Analysis of the regulatory phosphorylation site in Acanthamoeba myosin IC by using site-directed mutagenesis

The actin-activated ATPase activity of Acanthamoeba myosin IC is stimulated 15- to 20-fold by phosphorylation of Ser-329 in the heavy chain. In most myosins, either glutamate or aspartate occupies this position, which lies within a surface loop that forms part of the actomyosin interface. To investigate the apparent need for a negative charge at this site, we mutated Ser-329 to alanine, asparagine, aspartate, or glutamate and coexpressed the Flag-tagged wild-type or mutant heavy chain and light chain in baculovirus-infected insect cells. Recombinant wild-type myosin IC was indistinguishable from myosin IC purified from Acanthamoeba as determined by (i) the dependence of its actin-activated ATPase activity on heavy-chain phosphorylation, (ii) the unusual triphasic dependence of its ATPase activity on the concentration of F-actin, (iii) its K_m for ATP, and (iv) its ability to translocate actin filaments. The Ala and Asn mutants had the same low actin-activated ATPase activity as unphosphorylated wild-type myosin IC. The Glu mutant, like the phosphorylated wild-type protein, was 16-fold more active than unphosphorylated wild type, and the Asp mutant was 8-fold more active. The wild-type and mutant proteins had the same K_m for ATP. Unphosphorylated wild-type protein and the Ala and Asn mutants were unable to translocate actin filaments, whereas the Glu mutant translocated filaments at the same velocity, and the Asp mutant at 50% the velocity, as phosphorylated wild-type proteins. These results demonstrate that an acidic amino acid can supply the negative charge in the surface loop required for the actin-dependent activities of Acanthamoeba myosin IC in vitro and indicate that the length of the side chain that delivers this charge is important.     

Discovery of ultrafast myosin,its amino acid sequence,and structural features

Cytoplasmic streaming with extremely high velocity (∼70 μm s⁻¹) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 μm s⁻¹, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 μm s⁻¹, one-third of the cytoplasmic streaming velocity in Chara. Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 μm s⁻¹. These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

