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1.
Effect of 5''-(N-Ethylcarboxamido)adenosine on Adenosine Transport in Cultured Chromaffin Cells 总被引:4,自引:3,他引:1
Esmerilda G. Delicado Alexandra Rodrigues Raquel P. Sen Ana M. Sebastiao J. Alexandre Ribeiro M. Teresa Miras-Portugal 《Journal of neurochemistry》1990,54(6):1941-1946
Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin. 相似文献
2.
The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-l-thyronine (T3) throughout the prolonged period studied. The Vmax values of this transport obtained in absence and presence of 1 M T3 were 36.21±2.1 and 44.17±3.5 (means±SD) pmol/106cells/min respectively for 26 hours incubation-time with the hormone. The Km values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [3H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 M T3 for 26 hours incubation-time. The values of binding sites per cell were 33,500±3,000 and 40,153±3,700 in absence and presence of T3 respectively, without changing the Kd constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9±2.8 and 24.3±2.1 hours both in the presence or absence of T3 respectively. When cells were incubated in the presence of both T3 and cycloheximide, not only the activatory effect of T3 was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T3 activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism. 相似文献
3.
The transport of [3H]adenosine at 22°C was investigated in guinea pig cerebral cortical synaptosomes using an inhibitor-stop filtration method. Under these conditions adenosine was not significantly metabolized during the incubation period used to determine the initial rates of adenosine transport. The dose response curves for the inhibition of adenosine transport by nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole were biphasic—approx. 50–60% of the transport activity was inhibited with IC50 values of 0.7, 1 and 9 nM respectively, but the remaining activity was insensitive to concentrations as high as 1 μ M. Adenosine influx by both components was saturable (Km values of 17 ± 3 and 68 ± 8 μ M; Vmax values of 2.8 ± 0.3 and 6.1 ± 0.4 pmol/mg protein per s for NBMPR-sensitive and -insensitive components, respectively), and inhibited by other nucleosides and benzodiazepines. The two transport components also differed in their sensitivity to inhibition by other nucleosides and benzodiazepines indicating that the NBMPR-sensitive component of nucleoside transport in guinea pig synaptosomes exhibits a higher affinity than the NBMPR-insensitive component. However, both components have a broad specificity. Inhibition of adenosine transport by NBMPR was associated with high affinity binding of NBMPR to the synaptosomes (Kd 88 ± 6 pM). Binding of NBMPR to these sites was blocked by dilazep and dipyridamole with K1 values similar to those measured for inhibiting NBMPR-sensitive adenosine influx. These results, together with previous findings using NBMPR and dipyridamole as ligand probes, suggest that there are two components of nucleoside transport in mammalian cerebral cortical synaptosomes that differ in their sensitivity to inhibition by NBMPR and other transport inhibitors. 相似文献
4.
Adenosine transporters in chromaffin cells: subcellular distribution and characterization 总被引:4,自引:0,他引:4
Adenosine transporters in freshly isolated and cultured chromaffin cells were quantified by the [3H]dipyridamole binding technique, showing a maximal bound capacity of 0.4 +/- 0.05 pmol/10(6) cells (240,000 +/- 20,000 transporters by cell). Scatchard analysis showed a similar affinity for [3H]dipyridamole in isolated cells and subcellular fractions (Kd = 5 +/- 0.6 nM). For enriched plasma membrane preparations and chromaffin granule membranes, the maximal binding capacities were also very similar, 2.3 +/- 0.3 and 1.8 +/- 0.4 pmol/mg protein, respectively. When [3H]nitrobenzylthioinosine was employed as a radioligand, the maximal bound capacity in cultured chromaffin cells was 0.053 +/- 0.004 pmol/10(6) cells (32,000 +/- 3000 transporters per cell) with a high affinity constant (Kd = 0.25 +/- 0.03 nM); similar values were obtained in all subcellular fractions (Kd = 0.1 +/- 0.01). Also, plasma and chromaffin granule membranes showed similar maximal binding values (0.4 +/- 0.06 pmol/mg protein). Photoincorporation studies with [3H]nitrobenzylthioinosine into plasma membrane polypeptides showed the presence of three molecular species of 115 +/- 10; 58 +/- 6 and 42 +/- 5 kDa. Chromaffin granule membranes showed only the 105 +/- 9 and 51 +/- 4 molecular species. 相似文献
5.
Simone T. Hall Glenda A. Odgers Annette M. Gero 《International journal for parasitology》1993,23(8):1039-1044
Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent Km values for adenosine and guanosine were 9.34 ± 1.30 and 10.6 ± 2.60 μM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer,
-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae. 相似文献
6.
