Birgitte Bugge

The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor

When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing.

Effect of bacitracin on the biosynthesis of dolichol derivatives in calf pancreas microsomes.

Publisher Summary bacitracin is a cyclic peptide antibiotic that forms a complex with polyisoprenyl pyrophosphates in the presence of a divalent cation. In bacteria, the formation of this complex prevents the regeneration of undecaprenyl phosphate, which is essential for the biosynthesis of peptidoglycan in the cell wall. This chapter discusses an experiment in which the effect of bacitracin on the some enzymic reactions was investigated. In the experiment, the calf pancreas microsomes were incubated with labeled nucleotide sugars. The radioactivity incorporated into glycolipids extracted with chloroform–methanol (2:1) (CM extract) and chloroform–methanol–water (10:10:2.5) (CMW extract) and into the precipitate (Ppt) containing glycoproteins was measured. It was found from the experiment that bacitracin inhibited the formation of Dol-PP-GlcNAc and Dol-PP-(GlcNAc) 2 from UDP-N-acetyl-D-[ 14 C]lglucosamine and endogenous dolichyl phosphate and the incorporation of D-[14-4C]mannose into dolichyl pyrophosphate oligosaccharides. It also inhibited the incorporation of N-acetyl-D-[14C] glucosamine and D-[14C] mannose into the precipitate.

Synthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Man2GlcNAc2 and Man1GlcNAc2 are transferred from dolichol to protein in vivo

Transfer of truncated oligosaccharides to protein in vivo and the structure of Man2GlcNAc2 synthesized by intact yeast (Saccharomyces cerevisiae) were investigated in the alg2 mutant. At the nonpermissive temperature the alg2 mutant accumulates lipid-linked oligosaccharides that migrate on Bio-Gel P4 in the range expected for Man2GlcNAc2 and Man1GlcNAc2 (T.C. Huffaker and P.W. Robbins (1983) Proc. Natl. Acad. Sci. USA 80, 7466-7470). We characterized the oligosaccharides, derived from protein and lipid, by comigration with standards on HPLC and by Smith degradation followed by HPLC. Man2GlcNAc2 and Man1GlcNAc2 are found on protein in alg2, since their release from a protein-containing precipitate of alg2 cells is N-glycanase (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) dependent. Transfer also occurred in alg2/pAC3 cells, which carry ALG2 on a multicopy plasmid that confers partial correction of the oligosaccharide phenotype. The alg2/pAC3 cells are viable at 36 degrees C. Two isomers of Man2GlcNAc2, Man1----3ManGlcNAc2 and Man1----6ManGlcNAc2, were present on lipid and protein. The transfer of Man2GlcNAc2 and Man1GlcNAc2 to protein by intact cells supports topological models that postulate access by early intermediates to the lumen of the endoplasmic reticulum.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High‐pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of α‐mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N‐acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Induced mannosidosis‐excretion of oligosaccharides by locoweed‐intoxicated sheep

Daily urine samples were collected from a locoweed‐fed sheep, and the oligosaccharide content examined by thin‐layer and liquid chromatography. An unusual pattern of urine oligosaccharides was observed, which appears to be characteristic of loco intoxication. Changes in the pattern could be correlated with the onset of visible disease, which occurred approximately 5 weeks after the typical urine sugars were first detected. HPLC showed that these sugars consisted of two homologous series of oligosaccharides containing one and two residues of 2‐acetamido‐2‐deoxy‐D‐glucose, respectively.

Fluorescence enhancement of uridine diphosphogalactose 4-epimerase induced by specific sugars

Abstract It has been found that 5′-uridylic acid (5′-UMP) and some specific sugars, related to induction and repression of the biosynthesis of UDPGal-4-epimerase, exerted a concerted action on UDPGal-4-epimerase fluorescence. This is manifested by a severalfold increase of blue fluorescence due to bound DPNH of epimerase. Among the uridine nucleotides, 5′-UMP (at 10−4 m ) is highly active. Other 5′-mono- or diphosphonucleosides were much less active. Synthetic UDP-glucose and UDP-galactose were inactive. The specific sugars (10−2–10−3 m ) which show strong activity are d -fucose > d -galactose > d -glucose > d -xylose, and l -arabinose; l -fucose and sucrose are inactive.

