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Proteasome from rabbit skeletal muscle: Some properties and effects on muscle proteins
PII:
SO309-1740(96)00126-X
Meat Science, Vol. 45, No. 4, 451462, 1997 (%, 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/97 s17.oo+o.oo
ELSEVIER
Proteasome from Rabbit Skeletal Muscle: Some Properties and Effects on Muscle Proteins M. Matsuishi & A. Okitani Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 7-l Kyonan-cho, I-chome, Musashino-shi, Tokyo 180, Japan (Received 17 August 1996; accepted 20 October 1996)
ABSTRACT Rabbit proteasome, likely to be a 20s proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purtfied enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20 000-29 000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580000-800000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pHfor hydrolysis of succinylLeu-Leu-Val-Tyr-(I-methylcoumaryl-?-amide) (Sue-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH S-9 and up to 60°C at pH 7.2. The enzyme activity was inhibited by diisopropyl jluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaClz. Remarkably, SDS increased the V,,, value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, a-actinin, actin. tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION Proteolysis during meat conditioning is considered to contribute to the tenderization of meat and the improvement of its flavor. Intracellular proteases, e.g. cathepsins, calpains and various aminopeptidases, are assumed to participate in proteolysis during conditioning of meat. Proteasomes were recently discovered as the proteinases that play major roles in ATP-dependent proteolysis in living eukaryotic cells. However, the contribution of these proteinases to proteolysis during meat conditioning has yet to be clarified. Proteasomes have been classified into two isoforms with apparent sedimentation coefficients of 20s and 26S, respectively (Tanahashi et al., 1993). The 20s proteasome has been purified from various muscles (Dahlmann et al., 1985; Mykles, 1989; Koohmaraie, 1992) and other tissues (Rivett, 1989; Orlowski, 1990; 451
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Rechsteiner et al., 1993), and is well characterized. This enzyme, which has a molecular mass of 600000-760 000 Da, consists of multiple subunits with molecular masses of 21 OO@-32000 Da. Interestingly, it is activated by low concentrations of SDS and various fatty acids, but not by ATP. The 26s proteasome has been purified from rat liver (Yoshimura et al., 1993; Ugai et al., 1993), human kidney (Kanayama et al., 1992) and Xenopus oocytes (Peters et al., 1993), but not from muscles. This enzyme, which possesses a molecular mass of about 2 000 000 Da, is an assembly of a 20s proteasome and multiple components with molecular masses of 30 00&l 10 000 Da. The 20s proteasome is considered to be the core unit of proteinase activity of the 26s enzyme, and other components are assumed to be the subunits which make the proteinase activity of the 26s enzyme ATP-dependent. It is also shown that ATP is needed to keep the 26s complex and the depletion of ATP causes rapid dissociation of the 26s complex into the 20s enzyme and multiple components (Kanayama et al., 1992). Driscoll and Goldberg (1989) isolated the proteasome from rabbit muscles rapidly (probably within a few days post mortem) in the presence of glycerol, which is known to stabilize 26s proteasome complex. Although their enzyme was likely to be a 20s proteasome judging from its molecular mass, it could be activated by ATP for a few days after purification. However, in order to elucidate the role of proteasomes in conditioning of meat, in which ATP disappears, it is necessary to investigate the properties of the 20s enzyme which does not show ATP-dependence rather than the 26s enzyme and ATPactivatable 20s enzym. Moreover, studies of the rabbit muscle enzyme lack such general information as optimum pH, heat stability and action towards muscle proteins (except ATP-dependency). Thus, in this study we isolated non-ATP-sensitive proteasome from rabbit skeletal muscle in the absence of glycerol and investigated its enzymatic properties and effects on myofibrillar and sarcoplasmic proteins.
MATERIALS
AND METHODS
Materials
Rabbit skeletal muscle (longissmus dorsi) was obtained from the carcasses immediately after slaughter and, after removal of fat and connective tissue, it was minced with a meat chopper. DEAE-cellulose (DE-52) was purchased from Whatman (Maidstone, UK). DEAESephadex A-50 and Sephacryl S-300 were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Hydroxyapatite was from Seikagaku Kogyo Co (Tokyo, Japan). Ferritin, aldolase, bovine serum albumin, ovalbumin, chymotrypsinogen and cytochrome c, were purchased from Boehringer Mannheim GmbH (Germany). Thyroglobulin was from Sigma Chemical Co (St. Louis, MO, USA). Sue-LLVY-MCA, 7-amino-4-methylcoumarin (AMC), leupeptin, E-64, bestatin, chymostatin and pepstatin were purchased from the Peptide Institute (Osaka, Japan). Bio-Lyte 3/10 Ampholyte was purchased from Bio-Rad Laboratories (Richmond, VA, USA). Centriflo CF25 ultrafiltration membrane cones were obtained from Amicon Corp (Danvers, MA, USA). All other chemicals were of analytical grade. Assay for proteasome
Proteasome activity was measured with Sue-LLVY-MCA according to the method of Ishiura et al. (1985) with a modification. The enzyme was incubated with 0.05 mM
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Sue-LLVY-MCA at 37°C in 50 mM Tris-HCl buffer (pH 8.0) except the case of pH optimum measurement, in a total volume of 0.4 ml. After 1 hr the reaction was terminated by the addition of 0.6 ml of 1% SDS/20 mM Tris-HCl buffer (pH 9.0). The fluorescence of liberated AMC from the peptide was measured at an excitation of 370 nm and emission of 460 nm. Purification of rabbit proteasome Minced muscle (260 g) was homogenized in a blender with 2 vol. of 40 mM Tris-HCl buffer (pH 7.2)/4 mM NaN3 and centrifuged at 7500 g for 15 min. The supernatant was subjected to ammonium sulfate fractionation. The precipitate obtained between 25 and 65% ammonium sulfate saturation was collected and dialysed against 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaNs (buffer A). The dialysate was applied to a DEAEcellulose column (2x 16 cm) equilibrated with buffer A. The enzyme was eluted with a 0.14.4 M NaCl gradient in buffer A. The active fractions eluting at 0.2 M NaCl were concentrated with a Centriflo CF25 ultrafiltration membrane cone and loaded onto a Sephacryl S-300 column (2.7x89 cm) equilibrated with buffer A. The active fractions from gel filtration were pooled, dialysed against 5 mM potassium phosphate buffer (pH 7.0) applied to a hydroxyapatite column (2x8.2 cm), and then eluted with a 5-300 mM potassium phosphate buffer gradient. The active fractions eluting at 180 mM phosphate buffer were collected, dialysed against buffer A, applied to a DEAE-Sephadex A-50 column (2x 10.2 cm) equilibrated with the same buffer. Elution was performed with a O.l0.4 M NaCl gradient in buffer A. The active fractions eluting at 0.27 M NaCl were pooled and used as the purified enzyme. Preparation of myofibrils and sarcoplasmic proteins Myofibrils were prepared as described by Yang et al. (1970). Sarcoplasmic proteins were obtained as follows. Minced muscle (5 g) was homogenized in a blender with 40 ml of 40 mM Tri-HCl buffer (pH 7.2)/0.16 M KC1/4 mM NaNs. After the homogenate was centrifuged at 2300 g for 10 min, the supernatant obtained was used as the sarcoplasmic protein fraction. Electrophoresis Polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions was performed according to the method of Davis (1964) using a 7.5% gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (1970) using a 12.5% gel. Proteasome was stained with a Kanto Kagaku (Tokyo, Japan) silver-staining kit. Myofibrillar and sarcoplasmic proteins were stained with Coomassie brilliant blue R-250. Isoelectric focusing Isoelectric focusing was performed with a Rotofor cell (Bio-Rad Laboratories, Richmond, VA, USA), free solution isoelectric focusing apparatus according to the manufacturer’s instructions. Rabbit proteasome was electrophoresed with 2% Bio-Lyte3/10 Ampholyte in the focusing chamber at 12 W constant power for 4 hr. After the electrophoresis, the content of the focusing chamber was separated into twenty fractions to determine the Sue-LLVY-MCA hydrolyzing activity and the pH.
