Protein for Lp82 Calpain Is Expressed and Enzymatically Active in Young Rat Lens

Exp. Eye Res. (1998) 67, 221–229 Article Number : ey980515

Protein for Lp82† Calpain Is Expressed and Enzymatically Active in Young Rat Lens H. M Aa, M. S H I Ha, I. H A T Aa, C. F U K I A GE,a,b, M. A Z U MAa,b    T. R. S H E A R E Ra* a

Departments of Oral Molecular Biology, Biochemistry and Molecular Biology, and Ophthalmology, Oregon Health Sciences University, Portland, Oregon 97201, U.S.A. and b Laboratory of Biology, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani, Nishi-ku, Kobe 651-22, Japan (Received Rochester 18 February 1998 and accepted in revised form 4 April 1998) mRNA for a newly discovered isoform of calpain, termed Lp82, was recently discovered in young rat lens. The purpose of the present experiments was to test for expression of Lp82 protein. Casein zymography after incubation with calcium was used to detect Lp82 proteolytic activity in regions of lenses from young rats. Lp82 protein was detected by immunoblotting or by ELISA after DEAE-5PW chromatography using a polyclonal antibody generated to a peptide sequence in Lp82. Northern blot analysis assessed expression of Lp82 mRNA. Four results demonstrated expression of Lp82 protein ; (1) immunoblot reactivity at the predicted molecular mass of 82 kDa, (2) a unique band of calcium-activated lysis in casein zymograms, (3) partial purification and retention of activity from a single Lp82 peak on DEAE-5PW chromatography, and (4) positive immunoblotting and Northern blot analysis only in lens and not in other rat tissues. These results showed that Lp82 protein is lens-preferred, relatively abundant in young rats (especially nucleus), and enzymatically active. Proteolysis of crystallins in the nucleus of young rat lens during normal maturation and cataract formation, formerly attributed solely to m-calpain, may in fact be due to concerted action of both lens Lp82 and ubiquitous m-calpain. # 1998 Academic Press Key words : Lp82 protein ; calpain ; rat lens ; casein zymogram ; immunoblot.

1. Introduction Recently mRNA for a lens-specific form of calpain, termed Lp82, was discovered in young rat lens (Ma et al., 1998). mRNA for Lp82 was expressed only in lens and at levels equal to the abundant, calcium-activated protease, m-calpain. Calpains (EC 34 . 22 . 17) are a family of non-lysosomal cysteine proteases comprised of ubiquitous calpains (m-calpain and µ-calpain found in most tissues) and tissue-specific calpains (muscle type p94, stomach and smooth muscle types nCl-2 and nCl-2«) (Suzuki et al., 1995). In rodent lenses, calpaininduced proteolysis of lens structural proteins, α- and β-crystallins, is thought to be the fundamental mechanism for a variety of cataracts (Shearer et al., 1997). Calpain activity is also the cause of extensive proteolysis observed in the nucleus of rat lens during normal maturation (David, Azuma and Shearer, 1994). Currently, tissue specific calpains are under intense investigation because they are postulated to have specific functional roles in their tissue-preferred locations. For example, genetic mutations in muscletype calpain p94 cause limb-girdle muscular dystrophy type 2A in man (Richard et al., 1995). Remarkably, the cDNA product for lens-specific calpain Lp82 was * Correspondence : T. R. Shearer, Oregon Health Sciences University, 611 SW Campus Dr., Portland, Oregon 97201, U.S.A. † The nucleotide sequence data for Lp82 cDNA can be accessed at GenBank accession number U96367 and for Lp85 at AF052540.

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similar to muscle-type calpain p94, except a different exon 1 was present, and the nucleotides for all of exons 6, 15 and 16 were deleted in Lp82 (Ma et al., 1998). Thus, Lp82 is a splice variant of p94. In the translated enzyme, these important deletions would result in removal of the two unique insert regions termed IS1 and IS2 in p94 (Sorimachi et al., 1989) and replacement with a completely different Nterminal region (Fig. 1). The IS1 and IS2 regions in p94 are thought to confer muscle-specific functions (very low calcium requirements, rapid autodegradation, nuclear localization). Their deletion in Lp82 may be related to lens-specific functions. The data reported below are the first to demonstrate expression of abundant amounts of proteolytically active, Lp82 protein in young rat lens, notably in the nuclear region. 2. Materials and Methods Tissue Collection and Isolation of RNA Soft tissues and lenses without decapsulation were isolated from Sprague-Dawley (B & K Universal, Fremont, CA and Seattle, WA, U.S.A.) and Wistar (SLC Japan, Shizuoka) rats. Experimental animals were handled in compliance with the ‘ Guiding Principles in Care and Use of Animals ’ (DHEW Publication, NIH 86-23). Capsule}epithelium preparations were isolated by pooling dissections of the capsule with adhering epithelium, and the remaining lens was then dissected # 1998 Academic Press

