Joseph M. Steim

Phase transitions in mammalian membranes

Abstract Reversible thermotropic phase transitions, centered at 0°C, have been detected in rat liver mitochondria and microsomes by differential scanning calorimetry. Water dispersions of the isolated lipids undergo similar transitions which arise from an order-disorder change in the fatty acid chains within bilayers. The transition temperature of the isolated lipids is slightly lower than that of the membranes in both mitochondria and microsomes. In the membranes, no change in the transition occurs following irreversible protein denaturation. The bulk of the lipids in the membrane participate in the cooperative melting process.

Calorimetric detection of a membrane-lipid phase transition in living cells.

The membrane lipids in living Mycoplasma laidlawii exhibit a phase transition characteristic of that from crystal to liquid crystal within the bilayer conformation. The transition occurs at the same temperature in viable organisms, membranes isolated from the organisms, and isolated membrane lipids. The enthalpy of the transition in the membrane is compared with that of an aqueous suspension of isolated membrane lipids. The result is consistent with presence of an extended lipid bilayer in the native membrane.

Structure of Human Serum Lipoproteins: Nuclear Magnetic Resonance Supports a Micellar Model

High-resolution proton nuclear magnetic resonance spectra of low- and high-density lipoproteins from human serum closely resemble those of dispersions of lipoprotein lipids in water. Linewidths of hydrocarbon proton absorptions are not increased in the lipoproteins. In contrast, apolar binding of lysolecithin on serum albumin causes extensive line-broadening and an upfield chemical shift of the hydrocarbon proton resonances of lysolecithin. The results are consistent with a predominantly micellar structure for the lipoproteins rather than with extensive hydrophobic association of lipid and protein.

Chemical synthesis and surface properties of an analog of the pulmonary surfactant dipalmitoyl phosphatidylcholine

Abstract An analog, ( S )-[3-[[2,3-bis(hexadecyloxy)propoxyl]hydroxyphosphinyl]-propyl]trimethylammonium hydroxide, inner salt, hydrate (DPPC-analog), of dipalmitoyl phosphatidylcholine (DPPC) was synthesized. The analog differs from pulmonary DPPC in that it: (1) is a dipalmityl diether rather than a dipalmitoyl diester; (2) has a trimethylammonium propylene phosphono polar head instead of a trimethylammonium ethyleneoxy phosphate head; and (3) has an absolute configuration opposite that of pulmonary DPPC. The dynamic surface pressure-area (π-A) characteristics of the DPPC-analog were determined on a Wilhelmy surface balance and compared with those of DPPC at both 23° C and 37° C. The respreading of both surfactants upon compression past collapse was measured quantitatively by a collapse plateau ratio criterion on successive cycles. The post-collapse respreading ability of the DPPC-analog was found to be significantly better than that of DPPC at both 23°C and 37°C as a function of several surface initial conditions. To complement the dynamic surface pressure-area determinations, differential scanning calorimetry and dilatometry measurements were carried out on DPPC and DPPC-analog water dispersions. The results showed that the liquid-crystalline transition temperature of the DPPC-analog is 45° C, slightly higher than the 41° C found for DPPC. Thus, the superior interfacial respreading found for the DPPC-analog at 23° and 37°C indicates that its bulk phase liquid-crystalline transition temperature is not as directly related to its surface properties as it is in the case of DPPC.

Isolation and characterization of lung surfactant.

Abstract Lung surfactant was isolated from canine tracheal washings by differential centrifugation, equilibrium gradient centrifugation, and flotation on sucrose solutions. The preparation exhibits all the usual physical properties of surfactant, including a large hysteresis loop with low minimum surface tension, bubble clicking, bubble stability, and resistance to antifoam. It is a mixture of lipids, not a lipoprotein or a lipopolysaccharide. The total protein and carbohydrate content is less than 3%. and removal of these minor amounts of non-lipid materials does not affect surface properties. Phosphatidyl choline, phosphatidyl ethanolamine, cholesterol, and triglycerides are the major components. Other workers have extraceted surface-active phospholipids from tracheal washings and whole lung homogenates by using organic solvents, which would denature lipoproteins. Since our isolation does not employ such drastic techniques but yields a product which is essentially free of protein and carbohydrate but retains the properties of surfactant, we conclude that native surfactant is a mixture of lipids.

