Báo cáo khoa học: Interaction between catalytically inactive calpain and calpastatin Evidence for its occurrence in stimulated cells docx

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Báo cáo khoa học: Interaction between catalytically inactive calpain and calpastatin Evidence for its occurrence in stimulated cells docx

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Interaction between catalytically inactive calpain and calpastatin Evidence for its occurrence in stimulated cells Monica Averna, Roberto Stifanese, Roberta De Tullio, Enrico Defranchi, Franca Salamino, Edon Melloni and Sandro Pontremoli Department of Experimental Medicine (DIMES), Section of Biochemistry and Centre of Excellence for Biomedical Research (CEBR), University of Genova, Italy In recent years, information has accumulated on the 3D structure of l-calpain and m-calpain [1–10], as well as their isolated catalytic cores [11–16]. Much less is known about the process by which calpain is activated [3,4,6,17,18]. It is generally accepted that it is initiated by the binding of calcium to several sites localized in both calpain subunits and completed by a conforma- tional change in domain II [19–29]. However, the role of the two Ca 2+ -binding sites recently identified in this catalytic domain is still to be defined [23]. More intriguing is the possibility of detecting calpain activa- tion in vivo, which has not been previously possible because of the lack of reliable techniques for evaluat- ing active calpain species and their intracellular local- ization. Identification of autolyzed calpain forms by means of a specific monoclonal antibody does not seem to be of a general use, as recent structural acquisitions have suggested that calpain activation can also proceed through a reversible process [1,2,4]. The proposed pro- cedure involving the identification of calpain-degraded target proteins appears not to be sufficiently specific because of the very large number of calpain substrates present in the cell (for reviews see [3,4,9,30]). The recently devised fluorescence resonance energy transfer technology has greatly improved the sensitivity, but not the selectivity, required for the precise evaluation of calpain activation and activity [31]. In this paper we report that, by means of a specific monoclonal antibody that recognizes the calpain cata- lytic domain [32], it is possible to detect conformation- al changes in the calpain molecule that occur after it binds to its natural effectors. Two conformational states of calpain can be distinguished on the basis of their affinity for this mAb: the native state shows low affinity, whereas binding of specific ligands induces Keywords activation; calcium; calpain; calpastatin; conformational states Correspondence S. Pontremoli, DIMES-Section of Biochemistry, Viale Benedetto XV, 1–16132 Genova, Italy Fax: +39 010518 343 Tel: +39 010353 8128 E-mail: pontremoli@unige.it (Received 9 January 2006, accepted 15 February 2006) doi:10.1111/j.1742-4658.2006.05180.x Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably repre- sent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca 2+ , substrate or calpasta- tin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca 2+ ] or extensive diffusion of calpast- atin into the cytosol without affecting Ca 2+ homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain–calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action. 1660 FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS transition to a conformation with significantly higher affinity. The most extensive conformational change is induced by calpastatin; the addition of substrate or Ca 2+ proved to be less effective. Using this methodo- logy, we have shown similar molecular transitions in calpain in intact cells stimulated with agents known to induce either a limited increase in intracellular [Ca 2+ ] or extensive redistribution and accumulation of cal- pastatin in the cytosolic compartment. These data sug- gest a new role for calpastatin in controlling the extent of calpain translocation to and activation at specific sites of action. Results To study the interaction of calpain with its natural effectors, the purified protease isolated from human erythrocytes was immobilized on a nitrocellulose sheet and detected by a specific mAb that recognizes the DI-DII polypeptide in both native calpain and the fragment that accumulates during trypsin digestion [14,24] (Fig. 1A). In Fig. 1B we provide evidence that calpain bound to nitrocellulose reacts with mAb 56.3, generating a light signal the intensity of which is a function of the amount of mAb used, with a saturation value at a concentration equal to 0.5–0.75 lg antibody. After exposure of the immobilized calpain to a mixture of Ca 2+ and a digestible substrate, catalytic activity can be detected, demonstrating