- Email: [email protected]
Concurrent identification of calpains I and II from chicken skeletal muscle
~
Comp. Biochem. PhysioL Vol. 107B, No. 4, pp. 519-523, 1994 Elsevier ScienceLtd Printed in Great Britain 0305-0491/94 $6.00 + 0.00
Pergamon
0305-0491(93)E0005-Z
Concurrent identification of calpains I and II from chicken skeletal muscle S. G. Birkhold and A. R. Sams Department of Poultry Science, Texas A & M University System, College Station, TX 77843-2472, U.S.A.
A single anion-exchange column resolved two peaks of calcium-activated neutral protease activity, corresponding to the two calpain forms from chicken skeletal muscle. Multiple columns have previously been needed to resolve the two isoforms from avian tissue. Calcium requirement assays confirmed one form to require approximately 100pM Ca 2+ for half-maximal activity, while the other required approximately 500pM Ca ~+. Electrophoresis revealed that the enzymes were not purified to homogeneity. Key words: Calpain; Skeletal muscle. Comp. Biochem. Physiol. 107B, 519-523, 1994.
Introduction The calpain system is composed of calpains I and II, and their specific inhibitor, calpastatin (GoU et al., 1990). The calcium-activated neutral thiol proteases, calpains I and II, have been identified in several mammalian species and tissues including bovine, porcine, rat and rabbit skeletal muscle. The calcium requirement for one-half maximal activity of each isoform varies with species and tissue, but generally ranges from 2 to 75/~M for calpain I (#-calpain) and from 0.2 to 0.8 mM for calpain II (m-calpain) (Melloni and Pontremoli, 1989). Although these two proteases have similar molecular weights, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Dayton et al., 1981), they are two distinct enzymes that share a common 30 kDa subunit but have different 78-80 kDa subunits (Croall and Demartino, 1991). Post-translational modification was originally proposed to cause the formation of #-calpain from m-calpain (Suzuki et al., 1981); however, they were later Correspondence to: A. R. Sams, Department of Poultry Science, Texas A & M University System, College Station, TX 77843-2472, U.S.A. Tel.: (409) 845-4818; Fax: (409) 845-1921. Received 26 July 1993; accepted 22 October 1993.
519
reported to be two distinct isozymes (Croall and Demartino, 1991) that required different calcium concentrations for activation. Autolysis, however, appears to affect the Ca 2÷ sensitivity of the individual forms. Suzuki et al. (1981) reported that incubation of chicken skeletal muscle calpain at 0°C for 2 min with 0.5 mM Ca 2÷ reduced the Ca 2÷ requirement for halfmaximal activity from 400 to 30#M. They further noted that substrate may control the autodigestion, as the absence of substrate was required to attain autodigestion. SDS-PAGE of casein column-treated CANP showed that autodigestion of calpain caused the appearance of a 76 kDa band with a corresponding disappearance of the 80 kDa band (Suzuki et al., 1981). Similarly, Hathaway et al. (1982) demonstrated that limited autolysis reduced the Ca 2÷ requirement for half-maximal activity of chicken gizzard m-calpain from 150 to 50/~M. However, neither group reported the existence of two distinct isoforms in avian tissue. Chicken calpain was suggested as an intermediate prototype proteinase that evolved into /~-calpain and m-calpain in mammalian tissues (Emori et al., 1986). Although chicken skeletal muscle was initially thought to contain only
520
S.G. Birkholdand A. R. Sams
m-calpain with a calcium requirement between mammalian #- and m- calpains, the presence of #-calpain was demonstrated in avian tissue (Wolfe et al., 1989). These researchers identified three isoforms of CANP from mature laying hen breast muscles. A multicolumn procedure was used to identify a novel, high m-calpain that required 3800/~M Ca 2+ for half-maximal activation, in addition to/~-calpain and m-calpain that required 5.35 and 420 #M Ca 2+ for halfmaximal activation, respectively. Although their work demonstrated that multiple isoforms existed in avian skeletal muscle, they were unable to identify activity of the/~-calpain form from a single DEAE-anion exchange column. The extensive chromatographic steps necessary for purification of the calpains have delayed studies of this enzyme system. The three or more successive columns needed to achieve purity reduce enzyme yield. The present study was undertaken to evaluate the possible presence of both #-calpain and m-calpain in chicken skeletal muscle using the more rapid, one-column procedure developed for mammalian tissues (Wheeler and Koohmaraie, 1990).
