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PubMed1975

Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.

Adenosine Triphosphatases↗
PubMed1986

Elastic energy storage in rigored skeletal muscle cells under physiological loading conditions.

The capability of heavy meromyosin (HMM) to store energy in reversible deformations has been investigated previously; yet, whether HMM is the site of most elastic energy storage in skeletal muscle cells has not been established. We conducted dynamic loading tests on single rigored muscle cells over the physiological range of sarcomere lengths. These tests enabled us to calculate the energy stored in reversible deformations or dissipated in the cell during each cycle of oscillation. Our findings show that these cells are capable of storing approximately 0.5 J . kg-1 of elastic energy during the last 50 ms of passive extension in vivo by agonists and before their own active contraction. Possible sites of this energy storage are HMM subunit 2, the proximal portion of HMM subunit 1, and parallel structures. However, energy storage increases monotonically as myofilament overlap decreases in the physiological range. This negative correlation suggests that HMM subunits are not the primary sites of elastic energy storage. Our electron-microscopic observations show that collagen fibrils at the cell's surface become oriented parallel to the cell's long axis over the range of sarcomere lengths where energy storage increases. This provides a mechanism for the observed increases in elastic energy storage.

Animals↗
PubMed1986

Dexamethasone modulates lipoprotein metabolism in cultured human monocyte-derived macrophages. Stimulation of scavenger receptor activity.

Human monocyte-derived macrophages (HMM) play a key role in the formation of atherosclerotic plaques by accumulating cholesteryl ester (CE) to become foam cells. HMM have receptors for native low density lipoprotein (LDL) and acetylated-LDL (ALDL), and uptake of ALDL can promote substantial cellular CE accumulation. Furthermore, macrophages specifically and saturably bind glucocorticoids, which in turn modulate numerous macrophage functions. Preincubating HMM in dexamethasone-inhibited LDL degradation (230 +/- 12 vs. 515 +/- 21 ng/mg cell protein X 18 h, P less than 0.001) but stimulated ALDL degradation (5.3 +/- 0.5 vs. 2.5 +/- 0.3 micrograms/mg X 18 h, P less than 0.01). These effects were time- and dose-dependent, occurring maximally by 24 h and with 2.5 X 10(-8) M dexamethasone. Dexamethasone increased the maximum velocity for ALDL degradation (16.2 vs. 12.0 micrograms/mg X 18 h, P less than 0.01) without changing the apparent Michaelis constant. Progesterone, 11 alpha-epicortisol, and 17 alpha-OH progesterone (a competitive antagonist of the glucocorticoid receptor) had no effect on HMM ALDL degradation, but 17 alpha-OH progesterone abolished the stimulatory action of dexamethasone. In he presence of ALDL, incorporation of [14C]oleic acid into CE was enhanced over fourfold by dexamethasone (4015 +/- 586 vs. 943 +/- 91 cpm/mg X 2 h, P less than 0.01), and HMM incubated with ALDL and dexamethasone accumulated more free cholesterol (34.6 +/- 1.9 vs. 26.2 +/- 0.8 micrograms/mg, P less than 0.02) and CE (32.8 +/- 2.3 vs. 14.8 +/- 0.8 micrograms/mg, P less than 0.002) than did macrophages without dexamethasone. In cultured human umbilical vein endothelial cells, dexamethasone did not change ALDL degradation, but reduced LDL degradation by 30% (P less than 0.001). In summary, dexamethasone inhibits LDL receptor activity by both macrophages and endothelial cells, but stimulates ALDL receptor activity only in macrophages. These observations provide evidence for the regulation of macrophage endocytic receptors by glucocorticoid hormones.

17-alpha-Hydroxyprogesterone↗
PubMed1987

Validity of Hill's equation in an artificial actomyosin streaming system.

The rotation of an actomyosin motor, assembled from blades, one side onto which F-actin of uniform polarity was attached, suspended in a solution of heavy meromyosin (HMM), was modelled as due to sliding of HMM over the margins of the blades, whereby the work resulting from ATPase activity is used for pushing bulk fluid containing HMM from the leading surface of the blade over the force-generating filaments to the back surface, which leads to increased sliding velocity. The amount of HMM contributing to force-generation is divided into one component perpendicular to the filaments, which is diffusion-limited and regulated by a component parallel to the filaments, represented by the movement of the bulk fluid, the supply of new HMM and the observable velocity of rotation of the blade. Using Hill's equation which essentially states that the product of a virtual force and a virtual velocity is constant within the range of observable forces and velocities, the force can be expressed as velocity, giving a simple first order differential equation. A solution to this equation can be fitted to the observed data with physically meaningful constants in terms of mass, viscosity, the force-velocity constant and a constant expressing the quality of the preparation. Control experiments were performed, using fixed blades. In these experiments, bulk flow around the margins did not increase as a result of ATPase activity and no movement or streaming was observed. The results show that Hill's equation may be applicable to streaming caused by actomyosin irrespective of its confinement to skeletal muscle and independently of preferred molecular model, and leads to several verifiable predictions.

