Heavy Meromyosin Subfragment Research Articles

Characterization and functional analysis of a chitin synthase gene (HcCS1) identified from the freshwater pearlmussel Hyriopsis cumingii.

The triangle sail mussel, Hyriopsis cumingii, is the most important freshwater pearl mussel in China. However, the mechanisms underlying its chitin-mediated shell and nacre formation remain largely unknown. Here, we characterized a chitin synthase (CS) gene (HcCS1) in H. cumingii, and analyzed its possible physiological function. The complete ORF sequence of HcCS1 contained 6903 bp, encoding a 2300-amino acid protein (theoretical molecular mass = 264 kDa; isoelectric point = 6.22), and no putative signal peptide was predicted. A myosin motor head domain, a CS domain, and 12 transmembrane domains were found. The predicted spatial structures of the myosin head and CS domains were similar to the electron microscopic structure of the heavy meromyosin subfragment of chicken smooth muscle myosin and the crystal structure of bacterial cellulose synthase, respectively. This structural similarity indicates that the functions of these two domains might be conserved. Quantitative reverse transcription PCR results showed that HcCS1 was present in all detected tissues, with the highest expression levels detected in the mantle. The HcCS1 transcripts in the mantle were upregulated following shell damage from 12 to 24 h post-damage, and they peaked (approximately 1.5-fold increase) at 12 h after shell damage. These findings suggest that HcCS1 was involved in shell regeneration, and that it might participate in shell and nacre formation in this species via chitin synthesis. HcCS1 might also dynamically regulate chitin deposition during the process of shell and nacre formation with the help of its conserved myosin head domain.

Myosin II Folding Is Mediated by a Molecular Chaperonin

The folding pathway of the heavy meromyosin subfragment (HMM) of a skeletal muscle myosin has been investigated by in vitro synthesis of the myosin heavy and light chains in a coupled transcription and translation assay. Analysis of the nascent translation products for folding intermediates has identified a major intermediate that contains all three myosin subunits in a complex with the eukaryotic cytosolic chaperonin. Partially folded HMM is released from this complex in an ATP-dependent manner. However, biochemical and functional assays reveal incomplete folding of the myosin motor domain. Dimerization of myosin heavy chains and association of heavy and light chains are accomplished early in the folding pathway. To test for other factors necessary for the complete folding of myosin, a cytoplasmic extract was prepared from myotubes produced by a mouse myogenic cell line. This extract dramatically enhanced the folding of HMM, suggesting a role for muscle-specific factors in the folding pathway. We conclude that the molecular assembly of myosin is mediated by the eukaryotic cytosolic chaperonin with folding of the motor domain as the slow step in the pathway.

Immunological relationship between the class I epitope of streptococcal M protein and myosin.

The class I epitope of streptococcal M protein is an epidemiological marker for acute rheumatic fever (ARF)-associated serotypes of group A streptococci and is recognized by anti-M protein monoclonal antibody (MAb) 10B6. Using MAb 10B6, we determined the relationship between the class I epitope of M protein and the alpha-helical coiled-coil protein myosin. MAb 10B6 reacted by enzyme-linked immunosorbent assay and Western blotting with human cardiac myosin and rabbit skeletal myosin and its heavy meromyosin (HMM) subfragment. Overlapping synthetic peptides of M5 protein were used to identify the region of M5 protein recognized by MAb 10B6. Two C repeat peptides (C2A and C3) containing the amino acid sequence KGLRRDLDASREAK reacted with MAb 10B6. Partial sequence identity, RRDL, was found in the HMM fragment of myosin, which reacted with MAb 10B6. However, not all peptides of M5 protein and myosin containing the RRDL sequence reacted with MAb 10B6. ARF sera and sera from uncomplicated pharyngitis (UNC) reacted with C repeat region peptides of M protein, while acute glomerulonephritis sera were not as reactive. Affinity-purified human antibody to peptide C3 reacted with myosin. The data demonstrate that the class I epitope of M protein is immunologically cross-reactive with myosin and the HMM subfragment, and antibodies to peptide C3 and myosin were present in ARF and UNC sera.

Rapid and gentle extraction, reconstitution and characterization of microfilament and glia filament from rat astrocytes

We developed gentle and rapid methods for depolymerization and extraction of both microfilament and glia filament separately from a crude cytoskeletal fraction of rat astrocytes. Electron microscopy revealed that the filament reconstituted from the microfilament extract closely resembled F-actin that was formed from G-actin of rabbit skeletal muscle. It was found by immunoblotting analysis that even the reconstituted microfilament-like filaments, which had been purified by affinity chromatography with heavy meromyosin subfragment 1 (S1)-conjugated Sepharose, contained vimentin and glia fibrillary acidic protein (GFAP) besides actin, inferring the interaction between microfilament and glia filament. The filaments (9–10 nm thick) reconstituted from the glia filament extract were composed of actin and other minor components in addition to vimentin and GFAP. Actin, GFAP, 101, 34, 32.5, 30.5, 29.5 and 28 kDa proteins found in the reconstituted glia filament-like filaments were suggested to be glia filament-associated proteins.

