Slow-motion ESR study of order and dynamics in oriented lipid multibilayers: effects of unsaturation and hydration

Electron spin resonance (ESR) experiments were carried out on 3-doxyl-5 alpha-cholestane spin-label (CSL) molecules embedded in macroscopically oriented multibilayers of dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC) and dilinoleoylphosphatidylcholine (DLPC). For these lipids we studied the effects of temperature, hydration and unsaturation on the orientational order parameters and rotational motions of the probe molecules in the liquid crystalline phase. The experimental ESR spectra were simulated by a numerical solution of the stochastic Liouville equation (SLE) for the density matrix of a spin-label molecule. This allows extraction of detailed information about both molecular order and rotational dynamics. The data show that, in our temperature range, the lipid systems are in the slow-motion regime, thereby precluding a motional narrowing interpretation. This is illustrated by a simple model calculation which shows that a fast-motion interpretation seriously overestimates the order parameters. We have compared our results with data obtained independently from angle-resolved fluorescence depolarization (AFD) experiments on oriented bilayers in which 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules were used as fluorescent probes (Deinum et al., (1988) Biochemistry 27, 852-860). It is found that the orientational order and the rotational dynamics obtained with both techniques agree well. This shows that the probe molecules do not perturb the local bilayer structure to any large extent and that they indeed reflect the intrinsic behaviour of the lipid molecules. Upon increase in temperature or hydration, we observe faster reorientational motion and lower molecular ordering. In contrast, we do not find any systematic effect of unsaturation on molecular reorientational motion. Our results indicate that changes in membrane molecular order and reorientational dynamics have to be considered separately and are not necessarily correlated as implied by the common concept of membrane fluidity.     

Progesterone in uterine and arterial tissue and in jugular and uterine venous plasma of sheep

Concentrations of progesterone in uterine and arterial tissue and in uterine and jugular venous plasma were determined. Blood was collected on Days 4 and 9 postestrus from the jugular vein and the first and last venous branches draining each uterine cornu; uterine tissue and arteries were subsequently collected. Progesterone was greater (p less than 0.05) in the cranial third than in the middle or caudal thirds of the uterine horn adjacent to the corpus luteum (CL)-bearing ovary or in any third of the contralateral horn. Progesterone in uterine arterial segments adjacent to the CL-bearing ovary was higher (p less than 0.05) than in contralateral segments. Progesterone was higher (p less than 0.05) in blood from the first venous branch of the cranial third of the uterine cornu adjacent to the ovary with the CL, than in the last branch of the caudal third, or contralateral horn, or in jugular blood. When oviductal veins were resected on Day 9 postestrus, progesterone in the first vein draining the cranial third of the uterine cornu adjacent to the CL-containing ovary was not different (p greater than 0.05) 48 h after resection than in the same vessel in the opposite horn or in jugular blood. We concluded that progesterone and other ovarian products may be delivered to the uterus locally.     

The development of porcine zona pellucida using monoclonal antibodies: I. Immunochemistry and light microscopy

We established three monoclonal antibodies (Mabs) against the zonae pellucidae (ZP) of porcine oocytes, named STA-1, STA-2, and STA-3, and eventually we determined that they all reacted with the isolated ZP. Based on Western blotting without 2-mercaptoethanol (2-ME), STA-1 reacted with the 80,000-110,000 Mr component, STA-2 with the 42,000-63,000 Mr component, and STA-3 with the 40,000-80,000 Mr component of ZP. We immunohistochemically specified the components of porcine ZP reactive with the three Mabs during the course of follicular development. Each Mab reacted with both the ZP and the interfollicular cell space (IFCS). One ZP component, reactive with STA-2 and STA-3, was first produced in the primordial follicle and was not found at the cumulus follicle stage, which corresponds to the stage of large antral follicles more than 5 mm in diameter. Another ZP component, reactive with STA-1, was not produced until the secondary follicle stage, and was never found at the antral follicle stage. These results suggest that each ZP component is produced and secreted at a specific stage or stages of folliculogenesis.     

Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.     

Tilted view reconstruction in optical microscopy. Three-dimensional reconstruction of Drosophila melanogaster embryo nuclei.

