Isolation of Cytokinetic Actomyosin Rings from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Cytokinesis is the final stage of cell division, through which cellular constituents of mother cells are partitioned into two daughter cells resulting in the increase in cell number. In animal and fungal cells cytokinesis is mediated by an actomyosin contractile ring, which is attached to the overlying cell membrane. Contraction of this ring after chromosome segregation physically severs the mother cell into two daughters. Here we describe methods for the isolation and partial purification of the actomyosin ring from the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which can serve as in vitro systems to facilitate biochemical and ultrastructural analysis of cytokinesis in these genetically tractable model systems.

[Effect of electromagnetic field of extremely low frequency on ATPase activity of actomyosin].

The Mg2+/Ca2+ and K(+)-ATPase actomyosin activity of rabbit skeletal muscle was evaluated by the Fiske-Subbarow method during a five-hour exposition of protein solutions in electromagnetic field of extremely low frequency of 8 Hz and 25 microT induction. The results of the study of the ATPase activity of actomyosin upon electromagnetic exposure have shown statistically significant changes that are characterized by a rather complex time dynamics. After 1, 2 and 4 hours of exposure of protein solutions the effect of ELF EMF exposure inhibits the ATPase activity compared to control samples, which are not exposed to the magnetic field. By the third and fifth hours of exposure to the electromagnetic field, there is a significant increase in the ATPase activity of actomyosin. It should be noted that a similar pattern of change in enzyme activity was universal, both for the environment by Mg2+ and Ca2+, and in the absence of these ions in the buffer. This can evidence for Ca(2+)-independent ways of the infuence of electromagnetic field (EMP) on biologic objects. In our opinion, the above effects are explained by EMP influence on the dynamic properties of actomyosin solutions, which are based on the processes of spontaneous dynamic formation of structure.

Comparative studies on the biochemical characteristics of natural actomyosin isolated from PSE and normal pork.

Biochemical changes of natural actomyosin from fresh pale, soft, exudative (PSE) and normal pork were studied, and the effects of different storage temperatures and different incubation temperature and times on sample superprecipitation, total sulfhydryl (-SH) content, and ATP (adenosine triphosphate) sensitivity were investigated. The results demonstrated that ATPase activity and thermal stability of PSE actomyosin were lower than those of normal pork; and that PSE actomyosin had higher -SH content than that of normal pork at all incubation temperatures and times tested.

Expression and regulation of mutant forms of cardiac TnI in a reconstituted actomyosin system: role of kinase dependent phosphorylation.

When phosphorylated, the inhibitory subunit of troponin (TnI) causes a loss in calcium sensitivity and a decrease in actomyosin ATPase. To examine this process, we bacterially expressed wild type TnI and TnI mutants in which serine 22 and 23, a putative protein kinase A (PKA) site, and threonine 143, a putative protein kinase C (PKC) site, were replaced by alanine S22A/23A and TI43A. PKA dependent phosphorylation was approximately 90% reduced in the S22A/23A mutant and unaffected in T143A. PKC dependent phosphorylation was markedly reduced in T 143A relative both to a wild type construct and to S22A/23A, although some residual phosphorylation (likely at sites other than T143) was seen. The calcium sensitivity (i.e. inhibition of actomyosin ATPase in the presence of EGTA) and regulation of the reconstituted actomyosin system was preserved in the absence of phosphorylation using wild type TnI or either mutant. Calcium sensitivity was decreased by both PKA and PKC with the wild type TnI but was unaffected by PKA when the S22A/23A mutant was employed and by PKC when the T143A mutant was reconstituted. The calcium dependency of the ATPase curve was substantially right shifted when PKC phosphorylated wild type TnI was employed for regulation, and this was markedly attenuated when T143 A was reassociated (although a slight rightward shift and a reduction in maximal ATPase activity was still seen). These data confirm that phosphorylation of TnI by regulatory kinases plays a major role in the regulation of myofibrillar ATPase. The N-terminal serines (22 and 23) appear to be uniquely important for the PKA response whereas threonine 143 is involved in the PKC response although other residues may also have functional significance.

[Effect of temperature on superprecipitation of actomyosin of myometrial smooth muscle and reactions of ATP hydrolysis catalyzed by this protein]. / Vliianie temperatury na superpretsipitatsiiu aktomiozina gladkoi myshtsy miometriia i reaktsiiu gidroliza ATP, kataliziruemuiu étim belkom.