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A. Okitani
Protein determination The absorbance at 280 nm was used to detect the protein peaks on chromatography. The A,‘z’m’ at 280 nm was assumed to be 1.O. The protein concentration of myofibril was determined by the Biuret method (Gornall el al., 1949) using bovine serum albumin as a standard. RESULTS Purification of rabbit proteasome The results of the purification process are summarized in Table 1. Proteasome was purified 91.7-fold over crude extract, and 3.7 mg of enzyme was isolated from 260 g muscle with a yield of 1.9%. The proteasome thus obtained gave a single band on PAGE [Fig. l(A)]. TABLE 1 Purification of Rabbit Proteasome Puri$cation step
Total volume (ml)
Total protein (mg)
Muscle Crude extract (NH&SCkb DEAE-cellulose Sephacryl S-300 Hydroxyapatite DEAE-Sephadex
260” 482 99 77 33.2 76.4 33.0
18436 9943 131 42.3 12.8 3.7
A-50
Total activity (nmol AMC/min)
326 217 18.0 13.2 14.4 6.1
Yield of activity (%)
Specific activity (nmol Ah4C/ min/mg)
Purity (-fold)
100 66.6 5.5 4.0 4.4 1.9
0.018 0.022 0.137 0.312 1.13 1.65
1 1.2 7.6 17.3 62.8 91.7
“grams. b25-65% saturation.
Mr
Mr (K) 29.0 27.5 25.7 24.7 23.3 22.5 21.5 20.0
dye front-
(a)
(b)
Fig. 1. Gel electrophoresis of rabbit proteasome. (A) Rabbit proteasome (9 pg) was applied to a 7.5% gel under non-denaturing conditions. (B) The molecular mass standards [lane (a)] and rabbit proteasome [0.21 Kg, lane (b)] applied to a 12.5% gel under denaturing conditions. The molecular mass standards consist of bovine serum albumin (Mr =68000), ovalbumin (45 000), chymotrypsinogen (25 000) and cytochrome c (12 500).
Proteasomefrom rabbit skeletal muscle
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Some properties of the purified proteasome Molecular mass
The molecular mass of proteasome was estimated to be 580 00&800 000 Da on Sephacryl S-300 column chromatography (Fig. 2). The molecular mass could not be determined clearly, because not all the standard proteins were on the straight line. Purified proteasome gave eight bands ranging from 20 000 to 29 000 Da on SDS-PAGE [Fig. l(B)], indicating that this enzyme comprises multiple hetero-subunits with low molecular masses. Isoelectric point
The isoelectric point of proteasome was determined to be 5.5 by isoelectric focusing on an ampholine pH gradient (Fig. 3). pH optimum
The activity of proteasome was measured in 50 mM sodium acetate-HCl, Tris-acetate and Tris-HCl buffer of various pH. As shown in Fig. 4, the optimum pH was around 8. pH stability
After incubating the enzyme without a substrate in the presence of 59 mM buffer of various pH values at 37°C for 1 hr, the residual activity was measured at pH 8.0. Sodium acetate-HCl, Tris-acetate, Tris-HCl and glycine-NaOH buffers were used. This enzyme was almost stable in the range of pH 5-9 (Fig. 5). Heat stability
When the enzyme was left without a substrate at pH 7.2 and at various temperatures for 10 min, it was stable up to 60°C but completely inactivated at 70°C or higher (Fig. 6).
2.0 c Bovine serum albumin
p 3 >
1.5-
1.0 -
;‘ I
I
5
10
Aldolase Ferritin 'a Thyroglobulin
I 20
I
Proteasome . '
I111111 40
6080100
Molecular mass (X 10m4)
Fig. 2. Determination of molecular mass of rabbit proteasome. Rabbit proteasome, bovine serum albumin (Mr = 68 000) aldolase (158 000), ferritin (450000) and thyroglobulin (669000) were
applied to the Sephacryl S-300 column (2.7x89 cm) and eluted with 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaN3. The void volume (Vo) was determined with blue dextran and then the elution volume (Ve) for each protein was measured.
456
M. Matsuishi, A. Okitani
Fraction number
Fig. 3. Isoelectric focusing of rabbit proteasome with a Rotofor cell. Rabbit proteasome (specific activity, 0.059 nmol AMC/min/ml; 40 ml) was electrophoresed with 2% Bio-Lyte3/10 Ampholyte at 12 W constant power for 4 hr. After the electrophoresis, separated fractions were analyzed for their Sue-LLVY-MCA hydrolyzing activity (0) and their pH (0).
F
2
.r
E
3 % 2t 0.050 3 '5 .r +J z Jfo.025 2 z & 3 2 L
3
fl
o-
3
-'
4
I
I
I
I
5
6
7
89
I
I 10
-I :
PH
lz Fig. 4. Effect of pH on the activity of rabbit proteasome. Rabbit proteasome (0.78 pg) was incubated with 0.05 mM Sue-LLVY-MCA in 50 mM buffer at various pH values at 37°C for 60 min. 0, sodium acetate_HCl buffer; H, Tris-acetate buffer; A, Tris-HCI buffer.
Proteasome_from rabbit skeletal muscle
451
Eflects of various compounds
As shown in Table 2, the enzyme was inhibited by DFP and chymostatin. However, EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid and pepstatin did not inhibit the enzyme. The enzyme was activated about 8-fold at 0.01% SDS. The SDS concentration for maximum activation varied in the range of O.OlLO.O2%between different preparations (data not shown). CaClz and ATP did not activate the enzyme.
3
4
5
I
I
I
I
1
6
7
8
9
10
I
1
11
12
PH
Fig. 5. pH stability of rabbit proteasome. Rabbit proteasome (42 pg) in 0.425 ml of 59 mM buffer soiution of various pH was kept at 37°C for 1 hr. The residual activity was measured with 0.05 mM Sue-LLVY-MCA at pH 8.0, and expressed as a percentage of the value for the enzyme kept at pH 7.2 and 0°C for 1 hr. 0, sodium acetate-HCl buffer; 0, Tris-acetate buffer; A, Tris-HCl buffer; n , glycine_NaOH buffer.
” 3
.z c, x .r
2 T * z!
100 80 _ 60 40 20 0
Temperature ( “C ) Fig. 6. Heat stability of rabbit proteasome.
Rabbit proteasome (105 wg/ml) in 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaN3 was kept at various temperatures for 10 min. The residual activity was measured with 0.05 mM Sue-LLVY-MCA at pH 8.0, and expressed as a percentage of the original activity. Other conditions are described under Materials and Methods.