Actomyosin↗
PubMed1992

Hexamethylmelamine and low or moderate dose cisplatin with or without pyridoxine for treatment of advanced ovarian carcinoma: a study of the Eastern Cooperative Oncology Group.

A total of 248 analyzable patients with Stages III-IV ovarian epithelial cancer (114 with and 134 without prior chemotherapy) were randomized to one of four cisplatin (DDP)-hexamethylmelamine (HMM) regimens. In each, HMM, 200 mg/m2 was given orally daily on days 8-21 of each 21-day cycle. DDP was given i.v. on Day 1 at a dose of 37.5 mg/m2 (regimens A and B) or 75 mg/m2 (regimens C and D). In addition, since pyridoxine administration has been reported to reduce the neurotoxicity of HMM, that agent was given at a dose of 300 mg/m2 orally on Days 1-21 in regimens B and D. Randomization was stratified for performance status (0-1, 2-3) and largest tumor diameter at entry (greater than 2- less than or equal to 10 cm, greater than 10 cm) for previously untreated patients, and for performance status and time from initial diagnosis to entry on study (less than or equal to 1 year, greater than 1 year) for previously treated patients. The overall response rate (PR + CR) was 54%, with 25% of patients achieving a complete response. The 61% response rate with the higher dose DDP regimens was significantly greater than the 47% response rate with the lower dose regimens (p = 0.031). Multivariate analysis identified higher DDP dose, age less than 60 years, no prior chemotherapy, small tumor bulk and favorable tumor grade as significant prognosticators for response. The overall median response duration was 8.3 months (range 1-70 months). Prior chemotherapy, pyridoxine administration, recent diagnosis, and large tumor size were identified by multivariate analysis as factors adversely affecting response duration. Patients treated with the higher dose DDP regimens had more severe nausea, vomiting, and neurotoxicity. This study demonstrates that the combination of DDP + HMM is an effective regimen for advanced ovarian carcinoma that yields response rates comparable to other more complex regimens, and that there is a dose-response relationship for DDP in ovarian cancer. Although pyridoxine administration significantly reduced neurotoxicity, its adverse effect on response duration suggests that the agent should not be administered with DDP or HMM. The mechanism by which pyridoxine may unfavorably affect response duration deserves further investigation.

Altretamine↗
PubMed1992

Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle.

Phosphatase inhibitors microcystin-LR, tautomycin, and okadaic acid caused contraction and increased 20-kDa myosin light chain (MLC20) phosphorylation in Ca(2+)-free solutions in both phasic and tonic smooth muscle permeabilized with beta-escin, and inhibited the heavy meromyosin (HMM) phosphatase activity of smooth muscle homogenates with the same potency sequence: microcystin-LR greater than tautomycin greater than okadaic acid. The sensitivity to all three inhibitors was significantly higher, the half-times of relaxation and dephosphorylation were 4-6 times longer, and the HMM phosphatase and MLC20 kinase activity/smooth muscle cell wet weight was 2.0- and 1.9-fold lower in the tonic, femoral artery, than in the phasic, ileum or portal vein, smooth muscle. Preincubation with 0.2 microM inhibitor-2 decreased the HMM phosphatase activity by 35% in the ileum and by 60% in the femoral artery. The results suggest that the HMM phosphatases of smooth muscle have properties common to type 1 protein phosphatases, but are inhibited only partially by high concentrations of inhibitor-2, and that the lower HMM phosphatase activity of tonic smooth muscle may contribute to its greater sensitivity to phosphatase inhibitors and its slower rate of relaxation.

Animals↗
PubMed1977

Enzymatic properties of the heavy meromyosin subfragment of cardiac myosin from normal and thyrotoxic rabbits.

