removal of PMA, restoration of MEL cell sensitivity to HMBA is correlated with reaccumulation of protein kinase C activity; and (vW) MEL cells grown to density arrest are both depleted of protein kinase C activity and resistant to HMBA. Together, these results suggest that HMBA-mediated MEL cell differentiation involves a protein kinase C-related mechanism and the proteolytically activated form of the kinase, which does not require Ca2+ or phospholipid for its catalytic activity.Induction of murine erythroleukemia (MEL) cells to terminal erythroid differentiation has provided a model suitable for characterizing changes in gene expression during induced terminal differentiation (1). The mechanism by which hexamethylenebisacetamide (HMBA) induces the changes that initiate the process of terminal cell division and expression of the terminal differentiated phenotype is not known. HMBA causes a modulation in expression of genes related to the differentiated phenotype, such as the genes for the a1-and IBmaJ-globins (2); genes related to general metabolic functions, such as the rRNA genes (3); and genes that may have a role in regulating cell proliferation, such as the protooncogenes c-myb, c-myc, and c-fos (4). The tumor promoter phorbol 12-myristate 13-acetate (PMA) is a potent inhibitor of HMBAinduced MEL cell differentiation (5). A number of reports provide evidence for a role for protein kinase C (PKC) in signal transduction during cell proliferation and differentiation (6-19). There is evidence that tumor promoters such as TPA elicit responses through activation of PKC and that the kinase itself is a specific receptor for the tumor promoter (10)(11)(12)(13)(20)(21)(22)(23)(24)(25).We have examined the role of PKC in the pathway of HMBA induction. We report that induction of MEL cells by HMBA is associated with the appearance in the cytosol of a proteolytically activated form of PKC (PKM) that is catalytically active in the absence of Ca2+ and phospholipid (26)(27)(28)(29)(30)(31). Leupeptin (32), an inhibitor of the proteolytic enzyme calpain (33) (which can catalyze conversion of membrane-bound, native PKC to the soluble, activated PKM form of the kinase), blocks HMBA-induced ...