The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, exposure of thiol groups, far-UV circular dichroism, and the fluorescence emission intensity of myosin subfragment 1 (S-1) was studied under equilibrium conditions. It was found that the denaturation process involves several intermediate states. The enzymatic activity of S-1 is at first lost at very low concentrations of GdnHCl (lower than 0.5 M). At a slightly higher GdnHCl concentration (about 0.5 M), the light chains dissociate and this dissociation is closely followed by the formation of aggregates between the naked heavy chains of S-1 molecules in the guanidine hydrochloride range of concentrations 0.5-1 M. At GdnHCl concentrations above 1 M, aggregates gradually disappear and S-1 loses its secondary and tertiary structures. These phenomena are partly reversible, and ATPase activity is only partially recovered under highly limited conditions. These results are discussed in relation to the nature of myosin subunit assembly. The head fragment of 20 kDa is thus suggested to be implicated in the binding of light chain to heavy chain and in the self-association of free heavy chains.

We report here, for the first time, the expression of the muscle-specific isoform of the glycolytic enzyme, enolase (EC 4.2.1.11) (b enolase), in rabbit skeletal muscles. We have analysed the fast-twitch gastrocnemius and the slow-twitch soleus muscles during normal postnatal development and following denervation. We show that, in rabbit, as already described in rodents, b enolase gene expression behaves as a good marker of the fast-twitch fibers. In soleus muscle, the b enolase transcript level is 10±20% of that found in gastrocnemius. Denervation, performed at 8 postnatal days, induces an important drop of b enolase transcript levels in both developing soleus and gastrocnemius muscles, with a 80% decrease observed 1 week after denervation in the operated muscles, as compared to the corresponding contralateral muscles. Thereafter, the b enolase transcript level continues to decrease in the fast-twitch muscle, with the b enolase subunit being detectable only in the atrophic fast-twitch fibers. In contrast, the b transcript level tends to increase in the denervated slow-twitch muscle, reaching about 50% of that in contralateral soleus, at 7 weeks after surgery. The level of b enolase transcripts still expressed after denervation seems to stabilize at the same low level in both types of inactive muscles. This suggests that the small fraction of b enolase expression which is not controlled by the nerve, or by the contractile activity imposed by it, is independent of the muscle phenotype.

Recent reports by d'Albis et al. have shown that denervation of 8-day-old rabbit fast-twitch muscle (gastrocnemius) leads to the transformation of the muscle towards a slow phenotype but the changes towards slow-type myosin isoforms and contractile properties of the muscle were temporally uncoordinated. We analysed the time course of the effects of denervation of the gastrocnemius on the expression of the sarcoplasmic reticulum calcium pump isoforms (SERCA) and on the metabolic state of the muscle. Northern-blot analysis showed a rapid loss of the fast Ca2+ pump isoform (SERCA 1) mRNA from the denervated gastrocnemius which became of the oxidative type. The changes observed were complete as early as 35 days post-natal, i.e at the time when changes in contractile properties were previously observed. Denervation of the slow-twitch soleus led to a 50% decrease in the level of the slow Ca" pump isoform (SERCA 2 ) mRNA and was without effect on the metabolic state of the muscle.These findings extend previous results suggesting that in rabbit, continuous innervation is required for differentiation of fast-twitch muscles but is not an absolute requirement for differentiation of the slowtwitch muscle.Keywords: muscle denervation ; muscle metabolic state ; Ca2+-transporting ATPase ; glyceraldehyde-3-phosphate dehydrogenase.Innervation contributes to muscle specificity. In rat, mouse and cat, continuous innervation is believed to be required for differentiation of slow-twitch muscles but not of fast-twitch muscles (reviewed by Gunning and Hardeman, 1991). However, in rabbit, previous data indicate that denervation of fast and slow-twitch muscles at the age of 8 days leads to the transformation to the slow phenotype (d'Albis et al., 1994). Both denervated muscles contain slow-type myosin and have the properties of a slow-twitch muscle but there was an asynchrony between changes in muscle myosin isoform composition and contractile properties in the fast-twitch denervated muscle. Coinplete conversion to slow-type myosin occurred 52 days after denervation (60 days post-natal) whereas conversion to slow-twitch contractile features (muscle maximal shortening velocity V,,;,, and contraction time) occurred 27 days after denervation (35 days post-natal) (d'Albis et al., 1995b). These results suggest that modification of the phenotype of proteins other than myosin is involved in the alteration of the mechanical properties of the denervated gastrocnemius.We examined the expression of genes coding for proteins which contribute to the calcium regulatory mechanisms and toCorrespondence to M. Nozais, Gknes et protkines nzuscultr;re-es: ,stnrcture, ,foncrion et rigubtiorz, URA CNRS 11 31, Universite ParisSud,

