Addition Of Heavy Meromyosin Research Articles

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.

Chemical modification of tropomyosin with NBD-chloride.

Muscle tropomyosin was modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) at several different pH values. NBD-chloride reacts specifically with SH residue at neutral pH but it reacts with both SH residue and amino residues at alkaline pH. The polymerizability of tropomyosin at low ionic strength and the binding property of tropomyosin to F-actin were not affected by the modification of SH residues but they were lost rapidly by the modification of amino groups, in accordance with the previous report [Johnson, P. & Smillie, L.B. (1977) Biochemistry 16, 2264-2269]. By the addition of heavy meromyosin, labeled tropomyosin which could not bind to F-actin recovered the binding ability to F-actin and it could regulate the superprecipitation of actomyosin in the presence of troponin. Further modification of amino groups (labeling ratios more than 5) led to loss of the regulating ability completely.

Role of tropomyosin in actin filament formation in embryonic salamander heart cells.

Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.

Extraction and Functional Reformation of Thick Filaments in Chemically Skinned Molluscan Catch Muscle Fibers

A method for the almost complete extraction of myosin from smooth muscle fibers of the anterior byssal retractor muscle (ABRM) of Mytilus edulis was developed, and functional reformation of thick filaments in the fibers was achieved. Complete removal of myosin from the glycerol-extracted ABRM fibers with a solution containing 600 mM KCl, 5 mM MgCl2, and 5 mM ATP was difficult. However, successive treatments of the ABRM fibers with glycerol and saponin made the plasma membrane permeable to Mg-ATP and myosin. The extraction of myosin completely eliminated the tension induced by the addition of Mg-ATP. Partial recovery of tension development was observed by irrigation of myosin into fibers from which myosin had been extracted. Similar results were obtained using rabbit myosin instead of ABRM myosin. Addition of heavy meromyosin, on the other hand, had a suppressive effect on the tension development, as is the case in glycerinated rabbit psoas muscle fibers.

Morphological, Biochemical and Immunohistochemical Studies on Heart Development in Cardiac Mutant Axolotls,Ambystoma mexicanum

SYNOPSIS. A naturally-occurring genetic mutation, designated c for “cardiac lethal” in axolotls, Ambystoma mexicanum , is proving to be a useful model for studying myofibrillogenesis in differentiating heart cells. In this paper I describe morphological, biochemical and immunofluorescence studies which compare the contractile proteins in normal and mutant hearts. In addition, morphological studies on anterior endoderm, an important heart inductor tissue in salamanders, are reviewed. Detailed electron microscopic studies show that normal heart myocytes contain numerous well-organized myofibrils. Although mutant heart cells contain a few myosin and actin filaments, there are no organized myofibrils. Instead, amorphous proteinaceous collections are prominent in the peripheral cytoplasm of the cell where myofibrils would be expected to first form. SDS-polyacrylamide gel electrophoresis shows that actin is present in almost normal amounts in mutant hearts, myosin heavy chain is reduced and tropomyosin is virtually absent. Immunofluorescence studies reveal that myosin, α-actinin and tropomyosin are located prominently in theorganized myofibrils of normal heart cells. In mutant hearts myosin is localized almost exclusively in the amorphous collections at the cell peripheries, α-actinin also is distributed mainly in the peripheral cell cytoplasm. There is almost no staining for tropomyosin. Heavy meromyosin (HMM) binding experiments demonstrate that the actin in mutant heart cells is contained within the amorphous collections in a non-filamentous state and the addition of HMM causes its polymerization into filaments. In view of these findings, we undertook studies to determine whether there might be a causal relationship between theabsence of tropomyosin in mutants and the failure of actin to form into filaments. Our results indeed show that addition of tropomyosin to glycerinated mutant hearts or homogenates of mutant hearts causes the amorphous actin to form into filaments. Thus, this single gene mutation results in mutant heart cells having reduced, but significant, amounts of myosin and actin, even though non-filamentous, and substantial amounts of α-actinin. There is almost no tropomyosin. It is implied that the drastic reduction of tropomyosin in mutant cells is somehow related to the failure of normal myofilament formation, which in turn would seem to be an essential step in the normal organization of myofibrils.

