Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and post-spawned female hake (Merluccius hubbsi)

Myofibrils from pre- and post-spawned female hake were depleted of thin and thick filaments by KI-treatment and the cytoskeletal ultrastructure of the preparations was investigated by electron microscopy. Irrespective of the gonadal stage of the fish, transmission (TEM) and scanning (SEM) electron micrographs showed an extensive network of filaments connecting Z-structures, which appeared as two circular rings held together. Z-structures of KI-treated myofibrils from post-spawned hake were more compact than those from pre-spawned fish. This ultrastructural difference was absent when KI-treated myofibrils from hake in both gonadal conditions were prepared in the presence of a proteinase inhibitor cocktail (1 mM EDTA+1 mM PMSF+1 mM iodoacetic acid). The total lipid (TL) content of the I-Z-I fraction from fish in pre-spawned condition was about 3.7 mg/g I-Z-I and non-polar lipids (NPL) represent 86.5% of TL. TL and NPL contents of the I-Z-I fraction from post-spawned hake were about 50% and 78.2% lower than those obtained from this fraction in pre-spawning fish. No significant changes (p>0.05) were observed in phospholipids (PL) and acidic phospholipids (AP). No significant differences (p<0.01) were observed among the corresponding lipid fractions of I-Z-I purified from pre- and post-spawning fish in the presence of a protease inhibitor cocktail.     

Effect of triton X-100 treatment on the lipid composition,biochemical and functional properties of hake (Merluccius hubbsi) myofibrils purified from pre- and post-spawned fish

• 1.1. The effects of Triton X-100 treatment on the lipid contents and functional properties of hake myofibrils from pre- and post-spawned fish were investigated.
• 2.2. Differences in lipids, biochemical and functional properties of hake myofibrils related to the gonadal condition of fish were observed.
• 3.3. Triton X-100 treatment removed 65% of polar lipids in myofibrils from pre-spawned fish and only 10% in myofibrils from post-spawned fish.
• 4.4. Triton X-100 increased the Hill coefficient to 1.5 in an allosteric type of reaction for the myofibrillar Mg²⁺-ATPase from pre-spawned hake.
• 5.5. The detergent effect observed on the contraction response was greater in myofibrils from prespawned fish than in post-spawned fish.

     

Role of lipids in sarcoplasmic reticulum: A higher lipid content is required to sustain phosphoenzyme decomposition than phosphoenzyme formation

Enzyme preparations with variable phospholipid contents were obtained by removing lipids from sarcoplasmic reticulum with deoxycholate. Preparations containing from 90 to 37 phospholipids per enzyme showed normal values of both Ca²⁺-ATPase activity and steady-state phosphoenzyme levels. Fractions containing 37 to 23 phospholipids per enzyme had a reduced ATPase activity but normal phosphoenzyme levels, showing that in this range of lipid content the ATPase reaction is inhibited in a reaction step subsequent to phosphoenzyme formation but prior to phosphoenzyme decomposition. Delipidation below 23 lipids per enzyme caused a marked reduction of the amount of phosphoenzyme formed, so that although both reactions require lipids, fewer lipids are required for phosphoenzyme formation than for decomposition. The effect of lipid removal could be completely reversed by readdition of lipids to fractions containing more than 11 lipids per enzyme. It is proposed that phosphoenzyme formation requires full occupancy of a boundary domain of 23 lipids per enzyme, and that the selective inhibition of phosphoenzyme decomposition at higher lipid contents is caused by a decrease in the rotational mobility of the enzyme.     

Effect of phosphorylation on the actin-activated atpase activity of myosin

The purpose of this study was to test the hypothesis that the phosphorylation of myosin is solely responsible for the activation of the Mg²⁺-ATPase activity of gizzard actomyosin. Using a washed natural actomyosin and a reconstituted actomyosin it was shown that phosphorylation alone caused only a slight activation of ATPase activity. Full activity was obtained only when proteins in addition to the myosin light chain kinase were added. It is evident from these results that: 1) there is no simple relationship between the extent of myosin phosphorylation and the specific Mg²⁺-ATPase activity of actomyosin and 2) in order for full activation by actin of the Mg²⁺-ATPase activity of phosphorylated myosin additional factors are required.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Evidence for a role for cardiac myosin in regulating the contractile response.

Dinitrophenylated bovine cardiac myosin incorporates 1.3 mol of 1-fluoro-2,4-dinitro-benzene per 5 × 10⁵ g of protein. Concomitantly there was an activation of the Ca²⁺-ATPase activity and an inhibition of the K⁺(EDTA)-ATPase activity. The dinitrophenyl group is located in the smallest active proteolytic fragment, subfragment 1. Virtually all of the labeling occurs in the region containing the heavy chains of cardiac myosin as judged by dissociation experiments in sodium dodecyl sulfate. Dinitrophenylated myosin failed to form calcium-sensitive actomyosin when tested in an ATPase assay system containing actin, tropomyosin, troponin and ethylene glycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid. Thiolysis of the dinitrophenyl group from myosin with 2-mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. The Ca²⁺ and K⁺(EDTA)-ATPase activities were also restored to control values. These results indicate that cardiac myosin participates in the regulation of the interaction between the contractile proteins.     

