Chicken Gizzard Smooth Muscle Research Articles

Purification of Myosin from Bovine Tracheal Smooth Muscle, Filament Formation and Endogenous Association of Its Regulatory Complex.

Dynamic regulation of myosin filaments is a crucial factor in the ability of airway smooth muscle (ASM) to adapt to a wide length range. Increased stability or robustness of myosin filaments may play a role in the pathophysiology of asthmatic airways. Biochemical techniques for the purification of myosin and associated regulatory proteins could help elucidate potential alterations in myosin filament properties of asthmatic ASM. An effective myosin purification approach was originally developed for chicken gizzard smooth muscle myosin. More recently, we successfully adapted the procedure to bovine tracheal smooth muscle. This method yields purified myosin with or without the endogenous regulatory complex of myosin light chain kinase and myosin light chain phosphatase. The tight association of the regulatory complex with the assembled myosin filaments can be valuable in functional experiments. The purification protocol discussed here allows for enzymatic comparisons of myosin regulatory proteins. Furthermore, we detail the methodology for quantification and removal of the co-purified regulatory enzymes as a tool for exploring potentially altered phenotypes of the contractile apparatus in diseases such as asthma.

Inhibitory effect of potassium alum on smooth muscle contraction of rabbit and its mechanism.

To investigate the effects of potassium alum (Alunite) on smooth muscle contraction and phosphorylation of myosin light chain by myosin light chain kinase (MLCK) and to try to find out the clue of its mechanism. An isolated rabbit duodenum smooth muscle strip was selected to study the effects of potassium alum on its contractile activity under the condition of Krebs' solution using HW-400S constant temperature smooth muscle trough. The myosin and MLCK were purified from chicken gizzard smooth muscle. Myosin light chain phosphorylation was determined by glycerol-polyacrylamide gel electrophoresis; myosin Mg2+-ATPase activity was measured by inorganic phosphate liberation method. Potassium alum (2.5-20 mmol/L) inhibited the contraction on duodenum in a dose-related and a time-dependent manner; potassium alum could also inhibit the extent of phosphorylation of myosin light chain in a dose-related and a time-dependent manner; and potassium alum inhibited the extent of Mg2+-ATPase activity in a dose-related manner. Potassium alum inhibited smooth muscle contraction in a way of inhibiting phosphorylation of myosin light chain and Mg2+-ATPase activity. This has revealed the molecular mechanism of treatment of gastrointestinal spastic disorders by potassium alum.

Light Chain Kinase Specificity in Cardiac Myosin

Human ventricular cardiac myosin regulatory light chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain Ser15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (Ser20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine ventricular myosin, and chicken gizzard myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.

Bi-directional regulation of ginsenoside Re on smooth muscle myosin of gizzard

The ginsenoside Re is the major ingredient of ginsenosides, which was isolated from ginseng (genus Panax). The purpose of the study is to investigate into the effect of ginsenoside Re on Mg2+-ATPase activity of smooth muscle myosin and phosphorylation of myosin light chain by myosin light chain kinase (MLCK), and try to find out the regular pattern resulting from ginsenoside Re on myosin function. Myosin and MLCK used in our study were purified from the chicken gizzard smooth muscle. Myosin phorphorylation was determined by glycerol polyacrylamide gel electrophoresis (PAGE). Myosin Mg2+-ATPase activity was measured by Pi liberation method. The results showed that ginsenoside Re stimulates Mg2+-ATPase activities, promotes phosphorylation of partially phosphorylated myosin, inhibits Mg2+-ATPase activity and decrease phosphorylation of fully phosphorylated myosin in a dose dependent manner and in a time dependent manner. The findings demonstrated that the use of ginsenoside Re on various states of myosin phosphorylation produced different responses in a purified incubation system. Key words: Ginsenoside Re, myosin phosphorylation, myosin Mg2+-ATPase activity, bi-directional regulation.

