©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification of -Calpain by a Novel Affinity Chromatography Approach
NEW INSIGHTS INTO THE MECHANISM OF THE INTERACTION OF THE PROTEASE WITH TARGETS (*)

Maurizio Molinari , Masatoshi Maki (1), Ernesto Carafoli (§)

From the (1)Institute of Biochemistry, Swiss Federal Institute of Technology, 8092 Zurich, Switzerland Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A calmodulin-binding motif is a common structural feature of a number of calpain substrates(1) . Since a calmodulin-like domain has been identified in both subunits of the calpain molecule, the proposal was made that the domain(s) would recognize the calmodulin-binding motifs of the substrates prior to the enzymatic modification by calpain. In keeping with the proposal, a succesful attempt to purify µ-calpain from human erythrocytes was made by using an affinity chromatography approach in which the synthetic peptide C49, containing the calmodulin-binding domain of the plasma membrane Ca-ATPase, was coupled to a Sepharose matrix. The calmodulin-like domain of the catalytic subunit of human µ-calpain expressed in Escherichia coli was also retained by the C49-Sepharose column. Both µ-calpain and the calmodulin-like domain interacted with C49 in a Ca-dependent way and were eluted from the column by Ca-chelating agents. The finding confirmed the interaction between the calmodulin-binding domain of the plasma membrane Ca-ATPase and the calmodulin-like domain of µ-calpain. Experiments were performed to establish whether irreversibly inactivated µ-calpain or its expressed C-terminal portion containing the calmodulin-like domain could activate the hydrolysis of ATP by the plasma membrane Ca pump, in keeping with the evident ATPase stimulation of the same pump by calmodulin. A stimulation was observed, but it was much weaker than that induced by calmodulin.


INTRODUCTION

The cytosolic Ca-activated neutral protease calpain (CANP,()EC 3.4.22.17) appears to be involved in physiologically important processes such as cell division (2), signal transduction(3, 4) , and long term potentiation(5) . Its role in the pathological modifications of cells and tissues (e.g. degenerative diseases of muscle and nerve(3, 6, 7, 8, 9, 10) , development of hypertension, cataract formation) is also frequently discussed. In spite of the intense interest of CANP as an object of study, several fundamental questions on its structure (e.g. the role of the heterodimer), its mechanism of activation, and its recognition of target proteins remain obscure. The latter point, i.e. the identification of the CANP region responsible for the recognition of substrates, appears of particular interest, also considering that it could lead to the development of inhibitors alternative to those directed to the active site.

A striking characteristic of a number of CANP substrates is the presence of CaM-binding domains and of highly hydrophilic motifs (PEST sequences) near the cleavage site. Proteins with intracellular half-lives of less than 2 h are unusually rich in PEST regions, defined as sequences rich in proline (P), glutamic (and aspartic) acids (E), serine (S), and threonine (T), flanked by domains containing positively charged amino acids. PEST sequences are common in a number of proteins rapidly degraded by a non-ubiquitin-mediated process, which could involve CANP. This led to the proposal that PEST sequences would sequester Ca, thus creating the microenvironment of higher Ca concentration necessary for CANP activation (11, 12).

However, work in this laboratory (13) has shown that mutations lowering the PEST score of domains surrounding the CaM-binding region of the plasma membrane Ca-ATPase failed to influence the susceptibility of the latter to µ-CANP. By contrast, modifications of the CaM-binding domain of the substrate, e.g. by phosphorylation or its ``occupation'' by CaM, dramatically decreased the susceptibility of the ATPase to the protease. The proposal was thus made that an accessible CaM-binding region is critical for substrate (i.e. the Ca pump) proteolysis, i.e. the CaM-binding domain of the substrate would be important for recognition and interaction with the protease(13) .

