La Plataforma de Convergencia Democrática

Calpain 3 was first found in 1989 (Sorimachi et al., 1989) and called p94 in reference to its molecular mass. The intact form of calpain 3 was observed as a band at 94kDa and

fragmented calpain 3 was also observed. The most distinctive characteristic of calpain 3 is its very rapid autolysis, with a half-life of less than 10 min (Kinbara et al., 1997). It has been reported that autolysis of calpain 3 produced 60 and 58kDa fragments as intermediates before a stable 55kDa fragment appeared (Kinbara, Ishiura, et al., 1998).

In this experiment, muscle samples were pre-rigor (day-0) samples collected from ovine carcass immediately after slaughtering. The samples were immersed in liquid nitrogen, ground to powder, and kept at -80˚C to avoid autolysis and further proteolysis. The frozen muscle samples were treated at cold temperature until extraction of total protein with

appropriate buffer solutions to avoid a calpain 3 autolysis. Muscle homogenates were kept at 4°C for short term storage or -20°C overnight before denaturing with PDB. This process was the same for all buffer solutions. Therefore difference in the results correspond to the

effectiveness of a selected buffer solutions and extraction procedures.

Sorimachi-buffer protein samples showed two dominant bands at 94kDa and 55kDa (Figure 3.6). The band at 94kDa was the intact form of calpain 3 and 55kDa was the fragmented form. The 55kDa fragment has been reported as a product of calpain 3 autolysis (Kinbara, Ishiura, et al., 1998). The intensity of autolysed form of calpain 3 band was as strong as the band of the intact form, indicating quite a high degree of calpain 3 autolysis during this extraction procedure with Sorimachi-buffer. This buffer has been used to purify recombinant inactive calpain 3 expressed in COS cells, in which the active site cysteine residue was changed to serine (Kinbara, Ishiura, et al., 1998). Extractions with Sorimachi-buffer were not suitable for

characterising intact calpain 3 from crude protein, because of calpain 3 autolysis continued during the extraction.

Lamb-buffer contains high concentrations of the chelating agents, EDTA and EGTA (18mM EDTA and 50mM EGTA), the Sorimachi-buffer contains 1mM EDTA and 1mM EGTA, and the Fritz-buffer contains 2mM EGTA. The Lamb-buffer was developed to reduce the Ca2+

concentration in muscle homogenate to allow for analyse of autolysis of calpains 1 and 3 in human muscle (Murphy, Snow, et al., 2006). It was expected that calpain 3 autolysis would be avoided by reducing the Ca2+ _{concentration in muscle homogenates, which would lead to a}

dominant intact calpain 3 band at 94kDa. However, the intact calpain 3 band was observed as a very weak band (Figure 3.7). Dominant bands were observed at 60 and 55kDa. The study with human muscle (Murphy, Snow, et al., 2006) observed intact calpain 3 at 94kDa when muscle was homogenised with a buffer almost identical to the Lamb-buffer. However, calpain 3 autolysis was confirmed after 1 min exposure to as little as 2.5µM Ca2+_{. Extraction of}

muscle protein with Lamb-buffer did not avoid calpain 3 autolysis, and almost all the calpain 3 was fragmented.

Fritz-buffer was developed from a buffer solution to extract muscle protein for analysing titin (Fritz et al., 1993) with slight modification such as the addition of 2-mercaptoethanol (β-ME). The homogenate was centrifuged at 3000g for 5min, and the supernatant analysed. No bands were observed after staining with a calpain 3 antibody (anti-calpain 3 pIS2). The original procedure (Fritz et al., 1993) did not include centrifugation of the 1% (w/v) meat content homogenate, and Western blotting of the 1% (w/v) homogenates without centrifugation showed clear bands at 94kDa, which was the intact form of calpain 3 (Figure 3.8). Bands of the fragmented calpain 3 were barely visible. Muscle protein extraction with Fritz-buffer for 1% (w/v) meat content was settled on as optimum procedure for analysing calpain 3 from crude protein.

