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S Chacko

Publications and source records attributed to S Chacko.

At least 91 records · Page 5Linked to original sources
PubMed1987

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation.

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.

Actins↗
PubMed1987

Modulation of actomyosin ATPase by thin filament-associated proteins.

Phosphorylation of the myosin light chain is a prerequisite for actin-activation of the Mg-ATPase of smooth muscle myosin. However, maximal activation of the Mg-ATPase by actin requires stoichiometric binding of tropomyosin to actin filaments and Ca2+ at free Mg2+ below 3 mM. The requirement for Ca2+ for actin-activation is not due to a calcium-mediated binding of tropomyosin to actin since the binding of tropomyosin to actin is not dependent on Ca2+. Caldesmon, an actin and calmodulin binding protein, at caldesmon:actin molar ratio of 1:18, binds equally to pure actin and actin containing stoichiometric amounts of bound tropomyosin. The Mg-ATPase of myosin reconstituted with actin is not affected by the caldesmon; on the other hand, the activity of actomyosin containing tropomyosin is inhibited. The inhibition of activity by the caldesmon is reversed by the addition of calmodulin (caldesmon:calmodulin molar ratio, 1:8) in the presence of Ca2+. The amount of caldesmon bound to actin in the presence of calcium-calmodulin is 50% more when actin filaments contain tropomyosin, indicating that the release of inhibition of the activity inhibited by caldesmon does not require complete release of caldesmon from actin.

Actins↗
PubMed1986

Modulation of smooth muscle actomyosin ATPase by thin filament associated proteins.

Caldesmon binds equally to both gizzard actin and actin containing stoichiometric amounts of bound tropomyosin. The binding of caldesmon to actin inhibits the actin-activation of the Mg-ATPase activity of phosphorylated myosin only when the actin contains bound tropomyosin. The reversal of this inhibition requires Ca2+-calmodulin; but it occurs without complete release of bound caldesmon. Although phosphorylation of the caldesmon occurs during the ATPase assay, a direct correlation between caldesmon phosphorylation and the release of the inhibited actomyosin ATPase is not consistently observed.

Actins↗
PubMed1986

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.

Actins↗
PubMed1986

Regulation of actomyosin ATPase in smooth muscle.

Actomyosin in smooth muscle is in a quiescent state. The mechanism or mechanisms by which Ca2+ activates the actomyosin ATPase is not clear. There is sufficient evidence for the presence of enzyme systems which phosphorylate and dephosphorylate myosin light chains. The activity of the kinase that phosphorylates the myosin is regulated by cAMP-dependent protein kinase. Phosphorylated kinase has decreased affinity for calmodulin and lower activity when compared with unphosphorylated myosin light chain kinase. The activity of myosin light chain kinase is also regulated by calcium-calmodulin. In the presence of Ca2+, myosin is phosphorylated. In the absence of Ca2+, the phosphatase activity becomes dominant; the myosin remains in the unphosphorylated form under this condition. The Mg2+-ATPase of the phosphorylated myosin is activated by actin. The maximal activation of the Mg2+-ATPase by actin requires Ca2+ and tropomyosin, a protein located on the thin filament. Hence, the actin-activation of the Mg2+-ATPase requires Ca2+ even after phosphorylation by the calcium-calmodulin dependent kinase. The regulation of actin-activated ATPase activity by myosin light chain phosphorylation is depicted in the schematic diagram. Caldesmon, an actin-binding protein which also binds to calmodulin in the presence of Ca2+, has been shown to be present in thin-filaments isolated from smooth muscle. This protein inhibits actin-activated myosin ATPase activity. The release from this inhibition requires Ca2+ and calmodulin. The possibility that caldesmon is also involved in the calcium regulation of actomyosin in smooth muscle is presently under investigation in a number of laboratories.

Adenosine Triphosphatases↗
PubMed1985

The effect of leiotonin fraction on stably phosphorylated smooth muscle myosin.

This study was designed to determine the effect of leiotonin on the actin-activation once the myosin is stably phosphorylated. Gizzard myosin was stably phosphorylated by ATP-gamma-S using the gizzard light chain kinase. Addition of leiotonin preparation to phosphorylated myosin reconstituted with actin and tropomyosin did not alter the ATPase activity. Furthermore, leiotonin did not confer the calcium sensitivity of the ATPase activity. These experiments show that the actin-activated ATPase activity of stably phosphorylated gizzard myosin is not altered by leiotonin.

Actins↗
PubMed1985

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin.

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Actomyosin↗
PubMed1984

Effects of Ca2+ and Mg2+ on the actin-activated ATP hydrolysis by phosphorylated heavy meromyosin from arterial smooth muscle.