Myosins are motor proteins that convert chemical energy, ATP, to physical force to move actin filaments. Phylogenetic analyses of myosin motor domain (MD) sequences have shown that there are at least 79 myosin classes, with several subclasses under each class (1). Myosins of different classes and subclasses differ significantly in properties such as velocity, ATPase activity, and duty ratio (the proportion of the ATPase cycle in which the MD remains strongly bound to actin) and perform different intracellular functions (2). The diversity of properties of these classes and subclasses arise from differences in the rates of the binding and dissociation of ATP, ADP, and actin filaments (3).Plants have two plant-specific myosin classes, myosin VIII and myosin XI. Myosin VIII moves actin filaments at very slow velocities (4) and is involved in endocytosis, cell plate formation, and plasmodesmatal functioning in plants (5–7). Myosin XI produces an intracellular flow known as cytoplasmic streaming in plant cells by moving on actin filaments while binding organelles via its tail domain. Cytoplasmic streaming facilitates the distribution of molecules and vesicles throughout large plant cells (8–12). The velocities of myosin XI are generally high, and the molecule specializes in cytoplasmic streaming. Some cells of characean algae (Chara) are very large, being up to 10 cm long and 0.1 cm in diameter. Very fast cytoplasmic streaming, of up to 70 μm s⁻¹, is required for the dispersal of molecules and vesicles into the giant Chara cells (13).Based on the velocity of cytoplasmic streaming in Chara cells, it has long been suggested that Chara has a myosin moving on actin filaments at 70 µm s⁻¹ (13–17). This velocity is 10 times faster than the velocity of fast skeletal muscle myosin and the fastest of all myosins measured. A motor protein isolated from Chara cells moved actin filaments at 60 μm s⁻¹ (18). The development of approaches for cloning this ultrafast myosin is urgently needed. Details of the sequence of the protein and the ability to work with cloned myosin constructs will allow the investigation of the mechanisms that control the myosin velocity and facilitate investigation of the detailed chemical–mechanical conversion mechanism of myosin (19). Kashiyama et al. cloned the complementary DNA (cDNA) of Chara myosin from a Chara corallina cDNA library by immunoscreening using antibodies against purified C. corallina myosin (20). Morimatsu et al. also cloned the cDNA of Chara myosin using the same method as that used by Kashiyama et al. (21). The sequences of the MD of myosins cloned by the two groups were identical, and there was a 15 amino acid indel variation in the tail domain, a finding that indicates potential alternative splicing in the tail domain. The C. corallina myosin XI (CcXI) contains six isoleucine–glutamine (IQ) motifs, which are light chain–binding sites. It was not possible to express the protein and measure its velocity using the cloned CcXI, because the myosin light chains that bind to the six IQ motifs of CcXI have not been identified. Therefore, the functional expression of CcXI has been carried out using either a CcXI MD construct that did not have the myosin light chain–binding sites (IQ motifs) or chimeric full-length CcXI constructs in which IQ motifs and myosin light chains of CcXI were replaced with those of other myosins. The velocity of CcXI was then estimated from the velocity measured using these constructs. The estimated velocity of CcXI was about 20 µm/s⁻¹ or less at 25 °C (10, 22–26), which is less than about one-third of the velocity of cytoplasmic streaming observed in Chara cells. Three possibilities have been suggested as to why the velocities of CcXI obtained using the recombinant constructs were different from that expected from cytoplasmic streaming (1). The recombinant CcXI constructs do not have the same IQ motifs and myosin light chains as native CcXI, and this substitution may have affected the velocity (2). CcXI may undergo a posttranslational modification in Chara cells, which may increase the velocity of CcXI in cells (3). A myosin XI gene other than CcXI may be present in Chara cells, and this myosin XI may be responsible for cytoplasmic streaming with a velocity of 70 µm s⁻¹.Recently, a genome project (Chara braunii genome sequencing project, National Center for Biotechnology Information (NCBI) Bio Project ID: PRJDB3348) has been conducted for C. braunii (27). C. braunii is phylogenetically close to C. corallina (28–30), and both species have the same cytoplasmic streaming velocity, 70 µm s⁻¹. The Chara genome project revealed that the C. braunii genome contains four myosin XI genes.In this study, we cloned the four C. braunii myosin XIs and named them CbXI-1, CbXI-2, CbXI-3, and CbXI-4. Phylogenetic analyses indicated that the myosin XIs in Chara form a clade in streptophyte myosin XIs, expanded independently from seed plant myosin XIs, and gave rise to the four members in C. braunii. CbXI-4 may be an ortholog of CcXI. We show that the velocity of CbXI-1 (60 µm s⁻¹) is almost the same as the velocity of cytoplasmic streaming in Chara cells, the fastest currently known in the biological world. We also succeeded in crystallizing Arabidopsis myosin XI-2 (AtXI-2), an atomic structure of myosin XI and a valuable comparator for the Chara myosin. Structural analyses and mutation experiments suggest that the central regions that define Chara myosin XI''s fast movement are the actin-binding sites.     

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.     

Changes in rat cardiac myosin during development and in culture

Developmental changes in the subunit composition and ATPase activity of myosin isolated from rat ventricular myocardium and 5-day-old myocardial tissue cultures were examined. Electrophoretic analysis of cardiac myosin from 12-week-old adults, 9-, and 16-day neonates, and 5-day tissue cultures demonstrated two light chains (Mol. wt 25 500 and 20 000) with a molar ratio of 1: 1. In 21-day fetal myosin, three light chains were observed (Mol. wt: 25 500; 24 500; 20 000), with a molar ratio of 0.82:0.15: 1.0. Analysis of ATPase activity in the presence of three activators (Ca²⁺, K⁺, and F-actin) showed significant differences between these myosin preparations, though the time course of the change was activator specific. The change in K⁺-activated ATPase activity occurred soon after birth and correlated with the disappearance of the third light chain (Mol. wt 24 500) and a partial isozymic shift from V₃ to V₁ myosin. The Ca²⁺- and actin-activated ATPase activities increased more slowly and were accompanied by continuation of the V₃ to V₁ myosin shift. Thus, it appears that V₃ myosin is heterogeneous. Moreover, kinetic analysis of tissue culture myosin is consistent with the predominance of V₃ myosin with low K⁺-activated ATPase activity.     

