Characterization of calpain II in the retina and photoreceptor outer segments

Calpains (EC 3.4.22.17) are Ca²⁺-activated neutral cysteine proteases that appear to play critical roles in various cellular processes (reviewed by Murachi, 1989). They were first detected in the cytosol from rat brain (Guroff, 1964). Later studies identified two isozymes that are activated in vitro by micromolar (calpain I) or millimolar (calpain II) Ca²⁺ concentrations (Mellgren, 1980). Each isozyme has the same 25-30 kDa subunit plus a genetically distinct 72-82 kDa catalytic subunit (Murachi, 1983; Suzuki, 1987). In conjunction with an endogenous inhibitor, calpastatin (Drummond and Duncan, 1968; reviewed by Parkes, 1986), calpains constitute an extralysosomal proteolytic system found in both vertebrates and invertebrates (Murachi, 1989). Calpain proteolysis occurs at a limited number of sites, resulting in the cleavage of selected proteins into large and often functional polypeptides (Takahashi, 1990). The role that this irreversible post-translational modification has in cellular function is not well understood.

Calpains are abundant in neural tissues, where calpain II seems to be the predominant isozyme (Murachi et al., 1981; Nixon et al., 1986; Kawashima et al., 1988). Calpain II from brain has been characterized in homogeneously pure form (Malik et al., 1983; Kubota et al., 1986; Vitto and Nixon, 1986). Although its function(s) is largely unknown, brain calpain has been implicated in reorganization of the cytoskeleton, in neurotransmission, in long term potentiation, and in mediating degeneration (reviewed by Schlaepfer and Zimmerman, 1990; Seubert and Lynch, 1990).

Research on calpains in the retina has been limited to two studies, which identified calpains I and II in partially pure preparations from porcine (Yoshimura et al., 1984) and rat (Tsung and Lombardini, 1985) retinas. Tsung and Lombardini also detected Ca²⁺-activated neutral proteolytic activity in the soluble and particulate fractions of isolated rod photoreceptor outer segments, but they did not purify or characterize these activities any further, and there has been no other report on the distribution of calpain within the retina. The indication of calpain in the rod outer segment suggests a role for calpain in regulating the structure and function of this highly specialized compartment that is devoted entirely to phototransduction. In particular, calpain might function by modifying proteins involved in the phototransductive cascade, or by regulating the cytoskeleton that gives the outer segment its unique shape.

In the present study, we purified retinal calpain II to homogeneity and determined its catalytic properties. We found the enzyme to have similar characteristics to calpain II from brain. Biochemical and immunomicroscopical evidence was obtained showing that the enzyme is present in photoreceptor outer segments, where it appeared to be divided between the cytosolic and cytoskeletal fractions. As a first step towards addressing the possible function of calpain in regulating the outer segment cytoskeleton, we investigated which components of this cytoskeleton were susceptible to proteolysis by calpain in vitro. We focused on the actin filament-based cytoskeleton, which occupies a small domain within the connecting cilium and plays an essential role in the morphogenesis of the phototransductive disk membranes (see Williams, 1991, for review).

All procedures were performed at 0-4°C unless specified otherwise. Calpain II was purified from retina and brain by the same method, which was based on previously published procedures (Kubota et al., 1986; Vitto and Nixon, 1986). Three to five hundred frozen bovine retinas (180-300 g wet weight; purchased from JA and WL Lawson Company, Lincoln, Nebraska), were thawed in 50 mM Tris-HCl, pH 7.5, at 4°C, 10 mM ethylene glycol-bis-(β-amino-ethyl ether) N, N, N′, N′-tetraacetic acid (EGTA), 5 mM 2-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.02% NaN3 by two 30 s bursts in a 2 l blender. Four to eight fresh bovine brains (1-2 kg wet weight), obtained locally, were stripped of the meninges and blood vessels and homogenized by the same method. Cold (−20°C) acetone (analytical grade, Mallinckrodt) was added to the homogenates to 20% (v/v), the mixtures were stored at −20°C for 60 min, and then centrifuged at 10,000 g for 20 min in an IEC 872 rotor. The supernatants were adjusted to 50% (v/v) acetone, and stored overnight at −20°C. The mixtures were centrifuged, and the pellets were resuspended in a minimal volume of buffer A (20 mM Tris-HCl, pH 7.5, at 4°C, 2 mM EGTA, 5 mM 2-mercaptoethanol, 5% glycerol, and 0.02% NaN₃) with a Tekmar tissumizer. The resuspensions were centrifuged at 350,000 g for 50 min in a Beckman 60Ti rotor. The final supernatants were used as the crude extracts.