Actin-activated ATP hydrolysis by phosphorylated arterial myosin is Ca2+-dependent at Mg2+ concentrations which allow myosin to bind 2 mol of Ca2+ per mol (S. Chacko and A. Rosenfeld (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 292-296). The possible effects of myosin filament formation and conformational change involving the rod portion of the myosin molecule on actin-myosin interaction and actin-activated ATP hydrolysis was eliminated by utilizing soluble heavy meromyosin (HMM). HMM was prepared from phosphorylated arterial myosin by chymotryptic digestion and purified by gel filtration on Sepharose CL-6B. Actin-activated ATPase activity of the phosphorylated HMM was measured either at constant Mg2+ and variable Ca2+ or at constant Ca2+ and variable Mg2+ concentrations. At constant (0.05 M) ionic strength and pCa 5, the actin-activated ATPase activity increased until the free Mg2+ reached between 0.5-1 mM; this was followed by a decrease in activity to a very low level at 7 mM free Mg2+. Removal of Ca2+ at 1 mM free Mg2+ lowered the actin-activated ATPase activity (40-60% inhibition). These experiments demonstrated that Mg2+ and Ca2+ had a direct effect on actin-activated ATP hydrolysis. This effect was not due to myosin filament formation and was independent of the conformational change involving the rod portion of the myosin molecule.

Actins↗
PubMed1983

Diet- and hormone-induced lipid deposition in rat kidney: correlation with systolic blood pressure.

The influence of estradiol on deposition of cholesterol in tissues of ovariectomized rats on normal and high lipid diets was studied. Concomitantly the influence of a contraceptive steroid combination was studied in a similar manner in intact rats. It was found that the high lipid diet resulted in increased deposition of cholesterol in aorta, heart, liver and kidney. The presence of either endogenous or exogenous hormones accentuated the deposition of cholesterol in the kidney and resulted in significantly higher systolic blood pressures in these rats. In the rats on a high lipid diet, the concentration of cholesterol in the kidney correlated positively with systolic blood pressure. It is concluded that estrogen and high lipid diet exert a synergistic effect on deposition of cholesterol in kidney. The positive correlation between kidney cholesterol concentration and systolic blood pressure suggests a possible role for kidney lipid deposition in the hypertensive effect of estrogens.

Animals↗
PubMed1982

Regulation of actin-activated ATP hydrolysis by arterial myosin.

Myosin was isolated from the main pulmonary artery of swine and was phosphorylated or dephosphorylated by utilizing the endogenous kinase or phosphatase, respectively. The myosins, phosphorylated to various degrees, were purified free of kinase and phosphatase activities by gel filtration on Sepharose CL-4B agarose columns. The level of actin-activated ATPase activity was dependent upon the degree of myosin light chain phosphorylation. Fully phosphorylated myosin reconstituted with actin and tropomyosin (actin/tropomyosin = 61:1) had the highest ATPase activity (0.1 mumol of Pi/mg . min). The actin-activated ATPase activity showed maximal (60--65%) Ca2+ sensitivity at 2 mol of Ca2+ bound per mol of myosin. The actin-activated ATPase activity, Ca2+ binding, and Ca2+ sensitivity of arterial myosin were also dependent upon Mg2+ concentration. The ATPase activity was maximal at 2--3 mM Mg2+ and, at low (0.5 mM) Mg2+ concentration, the activity was only one-third of the maximal activity. Increasing the Mg2+ above 3 mM was not associated with a further increase in ATPase activity, but the Ca2+ binding and Ca2+ sensitivity decreased with increasing Mg2+ concentration. The maximal Ca2+ sensitivity was observed at 2--3 mM Mg2+, a concentration at which the myosin bound 2 mol of Ca2+/mol. Both the ATPase activity and the Ca2+ sensitivity were more remarkable when actin that contained tropomyosin was used to activate the ATPase activity. The data indicate that calcium regulates the actin-activated ATP hydrolysis not only by its effects on the phosphorylation system but also by direct binding to the myosin.

Actins↗
PubMed1981

Effects of phosphorylation, calcium ion, and tropomyosin on actin-activated adenosine 5'-triphosphatase activity of mammalian smooth muscle myosin.

Actomyosin isolated from bovine stomach muscle contains the endogenous light-chain kinase and phosphatase. Myosin can be separated from other proteins by gel filtration on a Sepharose 4B--agarose column. The amount of phosphate covalently bound to the 20 000-dalton light chains of purified myosin can be controlled by phosphorylation or dephosphorylation using the endogenous enzymes prior to column purification. The purified myosin can serve as a substrate for exogenously added light-chain kinase and phosphatase, but the myosin itself is free of the activities for both enzymes. The adenosine 5'-triphosphatase (ATPase) activity of myosin was activated by rabbit skeletal muscle actin only when the 20 000-dalton light chain was phosphorylated. The level of activation correlated with the amount of phosphate bound to the light chain. The maximum activation by pure actin was observed when the molar ratio of myosin to actin was 1:20. The activation was dependent on the amount of phosphate bound to the myosin light chain at all levels of actin concentrations. The actin-activated ATPase activity of stomach muscle myosin is not dependent on Ca2+ concentration once the myosin is phosphorylated and is free of kinase and phosphatase activity. The actin-activated ATPase activity was higher when the actin was complexed with tropomyosin. The highest level of activation was obtained when the myosin was fully phosphorylated and the actin was complexed with tropomyosin at a molar ratio of 1:6 (Tm/A). The potentiation of actin-activated ATP hydrolysis by tropomyosin is not dependent on Ca2+. These data indicate that tropomyosin plays a major role in the actin-activated ATP hydrolysis by smooth muscle myosin in the absence of other regulatory proteins.

