Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.     

Change of actin isomers during differentiation of smooth muscle

Changes of actin isomers during development and differentiation of chicken gizzard were investigated by polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis with SDS-polyacrylamide gels in the presence of urea as the second dimension clearly separated three actin isomers which appear during development of the smooth muscle. The three actin isomers change the relative concentrations during development as follows: gizzard-type gamma-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, nonmuscle-type gamma-actin exists only at earlier stages (before 15 days of embryogenesis), and the amount of beta-actin increases in proportion to the decrease of nonmuscle type gamma-actin, the amount of nonmuscle actin in gizzards then becoming constant. Actin composition of gizzard before 7 days of embryonic age was nonmuscle type and consisted of beta-actin and nonmuscle-type gamma-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.     

Change of actin isomers during differentiation of smooth muscle

Changes of actin isomers during development and differentiation of chicken gizzard were investigated by polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis with SDS-polyacrylamide gels in the presence of urea as the second dimension clearly separated three actin isomers which appear during development of the smooth muscle. The three actin isomers change the relative concentrations during development as follows: (1) gizzard-type γ-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, (2) nonmuscle-type γ-actin exists only at earlier stages (before 15 days of embryogenesis), and (3) the amount of β-actin increases in proportion to the decrease of nonmuscle type γ-actin, the amount of nonmuscle actin in gizzards then becoming constant. Actin composition of gizzard before 7 days of embryonic age was nonmuscle type and consisted of β-actin and nonmuscle-type γ-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.     

Role of tropomyosin in smooth muscle contraction: effect of tropomyosin binding to actin on actin activation of myosin ATPase

The binding of gizzard tropomyosin to gizzard F-actin is highly dependent on free Mg2+ concentration. At 2 mM free Mg2+, a concentration at which actin-activated ATPase activity was shown to be Ca2+ sensitive, a molar ratio of 1:3 (tropomyosin:actin monomer) is required to saturate the F-actin with tropomyosin to the stoichiometric ratio of 1 mol of tropomyosin to 7 mol of actin monomer. Increasing the Mg2+ could decrease the amount of tropomyosin required for saturating the F-actin filament to the stoichiometric level. Analysis of the binding of smooth muscle tropomyosin to smooth muscle actin by the use of Scatchard plots indicates that the binding exhibits strong positive cooperativity at all Mg2+ concentrations. Calcium has no effect on the binding of tropomyosin to actin, irrespective of the free Mg2+ concentration. However, maximal activation of the smooth muscle actomyosin ATPase in low free Mg2+ requires the presence of Ca2+ and stoichiometric binding of tropomyosin to actin. The lack of effect of Ca2+ on the binding of tropomyosin to actin shows that the activation of actomyosin ATPase by Ca2+ in the presence of tropomyosin is not due to a calcium-mediated binding of tropomyosin to actin.     

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma- enteric isoform in differentiating striated myocytes

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha- vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.     

Factors determining the subunit composition of tropomyosin in mammalian skeletal muscle.

Adult rat fast-twitch skeletal muscle such as extensor digitorum longus contains alpha- and beta-tropomyosin subunits, as is the case in the corresponding muscles of rabbit. Adult rat soleus muscle contains beta-, gamma- and delta-tropomyosins, but no significant amounts of alpha-tropomyosin. Evidence for the presence of phosphorylated forms of at least three of the four tropomyosin subunit isoforms was obtained, particularly in developing muscle. Immediately after birth alpha- and beta-tropomyosins were the major components of skeletal muscle, in both fast-twitch and slow-twitch muscles. Differentiation into slow-twitch skeletal muscles was accompanied by a fall in the amount of alpha-tropomyosin subunit and its replacement with gamma- and delta-subunits. After denervation and during regeneration after injury, the tropomyosin composition of slow-twitch skeletal muscle changed to that associated with fast-twitch muscle. Thyroidectomy slowed down the changes in tropomyosin composition resulting from the denervation of soleus muscle. The results suggest that the 'ground state' of tropomyosin-gene expression in the skeletal muscle gives rise to alpha- and beta-tropomyosin subunits. Innervation by a 'slow-twitch' nerve is essential for the expression of the genes controlling gamma- and delta-subunits. There appears to be reciprocal relationship between expression of the gene controlling the synthesis of alpha-tropomyosin and those controlling the synthesis of gamma- and delta-tropomyosin subunits.     

