Potentiation of actomyosin ATPase activity by filamin

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.     

Caldesmon specifically inhibits the effect of tropomyosin on actomyosin system in platelet

Caldesmon, a calmodulin and actin binding protein, has been shown to exist in platelet. In this report, it is shown that caldesmon specifically inhibits the effect of tropomyosin to enhance the actomyosin ATPase activity in platelet. Platelet tropomyosin enhances the MgATPase activity of platelet actomyosin. This effect is abolished by platelet caldesmon. In the absence of tropomyosin, however, caldesmon has no effect on the ATPase activity. The inhibition is not due to displacement of the binding of tropomyosin to F-actin by caldesmon. The result indicates that caldesmon is the specific inhibitor of tropomyosin in resting platelet.     

Calpain 1-gamma filamin interaction in muscle cells: a possible in situ regulation by PKC-alpha

Calpains are a family of calcium-dependent cysteine-proteases involved in cytoskeleton remodelling and muscle differentiation. In a recent study, we observed the presence of calpain 1 in the muscle contractile apparatus and specifically in the N1- and N2-lines. This calpain isoform was found to be involved in the degradation of muscle fibres via proteolysis of key proteins in Z-disk and costameric junctions. The goal of this study was to determine whether gamma-filamin--a specific muscle isoform of the filamin family--is a calpain 1 substrate and to characterise this interaction. Gamma-filamin is a major muscle architectural protein located in the Z-line and under the sarcolemmal membrane. This protein is a component of the chain binding the sarcolemma to the sarcomeric structure. In this study, we found that gamma-filamin formed a stable complex in vitro and in cells with calpain 1 in the absence of calcium stimulation. We also located the binding domains in the C-terminus of gamma-filamin with a cleavage site between serine 2626 and serine 2627 in the hinge 2 region. The catalytic (80 kDa) and regulatory (28 kDa) subunits of calpain 1 are both involved in high affinity binding at gamma-filamin. Moreover, we showed that phosphorylation of the filamin C-terminus domain by PKC alpha protected gamma-filamin against proteolysis by calpain 1 in COS cells. Stimulation of PKC activity in myotubes, prevented gamma-filamin proteolysis by calpain and resulted in an increase in myotube adhesion.     

Calpain I activation is not correlated with aggregation in human platelets.

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.     

N1-monoacetylation abolishes the inhibitory effect of spermine and spermidine in the reticulocyte lysate translation system

At optimum magnesium, the translation of rat heart mRNA in the nuclease treated rabbit reticulocyte lysate system was inhibited by low concentrations of spermidine or spermine but not of putrescine. Spermidine and spermine cause a general reduction in the translation of all the heart mRNAs since no differential effects were observed when the translation products were examined by gel electrophoresis. Spermine was a five times more potent inhibitor than spermidine but no inhibition was obtained with N1-acetylspermidine or N1-acetylspermine. Since analyses of endogenous polyamines demonstrate that the inhibitory concentrations of spermine could be obtained by converting a small fraction of the endogenous spermidine to spermine, these results indicate that interconversions of the polyamines might be a sensitive regulatory mechanism for protein synthesis.     

The effect upon actomyosin of stoichiometric amounts of adenosinetriphosphate regenerated in a coupled enzyme system

Pyruvate kinase and phosphoenolpyruvate, added to actomyosin, cause a maintenance of the response of the actomyosin to stoichiometric amounts of ATP. This steady state maintenance depends on the presence of Mg ions.     

Delipidated serum abolishes the inhibitory effect of serum on in vitro liposome-mediated transfection

All the standard in vitro lipofection has been routinely performed in serum-free medium as the transfection activity of liposome/DNA complexes is sensitive to the presence of serum. In this study, we have demonstrated that lipid-rich serum lipoprotein included in the transfection medium strongly inhibited the transfection activity of DC-chol liposome/DNA complexes in five different cell types (CHO, 293, A2780CP, A431 and SKBR3). The levels of inhibition by serum lipoprotein were rather greater than those by serum and varied with cell types. However, this inhibition was completely abolished by delipidation of serum. Thus, delipidated serum can be included in the transfection medium. The complexes formed in the presence of serum (zeta=-18.2+/-1.07 mV), delipidated serum (zeta=-19.6+/-0.54 mV), IgG (zeta=-21.6+/-1.92 mV) or serum lipoprotein (zeta=-10.5+/-2.33 mV) were as much negatively charged as those in serum-free medium (zeta=-21.3+/-1.60 mV). The results suggest that the inhibition of liposome-mediated transfection by serum was not associated with charges of serum proteins but with lipids or lipid-associated proteins present in serum.     