458
M. Matsuishi, A, Okitani
Eflects of SDS on K, and V,, values of proteasome In the absence of SDS, K,,, and V,,,,, values of proteasome were 0.26 mM and 1.50 nmol AMC/min/mg, respectively. In the presence of 0.015% SDS, K, and V,,, values were 0.41 mM and 48.2 nmol AMC/min/mg. These data indicated that SDS increases markedly V,,,,, value of proteasome. Action towara3 myofibrils Myofibrils were incubated with proteasome in 50 mM Tri-acetate buffer (pH 8.0 and 5.5) containing O-0.02% SDS at 37°C for 20 hr, and their changes were investigated by SDS-PAGE. The enzyme concentrations employed for the treatment of myofibrils were comparable to the values expected for the muscle homogenate on the basis of the purification data shown in Table 1. As shown in Fig. 7, remarkable changes were observed in the presence of SDS. The most intensive degradation of proteins at pH 8.0 occurred in the presence of 0.017% SDS (lane 4). At this concentration of SDS, the intensity of the bands of actin, troponins T and I, and tropomyosin decreased, and the bands of a-actinin and myosin light chains almost disappeared. The new band, which was supposed to be the degradation product of myosin heavy chain, also appeared closely under myosin heavy chain. At pH 5.5, the intensity of the bands of cr-actinin, actin, troponins T and I decreased markedly in the presence of 0.01% SDS (lane 8). The bands of tropomyosin, troponin C and myosin light chains almost disappeared at that concentration of SDS. However, in the absence of SDS, no changes were observed at either pH (lanes 2 and 6). Action towards sarcoplasmic proteins Sarcoplasmic proteins were incubated with proteasome in 50 mM Tris-acetate buffer (pH 5.5 and 8.0) containing 0 or 0.02% SDS at 37°C for 20 hr, and their changes were investigated by SDS-PAGE. No changes were observed in the presence or absence of SDS at either pH (data not shown). TABLE 2 Effect of Various Compounds on the Activity of Rabbit Proteasome [rabbit proteasome (10.5 pg/ml) was assayed with 0.05 mM Sue-LLVY-MCA in the presence of various compounds at pH 8.0. Other conditions are described under ‘Materials and Methods’]
Compounds @al cont.) None EDTA (1 mM) CaCls (1 mM) ATP (1 mM) and MgC12 (10 mM) Leupeptin (0.1 mM) E-64 (0.1 mM) Be&tin (0.1 mM) DFP (25 mM) Monoiodoacetic acid (5 mM) Chymostatin (0.1 mM) Pepstatin (0.1 mM) SDS (0.005%) (0.01%) (0.02%) (0.04%)
Relative activity (%) 100
130 115 99 101 145 135 6.4 114 50.4 129 113 834 8.5 0.2
459
Proteasome from rabbit skeletal muscle
DISCUSSION Rabbit muscle proteasome was purified about 90-fold over the crude extract by the purification method used in this study. The purified enzyme showed a single protein band on PAGE. Driscoll and Goldberg (1989) reported that four successive chromatographies in the presence of glycerol brought about 130-fold increase in specific activity of rabbit skeletal muscle proteasome and the purified enzyme showed a single protein band on PAGE; our enzyme was, therefore, considered to be as pure as theirs. Although their enzyme was activated by ATP for a few days after purification, our enzyme was not activated. It is assumed that 26s proteasome is easily dissociated by ammonium sulfate fractionation into 20s proteasome and regulatory subunits relating to ATP dependence, and that glycerol and ATP suppress this dissociation (Kanayama et al., 1992). Since Driscoll and Goldberg (1989) used glycerol, and did not perform ammonium sulfate fractionation during purification, regulatory subunits could partly remain in their purified enzyme, resulting in the retention of ATP-dependence of the enzyme activity. On the other hand, since we did not use glycerol and performed ammonium sulfate fractionation, the regulatory subunits were probably lost completely during purification, resulting in disappearance of ATP-dependence. This inference is supported by the fact that they could detect not only 22 000-34 000 Da subunits for 20s proteasome but also larger subunits in their enzyme preparations, while our purified enzyme showed only 20 00&29 000 Da subunits. The molecular mass of the purified enzyme in the present study resembles those (600 000-760 000 Da) previously reported for 20s proteasome from skeletal muscles and other tissues (Driscoll & Goldberg, 1989; Dahlmann et al., 1985; Koohmaraie, 1992; Yamamoto et al., 1986; Tanaka et al., 1986). Our enzyme showed a similar isoelectric point to that (5.1-5.2) reported for rat muscle proteasome (Dahlmann et al., 1985).
a-Actinin
-
Actin
-
Lane No.
:1
SDS cont. (%j: PH
Fig. 7. SDS-polyacrylamide
:
2 0
-a.o---J
0
3
4
5
6
7
8
9
10
0.017 0 0.010 0.017 0.007 0.013 0.013 0.020 ‘----4.5-
gel electrophoresis of myofibril treated with rabbit proteasome. Myofibril (2 mg/ml) was incubated with rabbit proteasome (426 pg/ml; specific activity, 0.704 nmol AMC/min/ml) at the pH and SDS concentration indicated below each lane and 37°C for 20 hr in 50 mM Tri-acetate buffer/5 mM NaNs. Lane 1 was without added rabbit proteasome and SDS. The incubated myofibril (13 pg) was applied to a 10% polyacrylamide gel. Bands have been tentatively identified based on published molecular masses.
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Optimum pH of the enzyme from rabbit muscle was similar to those (pH 7-10) of the 20s enzymes from porcine (Ishiura et al., 1985), rat (Dahlmann er ai., 1985) and ovine (Koohmaraie, 1992) muscle. Rabbit muscle proteasome was stable at pH 5-9, which is demonstrated for the first time in the present study. It indicates that this enzyme possibly keeps its activity at the ultimate pH, 5.5-5.7, of post-mortem muscles. Rabbit muscle proteasome was stable up to 60°C at pH 7.2 when it was pre-incubated in the absence of the substrate for 10 min. On the other hand, bovine lens proteasome was reported to lose 60% activity when it was pre-incubated at 53°C for 10 min (Wagner & Margolis, 1993). Furthermore, the proteasomes from ovine (Koohmaraie, 1992) and lobster (Mykles, 1989) muscles were reported to be activated from 1l- to 18-fold on heating at 60°C for l-2 min and almost completely inactivated at 60°C for 10 min. The higher thermostability of the rabbit enzyme than those of enzymes from other species might be ascribed to the species specificity. It is also probable that this discrepancy was caused by the difference in substrates, because we used a peptide substrate (Sue-LLVY-MCA), although other investigators used protein substrates (oz-crystallin or [‘4C]methylcasein). Further study is needed to elucidate the reason. In comparison with other proteinases from rabbit skeletal muscles, proteasome is more stable than cathepsin B (Okitani et al., 1988) and L (Okitani et al., 1980), and is as stable as calpain in the absence of Ca*+ (Inomata et al., 1984). The proteasome from rabbit skeletal muscle was not inhibited by cysteine proteinase inhibitors (E-64 and monoiodoacetic acid), a metallo proteinase inhibitor (EDTA) or a carboxyl proteinase inhibitor (pepstatin). DFP and chymostatin inhibited this enzyme, indicating that it is likely to be a serine proteinase. Also, this enzyme was activated by 0.01-0.02% SDS. These properties are almost the same as those of 20s proteasomes