The nucleoside transporter present in chromaffin tissue membranes has been studied by [3H]nitrobenzylthioinosine (NBTI) binding. This ligand presents a high affinity, with a Kd value of 2.1 +/- 0.2 nM and a Bmax of 1.7 +/- 0.2 pmol/mg protein. From the Scatchard and the semilogarithmic graphical representations a positive cooperativity was deduced, with a Hill coefficient of 1.7 +/- 0.4. In displacement studies of NBTI by the non labelled compound, the Hill coefficient was also higher than 1 (1.44 +/- 0.11) in the presence of ATP. This nucleotide seems necessary to maintain the number of high affinity binding sites. 相似文献
7.
M Torres E G Delicado M D Fideu M T Miras-Portugal 《Biochimica et biophysica acta》1992,1105(2):291-299
The dynamics of the nitrobenzylthioinosine (NBTI)-sensitive nucleoside transporter were studied in cultured chromaffin cells. Photolabelling of transporters with [3H]NBTI induced a down-regulation of this protein from the plasma membrane with a half-life value of 2.31 +/- 0.61 h, measured by specific isolation of plasma membrane on polycationic beads. In this internalization step 50-60% of transporters were destroyed. The remaining labelled protein reappeared in plasma membranes and underwent a new disappearance cycle with a longer half-life period (34.65 +/- 3.9 h). A similar pattern of internalization and reappearance of nucleoside transporters was observed in cells cross-linked with non-labelled NBTI, with a half value of reappearance of 33 h. Chromaffin cells cultured in the presence of the protein synthesis inhibitor, cycloheximide, had a component of disappearance for NBTI binding sites with a half-life value of 24.6 +/- 1.4 h. 相似文献
8.
9.
K Schmidt H P Baer A Shariff W A Ayer L Browne 《Canadian journal of physiology and pharmacology》1987,65(5):803-809
The binding of [14,15-3H]14,15-dihydroforskolin ([3H]DHF) to rat liver membranes has been further characterized and was compared with the stimulatory effect of forskolin on adenylate cyclase. The binding equilibrium dissociation constant (KD) for 14,15-dihydroforskolin obtained in inhibition experiments was 0.6 microM, with a maximal binding capacity (Bmax) of 114 pmol/mg protein. A similar KD value (0.5 microM) was derived from kinetics studies that revealed very rapid association and dissociation reactions. For structure-activity relationship studies several forskolin derivatives were synthesized and tested for their ability to inhibit [3H]DHF binding and increase adenylate cyclase activity. Among the tested compounds, forskolin itself was the most potent agonist (K1 = 0.2 microM). Further modification of the molecule in position 7 and (or) 1 decreased or abolished its agonist properties in both adenylate cyclase and binding studies. [3H]DHF binding was not affected by several nucleotides, carbohydrates, lectins, and hormone receptor agonists including isoproterenol, glucagon, and adenosine, but the steroids 17-beta-estradiol, progesterone, and testosterone showed slight inhibitory effects at unphysiologically high concentrations. [3H]DHF binding and forskolin-stimulated adenylate cyclase were sensitive to heat and N-ethylmaleimide treatment. Forskolin protected adenylate cyclase against inactivation by heat but not by N-ethylmaleimide. Preincubation of the membrane with trypsin decreased [3H]DHF binding. The results presented in this study demonstrate that the binding sites identified with [3H]DHF have a high specificity for forskolin and provide evidence that these binding sites are involved in the stimulation of adenylate cyclase by forskolin. 相似文献
10.
A D-2 dopamine receptor and a β2-adrenoceptor occur in the intermediate lobe of the rat pituitary gland (IL). Exposure of intact IL tissue to a D-2 agonist diminished the ability of dopaminergic agonists [but not 5′-guanylyl imidodiphosphate (Gpp(NH)p)] to inhibit adenylate cyclase activity. Conversely, exposure of intact IL tissue to a β-adrenergic agonist diminished the ability of a β-adrenergic agonist (but not forskolin) to stimulate adenylate cyclase activity. Treatment of ovariectomized rats with 17β-estradiol desensitizes the β2-adrenoceptor but not the D-2 receptor. Desensitization of the IL catecholamine receptors is discussed within the framework of a previously published “working model” of these receptors. 相似文献
11.
Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. 125I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (Kd = 0.11 ± 0.04 nM) and low (Kd = 1.3 ± 0.4 μM) affinity binding sites for 125I-amylin in HepG2 cells. The dissociation experiments also showed that 125I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing 125I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace 125I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing 125I[Tyr0]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of 125I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction. 相似文献
12.
Nucleoside transport in cultured mammalian cells. Multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine 总被引:12,自引:0,他引:12
The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents. 相似文献
13.
High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg−1, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg2+). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp3− (ao) AMp2− (ao)+ ATp− (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol −1, ΔH° = −1.5 ± 1. 5 kJ mol −1, °S ° = −17 ± 5 J mol−1 K−1, and ACPp°≈ = −46 J mo1l−1 K−1 at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg2+] = 1.35 × 10−3 mol kg−1, and I = 0.23 mol kg−1) the apparent equilibrium constant (KA′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]2) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10−4 mol kg−1 for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates. 相似文献
14.
