Developmental Regulation of Proteolytic Activities and Subunit Pattern of 20 S Proteasome in Chick Embryonic Muscle*

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleav- ing activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-&naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was ob- served with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number , but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the develop- ment. These results suggest that the alterations in the proteasome activities and subunit pattern are devel- opmentally regulated and may be correlated.

The proteasome is a symmetrical ring-shaped particle with an unusually large mass of 600-800 kDa (20 S) and is composed of nonidentical subunits with small molecular masses of 21-32 kDa Orlowski, 1990). This complex exhibits at least three distinct endopeptidase activities, cleaving bonds on the carboxyl side of hydrophobic, acidic, and basic amino acid residues (Rivett, 1989). In addition, it shows a latent proteolytic activity that can be activated by poly-L-lysine or fatty acids (Dahlmann et al., 1985;Tanaka et al., 1986). This enzyme particle has been suggested to be involved in an energy-dependent nonlysosomal proteolytic pathway Matthews et al., 1989;Eytan et al., 1989). On the other hand, there are increasing reports indicating that the proteasome is similar to the 20 S cylindrical particle, called prosome (Arrigo et al., 1988;Falkenburg et al., 1988), which functions in repression of mRNA translation (Kuehn et al., 1990) and tRNA processing (Cas-tan0 et al., 1986). The proteasome has therefore been implicated to play an essential role(s) in many cellular events, although the actual physiological significance of this enzyme complex, as well as its in uiuo regulation, is still unknown. *This work was supported by grants from Korea Science and Engineering Foundation, Korea Ministry of Education, and the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
11 To whom correspondence should be addressed.
Of additional importance is that the multifunctional proteasome can undergo functional and structural changes during the course of the development of tissues and organisms. A recent report has shown that in developing Drosophila, the proteasome undergoes changes in its subunit pattern, and post-translational modification is at least in part responsible for the changes (Haass and Kloetzel, 1989). It has also been shown that cell-specific accumulation of the complex occurs during the embryogenesis of Drosophila (Klein et al., 1990).
These studies imply that the multicatalytic activities of proteasome may be under developmental control and involved in the process(es) related to embryonic development, such as nuclear functions and morphogenic events (Klein et al., 1990).
In an attempt to elucidate the role of the proteasome in muscle development, we investigated the changes in the level of proteolytic activities of the enzyme complex in variously aged muscle tissues of chick embryo. We also examined whether the proteasome undergoes changes in its subunit pattern during muscle development. In the present studies, we demonstrated that the proteolytic activities of proteasome are under developmental control. We also showed that the enzyme complex undergoes development-specific alterations in its subunit pattern.

Materials-Proteasome
and creatine kinase were purified from adult chick skeletal muscle to apparent homogeneity as described (Tanaka et al., 1986;Eppenberger et al., 1967). Antisera against proteasome and creatine kinase were prepared by injecting the purified proteins into albino rabbits. IgGs were isolated from the antisera by sodium sulfate fractionation. The purified anti-proteasome anti-IgG was covalently attached to CNBr-activated Sepharose CL-4B (Axen et al., 1967).
["]Casein was prepared as described by Jentoft and Dearborn (1979). ['HIFormaldehyde and '*'II-protein A were purchased from Du Pont-New England Nuclear. The synthetic peptide substrates of proteasome were obtained from Peptide Institute Inc., Japan. All other reagents were purchased from Sigma.
Preparation of Embryonic Muscle Extracts-Extracts of chick embryonic breast muscle, brain, and liver were prepared from 8-, 11-, 14-, 17-, and 20-day-old chick embryos. The tissues were washed and homogenized in 50 mM Tris-HC1 buffer (pH 7.8) containing 1 mM dithiothreitol, 0.1 mM EDTA, and 20% (v/v) glycerol. The homogenates were then centrifuged for 1 h a t 12,000 X g, and the supernatants were dialyzed against the same buffer and kept frozen a t -70 "C until use.
Assays-Protein breakdown was measured using ['Hlcasein as the substrate in the presence and absence of 0.1 mg/ml poly-L-lysine. Reaction mixtures (0.1 ml) contained 100 pg of the muscle extracts, 50 mM Tris-HC1 (pH 8.0), 1 mM dithiothreitol, and 1Opg of [3H] casein (2 X lo4 cpm). The assays were then performed as described (Seol et al., 1989). The cleavage of fluorogenic peptides was determined in similar reaction mixtures, except that they contained 20 pg of the muscle extracts and 0.1 mM peptide substrates. The release of fluorophores was then measured as described (Seol et al., 1989). Proteins were assayed as described by Bradford (1976) using bovine albumin as a standard.
Zmmunoaffinity Purification of Proteasomes-Each extract (180 mg) of 11-and 17-day-old embryonic muscle was loaded on a DEAEcellulose column (1 X 5 cm) equilibrated with 50 mM Tris-HC1 (pH 7.8) buffer containing 1 mM dithiothreitol, 0.1 mM EDTA, and 20% (v/v) glycerol. Proteins bound to the column were eluted with the buffer containing 0.3 M NaC1. The eluate was then applied to an antiproteasome immunoaffinity column (1 X 3 cm). After washing the column extensively with the elution buffer, the proteasome was released with 0.1 M glycine HCl (pH 2.3) at a flow rate of 10 ml/h, neutralized to pH 7 with 1 M Tris, and concentrated using Centricon (Amicon Corp.).
Electrophoretic Analysis-Polyacrylamide gel electrophoresis was carried out in 12.5% (w/v) slab gels containing sodium dodecyl sulfate (SDS)' (Laemmli, 1970). Two-dimensional gel electrophoresis was performed by following the method of O'Farrell (1975) with slight modification. In the first dimension, the affinity-purified proteasome was separated by isoelectric focusing on tube gels (0.2 x 10 cm) containing 8 M urea and Ampholine producing a pH gradient of 4-9. The resulting gels were equilibrated in 2% SDS and subjected to gel electrophoresis in 15% slab gel as above. Proteins were visualized by silver staining (Merril et al., 1981).
Proteins in muscle extracts that had been separated by the electrophoresis in 10% slab gels were transferred onto nitrocellulose membranes. The membranes were incubated with anti-creatine kinase anti-IgG and then with '*'I-protein A, dried, and exposed to x-ray films (Towbin et al., 1979).