from porcine (Ishiura et al., 1985) ovine (Koohmaraie, 1992) rat (Dahlmann et al., 1985) and lobster (Mykles, 1989) muscles. SDS increased K,,, and V,,,,, of the rabbit muscle proteasome, but it is reported that SDS decreased K,,, and increased V,,, of the rat muscle proteasome (Dahlmann et al., 1993). These discrepancies may be caused by the difference in substrates (Suc-LLVYMCA for the rabbit enzyme and Sue-Ala-Ala-Phe-MCA for the rat enzyme). Rabbit muscle proteasome degraded some myofibrillar proteins in the presence of SDS. The optimum concentrations of SDS and the mode of degradation of proteins differed by pH, suggesting that the conformational change for activation induced by SDS was affected by pH. At pH 8, the activity towards myofibrillar proteins was stimulated in the presence of 0.017-0.02% SDS (Fig. 7), although the activity towards the peptide substrate was stimulated in the presence of 0.01% SDS and reduced in the presence of 0.02% SDS (Table 2). These discrepancies may be caused by the difference in substrates. These may also be explained by the difference in the proteasome concentrations (10.5 pg/ml towards the peptide substrate and 426 pg/ml towards myofibrillar proteins), because the optimally activating concentration of SDS was reported to depend on the proteasome concentration (Dahlmann et al., 1993). Lobster muscle proteasome was reported to digest myosin heavy chain to produce 9000&170 000 Da fragments in the presence of 0.03% SDS, but not to degrade a-actinin, tropomyosin and troponins (Mykles & Haire, 1991). Ovine muscle proteasome was reported to degrade only troponin C and myosin light chains-2 and -3 in the presence of 0.25 mM (= 0.007%) SDS (Koohmaraie, 1992). These discrepancies in the findings between present and previous reports were thought to be due to the species specificity of ‘this enzyme and/or differences in the concentrations of SDS required to stimulate this enzyme. On the other hand, rabbit proteasome did not degrade myofibrillar proteins in the absence of SDS. Similar results were reported for lobster muscle proteasome (Mykles & Haire, 1991). However, Taylor et al. (1995) reported that the 20s proteasome of calf liver
Proteasome.from rabbit skeletal muscle
461
degraded myofibrillar proteins in the absence of SDS. These discrepancies may be attributed to the tissue specificity of the proteasome, because we previously indicated that the mode of the degradation of myofibrillar proteins was different between muscle and liver cathepsin B (Matsuishi et al., 1992). Also, another possibility is that the calf liver proteasome might be the activated form even in the absence of SDS or contaminated by the unknown activating factors like PA28 described later, because Taylor et al. (1995) purified the proteasome by a different method from ours. None of sarcoplasmic proteins were degraded regardless of the presence of SDS. These results suggest that sarcoplasmic proteins may be resistant to the proteasome and/or sarcoplasmic proteins may include endogenous inhibitors of the proteasome. Inhibitor proteins of 20s proteasome were reported to exist in human (Murakami & Etlinger, 1986) and bovine (Chu-Ping et al., 1992b) blood cells. It is possible that rabbit muscle cells also contain endogenous inhibitors, but this was not investigated further. It seems unlikely that 26s proteasome, which is thought to degrade proteins ATPdependently, acts on muscle proteins in the post-mortem muscle after disappearance of ATP. On the other hand, the 20s proteasome isolated in this study, lacking ATPdependence, possibly degrades muscle proteins. However, taken together with the present and previous reports (Mykles & Haire, 1991; Koohmaraie, 1992), proteasome probably needs some endogenous stimulating factors such as SDS to degrade proteins in the postmortem muscles. Although PA28 (the proteinaceous activator) which was found in bovine red blood cells and hearts (Chu-Ping et al., 1992a) is considered to be the candidate for such proteasome activating factors, it is unknown whether those also exist in skeletal muscles and could enhance the degradation of muscle proteins by the proteasome. In order to clarify the contribution of proteasome to proteolysis during meat conditioning, these problems should be solved.
ACKNOWLEDGEMENTS The authors are grateful to Dr Hiromichi Kato, Emeritus Professor of the University of Tokyo for his encouragement. The authors also wish to thank Mr Hiromichi Ohgoshi and Mr Yoshimasa Harimoto for their excellent technical assistance.
REFERENCES Chu-Ping, M., Slaughter, C. A. & DeMartino, G. N. (1992a) J. Biol. Chem. 267, 10515. Chu-Ping, M., Slaughter, C. A. & DeMartino, G. N. (1992b) Biochim. Biophys. Acta 1119, 303. Dahlmann, B., Becher, B., Sobek, A., Ehlers, C., Kopp, F. & Kuehn, L. (1993) Enzyme Protein 47, 214. Dahlmann, B., Kuehn, L., Rutschmann, M. & Reinauer, H. (1985) Biochem. J. 228, 161. Davis, B. J. (1964) Ann. NY Acad. Sci. 121, 404. Driscoll, J. & Goldberg, A. L. (1989) Proc. Natl. Acad. Sci. USA 86, 787. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) J. Biol. Chem. 177, 751. Inomata, M., Nomoto, M., Hayashi, M., Nakamura, M., Imahori, K. & Kawashima, S. (1984) J. Biochem. 95, 1661. Ishiura, S., Sano, M., Kamakura, K. & Sugita, H. (1985) FEBS Letts 189, 119. Kanayama, H., Tamura, T., Ugai, S., Kagawa, S., Tanahashi, N., Yoshimura, T., Tanaka, K. & Ichihara, A. (1992) Eur. J. Biochem. 206, 567. Koohmaraie, M. (1992) J. Animal Sci. 70, 3697. Laemmli, U. K. (1970) Nature 227, 680. Matsuishi, M., Matsumoto, T., Okitani, A. & Kato, H. (1992) ht. J. Biochem. 24, 1967.
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Murakami, K. & Etlinger, J. D. (1986) Proc. Natl. Acad. Sci. USA 83, 758%. Mykles, D. (1989) Arch. Biochem. Biophys. 274, 216. Mykles, D. & Haire, M. F. (1991) Arch. Biochem. Biophys. 288, 543. Okitani, A., Matsuishi, M., Matsumoto, T., Kamoshida, E., Sato, M., Matsukura, U., Watanabe, M., Kato, H. & Fujimaki, M. (1988) Eur. J. Biochem. 171, 377. Okitani, A., Matsukura, U., Kato, H. & Fujimaki, M. (1980) J. Biochem. 87, 1133. Orlowski, M. (1990) Biochemistry 29, 10291. Peters, J.-M., Cejka, Z., Harris, J. R., Kleinschmidt, J. A. & Baumeister, W. (1993) J. Molec. Biol. 234,932.
Rechsteiner, M., Hoffman, L. & Dubiel, W. (1993) J. Biol. Chem. 268, 6065. Rivet& A. J. (1989) Biochem. J. 263, 625. Tanahashi, N., Tsurumi, C., Tamura, T. & Tanaka, K. (1993) Enzyme Protein 47, 241. Tanaka, K., Ii, K., Ichihara, A., Waxman, L. & Goldberg, A. L. (1986) J. Biol. Chem. 261, 15197. Taylor, R. G., Tassy, C., Briand, M., Robert, N., Briand, Y. & Ouali, A. (1995) Mot. Biof. Rep. 21, 71.
Ugai, S., Tamura, T., Tanahashi, N., Takagi, S., Komi, N., Chung, C. H., Tanaka, K. & Ichihara, A. (1993) J. Biochem. 113, 7.54. Wagner, B. J. & Margolis, J. W. (1993) Arch. Biochem. Biophys. 307, 146. Yamamoto, T., Nojima, M., Ishiura, S. & Sugita, H. (1986) Biochim. Biophys. Acta 882, 297. Yang, R., Okitani, A. & Fujimaki, M. (1970) Agric. Biol. Chem. 34, 1765. Yoshimura, T., Kanayama, K., Takagi, T., Ikai, A., Tokunaga, F., Koide, T., Tanahashi, N., Tamura, T., Cejka, Z., Baumeister, W., Tanaka, K. & Ichihara, A. (1993) J. Struct. Biol. 111, 200.