One obvious component of the proposal was the presence in both CANP subunits of Ca-binding domains with strong sequence similarity to CaM(14, 46) . The striking resemblance on the amino acid level between the Ca-binding domains of CANP and those of CaM, troponin C, and parvalbumin had already been emphazised 10 years ago(38) . CANP is not the only example of a protein derived from the fusion of a gene for a CaM-like protein and a gene controlling the expression of a protein having another activity (an enzyme). The first description of the primary structure of a kinase described as an hybrid of a Ser/Thr protein kinase catalytic site and a CaM-like domain (calmodulin-like domain protein kinases) appeared in 1991(39) . In the meantime, other enzymes sharing the same features have been characterized in plants (39, 40) and protists like Parameciumtetraurelia(41) . They share a number of properties with CANP. They bind Ca through a regulatory domain on the same polypeptide, which contains the catalytic domain, and they exhibit micromolar dependence on Ca for activity. The C-terminal portion of this family of kinases includes four Ca binding motifs similar to those of CaM and the C-terminal portion of both CANP subunits (domains IV and IV`). shows a comparison of the sequences of the Ca-binding loops of CaM, of the catalytic and regulatory subunits of different CANP isoforms, and of the calmodulin-like domain protein kinase. The underlined amino acids coordinate Ca in CaM; clearly, the canonical CaM sequence is conserved in both CANP subunits and in the hybrid protein kinase.

The proposed mechanism for the activation of calmodulin-like domain protein kinase could also fit CANP; in the presence of Ca, the CaM-like domain of the former interacts with a positively charged amphipatic -helical domain next to or within the autoinhibitory region. The interaction would remove the autoinhibition and thus activate the kinase(40) . The CaMLD of CANP could, however, be important not only in the activation pathway of the protease, but also for the substrate recognition and targeting process; for example, the CaMLD of CANP interacts with the endogenous inhibitor CALST(42, 47) . As shown in this contribution, it also interacts with substrates, particularly those containing CaM-binding domains; the CaM-binding site of the preferred CANP substrate in erythrocytes (the plasma membrane Ca-ATPase) indeed interacts with the CaM-like domain of the protease.


MATERIALS AND METHODS

The chemicals used were of the highest purity grade commercially available. µ-CANP was partially purified as described in Ref. 13. The CANP inhibitor Cbz-Leu-Leu-Tyr-CHN was synthesized as described previously(15) . SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli(16) .

Peptide Synthesis

The peptides used for the experiments were derived from the C-terminal portion of isoform 1CI (17) of the plasma membrane Ca-ATPase (hPMCA1CI). Peptides A18 (EEIPEEELAEDVEEIDHA, PEST sequence 1) (18) and C49 (EEIPEEELAEDVEEIDHAERELRRGQILWFRGLNRIQTQIRVVNAFRSS, PEST sequence 1 + the CaM-binding domain) (19) were synthesized on an Applied Biosystems (Foster City, CA) peptide synthesizer model 431A using the standard-scale FastMoc chemistry according to the manufacturer's instructions. The purity of all synthetic products was confirmed by electrospray mass spectrometry.

Expression of the CaMLD of Human µ-CANP in Escherichia coli

The cDNA coding for the CaMLD was obtained by PCR amplification of the corresponding cDNA region of human µ-CANP large subunit gene(20) . The 0.6-kb BamHI cDNA fragment was inserted into the BamHI site of the plasmid pET-3d (a vector for the expression by T7 RNA polymerase, Novagen, Madison, WI)(47) . The recombinant plasmid pET-L-CaMLD was then used to transform the protease-poor host strain of E. coli (BL21(DE3)pLysE). The CaMLD was expressed by this T7 RNA polymerase system using the following procedure. The cultures were induced by the addition of isopropyl-1-thio--D-galactopyranoside to a final concentration of 0.4 mM. After a 4-h incubation, they were collected by centrifugation and stored overnight at -80 °C. The cells were thawed, resuspended in 50 mM Tris buffer, 1 mM EDTA, 1 mM NaN, pH 7.5, and lysed by sonication on ice (50% pulse for 2 min). The supernatant containing the expressed protein was cleared by centrifugation and stored at -20 °C. N-terminal sequencing after affinity purification confirmed that the expressed protein contained, in addition to the CaMLD region (residues 516-714) of the human µ-CANP catalytic subunit, 11 additional amino acids derived from the expression plasmid.