Materials and Methods Approximately 75 g of pectoralis were excised immediately following death by cervical dislocation from each of three broiler-type chickens and frozen in liquid nitrogen. Unless otherwise specified, all remaining procedures were performed at 4°C. Each of the three samples was homogenized on ice in 2.5 vol extraction buffer (100 mM Tris, 5 mM EDTA, l0 mM 2-mercaptoethanol, pH 8.3). Following centrifugation at 22,000g for 2.5hr, the supernatant was adjusted to pH 7.5, and dialyzed (12,000 MWCO) overnight against dialysis buffer (20mM Tris, 5mM EDTA, 10mM 2-mercaptoethanol, pH 7.5). Following centrifugation at 22,000 g for 2.5 hr, the supernatant was loaded on to a 2.5 x 50 cm DEAE-anion exchange column (Sephacel, Pharmacia, Uppsala, Sweden) that had been previously equilibrated with elution buffer (20 mM Tris, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, pH 7.45). Two peaks of CANP activity were eluted with a linear gradient of 0-500 mM NaCl at a flow rate of 0.54 ml/min. Eighty 6.5-ml fractions were collected. Absorbance at 278nm of all fractions was measured. Fractions were assayed for CANP activity by incubating a 500-#1 aliquot with 1.5 ml of a buffer containing 100 mM Tris, 5 mM CaC12, 5 mg/ml casein, pH 7.5, for 1 hr at 25°C. The CaCl 2 was replaced in a tandem assay tube by 5 mM EDTA to determine calcium-independent
proteolysis. The reaction was stopped by addition of 1.0 ml 5% trichloroacetic acid (TCA). Following centrifugation at 3200 rpm for 15 min, each fraction was screened for calpain activity by measuring absorbance at 278 nm of TCA-soluble material released during incubation. Within each of the two peaks of CANP activity, fractions were pooled and concentrated using a 20,000 MWCO ultrafiltration stirred cell (Amicon, Dawes, MA). Calcium requirements for the two calpain pools were determined by incubating an aliquot of the concentrated eluent with 30,000 cpm ~4C-~t-casein (Sigma, St Louis, MO) and concentrations of Ca 2÷ varying from 0 to 70,000#M in 35 mM Tris, 1 mM dithiothreitol, pH 7.5, at 25°C for 20 min. Reaction tubes were prepared by adding, in the following order, 100 #1 enzyme-containing fraction, 30/tl ~4C-~-casein, and 10/tl Ca 2+ solution. Control tubes were included for background and nonspecific binding of labeled casein. The reaction was terminated with 1.0 ml 5% TCA containing 2 mg/ml bovine serum albumin. The TCA-soluble material released during incubation was determined by liquid scintillation counting of a 400 #1 aliquot of supernatant mixed with 5.0 ml scintillation cocktail. Purity of the muscle extract prior to loading on to the column, and purity of the concentrated CANP pools were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10-20% T-gradient gel, performed according to the procedures of Laemmli (1970). Protein was determined on all fraction pools with the Bio-Rad protein assay (Bio-Rad, Richmond, CA) using bovine serum albumin as the standard. A high molecular weight standard protein mixture (Sigma) was prepared according to the manufacturer's instructions and included for reference. Aliquots of the pre-column extract and the eluted fraction pools were diluted 1:4 with sample buffer (0.0625 M Tris-HCl, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.002% Bromphenol Blue, pH = 6.8), heated in a boiling water bath for 3min, and cooled immediately on ice. Twenty-five micrograms of protein were loaded into each lane. Gels were run at 16 mA through the stacking gel and 24 mA through the resolving gel. Following staining with Coomassie Blue, the gels were subjectively evaluated for heterogeneity.
