The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin binding to the insulin receptor initiates a cascade of cellular events that are responsible for regulating cell metabolism, proliferation, and growth. We have investigated the structure of the purified, functionally active, human insulin receptor using negative stain and cryo-electron microscopy. Visualization of the detergent-solubilized and vesicle-reconstituted receptor shows the ␣ 2 ␤ 2 heterotetrameric insulin receptor to be a three-armed pinwheel-like complex that exhibits considerable variability among individual receptors. The ␣-subunit of the receptor was labeled with an insulin analogue⅐streptavidin gold conjugate, which facilitated the identification of the receptor arm responsible for insulin binding. The gold label was localized to the tip of a single receptor arm of the threearmed complex. The ␤-subunit of the insulin receptor was labeled with a maleimide-gold conjugate, which allowed orientation of the receptor complex in the membrane bilayer. The model derived from electron microscopic studies displays a "Y"-like morphology representing the predominant species identified in the reconstituted receptor images. The insulin receptor dimensions are approximately 12.2 nm by 20.0 nm, extending 9.7 nm above the membrane surface. The ␤-subunit-containing arm is approximately 13.9 nm, and each ␣-subunit-containing arm is 8.6 nm in length. The model presented is the first description of the insulin receptor visualized in a fully hydrated state using cryo-electron microscopy.The insulin receptor is a well known transmembrane protein that has been the focus of extensive scientific study for over 3 decades. It is through this receptor that the peptide hormone insulin regulates a multitude of cellular processes including glucose transport and metabolism, fatty acid metabolism, DNA and protein synthesis, amino acid transport, and mitogenesis. Insulin binding to the extracellular domain of the insulin receptor results in receptor autophosphorylation and activation (reviewed in Ref. 1). The phosphorylated insulin receptor activates many intracellular signaling pathways (reviewed in Ref.2) through the insulin receptor substrate (IRS) 1 family members and phosphatidylinositol 3Ј-kinase, which is upstream of protein kinase C and protein kinase B/Akt enzymes (3). How these kinases are biochemically connected to ultimate targets, such as glucose transporters, remains obscure. Despite the enormous amount of information regarding the insulin signaling pathway, information detailing structural aspects of the insulin receptor itself is limited.The insulin receptor is a member of the receptor tyrosine kinase family (4). This receptor shares considerable sequence similarity and structural characteristics with the insulin like growth factor-1 (IGF-1) receptor (4). The insulin receptor is a glycosylated heterotetrameric protein composed of two ␣-subunits and two ␤-subunits. Each ␤-subunit is covalently linked to an ␣-subunit by class II disulfide bonds to form an ␣␤ heterodimer (5). Two ␣␤ heterodimers are covalently l...

Earlier studies have shown that ether phospholipids display phase-forming properties distinct from those of their ester phospholipid counterparts. Dihexadecylphosphatidylcholine (DHPC) forms an interdigitated bilayer when fully hydrated, and dihexadecylphosphatidylethanolamine (DHPE) is observed in the inverted hexagonal phase (HII) at elevated temperatures. In contrast, the acyl lipid analogues display these phases only under more extreme conditions. In the present study, we examine fully hydrated mixtures of DHPC and DHPE by X-ray diffraction and differential scanning calorimetry and describe the temperature--composition phase diagram for the binary phospholipid system, DHPC/DHPE. Addition of 7 mol % DHPE to DHPC abolishes the ability of DHPC to form an interdigitated bilayer gel phase (L beta I), whereas 10 mol % DHPC destabilizes the HII phase favored by DHPE by elevating (to > 100 degrees C) or removing the L alpha-->HII transition. Evidence for bilayer gel phase separation occurring in DHPC/DHPE mixtures is obtained. In conclusion, it is found that small amounts of the appropriate phospholipid can seriously compromise the formation of the L beta I and HII phases.

A method that enables temperature-composition phase diagram construction at unprecedented rates is described and evaluated. The method involves establishing a known temperature gradient along the length of a metal rod. Samples of different compositions contained in long, thin-walled capillaries are positioned lengthwise on the rod and "equilibrated" such that the temperature gradient is communicated into the sample. The sample is then moved through a focused, monochromatic synchroton-derived x-ray beam and the image-intensified diffraction pattern from the sample is recorded on videotape continuously in live-time as a function of position and, thus, temperature. The temperature at which the diffraction pattern changes corresponds to a phase boundary, and the phase(s) existing (coexisting) on either side of the boundary can be identified on the basis of the diffraction pattern. Repeating the measurement on samples covering the entire composition range completes the phase diagram. These additional samples can be conveniently placed at different locations around the perimeter of the cylindrical rod and rotated into position for diffraction measurement. Temperature-composition phase diagrams for the fully hydrated binary mixtures, dimyristoylphosphatidylcholine (DMPC)/dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE)/DPPC, have been constructed using the new temperature gradient method. They agree well with and extend the results obtained by other techniques. In the DPPE/DPPC system structural parameters as a function of temperature in the various phases including the subgel phase are reported. The potential limitations of this steady-state method are discussed.