Effects of severe hemodynamic pressure overload on the properties of canine left ventricular myosin: mechanism by which myosin ATPase activity is lowered during chronic increased hemodynamic stress.

Left ventricular myosin ATPase activity, expressed as enzymatic V_max values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K⁺ and Ca²⁺ activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .01) in the experimental animals. For normal K⁺ activated myosin the V_max values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca²⁺ activated myosin the V_max values were: right ventricle, 0.77; left ventricle 0.97, when assayed at 37°C. Myosin enzymatic activity in the left ventricle progressively declined following severe aortic banding, reaching a value similar to that observed for normal right ventricular myosin; NH₄⁺ activated left ventricular myosin ATPase activity remained unchanged (7.20 ± 0.4 μmol PO₄/mg.min). Left ventricular myosin from the hearts subject to severe stress simulated normal right ventricular myosin in ATPase activity, chain proportions and degree of calcium binding, Normal left ventricular myosin contained approximately 10% of the myosin protein concentration in the light chains; myosin from the left ventricles of the hemodynamically overloaded hearts contained 20% of the myosin protein concentration in the light chains (P < .001). With only one of the myosin light chains binding calcium left ventricular myosin from the stressed hypertrophied tissue bound approximately 2 mol Ca²⁺ mol^?1 myosin similar to myosin of the normal right ventricle; normal left ventricular myosin bound approximately 1 mol of Ca²⁺ mol^?1 myosin.     

Hormonal influences on cardiac myosin ATPase activity and myosin isoenzyme distribution

It has been recognized for a long time that changes in hormone secretion can influence cardiac function; however, the biochemical basis for these changes has only recently been clarified. In this review the influences of hormonal status on the contractile protein myosin is discussed. Myosin has a rod-like portion and a globular head and consists of two myosin heavy chains (MHC) and four light chains (LC), two of which are identical. The globular head is the site of an ATP-splitting enzyme, the myosin ATPase, and increases in myosin ATPase activity are closely related to an increased velocity of contraction of the heart. Myosin ATPase activity shows marked response to alterations in thyroid hormone, insulin, glucocorticoid, testosterone and catecholamine levels, but marked animal species differences in this response occur. Thyroid hormone administration to normal rabbits, for example, increases myosin ATPase activity markedly, but the myosin ATPase activity of hyperthyroid rats remains unchanged. In contrast, in hypothyroid rats myosin ATPase activity is markedly decreased but the hypothyroid rabbit shows no such response. These species-related differences in the hormonal response of myosin ATPase activity result from the predominance pattern of specific myosin isoenzymes. In the normal rat heart three myosin isoenzymes, v₁, V₂ and V₃, can be separated electrophoretically. Myosin V₁ predominates (70% of total myosin), and has the highest myosin ATPase activity, whereas in rabbits myosin v₃, which has a lower myosin ATPase activity, is the predominant isomyosin. Thyroid hormone administration to rabbits induces myosin V₁ predominance and therefore increases myosin ATPase activity, whereas in hyperthyroid rats only a small further increase in V₁ predominance can occur. The alterations in myosin isoenzyme predominance and myosin ATPase activity are closely correlated to changes in cardiac contractility. Hormone-induced alterations in myosin isoenzyme predominance are mediated through changes in the formation of two isoforms of myosin heavy chain. Changes in the expression of different myosin heavy chain genes are most likely responsible for the thyroid hormone and insulin-induced alterations in myosin isoenzyme predominance. Investigation of the control of myosin heavy chain formation can provide further insights into the hormonal control of a multigene family as well as broaden our understanding of the molecular events which result in altered cardiac contractility. It is currently unclear if androgens, glucocorticoids and catecholamines influence myosin ATPase activity through changes in myosin isoenzyme predominance resulting from alterations in myosin heavy chain gene expression. Post-translational modifications of myosin heavy chain and light chain polypeptides have also to be considered.     

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca²⁺/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A’s activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.     

Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II

In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin–myosin–ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1–SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1–SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.     