The effect of tropomyosin on the adenosine triphosphatase activity of desensitized actomyosin

1. Tropomyosin preparations of the Bailey type, and those prepared in the presence of dithiothreitol to prevent oxidation of protein thiol groups, inhibit the Ca²⁺-activated adenosine triphosphatase (ATPase) of desensitized actomyosin by up to 60%. 2. The inhibitory activity of myofibrillar extracts and tropomyosin survives various agents known to denature proteins but to the action of which tropomyosin is unusually stable, namely heating at 100° and mild tryptic digestion. It is destroyed by prolonged treatment with trypsin. 3. The ethylenedioxybis-(ethyleneamino)tetra-acetic acid (EGTA)-sensitizing factor present in extracts of natural actomyosin and myofibrils could be selectively destroyed, leaving unchanged the inhibitory effect on the Ca²⁺-activated ATPase. There was no correlation between the EGTA-sensitizing and the Ca²⁺-activated inhibitory activities of tropomyosin prepared under different conditions. 4. Optimum inhibition was achieved when tropomyosin and the myosin of desensitized actomyosin were present in approximately equimolar proportions. Tropomyosin had no effect on the Ca²⁺-activated ATPase of myosin measured under similar conditions. 5. Evidence is presented showing that the tropomyosin binds to desensitized actomyosin under the conditions in which the ATPase is inhibited.     

Calpain system dysregulation in rat brain at beta-amyloid-induced neurodegeneration

Experimental evidence of calcium-dependent proteolysis dysregulation in brain of the murine model of Alzheimer’s disease was obtained. Experimental treatment, consisting of intra-hippocampal injection of amyloid beta-peptide (Aβ_1–40), promoted activation of main calpain forms in murine brain along with a decrease in the content of a natural calpain inhibitor, calpastatin. As a result of a prognostic experiment on the correction of neurodegeneration induced in rats, neuroprotective properties of a steroid hormone estradiol were confirmed and a possible mechanism of the protective effect was suggested. The results allow consideration of both biochemical modifications in protein facilities of a pathology-affected brain and the mechanisms of neurodegeneration and neuroprotection.     

Polyphasic character of ATP hydrolysis in actomyosin system.

The interaction of 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) with the metal ions (M2+) Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ was studied by potentiometry and spectrophotometry in aqueous solution (I = 0.1 or 1.0 M, KNO3, 25 degrees C). Stability constants of the M(Tris)2+ complexes were determined; those constants which were measured by both methods agreed well. Ternary complexes containing ATP4- as a second ligand were also investigated and it is shown that in the presence of Tris, mixed-ligand complexes of the type M(ATP)(Tris)2- are formed. The values for delta log KM, where delta log KM = log KM(ATP)M(ATP)Tris--log KMM(Tris), are all negative, thus indicating that the interaction of Tris with M(ATP)2- is somewhat less pronounced than with M2+. However, it should be noted that even in mixed-ligand systems complex formation with Tris may still be considerable, hence great reservations should be exercised in employing Tris as a buffer in systems which also contain metal ions. Distributions of the complex species in dependence on pH are shown for several systems, and the structures of the binary M(Tris)2- and the ternary M(ATP)(Tris)2- complexes are discussed. The participation of a Tris-hydroxo group in complex formation is, at least for the M(Tris)2- species, quite evident.     