Moreover, the sensitivity of intact calpain 3 was tested on protein samples after different storage procedures (Figure 3.8). The Western blotting results showed a clear strong band of intact calpain 3 at 94kDa for all samples, and no band pattern differences among samples. Autolysis of calpain 3 was not observed for the homogenate, frozen before addition of PDB. These results confirmed that intact calpain 3 is stable in Fritz-buffer at -20°C.

Bands of intact and fragmented forms of calpain 3 were confirmed by the band pattern of frozen and thawed muscle samples. The ground frozen muscle samples at -80°C were thawed and aged for a week at 4˚C before protein extraction. Bands of fragmented calpain 3 were

expected from thawed samples arising from calpain 3 autolysis (Kinbara, Ishiura, et al., 1998), and densities of intact calpain 3 bands were expected to be decreased. A strong band was observed at 94kDa for frozen muscle samples, which was faded for thawed muscle samples (Figure 3.9). These results confirmed the band at 94kDa as the intact form of calpain 3 which is degraded by autolysis during thawing. When the band at 94kDa was faded, a strong band appeared at 60kDa in the thawed muscle samples, confirming that this band is fragmented calpain 3 formed from calpain 3 autolysis during thawing. A strong band was also observed at 140kDa in both frozen and thawed muscle samples. Because this band density was unchanged between frozen and thawed muscle samples, it is unlikely to be involved with calpain 3 autolysis during post-mortem storage. Another band at 50kDa also showed similar intensities in frozen and thawed muscle samples, and therefore it was also considered non- specific. This was confirmed by the Western blotting with the secondary antibody, anti-goat IgG only (Figure 3.10). Bands were observed at 50kDa and 30kDa, when the membrane was incubated with only the secondary antibody.

In characterising the band pattern of calpain 3, muscle protein samples were prepared with the addition of various concentrations of Ca2+_{. Calpain 3 autolysis has been reported after a 1 min}

exposure to as little as 2.5µM Ca2+ _{in human muscle prepared with a buffer almost identical}

to the Lamb-buffer (Murphy, Snow, et al., 2006). However, no effect of the addition of Ca2+

on the fragmentation of intact calpain 3 was observed in this study (Figure 3.11). The intact calpain 3 band was still observed and no band of fragmented calpain 3 in protein samples were incubated with 25µM Ca2+ _{for 1 hour.}

Although the addition of Ca2+ _{had no effect on the fragmentation of calpain 3, the lack of a}

chelating agent had some effect on calpain 3 autolysis (Figure 3.12). Protein samples were prepared with Fritz-buffer which contained 2mM EGTA and intact form of calpain 3 was confirmed as a dominant band at 94kDa. After 3 hours of incubation at room temperature, calpain 3 autolysis was observed as very weak band at 60kDa. Intact calpain 3 was also observed for the samples prepared without EGTA (no-EGTA buffer), but its intensity decreased during 24 hours of incubation. Calpain 3 autolysis was clearly confirmed by the appearance of the band at 60kDa after 1 hour incubation, and its intensity increased during 24 hours. Thus, the lack of a chelating agent allowed calpain 3 autolysis. Moreover, the addition of up to 1mM Ca2+ _{to protein samples without EGTA had no effect on calpain 3 autolysis.}

These results suggest that calpain 3 autolysis may not be regulated by Ca2+ _{but may be}

the addition of Ca2+ _{had no effect, it could be that the activation of calpain 1 is not involved in}

the proteolysis of calpain 3.

It was noted that the intensity of the uncharacterised band at 140kDa tended to decrease in the presence of Ca2+_{. The band at 140kDa had similar intensity to the intact calpain 3 band for}

samples prepared with Fritz-buffer. However, the intensity of the band at 140kDa was much weaker in samples prepared in no-EGTA buffer. Perhaps EGTA in the buffer reduced the amount of Ca2+_{, and influenced the intensity of the band at 140kDa. This is supported by the}

intensity of band at 140kDa being much weaker in samples incubated with 25µM Ca2+ _{than in}

2.5µM Ca2+_{, while the incubation time had no effect. Further research will be required to}

identify the band at 140kDa.