Determinants for the syncytium-inducing phenotype of HIV-1 subtype F isolates are located in the V3 region

The HIV-1 syncytium-inducing phenotype is determined by virus replication and the presence of cytopathic effects in MT-2 cells. There is a strong correlation between the syncytium-inducing/MT-2-tropic phenotype and positively charged amino acids at positions 306 and 320 in the V3 loop for HIV-1 subtypes A, B, D, and E. In contrast, a lack of correlation between signature amino acids and syncytium formation in MT-2 cells for subtype F viruses from Romania has been reported. Virus phenotype and V3 loop amino acid sequences from Romanian HIV-1 subtype F isolates were further investigated in the present study. While the determinants of MT-2 tropism are clearly harbored in the V3 loop of subtype F isolates from Romania, the induction of syncytium formation occurs in the presence or absence of positively charged amino acids at positions 306, 320, and/or 324. However, the net positive charge of V3 loop sequences derived from syncytium-inducing viruses was higher than that of the nonsyncytium-inducing isolate.     

The role of surface loops (residues 204-216 and 627-646) in the motor function of the myosin head.

A characteristic feature of all myosins is the presence of two sequences which despite considerable variations in length and composition can be aligned with loops 1 (residues 204-216) and 2 (residues 627-646) in the chicken myosin-head heavy chain sequence. Recently, an intriguing hypothesis has been put forth suggesting that diverse performances of myosin motors are achieved through variations in the sequences of loops 1 and 2 [Spudich, J. (1994) Nature (London) 372, 515-518]. Here, we report on the study of the effects of tryptic digestion of these loops on the motor and enzymatic functions of myosin. Tryptic digestions of myosin, which produced heavy meromyosin (HMM) with different percentages of molecules cleaved at both loop 1 and loop 2, resulted in the consistent decrease in the sliding velocity of actin filaments over HMM in the in vitro motility assays, did not affect the Vmax, and increased the Km values for actin-activated ATPase of HMM. Selective cleavage of loop 2 on HMM decreased its affinity for actin but did not change the sliding velocity of actin in the in vitro motility assays. The cleavage of loop 1 and HMM decreased the mean sliding velocity of actin in such assays by almost 50% but did not alter its affinity for HMM. To test for a possible kinetic determinant of the change in motility, 1-N6-ethenoadenosine diphosphate (epsilon-ADP) release from cleaved and uncleaved myosin subfragment 1 (S1) was examined. Tryptic digestion of loop 1 slightly accelerated the release of epsilon-ADP from S1 but did not affect the rate of epsilon-ADP release from acto-S1 complex. Overall, the results of this work support the hypothesis that loop 1 can modulate the motor function of myosin and suggest that such modulation involves a mechanism other than regulation of ADP release from myosin.     

Alterations of mechanical parameters in chemically skinned preparations of rat myocardium as a function of isoenzyme pattern of myosin

Summary Myofibrillar ATPase activity, maximum unloaded shortening velocity, and isometric tension development were evaluated in left ventricular preparations of 5-week-old rats with a high endogeneous level of thyroid hormones and hypothyroid rats after 4-week treatment with propylthiouracil (PTU). The range of possible alterations of the above functional parameters was defined in relation to myosin isoenzyme distribution. The mechanical behaviour of the ventricular preparations was investigated in native myocardium as well as in the glycerinated state.The essential result of the present study is that alterations of myofibrillar ATPase activity and mechanical v_max, evaluated in glycerinated preparations, are limited to a well-defined range of similar magnitude for both functional parameters: 32–40% of maximum values (obtained from rat myocardium with homogeneous myosin V₁). Isometric tension was only insignificantly decreased in glycerinated preparations of the PTU-treated group.The alteration in the apparent maximum shortening velocity of native myocardium (v₀) was of the same magnitude as changes in v_max of chemically skinned preparations. Physical training revealed a shift in the direction of V₁-type myosin with increased ATPase activity and shortening velocity; aging and pressure overload showed an opposite effect. The documented mechanical alterations do not contradict an adaptational interpretation of the myosin isoenzyme redistribution in pressure-induced hypertrophy.Supported by the Deutsche Forschungsgemeinschaft.     