The experiments carried out on the preparation of myometrium actomyosin were carried out. The reaction of superprecipitation of this protein and ATP hydrolysis were studied as affected by temperature. For the both processes the value of temperature optimum is 45 degrees C. The value of activation energy Ea is equal to 20 +/- 3 and 23 +/- 3 kJ/mol for the reaction of superprecipitation and ATP hydrolysis, respectively. Preincubation of actomyosin (45-60 degrees C, 1-5 min) in the medium not including ions of Ca, Mg and ATP with following introduction of the components to this medium and ten-minute incubation of protein in it causes the inhibition of the both reactions at the incubation temperature 45 degrees C and their stimulation at the incubation temperature 55 and 60 degrees C. Data obtained are discussed with regard for the structure organization of actomyosin of the smooth muscle.

The effect of cross-linking of the two heads of porcine aorta smooth muscle myosin on its conformation and enzymic activity.

The two heads of porcine aorta smooth muscle myosin can be cross-linked by a disulfide bridge between the two 17-kDa essential light chains with 5,5'-dithiobis(2-nitrobenzoic acid) [Katoh, T., Tanahashi, K., Hasegawa, Y. & Morita, F. (1995) Eur. J. Biochem. 227, 459-465]. When the cross-linked myosin sample was visualized by rotary shadowing, the two heads of myosin molecules appeared predominantly to adhere to each other. The cross-linking of dephosphorylated myosin in the presence of ATP was greatly inhibited by a decrease in the concentration of NaCl from 0.4 M to 0.15 M, suggesting that the cross-linking of the two heads was suppressed in 10S myosin. However, the fraction of dephosphorylated myosin in a filamentous state at 0.1 M NaCl in the presence of 1 mM ATP was increased from 33% to 83% by the cross-linking. The cross-linking of the two heads might inhibit the formation of the 10S conformation, leading to the increase in the fraction of filamentous myosin. The filaments of the cross-linked myosin sample were visualized by electron microscopy and appeared morphologically similar to those of uncross-linked myosin. The ATPase activity of the cross-linked dephosphorylated myosin sample was more than three times as high as that of an uncross-linked control. The increase in the activity may be related to the increase in the fraction of filamentous myosin caused by the cross-linking. The ATPase activity of dephosphorylated myosin in the presence of actin was increased more than twofold by the cross-linking, but the activity of phosphorylated myosin was affected only slightly. The degree of phosphorylation-dependent regulation of actin-activated ATPase activity decreased with an increase in the degree of cross-linking and was extrapolated to zero at 100% cross-linking. Superprecipitation of acto-cross-linked dephosphorylated myosin was activated, while that of acto-cross-linked phosphorylated myosin was inhibited only slightly. These results suggest that the freedom of each head in myosin molecules may be required to keep the ATPase activity and superprecipitation of acto-dephosphorylated myosin low but not for keeping these activity levels high in acto-phosphorylated myosin.

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins.

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.

Regulation of actomyosin interactions in Limulus muscle proteins.

Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.

A new Acanthamoeba myosin heavy chain. Cloning of the gene and immunological identification of the polypeptide.

An Acanthamoeba myosin heavy chain has been identified whose tail domain amino acid sequence distinguishes it from Acanthamoeba myosins IB, IC, and II. The gene for this novel myosin heavy chain spans approximately 6.8 kilobases, is split by 17 introns, and encodes a 177-kDa polypeptide. While the amino-terminal approximately 90 kDa of this polypeptide is highly similar to the globular head sequences of myosins I and II, its approximately 87-kDa tail domain shows essentially no similarity to the tail sequences of either type of myosin. The only exception to this is the carboxyl-terminal approximately 50-amino acid region of the polypeptide, which is homologous to the carboxyl termini of the myosins I. Interestingly, this approximately 50-residue segment has been shown to exist in a diverse family of cytoskeleton-associated proteins that include nonreceptor tyrosine kinases, phospholipase C gamma, and fodrin (Rodaway, A. R. F., Sternberg, M. J. E., and Bentley, D. L. (1989) Nature 342, 624). Sequence analysis indicates that the tail domain of this new myosin is incapable of forming a myosin II-like coiled-coil structure, implying that the protein is single-headed and nonfilamentous. For this reason we have tentatively classified it as a high molecular weight form of myosin I (HMWMI). To determine if HMWMI exists in cells, antiserum was raised against a bacterially expressed fusion peptide made using a cDNA clone encoding most of the unique HMWMI tail domain. This antiserum does not recognize Acanthamoeba myosins IB, IC, or II but does recognize a single polypeptide in whole cell extracts with the mobility predicted for the HMWMI heavy chain. This protein is precipitated from crude extracts using F-actin and released from the pellet by ATP, supporting its classification as a member of the myosin family of proteins.

