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PubMed1976

Energetics and mechanism of actomyosin adenosine triphosphatase.

Rate constants were determined for the reaction of actin with subfragment 1 (S1), S1-product complex, heavy meromyosin (HMM), and HMM-products complex for a range of temperatures, pH's, and ionic strengths. For actin concentrations up to 10 muM, the rate of reassociation of the product intermediate was equal to the rate of actomyosin subfragment 1 (acto-S1) or acto-HMM adenosine triphosphatase (ATPase). Therefore, under these conditions, the only important pathway for adenosine triphosphate hydrolysis is through the dissociation and recombination of S1 or HMM. The apparent rate constants for the association of S1 and S1-product with actin showed a similar large ionic strength dependence. The S1-product reaction had a large temperature dependence paralleling the rate of acto-S1 ATPase, while the reaction with S1 had a much smaller variation with temperature. The low value of the rate constant for the S1-product reaction and its relationship to the s1 areaction suggests that the apparent rate constant does not measure a simple second-order reaction. A plausible mechanism is a rapid equilibrium for the binding step, followed by a transition (product release) which increases the association constant. A refractory state could also reduce the apparent rate constant of recombination. An approximate assignment of equilibrium constants for the acto-S1 ATPase reaction was made based on the interpretation of the present evidence and equilibrium constnats for the S1 ATPase.

Actins↗

PubMed1975

Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate.

A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.

Adenosine Triphosphatases↗

PubMed1975

Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C.

When stoichiometric amounts of tropomyosin (TM) are bound to F-actin in the presence of 2 mM ATP, the MG2+-activated acto-heavy meromyosin (HMM) ATPase is inhibited by about 60% in 5 mM MgCl2-30 mM KCl. If the concentration of MgCl2 is reduced to 1 mM, the inhibition disappears because TM no longer binds to F-actin. Increasing the concentration of KCl to 100 mM restores both the binding and the inhibition. Thus, the binding of TM alone to F-actin causes significant inhibition of the ATPase provided that the HMM is saturated with ATP. (When the HMM is not saturated, TM activates the ATPase). When TM alone can bind stoichiometrically to F-actin, addition of troponin I (TN-I) increases the inhibition from 60% to about 85%, but the TM binding to F-actin is not affected. Under conditions such that TM alone neither inhibits the acto-HMM ATPase nor binds to F-actin, the inhibition caused by TN-I plus TM still approaches 100%. Direct binding studies under these conditions show that TN-I induces binding between TM and F-actin. A dual role for TN-I is proposed: first, TN-I can induce TM to bind to F-actin, causing inhibition of the ATPase; and second, TN-I can itself enhance the inhibition of the ATPase in a cooperative manner. The addition of TN-C in the absence of CA2+ has only a limited effect on the first role, but seems to be able to block completely the cooperative inhibition caused by TN-I such that the residual inhibition is a function only of the TM which remains bound.

Actins↗

PubMed1977

Effects of myosin and heavy meromyosin on actin-related gelation of HeLa cell extracts.

The gelation induced by warming (to 25 degrees C) the 100,000 g supernatant fraction (extract) of HeLa cells lysed in a buffer containing sucrose, ATP, DTE, EGTA, imidazole, and Triton X-100 was studied in the presence of myosin and heavy meromyosin (HMM). Myosin mixed with extract induces shrinkage of the gel, but jelled extract or myosin alone does not shrink. In the concentration range, 0.14-1.04 mg/ml of myosin, the degree of shrinkage is roughly proportional to the concentration of myosin. Supplementa MgCl2 also promotes shrinkage. HMM (0.4-0.8 mg/ml) can inhibit gel formation by extract in tubes or floated on a sucrose cushion. Gel electrophoresis of gels shrunken by added myosin or electrophoresis of the proteins which can be sedimented from extract after incubation in the presence of HMM indicate that both myosin and HMM interfere with the changes in sedimentability of the high molecular weight protein (HMWP) thought to participate (together with actin) in gel formation in HeLa cell extracts (R. R. Weihing, 1976. J. Cell Biol. 71:303-307). These results, together with previous results showing that actin is present and that HMWP is enriched in the plasma membrane fraction of HeLa cells (R. R. Weihing, 1976. Cold Spring Harbor Conf. Cell Proliferation. 3:671-684), point to the possibility of dynamic changes in the interactions of HMWP or myosin with actin in processes of movement occurring at the cell surface.

Actins↗

PubMed1976

Separation of subfragment-1 of H-meromyosin into two equimolar fractions with and without formation of the reactive enzyme-phosphate-ADP complex.

