Superprecipitation of an actomyosin-like complex isolated from Naegleria gruberi amoebae

A protein complex similar to muscle actomyosin and plasmodial myosin B has been isolated from Naegleria gruberi amoebae. This extract, which comprises approximately 0.7% of the total cell protein, has the solubility properties of actomyosin, displays Ca²⁺-activated, Mg²⁺-inhibited ATPase activity, forms microfilaments, and undergoes a strong superprecipitation reaction. Superprecipitation is initiated by ATP and is preceded by a very brief clearing phase. Although added Mg²⁺ is not essential for superprecipitation of the extract, the reaction proceeds maximally when 7 mM Mg²⁺ is provided. This extract does not appear to have a Ca²⁺ requirement, and superprecipitation is in fact inhibited by added Ca²⁺ ion at all concentrations greater than 0.1 mM. Both ATPase activity and superprecipitation of the actomyosin-like complex are inhibited by the sulfhydryl inhibitor salyrgan.     

Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

Inhibition of Mg ++ ATPase activity of actin-activated Acanthamoeba myosin by muscle troponin-tropomyosin: implications for the mechanism of control of amoeba motility and muscle contraction

Troponin-tropomyosin is known to inhibit the Mg⁺⁺ATPase activity of muscle actomyosin in the absence, but not in the presence, of Ca⁺⁺. In contrast, we have now found that muscle troponin-tropomyosin inhibits the Mg⁺⁺ATPase activity of muscle actin-activated $\text{Acanthamoeba}$ myosin both in the presence and the absence of Ca⁺⁺. Addition of purified tropomyosin and troponins-I, C and T demonstrated that it is troponin-T that acts differently in the two systems which differ only in the source of the myosin. These data suggest that myosin, as well as actin, plays a role in the troponin-tropomyosin control of muscle contraction and make it unlikely that control proteins identical to troponin-tropomyosin function in this amoeba.     

Studies on the origin of resting tension of skeletal muscle

Glycerol extracted frog skeletal muscle fibres at 2.2 μm sarcomere length (in situ-length) in a solution free of Ca⁺⁺ and Mg⁺⁺ but containing ATP, show a decrease in both their resting tension and their elastic modulus, if the ionic strength of the bathing solution is increased. This finding is compared with the behaviour of intact skeletal muscle fibres in hypertonic solution. It is concluded that the resting tension of intact skeletal muscle fibres at in situ-length is caused by the longitudinal sarcoplasmic reticulum as well as by interactions between the contractile filaments.     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

Calcium movements associated with peptide induced phagocytosis inAmoeba proteus

Summary Calcium controls the level of phagocytosis inAmoeba proteus which is inhibited by substances such as verapamil and flunarazine which interfere with Ca⁺⁺ movements in a variety of cell systems. Calcium ion movements in amoebae under control conditions appear to be primarily diffusive in response to activity and electrical potential gradients. Chemotactic peptides (n-formylmethionylleucylphenylalanine, NFMLP) at high concentrations (10^–5 M) induce phagocytosis in the amoeba and bring about a concomitant increase in⁴⁵Ca influx. Verapamil, flunarazine and La⁺⁺⁺ all block the increased⁴⁵Ca influx caused by NFMLP, as well as inhibiting phagocytosis. It is suggested that initiation of phagocytosis in the amoeba is associated with the movement of Ca⁺⁺ into the cytoplasm from the external medium resulting in a transient increase in the cytoplasmic Ca⁺⁺ ion activity.     

Actin and Myosin in pea tendrils

We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K⁺-EDTA and Ca²⁺ and is inhibited by Mg²⁺. At low ionic strength, the Mg²⁺-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Actomyosin-like ATPase extracted from plain synaptic vesicle fractions

Ca²⁺-sensitive Mg²⁺-dependent ATP phosphohydrolase (EC 3.6.1.3, ATPase) was extracted from the plain synaptic vesicle fractions that were virtually devoid of contamination. The protein pattern of the ATPase preparation on SDS polyacrylamide gel electrophoresis closely resembled that of actomyosin from skeletal muscle. The finding suggests that the main components of the ATPase are actin- and myosin-like proteins of the brain (stenin and neurin). Microsome and synaptosomal plasmalemma fractions were extracted under the same conditions to examine the possibility that the ATPase extracted derived from contaminating particulates. An entirely different ATPase was extracted from microsomes, and no protein from plasma membranes. Although Ca²⁺-sensitive Mg²⁺-dependent ATPase was extracted from coated vesicle fraction, the electrophoretic pattern was dissimilar to that of the ATPase from plain synaptic vesicle fractions. It may be inferred that the whole complex of neurostenin is located in plain synaptic vesicles from the brain.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

ATP und Ca2+ induzierte actomyosinartige Kontraktion glycerinisierter Makronuklei

Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca²⁺, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca²⁺ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca²⁺, Mg²⁺ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca²⁺, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP.

