Proteolysis rates of a myosin heavy chain site with papain Evidence for a combined LC2-filament-mediated mechanism

In striated muscle myosin, a proteolysis site at the 25–50 kDa junction, susceptible in the filament and efficiently protected by nucleotides, is similarly protected when myosin is monomeric. Kinetic studies at low ionic strength show a close relationship between LC2 cleavage or degradation rate and cleavage of the 25–50 kDa heavy chain site. The myosin-[(T)-LC2′] species forms normal reconstituted filaments but its 25–50 kDa site susceptibility is closer to that of monomeric myosin, thus becoming practically ionic strength-independent. In this species the absence of the LC2 N-terminal segment induces a significantly greater susceptibility of the papain-sensitive site in LC1. In an LC2-depleted myosin the 25–50 kDa site susceptibility also becomes ionic strength-independent, however, the cleavage rates are then closer to that of filaments. Susceptibility in HMM and S1 is also much less dependent on ionic strength with rates intermediary between those of filament and monomer. These observations show that the maximum susceptibility to papain of the 25–50 kDa site requires both the integrity of the LC2 light chain and the filament structure and furthermore provide evidence that: (i) the LC2 N-terminus interacts specifically with some part of the filament; (ii) this interaction induces a specific transconformation in a region close to the ATPase active site; (iii) there is an interrelationship between LC1 and LC2 light chain N-terminal extremities, at least in the filament structure.     

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.     

Fungal myosin-1 homologs as key molecular motors at the membrane–cytoskeleton interface

Class I myosin (myosin-1) is a small, single-headed myosin, distributing from lower eukaryotes (such as fungi) to higher eukaryotes (such as vertebrates). Being able to interact with actin via the motor domain and to bind to membrane via the tail domain, myosin-1 plays a number of fundamental functions at the membrane–cytoskeleton interface. In fungi, myosin-1 is essential for many cellular processes, including endocytosis, hyphal growth, endoplasmic reticulum (ER) remodeling, and pathogenicity. In this review, we summarize the recent progress of fungal myosin-1 research in the past few years. We first introduce basic structure and function of each fungal myosin-1 domains, then compare the recently solved three-dimensional structure of fungal myosin-1 with that of vertebrate myosin-1, and finally focus on how fungal myosin-1 might participate in endocytosis and ER remodeling.     

Interaction of the N-terminus of chicken skeletal essential light chain 1 with F-actin

Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Transient interaction between the N-terminal extension of the essential light chain-1 and motor domain of the myosin head during the ATPase cycle

The molecular mechanism of muscle contraction is based on the ATP-dependent cyclic interaction of myosin heads with actin filaments. Myosin head (myosin subfragment-1, S1) consists of two major domains, the motor domain responsible for ATP hydrolysis and actin binding, and the regulatory domain stabilized by light chains. Essential light chain-1 (LC1) is of particular interest since it comprises a unique N-terminal extension (NTE) which can bind to actin thus forming an additional actin-binding site on the myosin head and modulating its motor activity. However, it remains unknown what happens to the NTE of LC1 when the head binds ATP during ATPase cycle and dissociates from actin. We assume that in this state of the head, when it undergoes global ATP-induced conformational changes, the NTE of LC1 can interact with the motor domain. To test this hypothesis, we applied fluorescence resonance energy transfer (FRET) to measure the distances from various sites on the NTE of LC1 to S1 active site in the motor domain and changes in these distances upon formation of S1-ADP-BeF_x complex (stable analog of S1¹-AТP state). For this, we produced recombinant LC1 cysteine mutants, which were first fluorescently labeled with 1,5-IAEDANS (donor) at different positions in their NTE and then introduced into S1; the ADP analog (TNP-ADP) bound to the S1 active site was used as an acceptor. The results show that formation of S1-ADP-BeF_x complex significantly decreases the distances from Cys residues in the NTE of LC1 to TNP-ADP in the S1 active site; this effect was the most pronounced for Cys residues located near the LC1 N-terminus. These results support the concept of the ATP-induced transient interaction of the LC1 N-terminus with the S1 motor domain.     