RESULTS
Developmental Regulation of Proteasome Activities-To examine whether the proteolytic activities of the proteasome are under developmental control, muscle extracts were prepared from variously aged chick embryos and assayed for their ability to cleave Suc-Leu-Leu-Val-Tyr-AMC, Cbz-Ala-Arg-Arg-MNA, and [3H]casein. As shown in Fig. 1, the extents of the peptide hydrolyses gradually declined at later developmental stages. On the other hand, the casein hydrolysis in the presence of poly-L-lysine markedly increased, while that in its absence remained unchanged during the development (Fig.  2). We then tested whether the proteasome is indeed responsible for the peptide hydrolyses and poly-L-lysine-activated casein hydrolyses. The enzyme complex was precipitated by incubating the same muscle extracts with anti-proteasome anti-IgG and then with protein A-Sepharose. Table I   hydrolyses by proteasome in embryonic muscle Muscle extracts were prepared from 17-day-old chick embryo, and their ability to hydrolyze the peptides and protein was determined as described in Figs. 1 and 2, respectively, but in the presence and absence of 20 pg of anti-proteasome anti-IgG or preimmune IgG. The casein hydrolysis by proteasome was estimated by subtracting the activity seen in the absence of poly-L-lysine from that seen in its presence. In the absence of the agent, either IgG showed little effect on the casein-degrading activity. ~~ little or no effect. These results indicate that the proteolytic activities are revealed by proteasome and are under developmental regulation. Tissue-specific Changes in Proteasome Actiuities-To examine whether the development-dependent alterations in the proteasome activities are tissue-specific, extracts of brain and liver were also prepared from the developing chick embryo and assayed for their ability to cleave the peptides and [3H]casein. As shown in Table 11, little or no change of proteasome activities was evident in the brain extract. In embryonic liver, however, the peptidase activity against Cbz-Ala-Arg-Arg-MNA dramatically increased, while that against Suc-Leu-Leu-Val-Tyr-AMC gradually decreased with the time of development. On the other hand, the extents of casein hydrolyses that can be activated by poly-L-lysine remained similar at all stages of development. In addition, the immunoprecipitation with the anti-proteasome anti-IgG eliminated the peptide-and casein-cleaving activities from the brain and liver extracts, as it had from the muscle extracts (data not shown).
Thus, it appears likely that the peptidase activities of proteasome in embryonic liver are also under developmental control.

Developmental Regulation
Changes in the levels of peptides and casein hydrolyses by proteasomes in developing embryonic brain and liver Brain and liver extracts were prepared and assayed for their activities against the peptides and protein as in Table I. Similar data were obtained in three different trials of the same experiment. " LLVY, leucine-leucine-valine-tyrosine. ARR, alanine-arginine-arginine.