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten Vmax and KM for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

Electron Microscopy and Persistence Length Analysis of Semi-Rigid Smooth Muscle Tropomyosin Strands

The structural mechanics of tropomyosin are essential determinants of its affinity and positioning on F-actin. Thus, tissue-specific differences among tropomyosin isoforms may influence both access of actin-binding proteins along the actin filaments and the cooperativity of actin-myosin interactions. Here, 40 nm long smooth and striated muscle tropomyosin molecules were rotary-shadowed and compared by means of electron microscopy. Electron microscopy shows that striated muscle tropomyosin primarily consists of single molecules or paired molecules linked end-to-end. In contrast, smooth muscle tropomyosin is more a mixture of varying-length chains of end-to-end polymers. Both isoforms are characterized by gradually bending molecular contours that lack obvious signs of kinking. The flexural stiffness of the tropomyosins was quantified and evaluated. The persistence lengths along the shaft of rotary-shadowed smooth and striated muscle tropomyosin molecules are equivalent to each other (approximately 100 nm) and to values obtained from molecular-dynamics simulations of the tropomyosins; however, the persistence length surrounding the end-to-end linkage is almost twofold higher for smooth compared to cardiac muscle tropomyosin. The tendency of smooth muscle tropomyosin to form semi-rigid polymers with continuous and undampened rigidity may compensate for the lack of troponin-based structural support in smooth muscles and ensure positional fidelity on smooth muscle thin filaments.

Effect of lithium on smooth muscle contraction and phosphorylation of myosin light chain by MLCK

The aims of our study were to investigate into the effect of lithium on smooth muscle contraction and phosphorylation of myosin light chain (MLC20) by MLCK and to find out the clue of its mechanism. Isolated rabbit duodenum smooth muscle strips were used to study the effects of lithium on their contractile activity under the condition of Krebs' solution by means of HW-400S constant temperature smooth muscle trough. Myosin and MLCK were purified from the chicken gizzard smooth muscle. Myosin phosphorylation was determined by Glycerol-PAGE, myosin Mg2+ -ATPase activity was measured by Pi liberation method. Lithium (10-40 mM) inhibited the contraction in duodenum in a dose-related and time-dependent manner. Lithium could also inhibit the extent of myosin phosphorylation in a dose-related and time-dependent manner, whereas it inhibited Mg2+ -ATPase activity in a dose-related manner. Lithium inhibited smooth muscle contraction by inhibition of myosin phosphorylation and Mg2+ -ATPase activity.

TR-FRET Experiments and MD Simulations Resolve Structural States of Smooth Muscle Regulatory Light Chain

We have combined time-resolved fluorescence resonance energy transfer (TR-FRET) and molecular dynamics (MD) simulations to elucidate structural changes in the phosphorylation domain (PD) of chicken gizzard smooth muscle regulatory light chain (RLC) bound to smooth muscle myosin. The PD is absent in crystal structures, leaving uncertainty about the mechanism of regulation. Donor-acceptor pairs of probes were attached to three site-directed di-Cys mutants, each having one Cys at position 129 in the C-terminal lobe and the other at position 2, 3, or 7 in the N-terminal PD. Labeled RLCs, with and without phosphorylation at S19, were reconstituted into myosin S1. Time-resolved FRET identified two structural states of the RLC, closed and open, which are present in both unphosphorylated and phosphorylated biochemical states. Phosphorylation shifts the equilibrium toward the open state by 20-30%. Molecular dynamics simulations, guided by these and previous spectroscopic studies, confirm the existence of the open and closed structural states and produce atomic resolution models of these states. In the closed state, the PD interacts with the surface of the C-terminal lobe. In the open state, the PD is more helical and straight, resides farther from the C-terminal lobe, and is more mobile. Phosphorylation stabilizes the open state by forming a specific salt bridge between R16 and phosphorylated S19. This conformational shift is consistent with a mechanism of regulation that catalyzes large structural changes within myosin at a low energetic cost. This work was supported by grants from NIH (AR32961, AR07612) and the Minnesota Supercomputing Institute. We thank Igor Negrashov for excellent technical assistance.

Myosin Nucleotide Pocket Thermodynamics Measured by Epr Reveal How Energy Partitioning Relates Speed to Efficiency