Myosin from the hearts of thyrotoxic animals (myosin-T) exhibits elevated Ca2+-ATPase activity. To clarify the physiological significance of this increased activity, we have investigated the steady state kinetics of the interaction of actin and MgATP with the double-headed heavy meromyosin subfragment of cardiac myosin from thyrotoxic rabbits (HMM-T). The enhanced Ca2+-ATPase activity of myosin-T was completely retained in HMM-T. The Vmax for actin-activated MgATP hydrolysis by HMM-T (1.08 +/- 0.10 mumol of Pi/mg/min). Under physiological ionic conditions, the Vmax was 0.14 +/- 0.02 mumol of Pi/mg/min as compared with the normal value of 0.08 +/- 0.01 mumol of Pi/mg/min. Furthermore, the salt dependence of Vmax and Kapp for the actin-activated ATPase of HMM-T differed markedly from normal and resembled that usually associated with the single-headed (S1) cleavage product of myosin. These results suggest that the changes in enzymatic properties of myosin-T are responsible for the increased speed of contraction observed in the hearts of thyrotoxic animals. Also, the alteration in the interaction of HMM-T with actin suggests that a loss of cooperativity between the myosin heads may occur.

Adenosine Triphosphatases↗
PubMedDate not supplied

Mexiletine metabolism in vitro by human liver.

Human livers were used in investigations of mexiletine biotransformation in vitro. The major metabolic pathways of mexiletine oxidation, to form hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM), were characterized in liver cell preparations. The localization of reactions in the microsomal fraction, their heat lability, NADPH requirement and inhibition by prototype cytochrome P-450 (P-450) inhibitors (CO, SKF 525-A, metyrapone and quinidine) implied that they were catalyzed by P-450. Kinetic studies of reactions were performed in microsomes from five different livers. Eadie-Hofstee plots of data gave no indication of systematic deviation from linearity, suggesting that over the range of mexiletine concentrations examined (3.3-133.3 microM), HMM and PHM were formed by a single enzymatic site. Within a liver preparation, Km and Vmax values for HMM and PHM formation were similar. Between livers, Km values of reactions were similar with only a 1.8-fold range for each reaction, whereas Vmax values showed 7.2- and 7.8-fold ranges for HMM and PHM production, respectively. There was a very strong correlation between Vmax values for both reactions. These results, coupled with a parallel effect of inhibitors (SKF-525A, metyrapone, alpha-naphtoflavone and quinidine) on HMM and PHM formation, argue that both reactions are mediated by a common P-450 or closely related isozymes. In addition, the present in vitro results support the hypothesis that the genetically variable P-450 db 1 isozyme catalyzes the oxidation of mexiletine.

Biotransformation↗
PubMed1990

Dissociation of the effect of caldesmon on the ATPase activity and on the binding of smooth heavy meromyosin to actin by partial digestion of caldesmon.

We have proposed earlier that caldesmon inhibits the actin-activated ATPase activity of smooth muscle heavy meromyosin (HMM) by inhibiting the binding of the HMM.ATP complex to the productive site of actin (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1868-1885). This has been difficult to prove directly because caldesmon also binds to HMM and it is difficult to distinguish the actin-caldesmon-HMM complex from the actin-caldesmon complex in binding studies. We have eliminated the interaction between caldesmon and smooth HMM by digestion of caldesmon with chymotrypsin. This cleaved caldesmon inhibits the actin-activated ATPase rate of smooth HMM and this inhibition is correlated with a decrease in the binding of HMM.ATP to actin. Therefore, caldesmon functions by inhibiting the binding of the myosin-ATP complex to actin regardless of the source of myosin. We have also isolated the myosin-binding region of caldesmon and have performed a partial sequence. Comparison of this sequence with the derived sequence of caldesmon demonstrates, unequivocally, that the myosin-binding region of caldesmon begins at the amino terminus and extends beyond the first Cys residue.

Actins↗
PubMed1990

Characterization of caldesmon binding to myosin.

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.

Adenosine Triphosphate↗
PubMed1975

The effects of ionic conditions, temperature, and chemical modification on the fluorescence of myosin during the steady state of ATP hydrolysis. A comparison of the fluorescnece and electron spin resonance spectra of the spin-labeled enzyme.