Changes of carp myosin subfragment-1 induced by temperature acclimation

Heavy meromyosin subfragment-1 (S1) was prepared by α-chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca2+- and K+ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 μmol Pi·min-1·mg-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (KD) of S1 from cold-acclimated carp was 32.1x10-4· s-1, compared to 13.2x10-4·s-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg2+-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.

Stress Fibers in situ in Proximal Tubules of the Rat Kidney.

Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 microns. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal proximal tubule epithelial cells can be listed among the examples of stress fibers in situ.

Evaluation of mechanical parameters of the mechanochemically ‘active state’ of actin filaments on the basis of purely chemical data

Oosawa and his collaborators (cf. F. Oosawa, Biophys. Chem. 11 (1980) 443), employing various optical techniques, have shown that the flexibility of actin filaments increases upon interacting with the enzymatically active myosin fragments, particularly heavy meromyosin (HMM). It has been reported (S. Hitchcock, L. Carlsson and U. Lindberg, Cell 7 (1976)53) that HMM can accelerate the DNase 1-induced depolymerization of F-actin, provided MgATP is also present. Since, as we have demonstrated (cf. J. Borejdo and A. Oplatka, Biochim. Biophys. Acta 440 (1976) 241), HMM, like filamentous myosin, is endowed with mechanochemical capability, we made an attempt to correlate the enhanced rate of depolymerization with the decrease in rigidity of the G-G bonds in F-actin. On the basis of the chemical kinetic data of Hitchcock et al. we could derive the approximate value of the HMM-MgATP-induced change in rigidity which is a mechanical molecular parameter. Since interaction between HMM or HMM subfragment-1 and F-actin in the presence of MgATP leads to the movement of the myosin heads along the actin filaments, it is argued that the enzymic behavior of this system should not be analyzed on the basis of simple, equilibrium, complex formation.

The interaction of linoleic acid with myosin and its subfragments: a microcalorimetric study

The interaction of rabbit muscle myosin (M), heavy meromyosin (HMM) and light meromyosin (LMM) with the amphiphiles sodium linoleate (L) and sodium n-dodecyl-sulphate (SDS) in aqueous solutions has been investigated by microcalorimetry. At pH 6.4, M and LMM interact endothermically with L whereas for HMM at pH 6.4, and M, HMM and LMM at pH 8.0, the interaction is exothermic. A significant exothermic thermal transition is found at pH 6.4 for the interaction of M with L at a ratio of approximately 0.1 mmol L per mg M which can be associated with the HMM subfragment. The interaction enthalpy for the linoleate-myosin and SDS-myosin systems are not additively related to those for linoleate and SDS interaction with the isolated subfragments, indicating that within the myosin molecule the subfragments do not interact with these amphiphiles independently, particularly at high amphiphile to protein ratios.

Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation.

Smooth muscle heavy meromyosin (HMM) is phosphorylated by the Ca2+-activated phospholipid-dependent protein kinase, i.e. protein kinase C, at three sites on each 20,000-dalton light chain. Phosphorylation of three sites also is observed with isolated 20,000-dalton light chain and HMM subfragment 1. The phosphorylation sites are serine 1, serine 2, and threonine 9. Threonine is phosphorylated most rapidly followed by either serine 1 or 2. Phosphorylation of the third site occurs only on prolonged incubation. Phosphorylation is a random process. HMM phosphorylated at two sites per light chain by protein kinase C can be dephosphorylated, as shown using two phosphatase preparations. Increasing levels of phosphorylation of HMM by protein kinase C causes a progressive inhibition of the subsequent rate of phosphorylation of serine 19 by myosin light chain kinase and causes a progressive inhibition of actin-activated ATPase activity of HMM, prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of HMM for actin rather than a change in Vmax. Previous results with HMM and protein kinase C (Nishikawa, M., Sellers, J. R., Adelstein, R. S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814) examined effects induced by phosphorylation of the threonine residues. Our results confirm these and consider also the influence of higher levels of phosphorylation by protein kinase C.

Differential Behavior of Heavy Meromyosin and Heavy Meromyosin Subfragment One toward A Monomeric Actin Derivative

Differential Behavior of Heavy Meromyosin and Heavy Meromyosin Subfragment One toward A Monomeric Actin Derivative

Conformational stability of the myosin rod.

