J. Anim Sci. 2007. 85:3400-3414. doi:10.2527/jas.2007-0356
© 2007 American Society of Animal Science
Isolation and characterization of µ-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps1
J. P. Camou2,
S. W. Mares,
J. A. Marchello,
R. Vazquez,
M. Taylor,
V. F. Thompson and
D. E. Goll3
Muscle Biology Group, University of Arizona, Tucson 85721
 |
Abstract
|
|---|
Evidence has indicated that µ-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and µ-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of µ-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25°C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca2+ requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11–13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive µ-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active µ-calpain even though very little µ-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this µ-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive µ-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active µ-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of µ-calpain activity remains unknown.
Key Words: µ-calpain • m-calpain • calpastatin • protein purification • postmortem muscle
|
INTRODUCTION
|
|---|
The calpains are an important contributor to postmortem proteolytic tenderization (Koohmaraie, 1988
, 1992, 1996; Goll et al., 1992), and postmortem changes in the calpain system as they relate to tenderization have been studied extensively. Three members of the calpain system, µ-calpain, m-calpain, and calpastatin, have been implicated in postmortem tenderization. During the first 3 d of postmortem storage, mice lacking the µ-calpain gene experienced little degradation of nebulin, dystrophin, metavinculin, desmin, and troponin T, polypeptides degraded by the calpains and whose degradation is related to increased postmortem tenderization (Geesink et al., 2006). Furthermore, mice over-expressing calpastatin experienced little degradation of troponin T and desmin (Kent et al., 2004).
These findings and a wealth of correlative evidence have led to the suggestion that increased postmortem tenderization is related to low calpastatin activity and that µ-calpain but not m-calpain is responsible for proteolysis in postmortem muscle (Koohmaraie, 1996). The calpastatin polypeptide is nearly completely degraded during the first 7 d postmortem, and calpastatin activity declines to 
20 to 30% of its original level during this period (Boehm et al., 1998). Activity of m-calpain remains nearly constant or declines slightly during the first 7 d postmortem, but µ-calpain activity decreases rapidly during postmortem storage and is nearly zero after 3 d postmortem, even though the µ-calpain polypeptide is autolyzed but not degraded further and would be expected to be proteolytically active (Boehm et al., 1998). Thus, it is difficult to understand how µ-calpain could have a primary role in postmortem tenderization.
We have, therefore, initiated efforts to purify the 2 calpains and calpastatin from postmortem muscle to determine whether postmortem µ-calpain is indeed proteolytically inactive, and to learn the efficacy of postmortem calpastatin in inhibiting the postmortem calpains.
|
MATERIALS AND METHODS
|
|---|
Materials
Acrylamide (99.9%) was from ICN Biomedicals Inc., Aurora, OH; bisacyrlamide (99.99%) was from Swartz/ Mann Biotech, Cleveland, OH; SDS (99%) was from BioRad, Hercules, CA; Tris (ultrapure, 99.8%) and 2-(N-morpholino)ethane sulfonic acid, free acid, were from Mallinckrodt Baker, Phillipsburg, NJ; EDTA (free acid, 99%) was from BMD Biosciences, Gibbstown, NJ; 2-mercaptoethanol (MCE), fluorescein isothiocyanate, and the protease inhibitors (except for E-64) used in calpain homogenizing buffers (Edmunds et al., 1991; Thompson and Goll, 2000) were from Sigma Chemical, St. Louis, MO; E-64 was from Peptides International, Louisville, KY; hexyl-TSK 650C was from TOSOH Bioscience LLC, Montgomeryville, PA; DEAE TSK 650S was from Supelco, Bellefonte, PA; and BODIPY-FL was from Invitrogen Corp. (D-2184), Carlsbad, CA.
A monoclonal antibody specific for the 80-kDa subunit of µ-calpain was from Affinity Bioreagents (9A4H8D3), Golden, CO; a monoclonal antibody specific for the 80-kDa subunit of m-calpain (C3989) was from Sigma Chemical Co., St. Louis, MO; a monoclonal antibody specific for the 28-kDa subunit common to both µ- and m-calpain was from Calbiochem (208730), San Diego, CA. Monoclonal antibodies used for calpastatin were 1F7 (Affinity Bioreagents MA3–944, which labels domain IV of bovine calpastatin); 2G11 (Affinity Bioreagents MA3–945, which labels domain II of bovine calpastatin); 2B8 (which labels domain IV of bovine calpastatin); and 4D5 (which labels domain I of bovine calpastatin).
All other chemicals were reagent grade or purer. Unless indicated otherwise, all protein isolations and chromatographic procedures were done at 2 to 4°C using precooled instruments and solutions, and all experiments used doubly deionized water that had been passed through a filter to remove organic material and then through a 0.45-µm filter to remove pyrogens.
Animals and Muscle Samples
Use of animal tissues in these studies was approved by the University of Arizona Institutional Animal Care and Use Committee. Preliminary studies involving 8 animals were done to select the type and sequence of columns used. Nine animals were then used to obtain the results shown in this study; the figures are representative of the results obtained for the 9 animals. Chromatographic profiles were similar among the different animals, and the SDS-PAGE profiles of the partly purified proteins differed slightly. A 350- to 450-g sample of bovine semimembranosus muscle was removed after 11 to 13 d of postmortem storage at 2 to 4°C. The sample was immediately placed on ice and transported to the laboratory. The muscle was trimmed free of extraneous fat and connective tissue and was passed through a meat grinder. The ground muscle was homogenized in 4.3 volumes (wt/vol) of homogenizing buffer (20 mM Tris·HCl, pH 8.0; 5 mM EDTA; 0.1% MCE; 0.1 mg/mL ovomucoid containing ovoinhibitor-Sigma Type II-0; 2.5 µM E-64; 2.0 mM phenylmethysulfonyl fluoride; Thompson and Goll, 2000), and the homogenate was centrifuged at 15,970 x gmax for 15 min. An aliquot of the supernatant was used for zymogram analysis of calpain activity; the remainder was adjusted to 125 mM KCl by adding 2 M KCl and then loaded onto a hexyl-TSK hydrophobic interaction column. Details of the column chromatographic procedures will be described in the Results section.
Assays of Enzyme Activity
Zymography was done as described by Arthur and Mykles (2000) using Hammersten casein without fluorescein isothiocyanate (FITC) labeling. Three to eight microliters of muscle homogenate containing 15 µg of protein were loaded in each well of a 4% polyacrylamide stacking gel; the resolving gel was 10% polyacrylamide, 8 x 7 cm (width x height) and 0.75-mm thick. The E-64 in the sample buffer does not inhibit the calpains in the absence of Ca2+ (Thompson and Goll, 2000), and because it has no net charge at pH 8.5, it does not enter the gel and so does not interfere with activity of the calpains. The gel was prerun for 30 min, and the sample was then loaded. After electrophoresis for 3 h at 4°C, the gel was removed, rinsed with deionized water, and then incubated with shaking for 1 h at room temperature (22 to 24°C) in 50 mM Tris·HCl, pH 7.5; 5 mM CaCl2; and 0.1% MCE. The Ca2+-containing solution was replaced with fresh solution, and the gel was incubated for another hour at room temperature with shaking. The Ca2+-containing solution was replaced a third time, and the gel was incubated overnight at room temperature with shaking. The Ca2+-containing solution was then removed, and the gel was stained with Coomassie Brilliant Blue R250. Size of the clear areas indicating calpain activity was quantitated by using a UVP Epi Chemi II bioimaging system (Upland, CA). The FITC-casein assay for calpastatin activity was conducted as described by Wolfe et al. (1989), and the BODIPY calpain assay as described by Thompson et al. (2000).