We have used spin labeled ADP to investigate the dynamics of the nucleotide-binding pocket in myosins. In actomyosin•ADP the nucleotide-binding pocket is in an equilibrium between closed and open conformations, with the open conformation favored in slow myosins. In rabbit slow skeletal muscle fibers, the ∈″G for the closed to open equilibrium is −3.9 kJ mol−1. We found similar values for pig ventricle myosin, chicken gizzard smooth muscle myosin, and chicken myosin V. For faster myosins, the equilibrium shifts to favor a closed conformation, rising to −2.7 kJ mol−1 for Dictyostelium discoideum myosin II, −1.9 kJ mol−1 for pig atrial myosin, −1.1 kJ mol−1 for rabbit fast skeletal muscle fibers, and +2.9 kJ mol−1 for Drosophila fight muscle fibers. We believe this represents a destabilization of the open actomyosin•ADP state in the faster myosins, driving ADP release. van't Hoff analysis of the temperature dependence of this equilibrium reveals that the closed to open conformation has a significant positive enthalpy and entropy, with ∈″H and T∈″S of 40-50 kJ mol−1 for slow myosins. Both components are reduced in this equilibrium for faster myosins, decreasing to ∈″H = 17.7 kJ mol−1 and T∈″S at 25°C = 18.8 kJ mol−1 for rabbit fast skeletal fibers, and ∈″H = 10.4 kJ mol−1 and T∈″S at 25°C = 7.5 kJ mol−1. Our model is that the open actomyosin•ADP state represents a partitioning point between the free energy released during the myosin catalytic cycle. Because of this partitioning, fast myosins destabilize the actomyosin•ADP state, reducing the energy available to do work up until that point, but leaving more free energy in reserve to drive ADP release. This gives a mechanism for the correlation between increased speed and reduced efficiency in muscle.

Calponin control of cerebrovascular reactivity: therapeutic implications in brain trauma

Calponin (Cp) is an actin-binding protein first characterized in chicken gizzard smooth muscle (SM). This review discusses the role of Cp in mediating SM contraction, the biochemical process by which Cp facilitates SM contraction and the function of Cp in the brain. Recent work on the role of Cp in pathological states with emphasis on traumatic brain injury is also discussed. Based on past and present data, the case is presented for targeting Cp for novel genetic and pharmacological therapies aimed at improving outcome following traumatic brain injury (TBI).

Acidic Calponin controls ERK1/2 translocation and l-Caldesmon phosphorylation in fibroblasts

Calponin is an actin binding, troponin-like molecule, first isolated from chicken gizzard smooth muscle cells where it is involved in regulation of contractility. So far there are three known isoforms, basic, acidic and neutral Calponin, each encoded by a separate gene. Expression of the acidic Calponin isoform is, unlike basic Calponin, not restricted to smooth muscle cells but is expressed in a variety of tissues and cell types, especially in the brain. For basic Calponin it is known that the molecule affects smooth muscle cell contractility by regulation of the PKC/ERK signaling pathway. Here we could show that acidic Calponin co-precipitates together with ERK1/2 and PKCalpha in the cultured rat fibroblast cell line REF52.2. Moreover it colocalises with ERK1/2 and PKCalpha in podosome-like structures and at the cell cortex after Phorbol-12,13-Dibutyrate (PDBu) treatment. Knockdown of acidic Calponin expression in these cells results in inhibition of PDBu-mediated ERK1/2 translocation to these structures, whereas PKCalpha targeting is not affected. Both endogenous ERK1/2 activity and phosphorylation of the ERK1/2 substrate l-Caldesmon are blocked by acidic Calponin knockdown, indicating an important role for the acidic Calponin isoform in regulation of ERK1/2 function in REF52.2 cells.Abstract sponsored by John Gergely, Boston Biomedical Research Institute, Watertown 02472, MA, USASupport: NIH grants HL80003, HL86655

Biochemical Properties of Gastrokine-1 Purified from Chicken Gizzard Smooth Muscle

The potential role and function of gastrokine-1 (GNK1) in smooth muscle cells is investigated in this work by first establishing a preparative protocol to obtain this native protein from freshly dissected chicken gizzard. Some unexpected biochemical properties of gastrokine-1 were deduced by producing specific polyclonal antibody against the purified protein. We focused on the F-actin interaction with gastrokine-1 and the potential role and function in smooth muscle contractile properties.BackgroundGNK1 is thought to provide mucosal protection in the superficial gastric epithelium. However, the actual role of gastrokine-1 with regards to its known decreased expression in gastric cancer is still unknown. Recently, trefoil factors (TFF) were reported to have important roles in gastric epithelial regeneration and cell turnover, and could be involved in GNK1 interactions. The aim of this study was to evaluate the role and function of GNK1 in smooth muscle cells.Methodology/Principal FindingsFrom fresh chicken gizzard smooth muscle, an original purification procedure was used to purify a heat soluble 20 kDa protein that was sequenced and found to correspond to the gastrokine-1 protein sequence containing one BRICHOS domain and at least two or possibly three transmembrane regions. The purified protein was used to produce polyclonal antibody and highlighted the smooth muscle cell distribution and F-actin association of GNK1 through a few different methods.Conclusion/SignificanceAltogether our data illustrate a broader distribution of gastrokine-1 in smooth muscle than only in the gastrointestinal epithelium, and the specific interaction with F-actin highlights and suggests a new role and function of GNK1 within smooth muscle cells. A potential role via TFF interaction in cell-cell adhesion and assembly of actin stress fibres is discussed.