Results and Discussion Two peaks of calcium-dependent proteolysis were detected in fractions obtained from one elution of a single DEAE-anion exchange column (Fig. 1). Peak 1 activity eluted at
521
Concurrent identificationof calpains 0.6
Abs 278 nm (Cslpain Activity)
Abs 278 nm (Total Protein) 3.5
500
0.5 2.5
0.4
3O0
0.3 1.5
0.2
1
0.1
0.5
0
21
41 Fraction
100
~0
61
mM NaCl
-o-Ca2+ -+-EDTA -A-TotaIProtein ~[NaCII
Fig. 1. Elution profile of chicken skeletal muscle extract from an anion exchangecolumn. approximately 160-180 mM NaCI, while peak 2 activity eluted at approximately 325-380mM NaCI (Fig. 1). Peak 1 activity eluted later in the profile as compared to the 70-125mM KC1 elution range for for porcine skeletal muscle /t-calpain (Goll et al., 1990), and the 60 mM KCI elution reported by Mellgren (1980). This greater salt concentration needed for elution of chicken skeletal muscle #-calpain may have contributed to the failure of previous studies to detect p-calpain in chicken tissue (Ishiura et al., 1978; Suzuki et al., 1981). The calcium concentration required for halfmaximal activity for peak 1, presumed to represent /~-calpain, was 100#M Ca2+(Fig. 2). This value is closer to the 50/~M Ca 2+ required for rabbit skeletal /t-calpain (Inomata et al., 1983) and the 45 # M Ca 2÷ required for porcine skeletal muscle (Dayton et al., 1981) than to the
previously reported 5.2 # M Ca 2+ requirement for chicken skeletal muscle (Wolfe et al., 1989). Differences between the requirement observed in this study and the requirement reported by others (Wolfe et al., 1989) may result from reporting calcium requirements for autolyzed /~-calpain vs non-autolyzed /~-calpain. Suzuki et al. (1981) reported that brief incubation at 0°C for 2 min enhances the Ca 2+ sensitivity of chicken skeletal muscle #-calpain. Substrate availability appears to direct autolysis. Therefore, the order of enzyme, substrate and calcium additions into the reaction tube should be reported by researchers so that the reader can determine whether or not the reported calcium requirement is for autolyzed calpain or nonautolyzed calpain. SDS-PAGE analysis of peak 1 revealed that the fraction pool was a heterogeneous protein solution (Fig. 3). A
1oo
i
50
...............
0
+
. . . . . . . . . .
0
70
"
140 700 1400 3500 Ca 2+ Concentration (ldVl) "Peak I ~ P e a k 2
7000
J 70000
Fig. 2. Chicken skeletal muscle calcium-activatedneutral protease activityof two peaks from an anion exchange column assayed at various Ca-'+ concentrations.
522
S.G. Birkhold and A. R. Sams
A
B
C
205kD
29kD
from chicken breast muscle. The 500 #M Ca 2+ requirement for half-maximal activity for peak 2 (Fig. 2) is similar to the 420/~M Ca 2+ required for half-maximal activity of m-calpain from mature laying hen breast tissue (Wolfe et al., 1989), the 400/~M Ca 2+ required for half-maximal activity of chicken skeletal muscle calpain (Suzuki et al., 1981), and the 520#M Ca 2+ required for half-maximal activity of m-calpain from human platelets (Yoshida et al., 1983). Although less concentrated than the peak 1 fraction pool, SDS PAGE revealed that the peak 2 fraction pool was also a heterogeneous protein solution. This study demonstrated that chicken skeletal muscle contains two forms of CANP, presumably corresponding to the two forms of calpain. These calpains can be separated and their activities quantified with one DEAE-anion exchange column. However, this one-column procedure does not purify the proteins to homogeneity.