Other Procedures
Protein concentration was determined by using the Coomassie Brilliant Blue G250 assay as described by Bradford (1976), and bovine serum albumin was calibrated by Kjeldahl analysis to prepare calibration curves. The FITC casein was prepared as described by Wolfe et al. (1989), and BODIPY-FL casein was made according to the methods of Thompson et al. (2000). The SDS-polyacrylamide gel electrophoresis was conducted as described by Laemmli (1970) using minigels (Wolfe et al., 1989). Western blotting was done using the procedure described by Towbin et al. (1979) and a semidry transfer procedure described by Taylor et al. (1995). Dot-blot assays to detect proteolytically inactive µ-calpain were as described by Talbot et al. (1984) using a 10-µL sample spotted on a PVDF membrane rather than a nitrocellulose membrane. Dilutions (vol/vol) used for the antibodies were 1:10,000 for both the primary and the secondary antibodies used in Western blotting of the calpains and the dot-blot assays of the 80-kDa subunit of µ-calpain, respectively; 1:2,000 for the primary antibody and 1:50,000 for the secondary antibody used in Western blotting of the 28-kDa subunit; 1:10,000 for the 1F7 anticalpastatin antibody, 1:2,000 for the 2G11 and 2B8 anticalpastatin antibodies; and 1:10 for the 4D5 anticalpastatin antibody; the secondary antibody used for all calpastatin antibodies was a goat antimouse from CalBiochem and was diluted 1:10,000.
|
RESULTS
|
|---|
Some General Principles
The calpains and calpastatin in postmortem muscle have several unique properties that need to be considered when attempting to purify them. First, a number of studies have reported that calpastatin activity decreases (Vidalenc et al., 1983; Ducastaing et al., 1985; Koohmaraie et al., 1987; Boehm et al., 1998) and that the calpastatin molecule is degraded during postmortem storage (Boehm et al., 1998; Delgado et al., 2001). The calpastatin molecule in mammalian muscle contains 4 repeating, marginally homologous domains, numbered I, II, III, and IV, in addition to an N-terminal domain, the L domain, that varies in size due to alternative splicing events and different start sites of transcription (Goll et al., 2003). The repeating domains each contain approximately 140 amino acids (about 15,500 Da) and are each capable of inhibiting 1 calpain molecule, whereas the L domain has no calpain inhibitory activity (Goll et al., 2003). Domains I through IV each contain 3 subdomains, A, B, and C in order from N- to C-terminus of the domain; each subdomain has approximately 10 (subdomain C) to 14 amino acids (subdomains A and B). Subdomains A and C have no inhibitory activity, and any effective calpain inhibitor must contain subdomain B, although paradoxically, subdomain B by itself also is not an effective inhibitor of the calpains. The smallest effective calpain inhibitor contains subdomain B plus 9 amino acids N-terminal to subdomain B and 4 amino acids C-terminal to subdomain B, a 27-amino acid peptide (Wendt et al., 2004). This 27-mer has a molecular weight of 3,050 Da, but because calpastatin migrates anomalously in SDS-PAGE, it migrates as a peptide of 4,500 Da in an SDS-gel. Because calpastatin has this domain structure, it is possible, if postmortem degradation occurs at exactly the sites needed to produce the 27-mer, to have a fragment of 4,500 Da in SDS-PAGE that would inhibit the calpains. It would also be possible to have a fragment of 20,000 Da (in SDS-PAGE) that would have no inhibitory activity. Other even larger fragments may have weak inhibitory activity depending on how the postmortem degradation occurred. Hence, any attempt to isolate the calpastatin fragments produced during postmortem storage must use procedures that will purify small 3,000-Da fragments as well as larger calpastatin fragments. We have found that calpastatin fragments elute over a wide range from anion-exchange columns (Thompson et al., 2002), making purification of postmortem calpastatin very difficult.
Second, studies have shown that µ-calpain is autolyzed during postmortem storage, and may be almost entirely in the autolyzed state after 7 to 11 d postmortem (Boehm et al., 1998; Delgado et al., 2001; Veiseth et al., 2004). Autolysis increases the hydrophobicity of calpains and neither autolyzed µ- nor autolyzed m-calpain can be eluted from a phenyl Sepharose hydrophobic interaction column without using SDS (G. H. Geesink, University of Arizona, Tucson; V. F. Thompson; and D. E. Goll, unpublished data). Although the available evidence indicates that m-calpain is not autolyzed even after 15 d postmortem (Boehm et al., 1998; Veiseth et al., 2001), phenyl Sepharose, which produces a significant purification of the calpains, cannot be used to purify postmortem calpains without the risk of losing any autolyzed calpain.
Third, because an unknown number of the calpastatin fragments may have inhibitory activity, and because it is impossible to assay calpain activity in the presence of active calpastatin, the first step in any attempt to purify the calpains and calpastatin from postmortem muscle must separate all the calpastatin fragments from the calpains. Because the calpastatin fragments produced during postmortem storage elute from anion-exchange columns over a wide salt range, it is not possible to use anion-exchange chromatography as the first step in purification of postmortem calpains, although anion-exchange chromatography is the most commonly used first step in calpain/calpastatin purification. The only known chromatographic procedure that separates calpastatin and all calpastatin fragments from the calpains is hydrophobic interaction chromatography.
Fourth, it was reported several years ago that autolyzed µ-calpain lost its proteolytic activity when incubated in vitro in salt concentrations above 150 mM (Geesink and Koohmaraie, 1999, 2000). Studies have consistently shown that µ-calpain rapidly loses its activity during postmortem storage (Vidalenc et al., 1983; Ducastaing et al., 1985; Koohmaraie et al., 1987; Boehm et al., 1998), and it was suggested that the loss of µ-calpain activity during postmortem storage was an artifact resulting from exposure of autolyzed µ-calpain to salt concentrations above 150 mM during purification. Subsequently, it was shown that both autolyzed µ- and autolyzed m-calpain lose their proteolytic activity in the presence of salt concentrations above 150 mM (Li et al., 2004). The loss of activity results from dissociation of the 2 calpain subunits because autolysis removes a number of the noncovalent interactions between the subunits, and loss of these noncovalent interactions weakens the interaction between the subunits to the point that they dissociate at ionic strengths above 150 mM. The dissociated subunits aggregate irreversibly, and the aggregates are inactive proteolytically (Li et al., 2004). Because m-calpain evidently is not autolyzed during postmortem storage, postmortem m-calpain may not be affected by salt concentrations above 150 mM. The instability of autolyzed µ-calpain in the presence of ionic strengths above 150 mM, however, means that salt concentrations above 150 mM must be avoided when purifying postmortem calpains.
In sum, the nature of the calpains and calpastatin in postmortem muscle requires that purification of these molecules must use a hydrophobic interaction chromatography as a first step, that phenyl Sepharose hydrophobic interaction chromatography cannot be used during purification of the autolyzed calpains, and that the ionic strength must be maintained below 150 mM during the purification of autolyzed calpains.
