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Calcium influx is known to be prerequisite for membrane fusion of myoblasts. However, little is known about the channels that are responsible for the entry of calcium into the cells. Here we show that K(Ca) channels and stretch-activated channels are involved in the calcium influx. Upon analysis of single-channel recordings, calcium sensitivity of K(Ca) channels in myoblasts was found to be about sixfold higher than that in myotubes. Their density in myoblasts (1.68 micron(-2)) was also about sixfold higher than that in myotubes (0.27 micron(-2)). In addition, the opening of the calcium-permeable cationic channels in myoblasts was found to increase with membrane stretching and could be blocked by gadolinium. The density of stretch-activated channels was 0.22 micron(-2) for myoblasts, and the relative permeability of calcium to potassium was P(Ca)/P(K) approximately 3.6. The channels could generate inward calcium currents to open K(Ca) channels in physiological solution. Furthermore, the activation of K(Ca) channels by phloretin dramatically hyperpolarized the resting membrane potential of myoblasts and this effect could be reversed upon treatment of tetraethylammonium. While phloretin induced precocious fusion, tetraethylammonium or gadolinium blocked not only the phloretin-induced precocious fusion but also the spontaneous fusion of myoblasts. These results suggest that hyperpolarization generated by reciprocal activation of stretch-activated channels and K(Ca) channels is involved in the calcium influx that triggers myoblast fusion.

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The ATP-dependent protease Ti consists of two different components: ClpA containing ATP-cleaving sites and ClpP having serine active sites for proteolysis. The clpA gene has dual translational start sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa (referred to as ClpA84 and ClpA65, respectively). Here we show that ClpA84, but not ClpA65, is degraded in vitro by ClpP in the presence of ATP. The ClpP-mediated hydrolysis of ClpA84 could be prevented by casein, which is an excellent substrate of protease Ti (i.e. ClpA84/ClpP complex). Thus, it appears that free form of ClpA84 competes with casein for the degradation by ClpA/ClpP complex. Furthermore, ClpA65 inhibited the auto-degradation of ClpA84 by the complex. These results suggest that ClpA65 may play an important role in the control of the ClpA84 level and in turn in the regulation of ATP-dependent protein breakdown in E. coli.

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Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents. Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease Ci must be a zinc metalloprotease. Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed. Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain. Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate. Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain. However, it shows little or no activity against proteins larger than 15 kDa (e.g. ovalbumin and denatured bovine serum albumin). Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

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The ATP-dependent protease Ti (Clp) consists of two distinct components, ClpP containing the serine active sites for proteolysis and ClpA having two ATP-binding sites. A ClpA variant (ClpAT) carrying Thr in place of Met169 is highly soluble but indistinguishable from the wild-type ClpA in its ability to hydrolyze ATP and to support the ClpP-mediated proteolysis. Here we show that ATP hydrolysis is essential for assembly of ClpAT/ClpP complex upon analysis of the mixture of its components by gel filtration followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Either ADP or adenosine 5'-(beta,gamma-imido)-triphosphate could not support the complex formation. Furthermore, ClpAT/K501T which carries a mutation in the second ATP-binding site and therefore is unable to cleave ATP could not interact with ClpP. On the other hand, ClpAT/K220T carrying a mutation in the first site and ClpP could be assembled into a complex at 2 mM ATP but not at 0.5 mM, at which concentration the trimeric mutant protein can not form a hexamer. These results indicate that assembly of protease Ti requires hydrolysis of ATP by ClpA in addition to its binding for hexamer formation.

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Involvement of transglutaminase in myofibrillogenesis of chick embryonic myoblasts has been investigated in vitro. Both the activity and protein level of transglutaminase initially decreased to a minimal level at the time of burst of myoblast fusion but gradually increased thereafter. The localization of transglutaminase underwent a dramatic change from the whole cytoplasm in a diffuse pattern to the cross-striated sarcomeric A band, being strictly colocalized with the myosin thick filaments. For a brief period prior to the appearance of cross-striation, transglutaminase was localized in nonstriated filamental structures that coincided with the stress fiber-like structures. When 12-o-tetradecanoyl phorbol acetate was added to muscle cell cultures to induce the sequential disassembly of thin and thick filaments, transglutaminase was strictly colocalized with the myosin thick filaments even in the myosacs, of which most of the thin filaments were disrupted. Moreover, monodansylcadaverine, a competitive inhibitor of transglutaminase, reversibly inhibited the myofibril maturation. In addition, myosin heavy chain behaved as one of the potential intracellular substrates for transglutaminase. The cross-linked myosin complex constituted approximately 5% of the total Triton X-100-insoluble pool of myosin molecules in developing muscle cells, and its level was reduced to below 1% upon treatment with monodansylcadaverine. These results suggest that transglutaminase plays a crucial role in myofibrillogenesis of developing chick skeletal muscle.