The retinal crude extract was loaded at a flow rate of 6 ml/min on a 500 ml DEAE-cellulose (DE 52, Whatman) column equilibrated in buffer A and washed with the same buffer. The brain crude extract was first adjusted to 150 mM NaCl and then loaded onto the DEAE-cellulose column which had been equilibrated with 150 mM NaCl in buffer A. The columns were washed further with 150 mM NaCl in buffer A until the absorbance at 280 nm of the washes fell below 0.01 absorbance unit. The calpain II-enriched fractions were eluted with 500 mM NaCl in buffer A and loaded at a flow rate of 4 ml/min on a 200 ml Phenyl-Sepharose (Pharmacia) column equilibrated in 500 mM NaCl in buffer A. The column was washed with 100 mM NaCl in buffer A and eluted with buffer A. The eluate was adjusted to 500 mM NaCl and loaded at a flow rate of 1.5 ml/min on a 130 ml Reactive Red-agarose (Sigma) column equilibrated with 500 mM NaCl in buffer A. The latter column was washed with 100 mM NaCl in buffer A and eluted with buffer A. This eluate was directly applied to a Mono Q column (HR 5/5, Pharmacia) equilibrated with buffer B (20 mM MOPS, pH 7.5, at 4°C, 100 μM EGTA, 5 mM 2-mercaptoethanol, 10% glycerol, and 0.02% NaN3). After washing the Mono Q column with 200 mM NaCl in buffer B, a 10 ml gradient of 200-500 mM NaCl in buffer B was applied and 0.5 ml fractions were collected. The fractions were mixed with one volume of glycerol and stored at −20°C.

[14C]Casein (24-48 μCi/mg, 0.5-1.0 mg/ml) was prepared as described by Dottavio-Martin and Ravel (1978). Samples were assayed in the presence of either 2 mM CaCl₂ (Mallinckrodt) or 4 mM EGTA (Sigma) with 96-240 nCi of [14C]casein in 100 mM Tris-HCl, pH 6.9, at 25°C, 15 mM 2-mercaptoethanol, 0.02% NaN₃, in a final volume of 200 μl. After 60 min of gentle agitation at 25°C, 50 μl of 1% bovine serum albumin (BSA) and 750 μl of cold 10% trichloroacetic acid (4°C) were added sequentially to stop the reaction. After the mixture was centrifuged in a microfuge for 3 min, 0.5 ml of the supernatant was mixed with 5 ml of Biosafe II scintillation cocktail (Research Products International) and its radioactivity measured in an LKB Rackbeta 1211 liquid scintillation counter. Caseinolysis under these conditions was found to be linear with time up to 90 min (not shown). The difference between radiolabel released into the supernatant in the presence of CaCl₂ and EGTA was defined as Ca2+-activated neutral proteolytic activity. One unit of calpain II activity is defined as the amount of calpain required to hydrolyze 1 μg casein in 200 μl per h at 25°C.

Pure or partially pure calpain II samples were diluted with buffer to reduce the final EGTA concentration to less than 3 μM and then characterized with the caseinolysis assay under the following conditions. The Ca2+-sensitivity profile of calpain II was obtained by incubating samples with different final concentrations of CaCl2 ranging from 30 to 2000 μM. The divalent cation specificity of calpain activation was determined by assaying samples with 1 mM and 5 mM MnCl₂, MgCl₂, or ZnCl₂ and comparing to the caseinolysis induced by 1 mM CaCl2. For the pH profile experiments, Tris-HCl or MOPS-NaOH buffers of various pH values were prepared and adjusted to 150 mM NaCl to standardize the ionic strength. The pH indicated represents that measured after salt adjustment. Calpain II samples were diluted at least 20-fold with 100 mM buffer of various pH values containing 15 mM 2-mercaptoethanol and 0.02% NaN3 and assayed for caseinolysis in the presence of 1 mM CaCl₂. No significant difference in enzyme activity was observed between Tris and MOPS buffers of the same pH (not shown). The susceptibility of calpain II to inhibition by various cysteine protease inhibitors was determined by assaying the enzyme in the presence of 1 mM CaCl₂ with 1 μM leupeptin (Sigma) or L-trans-epoxysuccinyl-leucylamido(3-methyl)butane (Ep-475, from Taisho Pharmaceutical Company, Tokyo), or various concentrations of Z-Leu-nLeu-H (calpeptin, a gift from Dr David Hathaway, Krannert Institute of Cardiology, Indianapolis, Indiana). The kinetic parameters of calpain II were generated by fitting the experimental data to the Hill equation or to a bell-shaped double pKa curve by non-linear least squares regression (Leatherbarrow, 1989).