The effect of guanidine hydrochloride on the gel-filtration chromatography, viscosity, far ultraviolet circular dichroism and fluorescence emission intensity of the myosin rod was studied under equilibrium conditions. The normalized transition curves for each of these methods were comparable with a midpoint at a guanidine hydrochloride concentration of 1.75 -2 M. The curves were not, however, superposable, suggesting that the loss of helix content and the dissociation of the two chains of the myosin rod were not tightly linked. Furthermore, they were unexpectedly independent of the protein concentration over 0.05-20 pM. These phenomena are interpreted takmg into account the large size of the molecule. A step-wise process is proposed as a model for the unfolding of the myosin rod.Myosin is a major component of the contractile apparatus of vertebrate skeletal muscle. It is composed of two globular heads flexibly joined to a rod-like tail. This tail is generally referred to as the myosin rod and consists of two a-helical, heavy polypeptide chains wound round one another in a lefthanded coiled coil (McLachlan and Karn, 1982;Cohen and Parry, 1990). It is involved in myosin thick-filament formation and may also play a role in muscular contraction.The myosin rod and some of its subfragments (the myosin subfragment-2 and light meromyosin) can be purified after proteolytic cleavage of whole myosin. The structural stability and flexibility of the rod and of some rod regions have been questioned for many years. Harrington and Rodgers (1984; references therein) have suggested that a part of the myosin tail near the junction between the light meromyosin and the subfragment-2 (the hinge region) can easily melt at approximately physiological temperatures, possibly to a random-coil conformation. Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al., 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al., 1990;Sugi et al., 1992).It has recently been proposed that the myosin rod contains six independent domains (Privalov, 1982 ;Lopez-Lacomba et al., 1989;Bertazzon and Tsong, 1990). The Cterminus of the heavy chain has been shown to be unfolded and mobile , and to play a crucial role in myosin assembly Kalbitzer et al. (1991) have indicated that the two chains are not in exact register but are slightly staggered. Thus, the variability in the structure of the rod may affect its properties.Protein denaturatiodrenaturation experiments may indicate how a multidomain dimeric protein, such as the myosin rod, folds and how its subunits and domains acquire stability [for a review, see Jaenicke (1987)l. We studied the effect of guanidine hydrochl...

The endogenous essential light chain (LC17) of myosin from intestine smooth muscle was replaced with mutated essential light chains prepared using recombinant techniques. Complete exchange was observed with histidinetagged derivatives of LC17a, LC17b and E122A-LC17a (LC17a and LC17b are the usual constituants of smooth muscle myosin), with small changes in the ATPase activity of reconstituted myosins. Much less exchange was observed with the light-chain derivative lacking the last 12 amino acid residues, demonstrating the importance of this segment, which may act as one arm of a pair of pincers to bind the myosin heavy chain.Keywords: isoforms; mutants; myosin light chains; site-directed mutagenesis; smooth muscle.Myosin II is a major contractile protein composed of two heavy chains (< 200 kDa each) and two pairs of light chains (< 20 kDa each). One pair of light chains is described as regulatory (20 kDa) and the other as essential (17 kDa), based on their distinctive chemical and functional features. In smooth muscle myosin, the two 20-kDa regulatory chains (LC20) can be phosphorylated and phosphorylation generates high levels of actin-activated ATPase activity and actin motility. The other pair of myosin light chains consists of the two 17-kDa essential chains (LC17), the functional significance of which is unclear [1]. The regulatory and essential light chains are bound to an 8-nm stretch of myosin heavy chain, mostly a helix. This complex constitutes a regulatory domain [2,3] and is thought to act as a lever arm during force production by muscle motor proteins [4].The essential light chain, LC17, has two isoforms, the ratio of which depends on the tissue. The difference between these two species is five amino acid substitutions in the nine C-terminal residues (Fig. 1). In particular, one isoform, LC17a, has a charged glutamic acid residue at position 142, whereas the other, LC17b, has an alanine residue in the corresponding position [5]. An inverse correlation has been found between relative LC17b content and the maximal shortening velocity of skinned smooth muscle fibers [6] but this result is controversial [1]. Recently, weak but significant LC17 isoform-specific effects on the mechanical properties of smooth muscle cells [7] and strips [8] have been shown. In addition, the two isoforms have different subcellular distributions suggesting that they may have different functions [9].It has been shown that substitution mutations in the essential light chain of nonmuscle myosin from Dictyostelium may lead to decreases in actin-activated ATPase activity and motor function, as assessed by an in vitro motility assay [10].Katoh and co-workers [11,12] recently reported an efficient method for essential light chain exchange in smooth muscle myosin. We used this method to exchange LC17a, LC17b and mutated essential light chains with endogenous light chains from rabbit small-intestine myosin. The recombinant proteins were prepared ( Fig. 1; ELC1±6) with a histidine tag on their N-terminus to facilitate purification. In...

The kinetics of the unfolding and refolding of the myosin rod have been studied by fluorescence and circular dichroism techniques, at different concentrations of protein and guanidine hydrochloride. The unfolding of the myosin rod was fast and at least biphasic in 2-3 M denaturant, with an initial immediate phase followed by a slower low-amplitude first-order phase. The refolding of the rod in 0.4-2 M guanidine hydrochloride was also at least biphasic; an initial immediate phase preceded a slow second-order phase. At the final denaturant concentration of 0.8 M, the amplitude of the burst phase was weakly dependent on the protein concentration and the rate constant of the refolding slow phase was optimal. These data are incorporated into a folding mechanism with at least three states. The high rates of the first steps of unfolding and refolding may be relevant for the functioning of the native myosin molecule by allowing a transient separation of the two strands of the myosin tail.