Coupling of Peptide C49 to CNBr-activated Sepharose 4B

1 g of CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) was allowed to swell in 25 ml of 1 mM HCl (15 min). The resin was washed with 500 ml of 1 mM HCl. 10 mg of peptide C49 (1.7 µmol) were solubilized in 7.8 ml of coupling solution (100 mM NaHCO, 500 mM NaCl, 0.05 mM CaCl, pH 8.3), added to the wet resin, and incubated overnight at 4 °C. The resin was washed with the coupling solution and finally incubated overnight in 10 ml of blocking solution (1 M ethanolamine). The C49-resin was ready after an additional washing step with the washing buffer (50 mM Tris-HCl, 0.5 mM CaCl, 0.5 mM 2-mercaptoethanol, pH 7.5). The same procedure was used to couple peptide A18 to the resin.

Partial Purification of µ-CANP and Isolation of Ghosts from Human Erythrocytes

The crude µ-CANP used for the experiments described here was isolated from freshly collected human erythrocytes. One unit (350-500 ml) of freshly drawn venous human blood in citrate buffer was filtered through a Pall filter (Pall Schweiz AG, Muttenz, Switzerland) to eliminate the white cells. The red cells were lysed in hypotonic Tris buffer (10 mM Tris, pH 7.5) containing 2 mM Na-EDTA, and the cytosolic fraction was chromatographed on DEAE-Sepharose CL-6B (Pharmacia). The peak containing the CANP activity was eluted by a NaCl-linear gradient (for a more precise description of the method used see Ref. 13). The membrane fraction was collected after the lysis of the red cells, washed 5 times with 50 mM Tris, 0.5 mM Na-EDTA, pH 7.5, to eliminate the cytosolic proteins (especially endogenous CaM and µ-CANP), and stored at -80 °C. The ghosts obtained with this procedure were used to measure the stimulation of the activity of the plasma membrane Ca-ATPase by CaM, by the irreversibly inhibited µ-CANP, and by the purified CaMLD.

Purification of µ-CANP on the C49-Affinity Column

Affinity purification of CANP based on its interaction with the endogenous inhibitor (calpastatin) or with a partial sequence derived from the latter (e.g. the 27-residue peptide, DPMSSTYIEELGKREVTIPPKYRELLA, encoded by exon 1B of the human calpastatin gene(25) ) is absolutely Ca-dependent (i.e. CANP binds to the column in the presence of Ca and is eluted with a buffer containing Ca-chelating agents()). The attempt to purify CANP with the C49 affinity column was similarly based on the assumption of Ca-dependence of the interaction of the protease with the CaM-binding domain of the Ca-ATPase of the plasma membrane, the preferred CANP substrate in erythrocytes(26, 27) .

Since CANP rapidly autolyzes in the presence of Ca, the crude CANP preparation obtained as described above was treated with an irreversible, active site-directed CANP inhibitor (Cbz-Leu-Leu-Tyr-CHN) (15, 22) prior to incubation with Ca. 100 µM (final concentration) of the inhibitor was added to the preparation with gentle mixing at 4 °C. After 30 min, CaCl was added to a final concentration of 500 µM, and the reaction mixture was left to incubate on ice for 3 h. Under these conditions the inhibitor alkylated the essential cysteine residue at the active site of CANP. The treatment prevented autoproteolysis and blocked the enzyme in the native 80/30-kDa heterodimeric form. The inhibitor-modified crude enzyme preparation was loaded on the C49-affinity column (0.8 6 cm) previously equilibrated with the washing buffer. The column was washed extensively with 20 volumes of washing buffer containing 1 M NaCl and then with 20 volumes of washing buffer to remove nonspecifically bound proteins. The bound µ-CANP was eluted with 50 mM Tris-HCl, 2 mM Na-EDTA, 0.5 mM 2-mercaptoethanol, pH 7.5 (elution buffer).