Proteins were separated in SDS-polyacrylamide slab gels (Laemmli, 1970), and then stained with Coomassie blue or with silver (Wray et al., 1981), or electrophoretically transferred (Towbin et al., 1979) to Immobilon-P membrane (Millipore). M_r standards for Coomassie-stained gels were obtained from Sigma and for silver-stained gels from Bio-Rad. Western blots were labeled with the antibodies listed below. Protein was determined by the Bradford assay (Bio-Rad) with BSA (Sigma) as the standard.

All calpain II antibodies were against the large (catalytic) subunit of the enzyme. Polyclonal antibodies against brain calpain II were generated in New Zealand White rabbits and affinity-purified (Harlow and Lane, 1988). Polyclonal antibodies against calpain II from bovine aorta and chicken gizzard were gifts from Dr David Hathaway (Krannert Institute of Cardiology, Indianapolis, Indiana), and polyclonal antibodies against porcine skeletal muscle calpain II were gifts from Dr Margaret Wheelock (Wistar Institute, Philadelphia, Pennsylvania). A monoclonal antibody against chicken brain myosin II heavy chain, mAb 23, was kindly provided by Drs Gary Conrad and Abigail Conrad. This same antibody was used to determine the distribution of myosin in rat rod outer segments (Williams et al., 1992). It has been shown to crossreact with non-muscle myosins and chick cardiac myosins, but not with skeletal or smooth muscle myosins (Conrad et al., 1991). In the present study, however, we used it at 10 times the usual concentration for immunoblots (1:100 dilution of ascites serum) in order to improve the detection of the proteolyzed myosin. At this concentration, crossreaction with 240 kDa and 48 kDa polypeptides was also detected. A monoclonal antibody against actin, mAb JLA20 (made by Lin, 1981) was obtained from the Developmental Studies Hybridoma Bank (Baltimore, Maryland). A polyclonal antibody against α-actinin, pAb B8, was a gift from Dr S. J. Singer’s laboratory (University of California, San Diego). This antibody has been used to determine the distribution of α-actinin in the outer segment (Arikawa and Williams, 1989) and elsewhere in the outer retina (Arikawa and Williams, 1991).

Posterior segments of rat, bovine or porcine eyes were fixed and processed for semithin (0.5-1.0 μm) cryosectioning as described by Williams et al. (1990). In addition, detached neural retinas (i.e. removed from the retinal pigmented epithelium) were cut into pieces and shaken briefly in physiological saline (20 mM HEPES-NaOH, pH 7.2, at 4°C, 150 mM NaCl), before being processed in the same way. The brief shake of the neural retinas was sufficient to splay the photoreceptors at the edges of the retinal pieces, but not sufficient to dislodge any outer segments. Details of immunolabeling in the outer segment could be more clearly observed in sections of splayed photoreceptors. The rat retinas were obtained from light-adapted animals at noon, or dark-adapted animals at midnight. Eyes from the latter were dissected under dim red light. Bovine and porcine eyes were obtained from local slaughterhouses and processed in the light. Sections were labeled with affinity-purified primary antibodies at 10 μg/ml. The primary antibodies were detected with goat anti-rabbit IgG that was conjugated to Texas Red (Jackson Immunoresearch Laboratories).