Influence of the regulatory light chain of fast skeletal muscle myosin on its interaction with actin in the presence and absence of ATP

The effect of myosin LC2 modifications (phosphorylation or selective proteolytic removal of a seven-residue N-terminal peptide) and partial or complete removal of the whole LC2 was studied under various conditions. (1) Actin binding in the absence of ATP is not influenced by the nature of the myosin species (phosphorylated, dephosphorylated or devoid of LC2). (2) A 50% inhibition of K+/EDTA-ATPase was obtained with actin concentrations hardly different when phosphorylated and dephosphorylated myosins were compared (of the order of 5 microM), whereas both myosin devoid of LC2 and myosin in which the LC2 N-terminal peptide has been removed required significantly higher concentrations of actin (13.0 +/- 2 and 12.0 +/- 2.0 microM, respectively). (3) Dissociation of the actomyosin complex at high ionic strength with nucleotides is not influenced by phosphorylation. (4) Actin activation of Mg2+-ATPase is enhanced when LC2 is phosphorylated; no activation enhancement is observed with myosin devoid of LC2. (5) Translational diffusion coefficient measurements of myosin in high-ionic-strength solutions indicate a tendency for LC2-deprived myosin to form autoassociation oligomers. It thus appears that a structural modification (partial cleavage or removal of LC2) induces important structural changes in myosin, pointing to a role for LC2 in the intrinsic conformation of the molecule and its interaction potentialities. Effects of LC2 removal at high ionic strength are best explained by interactions bearing no relationship to physiological functions. A physiologically significant effect of LC2 phosphorylation requires a minimum degree of organization (actomyosin complex) to be expressed in which LC2 could play the role of a return-spring in the cross-bridge mechanism.     

Differential localization of unconventional myosin I and nonmuscle myosin II during B cell spreading

Cross-linking of CD44 in vitro promotes chemokinesis and actin-based dendrite formation in T and B cells. However, the mechanisms by which the adhesion molecule CD44 induces cytoskeleton activation in lymphocytes are still poorly understood. In this study, we have investigated whether myosin isoforms are involved in CD44-dependent dendrite formation in activated B cells. Pharmacological inhibition of myosin with 2,3-butanedione monoxime strongly affected spreading and dendrite formation, suggesting that these cellular motors may participate in these phenomena. Furthermore, immunofluorescence analysis showed differences in subcellular localization of class I and class II myosin during B cell spreading. In response to CD44 cross-linking, myosin-1c was polarized to lamellipodia, where F-actin was high. In contrast, the distribution of cytosplasmic nonmuscle class II myosin was not altered. Expressions of myosin-1c and II were also demonstrated in B cells by Western blot. Although the inhibition of PLCgamma, PI3K and MEK-1 activation affected the spreading and dendrite formation in activated B cells, only PLCgamma and MEK-1 inhibition correlated with absence of myosin-1c polarization. Additionally, myosin-1c polarization was observed upon cross-linking of other surface molecules, suggesting a common mechanism for B cell spreading. This work shows that class I and class II myosin are expressed in B cells, are differentially distributed, and may participate in the morphological changes of these cells.     

Engineered cysteine mutants of myosin light chain 2. Fluorescent analogues for structural studies

Site-directed mutagenesis has been used to insert cysteine residues at specific locations in the myosin light chain 2 (LC2) sequence. The aim was to modify these cysteines with one or more spectroscopic probes and to reconstitute myosin with labeled light chains for structural studies. Native LC2 has two endogenous cysteine residues at positions 126 and 155; a third sulfhydryl was added by replacing either Pro2, Ser73, or Pro94 with cysteine. By oxidizing the endogenous cysteines to an intramolecular disulfide bond (Katoh, T., and Lowey, S., (1989) J. Cell Biol. 109, 1549), it was expected that the new cysteine could be selectively labeled with a fluorescent probe. This proved more difficult to accomplish than anticipated due to the formation of secondary disulfide bonds between the newly engineered cysteines and the native ones. Nevertheless, the unpaired cysteines were labeled with 5-(iodoacetamido)fluorescein, and singly labeled species were purified by ion-exchange chromatography. Chymotryptic digestion of the light chains, followed by high performance liquid chromatography separation of the peptides, led to the identification of the fluorescein-labeled cysteines. After light chain exchange into myosin, the position of the thiols was mapped by antifluorescyl antibodies in the electron microscope. Rotary-shadowed images showed the antibody bound at the head/rod junction of myosin for all the mutants. These mapping studies, together with the finding that widely separated cysteines can form multiple disulfide bonds, support a model for LC2 as a flexible, globular molecule that resembles other Ca/Mg-binding proteins in tertiary structure.     

