人血浆HDL对动脉粥样硬化家兔肝细胞膜LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化(As)模型家兔通过静脉注射人血浆HDL制剂,观察HDL对As家兔肝细胞膜LDL受体活性的影响.结果发现,摄取高胆固醇饲料的As家兔,其肝细胞膜LDL受体K_d值虽无明显变化但B_max值显著减小（P＜0.01,与正常对照组比较);注射HDL制剂后,As家兔肝细胞膜LDL受体K_d值仍无明显改变,但B_max值却显著回升（P＜0.01,与高脂组比较).表明人血浆HDL具有增加As家兔肝细胞膜LDL受体活性的作用.     

胰岛素及胰高血糖素对HepG2细胞载脂蛋白CⅢ受体功能的影响

为寻求一种可替代人肝细胞研究载脂蛋白（apolipoprotein, apo）CⅢ受体的生理模型,并探讨apoCⅢ受体的生理功能及其在内源性高甘油三酯血症发病机制中的作用,首先以¹²⁵I标记的人apoCⅢ为配体,利用放射性配体结合分析法观察了人肝癌细胞系HepG2细胞是否有apoCⅢ受体存在.结果证实HepG2细胞上存在高亲和力的、可饱和的、特异的apoCⅢ受体结合位点,其受体的亲和力(K_d)和apoCⅢ受体结合容量(B_max)分别为(9.53±1.03)×10^-9 mol/L和(3.28±0.31) μg/g.随后又分别研究了胰岛素及胰高血糖素对HepG2细胞apoCⅢ受体功能的影响,结果表明：胰岛素可使HepG2细胞apoCⅢ受体结合容量（B_max）显著增加,但对受体的亲和力（K_d）无影响;胰高血糖素可使HepG2细胞apoCⅢ受体亲和力显著下降（即K_d值升高）,对受体的结合容量无影响.提示人肝apoCⅢ受体的功能可能受胰岛素及胰高血糖素的不同调节.     

肝靶向配体半乳糖基白蛋白和多聚谷氨酸

化学合成两类去唾液酸糖蛋白受体(ASGPR)的人工配体——半乳糖基白蛋白(Gal_nHSA)和半乳糖基多聚-L-谷氨酸(Gal_nPLGA), 并以 ¹²⁵I标记的去唾液酸胎球蛋白(ASF)为标准配体,测定了合成配体抑制 ¹²⁵I-ASF与大鼠肝细胞膜ASGPR结合的IC₅₀值. 结果表明,Gal₁₂HSA、Gal₁₅HSA、Gal₂₆HSA、Gal₃₀HSA和Gal₃₄PLGA均能够有效地抑制 ¹²⁵I-ASF与ASGPR的结合,且前者与ASGPR的亲和力随半乳糖基化程度的增加而增加. 这些合成配体来源丰富、制备简单,适合于作为药物或基因肝靶向运送的导向配体.     

大鼠肝细胞膜高密度脂蛋白受体的研究

以 ¹²⁵碘标记不含apoE的人HDL₃为配体,采用简便的聚乙二醇沉淀分离法,建立了纯化的大鼠肝细胞膜HDL受体分析法,并对膜HDL受体的性质进行了研究。     

蝎毒素BmkTXKβ对家兔心房肌细胞瞬间外向钾电流的抑制作用

为研究蝎毒素BmkTXKβ对家兔心房肌细胞瞬间外向钾电流(I_to)的影响,采用全细胞膜片钳技术记录应用BmkTXKβ前后的I_to电流. 结果显示BmkTXKβ（1 μmol/L）使心房肌细胞I_to（刺激电压为+50 mV）从（13.63±0.87）pA/pF减少到（7.98±0.78）pA/pF,抑制率为41.4% (n=16, P＜0.001).冲洗后, I_to部分恢复至(11.18±0.82)pA/pF (n=6, P＜0.01,与给药后比较).在0.01～100 μmol/L范围内BmkTXKβ呈浓度依赖性地抑制I_to,IC₅₀的均值为0.95 μmol/L (n=10, P＜0.01),但无频率依赖性(n=6,P＞0.05). 1 μmol/L的BmkTXKβ可使I_to通道的失活动力学曲线明显左移,V_1/2分别为（－23.6±2.7） mV和（－35.3±3.6） mV（n=8,P＜0.05）,曲线斜率基本不变.同时可使I_to通道的恢复过程明显减慢,恢复曲线右移,τ值从（51.2±8.5） ms延长至（93.5±13.4） ms（n=9,P＜0.01）,但不影响其激活过程.据此推断BmkTXKβ对家兔心房肌细胞I_to具有显著的抑制作用,主要作用于失活过程,延长该通道的恢复时间.     