SO309-1740(96)00126-X
Meat Science, Vol. 45, No. 4, 451462, 1997 (%, 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0309-1740/97 s17.oo+o.oo
ELSEVIER
Proteasome from Rabbit Skeletal Muscle: Some Properties and Effects on Muscle Proteins M. Matsuishi & A. Okitani Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 7-l Kyonan-cho, I-chome, Musashino-shi, Tokyo 180, Japan (Received 17 August 1996; accepted 20 October 1996)
ABSTRACT Rabbit proteasome, likely to be a 20s proteasome, was purified and its properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purtfied enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20 000-29 000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580000-800000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pHfor hydrolysis of succinylLeu-Leu-Val-Tyr-(I-methylcoumaryl-?-amide) (Sue-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH S-9 and up to 60°C at pH 7.2. The enzyme activity was inhibited by diisopropyl jluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaClz. Remarkably, SDS increased the V,,, value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, a-actinin, actin. tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS. 0 1997 Elsevier Science Ltd. All rights reserved
INTRODUCTION Proteolysis during meat conditioning is considered to contribute to the tenderization of meat and the improvement of its flavor. Intracellular proteases, e.g. cathepsins, calpains and various aminopeptidases, are assumed to participate in proteolysis during conditioning of meat. Proteasomes were recently discovered as the proteinases that play major roles in ATP-dependent proteolysis in living eukaryotic cells. However, the contribution of these proteinases to proteolysis during meat conditioning has yet to be clarified. Proteasomes have been classified into two isoforms with apparent sedimentation coefficients of 20s and 26S, respectively (Tanahashi et al., 1993). The 20s proteasome has been purified from various muscles (Dahlmann et al., 1985; Mykles, 1989; Koohmaraie, 1992) and other tissues (Rivett, 1989; Orlowski, 1990; 451
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Rechsteiner et al., 1993), and is well characterized. This enzyme, which has a molecular mass of 600000-760 000 Da, consists of multiple subunits with molecular masses of 21 OO@-32000 Da. Interestingly, it is activated by low concentrations of SDS and various fatty acids, but not by ATP. The 26s proteasome has been purified from rat liver (Yoshimura et al., 1993; Ugai et al., 1993), human kidney (Kanayama et al., 1992) and Xenopus oocytes (Peters et al., 1993), but not from muscles. This enzyme, which possesses a molecular mass of about 2 000 000 Da, is an assembly of a 20s proteasome and multiple components with molecular masses of 30 00&l 10 000 Da. The 20s proteasome is considered to be the core unit of proteinase activity of the 26s enzyme, and other components are assumed to be the subunits which make the proteinase activity of the 26s enzyme ATP-dependent. It is also shown that ATP is needed to keep the 26s complex and the depletion of ATP causes rapid dissociation of the 26s complex into the 20s enzyme and multiple components (Kanayama et al., 1992). Driscoll and Goldberg (1989) isolated the proteasome from rabbit muscles rapidly (probably within a few days post mortem) in the presence of glycerol, which is known to stabilize 26s proteasome complex. Although their enzyme was likely to be a 20s proteasome judging from its molecular mass, it could be activated by ATP for a few days after purification. However, in order to elucidate the role of proteasomes in conditioning of meat, in which ATP disappears, it is necessary to investigate the properties of the 20s enzyme which does not show ATP-dependence rather than the 26s enzyme and ATPactivatable 20s enzym. Moreover, studies of the rabbit muscle enzyme lack such general information as optimum pH, heat stability and action towards muscle proteins (except ATP-dependency). Thus, in this study we isolated non-ATP-sensitive proteasome from rabbit skeletal muscle in the absence of glycerol and investigated its enzymatic properties and effects on myofibrillar and sarcoplasmic proteins.
MATERIALS
AND METHODS
Materials
Rabbit skeletal muscle (longissmus dorsi) was obtained from the carcasses immediately after slaughter and, after removal of fat and connective tissue, it was minced with a meat chopper. DEAE-cellulose (DE-52) was purchased from Whatman (Maidstone, UK). DEAESephadex A-50 and Sephacryl S-300 were obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Hydroxyapatite was from Seikagaku Kogyo Co (Tokyo, Japan). Ferritin, aldolase, bovine serum albumin, ovalbumin, chymotrypsinogen and cytochrome c, were purchased from Boehringer Mannheim GmbH (Germany). Thyroglobulin was from Sigma Chemical Co (St. Louis, MO, USA). Sue-LLVY-MCA, 7-amino-4-methylcoumarin (AMC), leupeptin, E-64, bestatin, chymostatin and pepstatin were purchased from the Peptide Institute (Osaka, Japan). Bio-Lyte 3/10 Ampholyte was purchased from Bio-Rad Laboratories (Richmond, VA, USA). Centriflo CF25 ultrafiltration membrane cones were obtained from Amicon Corp (Danvers, MA, USA). All other chemicals were of analytical grade. Assay for proteasome
Proteasome activity was measured with Sue-LLVY-MCA according to the method of Ishiura et al. (1985) with a modification. The enzyme was incubated with 0.05 mM
Proteasome from rabbit skeletal muscle
453
Sue-LLVY-MCA at 37°C in 50 mM Tris-HCl buffer (pH 8.0) except the case of pH optimum measurement, in a total volume of 0.4 ml. After 1 hr the reaction was terminated by the addition of 0.6 ml of 1% SDS/20 mM Tris-HCl buffer (pH 9.0). The fluorescence of liberated AMC from the peptide was measured at an excitation of 370 nm and emission of 460 nm. Purification of rabbit proteasome Minced muscle (260 g) was homogenized in a blender with 2 vol. of 40 mM Tris-HCl buffer (pH 7.2)/4 mM NaN3 and centrifuged at 7500 g for 15 min. The supernatant was subjected to ammonium sulfate fractionation. The precipitate obtained between 25 and 65% ammonium sulfate saturation was collected and dialysed against 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaNs (buffer A). The dialysate was applied to a DEAEcellulose column (2x 16 cm) equilibrated with buffer A. The enzyme was eluted with a 0.14.4 M NaCl gradient in buffer A. The active fractions eluting at 0.2 M NaCl were concentrated with a Centriflo CF25 ultrafiltration membrane cone and loaded onto a Sephacryl S-300 column (2.7x89 cm) equilibrated with buffer A. The active fractions from gel filtration were pooled, dialysed against 5 mM potassium phosphate buffer (pH 7.0) applied to a hydroxyapatite column (2x8.2 cm), and then eluted with a 5-300 mM potassium phosphate buffer gradient. The active fractions eluting at 180 mM phosphate buffer were collected, dialysed against buffer A, applied to a DEAE-Sephadex A-50 column (2x 10.2 cm) equilibrated with the same buffer. Elution was performed with a O.l0.4 M NaCl gradient in buffer A. The active fractions eluting at 0.27 M NaCl were pooled and used as the purified enzyme. Preparation of myofibrils and sarcoplasmic proteins Myofibrils were prepared as described by Yang et al. (1970). Sarcoplasmic proteins were obtained as follows. Minced muscle (5 g) was homogenized in a blender with 40 ml of 40 mM Tri-HCl buffer (pH 7.2)/0.16 M KC1/4 mM NaNs. After the homogenate was centrifuged at 2300 g for 10 min, the supernatant obtained was used as the sarcoplasmic protein fraction. Electrophoresis Polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions was performed according to the method of Davis (1964) using a 7.5% gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by the method of Laemmli (1970) using a 12.5% gel. Proteasome was stained with a Kanto Kagaku (Tokyo, Japan) silver-staining kit. Myofibrillar and sarcoplasmic proteins were stained with Coomassie brilliant blue R-250. Isoelectric focusing Isoelectric focusing was performed with a Rotofor cell (Bio-Rad Laboratories, Richmond, VA, USA), free solution isoelectric focusing apparatus according to the manufacturer’s instructions. Rabbit proteasome was electrophoresed with 2% Bio-Lyte3/10 Ampholyte in the focusing chamber at 12 W constant power for 4 hr. After the electrophoresis, the content of the focusing chamber was separated into twenty fractions to determine the Sue-LLVY-MCA hydrolyzing activity and the pH.