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A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC was substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a non-denaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alpha NH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.

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ClpA is the ATPase component of the ATP-dependent protease Ti (Clp) in Escherichia coli and contains two ATP-binding sites. A ClpA variant (referred to as ClpAT) carrying threonine in place of the 169th methionine has recently been shown to be highly soluble but indistinguishable from the wild-type, 84-kDa ClpA in its ability to hydrolyze ATP and to support the casein-degrading activity of ClpP. Therefore, site-directed mutagenesis was performed to generate mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys220 or Lys501 with Thr). ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis 10-50% as well as ClpAT depending on ATP concentration, while ClpAT/K501T was unable to cleave ATP or to support the proteolysis. Without ATP, ClpAT and both of its mutant forms behaved as trimeric molecules as analyzed by gel filtration on a Sephacryl S-300 column. With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but ClpAT/K220T remained trimeric. With 2 mM ATP, however, ClpAT/K220T also behaved as a hexamer. These results suggest that the first ATP-binding site of ClpA is responsible for hexamer formation, while the second is essential for ATP hydrolysis. When trimeric ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel filtration as above, a majority of ClpAT/K220T ran together with ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and to support the ClpP-mediated proteolysis. Similar results were obtained in the presence of 2 mM ATP and also with the mixture with ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was significantly higher than the sum of that of each protein, particularly in the presence of 2 mM ATP, although its ability to support the proteolysis by ClpP remained unchanged. These results suggest that a rapid exchange of the subunits, possibly as a trimeric unit, occurs between the ClpAT proteins in the presence of ATP and leads to the formation of mixed hexameric molecules.

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The clpA gene that encodes the ATPase subunit of the ATP-dependent protease Ti (Clp) in Escherichia coli contains a putative internal translational initiation site. Here we show that mutagenesis of its 5'-end AUG codon resulted in an exclusive synthesis of the 65-kDa protein (ClpA65), while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produced only the 84-kDa protein (ClpA84T). On the other hand, the cells carrying the wild-type clpA gene produced both the 84- and 65-kDa proteins (ClpA84/65). While the purified ClpA84T and ClpA84/65 hydrolyzed ATP nearly as well as the 84-kDa ClpA alone (ClpA84), ClpA65 cleaved ATP at a rate less than 5% of that by ClpA84. Unlike ClpA84 and ClpA84T, ClpA65 could not support the casein-degrading activity of ClpP. Furthermore, ClpA65 inhibited the proteolysis by the mixture of ClpP with ClpA84 or ClpA84T but not that with ClpA84/65, which could support the proteolytic activity of ClpP only about 40% as well as ClpA84. Nevertheless, ClpA65 showed little or no effect on the basal or protein-activated ATPase activity of ClpA84, ClpA84T, or ClpA84/65 alone or in the presence of ClpP. These results suggest that ClpA65 may interfere the interaction of ClpA84 or ClpA84T with ClpP and, hence, impair their assembly into an active form of the ATP-dependent protease Ti.