Insights into the different catalytic activities of Clostridium neurotoxins

The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave vesicle-associated membrane protein 2 (VAMP2) at the same scissile bond but possess different catalytic activities and substrate requirements, which make them the best candidates for studying the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1', and P2') and fine alignment of sites P2 and P3 and sites P2 and P4 by LC/B and LC/T, respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K(168)E) increased the rate of native VAMP2 cleavage so that it approached the rate of LC/B, which explains the molecular basis for the lower k(cat) that LC/T possesses for VAMP2 cleavage relative to that of LC/B. This analysis explains the molecular basis underlying the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.     

Mammalian Myosin-18A,a Highly Divergent Myosin

The Mus musculus myosin-18A gene is expressed as two alternatively spliced isoforms, α and β, with reported roles in Golgi localization, in maintenance of cytoskeleton, and as receptors for immunological surfactant proteins. Both myosin-18A isoforms feature a myosin motor domain, a single predicted IQ motif, and a long coiled-coil reminiscent of myosin-2. The myosin-18Aα isoform, additionally, has an N-terminal PDZ domain. Recombinant heavy meromyosin- and subfragment-1 (S1)-like constructs for both myosin-18Aα and -18β species were purified from the baculovirus/Sf9 cell expression system. These constructs bound both essential and regulatory light chains, indicating an additional noncanonical light chain binding site in the neck. Myosin-18Aα-S1 and -18Aβ-S1 molecules bound actin weakly with K_d values of 4.9 and 54 μm, respectively. The actin binding data could be modeled by assuming an equilibrium between two myosin conformations, a competent and an incompetent form to bind actin. Actin binding was unchanged by presence of nucleotide. Both myosin-18A isoforms bound N-methylanthraniloyl-nucleotides, but the rate of ATP hydrolysis was very slow (<0.002 s⁻¹) and not significantly enhanced by actin. Phosphorylation of the regulatory light chain had no effect on ATP hydrolysis, and neither did the addition of tropomyosin or of GOLPH3, a myosin-18A binding partner. Electron microscopy of myosin-18A-S1 showed that the lever is strongly angled with respect to the long axis of the motor domain, suggesting a pre-power stroke conformation regardless of the presence of ATP. These data lead us to conclude that myosin-18A does not operate as a traditional molecular motor in cells.     

Myosin polymorphism in urodelan amphibian dorsal skeletal muscle

• 1.1. Myosin isoforms were analyzed in the dorsal skeletal muscle of four urodelan amphibian species using a modified pyrophosphate gel electrophoresis which allowed better discrimination than classical methods.
• 2.2. The three fast and the intermediate isomyosins were characterized by a specific heavy chain, respectively HCf and HCi, associated with different combinations of the fast light chains LC1f, LC2f and LC3f.
• 3.3. Slow myosin was characterized by one (P. waltlii, T. palmatus, S. maculata) or two (T. alpestris) isoforms, combining a specific slow myosin heavy chain (HCs) with slow light chains only in the case of P. waltlii, or with slow and fast light chains in the other species.

     

Functional characterization of the N-terminal region of myosin-2

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.     

Temperature-dependent conformational transition in the head-rod junctional region of the myosin molecule

The effects of temperature, Mg2+, ATP, and actin on the conformation of the neck region of the myosin head were studied by limited proteolysis of heavy meromyosin (HMM) and subfragment 1 (S1) preparations obtained by papain digestion of myosin in the presence of Mg2+ (Mg-S1) or EDTA (EDTA-S1). The preparations were fluorescently labelled at the SH1 thiol group to enable identification of the COOH-terminal fragments of the head portion of the heavy chain where this group is located. The results indicate that the head-rod junctional region of the myosin heavy chain contains at least three different sites readily susceptible to trypsin at 25 degrees C if the light chain LC2 or its LC2' fragment are absent. The susceptibility of one of these sites dramatically decreases when the temperature is lowered to 0 degree C, indicating a temperature-dependent conformational transition in the head-rod junction. With the method used, this transition is detectable only in LC2/LC'2-deficient preparations since all three sites are protected, although to different extents, by LC2 and its LC'2 derivative. It is, however, most probable that the effect of the light chain is confined to steric hindrance of trypsin access and that the temperature-dependent structural transition in the head-rod junction can occur in the presence of intact LC2 as well and may contribute to the temperature sensitivity of force generation in muscle.     

Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres.

Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide ''maps'' published in Cleveland. Fischer, Kirschner & Laemmli [(1977) J. Biol. Chem. 252, 1102-1106], allowed a classification of muscle fibres into four classes, corresponding to histochemical types I, IIA, IIB and IIC. Type I fibres with a pure slow-twitch-type of myosin were found to be characterized by a unique set of isoforms of troponins I, C and T, in agreement with the immunological data of Dhoot & Perry [(1979) Nature (London) 278, 714-718], by predominance of the beta-tropomyosin subunit and by the presence of a small amount of an additional tropomyosin subunit, apparently dissimilar from fast-twitch-fibre alpha-tropomyosin subunit. The myofibrillar composition of type IIB fast-twitch white fibres was the mirror image of that found for slow-twitch fibres in that the fast-twitch-fibre isoforms only of the troponin subunits were present and the alpha-tropomyosin subunit predominated. Type IIA fast-twitch red fibres showed a troponin subunit composition identical with that of type IIB fast-twitch white fibres. On the other hand, a unique type of myosin heavy chains was found to be associated with type IIA fibres. Furthermore, the myosin light-chain composition of these fibres was invariably characterized by a small amount of LC3F light chain and by a pattern that was either a pure fast-twitch-fibre light-chain pattern or a hybrid LC1F/LC2F/LC3F/LC1Sb light-chain pattern. By these criteria type IIA fibres could be distinguished from type IIC intermediate fibres, which showed coexistence of fast-twitch-fibre and slow-twitch-fibre forms of myosin light chains and of troponin subunits.     

Phosphorylated and dephosphorylated myosin light chains of Drosophila fly and larva

1. The present study confirmed that light chains of Drosophila adult fibrillar (flight) muscle myosin consist of Lf1, Lf2, Lf2' and Lf3, and tubular muscle myosin light chains contain Lt1, Lt2, Lt2' and Lt3, as revealed by two-dimensional (isoelectric focusing and SDS-gel electrophoresis) gel electrophoresis. 2. Larva myosin light chains were of all the tubular type. However, it was found that Lt1 and Lt2' are produced by phosphorylation of Lt2, and Lf1 is produced by phosphorylation of Lf2'. 3. Injection of radioactive phosphate into Drosophila fly resulted in phosphorylations of Lf1 and Lt1. When larva or late pupa myosin was incubated with myosin light chain kinase from chicken gizzard or adult flies, phosphorylation of Lt1, Lf2' and Lt2' occurred. Drosophila myosin light chain kinase phosphorylated Lf1 in addition to Lt1 and L2' (Lf2' + Lt2') of adult myosin. 4. Dephosphorylation of adult myosin by potato acid and calf intestine alkaline phosphatases led to the shift of Lf1 (34,000), Lt1 (31,000) and L2' (Lf2' + Lt2') (30,000) to L2 (Lf2 + Lt2) positions (30,000). 5. Peptide mapping analyses revealed that larva Lt1, Lt2', Lt2 and adult Lt1 were all the same; therefore, it is thought that a single species of Lt2 specific to the tubular type of myosin and its phosphorylated isoforms (Lt1, Lt2') exist. 6. The peptide map of Lf1 was slightly different from that of Lt1, but very similar to that of L2' in adult myosin. L2 and L2' of adult myosin showed very similar peptide maps, but there were several different peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of removal of light chain 2 on the ATPase activities of cardiac myosin from normal and thyrotoxic rabbits