     

Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1

Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca⁺⁺ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca⁺⁺ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca⁺⁺ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution.     

The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca²⁺-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca²⁺ in the medium. 4. The Ca²⁺-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca²⁺-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.     

The influence of Mg2+ on the phosphorylation and dephosphorylation of myosin by an actomyosin preparation from vascular smooth muscle

Previous work has shown that Mg²⁺ levels modulate the net level of myosin light chain phosphorylation in bovine aortic smooth muscle actomyosin preparations. The goal of this study was to determine the precise step, i.e. phosphorylation or dephosphorylation, where Mg²⁺ modulates the net phosphorylation reaction. The technique using [γ³⁵S]ATPγS to monitor the phosphorylating step yielded no effect of either Mg²⁺ or Ca²⁺. Unfortunately the lack of Ca²⁺-dependence did not allow conclusions about the influence of Mg²⁺ on myosin light chain kinase activity. The study of the effect of Mg²⁺ on dephosphorylation showed that phosphatase activity in the actomyosin preparation exhibited a Mg²⁺ modulation only when the actomyosin was previously exposed to activating levels (3×10^?5M) of Ca²⁺, suggesting the presence of a Ca²⁺ -regulation system for myosin light chain phosphatase.     

The effect of Mg ++ on the conformation of the Ca ++ -binding component of troponin

Mg⁺⁺ like Ca⁺⁺ induces a conformational change in the Ca⁺⁺-binding component of troponin. However, this change is only 36 % of the change in fluorescence intensity and 80 % of the change in optical rotation induced by Ca⁺⁺. The apparent binding constant of Mg⁺⁺ to the Ca⁺⁺-binding component is 5 × 10³ M⁻¹, much smaller than that of Ca⁺⁺. Circular dichroism measurements show that these changes are simple helix-coil transitions. Unlike the Ca⁺⁺-induced conformational change, the Mg⁺⁺-induced change cannot be propagated to other muscle proteins, and therefore has no physiological meaning.     

Histochemical and cytochemical demonstration of Ca++-ATPase activity in the stellate cells of the adenohypophysis of the guinea pig

Summary The histo- and cytochemical localization of Ca⁺⁺-ATPase activity in the adenohypophysis of the guinea pig was studied utilizing a newly developed method (Ando et al. 1981). An intense reaction was observed in the wall of the blood vessels and between non-secretory cells (stellate cells) and endocrine cells of the pars distalis. Under the electron microscope the Ca⁺⁺-ATPase reaction product was located extracellularly in relation to the plasmalemma of the stellate cells. This reaction was dependent on Ca⁺⁺ and the substrate, ATP, and reduced by the addition of 0,1 mM quercetin to the standard incubation medium. Preheating of the sections before incubation completely inhibited the enzyme activity. When Mg⁺⁺ in different concentrations were substituted for Ca⁺⁺ in the incubation medium the reaction was always reduced. Both Ca⁺⁺ and Mg⁺⁺ in the incubation medium also reduced the reaction. The plasmalemma of the endocrine cells contains no demonstrable amount of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity is discussed in relation to the regulation of the extracellular Ca⁺⁺ concentration which seems to be important with respect not only to the secretory process of the endocrine cells but also to the metabolism of the adenohypophysis.     

Viability of cells reconstituted by virus-induced fusion of minicells with anucleate cells

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

Volume decrease of glycerinated polymorphonuclear leukocytes induced by ATP and Ca : 1. Resemblances to actomyosin contraction

The addition of 1 mM ATP to rabbit peritoneal polymorphonuclear leukocytes suspended in a solution of glycerol causes a decrease in their volume by 4–17% within 3 min. The suspending medium must not only contain glycerol but be of low ionic strength for ATP to be effective. Divalent cations are also required. Ca²⁺ present alone can sustain the volume increase induced by ATP; in the presence of low concentrations of Ca²⁺, Mg²⁺ is also effective but not to the same extent as Ca²⁺. When the cell volume is contracted by the ATP in the presence of Ca²⁺ the addition of EGTA induces a reexpansion of the volume.The organic mercurial, salyrgan prevents the ATP induced reduction in the volume but ouabain has no effect. Guanosine triphosphate (GTP), uridine triphosphate (UTP), and adenosine diphosphate (ADP) can also decrease the volume of the glycerinated leukocytes but to a distinctly lesser extent than ATP. Adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) are without significant effect.The results indicate that the volume decrease caused by exogenous ATP is unlikely to be a passive osmotic or an active, ouabain-sensitive process. The similarities to the interaction of ATP with actomyosin suggest that the volume decrease might be a result of the contraction of the leukocyte actomyosin by ATP.     

Effect of exogenous ATP on the volume of TA3 ascites tumor cells

When exogenous ATP is added to suspensions of TA₃ ascites tumor cells suspended in Ca⁺⁺ and Mg⁺⁺ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca⁺⁺ and/or Mg⁺⁺ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.