References
Fig. 3. Electrophoretic gel of chicken skeletal muscle extract prior to column loading (lane A) and after elution of calcium activated neutral proteolytic activity of peak 1 (lane B) and peak 2 (lane C) from an anion exchange column. Arrow indicates dye front and numbers indicate approximate molecular weights based on standard protein bands.
heterogeneous environment could have artificially elevated the observed calcium requirement, due to the presence of calcium binding proteins. While the lack of homogeneity may interfere with characterization, Koohmaraie (1990) reported that this one-column procedure was accurate for CANP activity quantification. A final reason for the differences between the results of the present study and the report of Wolfe et al. (1989) may be that a radioisotopic assay was used in the present study and Wolfe et al. (1989) used a fluorescence assay. In preliminary studies it was determined that the ultraviolet-based assay used to screen fractions for CANP activity was not sufficiently sensitive to detect the low levels of activity expressed at the lower calcium concentrations in the calcium requirement assay (data not shown). Peak 2 activity, presumed to represent mcalpain, began to elute at approximately 325 mM NaC1 (Fig. 1). This is near tbe 350 mM NaC1 concentration reported by Ishiura et al. (1978) for calcium-activated neutral protease
Croalt D. E. and Demartino G. N. (1991) Calcium-activated neutral protease (calpain) system: structure, function and regulation. Physiol. Rev. 71, 813-847. Dayton W. R., Schollmeyer J. V., Lepley R. A. and Cortes L. R. (1981) A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease. Biochem. biophys. Res. Commun. 659, 48-61. Emori Y., Kawasaki H., Sugihara H., Imajoh S., Kawashima S. and Suzuki K. (1986) Isolation and sequence analysis of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease. J. biol. Chem. 261, 9465-9471. Goll D. E., Kleese W. C., Okitani A., Kumamoto T., Cong J. and Kapprell H.-P. (1990) Historical background and current status of the Ca2+-dependent proteinase system. In lntracellular Calcium-Dependent Proteolysis (Edited by Mellgren R. L. and Murachi T.), pp. 3 24. CRC Press, Boca Raton, FL. Hathaway D. R., Werth D. K. and Haeberle J. R. (1982) Limited autolysis reduces the Ca2+-requirement of smooth muscle Ca2+-activated protease. J. biol. Chem. 257, 9072-9077. Inomata M., Hayashi M., Nakamura M., Imahori K. and Kawashima S. (1983) Purification and characterization of a calcium-activated neutral protease from rabbit skeletal muscle which requires calcium ions of/~M order concentration. J. Biochem. 93, 291 294. Ishiura S., Murofushi H., Suzuki K. and Imahori K. (1978) Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization. J. Biochem. 84, 225-230. Koohmaraie M. (1990) Quantification of Ca2+-dependent protease activities by hydrophobic and ion-exchange chromatography. J. Anita. Sei. 68, 659-665. Laemmti U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Melloni E. and Pontremoli S. (1989) The calpains. Trends Neurosci. 12, 438~,44.
Concurrent identification of calpalns Mellgren R. L. (1980) Canine cardiac calcium-dependent proteases: resolution of two forms with different requirements for calcium. FEBS Lett. 109, 129-133. Suzuki K., Tsuji S., Kubota S., Kimura Y. and Imahori K. (1981) Limited autolysis of Ca2+-activated neutral protease (CAN-P) changes its sensitivity to Ca 2+ ions. J. Biochem. 90, 275--278. Wheeler T. L. and Koohmaraie M. (1990) A micro procedure for simultaneous extraction and subsequent assay of calcium-dependent and lysosomal
523
protease systems from muscle. J. Anita. Sci. 69, 1559-1561. Wolfe F. H., Sathe S. K., Goll D. E., Kleese W. C., Edmunds T. and Duperret S. M. (1989) Chicken skeletal muscle has three Ca2+-dependent proteinases. Biochim. biophys. Acta 998, 236-250. Yoshida N., Weksler B. and Nachman R. (1983) Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function. J. biol. Chem. 258, 7168-7174.