F. 1. Location of peptide sequence (heavy bar) used to generate antibody against Lp82 in the deduced amino acid sequences for Lp82 and p94 in rat tissues. Differences are shaded. Unique NS, IS1 and IS2 regions of p94 are bracketed, and active site amino acids are indicated (*). Modified from (Ma et al., 1998). Dotted line indicates a second peptide used to generate an antibody for immunoassays in rabbit lens Lp82.

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at approximately 50 % of the radius into cortical and nuclear regions. Casein Zymography Ten percent (1 mm thick) gels, copolymerized with 0±1 % casein (TEFCO, Japan), were pre-run with a buffer containing 25 m Tris (pH 8±3), 192 m glycine, 1 m EGTA, and 1 m dithiothreitol for 15 min at 4°C (Fukiage et al., 1997 ; Raser, Posner and Wang, 1995). Soluble proteins were then loaded and run. After electrophoresis, gels were incubated with slow shaking overnight at room temperature in 20 m Tris (pH 7±4), 10 m dithiothreitol, and 20 m calcium. Gels were stained with Coomassie Brilliant Blue. Bands of caseinolysis appeared white. Production of Lp82 Antibody Antibody to rat Lp82 was generated by first synthesizing a peptide containing 21 amino acids (Fig. 1, heavy bar). The peptide was chosen because it possessed these characteristics : present in Lp82, not present in m- or µ-calpains, relatively hydrophillic, and predicted to be on the surface of Lp82. Note that portions of the 21-mer sequence are also present in p94 calpain (Fig. 1). On immunoblots, this allowed visualization of rat Lp82 at 82 kDa or p94 at 94 kDa depending on the tissue blotted. The peptide was synthesized by FMOC protocol, HPLC purified, and attached to KLH by EDC conjugation. The first immunization was with Freund’s complete adjuvant, and subsequent immunizations were at four week intervals with Freund’s incomplete adjuvant. Rabbits were anaesthetized and exsanguinated, and the antibody was purified from serum using the peptide bound to Aminolink resin (Pierce, Rockford). The antibody produced was strongly reactive for Lp82 protein in young rat lens at 1 : 2000 dilution in immunoblots (Fig. 2) and at 1 : 2000 dilution in ELISA [Fig. 4(B)]. For immunoassays of proteins from rabbit lens, an antibody against a second peptide of 14 amino acids (Fig. 1, dotted line) was used because it provided stronger staining than the antibody used for rat tissues described above. Chromatography of Rat Lens Proteins Lens regions were homogenized in buffer containing 20 m imidazole (pH 6±8), 1 m EGTA, 1 m EDTA and 2 m DTE. The soluble and insoluble fractions were obtained by centrifugation at 16 000 g for 15 minutes. Insoluble pellet was resuspended in 8  urea for analysis. The soluble proteins were fractionated by HPLC using a 7±5 mm¬7±5 mm DEAE 5-PW column (TOSOH, Japan) with a linear 0±0–0±5  NaCl gradient in buffer A containing 20 m Tris (pH 7±5), 1 m EGTA, 1 m EDTA and 2 m DTE at 1 ml min−" flow rate. ELISA was performed by absorbing 25 ul each