Relative activity against Embryonic days SUC-LLVY-Cbz-ARR-
--36 ". These results also suggest that the developmental regulation of proteasome activities is tissue-specific.
Developmental Regulation of Proteasome Subunit Pattern-To test whether the proteasome undergoes alterations in its subunit pattern during the embryonic muscle development, the enzyme complex was isolated from the muscle extracts using an immunoaffinity chromatography and was subjected to polyacrylamide gel electrophoresis in the presence of SDS.
As shown in Fig. 3, the overall subunit pattern of the proteasome from 11-day-old muscle was nearly identical to that from 17-day-old. However, when the same protein samples were analyzed by two-dimensional gel electrophoresis followed by silver staining, a number of subunit spots were changed in their intensity at the later developmental stage (Fig. 4). In particular, the intensity of spots 11, 12, and 14 increased while that of spots 3 and 10 declined to significant extents. In general, the intensity of the spots with low abundance tends to change more notably than that of major spots. Thus, it appears likely that a number of specific subunits of proteasome, but not all, are under developmental control.
Using an i n vitro culture system, the levels of musclespecific proteins in embryonic muscle cells have been shown to change during myogenic differentiation (Nadal-Ginard,  Fig. 3. They were then subjected to two-dimensional electrophoretic analysis. Proteins were visualized by silver staining. Numbering was begun from the spot with most acidic PI to that with basic PI. The arrows indicate the subunits that were changed in their amounts to relatively high extents. 1978; Endo and Nadal-Ginard, 1987). Therefore, we tested whether the muscle tissues obtained a t various embryonic days were expressing the muscle-specific cellular proteins. Extracts were prepared from developing embryonic muscles and subjected to immunoblot analysis to measure their contents of creatine kinase. As shown in Fig. 5, the protein level markedly increased a t later developmental stages. These results clearly indicate that the system we used exhibits the development-specific alterations in the cellular events, such as the induction of muscle-specific proteins.

DISCUSSION
The present studies demonstrated that both the peptideand casein-degrading activities of the proteasome in chick embryonic muscle are under developmental control. Moreover, a number of subunits were found to change in their levels to significant extents with the time of muscle development. Therefore, it appears possible that the rise and fall of the proteolytic activities are correlated with the changes in the subunit pattern, although it is totally unknown whether the subunits with changes in their amounts are directly responsible for the alterations in the proteolytic activities. It is noteworthy that the changes in the spot intensity are rather confined to the subunits with less abundance. Therefore, it is interesting to speculate that the major subunits may play a role in the maintenance of a proteasome's overall structure, while the minor spots may be directly or indirectly involved in the catalytic functions and therefore are under regulation as necessary.
A recent report has illustrated that Drosophila proteasome undergoes changes in its subunit pattern during the fly development, and yet its overall structure remained unchanged (Haass and Kloetzel, 1989). Furthermore, we have found that the changes in the subunit pattern of proteasome in developing chick embryonic liver are also limited to the subunits with minor abundance' and yet accompanied by dramatic changes in the peptidase activities (Table 11). It is also interesting to note that the proteasome subunits, whose primary structures are known, are found with relatively high abundance and that computer-assisted comparisons have failed to demonstrate any regions homologous to the amino acid sequences of active sites in other known proteases (Tamura et al., 1991).
Post-translational modification, such as protein phosphorylation, has been suggested to be responsible for the development-dependent diversification of the subunit pattern of Drosophila proteasome (Haass and Kloetzel, 1989). Any modification that brings the changes in the net charge of proteasome subunits should also alter the position of the corresponding spots in an electrical field. However, the proteasome subunits in developing embryonic muscle undergo changes in their spot intensity, although limited to a certain number, but not in their overall position as analyzed by the two-dimensional gel electrophoresis, in which an isoelectric focusing was used for the first-dimensional separation of the subunits (Fig. 4). Therefore, the changes in the subunit pattern may be attributed to the development-specific alterations in the expression of the proteasome subunits in embryonic muscle.
Of particular interest was the finding that the proteasome activity on poly-L-lysine-activated casein hydrolysis dramatically increased during embryonic periods of days 11-17. Analysis of the number of nuclei in chick leg and pectoralis muscles has shown that by embryonic day 9, only 12% of all nuclei have fused to form myotubes, but that by day 18, about 80% of all the nuclei are in myotubes (Herrmann et al., 1957;Herrmann et al., 1970). In addition, it has been well documented that in using an in vitro culture system, the induction of muscle-specific proteins occurs concomitantly with the morphological changes of mononucleated myoblasts to multinucleated myotubes (Nadal-Ginard, 1978;Endo and Nadal-Ginard, 1987;Bischoff and Holtzer, 1969;O'Neill and Stockdale, 1972). We also showed that in uiuo synthesis of creatine kinase, which is one of the muscle-specific proteins, markedly increased during the development (Fig. 5). Furthermore, this increase occurred at about the same period and revealed the dramatic rise in the proteasome activity on poly-L-lysineactivated casein hydrolysis. These facts suggest an involvement of the proteasome in the skeletal muscle development. Intracellular proteolysis has been shown to be prerequisite for the differentiation of embryonic muscle cells, which accompanies the massive mobilization of membrane proteins and reorganization of cytoskeletal components (Fulton etal., 1981;Pauw and David, 1979). Therefore, it is possible that the increased proteolytic activity of the proteasome may be involved in the cellular processes. It is also possible that the J . Y. Ahn, S. 0. Hong, K. B. Kwak, K. Tanaka, A. Ichihara, D. B. Ha, and C. H. Chung, unpublished data. rise and fall of the protein and peptide hydrolyses by the proteasome may lead to alterations in the level of unknown but crucial cell protein(s) that mediates the regulation of myogenic differentiation. However, all these possibilities are speculative at present, and further studies are required for the clarification of the role of the proteasomes in the development of chick embryonic muscle.