that immobilization on the nitrocellulose sheet does not modify its catalytic properties. This is demonstrated by the data in Fig. 1C, which indicate that the catalytic activity of immobilized calpain, as a function of Ca 2+ concentra- tion, is 50% of the maximal at 25 lm Ca 2+ and maximal at 100 lm Ca 2+ , as occurs when soluble native enzyme is used [3,4,9,20,30]. Furthermore, inhibition of the immobilized enzyme by E64 or calpastatin was retained (Fig. 1D). The efficiency of both inhibitors was identical with that observed in a control assay using soluble enzyme (data not shown). Together these results indicate that immobilized cal- pain is an appropriate tool for the study of the effects of natural ligands in changing its conformation, and that these can be monitored by evaluating the intensity of the light signal generated by the binding of mAb 56.3. To perform these investigations, the two preferential ligands of calpain, Ca 2+ ions and calpastatin, were tes- ted. In the presence of Ca 2+ concentrations ranging from zero to 5 lm (close to physiological values), a twofold increase in the intensity of the signal was detected, in the absence of any appreciable proteolytic activity (Fig. 2). The concentrations of Ca 2+ used in these experiments were selected to avoid undesired and confusing changes in calpain structure such as mole- cular aggregations or dissociation of the oligomers, which have been shown to occur at higher concentra- tions of the metal ion [45]. The addition of E64, a syn- thetic inhibitor of calpain, did not modify the intensity of the signal observed in the presence of Ca 2+ alone. The increase in the mAb-binding capacity of calpain observed in these conditions can be ascribed to increased accessibility of the calpain epitope recognized by this mAb. As this conformational transition does not lead to the expression of catalytic activity, these structural changes must precede the calpain active state. As shown in Fig. 3, when calpain was exposed to increasing concentrations of recombinant calpastatin RNCAST104, there was a much greater increase in the A B CD Fig. 1. Properties of nitrocellulose-immobilized native human eryth- rocyte calpain. (A) Human erythrocyte calpain (10 lg) was incubat- ed in the absence (lane 1) or presence of trypsin in a calpain ⁄ trypsin ratio of 1000 : 1 (lane 2). Western blot analysis was performed [41] using mAb 56.3. (B) Immobilized calpain was incu- bated in 0.1 mL 50 m M sodium borate buffer, pH 7.5, containing 10 m M EDTA and the indicated amounts of mAb 56.3. The immu- noreactive spots (see inset) were quantified with a Shimadzu CS9000 densitometer and expressed as arbitrary units. (C) Immobil- ized calpain (0.5 lg) was assayed as described in [33] using human denatured globin as substrate for 60 min at 37 °C in the presence of the indicated [Ca 2+ ]. The nitrocellulose sheet was removed before the addition of trichloroacetic acid (7% final concentration). Calpain activity was quantified after the release of free NH 2 groups as in [33]. (D) Activity of immobilized calpain was assayed as in (C) in the presence of 1 m M Ca 2+ and 10 lM E64 or 25 nmol rat brain recombinant calpastatin RNCAST104. M. Averna et al. Calpain–calpastatin interaction in stimulated cells FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS 1661 light signal (7–8-fold) than when calpain was exposed to Ca 2+ alone (Fig. 2). Moreover, the maximum effect was reached with 10 nmol RNCAST104, which corres- ponds approximately to a 1 : 1 protease ⁄ inhibitor molar ratio. The addition of Ca 2+ even at a concen- tration of 5 lm did not affect the RNCAST104-medi- ated increase in light emission. Thus in contrast with what occurs in the presence of Ca 2+ , the addition of equimolar amounts of calpasta- tin induces an increase of approximately one order of magnitude in the affinity of calpain for mAb 56.3, indicating that, in these conditions, the mAb epitope on the protease has become more accessible. These data not only indicate that calpastatin induces a pronounced Ca 2+ -independent change in calpain conformation, but also provide strong support for pre- vious observations indicating that calpain and calpast- atin can associate in a 1 : 1 molar ratio, regardless of the presence of Ca 2+ (unpublished work). In addition to Ca 2+ and calpastatin, a number of digestible substrates can behave as calpain ligands and may accordingly induce changes in the conformation of the protease detectable by the mAb binding. To investigate this, we exposed immobilized calpain to digestible and nondigestible proteins and evaluated their efficiency in promoting conformational change in the protease. BSA, a protein not digested by calpain, had no effect at any concentration tested. Casein, a cal- pain substrate [4,9,30], induced a progressive increase in the binding of mAb 56.3 