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column fraction in 0±1  NaHCO buffer (pH 9±3) $ overnight onto 96 well flat bottom plates (Corning, New York, U.S.A.). The wells were then blocked with 5 % non-fat dry milk and incubated with Lp82 (1 : 2000 dilution) or m-calpain (1 : 2000) antibodies for 1 hr. This was followed by visualization with goat anti-rabbit alkaline phosphatase-conjugated secondary antibody and p-nitrophenol (Bio-Rad, Hercules, CA, U.S.A.) substrate. The Lp82 and m-calpain peaks from several DEAE-5PW runs were separately pooled and concentrated by ultrafiltration (Centricon-10, Amicon, Beverly, MA, U.S.A). Protein assays (micro) were performed with the Bio-Rad dye-binding reagent or the Pierce BCA assay. Electrophoresis and immunoblotting SDS-PAGE of the soluble and insoluble lens proteins was performed on 8 % mini-polyacrylamide gels. Immunoblots for Lp82 and m-calpain were performed by electro-transferring proteins from SDS-PAGE gels onto PVDF membrane at 30 volts (constant) for 80 minutes at ice-cold temperatures using Tris-glycine buffer. The Lp82 and m-calpain antibodies were used at 1 : 2000 dilution, and immunoreactivity was visualized with alkaline phosphates conjugated to anti-rabbit IgG secondary antibody and BCIP}NBT (Bio-Rad). Images of immunoblots were digitized on a flat bed-scanner, and image analysis was performed by NIH Image 1±57 software (Internet, via anonymous ftp from zippy.nimh.nih.gov). Northern Blot Analysis Total RNA was extracted from various rat tissues in TRIzol reagent (1 ml 100 mg−" tissue) according to the manufacturer’s instructions (GIBCO BRL, Grand Island, NY, U.S.A.). A riboprobe to Lp82 mRNA was made by insertion of a PCR fragment (nucleotides 703 to 1097) into pGEM-4Z vector (Promega, Madison, U.S.A.). These 395 nucleotides were specific for a sequence in Lp82 spanning and not containing the 144 nucleotides deleted from the IS1 region. Thus, under the high temperature conditions (70°C) for Lp82 hybridization, the probe would not be expected to hybridize to p94 mRNA since the p94 mRNA contains the 144 nucleotide IS1 region. A Sp6 BrightStar BIOTINscript4 kit (Ambion, Austin, U.S.A.) was then used to synthesize a biotin-labeled RNA probe in vitro. RNA samples (20 µg of total RNA) were electrophoresed on a 1 % agarose gel under denaturing conditions. After electrophoresis, the RNA was transferred onto a nylon membrane (Ambion) and fixed under UV light. Hybridization was performed using biotin-labeled RNA probes in Northern Max4 Hybridization solution (Ambion) at 70°C for 16 hr. The Blots were finally detected with BrightStar4 BioDetect4 kit (Ambion). Light emission was recorded on X-OMAT AR film (Kodak, Rochester, U.S.A.).

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3. Results Lp82 Protein As predicted from the cDNA of Lp82, immunoblots for Lp82 protein in young rats lens showed a major band at 82 kDa (Fig. 2). Immunostaining at 94 kDa was not observed, strongly suggesting that the Lp82 band is not a breakdown product of muscle type calpain p94. Note that appreciable amounts of the Lp82 protein were found in both the soluble (lanes 2 and 3) and insoluble fractions (lanes 4, 5 and 6). In the soluble fraction, the amount of Lp82 protein in the nucleus was approximately equal to cortex, while epithelium did not immunostain. In the insoluble fraction, very large amounts of Lp82 protein were found in the nucleus, followed by cortex and epithelium (Fig. 2, lanes 4, 5 and 6).

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antibody [Fig. 3(C)]. By this method, the uppermost band on the zymogram was established to be Lp82, and the lowermost band was m-calpain. The identity of the middle band of enzyme activity remains unknown, but could represent another splice variant of calpain or an unknown calcium-protease. The relative proteolytic activity for Lp82 in two week old rat lens was nucleus " cortex " epithelium. This distribution was the opposite of m-calpain, where epithelium was highest and nucleus was lowest. Since Lp82 caseinolytic activity was detected in lens epithelium, but Lp82 protein in the epithelium did not stain in immunoblots, casein zymography under our conditions appeared to be more sensitive than immunoblotting. In addition, the large amounts of Lp82 protein in the lens nucleus were found to be proteolytically active against casein. (Azuma, Fukiage and Shearer, unpublished).