Analysis of actin and tropomyosin in hearts of cardiac mutant axolotls by two-dimensional gel electrophoresis, western blots, and immunofluorescent microscopy

When homozygous, recessive mutant gene c in Ambystoma mexicanum results in a failure of embryonic heart function. This failure is apparently due to abnormal inductive influences from the anterior endoderm resulting in an absence of normal sarcomeric myofibril formation. Biochemical and immunofluorescent studies were undertaken to evaluate the contractile proteins actin and tropomyosin in normal and mutant hearts. For the immunofluorescent studies, cardiac tissues were fixed in periodate-lysine-paraformaldehyde, frozen sectioned, and immunostained by an indirect method with monospecific polyclonal antibodies produced against highly purified chicken heart actin and tropomyosin. In normal hearts, both antiactin and antitropomyosin stained the myofibrillar I-bands intensely. In mutant hearts, intensity of staining with antiactin antibody was similar to normal, although sarcomeric patterns were not observed. Staining intensity for tropomyosin with antitropomyosin antibody was significantly reduced in mutant hearts when compared to normal. Biochemical studies were used to evaluate antibody specificity, antigenic variability, and relative protein concentrations of actin and tropomyosin in normal and mutant cardiac tissues. Tissue homogenates were electrophoresed in two dimensions, and second-dimension slab gels were either Coomassie Blue silver-stained or transblotted onto nitrocellulose and the proteins stained with antibodies. Stained gels and immunoblots of cardiac proteins reveal that the amounts of actin isoforms are identical in normal and mutant hearts. However, these methods demonstrate a significantly reduced amount of tropomyosin in mutant tissue. This confirms earlier studies suggesting reduced amounts of tropomyosin in mutant hearts based upon immunological assays. Thus, failure of normal myofibrillogenesis in gene c mutant hearts does not appear to result from a change in actin isoform composition but may be related to a deficiency in tropomyosin.     

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

Actin concentration and monomer-polymer ratio in developing chicken skeletal muscle

The actin concentration and monomer-polymer ratio in developing chicken skeletal muscle were determined by means of a DNase I inhibition assay. The concentration of G-actin in embryonic muscle was much higher than the critical concentration for polymerization of purified actin. As muscle development progressed, the amount of total actin remarkably increased, whereas the concentration of G-actin markedly decreased, and finally in adults reached the critical concentration for polymerization of purified actin. When the monomeric actin in the soluble fraction of embryonic muscle was purified, the critical concentration for polymerization of the embryonic actin decreased to the same value as that of adult skeletal muscle actin. On the other hand, there was no difference between the crude and purified actin in the type of actin. They consisted of alpha-, beta-, and gamma-actins; their amounts were in the order, beta greater than gamma greater than alpha. Furthermore, polymerization of the monomeric actin in the soluble fraction of embryonic muscle was induced by the addition of myosin or HMM. The large amount of monomeric actin in the embryonic skeletal muscle may be due to the presence of some factor(s) which inhibits actin polymerization and also to an insufficiency of myosin.     

Acrylodan-labeled smooth muscle tropomyosin reports differences in the effects of troponin and caldesmon in the transition from the active state to the inactive state

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin. When S1 was rapidly detached from actin-smooth muscle tropomyosin, there was a rapid decrease in acrylodan-tropomyosin fluorescence as the intermediate state became populated. The rate constant for this process was >600 s(-1) at temperatures near 5 °C. In the presence of skeletal troponin and EGTA, the decrease in fluorescence was followed by the redevelopment of fluorescence as the inactive state became populated. The apparent rate constant for the fluorescence increase was 14 s(-1) at 5 °C. Substituting smooth muscle caldesmon for skeletal muscle troponin produced a similar decrease and re-increase in fluorescence, but the apparent rate constant for the increase was >10 times that observed with troponin. Furthermore, the fluorescence increase was correlated with an increase in the extent of caldesmon attachment as S1-ATP dissociated. Although the measured rate constant appeared to reflect the rate-limiting transition for inactivation, it is unclear if the fluorescence change resulted from caldesmon binding, the movement of tropomyosin over actin, or both.     

Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.     