Purification of the CaMLD of Human µ-CANP on the C49-Affinity Column

The crude E. coli extract was loaded on the C49 affinity column previously equilibrated with the washing buffer. The column was washed extensively as described above, and the overexpressed CaMLD was eluted with the elution buffer. The identity of the protein was confirmed by N-terminal sequencing.

Preparation of µ-CANP and of the CaMLD to Study Their Stimulation of the Plasma Membrane Ca-ATPase

The irreversibly inactivated catalytic subunit of human µ-CANP and the purified CaMLD were used to test their ability to stimulate the plasma membrane Ca-ATPase. The crude µ-CANP was modified with the irreversible inhibitor Cbz-Leu-Leu-Tyr-CHN in the presence of 0.5 mM Ca and was loaded on a 12% SDS-Lämmli gel. After gel staining with Coomassie Brilliant Blue, the band corresponding to the catalytic subunit was cut and electroeluted from the gel. The denatured, covalently inhibited protein isolated in this way was inactive as a protease when tested on the isolated plasma membrane Ca-ATPase and on a fluorogenic substrate (Suc-Leu-Tyr-AMC). The fraction containing the inhibitor-modified catalytic subunit of µ-CANP as well as the purified CaMLD was dialyzed against the incubation buffer to be used later for the measurement of the ATPase (30 mM Hepes, 120 mM KCl, 1 mM MgCl, 0.5 mM EGTA, pH 7.2(23) ).

Ca-ATPase Activity Assay

The Ca-ATPase was measured in erythrocyte ghosts by following the release of inorganic phosphate by the colorimetric method of Lanzetta et al.(23) . CaCl was added to the reaction mixture to yield a final free concentration of 7 µM (calculated with the help of a computer program(24) ). The reaction was performed in the presence of 1 mM ATP at 37 °C and quantified by measuring the absorption at 660 nm (Shimadzu dual-wavelength spectrophotometer, model UV-3000, Shimadzu, Kyoto, Japan). The stimulation of the Ca-ATPase by CaM was tested using CaM, µ-CANP, and CaMLD at concentrations between 0.01 and 2 µM.


RESULTS

Preparation of the C49 Affinity Column

The synthetic C49 peptide used for the affinity column was derived from the C-terminal portion of isoform 1CI of the plasma membrane Ca-ATPase (hPMCA1CI). The peptide contains the PEST sequence (A18) and the CaM-binding domain of the ATPase. To verify the accessibility of the latter domain in the peptide coupled to the Sepharose matrix, a preliminary assay was performed with CaM. As expected, the protein bound to the column in the presence of Ca and could be eluted with 50 mM Tris, 2 mM Na-EDTA, 0.5 mM 2-mercaptoethanol. Thus, the CaM-binding site of the peptide coupled to the matrix was fully accessible.

Purification of µ-CANP on the C49 Affinity Column

The crude erythrocyte extract containing the irreversibly inhibited µ-CANP was loaded on the C49 column previously equilibrated with the Ca-containing washing buffer (Fig. 1, laneL). The extensive washing allowed the elimination of the nonspecifically bound material in the flow-through (Fig. 1, laneFT). The protein bound to the peptide C49 coupled to the matrix was then eluted with the elution buffer containing a Ca-chelating agent (Fig. 1, lanes1-6) and was identified as CANP using a polyclonal antibody against the human platelet-m-CANP, which cross-reacted with erythrocytes µ-CANP (data not shown).