Contractile Proteins of a Benthic Fish. II. Composition and ATPase Properties of Actomyosin

SYNOPSIS. Actomyosin was extracted from skeletal muscle of Coryphaenoides,a benthic fish living at 2,200 meters depth, at a temperatureof 2°C, or less, and at pressure of 3,000 psi. On SDS-ureaelectrophoresis on acrylamide gel, the actomyosin extracts yieldcomponents of apparent molecular weight 210,000 (myosin heavychains), 47,000 (actin), 35,000 (tropomyosin and/or troponinsubunits), and 13,000 (myosin light chains). The Mg²⁺-ATPaseof Coryphaenoides actomyosin shows a complex Arrhenius plot,with marked denaturation at temperatures above 30°C, anddiminished temperature sensitivity at temperatures below 15°C.Mg²⁺-ATPase is inhibited by pressure, with activation volumesof + 160 cc/mole at 25°C, and + 230 cc/mole at 2°C.Ca²⁺-ATPase of actomyosin exhibits the same pH, temperature,and pressure dependence as Ca²⁺-ATPase of myosin. The overalldata would be consistent with a positive activation volume thatis independent of temperature (to first approximation) and isrelated to the interaction of actin and myosin, and a negativeactivation volume that is temperature dependent and is relateddirectly to activation of myosin ATPase. The net effect appearsto be an adaptive mechanism whereby Mg²⁺-ATPase of Coryphaenoidesactomyosin is relatively insensitive to pressure and temperatureunder conditions encountered by the living fish.     

Kinetic Characterization of the ATPase and Actin-activated ATPase Activities of Acanthamoeba castellanii Myosin-2

Phosphorylation of Ser-639 in loop-2 of the catalytic motor domain of the heavy chain of Acanthamoeba castellanii myosin-2 and the phosphomimetic mutation S639D have been shown previously to down-regulate the actin-activated ATPase activity of both the full-length myosin and single-headed subfragment-1 (Liu, X., Lee, D. Y., Cai, S., Yu, S., Shu, S., Levine, R. L., and Korn, E. D. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, E23–E32). In the present study we determined the kinetic constants for each step in the myosin and actomyosin ATPase cycles of recombinant wild-type S1 and S1-S639D. The kinetic parameter predominantly affected by the S639D mutation is the actin-activated release of inorganic phosphate from the acto myosin·ADP·P_i complex, which is the rate-limiting step in the steady-state actomyosin ATPase cycle. As consequence of this change, the duty ratio of this conventional myosin decreases. We speculate on the effect of Ser-639 phosphorylation on the processive behavior of myosin-2 filaments.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Studies on (Na+ + K+)-activated ATPase. XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na+ + K+)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na+ + K+)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na+ + K+)-ATPase activity. These results indicate that cholesterol is not essential for (Na+ + K+)-ATPase activity.     

Effect of griseofulvin on lipid composition and membrane integrity inMicrosporum gypseum

The effect of griseofulvin on lipid constituents and membrane permeability ofMicrosporum gypseum has been investigated. Mycelia grown in medium containing griseofulvin (IC₅₀ concentration) possessed a lower content of total lipids, phospholipids and sterols. This inhibitory effect was further supported by decreased incorporation of [¹⁴C] acetate in total lipids, total phospholipids and sterols. Decrease in total phospholipids was also reflected to a varying extent in all individual phospholipids. An increase in the unsaturated to saturated fatty acid ratio was observed in mycelia grown in medium containing griseofulvin. Membrane permeability was affected by griseofulvin as shown by increased K⁺-efflux and greater leakage of intracellular [³²P] labelled components from prelabelled cells. Our results suggest that the antifungal activity of griseofulvin is partially due to its secondary effect on lipid constituents ofMicrosporum gypseum.     

Disorders in lipid composition and properties of plasma membrane in epithelial cells of the placental villous chorion in chlamydial infection

Lipid composition of plasma membranes of placental epithelial cells of villous chorion of healthy women and those with chlamydiosis has been studied. Lipid composition of plasma membranes of ill women differs from that of healthy women by reduction of total phospholipids quantity, by the increase of the level of free cholesterol and free fatty acids. A disturbance in the ratio between certain lipid fractions and increasing quantity of lysophospholipids is observed. Two-fold oppression of plasma membranes Na+, K+ -ATPase activity of placental epithelial cells of villous chorion in ill women has been detected but Mg2+, Ca2+ -ATPase activity has not changed. Thus chlamydial infection causes significant disorders in lipid composition and functioning of epithelial cells membranes of chorion.     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

Variation in polar-group content in lipids of cowpea (Vigna unguiculata) Cell cultures as a mechanism of haloadaptation