Caseinolytic Activity of Lp82

Partial Purification of Lp82

All soluble lens regions from two week old rats showed bands of calcium-activated, caseinolysis [Fig. 3(A)]. Identity of the lytic bands on the zymogram was established by separate immunoblotting from native gels with Lp82 antibody [Fig. 3(B)] and m-calpain

HPLC using DEAE-5PW with a NaCl gradient separated rat lens soluble proteins into several major protein peaks [Fig. 4(A)]. Two separate ELISA assays [Fig. 4(B)] on the DEAE-5PW fractions were then performed using : (1) Lp82 antibody and (2) an

F. 2. Immunoblot for Lp82 in the soluble and insoluble regions of 12 day old rat lens. Epi ¯ capsule}epithelium preparation, Cor ¯ cortex, and Nuc ¯ nucleus. Numbers to the left are migration positions of a 10 kDa ladder. Arrow points to the major immunoreactive band for Lp82 at 82 kDa. All lanes contain 30 µg protein.

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F. 3. (A) Casein zymogram showing lytic bands for Lp82 and m-calpain in lens regions from 2 week old rats. All lanes contained 20 µg protein. (B) Immunoblot with Lp82 antibody from native PAGE as used in zymogram. (C) Immunoblot with m-calpain antibody. All lanes in immunoblots contained 100 µg protein.

antibody reacting with both m- and µ-calpains. Lp82 eluted as a sharp peak at 185 m NaCl well before the m-calpain peak at 317 m salt. Since the amount of contaminating proteins in the Lp82 peak was decreased, the single DEAE-5PW step partially purified the Lp82 enzyme. After concentrating the Lp82 and m-calpain peaks separately from several DEAE-5PW runs, the Lp82 fraction still exhibited a major band of Lp82 proteolytic activity on casein zymograms (Fig. 5, lane 2) and showed staining at 82 kDa on immunoblots from SDS-PAGE gels (Fig. 5, lane 4). Preliminary data also indicated that the concentrated Lp82 fraction from DEAE-5PW chromatography of the soluble proteins from the nucleus from forty 13 day old rat pups produced approximately 3±1 µg FITC-labeled casein fragments min ml−" while the m-calpain fraction produced 1±4 µg min−" ml−" (Shih and Shearer, unpublished).

Lens-specificity Northern blot analysis of mRNA from seven rat tissues using a riboprobe to Lp82 showed a single transcript at 2±9 kb only in lens [Fig. 6(A)]. Lensspecificity was observed even though relatively large amounts (20 µg) of RNA from all tissues were probed as indicated by the heavy staining for GAPDH mRNA [Fig. 6(B)]. This was similar to our previous results with RT-PCR showing full-length Lp82 mRNA only in lens and not in seven other rat tissues (Ma et al., 1998). Lens-specific distribution of Lp82 mRNA was also reflected in immunoblot analysis for Lp82 protein in various rat tissues (Fig. 7). Lp82 protein was found only in lens (Fig. 7, lane 8). Our antibody also allowed visualization of the muscle-type calpain (p94) at 94 kDa in rat muscle (Fig. 7, lane 6), which was well above (open arrow)

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F. 4. Partial purification of Lp82 by DEAE-5PW-HPLC (A) followed by ELISA (B). The Lp82 and m-calpain peaks were detected by two separate ELISA assays using Lp82 and m-calpain antibodies. Lp82 eluted as a sharp peak at 185 m NaCl well before m-calpain at 317 m NaCl.

F. 5. Casein zymogram of total soluble proteins from two week old rat lens (lane 1), the Lp82 peak after DEAE-5PW chromatography (lane 2), and the m-calpain peak after DEAE-5PW chromatography (lane 3). Lane 4 is an immunoblot using Lp82 peak after DEAE-5PW chromatography.

C A L P A IN-L P82 P R O T E I N I N L E N S

F. 6. Northern blot analysis for mRNAs for Lp82 (A) and glyceraldehyde-3-P dehydrogenase (B) in various tissues from 12 day old rats. 1 ¯ brain, 2 ¯ kidney, 3 ¯ lung, 4 ¯ muscle, 5 ¯ lens, 6 ¯ cornea, and 7 ¯ retina. Approximate RNA sizes are listed on the sides in kb.