Figure 1: Purification of human µ-CANP using the C49 affinity column. LaneS, low molecular weight standard; laneL, crude human erythrocytes lysate loaded on the affinity column; laneFT, flow-through (unbound protein); lanes1-6, elution peak containing the purified µ-CANP. Additional details are found under ``Materials and Methods.''



The possibility was considered that µ-CANP was trapped by the column by unspecific interactions with the matrix. The synthetic peptide (A18) from the C-terminal portion of isoform 1CI of the plasma membrane Ca-ATPase, located immediately upstream of the CaM-binding domain, was thus coupled to the matrix used for the C49 affinity column (CNBr-activated Sepharose 4B); the A18 column failed to bind CANP in a Ca-dependent manner.

Purification of the CaMLD of the Catalytic Subunit of µ-CANP Using the C49 Affinity Column

Since the heterodimeric form of µ-CANP could be purified by taking advantage of the affinity of the protease for the CaM-binding region of the substrate, it seemed very likely that the regions of the CANP molecule responsible for the interaction were the CaM-like domains of the protease (domain IV and/or domain IV`). The crude E. coli extract containing the expressed CaMLD was loaded on the C49 affinity column, previously equilibrated with the washing buffer (Fig. 2, laneL). The column was washed extensively (Fig. 2, laneFT) as described above, and the pure CaMLD could be eluted with the EDTA-containing buffer (Fig. 2, lanes1-3). N-terminal sequencing confirmed that the isolated protein was indeed the CaMLD of µ-CANP.


Figure 2: Purification of the CaM-like domain (CaMLD) of the catalytic subunit of human µ-CANP using the C49 affinity column. LaneS, low molecular weight standard; laneL, crude E. coli extract loaded on the affinity column; laneFT, flow-through (unbound protein); lanes1-3, elution peak containing the purified CaMLD. Additional details are found under ``Materials and Methods.''



Activation of the Ca-ATPase by µ-CANP and by Its Isolated CaMLD

The Ca-dependent ATP-hydrolysis by the plasma membrane Ca-ATPase can be reversibly stimulated by CaM (28-29) or irreversibly stimulated by CANP proteolysis(30) . µ-CANP cleaves the purified pump upstream to the CaM-binding domain, removing its autoinhibitory effect on the pump. Interestingly, the plasma membrane Ca-ATPase could be weakly activated by irreversibly blocked CANP and by its isolated CaMLD.

Fig. 3compares the stimulation of the Ca-ATPase of erythrocytes ghosts by incubation with CaM with the irreversibly inactivated µ-CANP and with the CaMLD of the major CANP subunit; maximal stimulation in the case of CaM was reached at about 1.7 µM, when the activity of the ATPase was 3.5 times higher than in the absence of the effector. The stimulation by µ-CANP was much less pronounced (1.7-fold), and that by the CaMLD even weaker (1.4-fold). Both reached maximal value at about 2 µM.


Figure 3: A comparison between the activation effects by CaM, by the irreversibly inactivated catalytic subunit of human µ-CANP and by its isolated CaMLD on the plasma membrane ATPase. A, stimulation of the plasma membrane Ca-ATPase by CaM; B, stimulation of the plasma membrane Ca-ATPase by irreversibly inactivated µ-CANP; C, stimulation of the plasma membrane Ca- ATPase by the CaMLD. Additional details are found under ``Materials and Methods'' and ``Discussion.''