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We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and treatment of calmodulin antagonists, such as trifluoperazine, blocks the fusion. Here, we show that cAMP treatment of primary cultures of chick embryonic myoblasts blocks 100-kDa protein phosphorylation. This effect is dose-dependent and can be reversed upon removal of the nucleotide from the culture media. However, cAMP shows little or no effect on accumulation of the 100-kDa protein. Furthermore, phosphorylation of the 100-kDa protein by the partially purified Ca2+/calmodulin-dependent protein kinase (CaM kinase III) from cAMP-treated cells occurs to a much lower extent than that from untreated cells. Nevertheless, cAMP-sensitive protein kinase does not seem to be directly involved in phosphorylation and inactivation of CaM kinase III, because preincubation of cAMP with the myoblast extracts lacking the endogenous 100-kDa protein does not show any effect on activity of CaM kinase III. Similar to its effect on 100-kDa protein phosphorylation, cAMP reversibly inhibits the fusion of cultured myoblasts. Moreover, treatment with forskolin or theophylline, which is known to elevate the intracellular cAMP level, also reversibly blocks both protein phosphorylation and myoblast fusion. On the other hand, cAMP shows little or no effect on accumulation of muscle-specific proteins, such as creatine kinase and tropomyosin. These results suggest that cAMP is involved in down-regulation of both 100-kDa protein phosphorylation and membrane fusion of cultured myoblasts. These results also suggest that the cAMP-mediated inhibition of 100-kDa protein phosphorylation may be associated with its inhibitory effect on myoblast fusion.

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An endoprotease in earthworm (Lumbricus rubellus) is purified to apparent homogeneity using 125I-lactalbumin as a substrate. The protease has a molecular mass of 27 kDa and is markedly activated by poly-L-lysine or poly-L-arginine. It is a chymotrypsin-like serine protease. Its activity is distributed to coelomic fluid but relatively little to coelomocytes.

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ClpX, an alternative ATP-binding subunit for protease Ti (also called Clp), has been shown to support the ATP-dependent hydrolysis of lambda O-protein by ClpP. clpX has also been reported to be in an operon with clpP, and therefore both are co-transcribed in a single mRNA using the promoter proximal to clpP. Here, we show that clpX can be expressed independently from clpP using its own promoter. The cells carrying clpX alone on a multicopy plasmid successively produced the 46-kDa ClpX protein. Moreover, in vitro translation analysis revealed that the recombinant plasmid containing clpX generates the 46-kDa protein that can be immunoprecipitated with anti-ClpX antibody. In addition, it has recently been reported that CipX, but not ClpP, is required for normal replication of bacteriophage Mu. Thus, it appears that clpX can be expressed alone and/or co-expressed with clpP in cells depending on physiological conditions.

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The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.

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Nitric oxide (NO) is a messenger molecule of vascular endothelial cells, macrophages, and neurons. Here, we demonstrate that the activity of NO synthase increases transiently but dramatically in chick embryonic myoblasts that are competent for fusion. This activity requires Ca2+, calmodulin, and NADPH. In addition, the increase in NO synthase activity coincides with an increase in cellular cGMP level. Furthermore, NO generated by treatment with sodium nitroprusside induces precocious myoblast fusion, while treatment with NG-monomethyl-L-arginine, a competitive inhibitor of NO synthase, or methylene blue, an inhibitor of guanylate cyclase, delays fusion. These results provide the first evidence for a strong association of NO with myoblast fusion.

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The subunit patterns of the proteasomes, that were purified from muscle, liver and brain, were found to be significantly different from one another. Furthermore, the proteasomes from adult and embryonic tissues of the same types also differed from each other in their subunit patterns. In addition, the specific activities of the purified proteasomes for peptide-cleavage, but not for casein-hydrolysis, appeared to be varied among the enzymes isolated from the different tissues. Thus, expression of a large number of proteasome subunits appears to be tissue-specific and under developmental control, although its relation with the multicatalytic activities of the proteasomes remains unclear.

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Myoblast surface proteins with binding activity toward the N-terminal 29-kDa fragment of fibronectin were identified by two different experimental techniques: one involves radioiodination of the cell surface proteins, followed by solubilization with Triton X-100 and affinity purification on a Sepharose column conjugated with the 29-kDa fragment, and the other involves cross-linking of the 29-kDa fragment to the cells metabolically labeled with [35S]methionine, followed by immunoprecipitation with anti-29-kDa IgG. Both approaches revealed that primary cultures of chick myoblasts contain the 66- and 48-kDa proteins that bind to the 29-kDa fragment. These binding proteins were then purified to apparent homogeneity by two successive chromatographies of the solubilized extracts of 12-day-old embryonic muscle on wheat germ agglutinin-agarose and 29-kDa fragment-Sepharose columns. However, the 48-kDa protein was found to be derived from contaminating fibroblasts upon immunoblot analysis of the myogenic cell lines, rat L8E63 and mouse C2A3, and cultured fibroblasts using the antibody raised against the 66-kDa protein. Anti-66-kDa IgG inhibited the binding of the 125I-29-kDa protein to the primary culture of myoblasts in a dose-dependent manner. On the other hand, the same antibody showed little or no effect on the initial binding of 125I-fibronectin to the cell surface, but dramatically inhibited its incorporation into deoxycholate-insoluble matrices. Furthermore, Fab fragments of anti-66-kDa IgG completely blocked the incorporation of fluoresceinated fibronectin into matrices but not its binding to the cell surface. These results suggest that fibronectin matrix assembly is mediated at least in part by the interaction of the 66-kDa protein with the N-terminal type I domain of fibronectin.