Zymograms
Figure 1 is a schematic diagram summarizing the procedures used in the initial steps attempting to purify µ- and m-calpain and calpastatin from postmortem muscle. A zymogram assay was done on every muscle sample used in this study to ensure that the muscle was undergoing the changes in calpain activity that have been reported previously; a rapid decrease in µ-calpain activity with only a slight decrease in m-calpain activity. Figure 2 shows a zymogram of one of the samples used. We found that calpain activity in 11-d postmortem muscle was highly variable; most samples contained small amounts of µ- and m-calpain activity, and sometimes there was no m-calpain detectable by zymogram assay after 11-d postmortem. The loss of m-calpain activity observed in the semimembranosus muscle used in this study is greater than that observed in earlier studies (Vidalenc et al., 1983; Ducastaing et al., 1985; Koohmaraie et al., 1987; Boehm et al., 1998). The reason for this is unclear. The earlier studies used bovine longissimus dorsi (Ducastaing et al., 1985; Koohmaraie et al., 1987) or semimembranosus (Boehm et al., 1998) muscle, so it seems unlikely that the difference is due to different muscles. The samples were taken from muscles that had remained on the carcass, which had been placed in a –1°C cooler immediately after slaughter, and then maintained at 2 to 4°C, so the muscle was not exposed to high temperatures and low pH values that might denature the calpains during the first day postmortem. The loss of m-calpain activity was consistently noted in all 9 animals used in this study. A very small amount of µ-calpain activity could be detected after 11 d postmortem in 1 or 2 of the muscle samples used in this study, but this activity was very small, and m-calpain activity was usually greater than µ-calpain activity.
View larger version (11K):
[in this window]
[in a new window]
|
Figure 1. Schematic diagram summarizing the steps used in the initial stages of attempts to purify µ- and m-calpain and calpastatin from postmortem muscle. The 5 fractions identified after the initial steps described in this paper are in bold. CAST I and CAST II are abbreviations for calpastatin I and calpastatin II.
|
|
View larger version (47K):
[in this window]
[in a new window]
|
Figure 2. Zymogram of bovine semimembranosus muscle sampled at death (0 d), after 5 d of postmortem storage (5d), and after 11 d of postmortem storage (11 d), as indicated. Lanes labeled 0 d, 5 d, and 11 d were loaded with 15 µg of semimembranosus muscle extract; the lane labeled µ was loaded with 30 ng of µ-calpain purified from bovine diaphragm muscle. On the left of the figure, µ= position of µ-calpain, and m = position of m-calpain. The postmortem muscle samples were removed from carcasses that had been chilled in a cooler at –1°C for 24 h and then placed at 2 to 4°C until sampled. Zymograms were done as described in the Materials and Methods.
|
|
The First Step; Hexyl-TSK Hydrophobic Interaction Chromatography
For the reasons described in the preceding paragraphs, phenyl Sepharose cannot be used to purify the calpains from postmortem muscle. At least 0.8 M (NH4)2SO4 is required for the calpains to bind to butyl Sepharose (Thompson and Goll, 2000), so butyl Sepharose also cannot be used to purify the calpains from postmortem muscle because the high ionic strength would dissociate the subunits of autolyzed calpain. After extensive testing, it was determined that both autolyzed and the unautolyzed calpains would bind quantitatively to a hexyl-TSK column in the presence of 125 mM KCl, and the autolyzed calpains could be eluted nearly quantitatively from a hexyl-TSK column by using 1 mM EDTA, 0.1% MCE, 0.1% Brij 35 (Figure 3). Including Brij 35 enhances complete elution of the calpains off this column. Brij 35 does not inhibit calpain activity (actually increases the specific activity of m-calpain by 1.6- to 2.0-fold; Tan et al., 1988), and the Brij 35 micelles are easily removed during the following anion-exchange chromatographic step. Calpastatin did not bind to hexyl-TSK in the presence of 125 mM of KCl (Figure 3). After elution with 1 mM EDTA, 0.1% MCE, 0.1% Brij 35, the hexyl-TSK column was flushed with 5% SDS, and the SDS eluate was subjected to Western analysis using antibodies specific for the 80-kDa subunit of µ-calpain and for the 28-kDa subunit common to µ- and m-calpain to determine whether the 1 mM EDTA, 0.1% Brij 35 had eluted all the calpain off this column (Figure 4). Autolyzed µ-calpain is the most hydrophobic among the 4 forms of the calpains, autolyzed µ-calpain, unautolyzed µ-calpain, autolyzed m-calpain, and unautolyzed m-calpain (G. H. Geesink University of Arizona, Tucson; V. F. Thompson; and D. E. Goll, personal observation; see also Karlsson et al., 1985). Therefore, the amount of autolyzed µ-calpain that remains bound to the hexyl-TSK column after elution with 1 mM EDTA, 0.1% Brij 35 would be greater than the amount of any of the other calpains that might remain bound and can be used as an indicator of whether 1 mM EDTA, 0.1% Brij 35 elutes all calpain from the hexyl-TSK column. Western blots of the SDS eluant (Figure 4) were compared quantitatively with standards of known amounts of µ-calpain by using the Epi-Chem bioimaging system. Based on our experience in purifying µ- and m-calpain from different bovine muscles, we estimate that bovine skeletal muscle contains approximately 30 to 40 mg of calpain/1,000 g of muscle, and that this calpain is approximately 50 to 60% µ-calpain and 40 to 50% m-calpain. Hence, 350 g of semimembranosus muscle contains 5,250 to 8,400 µg of µ-calpain. The amount of µ-calpain eluted from the hexyl-TSK column by SDS-PAGE as determined by scanning the Western blots of the 40 tubes collected was a total of 18.48 µg for all 40 tubes (this number is an average of 6 samples assayed), and 18.48/5,250 = 0.00352 bound or 99.648% eluted. Thus, based on this analysis, >99% of all µ-calpain in the original sample was eluted from the hexyl-TSK column. Hence, the calpains in postmortem muscle are nearly completely eluted from the hexyl-TSK column by 1 mM EDTA, 0.1% Brij 35.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 3. Elution profile of a muscle extract from 11-d postmortem bovine semimembranosus muscle from a hexyl-TSK hydrophobic interaction column. Conditions: 1,800 mL of a muscle extract obtained as described in the Materials and Methods from 420 g of bovine semimembranosus after 11 d postmortem was adjusted to 125 mM KCl by adding 2 M KCl and loaded directly into a 5.0 x 13.0 cm hexyl-TSK column. The column was equilibrated with 965 mL of 125 mM KCl; 20 mM Tris·HCl, pH 7.5; 1.0 mM EDTA; 0.1% 2-mercaptoethanol (MCE) and then eluted with 1,628 mL of 0.1% Brij 35, 1.0 mM EDTA; 0.1% MCE. The flow rate was 114 mL/h. As indicated, the collected tubes 10 to 101 = 1,742 mL (calpastatin fraction), tubes 102 to 153 = 985 mL (inactive µ-calpain fraction), and tubes 154 to 189 = 700 mL (calpain fraction).
|
|
View larger version (55K):
[in this window]
[in a new window]
|
Figure 4. Western analysis using an anti-µ-calpain antibody (upper panel) and an anti28-kDa antibody (lower panel) of fractions eluted from the hexyl-TSK column by 5% SDS after the column had been flushed with 1 mM EDTA, 0.1% Brij 35, 0.1% 2-mercaptoethanol. Ten-milliliter fractions were collected in 34 tubes; tube numbers are shown above each lane. Lanes 10 to 28 were each loaded with 20 µL of SDS eluant, whereas tube 34 was loaded with 10 µL. The lane labeled µ contained 100 ng of µ-calpain purified from bovine diaphragm muscle, and the lane labeled am contained 393 ng of autolyzed bovine diaphragm muscle m-calpain. Note that the 28-kDa subunit was not autolyzed in these samples, although the 80-kDa subunit was completely in the autolyzed 76-kDa form. The 80 kDa indicates the distance of migration of an 80-kDa polypeptide, 28-kDa the distance of migration of a 28-kDa polypeptide, and 18-kDa the distance of migration of an 18-kDa polypeptide.
|
|
Detection of Inactive µ-Calpain by Dot-Blot Assay.