All procedures were performed in the dark or under dim red light at 0-4°C. Rod outer segments were isolated from bovine retinas according to Zimmerman and Godchaux (1982) and Schnetkamp and Daemen (1982) with modifications. Sixty bovine eyes were obtained from a local slaughterhouse and kept in a light-tight icebox during transport to the laboratory (2.5 h). The eyes were hemisected with razor blades and the retinas were extracted from the posterior eyecups with forceps. Care was taken to avoid contamination from the retinal pigment epithelium. The retinas were distributed among six 50 ml conical tubes (Corning) each containing 5 ml of 20% sucrose in 20 mM Tris-HCl, pH 7.3, at 4°C, 2 mM EGTA, 2 mM MgCl₂, 1 mM ascorbic acid, 10 mM D-glucose, 20 μM leupeptin, and 100 μM PMSF. The conical tubes were vortexed for 30 s to break off the rod outer segments. The mixture was squeezed through a nylon mesh (no. 100, Sargent-Welch), and the filtrate (crude rod outer segments) was divided among six 26 ml 27-50% sucrose gradients in 20 mM Tris-HCl, pH 7.3, at 4°C, 2 mM EGTA, 2 mM MgCl₂, 10 mM D-glucose, and 1 mM ascorbic acid. The gradients were centrifuged at 157,000 g for 1.5 h in a Beckman SW-28 rotor. The band containing rod outer segments was first examined for purity with a phase microscope, and was then removed with a Pasteur pipet, diluted with 2 volumes of 20 mM Tris-HCl, pH 7.3, at 4°C, 2 mM EGTA, 2 mM MgCl₂, and 150 mM NaCl, and centrifuged at 44,000 g for 20 min in a Sorvall SS-34 rotor. The pellet was resuspended in 20 mM Tris-HCl, pH 7.3, at 4°C, 100 mM KCl, 2 mM MgCl₂, and 2 mM EGTA, frozen in liquid nitrogen, and thawed to lyse the rod outer segments. The lysate was centrifuged at 541,000 g for 22 min in a Beckman TLA 100.3 rotor. The supernatant of the lysate was centrifuged twice more to remove any contamination from the particulate fraction of the lysate, and the final supernatant was used as the cytosolic fraction. The particulate fraction of the lysate was washed 3 times in buffer containing 2% Triton X-100 and the final pellet was used as the Triton-insoluble fraction. The Triton washes were pooled and used as the Triton-soluble fraction. All fractions were precipitated with acetone and solubilized in sample buffer for SDS-PAGE, or stored at −20°C in 50% glycerol.

All buffers contained 5 mM 2-mercaptoethanol. The cytosolic fraction of isolated rod outer segments was prepared as described above. For the retinal cytosolic fraction, 5 frozen bovine retinas were homogenized in 20 mM Tris-HCl, pH 7.3, at 4°C, 100 mM KCl, 2 mM MgCl2, and 2 mM EGTA, with two 20 s bursts in a Tekmar tissumizer. The homogenate was centrifuged at 541,000 g for 22 min at 4°C in a Beckman TLA 100.3 rotor and the supernatant was centrifuged twice more. These cytosolic fractions were diluted with one volume of buffer A and applied to a Mono Q column equilibrated in buffer A. The column was washed with buffer A and then eluted with a gradient of 0-500 mM NaCl in buffer A. Caseinolytic fractions were pooled and characterized as described.

Rod outer segments were purified from 60 dark-adapted bovine retinas as described above, but lysed by resuspending the rod outer segment pellet in 3% Triton X-100 in 20 mM Tris-HCl, pH 7.3, at 4°C, 100 mM KCl, 2 mM EGTA, 1 mM MgCl 2, 10 μg/ml phalloidin, and 5 μM taxol, with a Dounce homogenizer under dim red light. The mixture was centrifuged at 541,000 g for 22 min at 4°C in a Beckman TLA100.3 rotor and washed twice more in the same buffer without taxol. The final pellet was resuspended in the same buffer without detergent or taxol, adjusted to 100 mM Tris and 15 mM 2-mercaptoethanol, and divided into 5 aliquots. The aliquots were incubated with CaCl₂ and pure brain calpain II as described in the legend of Fig. 8. To stop the reaction, leupeptin was added to each aliquot (final concentration of 200 μM), and the mixture was precipitated with acetone. Laemmli sample buffer was added to the pellets and the proteins were separated in 4-10% SDS-polyacrylamide gels. The gels were stained with Coomassie blue or transblotted. The western blots were sequentially immunolabeled with antibodies myosin II heavy chain, actin, and α-actinin. The Coomassie-stained gels were scanned with a Hoefer GS 300 densitometer. The M_r values were determined using Hoefer GS 370 software.