Comp. Biochem. PhysioL Vol. 107B, No. 4, pp. 519-523, 1994 Elsevier ScienceLtd Printed in Great Britain 0305-0491/94 $6.00 + 0.00
Pergamon
0305-0491(93)E0005-Z
Concurrent identification of calpains I and II from chicken skeletal muscle S. G. Birkhold and A. R. Sams Department of Poultry Science, Texas A & M University System, College Station, TX 77843-2472, U.S.A.
A single anion-exchange column resolved two peaks of calcium-activated neutral protease activity, corresponding to the two calpain forms from chicken skeletal muscle. Multiple columns have previously been needed to resolve the two isoforms from avian tissue. Calcium requirement assays confirmed one form to require approximately 100pM Ca 2+ for half-maximal activity, while the other required approximately 500pM Ca ~+. Electrophoresis revealed that the enzymes were not purified to homogeneity. Key words: Calpain; Skeletal muscle. Comp. Biochem. Physiol. 107B, 519-523, 1994.
Introduction The calpain system is composed of calpains I and II, and their specific inhibitor, calpastatin (GoU et al., 1990). The calcium-activated neutral thiol proteases, calpains I and II, have been identified in several mammalian species and tissues including bovine, porcine, rat and rabbit skeletal muscle. The calcium requirement for one-half maximal activity of each isoform varies with species and tissue, but generally ranges from 2 to 75/~M for calpain I (#-calpain) and from 0.2 to 0.8 mM for calpain II (m-calpain) (Melloni and Pontremoli, 1989). Although these two proteases have similar molecular weights, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Dayton et al., 1981), they are two distinct enzymes that share a common 30 kDa subunit but have different 78-80 kDa subunits (Croall and Demartino, 1991). Post-translational modification was originally proposed to cause the formation of #-calpain from m-calpain (Suzuki et al., 1981); however, they were later Correspondence to: A. R. Sams, Department of Poultry Science, Texas A & M University System, College Station, TX 77843-2472, U.S.A. Tel.: (409) 845-4818; Fax: (409) 845-1921. Received 26 July 1993; accepted 22 October 1993.
519
reported to be two distinct isozymes (Croall and Demartino, 1991) that required different calcium concentrations for activation. Autolysis, however, appears to affect the Ca 2÷ sensitivity of the individual forms. Suzuki et al. (1981) reported that incubation of chicken skeletal muscle calpain at 0°C for 2 min with 0.5 mM Ca 2÷ reduced the Ca 2÷ requirement for halfmaximal activity from 400 to 30#M. They further noted that substrate may control the autodigestion, as the absence of substrate was required to attain autodigestion. SDS-PAGE of casein column-treated CANP showed that autodigestion of calpain caused the appearance of a 76 kDa band with a corresponding disappearance of the 80 kDa band (Suzuki et al., 1981). Similarly, Hathaway et al. (1982) demonstrated that limited autolysis reduced the Ca 2÷ requirement for half-maximal activity of chicken gizzard m-calpain from 150 to 50/~M. However, neither group reported the existence of two distinct isoforms in avian tissue. Chicken calpain was suggested as an intermediate prototype proteinase that evolved into /~-calpain and m-calpain in mammalian tissues (Emori et al., 1986). Although chicken skeletal muscle was initially thought to contain only
520
S.G. Birkholdand A. R. Sams
m-calpain with a calcium requirement between mammalian #- and m- calpains, the presence of #-calpain was demonstrated in avian tissue (Wolfe et al., 1989). These researchers identified three isoforms of CANP from mature laying hen breast muscles. A multicolumn procedure was used to identify a novel, high m-calpain that required 3800/~M Ca 2+ for half-maximal activation, in addition to/~-calpain and m-calpain that required 5.35 and 420 #M Ca 2+ for halfmaximal activation, respectively. Although their work demonstrated that multiple isoforms existed in avian skeletal muscle, they were unable to identify activity of the/~-calpain form from a single DEAE-anion exchange column. The extensive chromatographic steps necessary for purification of the calpains have delayed studies of this enzyme system. The three or more successive columns needed to achieve purity reduce enzyme yield. The present study was undertaken to evaluate the possible presence of both #-calpain and m-calpain in chicken skeletal muscle using the more rapid, one-column procedure developed for mammalian tissues (Wheeler and Koohmaraie, 1990).