Callus and cell suspension cultures of cowpea (Vigna unguiculata) were induced with 2,4-dichlorophenoxyacetic acid and grown at different NaCl concentrations. The cell biomass yield and its total lipid content decreased with increasing salinity. However, while the hexose content in lipids was higher, the amount of lipid phosphorus was significantly lower in both agar and cell suspension cultures. Ion-transport rates with artificial membranes prepared with different lipid fractions showed that lipids from cells grown in a saline medium were less permeable to Na⁺ and to Cl^- than those grown in a non-saline medium. Also the permeability of membranes prepared with glycolipids was lower than those prepared with phospholipids and whole lipids. Apparently, the increase of hexose/phosphorus ratio in membrane lipids is induced in response to the halo-adaptation process.     

Gamma-ray-induced changes in hypodermal mesocarp tissue plasma membrane of pre- and post-storage muskmelon

Gamma irradiation (1.0 kGy) of intact, newly harvested, mature muskmelon fruit (Cucumis melo L. var. reticulatus Naud.) appears to have an immediate deleterious effect, but also a long-term beneficial effect, on the integrity and function of the plasma membrane (PM) of hypodermal mesocarp tissue. The initial consequences of gamma irradiation included an increase in the free sterol:phospholipid ratio, resulting at least in part from deglycosylation of steryl glycosides, a decrease in the spinasterol:7-stigmastenol ratio in each of the PM steryl lipids (free sterols, steryl glycosides, and acylated steryl glycosides), and a decrease in H⁺-ATPase activity. Irradiation did not increase protein loss, suggesting that the decrease in H⁺-ATPase activity resulted from either direct inactivation of the enzyme or altered PM ordering caused by the steryl lipid modifications. The long-term beneficial effects of irradiation, observed following 10 days of commercial storage, included greater retention of total PM protein, a diminished decline in total PM phospholipids (PL) and in the PL:protein ratio, and maintenance of greater overall H⁺-ATPase activity (activity was the same as in controls on a per mg protein basis, but there was > 30% more protein in the PM of stored irradiated fruit). These results indicate that 1 kGy gamma irradiation administered prior to storage slowes the progression of two key parameters of senescence, PM protein loss and PL catabolism.     

Ca2+- and calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin

Fodrin, a spectrin-like actin and calmodulin binding protein, was purified to electrophoretic homogeneity from a membrane fraction of bovine brain. The effect of fodrin on smooth muscle actomyosin Mg2+-ATPase activity was examined by using a system reconstituted from skeletal muscle actin and smooth muscle myosin and regulatory proteins. The simulation of actomyosin Mg2+-ATPase by fodrin showed a biphasic dependence on fodrin concentration and on the time of actin and myosin preincubation at 30 degrees C. Maximal stimulation (50-70%) was obtained at 3 nM fodrin following 10 min of preincubation of actin and myosin. This stimulation was also dependent on the presence of tropomyosin. In the absence of myosin light chain kinase, the fodrin stimulation of Mg2+-ATPase could not be demonstrated with normal actomyosin but could be demonstrated with acto-thiophosphorylated myosin, suggesting that fodrin stimulation depends on the phosphorylation of myosin. Fodrin stimulation was shown to require the presence of both Ca2+ and calmodulin when acto-thiophosphorylated myosin was used. These observations suggest a possible functional role of fodrin in the regulation of smooth muscle contraction and demonstrate an effect on Ca2+ and calmodulin on fodrin function.     

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca²⁺-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca²⁺-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca²⁺-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.     

Human platelet actin. Evidence of beta and gamma forms and similarity of properties with sarcomeric actin.

Human blood platelet actin was purified using 30% sucrose to extract actomyosin and potassium iodide to dissociate actomyosin and to depolymerize actin. Pure actin thus obtained resembles skeletic muscle actin in its polymerization properties, CD spectra and ability to activate myosin myosin Mg2+-ATPase. Isoelectric focusing gel analysis shows that human blood platelet actin exists in beta and gamma forms. The ratio of beta to gamma forms is of 5 in purified actin, in whole cell extract and in all the fractions studied.     

Influence of hyperthyroidism on the superprecipitation response and Ca-sensitivity of natural actomyosin in cardiac and skeletal muscle

In cardiac natural actomyosin prepared from hyperthyroid rabbits, the time of onset of the superprecipitation response was shortened by 58% and the rate of response was increased 4-fold compared with euthyroid animals. However, Ca²⁺-sensitivity of cardiac natural actomyosin prepared from either euthyroid or hyperthyroid rabbits was not changed over the range of 10⁻⁷ to 6.6·10⁻⁵ mM free Ca²⁺ concentrations. Skeletal natural actomyosin prepared from either euthyroid or hyperthyroid rabbits showed a far higher Ca²⁺-sensitivity than cardiac natural actomyosin, but there was no difference in either time of onset or rate of superprecipitation response.