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A. Okitani
Protein determination The absorbance at 280 nm was used to detect the protein peaks on chromatography. The A,‘z’m’ at 280 nm was assumed to be 1.O. The protein concentration of myofibril was determined by the Biuret method (Gornall el al., 1949) using bovine serum albumin as a standard. RESULTS Purification of rabbit proteasome The results of the purification process are summarized in Table 1. Proteasome was purified 91.7-fold over crude extract, and 3.7 mg of enzyme was isolated from 260 g muscle with a yield of 1.9%. The proteasome thus obtained gave a single band on PAGE [Fig. l(A)]. TABLE 1 Purification of Rabbit Proteasome Puri$cation step
Total volume (ml)
Total protein (mg)
Muscle Crude extract (NH&SCkb DEAE-cellulose Sephacryl S-300 Hydroxyapatite DEAE-Sephadex
260” 482 99 77 33.2 76.4 33.0
18436 9943 131 42.3 12.8 3.7
A-50
Total activity (nmol AMC/min)
326 217 18.0 13.2 14.4 6.1
Yield of activity (%)
Specific activity (nmol Ah4C/ min/mg)
Purity (-fold)
100 66.6 5.5 4.0 4.4 1.9
0.018 0.022 0.137 0.312 1.13 1.65
1 1.2 7.6 17.3 62.8 91.7
“grams. b25-65% saturation.
Mr
Mr (K) 29.0 27.5 25.7 24.7 23.3 22.5 21.5 20.0
dye front-
(a)
(b)
Fig. 1. Gel electrophoresis of rabbit proteasome. (A) Rabbit proteasome (9 pg) was applied to a 7.5% gel under non-denaturing conditions. (B) The molecular mass standards [lane (a)] and rabbit proteasome [0.21 Kg, lane (b)] applied to a 12.5% gel under denaturing conditions. The molecular mass standards consist of bovine serum albumin (Mr =68000), ovalbumin (45 000), chymotrypsinogen (25 000) and cytochrome c (12 500).
Proteasomefrom rabbit skeletal muscle
455
Some properties of the purified proteasome Molecular mass
The molecular mass of proteasome was estimated to be 580 00&800 000 Da on Sephacryl S-300 column chromatography (Fig. 2). The molecular mass could not be determined clearly, because not all the standard proteins were on the straight line. Purified proteasome gave eight bands ranging from 20 000 to 29 000 Da on SDS-PAGE [Fig. l(B)], indicating that this enzyme comprises multiple hetero-subunits with low molecular masses. Isoelectric point
The isoelectric point of proteasome was determined to be 5.5 by isoelectric focusing on an ampholine pH gradient (Fig. 3). pH optimum
The activity of proteasome was measured in 50 mM sodium acetate-HCl, Tris-acetate and Tris-HCl buffer of various pH. As shown in Fig. 4, the optimum pH was around 8. pH stability
After incubating the enzyme without a substrate in the presence of 59 mM buffer of various pH values at 37°C for 1 hr, the residual activity was measured at pH 8.0. Sodium acetate-HCl, Tris-acetate, Tris-HCl and glycine-NaOH buffers were used. This enzyme was almost stable in the range of pH 5-9 (Fig. 5). Heat stability
When the enzyme was left without a substrate at pH 7.2 and at various temperatures for 10 min, it was stable up to 60°C but completely inactivated at 70°C or higher (Fig. 6).
2.0 c Bovine serum albumin
p 3 >
1.5-
1.0 -
;‘ I
I
5
10
Aldolase Ferritin 'a Thyroglobulin
I 20
I
Proteasome . '
I111111 40
6080100
Molecular mass (X 10m4)
Fig. 2. Determination of molecular mass of rabbit proteasome. Rabbit proteasome, bovine serum albumin (Mr = 68 000) aldolase (158 000), ferritin (450000) and thyroglobulin (669000) were
applied to the Sephacryl S-300 column (2.7x89 cm) and eluted with 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaN3. The void volume (Vo) was determined with blue dextran and then the elution volume (Ve) for each protein was measured.
456
M. Matsuishi, A. Okitani
Fraction number
Fig. 3. Isoelectric focusing of rabbit proteasome with a Rotofor cell. Rabbit proteasome (specific activity, 0.059 nmol AMC/min/ml; 40 ml) was electrophoresed with 2% Bio-Lyte3/10 Ampholyte at 12 W constant power for 4 hr. After the electrophoresis, separated fractions were analyzed for their Sue-LLVY-MCA hydrolyzing activity (0) and their pH (0).
F
2
.r
E
3 % 2t 0.050 3 '5 .r +J z Jfo.025 2 z & 3 2 L
3
fl
o-
3
-'
4
I
I
I
I
5
6
7
89
I
I 10
-I :
PH
lz Fig. 4. Effect of pH on the activity of rabbit proteasome. Rabbit proteasome (0.78 pg) was incubated with 0.05 mM Sue-LLVY-MCA in 50 mM buffer at various pH values at 37°C for 60 min. 0, sodium acetate_HCl buffer; H, Tris-acetate buffer; A, Tris-HCI buffer.
Proteasome_from rabbit skeletal muscle
451
Eflects of various compounds
As shown in Table 2, the enzyme was inhibited by DFP and chymostatin. However, EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid and pepstatin did not inhibit the enzyme. The enzyme was activated about 8-fold at 0.01% SDS. The SDS concentration for maximum activation varied in the range of O.OlLO.O2%between different preparations (data not shown). CaClz and ATP did not activate the enzyme.
3
4
5
I
I
I
I
1
6
7
8
9
10
I
1
11
12
PH
Fig. 5. pH stability of rabbit proteasome. Rabbit proteasome (42 pg) in 0.425 ml of 59 mM buffer soiution of various pH was kept at 37°C for 1 hr. The residual activity was measured with 0.05 mM Sue-LLVY-MCA at pH 8.0, and expressed as a percentage of the value for the enzyme kept at pH 7.2 and 0°C for 1 hr. 0, sodium acetate-HCl buffer; 0, Tris-acetate buffer; A, Tris-HCl buffer; n , glycine_NaOH buffer.
” 3
.z c, x .r
2 T * z!
100 80 _ 60 40 20 0
Temperature ( “C ) Fig. 6. Heat stability of rabbit proteasome.
Rabbit proteasome (105 wg/ml) in 10 mM Tris-HCl buffer (pH 7.2)/0.1 M NaCl/l mM NaN3 was kept at various temperatures for 10 min. The residual activity was measured with 0.05 mM Sue-LLVY-MCA at pH 8.0, and expressed as a percentage of the original activity. Other conditions are described under Materials and Methods.