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of Pi liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1). The size of the initial burst of Pi liberation of acto-S-1 was 0.35 mole/mole of S-l at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/moleof S-1 at ATP concentrations above 1 mole/mole of S-1...

Actins↗

PubMed1976

Cooperative conformational change in F-actin filament induced by the binding of heavy meromyosin.

The binding of HMM to F-actin containing bound 1, N6-ethenoadenosine diphosphate (epsilon-ADP), a fluorescent analogue of ADP, caused a significant increase in the fluorescence intensity of epsilon-ADP at 410 nm on excitation at 340 nm. This increase is regarded as due to a conformational change in the actin molecule induced by HMM binding. The fluorescence intensity increase was not directly proportional to the amount of bound HMM. This phenomenon suggests that a conformational change in neighbouring actin molecules is induced cooperatively by the conformational change of the actin molecule binding HMM.

Actins↗

PubMed1977

Affinity chromatography of myosin, heavy meromyosin, and heavy meromyosin subfragment one on F-actin columns stabilized by phalloidin.

A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract.

Actins↗

PubMed1979

Disposition and metabolism of pentamethylmelamine and hexamethylmelamine in rabbits and humans.

The disposition and metabolism of pentamethylmelamine (PMM) and hexamethylmelamine (HMM) were studied in the rabbit, and the disposition of PMM was studied in humans. Parent compound and metabolites were identified by thin-layer chromatography, gas chromatography, and gas chromatography/mass spectrometry analyses. Plasma elimination in both species following i.v. administration of each drug was best described by a two-compartment open model. Both compounds were extensively demethylated with less than 1% of the total dose administered recovered in the urine over 24 hr. The areas under the plasma time-concentration curves of PMM and HMM following p.o. administration to rabbits were 5 and 25% of the areas following i.v. administration. Gastrointestinal absorption was rapid and efficient with 75 to 89% of drug equivalents recoverable in the urine after p.o. administration of [ring-14C]PMM or [ring-14C]HMM to rabbits. Reduced bioavailability of PMM and HMM p.o. appears to be a consequence of rapid metabolism presumably in the liver.

Altretamine↗

PubMed1979

Interaction of actin with myosin A and heavy meromyosin.

Ca2+ "free" actomyosin suspensions as well as actin heavy meromyosin (HMM) solutions in the presence of Ca2+ showed no contractile response (superprecipitation) and had low steady-state Mg2+-ATPase activity. Under the same experimental conditions both the enzymatic activity increased and contractile response was restored if the solubility of the proteins was depressed by the addition of polyethylene glycol 4000 (PEG-4000). The stability of the enzymatically active actomyosin or actin HMM complexes was 10 times lower in cleared solutions than in the insoluble actomyosin or actin HMM suspensions. It was concluded that soluble actomyosin or actin HMM solutions are inadequate test tube models for studying muscular contraction.

Adenosine Triphosphatases↗

PubMed1976

Hexamethylmelamine. An evaluation of its role in the therapy of cancer.

Hexamethylmelamine (HMM) has been undergoing clinical trials for about a decade under the sponsorship of the National Cancer Institute. It has been tested in Phase II and III cooperative group studies and has a wide spectrum of activity in solid tumors. Its activity is most marked in ovarian cancer, lymphomas, and carcinoma of the cervix; the drug is also active in bronchogenic carcinoma and carcinoma of the breast. Considerable clinical evidence suggests a lack of cross-resistance between HMM and alkylating agents. the currently popular dose is 300 mg/m2/day p.o. for indefinite periods if tolerated. The dose-limiting toxicity is neurologic, but gastrointestinal side effects and a moderate degree of myelosuppression are also observed. Combination chemotherapy using HMM is underway in ovarian and lung cancer. Futher exploration of its activity as a single agent in tumors such as those of the bladder, prostate, and uterus, and in combination chemotherapy in lymphomas, and mammary, cervical, and pulmonary tumors is warranted.

Altretamine↗

PubMed1976

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K+- and Ca2+-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.

Adenosine Triphosphatases↗

PubMed1978

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.

Actins↗

PubMed1976

Reexamination of the conformation of muscle proteins by optical activity.

