Changes in rabbit skeletal myosin and its subfragments under high hydrostatic pressure

The pressure-induced denaturation of rabbit skeletal myosin and its subfragments under hydrostatic pressure were investigated. Four nanometer of red shift of the intrinsic fluorescence spectrum was observed in myosin under a pressure of 400 MPa. The ANS fluorescence of myosin increased with elevating pressure. Changes in the intrinsic fluorescence spectra of myosin and its subfragments were quantified and expressed as the center of spectral mass. The center of spectral mass of myosin and its subfragments linearly decreased with elevating pressure, and increased with lowering pressure. The fluorescence intensity of the ANS-labeled rod did not change during pressure treatment. The present results indicate that the most pressure-sensitive portion of myosin molecule is the head. Hysteresis of the center of spectral mass of S1 appeared under pressures above 300 MPa. Changes in the center of spectral mass of S1 above 350 MPa showed stronger hysteresis. The center of spectral mass did not decrease above 350 MPa during the compression process, indicating that S1 was stable in a partially denatured state at 350 MPa under pressure. The changes in the relative intensities of ANS fluorescence of S1 were measured under pressures up to 400 MPa, and the ANS fluorescence intensity increased with elevating pressure but it did not change after pressure release. The ANS fluorescence intensity increased under constant pressure suggesting that the pressure-induced denaturation of myosin was accelerated during pressurization.     

Ruthenium-red staining of skeletal and cardiac muscles

Summary The effects of ruthenium red (RR) on amphibian and mammalian skeletal muscles and mammalian myocardium were examined. In skeletal muscle cells, a discrete pattern of staining can be brought about within the lumina of the terminal cisternae (junctional sarcoplasmic reticulum [SR]) by sequential exposure to RR and OsO₄. After prolonged immersion in RR solution, formation of pentalaminar segments (zippering) occurs at various points along the longitudinal (network) SR tubules. Zippering can be elicited in skeletal SR at any stage of preparation prior to postfixation with OsO₄. By means of dispersive X-ray analysis, both ruthenium and osmium were seen to be deposited in skeletal muscle junctional SR, and ruthenium was detected in the myoplasm as well. In skeletal muscles whose T tubules were ruptured by exposure to glycerol, the pattern of SR staining and zippering resulting from ruthenium-osmium treatment was not affected. These findings indicate that RR is capable of passage across the sarcolemma of skeletal muscle and that this passage does not occur solely under conditions in which the plasma membrane is damaged. In contrast, RR does not opacify or modify any region of the SR of cardiac muscle. However, after this treatment, randomly distributed opaque bodies, composed of parallel lamellar structures, appear throughout the myocardial cells. A few of these bodies are associated with lipid droplets, but the rest are of unknown origin. The failure of the SR of cardiac muscle to stain after exposure to ruthenium dye (even though this material enters these cells) suggests that the chemical composition of cardiac SR is significantly different from that of skeletal muscle SR.Supported in part by PHS grant HL-11155 (to N.S.) and American Heart Grant-in-Aid 78-753 (to M.S.F.). The authors are grateful to Drs. David Harder and Lawrence Sellin for their assistance with the preparation of frog skeletal muscle, to Dr. S.K. Jirge for his helpful suggestions and discussions, and particularly to Dr. Kenneth R. Lawless and Ms. Ann Marshall of the Department of Materials Sciences, University of Virginia School of Engineering, and Col. John M. Brady of the United States Army Institute of Dental Research, Walter Reed Army Medical Center, for their help with, and for the use of, the X-ray analysis equipment     

Investigation of the effect of high hydrostatic pressure on proteins and lipidic membranes by dynamic fluorescence spectroscopy

Dynamic fluorescence spectroscopy brings new insight into the functional and structural changes of biological molecules under moderate and high hydrostatic pressure. The principles of time-resolved fluorescence methods are briefly described and the resulting type of information is summarized. A first set of selected applications of the use of dynamic fluorescence on pressure effects on proteins in terms of denaturation, ternary and quaternary structure, aggregation and also interaction with DNA are presented. A second set of applications is devoted to the effect of pressure and of cholesterol on lateral heterogeneity of lipidic membranes.     

Action of insulin on the transport of 3-O-methylglucose in frog sartorius muscles

The initial rate of entry of 3-O-methyl-D-glucose into isolated frog sartorius muscles was measured at various concentrations of substrate, at 0, 9, 19, or 29 degrees C, after prior incubation at 19 degrees C with a maximally stimulating concentration of insulin. Control muscles were treated similarly, except for the omission of insulin. A saturable transport system provided for most of the entry of 3-O-methylglucose into muscle cells, but a small amount of penetration occurred by a nonsaturable route. The major effect of insulin was to produce a large increase in activity of the suturable system. The Vmax of entry increased, but there was no significant change in the apparent Km. The ratio of insulin-stimulated to basal Vmax was 10 when transport was measured at 29 degrees C but was 22 at 0 degrees C. These findings support the hypothesis that, although a large part of the effect of insulin on sugar transport can be accounted for by an increase in the number of functional transporters in the plasma membrane, there is a separate hormonal effect that permits a relatively greater activity of transporters at lower temperatures, compared with control rates. An additional effect of insulin was to produce a small but definite increase in the entry of sugar by the nonsaturable transport system.     