Because µ-calpain is proteolytically inactive or nearly proteolytically inactive after 11 d postmortem (Figure 2
), a dot-blot antibody assay was used to detect µ-calpain in the column effluent during isolation of the calpains in postmortem muscle. The effluent containing the calpastatin fraction that did not bind to the hexyl-TSK matrix (Figure 3) also contained a compound that was labeled with the secondary antimouse antibody used in our dot-blot assay, so it was necessary to do 2 dot-blot assays, one using only the secondary antibody and a second using both the anti-µ-calpain and the secondary antibody (Figure 5). The signal obtained in the first assay was subtracted from the signal obtained in the second assay to obtain a true response to the anti-µ-calpain antibody. Figure 5 shows the results of such an assay of the eluant from a hexyl-TSK column. A fraction that was labeled by the µ-calpain antibody was detected just at the end of the unbound calpastatin peak in all the samples used in this study, and this fraction was collected. The exact nature of this µ-calpain is unknown at the present state of this project. Whatever it is, it bound very weakly to the hexyl-TSK matrix, so its elution was delayed to the end of the calpastatin elution. Because the fraction coeluted with calpastatin, it was possible that the calpastatin masked the proteolytic activity of the putative µ-calpain in this fraction. Subsequent analysis (see later in this paper), however, showed that this putative µ-calpain, named the inactive µ-calpain fraction henceforth in this paper, was proteolytically inactive or nearly proteolytically inactive.
View larger version (15K):
[in this window]
[in a new window]
|
Figure 5. Dot-blot assay of fractions eluting off the hexyl-TSK column. The plot shows the difference, as determined by the UVP Epi-Chemi II bioimaging system, between the signal obtained from the secondary antibody alone and the anti-µ-calpain antibody, and the inset shows the dot blots obtained when using both the first and second antibody (first Ab) and when using the second antibody alone (second Ab). Tubes 74 to 87 were collected as the "inactive calpain" fraction in this preparation. IOD = integrated optical density.
|
|
Salting Out Postmortem Calpastatin.
The calpastatin fraction was salted out at 80% (NH4)2SO4 saturation in an attempt to ensure that all calpastatin fragments were salted out [the skeletal muscle 110- to 125-kDa calpastatin salts out at 50 to 55% (NH4)2SO4 saturation]. Initial experiments tested first salting out at 40% (NH4)2SO4 and then salting out at 80% (NH4)2SO4, but almost nothing precipitated at 40% (NH4)2SO4, so subsequent experiments simply went directly to 80% (NH4)2SO4. The salted-out calpastatin was dialyzed extensively using a Spectra/Por membrane having a 3,500 molecular weight cut-off to retain all calpastatin fragments larger than 3,500 Da. After extensive dialysis to remove all (NH4)2SO4, the P0–80 calpastatin was clarified at 186,400 x gmax for 90 min and then loaded onto an anion-exchange column (either DEAE-TSK or Q-Sepharose; results were identical for the 2 columns). One hundred milliliters of the supernatant remaining after salting out at 80% (NH4)2SO4 (the S80 fraction) was dialyzed extensively using a Spectra/Por membrane having a 500 Da cut-off because it seemed that any fragment not salting out at 80% (NH4)2SO4 saturation would be very small. The dialyzed sample was concentrated 30- to 35-fold by using a YM-1 membrane (1,000 Da cut-off), and the concentrated sample was assayed to determine whether it had any calpastatin activity. Calpastatin activity in these concentrated S80 samples ranged from 0 to 16% inhibition. The concentrated S80 supernatant contained a major peptide of 16 kDa (in SDS) and several other peptides (Figure 6). N-terminal sequencing of the 16-kDa peptide showed that this polypeptide was myoglobin. The concentrated S80 fraction was subjected to Western analysis using 3 different antibovine calpastatin monoclonal antibodies and an anti-human calpastatin polyclonal antibody. None of the peptides visible in the gels shown in Figure 6 were labeled by any of the antibodies used. The monoclonal antibodies whose epitope is in domains IV and I and an anti-human polyclonal antibody all very weakly labeled polypeptides at 75 and 45 kDa. Because these assays were done on samples that had been concentrated 30-to 35-fold, it is clear that nearly all the postmortem calpastatin was salted out at 80% (NH4)2SO4 concentration.
View larger version (80K):
[in this window]
[in a new window]
|
Figure 6. The SDS-PAGE of the concentrated supernatants remaining after salting out at 80% (NH)2SO4 concentration (S80 fraction). The S80 fractions from 3 preparations, labeled 1, 2, and 3, are shown. The MF is a myofibril from bovine skeletal muscle. Molecular weights are indicated on the right-hand margin. All 3 S80 fractions contained a prominent (after concentrating 30- to 35-fold) polypeptide of 16-kDa in SDS-PAGE. The gel was 15% acrylamide in a 8 x 10-cm "tall" minigel; 10 µg of protein was loaded on each lane.