The ATP-induced enhancement of the intrinsic fluorescence of myosin and heavy meromyosin (HMM) that persists during the steady state of hydrolysis has been investigated. To compare the substrate-induced changes in fluorescence with those in the electron spin resonance spectrum of the spin-labeled enzyme, we studied the influence of temperature, pH, and ionic strength, as well as the effect of chemical modification (spin labeling) of the SH-1 sulfhydryl groups. Changing the pH between 6 and 9 does not affect the enhancement of fluorescence of myosin or HMM; changing the ionic strength, which could be studied only with HMM, also has no effect; and decreasing the temperature from 20 to 5 degrees slightly diminishes the enhancement with both myosin and HMM. Chemical modification with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetamide, which blocks the SH-1 thiol groups, reduces the enhancement of fluorescence, induces a strong dependence on ionic strength and pH, and substantially increases the dependence on temperature. The enhancement with labeled myosin or labeled HMM increases with increasing pH, ionic strength, and temperature, closely paralleling the effects of these parameters on the electron spin resonance spectrum of spin-labeled myosin (SEIDEL, J.C. and GERGELY, J. (1973) Arch. Biochem. Biophys. 158, 853), suggesting that the same molecular change, induced by ATP and associated with formation of the MADP-P1 complex, underlies both the change in fluorescence and the change in ESR spectrum. Those analogues of ATP that produce the maximal enhancement of fluorescence (WERBER, M., SZENT-GYORGYL, A.G., and FASMAN, G. (1972) Biochemistry 11, 2872) also produce the maximal change in the ESR spectra. Both an amino group at position 6 of the substrate and an unmodified triphosphate chain are required for maximal change in either fluorescence or ESR spectra. The smaller enhancement of fluorescence produced by spin labeling the SH-1 groups persists after the nitroxide has been chemically changed to a diamagnetic species. Thus the small enhancement cannot be attributed to paramagnetic quenching of tryptophan fluorescence by the spin label. An initial burst of phosphate liberation accompanies the hydrolysis of ATP, cytidine 5'-triphosphate, uridine 5'-triphosphate, guanosine 5'-tryphosphate, iosine 5'-triphosphate, 2'-deoxyadenosine 5'-tryphosphate, adenosine 5'-tetraphosphate, and tripolyphosphate. The presence or absence of the burst does not correlate with the extent of the spectral change.

Adenosine Triphosphatases↗
PubMed1984

Regulation of molluscan actomyosin ATPase activity.

The interaction of myosin and actin in many invertebrate muscles is mediated by the direct binding of Ca2+ to myosin, in contrast to modes of regulation in vertebrate skeletal and smooth muscles. Earlier work showed that the binding of skeletal muscle myosin subfragment 1 to the actin-troponin-tropomyosin complex in the presence of ATP is weakened by less than a factor of 2 by removal of Ca2+ although the maximum rate of ATP hydrolysis decreases by 96%. We have now studied the invertebrate type of regulation using heavy meromyosin (HMM) prepared from both the scallop Aequipecten irradians and the squid Loligo pealii. Binding of these HMMs to rabbit skeletal actin was determined by measuring the ATPase activity present in the supernatant after sedimenting acto-HMM in an ultracentrifuge. The HMM of both species bound to actin in the presence of ATP, even in the absence of Ca2+, although the binding constant in the absence of Ca2+ (4.3 X 10(3) M-1) was about 20% of that in the presence of Ca+ (2.2 X 10(4) M-1). Studies of the steady state ATPase activity of these HMMs as a function of actin concentration revealed that the major effect of removing Ca2+ was to decrease the maximum velocity, extrapolated to infinite actin concentration, by 80-85%. Furthermore, at high actin concentrations where most of the HMM was bound to actin, the rate of ATP hydrolysis remained inhibited in the absence of Ca+. Therefore, inhibition of the ATPase rate in the absence of Ca2+ cannot be due simply to an inhibition of the binding of HMM to actin; rather, Ca2+ must also directly alter the kinetics of ATP hydrolysis.

Actins↗
PubMed1984

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin.

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.

Actins↗
PubMedDate not supplied

Pharmacokinetics of hexamethylmelamine and pentamethylmelamine in mice.

Plasma pharmacokinetics of hexamethylmelamine (HMM) and pentamethylmelamine (PMM) were investigated after ip administration of doses of 50 or 100 mg/kg to M5076/73A ovarian tumor-bearing C57BL/6J female mice. The half-life of HMM was 44.5-49 mins; the half-life of PMM was 7.6-8.7 mins. The area under the concentration-versus-time curve (AUC) was also much higher for HMM than for PMM. The AUC for the metabolite N2,N2,N4,N6-tetramethylmelamine (TMM) was seven to ten times higher than that of the parent compounds. After incubation with mouse liver microsomes and cofactors, the rates of metabolism of HMM and PMM were essentially the same. Plasma protein binding was 94%, 71%, and 50% for HMM, PMM, and TMM, respectively.

Altretamine↗
PubMed1982

Hexamethylmelamine distribution in patients with ovarian and other pelvic cancers.