F. 7. Immunoblot using Lp82 antibody in nine tissues from 12 day old rats. Lanes are : 1 ¯ brain, 2 ¯ heart, 3 ¯ kidney, 4 ¯ liver, 5 ¯ lung, 6 ¯ muscle, 7 ¯ cornea, 8 ¯ lens and 9 ¯ retina. Only lens showed a major immunoreactive band at 82 kDa (Closed arrow). Other tissues showed positive staining near p94 (open arrow), but not for Lp82. All lanes contained 30 µg protein.

the Lp82 signal (solid arrow). Heart, liver and kidney also stained at 94 kDa, and small amounts of p94 mRNA have been reported in other tissues in addition to muscle (Sorimachi et al., 1995). Note, however, that even though Lp82 is a splice variant of p94, the upper p94 protein band was not found in lens tissues (Fig. 7, lane 8). Further, RT-PCR using primers for nearly full length p94 has not detected p94 mRNA in rat lens (Ma and Shearer, unpublished). These data again indicate that Lp82 protein was not a degradation product of p94 protein and that Lp82 is at least lenspreferred in tissue distribution. Immunoreactive bands for Lp82 have also been noted in one week old rabbit lenses (Fig. 8). Rabbit lens Lp82 showed somewhat similar regional distribution in the epithelium, cortex and nucleus of the soluble and insoluble fractions as in rats.

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The identities of most other fainter bands in the immunoblots blots for Lp82 in lens were unknown, and they could be degradation products of Lp82, crossreactive proteins or other splice variants. Indeed, note that in rat lens, a fainter immunoreactive band at approximately 85 kDA was consistently observed above the Lp82 band in most lens fractions (Fig. 2), and it co-eluted with Lp82 after DEAE-5PW fractionation (Fig. 5, lane 4). The cDNA for this band was cloned and sequenced (GenBank accession number AF052540) and found to be identical to Lp82 except for a 84 nucleotide sequence inserted behind the IS2 region. This cDNA was predicted to code for a protein with a mass of 85±0 kDa, and was termed Lp85. Note that Lp82 is not a breakdown product of Lp85, since Lp82 does not contain the internal amino acid sequence coded for by the 84 base pair insertion. Thus, Lp85 is a less abundant isoform of calpain with properties to be reported elsewhere. 4. Discussion Until now, Lp82 protein was only deduced from our published cDNA sequence (Ma et al., 1998). The major finding of the present report was that the enzymatically active, Lp82 protein is indeed expressed in young rodent lenses. Several findings supported this conclusion : (1) Lp82 antibody showed a major protein band at 82 kDa after immunoblotting rat lens samples from SDS-PAGE gels. The migration position of the Lp82 protein coincided with the mass of 82±2 kDa predicted from the cDNA sequence. (2) Lp82 protein was proteolytically active against casein after incubation of zymograms in calcium buffer. Identity of the Lp82 lytic band was distinguished from calciumactivated m-calpain by migration position on immunoblots after a second type of electrophoresis—native gel electrophoresis. This established that young rat lens contains at least two types of abundant, calciumactivated, cysteine protease activity : Lp82 and mcalpain. Interestingly, Lp82 was most active in the nucleus, while m-calpain showed more activity in cortex and epithelium. (3) ELISA assay after HPLCDEAE-5PW resolved a partially purified Lp82 peak which retained caseinolytic activity, characteristic mobility on native PAGE gels, and immunoreactivity at 82 kDa after SDS-PAGE. (4) Lp82 protein was found by immunoblot analysis only in lens and not in eight other tissues from young rats. This lens-specificity at the protein level was logical since Northern blotting data and previous RT-PCR analysis (Ma et al., 1998) found mRNA for Lp82 and not in six other rat tissues. The Lp82 protein appears to be translated from a single mRNA transcript of approximately 2±9 kb. (5) Lp82 protein was not limited to rat, but has been detected by immunoblotting in lenses from young rabbit. Thus, ample evidence has established that relatively abundant amounts of enzymatically active Lp82 protein are expressed in young lenses from

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F. 8. Immunoblot for Lp82 in one week old rabbit lens. Epi ¯ capsule epithelium preparation, Cor ¯ cortex, and Nuc ¯ nucleus. All lanes contain 30 µg protein.