|
|
Anion-exchange Chromatography of Postmortem Calpastatin off Hexyl-TSK
The P0–80 calpastatin fraction that did not bind to the hexyl-TSK column eluted as 2 broad peaks of activity off an anion-exchange column (DEAE-TSK shown in Figure 7). The relative inhibitory abilities of these 2 peaks varied among the different preparations in this study. In approximately 50% of the preparations, the first peak had lower inhibitory activity than the second peak (as shown in Figure 7), but in other preparations, the first peak had slightly higher or equal inhibitory activity when compared with the second peak. Because the activities in Figure 7 are activities of fractions assayed in the column eluates, the lower activity could indicate simply that the calpastatin content of the fractions eluting in the first peak was lower than the calpastatin content in the fractions eluting in the second peak and does not necessarily infer that the specific inhibitory activity of the first peak is lower than the specific inhibitory activity of the second peak. The first peak, calpastatin I, did not bind to the DEAE-TSK column at low ionic strength (20 mM Tris·HCl, pH 7.5; 1 mM ETDA; 0.1% MCE), and inhibited calpain activity by only 35 to 70%, depending on the preparation. The second peak, calpastatin II, inhibited calpain activity by 35 to 100% (Figure 7). Concentrating the 2 calpastatin peaks 10- to 30-fold by using a YM-1 membrane increased the inhibitory ability of both peaks, but even after a 10-fold concentration, peak I inhibited calpain to less than 50% of its original activity in some instances and only twice inhibited over 90% of calpain activity. Concentrating the second peak by 10- to 30-fold enhanced its inhibitory ability to 80 to 100% inhibition. It is unclear why concentrating the 2 calpastatin peaks did not increase their inhibitory properties more; it is possible that none of the calpastatin degradation products in postmortem muscle contain all 3 subdomains, A, B, and C, and that the binding constants of these fragments is so high (weak binding) that none of them are effective calpain inhibitors regardless of how concentrated they are. In 2 of the 9 animals used in this study, the second inhibitory peak eluted as a doublet; the doublet had inhibitory properties similar to the single peak shown in Figure 7 (i.e., inhibited approximately 35 to 100% of the calpain activity in the assay used), and it was collected as a single calpastatin II peak. Calpastatin is unique in its resistance to denaturation by heat. Therefore, after concentrating calpastatin I and II 10- to 30-fold, the concentrated samples were brought to 100°C by heating for 40 to 80 s in a microwave oven, and then placed on ice. The heating step removed 50 to 65% of the protein in the calpastatin peak I and peak II samples and resulted in significant purification of the postmortem calpastatin. The heated samples were centrifuged at 40,000 x gmax for 20 min and the supernatant concentrated 4- to 6-fold by using a YM-1 membrane. Even after heating the concentrated calpastatin fractions to 100°C and removing the denatured protein by centrifugation, both calpastatin I and II fractions still contained a number of polypeptides (Figure 8). These polypeptides all migrated more rapidly than polypeptides having a molecular mass of 70-kDa in SDS-PAGE. Because they each inhibited the calpains, both calpastatin I and calpastatin II must contain calpastatin polypeptides that have the inhibitory subdomain B. Neither calpastatin I nor calpastatin II are pure, so it is difficult to determine their relative specific inhibitory activities. At-death calpastatin that has been only partly purified from skeletal muscle, however, can inhibit 100% of calpain activity. Hence, it seems likely that the fragments in calpastatin I and II either lack part of subdomain B or lack some of the additional 9 N-terminal and 4 C-terminal amino acids needed to produce an inhibitor as effective as at-death calpastatin. It also is very likely that both calpastatin I and calpastatin II contain more than 1 calpastatin fragment; indeed, it is surprising that postmortem calpastatin eluted in only 2 peaks of activity. The variability that we encountered in activity of the 2 postmortem calpastatin peaks is not surprising and is likely the result of varying degrees of postmortem degradation of the calpastatin polypeptide. Identification of the inhibitory polypeptides and N-terminal sequencing will be needed to determine the specific activity of postmortem calpastatin; this will be the focus of a subsequent paper.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 7. Elution profile from a DEAE-TSK column of the calpastatin fraction collected from the hexyl-TSK column. Conditions: salted out the 1,272 mL collected from tubes 12 to 73 from the hexyl-TSK column with 80% saturation (NH4)2SO4, dialyzed extensively against 1 mM KHCO3, 1 mM EDTA, 5 mM 2-mercaptoethanol (MCE), and then loaded 315 mL of the P0–80 fraction directly onto this 5.0 x 16.8 cm DEAE-TSK column that had been equilibrated with 20 mM Tris·HCl, pH 7.5; 1.0 mM EDTA, 0.1% MCE (TEM). Flushed with 440 mL of TEM, and then eluted with a linear 0 100 mM KCl gradient in TEM (750 mL of each) followed by flushing with 530 mL of 2.5 M KCl; 20 mM Tris·2-(N-morpholino)ethane sulfonic acid, pH 6.5; 1 mM EDTA; and 0.1% MCE. Flow rate was 116 mL/h. Collected tubes 13 to 35 = 438 mL (calpastatin peak I) and tubes 73 to 107 = 553 mL (calpastatin peak II).
|
|
View larger version (78K):
[in this window]
[in a new window]
|
Figure 8. The SDS-PAGE of postmortem calpastatin peaks I and II after successive hexyl-TSK and Q-Sepharose columns followed by a 10-fold concentration with a YM-1 membrane, then heating to 100°C, centrifugation, and a 4- to 6-fold concentration of the supernatant with a YM-1 membrane. Gel is an 8 to 16% polyacrylamide gradient gel; 15 µg of protein was loaded onto each lane. Peak I and peak II are calpastatin I and calpastatin II, respectively; numbers 8 and 9 are the numbers of the preparations from which these samples were taken.
|
|
DEAE-TSK Chromatography of Postmortem Calpain off Hexyl-TSK
The DEAE-TSK anion-exchange chromatography of the calpain fraction eluted off the hexyl-TSK column was done at pH 6.5, so the calpains would elute at ionic strengths less than 200 mM. The DEAE-TSK chromatography separated 2 peaks of calpain activity (Figure 9); the first peak of activity eluted at 65 to 90 mM KCl, slightly later than the position that µ-calpain normally elutes from DEAE-TSK at pH 6.5 (dot-blot analysis confirmed that it was µ-calpain), and the second peak eluted at 160 to 200 mM KCl, the normal KCl concentration that m-calpain elutes off these columns. We consistently found µ-calpain activity eluting off the DEAE-TSK column, even though very little µ-calpain activity could be detected in the muscle homogenates by zymogram assay (see Figure 2). The BODIPY assay is 2- to 3-fold more sensitive than the zymogram assay (Thompson et al., 2000), and the DEAE-TSK concentrated whatever proteolytically active µ-calpain remained in the sample, so these 2 factors may have combined to make it possible to detect proteolytically active µ-calpain after DEAE-TSK chromatograpy. Activity of the calpains eluting off the DEAE-TSK column was low, ranging from 60 to 600 fluorescence units in the BOD-IPY assay compared with >2,000 flourescence units normally observed for at-death bovine calpains after 2 columns. This is consistent with the low activities observed in the zymograms of these samples (Figure 2). In 5 instances out of the 9 animals studied, µ-calpain activity was as high as m-calpain activity, but m-calpain activity was 2- to 5-fold greater than µ-calpain activity in the other 4 animals (the latter is shown in Figure 9). Activity of both calpains was low in those instances where their activities were similar (60 to 150 fluorescence units in the BODIPY assay, compared with >6,000 fluorescence units for an equal amount of purified calpain in this assay). In 8 of the 9 samples, µ-calpain activity was 60 to 100 fluorescence units (FU) from the BODIPY casein assay, whereas m-calpain activity ranged from 60 to 5,900 FU. That the postmortem calpain was still very impure after 2 columns contributes to this low activity, and purification will be required to determine whether postmortem calpain actually has a lower specific activity than at-death calpain. In 2 of the 9 animals used in this study, the postmortem µ-calpain eluted as a doublet and in 2 other animals, it eluted as a broad peak (the broad peak is shown in Figure 9). It eluted as a single peak in the other 5 animals. We have no explanation for this variation. It may reflect variation in the environment in postmortem muscle. The 2 calpain peaks were concentrated by using a YM-10 membrane (10,000 molecular weight cut-off), and the concentrated fractions were subjected to SDS-PAGE and Western analysis (Figure 10).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 9. Elution profile from a DEAE-TSK column of the calpain fraction collected from the hexyl-TSK column. Conditions: loaded 1,218 mL collected from the hexyl-TSK column directly onto this 2.6 x 24.9 cm DEAE-TSK column that had been equilibrated in 20 mM Tris·2-(N-morpholino)ethane sulfonic acid (MES), pH 6.5; 1 mM EDTA; 0.1% 2-mercaptoethanol (MCE). The column was flushed with 120 mL of 20 mM KCl; 20 mM Tris·MES, pH 6.5; 1 mM EDTA; 0.1% MCE followed by elution with a linear 20 to 300 mM KCl gradient (600 mL of each) in 20 mM Tris·MES, pH 6.5; 1 mM EDTA; 0.1% MCE and then flushing with 110 mL of 2.0 M KCl; 20 mM Tris·MES, pH 6.5; 1 mM EDTA; 0.1% MCE. Flow rate = 58.9 mL/h. As indicated, tubes 127 to 152 = 375 mL were collected as µ-calpain and tubes 162 to 183 = 332 mL were collected as m-calpain. The ratio of µ-calpain to m-calpain activity in this figure was typical of most preparations done, but on several occasions, µ-calpain activity was nearly as great as m-calpain activity. Dot-blot analysis did not detect any inactive µ-calpain in the eluant from this column. The large spike in absorbance at tubes 95 to 99 was due to the beginning of the 20 mM KCl flush, and the dramatic increase in conductivity at tube 190 was due to the 2.0 M KCl flush.
