The role of the lipid matrix in the biosynthesis of dolichyl-linked oligosaccharides

The enzymes in the dolichol pathway are membrane-proteins that utilize a combination of hydrophilic and extremely hydrophobic substrates. The enzymes in this pathway that have been purified and characterized to any extent have either been shown to be stabilized by mixed phospholipid/detergent micelles, or else require a lipid matrix for catalytic activity. Further understanding of the mechanisms of these essential enzymes may require developing methods for the reconstitution of the glycosyltransferases and their hydrophobic substrates in appropriate lipid matrices. Abbreviations: CHO, Chinese hamster ovary; Dol, dolichol; DAG, diacylglycerol; DOPC, dioleolylphosphatidylcholine; DOPE, dioleolyphosphatidylethanolamine; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol This revised version was published online in November 2006 with corrections to the Cover Date.     

Gramicidin A-phospholipid model systems

Gramicidin A forms ion-conducting channels which can traverse the hydrocarbon core of lipid bilayer membranes. The structures formed by gramicidin A are among the best characterized of all membrane-bound polypeptides or proteins. In this review a brief summary is given of the occurrence, conformation, and synthesis of gramicidin A, and of its use as a model for ion transport and the interaction of proteins and lipids in biological membranes.     

Single chloride-permeable channels of large conductance in cultured cardiac cells of new-born rats

Large conductance channels were observed in the membrane of cultured cardiac cells of newborn rats studied with the patch-clamp technique in cell-attached and inside-out configurations. These channels were observed in 4% of the patches. In the cell-attached configuration they exhibited outward rectification and partial inactivation. In the inside-out configuration no rectification occurred but inactivation was present, mainly during hyperpolarizations. Two channels with large single unit conductances (400–450 pS) and one with a smaller conductance (200–250 pS) were frequently observed in the same patch. The two large channels generally had different kinetics. Under steady-state conditions the opening probability of the faster channel appeared to be voltage-independent. The slower channel was activated by depolarization. In asymmetrical solutions the permeability ratios P _Na/P _Cl were 0.03 and 0.24 for the larger and smaller channels, respectively; corresponding values for P _Ba/P _Cl were 0.04 and 0.09. It is proposed that in cardiac membranes the chloride permeability system is composed of widely dispersed microclusters forming grouped channels of different types and sizes.     

Depolarized fluorescence photobleaching recovery

The effects of the fact that the laser sources typically used in fluorescence photobleaching recovery (FPR) experiments in the most commonly employed in-line microscope imaging geometries, are highly linearly polarized, are examined in some detail. The implications of the results, in particular for the interpretation of FPR data in complex cell membrane systems in terms of laterally mobile and immobile sub-populations of the labelled molecular species of concern, are discussed. Methods of experimentally eliminating the potentially major rotational diffusion-based artifacts, different from those appropriate to three-dimensional (solution or suspension) systems which require other than in-line geometries, are delineated.Abbreviations FPR fluorescence photobleaching recovery - FRAP fluorescence recovery after photobleaching - 2- and 3-D two- and three-dimensional     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen peroxide permeation across liposomal membranes: a novel method to assess structural flaws in liposomes

Hydrogen peroxide permeation across large multilamellar vesicles of defined and complex lipid composition was shown to obey precise kinetic relationships for the activity of the occluded catalase. Careful assay conditions precluded simultaneous peroxidative damage to the lipids. The kinetic data was consistent with a barrier role for the bilayer for hydrogen peroxide permeation. More interestingly, hydrogen peroxide permeation across liposomes of complex lipid mixtures exhibited osmotic inhibition of permeation of hydrogen peroxide. On the other hand, purified egg lecithin vesicles did not exhibit any effect of external osmolality on hydrogen peroxide permeation in an experimentally defined non-lytic zone of external osmolarity. These results argue in favour of a heterogeneous, heteroporous structure of bilayers with complex lipid composition.     

The ultrastructure of chemolithoautotrophic Gallionella ferruginea and Thiobacillus ferrooxidans as revealed by chemical fixation and freeze-etching

By using sodium thioglycolate to dissolve the high amount of excreted stalk material in axenic cultures of the chemolithoautotrophic iron bacterium Gallionella ferruginea, the ultrastructure of Gallionella cells from pure cell suspensions could be studied without any loss of viability or disturbance by dense ferric stalk fibers, and compared with Thiobacillus ferrooxidans, also grown chemolithoautotrophically with ferrous iron as energy source. Both organisms were chemically fixed or freeze-etched. Particular structural differences between these iron-bacteria could be ascertained. G. ferruginea possesses intracytoplasmic membranes and soluble d-ribulose-1,5-bisphosphate-carboxylase, whereas T. ferrooxidans contains carboxysomes but no intracytoplasmic membranes; Gallionella forms poly--hydroxybutyrate and glycogen as storage material; T. ferrooxidans produces only glycogen. Both organisms also differ from each other with respect to the freeze fracture behaviour of the cell envelope layers. Whereas the cells of T. ferrooxidans exhibit a characteristic double cleavage, exposing the plasmic fracture face and exoplasmic fracture face of the outer membrane and cytoplasmic membrane, the exceptionally thin multilayered cell envelope of G. ferruginea revealed a particularly intimate association between the layers, resulting in a visualisation of the supramolecular organisation of only the inner fracture face of the cytoplasmic membrane. The results are discussed predominantly in relation to the extremely distinct environments of both organisms.     

Acyl-CoA: 1-acyl-glycero-3-phosphoethanolamine O-acyltransferase and liver plasma membrane fluidity

Investigations have been carried out on the influence of membrane lipid composition and physical state on acyl-CoA: 1-acyl-glycerol-3-phosphoethanolamine O-acyltransferase activity in rat liver plasma membranes. The lipid composition of the membranes was modified either by way of lipid transfer proteins or by partial delipidation with exogenous phospholipases and subsequent enrichment of the membranes with different phospholipids. The results indicated that membrane rigidification by enrichment of the membranes with DPPC or SM reduced the transfer of oleic and palmitic acid to lysophosphatidylethanolamine, whereas all phospholipids inducing membrane fluidization lead to acyltransferase activation. The eventual role of membrane fluidity in the deacylation-reacylation cycle is discussed.     

The Cellular Mechanism of Orcadian Rhythms-A View on Evidence, Hypotheses and Problems

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known “ultradian” oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process.

The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of “oscillatory” and “change of φ or τ after perturbation” seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain.

The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca²⁺ may be involved in either input or essential function. Ca²⁺ -calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.