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We have previously shown that Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein dramatically increases during the early period of myoblast fusion and inhibition of the protein phosphorylation prevents the fusion. Here, we show that the protein phosphorylation occurs exclusively at Thr residue(s) and the purified 100-kDa protein can be ADP-ribosylated upon treatment with diphtheria toxin. Furthermore, the 13 N-terminal amino acid sequence of the 100-kDa protein, N-Val-Asn-Phe-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp-Lys, exactly matches with that of elongation factor-2 from rat and hamster. These results indicate that the 100-kDa protein in chick embryonic myoblasts is identical to the eukaryotic elongation factor-2.

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The heat shock protein ClpB in Escherichia coli is a protein-activated ATPase and consists of two proteins with sizes of 93 and 79 kDa. By polymerase chain reaction-aided site-directed mutagenesis, both the proteins have been shown to be encoded by the same reading frame of the clpB gene, the 93-kDa protein (ClpB93) from the 5'-end AUG translational initiation site and the 79-kDa protein (ClpB79) from the 149th codon (an internal GUG start site). Both the purified ClpB93 and ClpB79 proteins behave as tetrameric complexes with a very similar size of about 350 kDa upon gel filtration on a Superose-6 column. Both appear to be exclusively localized to the cytosol of E. coli. Both show inherent ATPase activities and have an identical Km of 1.1 mM for ATP. The ATPase activity of ClpB93 is as markedly stimulated by proteins, including casein and insulin, as that of wild-type ClpB, but the same proteins show little or no effect on ClpB79. Because ClpB79 lacks the 148 N-terminal sequence of ClpB93 but retains the two consensus sequences for adenine nucleotide binding, the N-terminal portion appears to contain a site(s) or domain(s) responsible for protein binding. Furthermore, ClpB79 is capable of inhibiting the casein-activated ATPase activity of ClpB93 in a dose-dependent manner but without any effect on its inherent ATPase activity. In addition, ClpB93 mixed with differing amounts of ClpB79 behave as tetrameric molecules, although its protein-activated ATPase activity is gradually reduced. These results suggest that tetramer formation between ClpB93 and ClpB79 may be responsible for the inhibition of the activity.

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The 33 kDa IciA protein, an inhibitor of replication initiation of the Escherichia coli chromosome, was found to be specifically cleaved to 27 kDa fragment by protease Do, the htrA gene product. The 27 kDa polypeptide could no longer interact with the oriC region, and therefore the cleavage-site is likely to reside within the N-terminal DNA-binding domain of the IciA protein. In addition, protease Do was found to localize primarily to the cytoplasm although it also could bind to membranes through an ionic interaction. These results suggest that intracellular breakdown of the IciA protein by protease Do may provide a potential mechanism involving the regulation of initiation of DNA replication in Escherichia coli.

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Benzyloxycarbonyl(Z)-Leu-nLeu-H (calpeptin) and Z-Leu-Met-H, cell-penetrating inhibitors of calpain, were found to block myoblast fusion without any effect on cell proliferation and alignment along their bipolar axis. They also inhibited the accumulation of creatine kinase during myogenesis. These effects were dose-dependent, and could be reversed upon removal of the drug from the culture medium. Furthermore, treatment of the inhibitors prevented the hydrolysis of filamin, which is sensitive to cleavage by calpain in vitro and interferes with actin-myosin filament formation by cross-linking F-actin molecules. On the other hand, leupeptin, which can also inhibit calpain in vitro but can not penetrate into cells, showed little or no effect on both myoblast fusion and filamin clevage. These results suggest that calpain may play an important role in cytoskeletal reorganization that is requisite for myoblast fusion. The role of calpain on the expression of muscle-specific proteins remains unknown.

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