DISCUSSION

This contribution describes the purification of µ-CANP by a novel affinity chromatography approach. The results were obtained by taking advantage of the ability of CANP, and more precisely of its CaM-like domains, to interact with the CaM-binding region of the plasma membrane Ca-ATPase. The general preference of CANP for CaM-binding proteins as substrates in vivo and in vitro(1) and previous work showing that changes in the CaM-binding domains of substrates impaired their susceptibility to CANP digestion (13) led to the proposal of a direct interaction between CANP and CaM-like domains. This interaction was established by the following findings. (a) µ-CANP could be purified by affinity chromatography on a column of peptide C49, which contains the CaM-binding domain of the plasma membrane Ca-ATPase; (b) an expressed peptide containing the CaMLD of the catalytic subunit of human µ-CANP was isolated from crude E. coli extracts using the same C49 affinity column; (c) the activity of the Ca-ATPase of erythrocytes ghosts was stimulated, albeit only weakly, by irreversibly inactivated µ-CANP and by the purified CaMLD (subdomain IV of human µ-CANP). Although the work described here only relates to the plasma membrane Ca-ATPase, it appears likely that the CaMLDs of CANP will interact in a similar way with the CaM-binding regions of other CANP targets (e.g. spectrin).

The co-purification of both the catalytic and the regulatory subunits of CANP was a remarkable accomplishment. As reported recently(45) , the two subunits of CANP dissociate in the presence of Ca. In the chromatography approach described here, the protease was loaded onto the column in the presence of 500 µM Ca, a concentration inducing the dissociation. In spite of that, both subunits were retained in the column in a Ca-dependent way and eluted with Ca-chelating agents. This is further evidence that both subunits of CANP interacted with the CaM-binding domain of the ATPase. The finding that the subunits were not eluted in an equimolar ratio (Fig. 1, lanes1-6) could be traced back to the loss of a portion of the regulatory subunit during the preceding purification steps described under ``Materials and Methods.'' A comparison of lanesL and FT in Fig. 1reveals that nearly all of the loaded small subunit (band at 30 kDa) was bound to the column, with only a faint trace of it appearing in the flow through.

Previous work had established that CANP migrated from the cytosol to the membranes in the presence of Ca(27, 31, 32, 33, 34, 35, 36) . Although it is not yet conclusively established whether the interaction occurs with membrane/cytoskeletal proteins or with membrane phospholipids, recent experiments have shown that trypsinization, but not the treatment with phospholipase C, greatly reduced µ-CANP binding to inside out membrane vesicles(34) . Moreover, Kawasaki et al.(37) have reported that the binding of CANP to membranes was inhibited by a fragment of calpastatin that interacted with the CaMLDs of CANP. As discussed in this work, this region of the protease interacts with target proteins as well. The results presented here are relevant to the problem, i.e. they add weight to the suggestion that CANP binds to membrane or cytoskeletal substrate proteins (probably at physiological Ca concentrations as well). On the other hand, it must be mentioned that work in this laboratory has never identified the autolyzed µ-CANP (lacking the Gly-rich hydrophobic tail of the regulatory subunit, but containing the intact CaMLDs) bound to the membrane(27) .