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.

Actin filament localization and distribution in the young adult mouse cornea: a correlative immunofluorescent and cytochemical study

The localization and distribution of actin filaments was determined in the corneal epithelium and endothelium of the young adult Swiss Webster mouse by correlative indirect immunofluorescence using rabbit anti-skeletal muscle actin antiserum and by in situ labelling with heavy meromyosin subfragment-1 (HMM-S1). A diffuse fluorescent staining was observed in the cytoplasm of epithelial and endothelial cells and in stromal keratocytes. In addition, a highly fluorescent punctate cytoplasmic staining was seen only in the corneal epithelium. Ultrastructurally, HMM-S1 decorated actin filament bundles were generally distributed within the cytoplasm of glycerinated superficial, wing and basal epithelial cells and within pedicle-like processes on the posterior surface of superficial and wing cells. Actin filament bundles also were seen in close association with intermediate filament bundles in the epithelium. HMM-S1 labelled filaments were observed in the endothelial cytoplasm anteriorly near Descemet's membrane, posteriorly near the anterior chamber and adjacent to lateral cell borders. Actin filaments in epithelial and endothelial cells appeared to insert into the plasma membrane.

Identification of a microfilament-enriched, motile domain in amoeboflagellates of Physarum Polycephalum

ABSTRACT A distinctive, transient cytoplasmic domain was identified in haploid cells of Physarum polycephalum that are transforming from amoebae into flagellate swimming cells. As revealed by light microscopy, this region, termed the ridge, is a flattened cytoplasmic extension that displays a specific form of motility, characterized by undulations that propagate rapidly down its long axis. The ridge was further examined by scanning electron microscopy of whole cells, and by transmission electron microscopy of serial sections and negative stain preparations of amoeboflagellate cytoskeletons. We identified three distinct components possibly related to ridge motility: a short flagellum closely associated with the ridge surface, an array of micro-tubules that is part of a complex microtubular apparatus and a network of filaments that forms a well-defined laminar core within the ridge. Labelling studies with heavy meromyosin subfragment 1 (S-1) indicated that a major portion of the filament network is actin. Peripheral microfilaments appeared to have a uniform orientation in that arrowheads pointed towards the cell body. Other ordered filament arrangements were apparent with a modified extraction protocol using phalloidin. Similar microfilament organization has been reported in motile regions of other systems, such as the lamellipodia of tissue culture cells. However, the ridge can be distinguished from such other motile regions in that it forms independently of contact with a substratum and is only present in a specific region of amoeboflagellates. Moreover, the ridge appears at a specific time during the course of the transformation. The ridge constitutes a new system for studying the structural basis of cell motility. The arrangements of actin- and microtubule-containing ridge components suggest several testable hypotheses concerning their involvement in cytoskeletal or motile functions.