Smooth muscle type isoform of 20 kDa myosin light chain is expressed in monocyte/macrophage cell lineage

Myosin light chain genes of human hematopoietic cells have not been fully characterized. We previously reported the cloning of the full-length cDNAs of 20 kDa regulatory myosin light chain (MLC-2), named as MLC-2A, from Meg-01, a human megakaryoblastic leukemia cell line (J. Smooth Muscle Res. 37: 25-38, 2001). We now cloned another MLC-2 isoforms from human platelets and U937, a human monocytic leukemia cell line, named as MLC-2B and MLC-2C, respectively. Both MLC-2A and MLC-2B consisted of three exons, which were situated on gene loci 18p1.3. Analysis of the gene structure indicated that MLC-2A and MLC-2B utilized different exons. MLC-2C also consisted of three exons, which was situated on gene loci 20p12. Amino acid sequence of MLC-2C was, of interest, apparently almost the same as that of MLC-2 from chicken gizzard smooth muscle LC20-A (one amino acid's difference) and human vascular smooth muscle LC-20 (two amino acids' difference). All three protein kinase C phosphorylation residues (Ser-1, Ser-2, Thr-9) and both myosin light chain kinase phosporylation residues (Thr-18, Ser-19) are conserved in these three isoforms. The MLC-2A and MLC-2B mRNA were expressed constitutively in all of the human hematopoietic cell lines examined and their expression levels were almost the same. On the other hand, MLC-2C mRNA was expressed in untreated monocytic cell lines (U937 and A-THP-1) and HL-60 differentiated into monocyte/macrophage cell lineage by TPA treatment. These results indicate that smooth muscle type isoform, MLC-2C is the inducible isoform, and might play a crucial role in monocyte/macrophage cell lineage.

Calponins: Adaptable modular regulators of the actin cytoskeleton

Over 20 years ago Katsuhito Takahashi isolated a heat stable, calmodulin and actin binding protein from chicken gizzard smooth muscle. Considered initially as a mainly structural component of the vertebrate smooth muscle contractile machinery, the 34-kDa calcium- and calmodulin-binding troponin T-like protein, calponin quickly appeared to also be involved in a number of regulatory and signal transduction events in the actin cytoskeleton. Calponins regulate actomyosin contraction, and reduce metastatic cell motility and tissue invasion. From these various cellular functions the biological role of calponin is now slowly emerging, namely that of an actin filament-stabilizing molecule that contributes to physiological thin filament turnover rates in different cell types.

Regulatory and catalytic domain dynamics of smooth muscle myosin filaments.

Domain dynamics of the chicken gizzard smooth muscle myosin catalytic domain (heavy chain Cys-717) and regulatory domain (regulatory light chain Cys-108) were determined in the absence of nucleotides using saturation-transfer electron paramagnetic resonance. In unphosphorylated synthetic filaments, the effective rotational correlation times, tau(r), were 24 +/- 6 micros and 441 +/- 79 micros for the catalytic and regulatory domains, respectively. The corresponding amplitudes of motion were 42 +/- 4 degrees and 24 +/- 9 degrees as determined from steady-state phosphorescence anisotropy. These results suggest that the two domains have independent mobility due to a hinge between the two domains. Although a similar hinge was observed for skeletal myosin (Adhikari and Fajer (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 9643-9647. Brown et al. (2001) Biochemistry 40, 8283-8291), the latter displayed higher regulatory domain mobility, tau(r)= 40 +/- 3 micros, suggesting a smooth muscle specific mechanism of constraining regulatory domain dynamics. In the myosin monomers the correlation times for both domains were the same (approximately 4 micros) for both smooth and skeletal myosin, suggesting that the motional difference between the two isoforms in the filaments was not due to intrinsic variation of hinge stiffness. Heavy chain/regulatory light chain chimeras of smooth and skeletal myosin pinpointed the origin of the restriction to the heavy chain and established correlation between the regulatory domain dynamics with the ability of myosin to switch off but not to switch on the ATPase and the actin sliding velocity. Phosphorylation of smooth muscle myosin filaments caused a small increase in the amplitude of motion of the regulatory domain (from 24 +/- 4 degrees to 36 +/- 7 degrees ) but did not significantly affect the rotational correlation time of the regulatory domain (441 to 408 micros) or the catalytic domain (24 to 17 micros). These data are not consistent with a stable interaction between the two catalytic domains in unphosphorylated smooth muscle myosin filaments in the absence of nucleotides.