to calpain, as revealed by a 2.5–3-fold increase in the light signal at a concentra- tion of 2 mgÆmL )1 (Fig. 4). The observations so far reported indicate that cal- pain can exist in two freely convertible inactive confor- mations. The former is mostly present in the absence of any effector, and the other is induced, with different degrees of efficiency, by interaction with micromolar Fig. 2. Effect of Ca 2+ on binding of mAb 56.3 to nitrocellulose- immobilized calpain. Immobilized calpain was incubated in 0.1 mL 50 m M sodium borate buffer, pH 7.5, in the presence of 10 mM EDTA or the indicated Ca 2+ concentration (d). After saturation, mAb 56.3 was added and the binding of the mAb to calpain was measured as described in Experimental procedures and the legend to Fig. 1B and expressed as arbitrary units. Alternatively, immobi- lized calpain was exposed to Ca 2+ in the presence of 10 lM E64 (s). Immobilized calpain was also incubated in 0.1 mL 50 m M sodium borate buffer, pH 7.5, containing the indicated Ca 2+ concen- tration in the presence of human denatured globin as substrate, and its activity was measured as described in the legend to Fig. 1C (n). The results are expressed as the arithmetical mean ± SD from four different experiments. Fig. 3. Effect of calpastatin on the binding of mAb 56.3 to immobil- ized calpain. Immobilized calpain was exposed to increasing con- centrations of recombinant rat brain calpastatin RNCAST104 in the presence of 10 m M EDTA (h)or5lM Ca 2+ (d). The binding of mAb 56.3 to immobilized calpain was measured as described in Figs 1 and 2. The data obtained in these experiments were also analyzed by Scatchard plot (inset), using the relationship [B] ⁄ [F] ¼ k(B max – B), where [B] is the bound ligand concentration, [F] is the free ligand concentration, B max is the maximum ligand amount, and k is the affinity constant. +RNCAST104 Fig. 4. Effect of calpain digestible or nondigestible proteins on the binding of mAb 56.3 to immobilized calpain. Immobilized calpain was mixed with 10 m M EDTA (control) in the presence of 10 pmol rat brain recombinant calpastatin RNCAST104 (black bar) or the indic- ated amounts of BSA (light grey bars) or casein (heavy grey bars). The mAb bound to immobilized calpain was detected as described in Experimental procedures and quantified as reported in the legend to Fig. 2. Calpain–calpastatin interaction in stimulated cells M. Averna et al. 1662 FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS (physiological amounts) Ca 2+ concentrations, a digest- ible protein substrate, and finally calpastatin. The finding that calpastatin was the most efficient ligand at promoting calpain transition, detected as an increase in mAb that bound to calpain, is consistent with the fact that the protease has an affinity for its protein inhibitor that is more than 10 000-fold higher than that for its substrates. When native calpain was replaced with the autolyzed 75-kDa form, identical results were obtained in all the experimental conditions tested (data not shown). This indicates that the removal of part of the DI and DV domain from the calpain molecule does not abolish the conformational transition described above and may explain the Ca 2+ dependence of the autolyzed enzyme [34]. We then explored whether, in stimulated cells, cal- pain undergoes conformational changes that could be detected by mAb 56.3 binding. For this purpose, human neutrophils were stimulated with the chemotac- tic peptide f-Met-Leu-Phe, which is known to promote intracellular mobilization of Ca 2+ [46–48]. As shown in Fig. 5A,B and quantified in Fig. 5C, under these conditions, an approximately 10-fold increase in fluor- escence emission was detected, indicating that calpain had undergone a transition from the low to the high affinity mAb-binding form. Interestingly, these results obtained in vivo were almost superimposable on those obtained in vitro after exposure of native calpain to calpastatin. This suggests that the same process is operating in both experimental situations. To establish if the data for human neutrophils could be reproduced in a different cell line, we used murine erythroleukemia (MEL) cells stimulated with the Ca 2+ ionophore A23187. In resting cells, calpain was poorly stained by the mAb, indicating that it was mainly pre- sent in the low-affinity form (Fig. 6A). Scanning the fluorescence throughout the cell revealed that the pro- tease is quite homogeneously diffuse throughout the cytosol. After stimulation with the Ca 2+ ionophore (Fig. 6B), the intensity of calpain staining increased 7– 8-fold, indicating that the protease was now mainly present in the high-affinity form. In these conditions, although the highest amount of calpain is still in the cytosol, a small fraction is locali- zed at the membrane, as indicated by the two small fluorescent