Tropomyosin-caldesmon/actomyosin systems in platelets and arterial smooth muscle: results from exchange experiments

Tropomyosin and caldesomon reciprocally control the actomyosin system in smooth muscle and some non-muscle cells. To compare this mechanism between arterial smooth muscle and platelets, we carried out extensive exchange experiments. Actin, myosin, tropomyosin from arterial smooth muscle cells and platelets were recombined and the effects of two species of caldesmon ('caldesmon77' and 'caldesmon140') on the ATPase activities of both systems were examined and analyzed by the method of analysis of variance. (a) The actomyosin system itself is different between artery and platelets, the difference being determined by myosin (P less than 0.05) and not by actin. (b) Platelet tropomyosin differentiates platelet actin from arterial actin (P less than 0.01), while arterial tropomyosin does not. Neither does tropomyosin differentiate myosin. (c) The effect of caldesmon77 differentiates the origins of myosin (P less than 0.01), actin (P less than 0.05) and tropomyosin (P less than 0.05). The effect of caldesmon140 differentiates the origin of myosin (P less than 0.05) and the actin-myosin 'interaction' (combination) (P less than 0.01), but not the origin of tropomyosin (P greater than 0.1). (1) It is concluded that actomyosin/tropomyosin-caldesmon system is distinguishable between platelets and artery. (2) It is suggested that caldesmon is an actomyosin inhibitor which may interact with myosin, in addition to actin and tropomyosin.     

Cooperativity of the calcium switch of regulated rabbit actomyosin system

Summary The concentration range required for calcium activation of skeletal myofibrillar ATPase activity has previously been attributed to simultaneous binding of two calcium ions to each troponin. We present data representative of the majority of myofibrillar preparations and data with acto subfragment-1 (S-1) whose calcium activation of ATPase activity occurs over a much too narrow range of calcium concentrations to be so explained. S-1 binding significantly broadened the range of Ca²⁺ concentrations over which activation occurred but not to the extent that is associated with simultaneous binding of 2 calcium ions.     

Phosphoinositide signalling system in platelets of schizophrenic patients and the effect of neuroleptic therapy.

Alterations in the phosphoinositide signalling system have been proposed as a possible biological marker of schizophrenia. We studied the levels of inositol 1,4,5-trisphosphate (IP3), cytosolic Ca2+ concentrations ([Ca2+]i), and the incorporation of [32P]-orthophosphate into inositol phospholipids and phosphatidic acid (PA) in blood platelets of neuroleptic-treated schizophrenics in comparison with controls. The [Ca2+]i was significantly higher in platelets of one month neuroleptic-treated patients (155+/-5.8 nM) in comparison with controls (95+/-5.4 nM). Neuroleptic therapy decreased the [Ca2+]i, but even after long-term therapy it remained significantly higher (114+/-5.7 nM) than in controls. Differences were also found in the level of IP3 between controls (30+/-4.0 pmol/10(9) platelets), drug-free schizophrenics (52+/-9.0 pmol/10(9) platelets) and treated patients (50+/-6.0 pmol/10(9) platelets). The increased turnover of PA was observed in platelets of neuroleptic-treated schizophrenic patients. The study suggests that the regulation of calcium homeostasis and pathways involved in the phosphoinositide signalling system are altered in the platelets of schizophrenics. Neuroleptic therapy did not remove the observed changes in [Ca2+]i and IP3 levels.     

The cortical actomyosin system of cytochalasin D-treated lymphoblasts

Global cytoskeleton dynamics is likely to exist in animal cells and some experimental evidence for this has recently been obtained in cells from the human lymphoblastic cell line KE37. We have further investigated the dramatic and reversible microtubule-dependent cell elongation which occurs upon treatment of KE37 cells with cytochalasin D. This phenomenon results in a non-locomotory cell with definite polarity. It involves a sustained equatorial myosin II-dependent contraction of cortical, most of the myosin II being accumulated on segments of the main cellular extension. We report here that such a cell lengthening is energy-dependent and can be inhibited, or suppressed, by surface ligands such as wheat germ agglutinin but not by concanavalin A. Suppression of the cytochalasin D effect by wheat germ agglutinin is rapid and appears to be collapse of the cell extension and relocalization of the contracted actomyosin as a whole. It suggests that the binding of the wheat germ agglutinin to the cell surface results in the transient disassembly of microtubules, a possibility also raised by the potent antagonist effect of taxol on wheat germ agglutinin action. Taken together, the data are consistent with a specific role of microtubules in the control of the activity of the cortical actomyosin system.