A temporal dissociation of energy liberation and high energy phosphate splitting during shortening in frog skeletal muscles

Measurements of the time course of high energy phosphate splitting and energy liberation were performed on rapidly shortening Rana pipiens skeletal muscles. In muscles contracting 30 times against small loads (less the 0.02P), the ratio of explained heat + work (H + W) (calculated from the measured high energy phosphate splitting) to observed H + W (from myothermal and mechanical measurements) was 0.68 +/- 0.08 and is in agreement with results obtained in isometric tetani of R. pipiens skeletal muscle. In lightly afterloaded muscles which were tetanized for 0.6a and whose metabolism was arrested at 3.0 s after the beginning of stimulation, a similar ratio of explained H + W to observed H + W was obtained. However, in identical contractions in which metabolism was arrested at 0.5-0.75 s after the beginning of stimulation, the ratio of explained H + W to observed H + W declined significantly to values ranging from 0.15 to 0.40. These results suggest that rapid shortening at the beginning of contraction induces a delay between energy production and measurable high energy phosphate splitting. This interpretation was tested and confirmed in experiments in which one muscle of a pair contracted isometrically while the other contracted against a small afterload. The afterload and stimulus pattern were arranged so that at the time metabolism was arrested, 0.5 s after the beginning of stimulation, the total energy production by both muscles was the same. Chemical analysis revealed that the isotonically contracting muscle spilt only 25% as much high energy phosphate as did the isometrically contracting muscle.     

Histochemical study of skeletal muscles of the frog, Rana temporaria

Histochemical profiles of muscles were identified based on staining for myosin ATPase activity. They reveal typical arrangement of muscular fibres with a zoned pattern. Tonic fibres have a unique histochemical profile and are mixed with the most oxydative fast fibres to form toxic zones. Muscles show fast profiles in thigh and tonic or mixed profiles in fore-arm.     

Water compartments in living,glycerinated and fixed skeletal muscles of the frog

Summary The kinetics of water replacement with heavy water (deuterium oxide) in the gastrocnemius and sartorius muscles of the frog under isotonic conditions, studied both gravimetrically and by infrared photometry, reveals three water compartments: (i) non-exchangeable ( 80 ml/kg fresh weight), (ii) slowly exchanging ( 500 ml/kg fresh weight), (iii) rapid exchanging — extracellular ( 200 ml/kg fresh weight). Exposure to both glycerol and glutaraldehyde increases the permeability coefficients and the amount of rapid exchanging water; glutaraldehyde also increases the amount of nonexchangeable water. Approximately 90% of the water is kept in the tissue only by weak intermolecular forces, the energies of which amount to 1 kcal/mol. The amount of non-exchangeable water is equivalent to about six continuous adsorption layers covering the myofilaments. Approximately 70 % of the tissue water appears to be replaced by glutaraldehyde during standard fixation.     

Effects of high hydrostatic pressure on Clostridium sporogenes spores

Spores of Clostridium sporogenes were found to be resistant to ultra high pressure, with treatments of 600 MPa for 30 min at 20 °C causing no significant inactivation. Combination treatments including heat and pressure applied simultaneously (e.g. 400 MPa at 60 °C for 30 min) or sequentially (e.g. 80 °C for 10 min followed by 400 MPa for 30 min) proved more effective at inactivating spores. Pressure cycling (e.g. 60 MPa followed by 400 MPa at 60 °C) also reduced spore numbers. Overall, these pressure treatments resulted in less than a 3 log reduction, and it was concluded that the spores could not be inactivated by pressure alone. This could indicate that for the effective inactivation of bacterial spores, high pressure technology may have to be used in combination with other preservation methods.     

超高压处理对微生物生理特性的影响

单核增生李斯特菌(Listeriamonocytogenes)NCTC11994和大肠杆菌(Escherichiacoli)ATCC80739经高压处理,其生理特性发生了深刻的变化,主要表现是400MPa以上的压力处理10min,微生物数量下降7个对数单位,压力处理还会导致细胞内pH值的变化,使膜电位下降,细胞内钾流失,ATP浓度降低。     

Glycogen metabolic enzymes in the skeletal muscles of the developing chick embryo

In the skeletal muscles of the chick embryo from the 10th till the 15th day of embryogenesis, phosphorylase (EC. 2.4.1.1) is represented by two isozymes one of which corresponds, by electrophoretic mobility, to the liver phosphorylase and another to phosphorylase of the skeletal muscles of the adult rat. From the 17th day of embryogenesis on only one isozyme of phosphorylase is found in the skeletal muscles which is identical with that of the skeletal muscles of the adult bird. The isozyme spectrum of phosphorylase of the whole 4 days old embryo contains, besides phosphorylase L, a special "embryonic" isozyme which differs from that of the skeletal muscles by immunochemical characteristics and electrophoretic mobility. From the 10th day of embryogenesis till hatching, the activity of phosphorylase of the skeletal muscles increases more than 50 times and that of glycogen synthetase (EC. 2.4.1.11) only 4 times.