Pressure versus distance relationships have been obtained for egg phosphatidylcholine bilayers containing a range of cholesterol concentrations. Water was removed from between adjacent bilayers by the application of osmotic pressures in the range of 0.4-2600 atm (4 x 10(5)-2.6 x 10(9) dyn/cm2), and the distance between adjacent bilayers was obtained by Fourier analysis of X-ray diffraction data. For applied pressures up to about 50 atm and bilayer surface separations of 15-5 A, the incorporation of up to equimolar cholesterol has little influence on plots of pressure versus bilayer separation. However, for the higher applied pressures, cholesterol reduces the interbilayer separation distance by an amount that depends on the cholesterol concentration in the bilayer. For example, the incorporation of equimolar cholesterol reduces the distance between bilayers by as much as 6 A at an applied pressure of 2600 atm. At this applied pressure, electron density profiles show that the high-density head-group peaks from apposing bilayers have merged. This indicates that equimolar concentrations of cholesterol spread the lipid molecules apart in the plane of the bilayer enough to allow the phosphatidylcholine head groups from apposing bilayers to interpenetrate as the bilayers are squeezed together. All of these X-ray and pressure-distance data indicate that, by reducing the volume fraction of phospholipid head groups, cholesterol markedly reduces the steric repulsion between apposing bilayers but has a much smaller effect on the sum of the longer ranged repulsive hydration and fluctuation pressures. Increasing concentrations of cholesterol monotonically increase the dipole potential of egg phosphatidylcholine monolayers, from 415 mV with no cholesterol to 493 mV with equimolar cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

The change in pressure needed to bring egg phosphatidylcholine bilayers into contact from their equilibrium separation in excess water has been determined as a function of both distance between the bilayers and water content. A distinct upward break in the pressure-distance relation appears at an interbilayer separation of about 5 A, whereas no such deviation is present in the pressure-water content relation. Thus, this break is not a property of the dehydration process per se, but instead is attributed to steric repulsion between the mobile lipid head groups that extend 2-3 A into the fluid space between bilayers. That is, electron density profiles of these bilayers indicate that the observed break in the pressure-spacing relation occurs at a bilayer separation where extended head groups from apposing bilayers come into steric hindrance. The pressure-spacing data are used to separate steric pressure from the repulsive hydration pressure, as well as to quantitate the range and magnitude of the steric interaction. An appreciable fraction of the measured steric energy can be ascribed to a decrease in configurational entropy due to restricted head-group motion as adjacent bilayers come together.

The tension that develops when relaxed muscles are stretched is the resting (or passive) tension. It has recently been shown that the resting tension of intact skeletal muscle fibers is equivalent to that of mechanically skinned skeletal muscle fibers. Laser diffraction measurements of sarcomere length have now been used to show that the exponential relation between resting tension and sarcomere length for whole frog semitendinosus muscle is similar to that of single fibers. Slack sarcomere lengths and the rates of stress relaxation in these muscles were similar to those in skinned fibers, and sarcomere length remained unchanged during stress relaxation, as in skinned fibers. Thus, in intact semitendinosus muscle of the frog up to a sarcomere length of about 3.8 micrometers, resting tension arises, not in the connective tissue as is commonly thought, but in the elastic resistance of the myofibrils.

Well-ordered multilamellar arrays of liquid-crystalline phosphatidylcholine and equimolar phosphatidylcholine-cholesterol bilayers have been formed in the nonaqueous solvents formamide and 1,3-propanediol. The organization of these bilayers and the interactions between apposing bilayer surfaces have been investigated by X-ray diffraction analysis of liposomes compressed by applied osmotic pressures up to 6 X 10(7) dyn/cm2 (60 atm). The structure of egg phosphatidylcholine (EPC) bilayers in these solvents is quite different than in water, with the bilayer thickness being largest in water, 3 A narrower in formamide, and 6 A narrower in 1,3-propanediol. The incorporation of equimolar cholesterol increases the thickness of EPC bilayers immersed in each solvent, by over 10 A in the case of 1,3-propanediol. The osmotic pressures of various concentrations of the neutral polymer poly(vinylpyrrolidone) dissolved in formamide or 1,3-propanediol have been measured with a custom-built membrane osmometer. These measurements are used to obtain the distance dependence of the repulsive solvation pressure between apposing bilayer surfaces. For each solvent, the solvation pressure decreases exponentially with distance between bilayer surfaces. However, for both EPC and EPC-cholesterol bilayers, the decay length and magnitude of this repulsive pressure strongly depend on the solvent. The decay length for EPC bilayers in water, formamide, and 1,3-propanediol is found to be 1.7, 2.4, and 2.6 A, respectively, whereas the decay length for equimolar EPC-cholesterol bilayers in water, formamide, and 1,3-propanediol is found to be 2.1, 2.9, and 3.1 A, respectively. These data indicate that the decay length is inversely proportional to the cube root of the number of solvent molecules per unit volume.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the cholesterol analog 5 alpha-cholestan-3 beta-ol-6-one (6-ketocholestanol) on bilayer structure, bilayer cohesive properties, and interbilayer repulsive pressures have been studied by a combination of x-ray diffraction, pipette aspiration, and dipole potential experiments. It is found that 6-ketocholestanol, which has a similar structure to cholesterol except with a keto moiety at the 6 position of the B ring, has quite different effects than cholesterol on bilayer organization and cohesive properties. Unlike cholesterol, 6-ketocholestanol does not appreciably modify the thickness of liquid-crystalline egg phosphatidylcholine (EPC) bilayers, and causes a much smaller increase in bilayer compressibility modulus than does cholesterol. These data imply that 6-ketocholestanol has both its hydroxyl and keto moieties situated near the water-hydrocarbon interface, thus making its orientation in the bilayer different from cholesterol's. The addition of equimolar 6-ketocholestanol into EPC bilayers increases the magnitude, but not the decay length, of the exponentially decaying repulsive hydration pressure between adjacent bilayers. Incorporation of equimolar 6-ketocholestanol into EPC monolayers increases the dipole potential by approximately 300 mV. These data are consistent with our previous observation that the magnitude of the hydration pressure is proportional to the square of the dipole potential. These results mean that 6-ketocholestanol, despite its location in the bilayer hydrocarbon region, approximately 10 A from the physical edge of the bilayer, modifies the organization of interlamellar water. We argue that the incorporation of 6-ketocholestanol into EPC bilayers increases the hydration pressure, at least in part, by increasing the electric field strength in the polar head group region.