Materials and Methods Approximately 75 g of pectoralis were excised immediately following death by cervical dislocation from each of three broiler-type chickens and frozen in liquid nitrogen. Unless otherwise specified, all remaining procedures were performed at 4°C. Each of the three samples was homogenized on ice in 2.5 vol extraction buffer (100 mM Tris, 5 mM EDTA, l0 mM 2-mercaptoethanol, pH 8.3). Following centrifugation at 22,000g for 2.5hr, the supernatant was adjusted to pH 7.5, and dialyzed (12,000 MWCO) overnight against dialysis buffer (20mM Tris, 5mM EDTA, 10mM 2-mercaptoethanol, pH 7.5). Following centrifugation at 22,000 g for 2.5 hr, the supernatant was loaded on to a 2.5 x 50 cm DEAE-anion exchange column (Sephacel, Pharmacia, Uppsala, Sweden) that had been previously equilibrated with elution buffer (20 mM Tris, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, pH 7.45). Two peaks of CANP activity were eluted with a linear gradient of 0-500 mM NaCl at a flow rate of 0.54 ml/min. Eighty 6.5-ml fractions were collected. Absorbance at 278nm of all fractions was measured. Fractions were assayed for CANP activity by incubating a 500-#1 aliquot with 1.5 ml of a buffer containing 100 mM Tris, 5 mM CaC12, 5 mg/ml casein, pH 7.5, for 1 hr at 25°C. The CaCl 2 was replaced in a tandem assay tube by 5 mM EDTA to determine calcium-independent
proteolysis. The reaction was stopped by addition of 1.0 ml 5% trichloroacetic acid (TCA). Following centrifugation at 3200 rpm for 15 min, each fraction was screened for calpain activity by measuring absorbance at 278 nm of TCA-soluble material released during incubation. Within each of the two peaks of CANP activity, fractions were pooled and concentrated using a 20,000 MWCO ultrafiltration stirred cell (Amicon, Dawes, MA). Calcium requirements for the two calpain pools were determined by incubating an aliquot of the concentrated eluent with 30,000 cpm ~4C-~t-casein (Sigma, St Louis, MO) and concentrations of Ca 2÷ varying from 0 to 70,000#M in 35 mM Tris, 1 mM dithiothreitol, pH 7.5, at 25°C for 20 min. Reaction tubes were prepared by adding, in the following order, 100 #1 enzyme-containing fraction, 30/tl ~4C-~-casein, and 10/tl Ca 2+ solution. Control tubes were included for background and nonspecific binding of labeled casein. The reaction was terminated with 1.0 ml 5% TCA containing 2 mg/ml bovine serum albumin. The TCA-soluble material released during incubation was determined by liquid scintillation counting of a 400 #1 aliquot of supernatant mixed with 5.0 ml scintillation cocktail. Purity of the muscle extract prior to loading on to the column, and purity of the concentrated CANP pools were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 10-20% T-gradient gel, performed according to the procedures of Laemmli (1970). Protein was determined on all fraction pools with the Bio-Rad protein assay (Bio-Rad, Richmond, CA) using bovine serum albumin as the standard. A high molecular weight standard protein mixture (Sigma) was prepared according to the manufacturer's instructions and included for reference. Aliquots of the pre-column extract and the eluted fraction pools were diluted 1:4 with sample buffer (0.0625 M Tris-HCl, 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.002% Bromphenol Blue, pH = 6.8), heated in a boiling water bath for 3min, and cooled immediately on ice. Twenty-five micrograms of protein were loaded into each lane. Gels were run at 16 mA through the stacking gel and 24 mA through the resolving gel. Following staining with Coomassie Blue, the gels were subjectively evaluated for heterogeneity.