certain species. To date, our evidence indicates that the protein is at least lens-preferred. The function of Lp82 protein in young rat lens is presently speculative and will be determined after purification of the Lp82 enzyme. For purification of Lp82 from rat lens, the elution position on DEAE-5PW was particularly advantageous. The Lp82 peak was well before m-calpain, after µ-calpain and calpastatin inhibitor peaks at approximately 100–150 m NaCl (David and Shearer, 1986), (Shearer et al., 1990), and in an area showing very low amounts of other lens proteins. The Lp82 was stable to DEAE-5PW chromatography and concentration, and it may already be highly enriched after this one step purification. Rabbit lens may possibly serve as a larger tissue source for further Lp82 purification. The present finding of high amounts of Lp82 enzyme activity in the nucleus of young rats was provocative because the nucleus of young rat lens is sensitive to cataract formation. For example, selenite cataract forms rapidly in the nucleus of 12 day old rats, while the majority of the cortex remains clear (Shearer et al., 1983). Massive precipitation of truncated crystallins is also observed in young rat lens during formation of a

variety of other types of cataract (Shearer et al., 1997). Further, the nucleus is the major site for accumulation of truncated crystallins during normal maturation of young rat lens (David, Azuma and Shearer, 1994). These data suggest possible synergistic relationships between Lp82 and m-calpain. For example, the proteolysis occurring during cataract formation and during normal lens maturation, long attributed solely to m-calpain, may in fact be partially due to the proteolytic activity of Lp82 protease described in the present report. Acknowledgements Supported in part by NIH grant EY05786 to TRS.

References David, L. L. and Shearer, T. R. (1986). Purification of calpain II from rat lens and determination of endogenous substrates. Exp. Eye Res. 42(3), 227–38. David, L. L., Azuma, M. and Shearer, T. R. (1994). Cataract and the acceleration of calpain-induced beta-crystallin insolubilization occurring during normal maturation of rat lens. Invest. Ophthalmol. Vis. Sci. 35(3), 785–93.

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Fukiage, C., Azuma, M., Nakamura, Y., Tamada, Y. and Shearer, T. R. (1997). Calpain-induced light scattering in crystallins from three rodent species. Exp. Eye Res. 65, 757–70. Ma, H., Fukiage, C., Azuma, M. and Shearer, T. R. (1998). Cloning and expression of mRNA for calpain Lp82 from rat lens : splice variant of p94. Invest. Ophthalmol. Vis. Sci. 39, 454–61. Raser, K. J., Posner, A. and Wang, K. K. (1995). Casein zymography : a method to study mu-calpain, m-calpain, and their inhibitory agents. Arch. Biochem. Biophys. 319(1), 211–16. Richard, I., Broux, O., Allamand, V., Fougerousse, F., Chiannilkulchai, N., Bourg, N., Brenguier, L., Devaud, C., Pasturaud, P., Roudaut, C. et al. (1995). Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy 2A. Cell 81(1), 27–40. Shearer, T. R., Anderson, R. S., Britton, J. L. and Palmer, E. A. (1983). Early development of selenium-induced cataract : slit lamp evaluation. Exp. Eye Res. 36(6), 781–8.

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Shearer, T. R., Azuma, M., David, L. L., Yamagata, Y. and Murachi, T. (1990). Calpain and calpastatin in rabbit corneal epithelium. Curr. Eye Res. 9(1), 39–44. Shearer, T. R., Ma, H., Fukiage, C. and Azuma, M. (1997). Selenite nuclear cataract : Review of the model. Mol. Vis. 35, 8. Sorimachi, H., Imajoh-Ohmi, S., Emori, Y., Kawasaki, H., Ohno, S., Minami, Y. and Suzuki, K. (1989). Molecular cloning of a novel mammalian calcium-dependent protease distinct from both m- and mu-types. Specific expression of the mRNA in skeletal muscle. J. Biol. Chem. 264(33), 20106–11. Sorimachi, H., Tsukahara, T., Okada-Ban, M., Sugita, H., Ishiura, S. and Suzuki, K. (1995). Identification of a third ubiquitous calpain species-chicken muscle expresses four distinct calpains. Biochim. Biophys. Acta 1261(3), 381–93. Suzuki, K., Sorimachi, H., Yoshizawa, T., Kinbara, K. and Ishiura, S. (1995). Calpain : novel family members, activation, and physiologic function. Biol. Chem. Hoppe Seyler 376(9), 523–9.