458
M. Matsuishi, A, Okitani
Eflects of SDS on K, and V,, values of proteasome In the absence of SDS, K,,, and V,,,,, values of proteasome were 0.26 mM and 1.50 nmol AMC/min/mg, respectively. In the presence of 0.015% SDS, K, and V,,, values were 0.41 mM and 48.2 nmol AMC/min/mg. These data indicated that SDS increases markedly V,,,,, value of proteasome. Action towara3 myofibrils Myofibrils were incubated with proteasome in 50 mM Tri-acetate buffer (pH 8.0 and 5.5) containing O-0.02% SDS at 37°C for 20 hr, and their changes were investigated by SDS-PAGE. The enzyme concentrations employed for the treatment of myofibrils were comparable to the values expected for the muscle homogenate on the basis of the purification data shown in Table 1. As shown in Fig. 7, remarkable changes were observed in the presence of SDS. The most intensive degradation of proteins at pH 8.0 occurred in the presence of 0.017% SDS (lane 4). At this concentration of SDS, the intensity of the bands of actin, troponins T and I, and tropomyosin decreased, and the bands of a-actinin and myosin light chains almost disappeared. The new band, which was supposed to be the degradation product of myosin heavy chain, also appeared closely under myosin heavy chain. At pH 5.5, the intensity of the bands of cr-actinin, actin, troponins T and I decreased markedly in the presence of 0.01% SDS (lane 8). The bands of tropomyosin, troponin C and myosin light chains almost disappeared at that concentration of SDS. However, in the absence of SDS, no changes were observed at either pH (lanes 2 and 6). Action towards sarcoplasmic proteins Sarcoplasmic proteins were incubated with proteasome in 50 mM Tris-acetate buffer (pH 5.5 and 8.0) containing 0 or 0.02% SDS at 37°C for 20 hr, and their changes were investigated by SDS-PAGE. No changes were observed in the presence or absence of SDS at either pH (data not shown). TABLE 2 Effect of Various Compounds on the Activity of Rabbit Proteasome [rabbit proteasome (10.5 pg/ml) was assayed with 0.05 mM Sue-LLVY-MCA in the presence of various compounds at pH 8.0. Other conditions are described under ‘Materials and Methods’]
Compounds @al cont.) None EDTA (1 mM) CaCls (1 mM) ATP (1 mM) and MgC12 (10 mM) Leupeptin (0.1 mM) E-64 (0.1 mM) Be&tin (0.1 mM) DFP (25 mM) Monoiodoacetic acid (5 mM) Chymostatin (0.1 mM) Pepstatin (0.1 mM) SDS (0.005%) (0.01%) (0.02%) (0.04%)
Relative activity (%) 100
130 115 99 101 145 135 6.4 114 50.4 129 113 834 8.5 0.2
459
Proteasome from rabbit skeletal muscle
DISCUSSION Rabbit muscle proteasome was purified about 90-fold over the crude extract by the purification method used in this study. The purified enzyme showed a single protein band on PAGE. Driscoll and Goldberg (1989) reported that four successive chromatographies in the presence of glycerol brought about 130-fold increase in specific activity of rabbit skeletal muscle proteasome and the purified enzyme showed a single protein band on PAGE; our enzyme was, therefore, considered to be as pure as theirs. Although their enzyme was activated by ATP for a few days after purification, our enzyme was not activated. It is assumed that 26s proteasome is easily dissociated by ammonium sulfate fractionation into 20s proteasome and regulatory subunits relating to ATP dependence, and that glycerol and ATP suppress this dissociation (Kanayama et al., 1992). Since Driscoll and Goldberg (1989) used glycerol, and did not perform ammonium sulfate fractionation during purification, regulatory subunits could partly remain in their purified enzyme, resulting in the retention of ATP-dependence of the enzyme activity. On the other hand, since we did not use glycerol and performed ammonium sulfate fractionation, the regulatory subunits were probably lost completely during purification, resulting in disappearance of ATP-dependence. This inference is supported by the fact that they could detect not only 22 000-34 000 Da subunits for 20s proteasome but also larger subunits in their enzyme preparations, while our purified enzyme showed only 20 00&29 000 Da subunits. The molecular mass of the purified enzyme in the present study resembles those (600 000-760 000 Da) previously reported for 20s proteasome from skeletal muscles and other tissues (Driscoll & Goldberg, 1989; Dahlmann et al., 1985; Koohmaraie, 1992; Yamamoto et al., 1986; Tanaka et al., 1986). Our enzyme showed a similar isoelectric point to that (5.1-5.2) reported for rat muscle proteasome (Dahlmann et al., 1985).
a-Actinin
-
Actin
-
Lane No.
:1
SDS cont. (%j: PH
Fig. 7. SDS-polyacrylamide
:
2 0
-a.o---J
0
3
4
5
6
7
8
9
10
0.017 0 0.010 0.017 0.007 0.013 0.013 0.020 ‘----4.5-
gel electrophoresis of myofibril treated with rabbit proteasome. Myofibril (2 mg/ml) was incubated with rabbit proteasome (426 pg/ml; specific activity, 0.704 nmol AMC/min/ml) at the pH and SDS concentration indicated below each lane and 37°C for 20 hr in 50 mM Tri-acetate buffer/5 mM NaNs. Lane 1 was without added rabbit proteasome and SDS. The incubated myofibril (13 pg) was applied to a 10% polyacrylamide gel. Bands have been tentatively identified based on published molecular masses.
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Optimum pH of the enzyme from rabbit muscle was similar to those (pH 7-10) of the 20s enzymes from porcine (Ishiura et al., 1985), rat (Dahlmann er ai., 1985) and ovine (Koohmaraie, 1992) muscle. Rabbit muscle proteasome was stable at pH 5-9, which is demonstrated for the first time in the present study. It indicates that this enzyme possibly keeps its activity at the ultimate pH, 5.5-5.7, of post-mortem muscles. Rabbit muscle proteasome was stable up to 60°C at pH 7.2 when it was pre-incubated in the absence of the substrate for 10 min. On the other hand, bovine lens proteasome was reported to lose 60% activity when it was pre-incubated at 53°C for 10 min (Wagner & Margolis, 1993). Furthermore, the proteasomes from ovine (Koohmaraie, 1992) and lobster (Mykles, 1989) muscles were reported to be activated from 1l- to 18-fold on heating at 60°C for l-2 min and almost completely inactivated at 60°C for 10 min. The higher thermostability of the rabbit enzyme than those of enzymes from other species might be ascribed to the species specificity. It is also probable that this discrepancy was caused by the difference in substrates, because we used a peptide substrate (Sue-LLVY-MCA), although other investigators used protein substrates (oz-crystallin or [‘4C]methylcasein). Further study is needed to elucidate the reason. In comparison with other proteinases from rabbit skeletal muscles, proteasome is more stable than cathepsin B (Okitani et al., 1988) and L (Okitani et al., 1980), and is as stable as calpain in the absence of Ca*+ (Inomata et al., 1984). The proteasome from rabbit skeletal muscle was not inhibited by cysteine proteinase inhibitors (E-64 and monoiodoacetic acid), a metallo proteinase inhibitor (EDTA) or a carboxyl proteinase inhibitor (pepstatin). DFP and chymostatin inhibited this enzyme, indicating that it is likely to be a serine proteinase. Also, this enzyme was activated by 0.01-0.02% SDS. These properties are almost the same as those of 20s proteasomes