Dissociation of myosin light chains and decreased myosin ATPase activity with acidification of synthetic myosin filaments: Possible clues to the fate of myosin in myocardial ischemia and infarction

The effect of mild acidification of synthetic (reconstituted) myosin filaments was studied in order to gain insight into some of the possible effects of ischemia-induced intracellular acidosis on the structure and function of myosin following myocardial infarction and myocardial ischemia. Degradation products of myosin that are soluble (at physiologic ionic strength and pH) would be of potential diagnostic value for myocardial infarction. Acidification of rabbit skeletal synthetic myosin filaments led to a pH dependent partial dissociation of the heaviest (LC₁) and lightest (LC₃) of the 3 light chains. Dissociation was detected from pH 5.0 to 6.5 and was maximal at pH 6.0, at which 30% of LC₁ was dissociated. Acidification of canine cardiac synthetic myosin filaments led to partial dissociation of both light chains; but more LC₁ than LC₂ was dissociated. Light chains reassociated with heavy chains upon return of the pH to 7. Light chains of myosin have recently been reported to appear in the peripheral blood after myocardial infarction but the small amount of free light chains in the heart is insufficient to account for the amount that appears in the blood. Acid-mediated dissociation of light chains in vitro suggests that circulating light chains after myocardial infarction may arise as a result of the intracellular acidosis of ischemic myocytes. The mechanisms responsible for the acidification-induced decrease in myofibrillar actomyosin adenosine triphosphatase (ATPase) activity are unclear. One possibility is that the decreased myofibrillar ATPase activity is due in part to an acid-induced decrease of the myosin ATPase of the myofibril irrespective of the effect of acid on the troponin-tropomyosin regulatory system. This possible mechanism is supported by the observations that acidification of rabbit skeletal and human and canine cardiac synthetic myosin filaments resulted in a reduction of ATPase activity (measured at pH 7.5) of the redissolved myosin which was progressive with greater acidification. The reduction in ATPase activity occurred whether the return of the myosin to pH 7.5 was accomplished in the presence or absence of dissociated light chains.     

Structural and functional properties of myosin associated with the compensatory cardiac hypertrophy in the rabbit.

Right ventricular hypertrophy was produced in rabbits by pulmonary artery constriction. Measurement of intraventricular pressure, ventricular wet and dry weights and histologic examination of the liver demonstrated substantial hypertrophy with no signs of congestive failure 3 to 6 weeks following constriction. Right ventricular papillary muscle from these hearts exhibited a depressed force-velocity relationship and an increased time to peak tension but otherwise were normal. Myosin prepared from control and hypertrophied hearts was of comparable purity and yield. Quantitative densitometry of SDS polyacrylamide gels showed the light chains of hypertrophy myosin were unchanged in number, molecular weight and stoichiometry. Total protein sulfhydryl content was not different for control and hypertrophy myosins. Calcium-activated ATPase activity of hypertrophy myosin was significantly decreased shen measured at varying ATP concentration. By Lineweaver-Burk analysis, V_max of hypertrophy myosin was depressed by 50%, while the apparent K_m was 10% lower. When sulfhydryl-dependent ATPase activity was measured as a function of p-chloromercuribenzoate (PCMB) concentration, hypertrophy myosin exhibited a normal activation profile. The ATPase activity of hypertrophy myosin was initially depressed by 50% and was significantly reduced at low PCMB concentrations. However the difference was absent at peak activation, suggesting that the SH₁ sulfhydryls were capable of activation by PCMB to the same level as for control myosin. These data were consistent with a subtle conformational change at or near the active site, rather than with a change in the composition of major subunits of myosin from hypertrophied hearts.     

Polymorphic myosin as the common determinant of myofibrillar ATPase in different haemodynamic and thyroid states