Solute interactions with membrane proteins can be analyzed by biomembrane affinity chromatography (BAC), previously applied to the human red cell glucose transporter. As a novel example, frontal BAC analysis of interactions between the nucleoside transport inhibitor nitrobenzylthioinosine (NBTI) and immobilized reconstituted nucleoside and glucose transporters from human red cells revealed two binding sites, presumably corresponding to the two transporters. The affinities and amounts of sites were determined by use of a double rectangular hyperbolic equation. The Kd value for NBTI binding to the nucleoside transporter in egg phospholipid proteoliposomes was 0.38 +/- 0.08 nM (22 degrees C, I = 0.16, pH 7.4), lower than previously reported for reconstituted systems. The molar ratio between the amounts of nucleoside transporter sites for NBTI and glucose transporter sites for cytochalasin B was 4.5 +/- 0.6%. 相似文献
15.
16.
Christoph H. Gleiter Jurgen Deckert David J. Nutt Paul J. Marangos 《Journal of neurochemistry》1989,52(2):641-646
The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin. 相似文献
17.
The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS). 相似文献
18.
Navolotskaya EV Kovalitskaya YA Zolotarev YA Kudryashova NY Goncharenko EN Kolobov AA Kampe-Nemm EA Malkova NV Yurovsky VV Lipkin VM 《Peptides》2003,24(12):1941-1946
β-Endorphin-like peptide immunorphin (SLTCLVKGFY), a selective agonist of nonopioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (Kd=31.6±0.2 nM, Bmax=37.4±2.2 pmol/mg protein). Immunorphin at concentrations of 10−9 to 10−6 M was found to inhibit the adenylate cyclase activity in adrenal cortex membranes, while intramuscular injection of immunorphin at doses of 10–100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream. 相似文献
19.
Azim MK Ahmed W Khan IA Rao NA Khan KM 《Bioorganic & medicinal chemistry letters》2008,18(9):3011-3015
We have identified acridinyl derivatives as potent aspartic protease inhibitors by virtual screening of in-house library of synthetic compounds. Enzyme inhibition experiments showed that both compounds inhibit human cathepsin D and Plasmodium falciparum plasmepsin-II in nanomolar ranges. The IC50 values against cathepsin D and plasmepsin-II of compound-Nar103 were found to be 9.0 ± 2.0 and 4.0 ± 1.0 nM and of compound-Nar110 were 0.5 ± 0.05 and 0.13 ± 0.03 nM, respectively. Ligand docking predicted the binding of acridinyl derivatives at the substrate-binding cleft, where hydrazide part of the inhibitors interact with the S1–S1′ subsite residues including catalytic aspartates. The phenyl ring and acridinyl moiety of the inhibitors were predicted to interact with S2/S3 and S2′/S3′ subsite residues. 相似文献
20.
Bovine adrenal chromaffin cells were incubated with inorganic thiophosphate, using a protocol similar to experiments with inorganic phosphate, in order to determine the source of previously observed thiophosphoproteins. Incubation of cultured cells with [35S]thiophosphate resulted in its incorporation into cell constituents within 2 min. SDS PAGE of the treated cells showed incorporation of label into a broad 97–121 kDa band that was evident after 5 min of treatment and increased progressively to the 40 min exposure limit. Monolayers of chronically treated cells were fractionated into subcellular constituents. The only particulate fraction containing radiolabelled proteins was the chromaffin vesicle fraction. Two-dimensional electrophoresis of the treated cells and isolated chromaffin vesicles showed a majority of proteins in the acidic region of the first dimension gel. A fluorogram of the gel revealed two regions of radiolabelled proteins at acidic and neutral regions of the 2-D gel. These were within the boundaries of the 97–121 kDa band. The thiophosphorylated proteins were released as soluble proteins upon osmotic or freeze-thaw lysis of the vesicles. Chromaffin vesicles isolated from either cultured cells or adrenal medulla tissue were energized by 2 mM ATP but not by the analog adenosine 5′-O-(3-thiotriphosphate). The 97–121 kDa proteins in intact or lysed vesicles prepared from adrenal medulla tissue were not thiophosphorylated by either inorganic thiophosphate or adenosine 5′-O-(3-thiotriphosphate) in the presence or absence of energization by ATP. Nearly complete loss of radiolabel from matrix proteins treated with chondroitinase ABC suggests that it is a component of vesicle proteoglycans.
The results demonstrate that chromaffin vesicle matrix proteins are rapidly and intensely thiophosphorylated in cultured chromaffin cells but not in isolated vesicles. The data suggest that phosphorylation must play an important role in the normal function of these vesicle proteins. 相似文献