from porcine (Ishiura et al., 1985) ovine (Koohmaraie, 1992) rat (Dahlmann et al., 1985) and lobster (Mykles, 1989) muscles. SDS increased K,,, and V,,,,, of the rabbit muscle proteasome, but it is reported that SDS decreased K,,, and increased V,,, of the rat muscle proteasome (Dahlmann et al., 1993). These discrepancies may be caused by the difference in substrates (Suc-LLVYMCA for the rabbit enzyme and Sue-Ala-Ala-Phe-MCA for the rat enzyme). Rabbit muscle proteasome degraded some myofibrillar proteins in the presence of SDS. The optimum concentrations of SDS and the mode of degradation of proteins differed by pH, suggesting that the conformational change for activation induced by SDS was affected by pH. At pH 8, the activity towards myofibrillar proteins was stimulated in the presence of 0.017-0.02% SDS (Fig. 7), although the activity towards the peptide substrate was stimulated in the presence of 0.01% SDS and reduced in the presence of 0.02% SDS (Table 2). These discrepancies may be caused by the difference in substrates. These may also be explained by the difference in the proteasome concentrations (10.5 pg/ml towards the peptide substrate and 426 pg/ml towards myofibrillar proteins), because the optimally activating concentration of SDS was reported to depend on the proteasome concentration (Dahlmann et al., 1993). Lobster muscle proteasome was reported to digest myosin heavy chain to produce 9000&170 000 Da fragments in the presence of 0.03% SDS, but not to degrade a-actinin, tropomyosin and troponins (Mykles & Haire, 1991). Ovine muscle proteasome was reported to degrade only troponin C and myosin light chains-2 and -3 in the presence of 0.25 mM (= 0.007%) SDS (Koohmaraie, 1992). These discrepancies in the findings between present and previous reports were thought to be due to the species specificity of ‘this enzyme and/or differences in the concentrations of SDS required to stimulate this enzyme. On the other hand, rabbit proteasome did not degrade myofibrillar proteins in the absence of SDS. Similar results were reported for lobster muscle proteasome (Mykles & Haire, 1991). However, Taylor et al. (1995) reported that the 20s proteasome of calf liver
Proteasome.from rabbit skeletal muscle
461
degraded myofibrillar proteins in the absence of SDS. These discrepancies may be attributed to the tissue specificity of the proteasome, because we previously indicated that the mode of the degradation of myofibrillar proteins was different between muscle and liver cathepsin B (Matsuishi et al., 1992). Also, another possibility is that the calf liver proteasome might be the activated form even in the absence of SDS or contaminated by the unknown activating factors like PA28 described later, because Taylor et al. (1995) purified the proteasome by a different method from ours. None of sarcoplasmic proteins were degraded regardless of the presence of SDS. These results suggest that sarcoplasmic proteins may be resistant to the proteasome and/or sarcoplasmic proteins may include endogenous inhibitors of the proteasome. Inhibitor proteins of 20s proteasome were reported to exist in human (Murakami & Etlinger, 1986) and bovine (Chu-Ping et al., 1992b) blood cells. It is possible that rabbit muscle cells also contain endogenous inhibitors, but this was not investigated further. It seems unlikely that 26s proteasome, which is thought to degrade proteins ATPdependently, acts on muscle proteins in the post-mortem muscle after disappearance of ATP. On the other hand, the 20s proteasome isolated in this study, lacking ATPdependence, possibly degrades muscle proteins. However, taken together with the present and previous reports (Mykles & Haire, 1991; Koohmaraie, 1992), proteasome probably needs some endogenous stimulating factors such as SDS to degrade proteins in the postmortem muscles. Although PA28 (the proteinaceous activator) which was found in bovine red blood cells and hearts (Chu-Ping et al., 1992a) is considered to be the candidate for such proteasome activating factors, it is unknown whether those also exist in skeletal muscles and could enhance the degradation of muscle proteins by the proteasome. In order to clarify the contribution of proteasome to proteolysis during meat conditioning, these problems should be solved.
ACKNOWLEDGEMENTS The authors are grateful to Dr Hiromichi Kato, Emeritus Professor of the University of Tokyo for his encouragement. The authors also wish to thank Mr Hiromichi Ohgoshi and Mr Yoshimasa Harimoto for their excellent technical assistance.
REFERENCES Chu-Ping, M., Slaughter, C. A. & DeMartino, G. N. (1992a) J. Biol. Chem. 267, 10515. Chu-Ping, M., Slaughter, C. A. & DeMartino, G. N. (1992b) Biochim. Biophys. Acta 1119, 303. Dahlmann, B., Becher, B., Sobek, A., Ehlers, C., Kopp, F. & Kuehn, L. (1993) Enzyme Protein 47, 214. Dahlmann, B., Kuehn, L., Rutschmann, M. & Reinauer, H. (1985) Biochem. J. 228, 161. Davis, B. J. (1964) Ann. NY Acad. Sci. 121, 404. Driscoll, J. & Goldberg, A. L. (1989) Proc. Natl. Acad. Sci. USA 86, 787. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) J. Biol. Chem. 177, 751. Inomata, M., Nomoto, M., Hayashi, M., Nakamura, M., Imahori, K. & Kawashima, S. (1984) J. Biochem. 95, 1661. Ishiura, S., Sano, M., Kamakura, K. & Sugita, H. (1985) FEBS Letts 189, 119. Kanayama, H., Tamura, T., Ugai, S., Kagawa, S., Tanahashi, N., Yoshimura, T., Tanaka, K. & Ichihara, A. (1992) Eur. J. Biochem. 206, 567. Koohmaraie, M. (1992) J. Animal Sci. 70, 3697. Laemmli, U. K. (1970) Nature 227, 680. Matsuishi, M., Matsumoto, T., Okitani, A. & Kato, H. (1992) ht. J. Biochem. 24, 1967.
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Murakami, K. & Etlinger, J. D. (1986) Proc. Natl. Acad. Sci. USA 83, 758%. Mykles, D. (1989) Arch. Biochem. Biophys. 274, 216. Mykles, D. & Haire, M. F. (1991) Arch. Biochem. Biophys. 288, 543. Okitani, A., Matsuishi, M., Matsumoto, T., Kamoshida, E., Sato, M., Matsukura, U., Watanabe, M., Kato, H. & Fujimaki, M. (1988) Eur. J. Biochem. 171, 377. Okitani, A., Matsukura, U., Kato, H. & Fujimaki, M. (1980) J. Biochem. 87, 1133. Orlowski, M. (1990) Biochemistry 29, 10291. Peters, J.-M., Cejka, Z., Harris, J. R., Kleinschmidt, J. A. & Baumeister, W. (1993) J. Molec. Biol. 234,932.
Rechsteiner, M., Hoffman, L. & Dubiel, W. (1993) J. Biol. Chem. 268, 6065. Rivet& A. J. (1989) Biochem. J. 263, 625. Tanahashi, N., Tsurumi, C., Tamura, T. & Tanaka, K. (1993) Enzyme Protein 47, 241. Tanaka, K., Ii, K., Ichihara, A., Waxman, L. & Goldberg, A. L. (1986) J. Biol. Chem. 261, 15197. Taylor, R. G., Tassy, C., Briand, M., Robert, N., Briand, Y. & Ouali, A. (1995) Mot. Biof. Rep. 21, 71.
Ugai, S., Tamura, T., Tanahashi, N., Takagi, S., Komi, N., Chung, C. H., Tanaka, K. & Ichihara, A. (1993) J. Biochem. 113, 7.54. Wagner, B. J. & Margolis, J. W. (1993) Arch. Biochem. Biophys. 307, 146. Yamamoto, T., Nojima, M., Ishiura, S. & Sugita, H. (1986) Biochim. Biophys. Acta 882, 297. Yang, R., Okitani, A. & Fujimaki, M. (1970) Agric. Biol. Chem. 34, 1765. Yoshimura, T., Kanayama, K., Takagi, T., Ikai, A., Tokunaga, F., Koide, T., Tanahashi, N., Tamura, T., Cejka, Z., Baumeister, W., Tanaka, K. & Ichihara, A. (1993) J. Struct. Biol. 111, 200.