Retinal and brain calpain II were purified by the same procedure. Data obtained from a typical preparation of the retinal enzyme are summarized in Table 1. The calpain activity in the homogenates was precipitated between 20%-50% acetone (v/v). Calpain II was separated on a DEAE-cellulose column from calpain I and calpastatin, which eluted with 200 mM NaCl; calpain II was eluted with 500 mM NaCl (Mellgren, 1980). Calpain I and calpastatin coeluted with protein kinase C from the DEAE-cellulose column, even when a salt gradient was employed (cf. Newton, 1993). Retinal and brain calpain II were then purified to apparent homogeneity by chromatography on Phenyl-Sepharose, Reactive Red-agarose, and Mono Q columns. Both of the purified proteases appeared to be heterodimers consisting of a 75 kDa and a 29 kDa polypeptide (Fig. 1a), which coeluted from a Superose 6 gel filtration column (not shown).

From 180 g wet weight of retina, 0.14 mg of pure calpain II was obtained with a specific activity of 10 units/μg (Table 1), amounting to 4.7 units of pure calpain II per retina, or 7.8 units per gram wet weight.

A polyclonal antibody generated against the catalytic subunit of brain calpain II crossreacted with the retinal subunit (Fig. 1b,c), as did polyclonal antibodies against the catalytic subunit of bovine aorta and chicken gizzard calpain II (data not shown). The Ca²⁺ sensitivity profile of both the retinal and brain enzymes indicated no detectable activity in the presence of 30 μM Ca²⁺ (Fig. 2a); at this concentration of Ca²⁺, any calpain I should have been fully activated (Mellgren, 1980). Maximal activity occurred near 1 mM Ca²⁺. The Ca²⁺ concentrations required for half-maximal caseinolytic activity (EC50) for retinal and brain calpain II were 262 ± 10 μM and 311 ± 24 μM Ca²⁺, respectively, with Hill coefficients of 2.8 ± 0.2 and 2.2 ± 0.3, respectively. Fig. 2c shows the inhibition profile of retinal and brain calpain II by calpeptin, one of the more potent and more specific inhibitors of calpains (Tsujinaka et al., 1988). The concentrations of calpeptin required for 50% inhibition of the maximal caseinolytic activity (IC50) were 10 ± 1 nM and 21 ± 3 nM for retinal and brain calpain II, respectively, with a Hill coefficient of 0.6 ± 0.1 for both. The optimal pH was 6.9 for both proteases (Fig. 2b). At a concentration of 1 mM, Ca²⁺ was the only divalent cation that activated retinal or brain calpain II; MnCl2, MgCl2, and ZnCl2 were ineffective, although in the presence of 5 mM MnCl2, 25-30% of the activity induced by 1 mM CaCl2 was detected. Both proteases were inhibited to the same extent by cysteine protease inhibitors. At 1 μM, Ep-475 inhibited retinal and brain calpain II by 49% ± 5% and 46% ± 5%, respectively; leupeptin inhibited them both by 67% ± 4%; and calpeptin, the most potent, inhibited 92% ± 12% and 92% ± 7% of the respective activities.

Several different antibodies against calpain II (see Materials and Methods) were used on semithin cryosections of rat, bovine and porcine retinas. All antibodies immunolabeled specifically the 75 kDa subunit on western blots of retinal proteins (e.g. Fig. 1c). Retinal sections that were probed with these antibodies showed a similar distribution of label. Dark-adapted retinas showed most intense labeling in the photoreceptor outer segments and in the inner plexiform layer (Fig. 3). The latter contains processes of the Müller cells and synaptic contacts between the bipolar cells, the ganglion cells, and the amacrine cells. Punctate labeling was also evident in the outer plexiform layer, possibly in the photoreceptor cells synaptic terminals. Immunolabel was also present in the retinal pigmented epithelium (Fig. 3).

In light-adapted retinas, the immunolabel was less intense in the photoreceptor outer segments, but more intense in the photoreceptor inner segments (Fig. 4). In the outer segment, label was restricted to the basal region and concentrated along one side; it appeared to coincide with the connecting cilium. This labeling of the connecting cilium was seen more clearly in retinas whose photoreceptors had been splayed (Fig. 5).