壳聚糖固定化半纤维素酶的研究

从青霉菌m8提取出半纤维素酶,将其固定在用戊二醛交联的壳聚糖载体上.0.5 g壳聚糖与4%的戊二醛结合固定2.5 mg蛋白质,酶活回收率为45.6%. 原酶的最适pH为4.6,固定化酶为pH 3.6.原酶的最适温度为55℃,固定化酶在60～75℃都具有较高活性.固定化酶的耐热性优于原酶. 以半纤维素为底物,固定化酶的表观K_m值略低于原酶,前者为5.0×10^－2 g/L,后者为3.58×10^－2 g/L.     

人T细胞系Jurkat表达特异性的C1q受体

采用酶联免疫吸附试验、流式细胞术、放射配体结合试验及蛋白质印迹等方法,首次证实了人Ｔ细胞系Jurkat表达特异性C1q受体,并对其特性进行了分析Jurkat可为C1q或抗人C1q受体抗体112识别,其对异硫氰酸荧光黄标记C1q的结合能被未标记C1q所抑制．在生理离子强度和温度条件下,Jurkat与 ¹²⁵I－C1q的结合呈持异、剂量依赖、可饱和及可逆性,每个细胞C1q结合位点数为1.1×10⁶,对C1q的K_a值为1.5×10⁷mol^-1,Hill系数为0.9643．Jurkat C1q受体识别C1q的胶原样区．该受体是分子量约70000的膜蛋白分子     

人肌型特异烯醇化酶放免分析法及初步应用

建立了人肌型特异烯醇化酶(hMSE)的放免分析法,抗血清的亲和常数为5.1×10⁹L/mol,采用改良的BHR法制备了 ¹²⁵I-hMSE,后者非特异结合率为3%,与抗血清(1∶10³)结合率达50.16%;批内和批间CV分别为8.6%和13%.回收率为95%-105%.标准曲线范围为5-320μg/L.最小检出率为5μg/L.最佳反应条件:0.1mol/LpH7.4PBS(含5mmol/L MgSO₄,0.1%吐温-20,0.1%NaN₃);反应温度和时间:37℃反应0.5h和4℃,0.5h,作为快速测定;或选用4℃反应24h.65例健康人血清hMSE浓度为23.9±10.9μg/L(x±s).hMSE超过57μg/L(x+3s).为阳性值.测定了AMI和肌病患者,血清hMSE明显升高.     

马肝醇脱氢酶催化有机硅酮不对称还原反应动力学

探讨了马肝醇脱氢酶(HLADH)催化三甲基硅乙酮及其碳结构类似物不对称还原反应动力学.结果表明,在酶浓度低于150 mg/L时,底物浓度与反应初速度的关系符合米氏动力学方程;HLADH催化三甲基硅乙酮不对称还原反应的K_m、v_max和E_a分别为2.67 mmol/L、0.118 mmol/(L·min·mg)和37 kJ/mol, 其碳结构类似物的相应值分别为3.56 mmol/L、0.084 mmol/(L·min·mg)和61 kJ/mol.     

