Functional regions in the essential light chain of smooth muscle myosin as revealed by the mutagenesis approach.

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 histidine-tagged 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.

Role of the C-terminal extremities of the smooth muscle myosin heavy chains: implication for assembly properties.

The two light meromyosin isoforms from rabbit smooth muscle were prepared as recombinant proteins in Escherichia coli. These species which differed only by their C-terminal extremity showed the same circular dichroism spectra and endotherms in measurements of differential scanning calorimetry. Their solubility properties were different at pH 7.0 in the absence of monovalent salts. Their paracrystals formed at low pH differed by their aspect and number. These data suggest a role for the C-terminal extremity of myosin heavy chains in the assembly of myosin molecules in filaments and consequently in the contractility of smooth muscles.

Kinetics of folding of the myosin rod.

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.

The effect of pH on the folding and stability of the myosin rod.

The far-ultraviolet circular dichroism and fluorescence emission intensities of the myosin rod were studied at pH 2-11, in the absence and presence of guanidine hydrochloride. The protein kept its helicity in this pH range. Its stability in the denaturant was higher at acidic pH than at pH 7. This may be due to favorable interactions involving protonated glutamic acid residues at the interface of the two alpha-helical chains of the molecule. At alkaline pH, the fluorescence of the myosin rod was quenched, and the tryptophan region of the protein became less stable in the presence of guanidine hydrochloride, due to ionization of tyrosine residues or other amino acids close to tryptophans in the double-helix arrangement.

Unfolding/refolding studies of the myosin rod.

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 microM. These phenomena are interpreted taking into account the large size of the molecule. A step-wise process is proposed as a model for the unfolding of the myosin rod.

A model system of coupled activity of co-immobilized creatine kinase and myosin.

Myosin and creatine kinase were co-immobilized onto Immunodyne films to mimic the behaviour of creatine kinase bound to the M-line of myofilaments. The Mg-ATPase activity of bound myosin was studied by a coupled enzymatic assay, which detects Mg-ADP in the bulk solution by means of pyruvate kinase and lactate dehydrogenase. The competition for Mg-ADP between pyruvate kinase and creatine kinase either free in solution or co-immobilized with myosin was studied at various creatine phosphate concentrations. Bound creatine kinase competed efficiently when present in very low amounts, corresponding to an activity ratio higher than 1:20,000 between creatine kinase and pyruvate kinase and a molar ratio higher than 1:1000 between creatine kinase and myosin. The Mg-ADP produced by myosin ATPase in the vicinity of the film did not diffuse into the bulk solution but, in the presence of creatine phosphate, was recycled into Mg-ATP by the neighbouring creatine kinase. The existence of an unstirred layer near the surface of the film is sufficient to explain the channeling of ADP (or ATP) between co-immobilized myosin and creatine kinase, without direct interaction or 'intimate coupling' between the enzymes. The problem now is to determine the importance of this kind of facilitated diffusion in the myofilaments in vivo.

Inactivation, subunit dissociation, aggregation, and unfolding of myosin subfragment 1 during guanidine denaturation.

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.

Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits.

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared. Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment-1 species, and trypsin-digested derivatives showed that the neonatal protein contained less alpha-helices than the adult form. The Mg2(+)-ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment-1, it was found that the binding of ATP analogues such as adenosine 5'-[beta, gamma-imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KmATP) to neonatal subfragment-1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP - neonatal-subfragment-1 complex was weaker. These features must be expressed in the cyclical actin-myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP-hydrolysis-products - myosin complex.

Diffusion-limited kinetics of immobilized myosin ATPase.

To test the possibility that ATP diffusion limits the kinetics of myosin ATPase (EC. 3.6.1.3) in situ, myosin was covalently bound to the surface of 2 kinds of films: collagen and Immunodyne. On collagen films, it was bound either with 1-ethyl-3 (3 dimethyl-aminopropyl)carbodiimide (EDC) or with dimethyl-3,3'-dithiobis(propionimidate) (DTP). The apparent Km for K+-ATP rose from 0.26 mM for free myosin in solution to 2-5 mM for covalently bound myosin, and maximum K+-ATPase activity was very low. With the other film, Immunodyne from Pall, the maximum activity of bound myosin was 170 nmol per min per 1.5 cm2 film. The apparent Km for K+-ATP was 2.1 mM when the incubation mixture was vigorously stirred, and the effect of stirring indicated that the kinetics of K+-ATP hydrolysis are limited by external diffusion. The large amount of myosin bound per unit of Immunodyne film surface permitted the study of Mg2+-ATPase activity, although it was 400-500 times less than the K+-ATPase activity. The apparently non-Michaelian kinetics of Mg2+-ATP hydrolysis are attributable to the external diffusion. The apparent Michaelis constant observed at low Mg2+-ATP concentrations rose from 0.27 microM for myosin in solution to 5 microM for myosin bound to Immunodyne film.

Prediction of the secondary structure of myosin light chains from comparison of homologous sequences. Implications for the interaction between myosin heavy and light chains.