Characterization of oligosaccharides from the urine of loco-intoxicated sheep

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed‐fed sheep. Analysis by gas‐liquid chromatography and mass‐spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with α‐D‐mannosidase and endo‐β‐N‐acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo‐β‐N‐acetylglucosaminidase was without effect.

Barbiturate action is dependent on the conformational state of the acetylcholine receptor.

BackgroundBarbiturates act on many neuronal ion channels by poorly understood mechanisms. The authors investigated the hypothesis that barbiturates inhibit the transient open-channel conformation of the nicotinic acetylcholine receptor (nAchR) by binding to a discrete site. MethodsInhibition curves of the agonist-stimulated efflux of 86Rb+ from nAchR-rich membrane vesicles prepared from the electric tissue of Torpedo nobiliana were obtained for 14 barbiturated using a filter assay. ResultsWhen added simultaneously with agonist, all agents inhibited the ion efflux with half-inhibitory concentrations (IC50), varying from 23 μM for pentobarbital to 880 μM for barbital, and with Hill coefficients of one. The effect of several barbiturates on the agonist concentration-response curve for carbachol-stimulated efflux indicated that this inhibitory action was not competitive. ConclusionsThe IC50s of these agents did not correlate with their octanol/water partition coefficients, nor with general anesthetic potency, although a degree of channel inhibition occurred with many agents at general anesthetic concentrations. The existence of a barbiturate-inhibitory site of action was indicated by the structural specificity. This conclusion was supported by the Hill coefficient of one, and by the high inhibitory potencies, which ruled out membrane perturbations as a mechanism. This site on the transient open-channel conformation exhibits different structure-activity relationships than an allosteric site established by equilibrium barbiturate binding on the resting conformation of the AchR. Thus, barbiturate action depends on the nAchRs conformational state

Stereoselectivity of Channel Inhibition by Secondary Alkanol Enantiomers at Nicotinic Acetylcholine Receptors

BackgroundAt the nicotinic acetylcholine receptor, long chain alkanols reduce, whereas short chain alkanols augment endplate currents. Using the enantiomers of five members of a homologous series of secondary alkanols (2-butanol through 2-octanol), we tested the hypothesis that these actions occur at a single hydrophobic site in the lumen of the channel. Small alkanols would bind to this site without blocking the channel, stabilizing the open state and enhancing the apparent affinity of the agonist for channel opening. Long chain alkanols would bind the same site and simply inhibit without affecting the agonists apparent affinity. MethodsAgonist-stimulated 86Rb+ efflux from acetylcholine receptor-rich vesicles from Torpedo nobiliana was studied by adding agonist and allowing efflux to proceed for 10 s before termination by filtration. ResultsAll of the 2-alkanols inhibited 86Rb+ efflux elicited by a maximally stimulating concentration of agonist. Inhibitory potency increased logarithmically with the number of carbon atoms in the hydrocarbon chain of the alkanol. The inhibitory potency of the enantiomers of 2-butanol differed twofold, but the other enantiomers exhibited no stereoselectivity. The enantiomers of 2-octanol caused a concentration-dependent depression of carbamylcholine-stimulated 86Rb+ efflux without significantly altering the agonists apparent dissociation constant. In contrast, the enantiomers of 2-butanol caused: (1) a nonstereoselective decrease in carbachols apparent dissociation constant and (2) the expected stereoselective decrease in maximal carbamylcholine-stimulated 86Rb+ efflux. ConclusionsThe alkanol site that modulates the apparent agonist affinity for channel opening is distinct from the site that results in inhibition of cation flux through the channel.

The accumulation of oligosaccharides in tissues and body fluids of cats with α-mannosidosis

Oligosaccharides were extracted from tissues and body fluids of five kittens with alpha-mannosidosis, three being from the same litter. The kittens were all of different ages at death and were compared to normal and heterozygote cats. The oligosaccharides were analyzed by high-pressure liquid chromatography after perbenzoylation and were identified by comparison with compounds of known structure. This provided a detailed picture of the distribution of oligosaccharides in each tissue, and a method for quantitation of the total oligosaccharides. With the exception of the youngest animal (death at day 2), the oligosaccharide elution profiles were broadly similar for all tissues and fluids, and were typical of feline alpha-mannosidosis. In contrast, concentrations of total oligosaccharides diverged widely from one source to another, from a high of 17.3 mumol/g to a low of 0.04 mumol/g. The results are interpreted in the context of glycoprotein catabolism.