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Role of Protein Phosphatase Type 1 in Contractile Functions: Myosin Phosphatase

Unzipping the Role of Myosin Light Chain Phosphatase in Smooth Muscle Cell Relaxation

Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p<0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.

Regulation of E-kinase mediated cell-adhesion molecule(s) function by E-NTPase(s): imminent regulatory inter-dependency for cell adhesion

E-NTPase is a cell membrane bound enzyme, suggested to play a role in maintaining the [NTP] o and cell adhesion. Attempts to purify this enzyme resulted identification of many proteins co-purifying with E-NTPase. The amino acid sequence of chicken gizzard smooth muscle E-NTPase has been shown to have considerable amino acid sequence homology with mammalian CD39. On the other hand, the purified rat cardiac sarcolemmal E-NTPase showed identical sequence homology with platelet CD36. In this communication, an effort was made to provide rationale for the functional relevance of E-NTPase with reference to cell-adhesion process.

Expression and purification of the h1 and h2 isoforms of calponin

Three homologous calponin isoforms, named h1, h2, and acidic calponins, have been found in birds and mammals. Based primarily on studies of chicken gizzard smooth muscle (h1) calponin, calponin has been identified as a family of actin-associated proteins that inhibit actomyosin ATPase activity. Evolutionary divergence of the calponin isoforms suggests differentiated function. While the role of h1 calponin in smooth muscle contraction is under investigation, h2 calponin has been shown regulating the function of actin cytoskeleton. Using cloned cDNA, we expressed mammalian h1 and h2 calponins in Escherichia coli. We have developed effective methods to purify biologically active h1 and h2 calponin proteins from transformed bacterial culture. The purified calponin isoform proteins were used to generate monoclonal antibodies that reveal epitopic structure difference between h1 and h2 calponins. Together with their differential expression in tissues and during development, the structural diversity of h1 and h2 calponins suggests non-redundant physiological function. Nevertheless, h1 and h2 calponins bind F-actin with similar affinity, indicating a conserved mechanism for their role in regulating actin filaments in smooth muscle and non-muscle cells.

Smitin, a novel smooth muscle titin-like protein, interacts with myosin filaments in vivo and in vitro.

Smooth muscle cells use an actin–myosin II-based contractile apparatus to produce force for a variety of physiological functions, including blood pressure regulation and gut peristalsis. The organization of the smooth muscle contractile apparatus resembles that of striated skeletal and cardiac muscle, but remains much more poorly understood. We have found that avian vascular and visceral smooth muscles contain a novel, megadalton protein, smitin, that is similar to striated muscle titin in molecular morphology, localization in a contractile apparatus, and ability to interact with myosin filaments. Smitin, like titin, is a long fibrous molecule with a globular domain on one end. Specific reactivities of an anti-smitin polyclonal antibody and an anti-titin monoclonal antibody suggest that smitin and titin are distinct proteins rather than differentially spliced isoforms encoded by the same gene. Smitin immunofluorescently colocalizes with myosin in chicken gizzard smooth muscle, and interacts with two configurations of smooth muscle myosin filaments in vitro. In physiological ionic strength conditions, smitin and smooth muscle myosin coassemble into irregular aggregates containing large sidepolar myosin filaments. In low ionic strength conditions, smitin and smooth muscle myosin form highly ordered structures containing linear and polygonal end-to-end and side-by-side arrays of small bipolar myosin filaments. We have used immunogold localization and sucrose density gradient cosedimentation analyses to confirm association of smitin with both the sidepolar and bipolar smooth muscle myosin filaments. These findings suggest that the titin-like protein smitin may play a central role in organizing myosin filaments in the contractile apparatus and perhaps in other structures in smooth muscle cells.