Most of the Ca²⁺-activated neutral caseinolytic activity in the cytosolic fractions of both isolated rod outer segments and neural retina was eluted by 350-363 mM NaCl from a Mono Q column that was developed with a linear salt gradient (Fig. 6; Table 2). This caseinolytic activity was identified as calpain II activity, based on its Ca²⁺ requirement (Fig. 7) and inhibition by calpeptin (see below) at neutral pH. A small amount of caseinolytic activity, which eluted between 130-200 mM NaCl, was detected in the retinal sample; it was probably due to some calpain I that eluted before calpastatin.

The partially pure calpain II from rod outer segments and from neural retina were found to be indistinguishable with respect to chromatographic behavior, Ca²⁺-sensitivity, and sensitivity to calpeptin (Table 2). The Ca²⁺-sensitivity of the partially pure calpain II (EC50 of 468-471 μM; Table 2) was notably lower than the pure enzymes (EC50 of 261-311 μM; Fig. 2a), which may be due to the presence of an ‘inactivating factor’ having the opposite effect of the activator protein reported in studies of brain calpain (DeMartino and Blumenthal, 1982; Takeyama et al., 1986).

Bovine rod outer segments were isolated from dark-adapted retinas and fractionated into cytosolic and particulate fractions. The particulate fraction was fractionated further with buffer containing 2% Triton X-100, as described in Materials and Methods. Fig. 8 shows a stained gel and a western blot of these fractions, each derived from the same amount of rod outer segment homogenate. The blot was probed with an affinity-purified antibody against the catalytic subunit of brain calpain II. Similar immunolabeling was detected in the cytosolic (S) and the Triton-insoluble (TI) fractions. Only minor labeling was detected in the Triton-soluble fraction (TS).

The cytoskeletal fraction of isolated bovine rod outer segments was prepared in the presence of phalloidin (to stabilize the actin filament cytoskeleton), and then incubated with or without purified calpain II in the presence of Ca²⁺ and analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 9a,b). Ca²⁺ alone was not sufficient to effect proteolysis; the lack of endogenous calpain activity was most likely because of the negating presence of calpastatin. Two major polypeptides of Mr 240,000 and 56,000 were proteolyzed completely within 2 minutes (Fig. 9a,b). After 50 minutes, three other prominent polypeptides (Mr 113,000, 62,000 and 53,000) had been significantly proteolyzed, whereas a major polypeptide of Mr 42,000 was not proteolyzed. Other polypeptides that were detectable on Coomassie-stained gels and proteolyzed completely within 2 minutes included three of Mr 340,000-370,000, one of Mr 212,000, and several of Mr 66,000-100,000. After 50 minutes of proteolysis there was a noticeable increase in polypeptides of Mr less than 40,000, resulting from the cleavage of higher Mr polypeptides (note middle panel in Fig. 9b). The same pattern of proteolysis was observed with brain or retinal calpain II.

Immunoblots were used to determine whether or not the known components of the actin-based cytoskeleton in the connecting cilium were substrates for calpain in vitro. Fig. 9c shows a western blot that was labeled sequentially with antibodies against myosin II heavy chain, α-actinin, and actin. Significant proteolysis of myosin II heavy chain had occurred by 2 minutes; no proteolytic products were detected by the monoclonal antibody. Significant degradation of α-actinin became evident at 50 minutes; a highly immunoreactive product, about 1-2 kDa smaller, and two other minor polypeptides of Mr 50,000 and 60,000 were immunostained by the polyclonal antibody. Actin was not proteolyzed even after 50 minutes.

We have described the purification to homogeneity and characterization of retinal calpain II. The purification procedure included acetone fractionation and four chromatographic steps on DEAE-cellulose, Phenyl-Sepharose, Reactive Red-agarose, and Mono Q columns. Acetone fractionation has not been used before in the purification of calpain. We found that calpain activity was not affected by acetone, and that acetone fractionation proved to be a helpful step. It significantly reduced the sample volume for the high speed centrifugation and the first chromatographic separation. In addition, calpain could be stored conveniently in 50% acetone at −20°C for at least a week; freezing results in the loss of some calpain activity.