|
|
View larger version (48K):
[in this window]
[in a new window]
|
Figure 10. The SDS-PAGE and Western blots of µ-calpain and m-calpain after passing through successive hexyl-TSK hydrophobic interaction and DEAE-TSK anion-exchange columns. (A) SDS-PAGE gel was a 816% acrylamide gel; 15 µg of protein was loaded onto each lane. MF is a bovine myofibril standard, numbers above each lane are the number of the preparation from which the samples were taken, and numbers to the left of the gel are the approximate molecular weights of proteins migrating at that position. (B) Western analysis of samples from preparations 6 (µ-calpain) and 5 (m-calpain). For the 80-kDa blot: the µ-calpain standard (µ above lane 1) was loaded with 250 ng of purified human placenta µ-calpain; the autolyzed µ-calpain standard (aµ, lane 2) was loaded with 600 ng of autolyzed µ-calpain; the m-calpain standard (m, lane 4) was loaded with 400 ng of purified human placenta m-calpain; the autolyzed m-calpain standard was loaded with 547 ng of autolyzed m-calpain; the µ-calpain sample (preparation 6) was loaded with 30 µg of protein; the m-calpain sample (preparation 5) was loaded with 9 µg of protein. For the 28-kDa blot: the µ-calpain standard was loaded with 250 ng of purified µ-calpain; the autolyzed µ-calpain standard was loaded with 625 ng of purified µ-calpain; the m-calpain standard was loaded with 400 ng of purified m-calpain; the autolyzed m-calpain standard was loaded with 400 ng of autolyzed m-calpain; the µ-calpain (preparation 6) was loaded with 13 µg of protein; and the m-calpain (preparation 5) was loaded with 15 µg of protein. Note that the 80-kDa blots and the 28-kDa blots involve the same protein (either preparation 6 for µ-calpain or preparation 5 for m-calpain), so the partly autolyzed 28-kDa subunit is part of the same molecule as the completely autolyzed 76-kDa polypeptide for µ-calpain.
|
|
SDS-PAGE and Western Analysis of Postmortem Calpains.
The SDS-PAGE shows that both postmortem calpains still contain a large number of polypeptides after 2 columns; the large and small calpain subunits cannot be detected in the SDS gels of these calpain fractions after 2 column chromatographic procedures. Western analysis shows that postmortem m-calpain contains 80- and 28-kDa subunits, suggesting that m-calpain is not autolyzed even after 11 to 13 d of postmortem storage (the 78-kDa autolyzed form of the m-calpain 80-kDa subunit is not easily distinguished from the 80-kDa unautolyzed subunit in Western blots, and any determination of autolysis cannot be based only on detection of a 78-kDa polypeptide). Postmortem µ-calpain contains an autolyzed 76-kDa form of its large subunit and a mixture of 22-, 24-, and 28-kDa forms of its small subunit (Figure 10). An earlier study also reported that the small subunit of µ-calpain is not autolyzed completely to the 18-kDa form during postmortem storage, even though the 80-kDa large subunit is completely autolyzed (Camou et al., 2007). Subsequent studies will attempt to purify these 2 postmortem calpains.
DEAE-TSK Chromatography of the Inactive µ-Calpain Fraction off Hexyl-TSK
There was also variation in elution of the fraction that was detected by the anti-µ-calpain antibodies near the end of the calpastatin peak off the hexyl TSK column (Figure 5). In 4 of the 9 animals that we analyzed, elution of samples that reacted with the µ-calpain antibody in a dot-blot assay extended from the end of the calpastatin peak all the way to the calpain peak (see Figure 3), whereas in the other 5 samples, elution of the samples reacting with the anti-µ-calpain antibody ended before the proteolytically active calpain was eluted from the hexyl-TSK column (see Figure 5). For those samples where elution of the inactive calpain extended to the calpain elution, it seemed possible that elution of some of the proteolytically inactive µ-calpain may have overlapped with elution of the active calpain discussed in the preceding section. Therefore, for the 4 animals where elution of calpain detected only by dot-blot assay extended to the proteolytically active calpain elution, the eluant from the DEAE-TSK column of calpain (Figure 9) was also analyzed with the anti-µ-calpain dot-blot assay. Protein that was labeled by the antiµ-calpain antibody in the dot-blot assays was detected in all 4 of these samples; the inactive µ-calpain eluted from these columns during the 20 mM KCl flush before the proteolytically active µ-calpain. Hence, this inactive µ-calpain elutes from an ion-exchange column at a lower KCl concentration than µ-calpain from at-death muscle does and was named peak I inactive µ-calpain. The sample detected by dot-blot assay off the hexyl-TSK column was dialyzed to reduce the salt concentration to less than 10 mM and then was applied to a DEAE-TSK anion exchange column (Figure 11). Two peaks of calpastatin activity corresponding to the 2 peaks of calpastatin activity that eluted from the anion-exchange (DEAE-TSK or Q-Sepharose) column of the P0–80 calpastatin fraction were detected. These 2 peaks of calpastatin activity were combined with the 2 peaks from the previous calpastatin anion-exchange column, and the combined calpastatins I and II were subjected to heating and centrifugation as described previously. Dot-blot assays of the eluant from the DEAE-TSK column of inactive µ-calpain identified a proteolytically inactive µ-calpain that eluted at 75 mM KCl off this column (Figure 11). Flushing the column with 2.5 M KCl at pH 6.5 and then with 0.15 N KOH did not elute any additional polypeptides that were labeled with the anti-µ-calpain antibody. Hence, 2 kinds of inactive µ-calpain have been detected: 1 kind that coeluted with the calpain peak off the hexyl-TSK column and elutes at 20 mM KCl off a DEAE-TSK column eluted at pH 6.5 (peak I), and a second kind that elutes off the hexyl-TSK column at the end of calpastatin peak and that elutes at 75 mM KCl from a DEAE-TSK column at pH 6.5 (peak II). The 2 inactive µ-calpains were each concentrated by using a YM-10 membrane. The SDS-PAGE showed that the concentrated inactive µ-calpain fractions also contained a number of polypeptides just as the µ- and m-calpain samples did at this stage of purification, although the polypeptide composition of peak I and peak II inactive µ-calpains were different (Figure 12). Western analysis showed that inactive µ-calpain had an autolyzed 76-kDa large subunit and a partly degraded 28-kDa subunit (Figure 12; only peak II is shown), similar to that observed for the proteolytically active µ-calpain (Figure 10). In 4 of the 9 animals studied, the BODIPY assay detected a small amount of calpain activity (10 to 30 fluorescence units in the BODIPY assay) in the "inactive" µ-calpain fraction, suggesting that after sufficient concentration and a sensitive assay, "inactive" µ-calpain may possess a very small amount of proteolytic activity. Future studies will attempt additional purification of this inactive µ-calpain to learn why it is no longer proteolytically active (or has such a low proteolytic activity).