Summary Chronic alterations in haemodynamic load or thyroid state affect the ATPase activity of myofibrils from the rat heart. In order to determine the limits of such an adaptational reaction, the Ca²⁺-dependent activation of myofibrils containing only myosin V₁ or V₃ was investigated. These limiting states were achieved by changes in the thyroid state, i.e., myosin V₁ myofibrils were obtained from 5-week-old rats, and myosin V₃ myofibrils from hypothyroid rats. Analysis of the activation of myofibrillar ATPase by Ca²⁺ using the model-independent Hill equation showed that transition of the myosin isoenzymes neither affected Ca²⁺ sensitivity (pCa_50%=6.52) nor positive co-operativity (n=2.4). The ATPase activity at saturating Ca²⁺ concentrations was 192 nMole P mg^–1 min^–1 in myosin V₁-myofibrils and was reduced to 131 nMole P mg^–1 min^–1 in the case of myosin V₃-myofibrils. Beside changes in the thyroid hormone state, polymorphic myosin is also influenced by chronic pressure overload and endurance training of a swimming routine. Such haemodynamically induced changes in myofibrillar ATPase were accounted for on the basis of the altered myosin isoenzyme pattern and the ATPase activities of isoenzymatically homogeneous myofibrils obtained by changes in the thyroid state. It can therefore be concluded that both haemodynamic load and thyroid state affect myofibrillar ATPase by a common mechanism, namely, by inducing a change in the isoenzyme pattern of myosin due to a redirected expression of myosin genes. Myosins from other mammals were compared with rat myosin on pyrophosphate gels. The Ca²⁺-dependent activation of myofibrillar ATPase from euthyroid and hypothyroid adult mice suggested the existence of a polymorphic myosin which was verified by the pyrophosphate gel technique. The mouse myosin isoenzymes were, however, less separated on pyrophosphate gels when compared with the rat myosins. It therefore seems advisable to consider both the activation properties of myofibrillar ATPase, as well as electrophoretic properties, for defining the adaptational state of myocardium of higher mammalian species.

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Zusammenfassung Die ATPase-Aktivität von Myofibrillen aus Rattenherz wird durch chronische Veränderungen in der hämodynamischen Belastung oder im Status der Schilddrüsenhormone beeinflußt. Um die Grenzen solcher Anpassungsmechanismen abzustecken, wurde die Ca²⁺-abhängige Aktivierung von Myofibrillen, die entweder Myosin V₁ oder Myosin V₃ enthielten, untersucht. Diese den Anpassungsbereich begrenzenden Zustände wurden durch Veränderungen im Spiegel der Schilddrüsenhormone erzeugt: Myosin V₁-Myofibrillen wurden aus 5 Wochen alten Ratten und Myosin V₃-Myofibrillen wurden aus hypothyreoten Ratten isoliert. Eine Analyse der Ca²⁺-abhängigen Aktivierung der Myofibrillen auf der Grundlage der Hill-Gleichung zeigte, daß weder die Ca²⁺-Sensitivität (pCa_50%=6.52) noch die positive Kooperativität (n=2.4) verändert ist. Die ATPase-Aktivität bei Vollaktivierung war 192 nMol P mg^–1 min^–1 bei Myosin V₁-Myofibrillen und 131 nMol P mg^–1 min^–1 bei Myosin V₃-Myofibrillen. Neben Veränderungen im Status der Schilddrüsenhormone wird das polymorphe Myosin auch durch eine chronische Druckbelastung und durch das Ausdauertraining eines Schwimmprogrammes beeinflußt. Solche Veränderungen in der myofibrillären ATPase lassen sich erklären auf Grund des veränderten Isoenzymmusters und der ATPase-Aktivitäten von Myofibrillen, die homogen sind bezüglich der Isoenzyme, die durch Veränderungen im Status der Schilddrüsenhormone induziert wurden. Die hämodynamische Belastung und die Schilddrüsenhormone beeinflussen daher die myofibrilläre ATPase in einem beiden gemeinsamen Mechanismus: Sie führen zu einer Veränderung im Isoenzymmuster des Myosins als Folge einer veränderten Genexpression.Die Myosine anderer Säugetiere wurden mit Hilfe der Pyrophosphat-Gel-Elektrophorese mit dem Myosin der Ratte verglichen. Die Ca²⁺-abhängige Aktivierung der myofibrillären ATPase von euthyreoten und hypothyreoten, erwachsenen Mäusen legte ein polymorphes Myosin nahe, das mit Hilfe der Pyrophosphat-Gel-Elektrophorese bestätigt wurde. Die Auftrennung der Myosin-Isoenzyme der Maus war jedoch geringer als die der Ratte. Es wird daher empfohlen, die Ca²⁺-abhängige Aktivierung der myofibrillären ATPase neben elektrophoretischer Eigenschaften für die Charakterisierung des myokardialen Anpassungszustandes höherer Säugetiere heranzuziehen.