The distribution of hexamethylmelamine (HMM) was investigated in surgical biopsies from 12 patients with pelvic cancer who were given an oral dose of 200 mg at 2-24 hours before the operation. The highest concentrations of HMM were found in tissues, such as omentum and subcutaneous tissue, that have a predominant lipidic component. HMM concentrations in the primary tumor were lower than in other tissues and were similar to those in plasma. HMM concentrations in metastases appeared to be higher than in the primary tumor, and in metastases with a diameter less than 3 mm, drug levels were two to ten times those in larger ones. Plotting the HMM concentrations versus time, utilizing the data from different patients, the elimination half-lives were found to be 9.05, 8.82, and 8.53 in plasma, tumor, and subcutaneous tissue, respectively.

Adipose Tissue↗
PubMed1981

The mechanism of limited tryptic proteolysis of heavy meromyosin as revealed by peptide analysis.

To elucidate some ambiguous details in the tryptic fragmentation scheme of HMM as given by Bálint et al. (J. Biol. Chem. 250 (1975) 6168; Arch. Biochem. Biophys. 190 (1978)793), the peptide fragments were isolated by a milligram scale preparative gel electrophoresis procedure. The dansyl-peptide map of the 20 kDal tryptic fragment obtained from tryptic heavy meromyosin (HMM) and that of a similar fragment from papainic subfragment-1 (S-1) were found to be nearly identical. This finding gives unequivocal proof of the location of the 17 kDal peptide stretch lost during digestion in the form of small peptides, at the C terminal part of the heavy chain backbone of HMM. The N terminals of the 150, 74, and 25 kDal fragments of the heavy chain isolated from HMM digested by trypsin under widely differing conditions were shown to be acetylated. The N terminal amino group of the other peptide fragments of HMM remains the same under widely differing conditions of digestion. We conclude that all the fragments are well defined polypeptides and digestion progresses by splitting from the C terminals formed by the primary splits.

Acetylation↗
PubMed1995

Significance of the N-terminal fragment of myosin regulatory light chain for myosin-actin interaction.

The influence of myosin regulatory light chains (LC2s) lacking the 2kD N-terminal portions of these chains, on internal organization of myosin heads and on actin-myosin interaction was studied with limited proteolysis and polarized fluorescence methods. For these studies heavy meromyosin (HMM) preparations were used: HMM containing intact LC2s (phosphorylated or dephosphorylated) and HMM containing LC2s lacking the 2kD N-terminal portions (including serine which can be phosphorylated). It was found that the susceptibility of the heavy chain cleavage site to trypsin and the alkali light chain (LC1) site to papain of myosin containing shortened regulatory LC2s, is not dependent on saturation of LC2s with Ca2+ or Mg2+ ions. This is in contrast to the myosin containing intact LC2s where Ca2+ or Mg2+ ion saturation does demonstrate a dependence. Similarly, in spectroscopic experiments, dephosphorylated HMM containing intact LC2s causes decrease or increase of actin filament flexibility depending on whether Mg2+ or Ca2+ are bound to LC2s. Correspondingly, HMM with shortened LC2s induces only increase of actin flexibility despite cations being bound. We conclude that the N-terminal fragment of LC2 is important for ensuring a proper Ca2+ dependent conformation of myosin head in the course of its actin-activated ATP hydrolysis.

Actins↗
PubMed1984

Actin microfilaments in paramecium: localization and role in intracellular movements.

Using heavy meromyosin (HMM) or the fragment S1 of myosin as probes for actin microfilaments, we studied their organization in Paramecium both by fluorescence and electron microscopy. In interphasic cells, HMM decorates (a) most prominently the periphery of nascent and young food vacuoles and their route during the early phase of their intracellular transit; (b) a thin meshwork radiating from the gullet throughout the cytoplasm; (c) a small area beneath the pore of contractile vacuoles and beneath the cytoproct when open to release food residues. Most of these HMM-decorated structures are in close contact with microtubular arrays. All HMM decoration disappears in dividing cells and in cytochalasin-treated cells. In vivo, the drug immediately blocks food vacuole formation but does not affect cytokinesis, cyclosis, contractile vacuole pulsation, defecation, or nuclear movements. The data show that, as in the cells of other organisms, actin microfilaments form defined arrays that undergo physiologically controlled cycles of assembly/disassembly. These arrays contribute (at least in the phagocytotic process) to diverse types of movement: constriction, membrane fusion, and migration of food vacuoles. However, aside from their massive concentration along the phagocytotic tractus, actin microfilaments are neither major structural components of Paramecium cytoplasm nor the only cytoskeletal components ensuring motility or contractility processes.

Actins↗