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1.
Synaptosomal membranes were fused with liposomes using the hydration technique to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters.
Offprint requests to: H. Breer 相似文献
2.
Mohammed Sbia Marie-Francoise Diebler Nicolas Morel Maurice Israël 《Journal of neurochemistry》1992,59(4):1273-1279
The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization. 相似文献
3.
A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed. 相似文献
4.
Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed. 相似文献
5.
Heidi LandmesserAnke Stein Bettina BlüschkeMelanie Brinkmann Sabine HunkeErwin Schneider 《生物化学与生物物理学报:生物膜》2002,1565(1):64-72
The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK2) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK2 complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a Vmax of 1.25 μmol Pi/min/mg and a Km of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIAGlc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes. 相似文献
6.
Marek M Milles S Schreiber G Daleke DL Dittmar G Herrmann A Müller P Pomorski TG 《The Journal of biological chemistry》2011,286(24):21835-21843
The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. 相似文献
7.
T. Lockwich J. Chauthaiwale S. V. Ambudkar I. S. Ambudkar 《The Journal of membrane biology》1995,148(3):277-285
We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289–296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3–5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent K
ca=242 ± 50.9 m and V
max=13.5 ± 1.14 nmoles Ca2+/mg protein/ minute. The K
ca of Ca2+-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the V
max was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+>Mn2+>Co2+=Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40–50% decrease in V
max of Ca2+ transport without an alteration in K
ca. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.We thank Dr. Bruce Baum for his constant support and encouragement. We also thank Ms. Grace Park and all our colleagues for their assistance during the course of this work. 相似文献
8.
Katrin Kuehnle Maria D. Ledesma Lucie Kalvodova Alicia E. Smith Arames Crameri Fabienne Skaanes-Brunner Karin M. Thelen Luka Kulic Dieter Lütjohann Frank L. Heppner Roger M. Nitsch M. Hasan Mohajeri 《Neurochemical research》2009,34(6):1167-1182
Cholesterol is a prominent modulator of the integrity and functional activity of physiological membranes and the most abundant
sterol in the mammalian brain. DHCR24-knock-out mice lack cholesterol and accumulate desmosterol with age. Here we demonstrate
that brain cholesterol deficiency in 3-week-old DHCR24−/− mice was associated with altered membrane composition including disrupted detergent-resistant membrane domain (DRM) structure.
Furthermore, membrane-related functions differed extensively in the brains of these mice, resulting in lower plasmin activity,
decreased β-secretase activity and diminished Aβ generation. Age-dependent accumulation and integration of desmosterol in
brain membranes of 16-week-old DHCR24−/− mice led to the formation of desmosterol-containing DRMs and rescued the observed membrane-related functional deficits. Our
data provide evidence that an alternate sterol, desmosterol, can facilitate processes that are normally cholesterol-dependent
including formation of DRMs from mouse brain extracts, membrane receptor ligand binding and activation, and regulation of
membrane protein proteolytic activity. These data indicate that desmosterol can replace cholesterol in membrane-related functions
in the DHCR24−/− mouse.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
An erratum to this article can be found at 相似文献
9.
Carolina Fortes Rigos Hérica de Lima Santos Juliana Sakamoto Yoneda Guillermo Montich Bruno Maggio Pietro Ciancaglini 《Biophysical chemistry》2010
We studied the thermal dependence of amide I′ infrared absorption and fluorescence emission of Trp residues in the Na,K-ATPase of rabbit kidney. We studied the whole enzyme solubilized with detergent, the whole enzyme reconstituted in proteoliposomes and the protein fraction that remained in the lipid membrane after the trypsin digestion of the proteoliposomes. Cooperative unfolding and aggregation with increasing temperature were observed in the whole protein, whether solubilized or reconstituted, but not in the fraction remaining after trypsinization. The protein influenced the physical state of the lipid, decreasing the temperature of the gel to liquid-crystalline phase transition and the degree of cooperativity. This study provides new information for the understanding of the processes controlling the association mechanisms that are important for enzyme function in natural membranes. 相似文献
10.
J.-L. Rigaud Daniel Levy Gervaise Mosser Oliver Lambert 《European biophysics journal : EBJ》1998,27(4):305-319
Detergent removal from lipid-protein-detergent micellar solutions is the most successful strategy for reconstitution of integral
membrane proteins into proteoliposomes or into two-dimensional crystals. This review establishes the potential of polystyrene
beads as a simple alternative to other conventional detergent removing strategies such as dialysis, gel chromatography and
dilution. Kinetics and equilibrium aspects of removal of different detergents by hydrophobic adsorption onto polystyrene beads
have been systematically investigated. A mechanism of adsorption onto polystyrene beads is proposed and provide useful information
about the use of these beads in reconstitution experiments. The usefulness of this detergent removal strategy to produce quasi-ideal
proteoliposomes is evaluated by considering the morphology and the size of the reconstituted vesicles, the homogeneity in
size and protein distribution, the final protein orientation and the permeability of resulting proteoliposomes. Finally, the
advantages of detergent removal by polystyrene beads as an alternative to conventional dialysis in two-dimensional crystallization
trials are evaluated through review of recent structural reconstitution studies.
Received: 1 December 1997 / Revised version: 6 February 1998 / Accepted: 6 February 1998 相似文献