     

Structural and functional changes of the contractile proteins in experimentally-induced cardiac hypertrophy in animals, and heart failure in man

During the first acute stage of heart over-loading after experimentally-induced aortic stenosis, the ATPase activity of isolated actomyosin and the contractility of glycerinized fibrils from the left ventricle decrease. The restoration of the initial ATPase activity level and contractility of the fibrils takes place with the development of hypertrophy, during 3 months. The second stage—relatively stable cardiac hyperfunction (6 to 8 months after coarctation of aorta in dogs)—is characterized by an increased enzymic activity of the contractile proteins and some changes in their physicochemical properties (increase of absorption in the u.v. region, decrease of K_m and V_max for the reaction of tryptic digestion, values of Δn and “melting” point of myosin). At the same time, a significant activation is observed in the ATPase of the contractile proteins by strophanthin, as well as a marked myosin “anomaly” in neutral pH medium. Our results indicate the production of local changes in the HMM region of myosin. The detected phenomena suggest the possibility of the synthesis of a new myosin population (ATPase isozyme), at the stage of relatively stable cardiac hypertrophy.The investigation of the contractile proteins of biopsied left auricles from patients with mitral stenosis has demonstrated significant changes in their enzymic activity and other characteristics in accordance with the reduction in the degree of circulatory adaptability to exercise.     

Electrophoretic analysis of multiple forms of rat cardiac myosin: Effects of hypophysectomy and thyroxine replacement

Electrophoretic analysis in pyrophosphate gels of intact myosin of adult rat myocardium revealed the presence of five distinct components, two in atrial myosin (A₁, A₂) and three in ventricular myosin (V₁, V₂, V₃). Analysis of Ca²⁺-activated myosin ATPase activity in the gels revealed that A₁, A₂ and V₁ had about the same specific activity; V₃ had the lowest activity, while that of V₂ was intermediate. At 3 weeks of age, ventricular myosin was exclusively V₁, there being a slow age-dependent shift in myosin distribution toward the adult pattern. Hypophysectomy of juvenile rats caused a shift towards V₃, which by 45 days after operation accounted for 90% of total myosin. This shift was prevented by daily administration of 5 μg of thyroxine. When chronically hypophysectomized rats were similarly treated, there was a rapid shift of myosin components towards V₁. These changes in distribution of myosin components were associated with appropriate changes in Ca²⁺-activated ATPase activity of electrophoretically purified ventricular myosin as measured in the gel. Sodium dodecyl sulphate gel analysis of purified myosin showed little or no contaminants. For both V₁ and V₃ rich myosins, the ratio of the two ventricular light chains (V-LC₁, V-LC₂) and the ratio of light chains to heavy chains are consistent with the model that each intact molecule contains two each of V-LC₁ and V-LC₂. Sodium dodecyl sulphate electrophoresis of purified atrial myosin revealed two types of light chains: A-LC₁ (mol. wt 27 000, not resolved from V-LC₁) and A-LC₂ (mol. wt 22 000), the latter being distinct from V-LC₂.     

Alterations of subunit composition and ATPase activity of myosin in early hypertrophied right ventricles of dogs with mild experimental pulmonic stenosis.

Mild pulmonic stenosis was performed in dogs by banding of the pulmonary artery to evaluate the effect of systolic pressure overload on the enzymatic activity and subunit composition of myosin in early hypertrophied right ventricles. Three weeks following pulmonary constriction, 12 hypertrophied dogs were sacrificed and compared to 12 control animals. The weight of the hypertrophied right ventricles (HRV) relative to body weight was 46% greater than the weight of normal right ventricles (NRV) (P < .001) in dogs of similar body weights. Myosin ATPase activity (V_max values in μmol phosphate/mg min) was 25% higher in the stressed ventricles for both K⁺ and Ca²⁺ activated myosin (P < .001). Since the V_max values for the enzymatic activity of myosin with NH₄⁺ or Mn²⁺, as the activator cations, was the same in HRV and NRV, the augmented K⁺ and Ca²⁺ activity in HRV was not due to an increased concentration of more enzymatically active myosin. Associated with the increase in myosin activity there was a 33% decrease in the percent of light chains present in myosin from HRV as compared to myosin from NRV (P < .001). There was approximately 4 mol of myosin light chains/mol of myosin in NRV; in contrast, there was approximately 2 mol of myosin light chains/mole of myosin in HRV, similar to the proportion observed in NLV. The proportion of light chain C₁ to light chain C₂ did not change in myosin from HRV. Of the C₁ light chains analyzed on 2-dimensional gel electrophoresis, there was significantly less C_1d as compared to C_1c in HRV.     