The circular dichroism and optical rotatory dispersion of muscle proteins are reexamined. By the method of Chen et al. (Chen, Y.H., Yang, J.T., and Chau, K.H. (1974), Biochemistry 13, 3350), the estimated helical contents of myosin (78%), heavy meromyosin (HMM) (70%) subfragment 1 (SF-1) (60%), and G-actin (45%) are higher than hitherto reported. Tropomyosin (TM) and light meromyosin fraction I (LMM Fr. I) possess more than 90% helix in agreement with the values based on the bo method. HMM, SF-1, and G-actin also contain about 8, 16, and 27% beta form. The three troponins (TN) and three light chains (LC) of myosin have moderate amounts of helices (29 to 51%) and some beta form (13 to 23%). If the light chains are intact in HMM and SF-1, myosin would have 3-5% beta form, which is difficult to detect with the present method. For comparison, the predictive method based on amino acid sequence gives similar estimates for TM, G-actin, and TN-C with bound calcium, but slightly higher helical contents than our results for TN-I and the light chains.

Actins↗

PubMed1978

Heavy meromyosin labeling of intermediate filaments in cultured connective tissue cells.

Mild treatment with trypsin causes a radical change in the heavy meromyosin (HMM) binding properties of intermediate filaments in glycerinated, myosin-extracted cultured chick embryo connective tissue cells. In non-trypsin-treated cells, HMM labeling of filaments was often indistinct and variable in its distribution. By contrast, in cells treated with trypsin (under conditions which allowed most intermediate filaments to survive), virtually all filaments, including those of intermediate size, decorated with HMM to give distinct arrowhead patterns. We suggest that most intermediate filaments in such cells contain a core of F-actin masked by trypsin-labile accessory proteins.

Actins↗

PubMed1976

Reaction intermediates of H-meromyosin-ATPase and ultraviolet difference spectrum of H-meromyosin induced by ATP.

UV Difference spectra of H-meromyosin (HMM) during the steady state of the myosin-ATPase reaction [EC 3.6.1.3] were measured in 1.5 and 0.05M KC1 in the presence of 5mM MgC1(2) and 20mM Tris-HC1 at pH 8.0 and 24 degrees, using pyruvate kinase [EC 2.7.1.40] and phosphoenolpyruvate to regenerate ATP. It was found that the difference spectrum and its dependence on ATP concentration were the same in 1.5M KC1 as in 0.05M KC1. On the bases of these and other results, the nature of the intermediates of HMM ATPase in the steady-state reaction of HMM ATPase was discussed.

Adenosine Triphosphatases↗

PubMed1976

2,4-Dinitrophenol as a specific inhibitor of the breakdown of the actomyosin-phosphate-ADP complex.

2,4-Dinitrophenol (DNP) was found to cause a "clearing response" of myosin B in a medium in which "superprecipitation" of myosin B would otherwise take place. The effect of actin concentration on Mg-ATPase [EC 3.6.1.3] of HMM was studied in the presence and absence of DNP. The results indicate that DNP causes an increase rather than a decrease in the affinity of HMM for actin, and that it causes a decrease only in the actin-activated portion of the Mg-ATPase activity. Using a light-scattering technique, it was shown that neither the ATP-induced dissociation of acto-HMM nor subsequent reassociation is significantly affected by the presence of DNP. As for the formation of the myosin-phosphate-ADP complex in the myosin-ATPase reaction, it was shown that formation of the reactive complex is not affected by DNP. It can thus be concluded that DNP inhibits the decomposition of the actomyosin-phosphate-ADP complex, which is thought to be coupled with superprecipitation.

Actins↗

PubMed1977

Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.

Actins↗

PubMedDate not supplied

Actin like filaments in the peritubular cells of human testis chemical extraction and binding with heavy meromyosin.

Two types of the cytoplasmic filaments are found in human peritubular cells; thin filaments (50-80 A) and 100 A filaments. The thin filaments are numerous and tightly packed in cytoplasma of peripheral cell processes, and form bundles. These bundles are arranged in parallel with the long axis of the cells. Dense areas are occasionally seen among the filaments, or underneath the cell membrane to which the thin filaments seem to be anchored. The 100 A filaments are generally located around the perinuclear region or in the middle region of cytoplasma. The thin filaments are not altered after incubation with Hasselbach-Schneider's solution. These filaments, however, disappear in cytoplasma when extracted with 0.6 M KI solution. And also, the thin filaments form chracteristic arrowhead complexes, when incubated with HMM, while these complexes are never found, when incubated with HMM plus ATP. The 100 A filaments do not react to HMM. Thus it is suggested that the thin filaments are actin or actin like and that a contractile system exists in human peritubular cells, which may be involved in transport of nonmotile sperm or permeability of the seminiferous walls.

Adult↗