Actins↗
PubMed1981

Emergence of beta-adrenergic sensitivity in the developing chicken heart.

Muscle cells dissociated from 5-day embryonic chicken hearts showed dose-dependent increases in both chronotropic rates of contraction and cyclic AMP (cAMP) levels in response to epinephrine (EPI), an effect that could be blocked by beta-adrenergic antagonists. However, 2- to 2.5-day embryonic chicken myocardial cells, although similar to 5-day heart cells with respect to the organization of myofibrils, failed to respond to EPI either by increased rates of contraction or by elevated levels of intracellular cAMP. Development of beta-adrenergic sensitivity in 2- to 2.5-day cells did not occur even after several days of growth in culture. However, addition of an extract prepared from 11-day chicken embryos to 2- to 2.5-day muscle cell cultures at any point during in vitro growth resulted in the development of sensitivity to EPI as measured by increases in both the beating frequency and cAMP levels after a 48-hr incubation in embryo extract (EE). The basal level of cAMP in cells unresponsive to EPI is 5 times that in EPI-sensitive muscle cells from older hearts (5 days). The high basal level of cAMP in these cells is reduced to a level characteristic of cells from older hearts when treated with the EE. Once sensitivity was acquired, it was retained as a stable trait of the muscle cells in culture. Furthermore, EE-treated 2- to 2.5-day cells showed less reduction of positive chronotropy in response to multiple doses of EPI than did cells prepared from 5-day hearts. Fractionation of EE on Sephadex G-200 showed that the activity was present in the large-molecule fractions. It is concluded that the development of beta-adrenergic sensitivity can be induced in unresponsive heart cells cultured from 2- to 2.5-day embryos by a factor(s) in the EE that is not adsorbed on Sephadex G-200.

Adrenergic beta-Antagonists↗
PubMed1977

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.

Actins↗
PubMed1977

Localization of bovine brain filament antibody on intermediate (100 A) filaments in guinea pig vascular endothelial cells and chick cardiac muscle cells.

Guinea pig vascular endothelial cells contain naturally occurring rings of intermediate filaments that completely encircle the nucleus. Indirect immunofluorescence staining showed that these perinuclear rings bound antibody prepared against protein from bovine brain 9-nm filaments. In endothelial cells grown in the presence of 1 muM demecolcine (Colcemid) the perinuclear ring "coils" into a juxtanuclear "cap". Throughout this process we could demonstrate staining of the intermediate filaments. Chick cardiac muscle cells in culture stained diffusely with the antibody. After treatment for 24 hr with 1 muM demecolcine the cardiac cells accumulated large bands of intermediate filaments. These bands stained intensely with the antibody. Our findings suggest that intermediate filaments in guinea pig endothelial cells and those induced in chick cardiac muscle cells are antigenically similar to bovine brain filaments. The staining of these filaments is not affected by treatment with demecolcine.

Animals↗
PubMed1976

The genetics and pathology of discrete subaortic stenosis in the Newfoundland dog.

Breeding experiments confirm that discrete subaortic stenosis (SAS) in Newfoundland dogs is a specific inherited trait. Specificity of the morphogenetic abnormality is not complete, however, since matings between Newfoundlands with SAS occasionally produced pups with valvular and subvalvular pulmonic stenosis as well as SAS. The spectrum of severity of SAS ranged from a subclinical forme fruste to a severe form causing death before maturity. Well-developed subvalvular stenotic rings consisted of a base of loosely arranged fibrous connective tissue and a subendocardial region of cartilagenous tissue. Severely affected dogs, some of which died suddenly, had foci of necrosis and fibrosis in the left ventricular myocardium, associated with thickening of the intramural coronary arteries. The lesions of SAS were not found in dogs before 3 weeks of age, and the mildest form was seen only in dogs between 3 and 12 weeks of age, suggesting that SAS is not a true congenital defect but develops postnatally. It is hypothesized that the fibrocartilagenous ring of SAS is derived from persistent embryonal endocardial tissue which retains its proliferative capacity and has chondrogenic potential for some time after birth. The results of breeding experiments were not consistent with any simple genetic hypothesis, and indicate that SAS is inherited as a polygenic trait or as an autosomal dominant trait with modifiers.

Age Factors↗