The resolution along the optical axis (z) is much less than the in-plane resolution in any current optical microscope, conventional or otherwise. We have used mutually tilted, through-focal section views of the same object to provide a solution to this problem. A tilting specimen stage was constructed for an optical microscope, which with the use of a coverslip-free water immersion lens, allowed the collection of data sets from intact Drosophila melanogaster embryos at viewing directions up to 90 degrees apart. We have devised an image processing scheme to determine the relative tilt, translation, and sampling parameters of the different data sets. This involves the use of a modified phase cross-correlation function, which produces a very sharp maximum. Finally the data sets are merged using figure-of-merit and local area scaling techniques borrowed from x-ray protein crystallography. We demonstrate the application of this technique to data sets from a metaphase plate in an embryo of Drosophila melanogaster. As expected, the merged reconstruction combined the highest resolution available in the individual data sets. As estimated from the Fourier transform, the final resolution is 0.25 microns in x and y and 0.4 microns in z. In the final reconstruction all ten chromosome arms can be easily delineated; this was not possible in the individual data sets. Within many of the arms the two individual chromatids can be seen. In some cases the chromatids are wrapped around each other helically, in others they lie alongside each other in a parallel arrangement.     

Nature of forces stabilizing the transmembrane protein bacteriorhodopsin in purple membrane

Analysis of the far-ultraviolet solution and the oriented-film circular dichroic (CD) spectra of the purple membrane (PM) has indicated that the α-helical segments of its sole protein bacteriorhodopsin (bR) can undergo a significant tilting from the normal to the membrane plane during light-dependent hydroxylamine-mediated bleaching of the bR. However, this drastic change in tertiary structure is free of any observable secondary structural changes. This phenomenon can provide an excellent means for studying the relative contributions of forces responsible for the stability of this transmembrane protein within the membrane bilayer. Perturbation of the PM by varying degrees of papain digestion (resulting in changes in the bR ranging from only an elimination of the long COOH-terminal tail to the additional eliminations of the short NH₂-terminal tail and a number of linkage amino acids between the helical segments of the bR) and by chemical cross-linking with dimethyl adipimidate (resulting primarily in the formation of intramolecular cross-links) resulted in a significant increase in this bleaching-induced tilting in all cases except the one in which only the COOH-tail was eliminated. The most severe perturbation (2-wk papain digestion) increased the net tilt angle per segment from 24 to 39° with no indication of any secondary structural changes. Although these perturbations drastically reduced the structural stability of the bR to bleaching, they caused virtually no observable changes in the intramolecular structure of the bR or the supramolecular structure of the PM based on analysis of extensive absorption, linear dichroic, and CD spectra. In addition, study of the bleaching rates for the perturbed PM samples indicated that a linear correlation exists between the calculated initial bleaching rates and the net tilt angles.

Considering the forces generally assumed to account for the stability of transmembrane proteins in membranes, (a) intersegmental hydrogen bonding and electrostatic interactions, (b) electrostatic interactions between hydrophilic polypeptide segments extending outside the bilayer and the many charged lipid heads of the bilayer, and (c) hydrophobic interactions, it is clear that the results of the bleaching experiments eliminate all but perhaps the last as contributing significantly to the bR stability in the PM. Furthermore, they provide more compelling evidence than previously available that the bR is capable of undergoing relatively large retinyldiene-controlled tertiary structural changes and that the chromophoric retinal serves as the most important factor in the native bR structural stability. This dynamic view of the bR bears directly on models proposed for bR function, favoring those in which protein structural metastability, rather than rigidity, is an essential factor. The proteinquake or deformation wave model proposed by this laboratory falls into this category.

     

Mg2+-paracrystal formation of tropomyosin as a condensation phenomenon. Effects of pH, salt, temperature, and troponin binding.