Activation of smooth muscle myosin by smooth and skeletal muscle actins

FEBS LettersVolume 134, Issue 2 p. 197-202 Full-length articleFree Access Activation of smooth muscle myosin by smooth and skeletal muscle actins Correction(s) for this article Corrigenda Volume 135Issue 2FEBS Letters pages: 309-309 First Published online: October 23, 2001 Hanna Strzelecka-Golaszewska, Hanna Strzelecka-Golaszewska Department of Biochemistry of the Nervous System and Muscle, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, PolandSearch for more papers by this authorApolinary Sobieszek, Apolinary Sobieszek Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstr. 11, 5020 Salzburg, AustriaSearch for more papers by this author Hanna Strzelecka-Golaszewska, Hanna Strzelecka-Golaszewska Department of Biochemistry of the Nervous System and Muscle, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, PolandSearch for more papers by this authorApolinary Sobieszek, Apolinary Sobieszek Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstr. 11, 5020 Salzburg, AustriaSearch for more papers by this author First published: November 16, 1981 https://doi.org/10.1016/0014-5793(81)80601-1Citations: 22AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 M.P. Sheetz, R.G. Painter, S.J. Singer, Biochemistry, 15, (1976), 4486– 4492. 2 E. Próchniewicz, H. Strzelecka-Gołaszewska, Eur. J. Biochem., 106, (1980), 305– 312. 3 J.V. Small, A. Sobieszek, Int. Rev. Cytol., 64, (1980), 241– 306. 4 U. Mrwa, J.C. Rüegg, FEBS Lett., 60, (1975), 81– 84. 5 A. Sobieszek, J.V. Small, J. Mol. Biol., 102, (1976), 75– 92. 6 U. Mrwa, J.C. Rüegg, R. Casteels Excitation—Contraction Coupling in Smooth Muscle (1977), Elsevier/North-Holland Amsterdam, New York 353– 357. 7 S.B. Marston, E.W. Taylor, FEBS Lett., 86, (1978), 167– 170. 8 S.B. Marston, E.W. Taylor, J. Mol. Biol., 139, (1980), 573– 600. 9 J.C. Seidel, Biochem. Biophys. Res. Comm., 85, (1978), 107– 113. 10 S.S. Margossian, S. Lowey, J. Mol. Biol., 74, (1973), 313– 330. 11 H. Strzelecka-Golaszewska, E. Próchniewicz, E. Nowak, S. Zmorzyński, W. Drabikowski, Eur. J. Biochem., 104, (1980), 41– 52. 12 G. Feuer, F. Molnár, E. Pettkó, F.B. Straub, Hung. Acta Physiol., 1, (1948), 150– 163. 13 K. Bailey, Biochem. J., (1948), 271– 273. 14 A. Sobieszek, J.V. Small, Eur. J. Biochem., 118, (1981), 533– 539. 15 C.H. Fiske, Y. SubbaRow, J. Biol. Chem., 66, (1925), 375– 400. 16 A.G. Gornall, C.J. Bardawill, M.M. David, J. Biol. Chem., 177, (1949), 751– 766. 17 K. Weber, M. Osborn, J. Biol. Chem., 244, (1969), 4406– 4412. 18 A. Sobieszek, R.D. Bremel, Eur. J. Biochem., 55, (1975), 49– 60. 19 J.C. Seidel, Biochem. Biophys. Res. Commun., 85, (1978), 107– 113. 20 H. Onishi, S. Watanabe, J. Biochem. (Tokyo), 85, (1979), 457– 472. 21 A. Sobieszek, B. Barylko, Proc. Symp. ‘The Biochemistry of Smooth Muscle’, Winnipeg, August 5–7, 1981, (1981), 22 E.W. Taylor, CRC Crit. Rev. Biochem., 4, (1979), 103– 164. 23 M. Bárány, J. Gen. Physiol., 50, (1967), 197– 218. suppl. 24 A. Sobieszek, J.V. Small, J. Mol. Biol., 112, (1977), 559– 576. 25 S. Chacko, M.A. Conti, R.S. Adelstein, Proc. Natl. Acad. Sci. USA, 74, (1977), 129– 133. 26 D.J. Hartshorne, A. Górecka, M.O. Aksoy, R. Casteels Excitation—Contraction Coupling in Smooth Muscle (1977), Elsevier/North-Holland Amsterdam, New York 377– 384. 27 R. Dabrowska, J. Sosiński, W. Drabikowski, D. Pette Plasticity of Muscle (1980), Walter de Gruyter Berlin, New York 225– 239. 28 Y. Nonomura, S. Ebashi, Biomed. Res., 1, (1980), 1– 14. 29 Y.-Z. Jong, E.D. Korn, E. Eisenberg, J. Biol. Chem., 254, (1979), 2084– 2088. 30 R. Dabrowska, E. Nowak, W. Drabikowski, Comp. Biochem. Physiol., 65B, (1980), 75– 83. Citing Literature Volume134, Issue2November 16, 1981Pages 197-202 ReferencesRelatedInformation

Stabilization and the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition.

Treatment of epithelial BSC-1 cells with low concentrations of the detergent Brij 58 results in partial or complete removal of the plasmalemma and partial extraction of internal membrane-bound organelles without causing massive release of "cytosolic" proteins from the cytoplasmic ground substance. Stereoscopic high-voltage electron microscopy of such extracted and fixed cells demonstrates a system of slender (4-20 nm) strands in a three-dimensional "microtrabecular" arrangement similar to that observed in unextracted whole-mount preparations. Extraction of Brij-extracted cells with Triton X-100 dissolves many of the microtrabecular strands, leaving, as a more stable structure, a characteristic cytoskeletal network composed of various filaments and microtubules. Two-dimensional polyacrylamide gel electrophoresis of 35S-labeled polypeptides performed concurrently with the morphological studies demonstrates that Triton extraction of Brij-extracted cells releases a large number of polypeptides. This release parallels the loss of structural components observed by electron microscopy. Labeling of Brij-extracted cells with heavy meromyosin subfragment 1 decorates actin filaments with characteristic arrowhead complexes which are readily visualized only after subsequent Triton extraction. These observations support the concept that many cytoplasmic proteins are structure-bound and, in addition to the components comprising the cytoskeleton, are structure-forming. We conclude that a metastable association of various proteins of the cytoplasmic ground substance exists whose morphological integrity is maintained, at lest temporarily, after removal of the plasmalemma in solutions containing Brij 58.