Actin and tropomyosin variants in smooth muscles. Dependence on tissue type

Actin was found to be the major source of myofibrillar protein heterogeneity in smooth muscles. Three isoelectric variants, alpha-smooth muscle (alpha-SM), beta-non-muscle (beta-NM), and gamma-actins (gamma-SM and gamma-NM) were measured in 15 different smooth muscles, alpha-SM and gamma-actin contents displayed an inverse relationship in a given smooth muscle, some of which contained primarily alpha-SM actin while gamma-actins dominated in others. alpha-SM actin and gamma-actin distributions were tissue-specific, independent of species. A greater proportion of alpha-SM actin appears to be associated with tissues having a high degree of tonic activity. beta-Nonmuscle actin was a significant, and relatively constant, component of all smooth muscle tissues. The high NM-actin content of these tissues may reflect the importance of proliferative, synthetic, or secretory activities in smooth muscle, because the alpha-SM actin disappeared in tissue culture with a time course paralleling the modulation of phenotype from a contractile to a proliferative cell. Two tropomyosin subunits were present in approximately equal amounts in all smooth muscle tissues studied. One tropomyosin subunit exhibited identical mobility on two-dimensional gel electrophoresis, while the other was characterized by some species-specific variation which was unrelated to actin variant distribution. No variants of the 20,000-dalton regulatory light chain of myosin were observed. These results suggest that SM-specific actin variants are associated with functional diversity among smooth muscles.     

Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, we made the first kinetic measurements of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform. The in vitro actin sliding velocity on the flight muscle isoform (6.4 microm x s(-1) at 22 degrees C) is among the fastest reported for a type II myosin and was 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increased 6-fold, whereas that on the flight muscle myosin slightly decreased. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Developmental changes in myosin isoforms from slow and fast latissimus dorsi muscles in the chicken

In the course of muscle differentiation, changes in fibre-type population and in myosin composition occur. In this work, the expression of native myosin isoforms in developing fast-twitch (posterior latissimus dorsi; PLD) and slow-tonic (anterior latissimus dorsi; ALD) muscles of the chick was examined using electrophoresis under nondissociating conditions. The major isomyosin of 11-day-old embryonic PLD comigrated with the adult fast myosin FM3. Two additional components indistinguishable from adult fast FM2 and FM1 isomyosins appeared successively during the embryonic development. The relative proportion of these latter isoforms increased with age, and the adult pattern was established by the end of the 1st month after hatching. Between day 11 and day 16 of embryonic development, PLD muscle fibres also contained small amounts of slow isomyosins SM1 and SM2. This synthesis of slow isoforms may be related to the presence of slow fibres within the muscle. At all embryonic and posthatch stages, ALD was composed essentially of slow isomyosins that comigrated with the two slow components SM1 and SM2 identified in adult. Several studies have reported that the SM1:SM2 ratio decreases progressively throughout embryonic and posthatching development, SM2 being predominant in the adult. In contrast, we observed a transient increase in SM1:SM2 ratio at the end of embryonic life. This could reflect a transitional neonatal stage in myosin expression. In addition, the presence in trace amounts of fast isomyosins in developing ALD muscle could be related to the presence of a population of fast fibres within this muscle.     

Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties

Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.     

Cooperativity of actin-activated ATPase of gizzard heavy meromyosin in the presence of gizzard tropomyosin

The mechanism for the potentiation of the actin-activated ATPase of smooth muscle myosin by tropomyosin is investigated using smooth muscle actin, tropomyosin, and heavy meromyosin. In the presence of tropomyosin, an increase in Vmax occurs with no effect on KATPase and Kbinding at 20 mM ionic strength. Utilizing N-ethylmaleimide-treated subfragment-1, which forms rigor complexes with actin in the presence of ATP but does not have ATPase activity, experiments were carried out to determine if the tropomyosin-actin complex exists in both the turned-off and turned-on forms as in the skeletal muscle system. At both 60 and 100 mM ionic strengths, the presence of rigor complexes on the smooth muscle actin filament containing bound tropomyosin causes a 2-3-fold increase in Vmax and about a 3-fold increase in KATPase, resulting in about a 4-fold increase in ATPase activity at moderate actin concentration. The increase in KATPase is correlated with an increase in Kbinding. The finding that rigor complexes increase Vmax and the binding constant for heavy meromyosin to tropomyosin-actin at an ionic strength close to physiological conditions indicates that the tropomyosin-actin complex can be turned on by rigor complexes in a cooperative manner. However, in contrast to the situation in the skeletal muscle system, the increase in KATPase is associated with a corresponding increase in Kbinding. Furthermore, there is only a 3-fold increase in KATPase in the smooth muscle system rather than a 10-fold increase as in the skeletal muscle system.     

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.