Steady-state ATPase activities of cardiac myosin from thyroxine-treated rabbit hearts have been determined before and after removal of the 18-kDa light-chain subunit (LC2) of myosin. LC2 was selectively removed from myosin by treatment with a myofibrillar protease according to the method of Kuo and Bhan (Biochem. Biophys. Res. Commun. 92, 570-576 (1980) ). The effects of removal of LC2 on the enzymatic properties of thyrotoxic myosin were compared with the results obtained for cardiac myosin from normal rabbits by parallel studies. It has been found that removal of LC2 does not affect the Ca2+- and K+ (EDTA)-ATPase activities of these myosins. The actin-activated myosin Mg2+-ATPase activities of intact and LC2-deficient thyrotoxic myosin were 0.18 +/- 0.03 and 0.36 +/- 0.03 mumol Pi/mg per min, respectively, whereas the actin-activated myosin Mg2+-ATPase activities of intact and LC2-deficient normal myosin were 0.12 +/- 0.02 and 0.18 +/- 0.03 mumol Pi/mg per min, respectively. Thus, removal of LC2 increases the actin-activated myosin Mg2+-ATPase activity of thyrotoxic myosin by 100%, and the same activity is increased about 50% for normal myosin, indicating that the degree of potentiation of actin-activated myosin Mg2+-ATPase activity as a result of LC2 removal is 2-fold greater in thyrotoxic myosin than that obtained for normal myosin. These results suggest that LC2 does not influence the increased actomyosin ATPase activity of thyrotoxic myosin and that potentiation of actomyosin ATPase following LC2 removal may depend on the variations of the heavy-chain domain where LC2 interacts.     

Myosin structure and thyroidian control of myosin synthesis in urodelan amphibian skeletal muscle

Electrophoretic analysis in non-dissociating conditions reveals three types of myosin in adult urodelan amphibian skeletal muscles: 3 isoforms of fast myosin (FM), one isoform of intermediate myosin (IM) and one or two isoforms of slow myosin (SM). Each type is characterized by a specific heavy chain HCf (FM), HCi (IM) and HCs (SM), respectively. In all urodelan species, as in mammals, fast isomyosins associate HCf and the three fast light chains LC1f, LC2f, and LC3f. In most urodelan species the intermediate myosin contains LC1f and LC2f and can be considered as an homodimer of the alkali LC1f. However, in Euproctus asper, IM is characterized by the association of both slow and fast LC with HCi. Slow myosin is a hybrid molecule associating HCs with slow and fast LC. During metamorphosis, a myosin isoenzymic transition occurs consisting in the replacement of three larval myosins (LM) characterized by a specific heavy chain (HCI), by the adult isomyosins with lower electrophoretic mobilities. At the same time there is a change in the ATPase myofibrillar pattern, with the larval fiber types being replaced by adult fibers of types I, IIA and IIB. In the neotenic and perennibranchiate species, which do not undergo spontaneous metamorphosis, sexually mature larval animals present a change in the myosin isoenzymic profile, but no complete transition. The coexistence of larval and adult isomyosins and the persistence of transitional fibers of type IIC in the skeletal muscle are demonstrated. Experimental hypo- and hyperthyroidism indicate that thyroid hormone stimulates the regression of the larval isomyosins, possibly through indirect pathways. In contrast, the appearance and the persistence of the adult isomyosins seem to be independent of thyroid hormone. Thus, the control of the isoenzymic transition in the skeletal muscle of urodelan amphibians appears to imply indirect mechanisms, operating differently on each of the two phases of the complete transition.     

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the myosin head in solution and the effect of light chain 2 removal.

Structural properties of rabbit skeletal myosin head (S1) and the influence of the DTNB light chain (LC2) on the size and shape of myosin heads in solution were investigated by small angle x-ray scattering. The LC2 deficient myosin head, S1 (-LC2), and the S1 containing LC2 light chain, S1 (+LC2) were studied in parallel. The respective values of the radius of gyration were found to be (40.2 +/- 0.5) A and (46.7 +/- 1) A, while the maximum dimension was (190 +/- 15) A for both species. The large difference between the two Rg values suggest that LC2 is located close to one extremity of the myosin head, in agreement with most electron microscopy observations. All models derived from the x-ray scattering pattern of the native myosin head share a common overall morphology, showing two main regions, an asymmetric globular portion which tapers smoothly into a thinner domain of roughly equivalent length making an angle of approximately 60 degrees, with a contour length of approximately 210 A.