Structural interaction of cytoskeletal components.

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the "microtrabecular network" (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).

Localization and organization of actin in melanophores.

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.

Influence of myosin heavy chains on the Ca2+-binding properties of light chain, LC2.

The association of myosin light chains with heavy chains, i.e. the intact oligomeric structure, profoundly affects the Ca(2+)-binding properties of the light chains. The Ca(2+)-binding affinity of the light chains is more than two magnitudes higher in the presence of heavy chains than in its absence. Modification of the reactive SH(2) thiol of myosin results in an alteration in the conformation of heavy chains of the molecule that influences the Ca(2+)-binding properties of light chains and generation of tension. When the SH(2) moiety is blocked with N-ethylmaleimide the influence of the heavy chains on the Ca(2+)-binding properties of light chain LC(2) is lost; under these conditions the Ca(2+)-binding affinity value of SH(2)-N-ethylmaleimide-blocked myosin (3.3x10(4)m(-1)) decreases to near that expressed with the dissociated light chain LC(2) (0.7x10(4)m(-1)). Conversely, the presence of actin, nucleotides or modification of either the reactive lysyl residue or SH(2) thiol does not affect Ca(2+) binding. The native secondary and tertiary structure of myosin seem to be required for Ca(2+) binding; binding does not occur in the presence of 6m-urea with either native myosin or the dissociated light chains. With SH(2)-N-ethylmaleimide-blocked myosin normal Ca(2+)- and (Mg(2+)+actin)-stimulated ATPase activities are expressed; however, there is a loss in K(+)-stimulated ATPase activity and the synthetic actomyosin threads of such myosin express no isometric tension. There are also variances in the binding of Ca(2+) with alterations in pH values. In the absence of Ca(2+)/EGTA buffer the biphasic Ca(2+)-binding affinity of myosin is twice as high at pH7.4 (site one: 1.2x10(6)m(-1) and site two: 0.4x10(6)m(-1)) as compared with values obtained at pH6.5 (site one: 0.64x10(6)m(-1) and site two: 0.2x10(6)m(-1)). The Ca(2+)-binding affinity of light chain LC(2) and S(1), where the (S-1)-(S-2) junction was absent, were not influenced by changes in pH values. Both expressed a low Ca(2+)-binding affinity, approx. 0.7x10(4)m(-1), whereas heavy meromyosin, where both (S-1) and (S-2) myosin subfragments were present, expressed a Ca(2+)-binding affinity value similar to that of native myosin, but was not biphasic. However, it is important to point out than in preparation of S(1) myosin subfragment light chain LC(2) was lost and thus was added back to the purified S(1) fraction. Light chain LC(2) was not, however, added to the heavy meromyosin fraction because it was not lost during preparation of the heavy meromyosin subfragment. In conclusion, it appears that the (S-1)-(S-2) junction is needed for the positioning of light chain LC(2) and thus influences its essential conformation for Ca(2+) binding.

Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers.

The binding of fluorescently labeled heavy meromyosin (HMM) and heavy meromyosin subfragment-1 (S-1) to thin filaments of myofibrils and of rabbit psoas muscle fibers was measured under conditions of rigor and contraction. The fragments diffused rapidly into the myofibrillar space and bound specifically to the thin filaments. The fragments bound strongest and in a uniform fashion to myofibrils in which the competition from indigenous myosin was abolished by removing it with Hasselbach-Schneider solution. Under these conditions, the rigor Ka values for HMM and S-1 were 1.5 x 10(6) M-1 and 4.8 x 10(4) M-1, respectively. The stoichiometry of binding was measured by independently estimating the concentration of actin sites. S-1 was found to be capable of saturating all available actin sites in a myofibril or a fiber, but HMM could only occupy 50% of the sites.

Interaction of studies of a 100,000 dalton chymotryptic fragment of rabbit skeletal M-line protein with the S2 subfragment of myosin.

Controlled chymotryptic digestion of the 165,000 dalton component of the M-line of rabbit skeletal muscle, followed by Biogel P-150 chromatography of the digest, has led to the isolation of a homogeneous 100,000 dalton species. This fragment was found, by both sedimentation equilibrium and gel filtration chromatography, to interact with heavy meromyosin subfragment 2 (HMM-S2). The persistence of this fragment, after chymotryptic digestion, to bind HMM-S2, along with the known insensitivity of the M-line to proteolysis, suggests a structural role for the parent 165,000 dalton component along the lines of the M-filament, as suggested by the Knappeis and Carlsen model for M-line structure.