The profiles and kinetic parameters for the sensitivity of brain and retinal calpain II to Ca²⁺, pH, and calpeptin were computed by a non-linear regression least squares curve-fitting algorithm (Leatherbarrow, 1989). The Hill coefficients determined from the Ca²⁺-sensitivity indicate positive cooperativity. Overall, the data showed that the biochemical properties of pure retinal and brain calpain II are similar. Indeed, the properties of these enzymes are similar to those reported for calpain II from non-neuronal tissues. The obtained values for the EC50 of Ca²⁺-activation and the pH optimum are comparable to those reported previously for pure brain calpain II (Malik et al., 1983; Kubota et al., 1986; Vitto and Nixon, 1986) and pure calpain II from other sources including skeletal muscle (Penny et al., 1985), cardiac muscle (Hara et al., 1983), kidney (Yoshimura et al., 1983; Kitahara et al., 1984), and liver (DeMartino and Croall, 1983). The IC50 for calpeptin inhibition is consistent with reported values for calpain II from kidney (Tsujinaka et al., 1988). The cation specificity of calpain II reported here is in agreement with previous studies of brain calpain II, which reported only modest activation of the enzyme by Mn²⁺ and no activation by Mg²⁺ or Zn ²⁺ (Malik et al., 1983; Vitto and Nixon, 1986).

Immunocytochemistry showed that calpain II is distributed in different regions of the retina, particularly in the plexiform layers (the synaptic regions) and the distal parts of the photoreceptor cells. A proposed role for calpain in postsynaptic terminals of hippocampal neurons is in regulating the number of glutamate receptors by cleavage of fodrin (Siman et al., 1985). Glutamate receptors are present in a number of different types of retinal cells (Massey, 1990), so that colocalization with calpain seems likely, consistent with the possibility of a similar role for calpain in retinal synaptic transmission.

A notable aspect of the immunolocalization of calpain II in the distal parts of the photoreceptor cells is that it varied depending on whether or not the retina had been exposed to light. It is unclear whether this variation results from movement of calpain between the inner and outer segments in response to light or darkness. Such translocation has been suggested for three soluble proteins that are involved in phototransduction (arrestin, transducin, and phosducin), based on immunocytochemical results (Brann and Cohen, 1987; Philip et al., 1987; Mangini and Pepperberg, 1988; Whelan and McGinnis, 1988). However, calpain was observed to be always present in the outer segment; it was either distributed throughout the outer segment (in the dark), or more prominent in the connecting cilium (in the light). This type of variation within the outer segment has not been reported for any other protein. Calpain II might not return to the inner segment from the outer segment; its detection in the inner segment after light exposure could result from a light-induced increase in its synthesis.

Immunolabeling of the connecting cilium indicates the presence of calpain II in a domain that contains both a microtubule-based and an actin-based cytoskeleton. Association of calpain II with an actin-based cytoskeleton has been described in other types of cells. Beckerle et al. (1987) showed that calpain II was concentrated in the adhesion plaques of cell-substrate junctions in a variety of cultured cells.

Partially pure preparations of calpain II from outer segments and from whole retinas were compared to see whether outer segment calpain II might be different from that in the rest of the retina. The biochemical characteristics of calpain II from the two sources were found to be the same. This result, however, does not preclude the possibility that the outer segment calpain II is a different isozyme; a novel isozyme from brain has been reported to have nearly the same Ca²⁺ sensitivity as calpain I but a different substrate specificity (Yoshihara et al., 1990).

Calpain II was found in both the cytosolic and cytoskeletal fractions of rod outer segments. Calpains have generally been described as soluble proteins, although calpain II has been detected in the detergent-insoluble fractions of other tissues (Ishizaki et al., 1983; Banik et al., 1991). The presence of calpain II in the cytosol and cytoskeleton of rod outer segments implies that the enzyme could function in regulating proteins involved in phototransduction or in the specialized cytoskeleton that gives the outer segment its unique structure. Some of the proteins involved in phototransduction, such as arrestin, have regions that are enriched in proline, glutamate, serine and threonine residues (Mangini, 1991), so-called ‘PEST’ sequences, which have been described as likely sites for calpain binding, and suggested to be indicative of calpain substrates (Wang et al., 1989). Indeed, a Ca²⁺-dependent protease has been shown to cleave the carboxy terminus of squid rhodopsin (Oldenburg and Hubbell, 1990). In the present study, we focused on the potential function of calpain in the cytoskeleton, prompted in part by: (1) a previous study in which a higher yield of sealed rat rod outer segments was obtained when a cysteine protease inhibitor, Ep-475, was included in the purification procedure, suggesting that the rod outer segment infrastructure is vulnerable to breakdown by a neutral cysteine protease (Williams et al., 1989); and (2) the present observation that after light adaptation, calpain II in the outer segment appeared to be concentrated in the connecting cilium.