View larger version (62K):
[in this window]
[in a new window]
|
Figure 12. The SDS-PAGE and Western blots of inactive µ-calpain after passing through successive hexyl-TSK hydrophobic interaction and DEAE-TSK anion-exchange columns. (A) The SDS-PAGE gel was an 8 to 16% gradient gel; each lane was loaded with 15 µg of protein. The numbers above each lane are the number of the preparation from which the samples were taken. PI and PII refer to the peak I and peak II of inactive µ-calpain that was detected in 4 of the animals used. (B) Western analysis of peak II samples taken from preparations 4 and 5. For the 80-kDa blot: µ is 500 ng of µ-calpain purified from human placenta; aµ is 625 ng of autolyzed µ-calpain; 5 is 15 µg of inactive µ-calpain from preparation 5. For the 28-kDa blot: m is 400 ng of purified m-calpain; am is 400 ng of autolyzed m-calpain; and 4 is 15 µg of inactive µ-calpain from preparation 4.
|
|
|
DISCUSSION
|
|---|
The initial steps in purifying µ-calpain, m-calpain, and calpastatin from postmortem bovine muscle described in this manuscript have separated 5 fractions of the calpain system in postmortem muscle: 1) calpastatin I that does not bind to an ion-exchange matrix at pH 7.5, and that has limited inhibitory activity, inhibiting calpain to 35 to 70% of its original activity; 2) calpastatin II that elutes from a DEAE ion-exchange column at 70 to 80 mM KCl and that is capable of completely inhibiting calpain after concentration; 3) autolyzed µ-calpain that is proteolytically active and that contains an autolyzed 76-kDa subunit and a partly autolyzed small subunit; 4) m-calpain that seems to be unautolyzed and equivalent of at-death m-calpain, although it is not completely active in zymogram assays; and 5) a proteolytically inactive µ-calpain that is detected by dot-blot assays and that contains an autolyzed 76-kDa large subunit and a partly degraded small subunit. These initial steps have not yet purified any of the members of the calpain system from postmortem muscle, but they have provided new information on several properties of the calpain system in postmortem muscle.
First, Western analysis shows that m-calpain does not seem to be autolyzed even after 11 d postmortem in bovine muscle. This observation confirms earlier findings on 7-d postmortem bovine muscle (Boehm et al., 1998) and 15-d postmortem ovine muscle (Veiseth et al., 2001). It is unclear why postmortem m-calpain was not as active as at-death m-calpain in zymogram assays. Postmortem m-calpain eluted from anion-exchange columns at the same salt concentration as at-death m-calpain does.
Second, as was observed previously (Camou et al., 2007), the small, 28-kDa subunit of µ-calpain is not autolyzed to an 18-kDa fragment after 11 d postmortem even though the 80-kDa subunit is completely autolyzed to the 76-kDa state. The partial degradation/autolysis of the µ-calpain small subunit results in 24- and 22-kDa fragments and a small amount of undegraded 28-kDa subunit. That these different fragments of the small subunit all eluted together with the 76-kDa large subunit off a hydrophobic interaction column and an anion-exchange column indicates that postmortem µ-calpain consists of a mixture of 22/76, 24/76, and 28/76 molecules. The catalytic properties of such µ-calpain molecules have not been studied. The inactive µ-calpain that was detected by dot-blot analysis also was autolyzed to a 76-kDa large subunit and to 24- and 22-kDa fragments and some unautolyzed 28-kDa subunit.
Third, both autolyzed µ-calpain and the inactive µ-calpain contain both the large subunit and the small subunit after several chromatographic steps. Hence, these subunits are not separated during postmortem storage in situ, although the subunits of autolyzed µ-calpain are dissociated in vitro by ionic strengths above 300 mM (Li et al., 2004). This dissociation is responsible for the salt-induced loss of µ-calpain activity observed in vitro (Geesink and Koohmaraie, 1999, 2000). Therefore, it seems unlikely that the loss of µ-calpain proteolytic activity that occurs during postmortem storage is caused by a salt-induced dissociation of the µ-calpain subunits.
Fourth, we found µ-calpain proteolytic activity after a DEAE-TSK column even though very little µ-calpain activity was detected by zymogram assay of crude homogenates of postmortem muscle. The BODIPY microplate assay we used for assay of the fractions eluting from the DEAE-TSK column is 2- to 3-fold more sensitive than the zymogram assay, and the DEAE-TSK column undoubtedly concentrated any µ-calpain present. Hence, postmortem muscle has µ-calpain activity after 11 d postmortem, even if it cannot be detected by zymogram assay. Such activity is small, however, and it remains unclear whether it could make a significant contribution to postmortem tenderization, especially at the temperatures (2 to 4°C) and pH values (5.6 to 5.9) that exist in postmortem muscle after 11 d (Camou et al., 2007). Because the activity of postmortem µ-calpain varied significantly among different preparations, it is possible that those preparations that had higher µ-calpain activity also were more tender, supporting the concept that µ-calpain has a primary role in postmortem tenderization, even though very little activity can be detected by ordinary zymogram assays. Tenderness was not measured in this study, so any relation between tenderness and the activity of postmortem µ-calpain detected in this study remains uncertain.
Fifth, the amount of inactive µ-calpain detected by our dot-blot assays is small compared with the amount of active µ-calpain that eluted from the DEAE-TSK column with m-calpain (Figure 9). We had expected all or nearly all the µ-calpain to be proteolytically inactive based on the zymogram assays. It seems based on the results thus far that the proteolytic activity of µ-calpain in postmortem muscle decreases to a level that cannot be detected by ordinary assays of muscle extracts. Additional purification of the postmortem µ-calpain to the point that its specific activity can be determined will be required to determine whether postmortem µ-calpain is proteolytically inactive or simply has a much reduced proteolytic activity compared with at-death µ-calpain. Such purification may also allow analysis of what causes the decrease in specific activity of µ-calpain.
Sixth, calpastatin is degraded to polypeptides smaller than 70 kDa after 11 d of postmortem storage. This degradation seems to decrease the ability of calpastatin to inhibit the calpains, but because of the multiheaded nature of the calpastatin molecule, small fragments retain some inhibitory activity. Additional purification and N-terminal amino acid analysis will be required to learn about the nature of these fragments and their ability to inhibit the calpains.
|
Footnotes
|
|---|
1 This work was supported by grants from the USDA National Research Initiative Competitive Grants Program, 2005-35206-15268; the Muscular Dystrophy Association, MDA4133; the NIH, AR52108-02; and the Arizona Agriculture Experiment Station, Project 28, a contributing project to USDA Regional Research Project NC-1131. We thank Janet Christner for help in preparing the manuscript, and Hamdi Ahmad and his associates at the University Livestock and Meats Complex for their help in obtaining the muscle samples needed for this research. 
2 Present address: Centro de Investigación en Alimentación y De-sarrollo, A.C., Hermosillo, Sonora, Mexico.
3 Corresponding author: darrel.goll{at}arizona.edu
Received for publication June 14, 2007.