The primary sequences of seventeen essential and seventeen regulatory myosin light chains were analyzed and compared, using algorithms based on the different structural properties of their amino acid residues. This process allowed estimation of the structural homology between the proteins studied, and improved the prediction of their mean secondary structure and functionally important segments or residues. On the basis of the crystal structure of troponin C, a model of the myosin essential light chain with a fairly compact form is proposed. The possible sites of interaction between myosin light and heavy chains from rabbit skeletal muscle were also investigated by a complementarity method adapted to helix-rich proteins. Segments 139-149 and 65-75 in the essential light chain and segments 27-37, 67-77 and 97-107 in the regulatory light chain are suggested to constitute some of these sites, as most of them were found to have the features of surface-seeking helices.

Correlation among the proton charges and molecular masses of myosin subunits.

The molecular masses and isoelectric points of myosin light and heavy chains were calculated from their known primary sequences and their respective distribution in a two-dimensional graph is displayed. Implications for the electrophoretic study of myosin subunits are inferred from this analysis.

Active-site titration of enzymes at high concentration. Application to myosin ATPase.

The number of active sites of soluble and filamentous myosin and of its subfragments, heavy meromyosin and subfragment-1, has been determined. The titration involves steady-state kinetic measurements at a high enzyme concentration and varying substrate concentrations (or vice versa), in the presence of a substrate-regenerating system. Some practical and theoretical conditions for its execution are given, and, in particular, the effect of a possible heterogeneity of the active sites on the titration curves is analysed. Under the experimental conditions of the study, the number of active sites is close to that of myosin heads, and the heads seem to be functionally identical; the catalytic constants kcat and Km characterizing each active site are similar within some limits (1-2 for the ratio of kcat values; 1-5 for that of Km values).

Estradiol activates methylating enzyme(s) involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in rat pituitary membranes.

17 beta-Estradiol (E2) affects the sensitivity of pituitary cells to several neurohormones as LHRH, TRH, or dopamine, presumably by modulating receptor coupling mechanisms. We attempted to pinpoint the membrane processes underlying this modulation and studied the effect of E2 on pituitary membrane phospholipid methylation. Anterior pituitary membranes prepared from ovariectomized (ovx) or ovx plus E2-treated rats were assayed for phospholipid methylation. Methylated phospholipids were separated by TLC. Incorporation of [3H]methyl groups into phospholipids increased with membrane concentration and incubation time with S-adenosyl-L-methyl [3H]methionine; it was not Mg2+ dependent and was inhibited in a dose-dependent manner by S-adenosyl-L-homocysteine, methyltransferase inhibitor. pH was found to be critical. Formation of phosphatidyl-monoethanolamine, phosphatidyl-dimethylethanolamine, and phosphatidylcholine was markedly stimulated by treatment with E2. The effect increased progressively when animals were killed 15 h to 5 days after E2 implantation. The response involved a shift in the maximum velocity (Vmax) although there was no change in the available substrate for the methylating enzyme. This change in Vmax probably reflects changes in the amount of the methylating enzyme itself. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2 was ineffective, pointing to a stereospecific interaction. After differential centrifugation of pituitary membranes, the highest specific methyltransferase activity was found in light mitochondrial (L) and microsomal (P) fractions and the lowest in nuclei (N) and the heavy mitochondrial (M) fractions. After sucrose density gradient centrifugation, methylated phospholipids were preferentially recovered from fractions corresponding to the endoplasmic reticulum and/or secretory granules. E2 treatment for 5 days did not modify the subcellular distribution of methyltransferase activity but stimulated it in all fractions; in contrast, it did not modify the activity of the other enzymes measured as fraction markers. Under the same experimental conditions, phospholipid methylation in membranes prepared from cortex, and anterior and mediobasal hypothalamic structures was not affected by the steroid, with the exception of a slight increment of [3H]methyl incorporation into mediobasal hypothalamic membrane phospholipids after 5 days of E2 treatment. These results indicate that E2-induced changes in pituitary responsiveness might be concomitant with selective effects of the steroid on specific membrane enzymatic activities involved in coupling mechanisms.

Comparison of myosins from the masseter muscle of adult rat, mouse and guinea-pig. Persistence of neonatal-type isoforms in the murine muscle.

Adult rat, mouse, and guinea-pig masseter muscles display distinct myosin electrophoretic patterns. The rat muscle contains four main forms which by reference to the myosins of the IIB tensor fasciae latae, of the IIA mylohyoid, and of the red and white portions of the sternomastoid muscles, correspond respectively to the intermediate-type and to the three fast-type isoforms. The mouse masseter muscle contains only three main myosins, the intermediate-type and two fast-type isoforms. The guinea-pig muscle also displays only three bands, whose assignment is, however, less straightforward than in the murine species; their electrophoretic mobilities are not strictly the same as those of their homologous forms in rat and mouse. Comparison with the myosins of the tensor fasciae latae and of the sternomastoid muscles of guinea-pig allows their identification as intermediate and fast-type myosins. In addition to these typical adult-type forms, adult murine masseter muscles are observed to contain between zero and 30% of neonatal-type myosins. The comparison of the developmental transitions of myosins in the rat masseter with those in the skeletal muscles of the same animal indicates a delay in the appearance of the adult as well as in the disappearance of the neonatal-type myosins in the masseter muscle. Both the variability in myosin types with the animal species and the atypical presence of neonatal forms in the murine adults suggest that myosin expression in the masseter muscle is subjected to unusual regulations.