Tropomyosin (Tm) paracrystal formation induced by Mg2+ was studied by monitoring increases in light scattering. Paracrystals formed above a critical Tm concentration with lag phases in the time courses at pH 7.5 and 6.0, indicating that condensation polymerization processes are involved. The kinetic data at pH 7.5 reasonably fit a model in which nucleation and elongation are taken into account. The rate and extent of light scattering increased at low [Mg2+] and decreased at high [Mg2+] with a maximum at [Mg2+] = 15 mM, indicating different effects of Mg2+ in the two [Mg2+] ranges. The paracrystals were destabilized by increasing the salt concentration and decreasing the temperature. Mg2+ produces paracrystals at pH 6.0 and pH 7.5 by different kinetic mechanisms. Different Tm intermolecular interactions at the two pH values were indicated by studies of the excimer fluorescence of pyrene-labeled Tm and by effects of salt and temperature on the kinetics. At pH 6.0 Tm more readily formed paracrystals with decreased electrostatic effects. Effects of troponin on Mg2+-paracrystal formation of Tm at the two pH values correlated with the known differences in paracrystal structure when troponin is bound to Tm.     

Absolute absorption spectra of batho- and photorhodopsins at room temperature. Picosecond laser photolysis of rhodopsin in polyacrylamide.

Picosecond laser photolysis of rhodopsin in 15% polyacrylamide gel was performed for estimating absolute absorption spectra of the primary intermediates of cattle rhodopsin (bathorhodopsin and photorhodopsin). Using a rhodopsin digitonin extract embedded in 15% polyacrylamide gel, a precise percentage of bleaching of rhodopsin after excitation of a picosecond laser pulse was measured. Using this value, the absolute absorption spectrum of bathorhodopsin was calculated from the spectral change before and 1 ns after the picosecond laser excitation (corresponding to the difference spectrum between rhodopsin and bathorhodopsin). The absorption spectrum of bathorhodopsin thus obtained displayed a lambda max at 535 nm, which was shorter than that at low temperature (543 nm) and a half band-width broader than that measured at low temperature. The oscillator strength of bathorhodopsin at room temperature was smaller than that at low temperature. The absolute absorption spectrum of photorhodopsin was also estimated from the difference spectrum measured at 15 ps after the excitation of rhodopsin (Shichida, Y., S. Matuoka, and T. Yoshizawa. 1984. Photobiochem. Photobiophys. 7:221-228), assuming a sequential conversion of photorhodopsin to bathorhodopsin. Its lambda max was located at approximately 570 nm, and the oscillator strength was smaller than those of rhodopsin and bathorhodopsin.     

Connecting incremental shear modulus and Poisson's ratio of lung tissue with morphology and rheology of microstructure

The width and curvature of the collagen and elastin fiber bundles in the human pulmonary interalveolar septa and alveolar mouths are measured. The data, together with the known mechanical properties of collagen and elastin fibers, are used to derive the incremental elastic moduli of the lung tissue. The constitutive equation for small incremental stress and strain superposed on a homeostatic inflated lung is linear and isotropic, and characterized by two material constants.     

Ca2+ transport through lipid membrane by diastereomer cyclic octapeptides

Effects of the nature and orientation of a side chain in cyclic octapeptides on Ca2+ transport were examined by using cyclo[L-Lys(Z)-Sar-L-Leu-Sar]2 (C8-L), cyclo[L-Lys(Z)-Sar]4 (C8KS), and their diastereomer cyclic octapeptides, cyclo[L-Lys(Z)-Sar-D-Leu-Sar]2 (C8-D) and cyclo[L-Lys(Z)-Sar-D-Lys(Z)-Sar]2 (C8Kk). All these cyclic octapeptides were found to take a single conformation in CDCl3, and the conformation was C2-symmetric for C8-L and C8-D, and C4-symmetric for C8KS and C8Kk. They formed a complex with Ca2+. Upon complexation, C8KS accompanied isomerization of peptide bonds, but C8-D retained the arrangement of peptide bonds. The amount of Ca2+ extracted from an aqueous solution to a chloroform solution by all L cyclic octapeptide C8-L or C8KS was about twice that of Na+, but 6-8-fold smaller than that by C8-D or C8Kk including D units. These cyclic octapeptides were capable of transporting Ca2+ through a lipid membrane above the phase transition temperature, and the transport rate decreased in the order of C8Kk-C8KS greater than C8-D greater than C8-L.