Monogalactosyl diglyceride: A new neurolipid

Abstract 1. 1. Monogalactosyl diglyceride was isolated from bovine spinal cord and shown to be identical with the same lipid from spinach, except for fatty acid analysis. 2. 2. In particular, unequivocal evidence is presented for the presence of the β-glycosidic bond and the D configuration of the glyceryl moiety. 3. 3. In bovine spinal cord monogalactosyl diglyceride palmitic and oleic acids are dominant, together comprising 75% of the total fatty acids present. 4. 4. The lipid is present in the brains of all animals examined (cow, cat, pig, rat, human), but is absent from mammary gland, spleen, intestine and liver. A small amount was tentatively identified in kidney. 5. 5. In the brain the lipid is restricted to white matter, and is suggested to be a component of myelin.

An automated differential scanning dilatometer

Abstract A sensitive scanning dilatometer has been constructed which continuously records the apparent partial specific volume of small biological samples (10–100 mg) as a function of temperature. Changes in volume of 0.02 μl can be resolved. Tedious calibrations with their inherent errors are eliminated by employing a differential approach in which a recording electrobalance responds to the difference in buoyancy of identical sample and reference tubes. Agreement with the literature values for the specific volume of n -eicosane and the apparent partial specific volume of KCl was excellent. Apparent partial specific volumes obtained for dipalmitoyl lecithin, egg albumin, and bovine serum albumin agreed well with published values.

Control of fatty acid composition of Acholeplasma laidlawii membranes.

The temperature-dependent pattern of incorporation of palmitate and oleate from the growth medium into Acholeplasma laidlawii membrane lipids correlates with the physical state of the membrane defined by calorimetry. Both the pattern and the state can be changed at will by changing the fatty acid composition of the membrane lipids. The ratio of palmitate to oleate incorporated is independent of temperature when the membrane bilayer is below its transition and fully ordered, but becomes temperature dependent upon the onset of the transition and continues to be temperature dependent when the membrane is above its transition and fully fluid. This behavior is mimicked by the physical binding of palmitate and oleate to bilayers of extracted membrane lipids and to bilayers of lecithin. Selective binding by membranes may provide a means for controlling lipid fatty acid composition without invoking an enzymatic mechanism.

Thermal techniques in biomembrane and lipoprotein research

Abstract In structural determinations of inanimate materials, thermal analysis has long proven to be a powerful technique, particularly when coupled with X-ray diffraction or spectroscopic methods. In molecular biology, thermal analysis, frequently combined with other techniques, similarly shows itself to be a powerful tool. Its importance is not limited solely to understanding biological structures but extends to understanding physiological processes as well. This paper describes the application to molecular biology of differential scanning calorimetry (DSC) and the complementary technique of differential scanning dilatometry (DSD). The modification and operation of two commercial differential scanning calorimeters for use in molecular biology is discussed, as well as the design and operation of a novel differential scanning dilatometer together with its auxiliary techniques. Some examples are given of the uses of these methods in understanding the structure, thermodynamic state, and physiological functions of biological membranes. A further illustration of the type of approach taken in applying thermal analysis to molecular biology is provided by a description of an ongoing study. This study uses DSC and DSD in correlation with the temperature dependence of ultraviolet circular dichroism (CD) to elucidate the structures of human high density (HDL) and low density (LDL) serum lipoproteins.

The physical state of quick-frozen membranes and lipids

Lipid bilayers and biomembranes produce nearly identical calorimeter scans regardless of whether they are slowly cooled under near-equilibrium conditions or rapidly frozen at rates used in freeze-fracture electron microscopy. Except for the melting of ice at 273 K, for both cooling regimens no significant thermal events occur from 100 K to the usual gel to liquid crystal transition. The gel to liquid crystal transition itself is somewhat altered by rapid cooling when bilayers contain mixed lipid species. Combined with X-ray diffraction studies, the results indicate that quickly frozen bilayers are crystalline, but that the crystalline domains are quite small or otherwise disordered. In contrast to the behavior of lipids in bilayers, hexagonal-phase calcium cardiolipin easily forms a glass upon cooling.