Studies of muscle proteins in embryonic myocardial cells of cardiac lethal mutant mexican axolotls (Ambystoma mexicanum) by use of heavy meromyosin binding and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

In the Mexican axolotl Ambystoma mexicanum recessive mutant gene c, by way of abnormal inductive processes from surrounding tissues, results in an absence of embryonic heart function. The lack of contractions in mutant heart cells apparently results from their inability to form normally organized myofibrils, even though a few actin-like (60-A) and myosin-like (150-A) filaments are present. Amorphous "proteinaceous" collections are often visible. In the present study, heavy meromyosin (HMM) treatment of mutant heart tissue greatly increases the number of thin filaments and decorates them in the usual fashion, confirming that they are actin. The amorphous collections disappear with the addition of HMM. In addition, an analysis of the constituent proteins of normal and mutant embryonic hearts and other tissues is made by sodium dodecyl sulfate (SDS) gel electrophoresis. These experiments are in full agreement with the morphological and HMM binding studies. The gels show distinct 42,000-dalton bands for both normal and mutant hearts, supporting the presence of normal actin. During early developmental stages (Harrison's stage 34) the cardiac tissues in normal and mutant siblings have indistinguishable banding patterns, but with increasing development several differences appear. Myosin heavy chain (200,000 daltons) increases substantially in normal hearts during development but very little in mutants. Even so the quantity of 200,000-dalton protein in mutant hearts is significantly more than in any of the nonmuscle tissues studied (i.e. gut, liver, brain). Unlike normal hearts, the mutant hearts lack a prominent 34,000-dalton band, indicating that if mutants contain muscle tropomyosin at all, it is present in drastically reduced amounts. Also, mutant hearts retain large amounts of yolk proteins at stages when the platelets have virtually disappeared from normal hearts. The morphologies and electrophoresis patterns of skeletal muscle from normal and mutant siblings are identical, confirming that gene c affects only heart muscle differentiation and not skeletal muscle. The results of the study suggest that the precardiac mesoderm in cardiac lethal mutant axolotl embryos initiates but then fails to complete its differentiation into functional muscle tissue. It appears that this single gene mutation, by way of abnormal inductive processes, affects the accumulation and organization of several different muscle proteins, including actin, myosin, and tropomyosin.

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry: I. Conformation changes around the fluorescence-labeled sulfhydryl group of actin

Whether the conformation of the region containing the maleimide-reactive sulfhydryl group(s) in actin is associated with the functional states of the molecule was examined by means of fluorescence-label technique using a fluorescent sulfhydryl reagent, N-( p-(2-benzimidazolyl)phenyl)maleimide. 1. 1.|When G-actin labeled with the maleimide was polymerized to F-actin, the fluorescence intensity and the efficiency of the energy transfer from tryptophan residue(s) to the attached dye increased to a certain extent without shift of the emission maximum. 2. 2.|Nucleotides quenched the fluorescence of both maleimide-labeled G- and F-actins. Divalent cations, Mg 2+ and Ca 2+, antagonized this effect on G-actin, but not on F-actin. 3. 3.|On addition of heavy meromyosin, the fluorescence intensity of BIPM-labeled F-actin increased by 20% with a slight blue shift of the emission maximum. The addition of ATP to this system put these changes in the fluorescence properties back, but not completely, to those of the free BIPM-actin. Based on the results obtained, a possible role of the region containing BIPM-labeled sulfhydryl group(s) was discussed.

Synthesis of 2-(Dansylamino)ethyl triphosphate and its properties as a fluorescent substrate of heavy meromyosin-ATPase

2-(Dansylamino)ethanol, which was obtained after the reaction of dansyl chloride with ethanolamine, was phosphorylated by using tetrachloropyrophosphate, and the resulting 2-(dansylamino)ethyl monophosphate was further phosphorylated by using P i and N, N′-dicyclohexylcarbodiimide to obtain 2-(dansylamino)ethyl triphosphate (DTP). DTP was hydrolyzed by heavy meromyosin-ATPase at a rate similar to the hydrolysis of ATP in the presence of Ca 2+. In the presence of 10 mM Mg 2+, the K m of the DTP hydrolysis by heavy meromyosin was 1.9·10 −5 M, the hydrolysis apparently being abolished by the addition of ATP. Marked increases in the intensity of the maxima of the excitation and emission spectra of DTP were observed after the addition of heavy meromyosin in the presence of 83.4 mM Mg 2+ at 10°. The peak of the emission spectrum shifted from 540 to 530 nm accompanied by an increase in intensity, thus suggesting that the polarity around DTP became rather hydrophobic. Energy transfer from tryptophan and/or tyrosine to the dansyl group of DTP was assumed.