Results and Discussion Two peaks of calcium-dependent proteolysis were detected in fractions obtained from one elution of a single DEAE-anion exchange column (Fig. 1). Peak 1 activity eluted at
521
Concurrent identificationof calpains 0.6
Abs 278 nm (Cslpain Activity)
Abs 278 nm (Total Protein) 3.5
500
0.5 2.5
0.4
3O0
0.3 1.5
0.2
1
0.1
0.5
0
21
41 Fraction
100
~0
61
mM NaCl
-o-Ca2+ -+-EDTA -A-TotaIProtein ~[NaCII
Fig. 1. Elution profile of chicken skeletal muscle extract from an anion exchangecolumn. approximately 160-180 mM NaCI, while peak 2 activity eluted at approximately 325-380mM NaCI (Fig. 1). Peak 1 activity eluted later in the profile as compared to the 70-125mM KC1 elution range for for porcine skeletal muscle /t-calpain (Goll et al., 1990), and the 60 mM KCI elution reported by Mellgren (1980). This greater salt concentration needed for elution of chicken skeletal muscle #-calpain may have contributed to the failure of previous studies to detect p-calpain in chicken tissue (Ishiura et al., 1978; Suzuki et al., 1981). The calcium concentration required for halfmaximal activity for peak 1, presumed to represent /~-calpain, was 100#M Ca2+(Fig. 2). This value is closer to the 50/~M Ca 2+ required for rabbit skeletal /t-calpain (Inomata et al., 1983) and the 45 # M Ca 2÷ required for porcine skeletal muscle (Dayton et al., 1981) than to the
previously reported 5.2 # M Ca 2+ requirement for chicken skeletal muscle (Wolfe et al., 1989). Differences between the requirement observed in this study and the requirement reported by others (Wolfe et al., 1989) may result from reporting calcium requirements for autolyzed /~-calpain vs non-autolyzed /~-calpain. Suzuki et al. (1981) reported that brief incubation at 0°C for 2 min enhances the Ca 2+ sensitivity of chicken skeletal muscle #-calpain. Substrate availability appears to direct autolysis. Therefore, the order of enzyme, substrate and calcium additions into the reaction tube should be reported by researchers so that the reader can determine whether or not the reported calcium requirement is for autolyzed calpain or nonautolyzed calpain. SDS-PAGE analysis of peak 1 revealed that the fraction pool was a heterogeneous protein solution (Fig. 3). A
1oo
i
50
...............
0
+
. . . . . . . . . .
0
70
"
140 700 1400 3500 Ca 2+ Concentration (ldVl) "Peak I ~ P e a k 2
7000
J 70000
Fig. 2. Chicken skeletal muscle calcium-activatedneutral protease activityof two peaks from an anion exchange column assayed at various Ca-'+ concentrations.
522
S.G. Birkhold and A. R. Sams
A
B
C
205kD
29kD
from chicken breast muscle. The 500 #M Ca 2+ requirement for half-maximal activity for peak 2 (Fig. 2) is similar to the 420/~M Ca 2+ required for half-maximal activity of m-calpain from mature laying hen breast tissue (Wolfe et al., 1989), the 400/~M Ca 2+ required for half-maximal activity of chicken skeletal muscle calpain (Suzuki et al., 1981), and the 520#M Ca 2+ required for half-maximal activity of m-calpain from human platelets (Yoshida et al., 1983). Although less concentrated than the peak 1 fraction pool, SDS PAGE revealed that the peak 2 fraction pool was also a heterogeneous protein solution. This study demonstrated that chicken skeletal muscle contains two forms of CANP, presumably corresponding to the two forms of calpain. These calpains can be separated and their activities quantified with one DEAE-anion exchange column. However, this one-column procedure does not purify the proteins to homogeneity.