peaks detectable at both sides of the cell scan. This further confirms that translocation to the A C B Fig. 5. Binding of mAb 56.3 to calpain in human neutrophils stimul- ated with f-Met-Leu-Phe. Purified neutrophils (10 7 cells) were incub- ated at 37 °C for 10 min in 10 m M Hepes, pH 7.5, (10 mL), containing 140 m M NaCl, 5 mM MgCl 2 ,5mM glucose and 50 lM Ca 2+ in the absence (A) or presence (B) of 1 lM f-Met-Leu-Phe. Cells were fixed and permeabilized as described in Experimental procedures. They were then exposed to mAb 56.3, and its binding to calpain was detected by confocal microscopy, after incubation with fluorescein-labeled secondary antibody. (C) Fluorescence was measured as described in Experimental procedures. The data repre- sent the arithmetical mean ± SD of four different experiments. A B Fig. 6. Binding of mAb 56.3 to calpain in MEL cells loaded with Ca 2+ . MEL cells (10 7 cells) were incubated at 37 °C for 10 min in 10 m M Hepes, pH 7.5 (10 mL), containing 140 mM NaCl, 5 mM MgCl 2 ,5mM glucose and 50 lM Ca 2+ in the absence (A) or pres- ence (B) of 1 l M A23187 Ca 2+ ionophore. Cells were fixed and per- meabilized as described in Experimental procedures. They were then exposed to mAb 56.3, and its binding to calpain was detected by confocal microscopy, after incubation with fluorescein-labeled secondary antibody. The fluorescence detected in each section (0.5 lm) is shown at the right of each picture. The arrow points to the calpain ring around the cell. M. Averna et al. Calpain–calpastatin interaction in stimulated cells FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS 1663 plasma membrane is an obligatory step in the directing of the protease to its sites of action, the preferred cal- pain substrates being transmembrane or membrane- associated proteins [3,4,30]. We still required direct evidence of the nature of the ligand responsible for the observed conformational transition. Thus, to discriminate between the effect due to the rise in free Ca 2+ from that induced by the inter- action of calpain with calpastatin, we stimulated Jur- kat cells with arachidonate, which is known to induce apoptosis without producing, during the early phase of stimulation, appreciable changes in intracellular free [Ca 2+ ] [49,50]. As previously observed (Fig. 7B) in these cells, stimulation with ionophore A23187 promoted a calpain-mediated fluorescence increase of 7–8-fold. However, a sixfold increase in fluorescence intensity was observed after brief stimulation with arachidonate, indicating that a similar conformational transition of calpain can be obtained in conditions in which intracellular Ca 2+ homeostasis is almost unaf- fected [49,50]. These findings excluded the involvement of Ca 2+ in the conformational change in calpain, strongly suggesting that it is the interaction with cal- pastatin that is responsible for the observed effects. Other ligands such as digestible substrates were exclu- ded a priori because they would never be present in the cytosol at suitable concentrations. The different calpain fluorescence observed in control (Fig. 7A) and arachidonate-stimulated (Fig. 7C) cells after detection with the calpain mAb can be ascribed to the presence of large amounts of calpastatin which, after stimulation, becomes freely available in the cyto- sol for interaction with calpain. This hypothesis is confirmed by the effect of arachi- donate treatment on the intracellular distribution of calpastatin (Fig. 8). In untreated cells, cytosol contains a very limited amount of calpastatin, the bulk of the inhibitor being localized in perinuclear aggregates. After stimulation with arachidonate, the cell image is completely reversed, as calpastatin becomes freely diffuse in the cytosol and only traces of aggregates remain located in the perinuclear region. Thus, the increased availability of calpastatin, occurring in con- ditions of unmodified Ca 2+ homeostasis, clearly indi- cates that the ligand responsible for the changes in intracellular calpain conformation is its natural inhib- itor, calpastatin. The new conformational state acquired by calpain in these experimental conditions may represent an interme- diate, but still inactive, form which is stabilized by inter- action with ligands, the most efficient being calpastatin. Discussion A major problem in understanding the physiological role of calpain is the reliability of techniques capable Fig. 7. Binding of mAb 56.3 to calpain in Jurkat cells loaded with Ca 2+ or stimulated with arachidonate. Jurkat cells (10 7 cells) were incubated at 37 °C for 10 min in 10 m M Hepes, pH 7.5 (10 mL), containing 140 m M NaCl, 5 mM MgCl 2 ,5mM glucose and 50 lM Ca 2+ in the absence (A) or presence of (B) 1 lM A23187Ca 2+ -iono- phore or (C) 100 l M arachidonate. Cells were fixed and permeabil- ized as described in Experimental