Peptide modulators of myosin light chain kinase affect smooth muscle cell contraction.

To examine the importance of myosin light chain kinase (MLCK) in the initiation of contraction in smooth muscle, we used a constitutively active form of MLCK (IMLCK) and two specific peptide inhibitors of MLCK to study the activation of skinned single smooth muscle cells. Although unregulated by Ca-calmodulin, IMLCK, in vitro, was found to have biochemical properties like those of MLCK. Upon photolysis of caged ATP, IMLCK caused Ca-free shortening of skinned cells similar in time course and extent to that induced by Ca2+. Two peptide probes, RS-20 and SM-1, patterned after the Ca-calmodulin binding site and a pseudosubstrate inhibitory site, respectively, of the native MLCK molecule, were shown to specifically inhibit MLCK in in vitro experiments. Both peptides dose dependently inhibited Ca-induced shortening of skinned single cells. These results indicate that MLCK plays an essential role in the activation process in the smooth muscle cell in that activation of this enzyme is both necessary and sufficient for the initiation of contraction.

Properties of desensitized myosin B of chicken gizzard.

In order to explore the regulatory mechanism of smooth muscle contraction, properties of desensitized myosin B (natural actomyosin) prepared from chicken gizzard was investigated. Myosin B desensitized by the method of Ebashi (1988) was resensitized by the addition of the smooth muscle extract. Troponin of rabbit skeletal muscle did not resensitize the desensitized myosin B, but it increased the extent of resensitization when the desensitized myosin B was resensitized incompletely by the addition of small amount of the extract. Among three components (C, I, T) of troponin, this action in promoting the resensitization of desensitized myosin B was found only in troponin T. This finding suggests that troponin T-like factor is present in smooth muscle myosin B.

[Formation of muscle proteins during freezing]. / Strukturoobrazovanie myshechnykh belkov v protsesse zamorazhivaniia.

The effect of different conditions on the formation and properties of cryogels prepared by the freezing-thawing procedure from suspensions and solutions of the carp (Cyprinus carpio) myofibrillar proteins was studied. The freezing of water solutions and suspensions of the native myofibrillar proteins resulted in the formation of the structures mainly stabilized by non-covalent bonds. When muscle proteins were denatured prior to the freezing they formed the structures stabilized by both non-covalent and covalent disulfide bonds.

Differences in regulatory mechanisms of atrial and ventricular muscle contraction in bovine heart.

The purpose of this study was to characterize the regulatory mechanisms of atrial muscle contraction. Natural actomyosin (NAM) and tropomyosin-troponin (TM-TN) complex were prepared from atrial and ventricular muscle of the same bovine heart. The results were as follows: (1) Atrial NAM was more sensitive to Ca2+ than was ventricular NAM: the pCa required for 50% ATPase activation was 5.96 +/- 0.10 vs. 5.63 +/- 0.07, (mean +/- SE; n = 6; p less than 0.01); (2) reconstitution of desensitized actomyosin of rabbit skeletal muscle plus atrial or ventricular TM-TN complex produced higher Ca2+ sensitivity in atrial muscle than in ventricular muscle: the pCa required for 50% ATPase activation was 6.48 +/- 0.10 vs. 6.23 +/- 0.15 (n = 3; p less than 0.05); (3) the amount of inorganic phosphate covalently bound to atrial NAM was equivalent to that bound to ventricular NAM; (4) SDS-polyacrylamide gel electrophoresis of the two NAMs revealed several protein bands of different mobility from 16,000 to 30,000 daltons; and (5) the superprecipitation response of atrial NAM was characterized by a stepwise change in turbidity after the addition of MgATP, in contrast to the biphasic pattern of ventricular NAM. These data suggest that the free Ca ion concentration required for atrial muscle contraction is lower than that required for ventricular muscle contraction and that the difference is attributable to differences in atrial and ventricular regulatory proteins.

Inhibitory effect of squid (Todarodes pacificus) paramyosin on actomyosin ATPase and on superprecipitation.