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Pressure versus distance relations have been obtained for solid (gel) and neat (liquid-crystalline) phase uncharged lipid bilayers by the use of x-ray diffraction analysis of osmotically stressed monoglyceride aqueous dispersions and multilayers. For solid phase monoelaidin bilayers, the interbilayer repulsive pressure decays exponentially from a bilayer separation of approximately 7 A at an applied pressure of 3 x 10(7) dyn/cm2 to a separation of approximately 11 A at zero applied pressure, where an excess water phase forms. The decay length is approximately 1.3 A, which is similar to the value previously measured for gel phase phosphatidylcholine bilayers. This implies that the decay length of the hydration pressure does not depend critically on the presence of zwitterionic head groups in the bilayer surface. For liquid-crystalline monocaprylin, the repulsive pressure versus distance curve has two distinct regions. In the first region, for bilayer separations of approximately 3-8 A and applied pressures of 3 x 10(8) to 4 x 10(6) dyn/cm2, the pressure decays exponentially with a decay length of approximately 1.3 A. In the second region, for bilayer separations of approximately 8-22 A and applied pressures of 4 x 10(6) to 1 x 10(5) dyn/cm2, the pressure decays much more gradually and is inversely proportional to the cube of the distance between bilayers. These data imply that two repulsive pressures operate between liquid-crystalline monocaprylin bilayers, the hydration pressure, which dominates at small (3-8 A) bilayer separations, and the fluctuation pressure, which dominates at larger bilayer separations (greater than 8 A) and strongly influences the hydration properties of the liquid-crystalline bilayers. Thus, due primarily to thermally induced fluctuations, monocaprylin bilayers imbibe considerably more water than do monoelaidin bilayers. For both monoelaidin andmonocaprylin, the measured magnitude of the hydration pressure is found to be proportional to the square of the dipole potential.

Whole frog sartorius muscles can be chemically skinned in approximately 2 h by relaxing solutions containing 0.5% Triton X-100. The intensity and order of the X-ray diffraction pattern from living muscle is largely retained after such skinning, indicating good retention of native structure in fibrils and filaments. Best X-ray results were obtained using a solution with (mM): 75 K acetate; 5 Mg acetate; 5 ATP; 5 EGTA; 15 K phosphate, 2% PVP, pH 7.0. Equatorial X-ray patterns showed that myofibrils swell after detergent skinning, as also observed after mechanical skinning. This swelling could be reversed by adding high molecular weight colloids (PVP or dextran) to the extracting solution. By finding the colloid osmotic pressure needed to restore the in vivo interfilament spacing (3% PVP, 4 X 10(4) mol wt) the swelling pressure was estimated as 35 Torr in a standard KCl-based relaxing solution. The swelling pressure and the extent of swelling were less than acetate replaced chloride as the major anion. Detergent-skinned muscle lost the constant-volume relation between sarcomere length and lattice spacing seen in intact muscle. Changes in A band spacing were paralleled by changes in I and band-Z line spacing at a constant sarcomere length. After detergent skinning, I1,0 rose while I1,1 fell, a change in the relaxing direction. Since raising the calcium ion concentrations from pCa 9 to PCa 6.7 was without effect on equatorial or axial X-ray patterns, we concluded that these intensity changes were not due to calcium-dependent cross-bridge movement but rather to disordering of thin filaments in the A band.