References
Fig. 3. Electrophoretic gel of chicken skeletal muscle extract prior to column loading (lane A) and after elution of calcium activated neutral proteolytic activity of peak 1 (lane B) and peak 2 (lane C) from an anion exchange column. Arrow indicates dye front and numbers indicate approximate molecular weights based on standard protein bands.
heterogeneous environment could have artificially elevated the observed calcium requirement, due to the presence of calcium binding proteins. While the lack of homogeneity may interfere with characterization, Koohmaraie (1990) reported that this one-column procedure was accurate for CANP activity quantification. A final reason for the differences between the results of the present study and the report of Wolfe et al. (1989) may be that a radioisotopic assay was used in the present study and Wolfe et al. (1989) used a fluorescence assay. In preliminary studies it was determined that the ultraviolet-based assay used to screen fractions for CANP activity was not sufficiently sensitive to detect the low levels of activity expressed at the lower calcium concentrations in the calcium requirement assay (data not shown). Peak 2 activity, presumed to represent mcalpain, began to elute at approximately 325 mM NaC1 (Fig. 1). This is near tbe 350 mM NaC1 concentration reported by Ishiura et al. (1978) for calcium-activated neutral protease
Croalt D. E. and Demartino G. N. (1991) Calcium-activated neutral protease (calpain) system: structure, function and regulation. Physiol. Rev. 71, 813-847. Dayton W. R., Schollmeyer J. V., Lepley R. A. and Cortes L. R. (1981) A calcium-activated protease possibly involved in myofibrillar protein turnover. Isolation of a low-calcium-requiring form of the protease. Biochem. biophys. Res. Commun. 659, 48-61. Emori Y., Kawasaki H., Sugihara H., Imajoh S., Kawashima S. and Suzuki K. (1986) Isolation and sequence analysis of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease. J. biol. Chem. 261, 9465-9471. Goll D. E., Kleese W. C., Okitani A., Kumamoto T., Cong J. and Kapprell H.-P. (1990) Historical background and current status of the Ca2+-dependent proteinase system. In lntracellular Calcium-Dependent Proteolysis (Edited by Mellgren R. L. and Murachi T.), pp. 3 24. CRC Press, Boca Raton, FL. Hathaway D. R., Werth D. K. and Haeberle J. R. (1982) Limited autolysis reduces the Ca2+-requirement of smooth muscle Ca2+-activated protease. J. biol. Chem. 257, 9072-9077. Inomata M., Hayashi M., Nakamura M., Imahori K. and Kawashima S. (1983) Purification and characterization of a calcium-activated neutral protease from rabbit skeletal muscle which requires calcium ions of/~M order concentration. J. Biochem. 93, 291 294. Ishiura S., Murofushi H., Suzuki K. and Imahori K. (1978) Studies of a calcium-activated neutral protease from chicken skeletal muscle. I. Purification and characterization. J. Biochem. 84, 225-230. Koohmaraie M. (1990) Quantification of Ca2+-dependent protease activities by hydrophobic and ion-exchange chromatography. J. Anita. Sei. 68, 659-665. Laemmti U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Melloni E. and Pontremoli S. (1989) The calpains. Trends Neurosci. 12, 438~,44.
Concurrent identification of calpalns Mellgren R. L. (1980) Canine cardiac calcium-dependent proteases: resolution of two forms with different requirements for calcium. FEBS Lett. 109, 129-133. Suzuki K., Tsuji S., Kubota S., Kimura Y. and Imahori K. (1981) Limited autolysis of Ca2+-activated neutral protease (CAN-P) changes its sensitivity to Ca 2+ ions. J. Biochem. 90, 275--278. Wheeler T. L. and Koohmaraie M. (1990) A micro procedure for simultaneous extraction and subsequent assay of calcium-dependent and lysosomal
523
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