procedures. They were then exposed to mAb 56.3, and its binding to calpain was detected by confocal microscopy, after incubation with fluorescein-labeled sec- ondary antibody. The data represent the arithmetical mean ± SD of four different experiments. The arrow points to the calpain ring around the cell. Fig. 8. Effect of arachidonate on the intracellular distribution of cal- pastatin in Jurkat cells. Jurkat cells were incubated with arachido- nate as described in the legend to Fig. 7C. After 30 min of incubation, cells were fixed, and calpastatin was probed with 7 lgÆmL )1 mAb 35.23 [35] followed by a fluorescein-labeled second- ary antibody. Fluorescence was quantified using the software as described in Experimental procedures. Cell nuclei were stained with propidium iodide. The arrows indicate the perinuclear calpasta- tin aggregates. C ¼ control; Ar. ¼ arachidonate. Calpain–calpastatin interaction in stimulated cells M. Averna et al. 1664 FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS of detecting the changes in its conformation that accompany its activation and regulation in specific cell compartments. In spite of several attempts to solve this problem [19–29], no precise information is available, because of the inadequacy of the methods so far pro- posed for evaluating the interaction of calpain with natural ligands, a process that must occur in defined intracellular compartments and presumably precedes activation of the protease. We explored the possibility of approaching this problem by taking advantage of the different accessibility of a calpain epitope to a spe- cific mAb. We established that this short amino-acid sequence is confined to the catalytic domain of the protease, a region known to undergo profound con- formational rearrangements [21–23], leading to expres- sion of the catalytic activity. In preliminary experiments we demonstrated that the affinity of calpain for its mAb increases after expo- sure to various ligands. These changes in the condi- tions preserving the native conformation of the protease were interpreted as the result of a molecular transition converting the native form into a state in which the mAb epitope sequence becomes more acces- sible. In this paper we report that the exposure of calpain to micromolar physiological concentrations of Ca 2+ , or a digestible protein substrate, or calpastatin is fol- lowed by a change in its conformation, which can be monitored by an increase in its affinity for its mAb. Calpain does not show catalytic activity in any of these conditions, indicating that this molecular transition precedes the onset of the active enzyme form. Using a Scatchard plot as a calibration curve, we established that calpastatin promotes the conversion of almost all the calpain molecules into the high-affinity conforma- tion, whereas the other ligands promote the transition of only 20–30% of the calpain molecules. Thus, this procedure provides a tool for the identification of the calpain states generated by its interaction with natural ligands. This methodology was successfully applied to intact cells, and the results show that similar conformational changes in calpain occur after stimulation with appro- priate effectors. In human neutrophils and MEL cells, even though the limited increase in Ca 2+ could be regarded as the event that promoted these changes, two relevant find- ings suggest a different conclusion. The first concerns the extent of the increase in calpain fluorescence, which could not be induced by Ca 2+ alone as indicated in the in vitro experiments (Figs 2 and 3). The second is rela- ted to the availability of calpastatin in the cytosol, the concentration of which increases when Ca 2+ homeosta- sis is perturbed, as previously reported [43]. Thus, the pronounced change in calpain conformation revealed by the high fluorescence reached can only be attributed to the interaction of calpain with calpastatin. Stimulation of Jurkat cells with arachidonate provi- ded experimental evidence in favor of this hypothesis as it simultaneously promoted mobilization of calpast- atin accompanied by a marked transition in the cal- pain molecule, suggesting its interaction with the inhibitor. Moreover, these events occur in the early phase of Jurkat cell stimulation, in which no evidence for alteration in Ca 2+ homeostasis has been obtained [49,50]. Thus, the effect of arachidonate suggests that conditions promoting calpain–calpastatin interaction in the cytosol can be Ca 2+ independent and precede cal- pain activation. Additional information was obtained using this methodology in experiments with MEL cells stimulated with a Ca 2+ ionophore. Analysis of the dis- tribution of calpain in the cells revealed that, in addi- tion to the massive conformational change in the enzyme present in the cytosol, an increase in intracellu- lar [Ca 2+ ] also induces translocation of a small amount of the protease to the plasma membrane. These results are in agreement with the accepted evi- dence that an increase in intracellular [Ca 2+ ] induces degradation of some proteins specifically localized at the inner surface of the plasma membrane [3,4,30] and with the observation that the extent of calpain activa- tion at the plasma membrane is a function of the amount of cytosolic calpastatin [43]. Taken together, these findings suggest that the for- mation of a calpain–calpastatin complex before the onset of calpain activation is functionally relevant, not only for the modulation of calpain activation in the cytosol, but also for controlling the amount of calpain translocated to and activated at the plasma membrane. Experimental procedures Materials Ca 2+ ionophore A23187, f-Met-Leu-Phe, arachidonic acid, BSA, casein, nonfat skimmed milk powder, trypsin, and E64 were purchased from Sigma Aldrich (Milan, Italy). Purification of human erythrocyte calpain and recombinant rat brain calpastatin Human erythrocyte calpain was purified and assayed as reported in [33]. Autoproteolyzed human erythrocyte cal- pain (75 kDa) was prepared as described in [34]. Rat brain recombinant calpastatin RNCAST104 (GenBank accession number Y13588) was prepared as indicated in [35]. M. Averna et al. Calpain–calpastatin interaction in stimulated cells FEBS Journal 273 (2006) 1660–1668 ª 2006 The Authors Journal compilation ª 2006 FEBS 1665 Monoclonal antibodies The mAb against calpain (mAb 56.3) was obtained as reported in [32]. It showed inhibitory properties when added to a calpain activity assay mixture [32,36]. The mAb against calpastatin (mAb 35.23) was produced as described in [35]. Cell culture and human neutrophil isolation MEL cells were obtained and cultured as specified in [37]. Jurkat cells (human T-lymphocyte line) were cultured at 37 °C (5% CO 2 ) in RPMI 1640 (Sigma Aldrich) growth medium containing 10% fetal bovine serum (Euroclone Ltd, UK), 10 UÆmL )1 penicillin (Sigma Aldrich), 100 lgÆmL )1 streptomycin (Sigma Aldrich) and 4 mml-glu- tamine. Human neutrophil isolation was based on a modifi- cation [38] of the procedure described in [39]. Calpain digestion by trypsin Human erythrocyte calpain (10 lg) was incubated in 50 lL 50 mm sodium borate buffer, pH 7.5, containing 0.5 mm 2- mercaptoethanol and 1 mm EDTA for 60 min at room tem- perature with or without trypsin in a calpain ⁄ trypsin ratio of 1000 : 1 [14]. After 60 min, 50 lL 120 mm Tris ⁄ HCl, pH 6.8, containing 4% SDS, 4% 2-mercaptoethanol and 20% glycerol was added to the incubation mixture and then heated for 3 min at 100 °C. Samples (50 lL) were submitted to SDS ⁄ PAGE (10% gel) [40], and western blot was per- formed as indicated in [41]. Proteins were probed with mAb 56.3. The immunoreactive material was revealed as reported in [42]. Calpain immobilization The procedure for immobilization of human erythrocyte calpain is summarized in Scheme 1. The purified enzyme (0.5 lgin5lL50mm sodium borate buffer, pH 7.5, con- taining 0.1 mm EDTA) was spotted on a nitrocellulose sheet (0.5 cm · 0.5 cm; Bio-Rad Laboratories, Bio-Rad Ita- lia, Milan, Italy) and left for 15 min at 4 °C in a humidified chamber. The sheet was then washed with 1 mm EDTA and saturated with 5% nonfat skimmed milk powder. The nitrocellulose sheet was then incubated in 0.1 mL sodium borate buffer, pH 7.5, containing the calpain ligands in the conditions specified elsewhere. The mixtures were incubated at 4 °C for 30 min. mAb 56.3 (0.2 lg) was then added. Calpain was detected using a peroxidase-conjugated secon- dary antibody [42] developed with an ECL Ò detection sys- tem (Amersham Pharmacia Biotech). Immunoreactive material was detected by subjecting the probed nitrocellulose sheets to autoradiography and quanti- fied with a Shimadzu CS9000 densitometer using a fixed wavelength of 590 nm. Binding of mAb 56.3 to intracellular calpain revealed by confocal microscopy and fluorescence quantification Control or stimulated MEL cells, Jurkat cells or human neu- trophils were washed with NaCl ⁄ P i . Calpain and calpastatin were detected by confocal analysis as described in [43], using mAb 56.3 or mAb 35.23 respectively as primary antibody and a fluorescein isothiocyanate-conjugated sheep anti- mouse IgG as secondary antibody (Amersham Biosciences Europe Gmbh, Milan, Italy). The excitation ⁄ emission wave- lengths were 488 ⁄ 522 nm for fluorescein-labeled antibodies and 488–568 ⁄ 605 nm for propidium iodide-stained chroma- tin [44]. Fluorescence was quantified with Laser Pix Software (Bio-Rad Bioscience). Acknowledgements This work was supported in part by grants from MIUR, FIRB and PRIN projects, and from the Uni- Scheme 1. Calpain–calpastatin interaction in stimulated cells M. 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