Accepted for publication August 14, 2007.
|
LITERATURE CITED
|
|---|
Arthur, J. S. C., and D. L. Mykles. 2000. Calpain zymography with casein or fluorescein isothiocyanate casein. Pages 109–116 in Methods in Molecular Biology, vol. 144: Calpain Methods and Protocols. J. S. Elce, ed. Humana Press Inc., Totowa, NJ.
Boehm, M. L., T. L. Kendall, V. F. Thompson, and D. E. Goll. 1998. Changes in the calpains and calpastatin during postmortem storage of bovine muscle. J. Anim. Sci. 76:2415–2434.[Abstract/Free Full Text]
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72:248–254.[CrossRef][Medline]
Camou, J. P., J. A. Marchello, V. F. Thompson, S. W. Mares, and D. E. Goll. 2007. Effect of postmortem storage on activity of µ-and m-calpain in five different bovine muscles. J. Anim. Sci. 85:2660–2667.[Abstract/Free Full Text]
Delgado, E. F., G. H. Geesink, J. A. Marchello, D. E. Goll, and M. Koohmaraie. 2001. The calpain system in three muscles of normal and callipyge sheep. J. Anim. Sci. 79:398–412.[Abstract/Free Full Text]
Ducastaing, A., C. Valin, J. Schollmeyer, and R. Cross. 1985. Effects of electrical stimulation on postmortem changes in the activities of two Ca dependent neutral proteinases and their inhibitor in beef muscle. Meat Sci. 15:193–202.[CrossRef]
Edmunds, T., P. A. Nagainis, S. K. Sathe, V. F. Thompson, and D. E. Goll. 1991. Comparison of the autolyzed and unautolyzed forms of µ- and m-calpain from bovine skeletal muscle. Biochim. Biophys. Acta 1077:197–208.[CrossRef][Medline]
Geesink, G. H., and M. Koohmaraie. 1999. Effect of calpastatin on degradation of myofibrillar proteins by µ-calpain under postmortem conditions. J. Anim. Sci. 77:2685–2692.[Abstract/Free Full Text]
Geesink, G. H., and M. Koohmaraie. 2000. Ionic strength-induced inactivation of µ-calpain in postmortem muscle. J. Anim. Sci. 78:2336–2343.[Abstract/Free Full Text]
Geesink, G. H., S. Kuchay, A. H. Chirhti, and M. Koohmaraie. 2006. µ-Calpain is essential for postmortem proteolysis of muscle proteins. J. Anim. Sci. 84:2834–2840.[Abstract/Free Full Text]
Goll, D. E., R. G. Taylor, J. A. Christiansen, and V. F. Thompson. 1992. Role of proteinases and protein turnover in muscle growth and meat quality. Pages 25–36 in Proc. 44th Annu. Recip. Meat Conf., Natl. Live Stock and Meat Board, Chicago, IL.
Goll, D. E., V. F. Thompson, H. Li, W. Wei, and J. Cong. 2003. The calpain system. Physiol. Rev. 83:731–801.[Abstract/Free Full Text]
Karlsson, J.-O., S. Gustavsson, C. Hall, and E. Nilsson. 1985. A simple one-step procedure for separation, of calpain I, calpain II, and calpastatin. Biochem. J. 231:201–204.[Medline]
Kent, M. P., M. J. Spencer, and M. Koohmaraie. 2004. Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin. J. Anim. Sci. 82:794–801.[Abstract/Free Full Text]
Koohmaraie, M. 1988. The role of endogenous proteases in meat tenderness. Pages 89–100 in Proc. 41st Annu. Recip. Meat Conf., Natl. Live Stock and Meat Board, Chicago, IL. Natl. Live Stock and Meat Board, Chicago, IL.
Koohmaraie, M. 1992. The role of Ca2+-dependent proteinases (calpains) in postmortem proteolysis and meat tenderness. Biochimie 74:239–245.[Medline]
Koohmaraie, M. 1996. Biochemical factors regulating the toughening and tenderization process of meat. Meat Sci. 43:193–201.[CrossRef]
Koohmaraie, M., S. C. Seideman, J. E. Schollmeyer, T. R. Dutson, and J. D. Crouse. 1987. Effect of postmortem storage on Ca-dependent proteases, their inhibitor and myofibril fragmentation. Meat Sci. 19:187–196.[CrossRef]
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.[CrossRef][Medline]
Li, H., V. F. Thompson, and D. E. Goll. 2004. Effects of autolysis on properties of µ- and m-calpain. Biochim. Biophys. Acta 1691:91–103.[Medline]
Talbot, P. J., R. L. Knobler, and M. J. Buchmeier. 1984. Western and dot immunoblotting analysis of viral antigens and antibodies: Application to murine hepatitis virus. J. Immunol. Meth. 73:177–188.[CrossRef][Medline]
Tan, F. C., D. E. Goll, and Y. Otsuka. 1988. Some properties of the millimolar Ca2+-dependent proteinase from bovine cardiac muscle. J. Mol. Cell. Cardiol. 20:983–997.[CrossRef][Medline]
Taylor, R. G., G. H. Geesink, V. F. Thompson, M. Koohmaraie, and D. E. Goll. 1995. Is Z-disk degradation responsible for postmortem tenderization? J. Anim. Sci. 73:1351–1367.
Thompson, V. F., and D. E. Goll. 2000. Purification of µ-calpain, m-calpain, and calpastatin from animal tissues. Pages 3–16 in Methods in Molecular Biology, vol. 144: Calpain Methods and Protocols. J. S. Elce, ed. Humana Press Inc., Totowa, NJ.
Thompson, V. F., S. Saldaña, J. Cong, and D. E. Goll. 2000. A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases. Anal. Chem. 279:170–178.
Thompson, V. F., S. Saldaña, J. Cong, D. M. Luedke, and D. E. Goll. 2002. The calpain system in human placenta. Life Sci. 70:2493–2508.[CrossRef][Medline]
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350–4354.[Abstract/Free Full Text]
Veiseth, E., S. D. Shackelford, T. L. Wheeler, and M. Koohmaraie. 2001. Effect of postmortem storage on µ-calpain and m-calpain in ovine skeletal muscle. J. Anim. Sci. 79:1502–1508.[Abstract/Free Full Text]
Veiseth, E., S. D. Shackelford, T. L. Wheeler, and M. Koohmaraie. 2004. Indicators of tenderization are detectable by 12 h postmortem in ovine muscle. J. Anim. Sci. 82:1428–1436.[Abstract/Free Full Text]
Vidalenc, P., P. Cottin, N. Merdaci, and A. Ducastaing. 1983. Stability of two Ca2+-dependent neutral proteinases and their specific inhibitor during postmortem storage of rabbit skeletal muscle. J. Sci. Food Agric. 34:1241–1250.[CrossRef][Medline]
Wendt, A., V. F. Thompson, and D. E. Goll. 2004. Interaction of calpain with calpastatin: A review. Biol. Chem. 385:465–472.[CrossRef][Medline]
Wolfe, F. H., S. K. Sathe, D. E. Goll, W. C. Kleese, T. Edmunds, and S. M. Duperret. 1989. Chicken skeletal muscle has three Ca2+-dependent proteinases. Biochim. Biophys. Acta 998:236–250.[CrossRef][Medline]
Top
Abstract
INTRODUCTION