1. Paramyosin from squid mantle muscle inhibited the Mg-ATPase and the superprecipitation activities of actomyosin. 2. The inhibition was detected only when paramyosin forms a cofilament with myosin. 3. ATP-induced changes in the morphology of the cofilament of myosin and paramyosin are involved in the inhibition by paramyosin.

Calcium sensitivity of cytoplasmic actomyosin from Physarum polycephalum following different purification procedures.

Cytoplasmic actomyosins purified from the acellular slime mold physarum polycephalum by application of two different procedures (Hatano and Tazawa, 1968; Kohama and Kendrick-Jones, 1986) were compared by SDS-PAGE and contraction experiments. In contrast to the 'Hatano actomyosin', 'Kohama actomyosin' contracts in a calcium sensitive manner, i.e., contraction occurs from zero calcium up to pCa4, and is inhibited at greater than or equal to pCa 3. Distinct differences in SDS gels are discussed.

[Changes in the mechano-chemical properties of actomyosin during desensitization]. / Izmenenie mekhanokhimicheskikh svoistv aktomiozina pri desensibilizatsii.

The loss of Ca2+-sensitivity by natural actomyosin (desensitisation) after treatment with low ionic strength solutions results in marked deceleration of protein superprecipitation. This phenomenon is not due to the removal of minor proteins, since a similar effect was observed during "desensitisation" of synthetic actomyosin containing only myosin and actin. However, addition to desensitised actomyosin of tropomyosin, especially in combination with alpha-actinin markedly restores the initial parameters of superprecipitation and ATPase activity. It was assumed that desensitisation has a direct modifying influence on actomyosin, whose effect is weakened in the presence of tropomyosin and alpha-actinin.

Isolation and purification of actomyosin ATPase from mammalian brain.

A simple technique for the isolation and purification of mammalian brain actomyosin, based on extraction of whole brains in low ionic-strength buffer, is described. The final preparation of brain actomyosin is obtained in good yield, has relatively high K+-EDTA and Ca2+-ATPase activities, and is substantially free of other ATPases and tubulin. The preparation is useful for initial enzymatic studies and/or as an enrichment step toward purification of the individual protein components. The Mg2+-ATPase and K+-EDTA ATPase activities are strongly inhibited by the sulfhydryl blocking reagent, pHMB. Interaction between the actin and myosin components can be demonstrated. Brain actomyosin had a distinct electrophoretic profile and enzymatic activity when compared with smooth muscle actomyosin from the aorta.

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles.

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.

[Actomyosin from a low-differentiation skeletal muscle tumor]. / Aktomiozin iz nizkodifferentsirovannoi skeletno-myshechnoi opukholi.

Actin and subunits of myosin were identified in actomyosin preparations isolated from a low-differentiated rhabdomyosarcoma. Determination was made of Ca2+-ATPase activity and of the ratio of concentrations of tumor myosin light chains. Aggregates were obtained bearing similarity with synthetic filaments. The tumor myosin has all the light chains characteristic of the myosin of definitive fast skeletal muscles, and does not have light chains corresponding to any other myosin isoforms. Quantitative peculiarities of light chain composition of tumor myosin may be explained by peculiarities of cell composition of the tumor. The data obtained indicate that the mechanism coordinating myosin gene expression is extremely resistant to tumoral discoordinating factors. Peculiarities of coordination of the expression of genes coding tissue-specific polypeptides are discussed.

ATPase characteristics of myosin from nematode Caenorhabditis elegans purified by an improved method. Formation of myosin-phosphate-ADP complex and ATP-induced fluorescence enhancement.

Myosin was purified rapidly from the nematode Caenorhabditis elegans by an improved method. Crude actomyosin was extracted from the worms at low ionic strength. Paramyosin was removed by repeating the precipitation of myosin filaments in the presence of Mg2+ and the dissolution of them in 0.6 M NaCl. Actin was removed by ultracentrifugation in the presence of Mg-ATP and finally by column chromatography on DEAE-cellulose. This method gave a good yield of myosin (20-30 mg from 50 g wet weight of worms), and its EDTA(K+)-ATPase activity was about 3-fold higher than that of myosin prepared by the method of Harris and Epstein (1979). ATP hydrolysis by nematode myosin showed an initial Pi-burst due to formation of the myosin-phosphate-ADP complex. Tryptophan fluorescence of myosin was enhanced about 8% by ATP. The relationship between the structure and function of myosin is discussed based on the above results and the amino acid sequences of myosins from rabbit skeletal muscle and Caenorhabditis elegans.