SDS-polyacrylamide gel electrophoresis analysis of rod outer segment cytoskeletal proteins after incubation with pure calpain II showed that some proteins were proteolyzed quickly, some slowly, and some not at all -exemplifying the selectivity of calpain. The rapidly degraded polypeptide with Mr 240,000 was probably α-spectrin. An α-spectrinlike protein has been reported to be sensitive to endogenous proteolysis during purification from rod outer segments; in the absence of chelators, fragments of Mr 150,000, 120,000 and 95,000 are produced (Wong and Molday, 1986; S.M.A. and D.S.W., unpublished results). Fragments of similar sizes are produced by in vitro digestion of spectrin with calpain (Siman et al., 1984; Harris and Morrow, 1990). Another rapidly-degraded substrate was a 56 kDa polypeptide, which was probably vimentin. Vimentin from inner retinal cells has been found to be a contaminant in cytoskeletal fractions of rod outer segment preparations (own unpublished results). The most densely-stained band on SDS-polyacrylamide gel was a polypeptide of Mr 53,000. This band represents αand β-tubulin, some of which was slowly proteolyzed.

Using immunoblot analysis, we focused on known components of the actin-based cytoskeleton in the outer segment connecting cilium. This cytoskeleton has been shown to play an important role in the morphogenesis of disk membranes (Williams et al., 1988). It consists of a small cluster of actin filaments that radiate from the center of the axoneme, adjacent to the site of new disk formation, out to the plasma membrane, where the plus ends of the filaments are located (Arikawa and Williams, 1989; Chaitin and Burnside, 1989). Myosin II (Chaitin and Coelho, 1992; Williams et al., 1992) and α-actinin (Arikawa and Williams, 1989) are associated with these actin filaments. It has been suggested that bipolar myosin filaments interact with the actin filaments to effect a tuck in the plasma membrane, thus initiating the formation of a flattened membrane disk (Williams et al., 1992). (Subsequent outgrowth of the disk is independent of actin filaments; Williams et al., 1988). Of the three polypeptides, actin, α-actinin, and myosin II heavy chain, that were examined by immunoblots, myosin II heavy chain was clearly the best in vitro substrate for calpain II. Myosin II heavy chain from skeletal muscle has been previously reported to be an in vitro substrate for calpain (Pemrick and Grebenau, 1984). The rapid proteolysis of myosin II heavy chain, combined with the localization of calpain II in the connecting cilium, suggests that proteolysis of myosin could be physiologically relevant, perhaps playing an important role in the regulation of disk membrane morphogenesis. The apparent concentration of calpain II in the connecting cilium in response to light exposure suggests that this proteolysis of myosin could in turn be controlled by light. Consistent with this notion is the observation that the rate of disk membrane morphogenesis is affected by light (Besharse et al., 1977).

This work was supported by Grant EY07042 from the NIH to DSW. CLS received a Fight-for-Sight/NSPB postdoctoral fellowship (PD90025) during part of the study. We thank Drs Alexandra Newton and Terry Jenkins (Indiana University) for helpful discussions on kinetic analysis and for critical suggestions for the manuscript. Dr Wayne Weaver’s (Indiana University) help in the initial stages of calpain II purification was greatly appreciated. We are grateful to Dr David Hathaway (Krannert Institute of Cardiology) for calpeptin and for the calpain II antibodies that were used in comparisons with our antibodies. Dr David Blest (Australian National University) generously supplied us with Ep-475 as part of a gift he received from the Taisho Pharmaceutical Company (Japan). We are also grateful to Drs Gary Conrad and Abigail Conrad (Kansas State University) for the myosin antibody, to the Developmental Studies Hybridoma Bank (supported by NICHD contract N01-HD-6-2915) for the actin antibody, to Dr S. J. Singer (UC San Diego) for the α-actinin antibody, and to Dr Margaret Wheelock (Wistar Institute) for calpain II antibodies.