Charge replacement near the phosphorylatable serine of the myosin regulatory light chain mimics aspects of phosphorylation.

Phosphorylation of the myosin regulatory lightchains (RLCs) activates contraction in smooth muscle and modulates forceproduction in striated muscle. RLC phosphorylation changes the net charge in acritical region of the N terminus and thereby may alter interactions between theRLC and myosin heavy chain. A series of N-terminal charge mutations in the humansmooth muscle RLC has been engineered, and the mutants have been evaluated fortheir ability to mimic the phosphorylated form of the RLC when reconstitutedinto scallop striated muscle bundles or into isolated smooth muscle myosin.Changing the net charge in the region from Arg-13 to Ser-19 potentiates force inscallop striated muscle and maintains smooth muscle myosin in an unfoldedfilamentous state without affecting ATPase activity or motility of smooth musclemyosin. Thus, the effect of RLC phosphorylation in striated muscle and itsability to regulate the folded-to-extended conformational transition in smoothmuscle may be due to a simple reduction of net charge at the N terminus of thelight chain. The ability of phosphorylation to regulate smooth musclemyosin's ATPase activity and motility involves a more complexmechanism.     

Effects of extracellular calcium concentrations on myosin P light chain phosphorylation in hearts from running-trained rats

Certain biochemical responses to clevated perfusate calcium concentrations were studied in the isolated hearts of sedentary and running-trained rats from which natural actomyosin was prepared. Both the V_max and Ca²⁺ sensitivity of the Ca²⁺-stimulated, Mg²⁺-dependent actomyosin adenosine triphosphatase (ATPase) were similar in the hearts of trained and sedentary animals, and were not altered when the concentration of extracellular Ca²⁺ was increased. Enhanced myosin Ca²⁺-ATPase activities and phosphate contents of myosin P light chains were found in the hearts of trained animals compared with controls, and these differences were still maintained when perfusate Ca²⁺ concentrations were increased. Although treatment of the perfused hearts with isoproterenol also increased both parameters in the trained and sedentary series, the V_max for myosin Ca²⁺-ATPase and the alkali-labile phosphate contents of myosin P light chains remained, throughout, significantly greater in the hearts of trained rats than in their sedentary counterparts. These differences were not eliminated by the combined use of isoproterenol and high perfusate Ca²⁺. The results suggest that an enhanced capacity for trans-sarcolemmal Ca²⁺ flux in the hearts of trained animals may be responsible for enhanced Ca²⁺-dependent phosphorylation of myosin P light chains, and thus improving cardiac function.     

Increased cardiac myosin ATPase activity as a biochemical adaptation to running training: enhanced response to catecholamines and a role for myosin phosphorylation

The effects were studied of prior running training on protein phosphorylation and adenosine triphosphatase (ATPase) activities of natural actomyosin isolated from perfused rat hearts. Myosin Ca²⁺-ATPase activities were significantly higher in running-trained hearts than in controls, whereas the Ca²⁺-stimulated, Mg²⁺-dependent ATPase activities of natural actomyosin were not changed. After treatment of isolated perfused hearts with the β-agonist isoproterenol, both troponin-I and myosin P light chains became phosphorylated. Troponin-I phosphorylation (1 mol/mol) was the same in both sets of hearts and was accompanied by similar changes in cardiac cyclic AMP contents. The V_max values for myosin Ca²⁺-ATPase activity were increased after isoproterenol treatment in all the perfused hearts, but to a significantly greater extent in the hearts of running trained animals; this was correlated with enhancement of both the rate and extent of myosin P light chain phosphorylation. Enhanced Ca²⁺-dependent myosin P light chain phosphorylation, further enhanced by β-adrenergic stimulation, represents, at the molecular level, a biochemical response to running training.