The results of the experiments on the stimulation of the plasma membrane Ca-ATPase by CANP are of interest. It was already known that limited cleavage of the ATPase by CANP led to its stimulation due to the removal of the autoinhibitory CaM-binding domain of the pump. The present work is the first report of a stimulation of the pump by CANP, independent from proteolysis. Since the activation by inactive CANP or by its CaMLD was far lower than in the case of CaM, a word of caution on this matter is in order; the results nonetheless strenghten the proposal of a direct interaction between the CaMLD of CANP and the CaM-binding regions of substrates. That the stimulation, however limited, was not due to the presence of traces of proteolytical activity in the preparation of irreversibly inactivated, electroeluted, catalytic subunit of µ-CANP was established by appropriate control experiments using a fluorogenic substrate (Suc-Leu-Tyr-AMC), which is particularly sensitive to CANP. Another point that could be mentioned is that the work described here has considered only the catalytic subunit or the CaMLD located in the C-terminal portion of it. It is in principle possible that the slightly different CaMLD of the regulatory subunit is principally responsible for the interaction with substrates. The finding that CaM-binding proteins are preferred substrates of CANP in vivo(26, 27, 43, 44) indicates that the protease may be able to interact with them even in a system containing high concentrations of CaM, i.e. to compete with the latter for the same binding site in vivo. It could thus be speculated that the binding of CANP to the target protein would not be necessarily followed by its cleavage. In the particular case of the Ca-ATPase of the plasma membrane, the in vitro experiments shown here have documented that the irreversibly inactivated CANP or its CaMLD could qualitatively mimic the action of CaM, activating the pumping of Ca out of the cell. It could then be further speculated that, following the return of the intracellular Ca concentration to normal, CANP would leave the pump and return to the quiescent, soluble form. However, should the intracellular Ca concentration rise excessively, or be consistently maintained at a higher than normal level, CANP would cleave the (CaM-binding) autoinhibitory domain of the ATPase, permanently activating the latter. An activation pathway alternative to that generally accepted (21) thus can be postulated (Fig. 4). (a) The increased Ca-concentration in a limited portion of the cytosol (Fig. 4, a and b) would allow CANP to bind to a few molecules of CaM-binding proteins (e.g. the Ca-ATPase) embedded in the membrane; (b) the substrate-associated protease could partially digest the target (Fig. 4, b and c). Importantly, this may occur in the absence of CANP autolysis(27) ; (c) if the target is the Ca-ATPase, the binding of CANP to its CaM-binding domain (Fig. 4b) and/or its partial proteolysis (Fig. 4c) would induce activation of the enzyme to restore the physiological intracellular Ca concentration; (d) at this point, CANP would dissociate from the substrate and return to the cytoplasm in the native form, ready to be reactivated in the case of a new increase in Ca concentration. The partially proteolyzed pump (Fig. 4c) is then in all likelihood further proteolyzed by other cellular proteases specialized in the elimination of partially modified proteins(26) .


Figure 4: A proposal for the effect of CANP on the plasma membrane Ca-ATPase. a, resting state; b, the increase of the Ca concentration in the cytosol allows the binding of CANP to the ATPase molecule; possibly, CANP acts as ``pseudo CaM'' when the Ca concentration is insufficient to trigger the activation of the protease. The binding of CANP, perse, would be sufficient to partially activate the ATPase; c, CANP (the intact or the autolytically modified form) cleaves the pump, eliminating the autoinhibitory function of the CaM-binding domain; the Ca-ATPase would be permanently activated and then further degraded by the cellular proteolytical machinery. This pathway eventually implies the loss of the ATPase molecule and the irreversible activation of CANP by autolysis. d, if the Ca concentration in the cytosol would rapidly decrease, the CANP and the plasma membrane Ca-ATPase would both return, unmodified, to the resting state. Additional details are found under ``Discussion.''



  
Table: Sequence similarity between some E-F-hand Ca-binding proteins, the CaM-like domains of the two subunits of different CANP isoforms, and the CaM-like domain of a hybrid protein kinase from plants

The underlined amino acids, also reported in the canonical sequence at the top of the table, have been shown to be responsible for the coordination of Ca in CaM; the amino acids corresponding to X, Y, Z and -Z do so through the oxygen of their (carboxylic) side chain; -Y through its peptide-bond carbonyl oxygen; -X through the oxygen of a water molecule. G represents a conserved glycine in the middle of the Ca-binding loops of all EF-hand type proteins (see ``Discussion'').



FOOTNOTES

*
This work was supported by Swiss National Science Foundation Grant 31-30858.91). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Inst. of Biochemistry III, Swiss Federal Inst. of Technology (ETH), Universitätstrasse 16, 8092 Zurich, Switzerland. Tel.: 41-1-632-30-11; Fax: 41-1-632-12-13.

The abbreviations used are: CANP, Ca-activated neutral protease (calpain); CaM, calmodulin; CaMLD, calmodulin-like domain; Cbz, benzyloxycarbonyl.

M. Molinari, M. Maki, and E. Carafoli, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Paola Dainese for the sequence analysis of the affinity purified CaMLD and Carmela Galli for the drawing of Fig. 4.


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