Myosin switches in skeletal muscle development of an urodelan amphibian, Pleurodeles waltlii. Comparison with a mammalian, Mus musculus.

The isomyosins from dorsal axial muscle, which appear successively through metamorphosis of P.waltlii, are shown to be composed of identical fast-type light chains but of distinct heavy subunits. We observe that this modification goes with a change in ATPase activity as also in the case of mouse. Metamorphosis in amphibian as well as birth in mammalian are thus both accompanied by the synthesis of new myosins of higher catalytic efficiency.

Effect of the filamentous structure of myosin on the actomyosin ATPase activity.

The ATPase activities of acto-heavy meromyosin and of acto-myosin minifilaments have been compared under the same conditions at low ATP (0.1 mM) and at several KC1 concentrations. The activities, which are strongly salt-dependent in both systems, have been found to be similar at high ionic strength (about 0.16 M) but different at lower ionic strength (0.06-0.07 M). Under this last condition, the catalytic constants kcat and Km are lower for acto-myosin minifilaments than for acto-heavy meromyosin ATPase. In addition, at low ionic strength, any decrease in the concentration of any of the ionic species (ATP, citrate, etc.) induces an increase in the interaction strength between myosin and actin filaments, as revealed by the Km changes. The presence of the troponintropomyosin complex and of Ca2+ also enhances the strength of this interaction. On the other hand, the occurrence of particular interactions between F-actin and myosin minifilaments is further substantiated by the phenomenon of superprecipitation which occurs when the ATP concentration decreases. The favourable effect of the organized structure of the myosin minifilaments on the ATPase activity of actomyosin is discussed.

Myosin isoenzymes in adult rat skeletal muscles.

The myosin isoenzymic content of several adult rat muscles has been analyzed by electrophoresis under non-dissociating conditions. Fast-twitch fibres, whether of the oxidative type, such as the red masseter, or of the glycolytic type, such as the white tensor fasciae latae, are all shown to contain three isomyosins with respective mobilities identical in both types of muscles. These three isoenzymes, which, as in the case of myosins from avian fast muscles, represent alkali light chain hetero-and homodimers with similar large subunits, occur in somewhat variable relative proportions depending on the muscle. No obvious correlation was established between the type of the fast fibres--either oxidative or glycolytic--and the type of the myosins. Besides the three fast isoenzymes, other muscles, such as the predominantly fast latissimus dorsi and the mixed diaphragm, are shown to contain one myosin species of lower electrophoretic mobility; this supplementary isoenzyme comigrates with the major component of the predominantly slow-twich soleus muscle, but differs from the avian slow-tonic isoform. Ca2+ ATPase determinations on gel indicate that the fast isomyosins all display similar activity, which is five to ten times higher, depending on the experimental conditions of the assay, than the activity shown by the slow isoenzymes. Altogether, at least five isoenzymes, corresponding to one "slow-twich", one "slow-tonic", and three "fast-twitch" myosin species, were detected in rat skeletal muscles.U

Magnesium ion dependent adenosine triphosphatase activity of heavy meromyosin as a function of temperature between +20 and -15 degrees C.

The hydrolysis of Mg2+-adenosine 5'-triphosphate (ATP) by heavy meromyosin has been studied between +20 and -15 degrees C, especially in the low-temperature range, in a medium containing 30% (v/v) ethylene glycol by fluorometric, spectrophotometric, and potentiometric measurements. The time course of the fluorescence changes of the enzyme during the reaction depends markedly on the temperature in consequence of large differences between the activation energies of the various steps. The observed kinetics have been analyzed according to the simplified scheme of Bagshaw & Trentham [Bagshaw, C. R., & Trentham, D. R. (1974) Biochem. J. 141, 331-349]. The following results have been obtained. (1) The rate-limiting step of the reaction changes in this temperature range; at 20 degrees C M**.ADP.Pi is the predominant steady-state complex, and M*.ADP predominates at -15 degrees C, with a half-life of approximately 10 min. (2) As expected, on the basis that it is the dissociation of the M*.ADP complex which becomes rate limiting at low temperature, one observes, in the pre-steady-state below 0 degrees C, both a proton burst and a lag phase in ADP release. (3) At low temperature, the equilibrium M*.ATP in equilibrium M**.ADP.Pi is displaced to the left All the kinetic data obtained in this study are compatible with a simple pathway for the Mg2+-ATP hydrolysis by myosin and with sequential release of the reaction products.