来源于马赛菌(Massilia sp.) UMI-21的ACSMU和PhaCMU体外表达及功能研究

【目的】构建马赛菌(Massilia sp.) UMI-21来源乙酰辅酶A合成酶ACS_MU和聚羟基脂肪酸酯(polyhydroxyalkanoate, PHA)合酶PhaC_MU的体外重组表达体系并过表达2种酶,利用体外合成体系确定2种酶在Massilia sp. UMI21聚3-羟基丁酸(polyhydroxybutyrate, PHB)合成途径中的主要功能。【方法】利用无缝克隆技术将来源于Massilia sp. UMI-21的乙酰辅酶A合成酶基因acs_MU和PHA合酶基因phaC_MU扩增后与pQE-80L质粒连接,转导大肠杆菌(Escherichia coli) BL21(DE3)构建2个基因的重组表达体系;利用6×His标签纯化蛋白ACS_MU和PhaC_MU,并采用5,5′-二硫双(2-硝基苯甲酸) [5,5′-dithiobis-(2-nitrobenzoic acid), DTNB]法测定其活性;使用体外单相合成系统(one-phase reaction system, OPRS),以(R)-3HB为底物,验证ACS_MU和PhaC_MU这2种酶在合成PHB途径中的功能。【结果】成功构建了ACS_MU和PhaC_MU蛋白重组表达菌株BL21-pQE-80L-acs_MU和BL21-pQE-80L-phaC_MU,提纯得到过表达蛋白ACS_MU和PhaC_MU产率分别为24.8 mg/L和25.6 mg/L;ACS_MU酶比活力为(0.148±0.011) U/mg。PhaC_MU酶对(R)-3HBCoA的比活力为(0.102±0.011) U/mg;核磁共振氢谱(nuclear magnetic resonance hydrogen spectroscopy, ¹H-NMR)分析结果表明,使用ACS_Pt-PCT_CP-PhaC_Re、ACS_MU-PCT_CP-PhaC_Re和ACS_MU-PCT_CP-PhaC_MU这3条OPRS途径均能合成PHB,产量分别为0.62、0.76和0.64 g/L。【结论】acs_MU和phaC_MU基因可利用大肠杆菌表达体系过表达并可获得具有活性的可溶性蛋白;对比ACS_Pt-PCT_CP-PhaC_Re合成体系,ACS_MU替代ACS_Pt合成PHB产量增加22.58%,在聚合酶相同的情况下,PHB的合成产量依赖乙酰辅酶A合成酶(acetyl-CoA synthase, ACS)合成乙酰辅酶A的稳定性。使用PhaC_MU代替PhaC_Re,对比ACS_MU-PCT_CP-PhaC_Re组合,合成PHB产量减少了15.79%。在聚合前体浓度相同的情况下,PHB合成量依赖聚合酶的活性。     

[125I] gonadotropin binding to the ovary of an Indian major carp,Catla catla,at different stages of reproductive cycle

At different stages of the annual reproductive cycle ofCatla catla, a major Indian carp, specific binding of gonadotropic hormone to the plasma membrane receptors was demonstrated. Maximum specific binding of [¹²⁵I]Catla gonadotropic hormone was obtained at 30‡C and pH 7.5 during 2 h of incubation.Catla gonadotropic hormone binding was saturable with high affinity. Competitive inhibition experiment showed that binding site was specifically occupied by piscine gonadotropic hormone,Catla gonadotropic hormone and murrel gonadotropic hormone, human chorionic gonadotropin was a weak competitor while bovine thyroid stimulating hormone, bovine prolactin and ovine follicle stimulating hormone had no effect. Scatchard analysis ofCatla gonadotropic hormone binding to the plasma membrane preparation from the carp oocytes of different reproductive stages showed that the range of dissociation constant(K _d ) varied from 0.78 to 0.97 x 10^-10 M. However, maximum binding capacity (B-max) varied remarkably between the different stages of reproductive cycle, it was 6.11 ± 0.36 fmol/mg protein in the preparatory stage which increased to about three-fold in prespawning stage of reproductive cycle (17.0 ± 0.29 fmol/mg protein) and spawning (18.7 ± 0.17 fmol/mg protein) and lowest in postspawning stage of reproductive cycle (5.28 ± 0.28 fmol/mg protein). Fluctuation in the number of gonadotropic hormone binding site at different stages of annual reproductive cycle was found to be coincided well with the pattern of ovarian steroidogenesis in response toCatla gonadotropic hormone as determined by the formation of progesterone from pregnenolone.     

Endogenous Expression of Adenosine A<Subscript>1</Subscript>, A<Subscript>2 </Subscript>and A<Subscript>3</Subscript> Receptors in Rat C6 Glioma Cells

Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [³H]DPCPX, selective A₁R antagonist, revealed a single binding site with a K _D = 9.4 ± 1.4 nM and B _max = 62.7 ± 8.6 fmol/mg protein. Binding of [³H]DPCPX in intact cell revealed a K _D = 17.7 ± 1.3 nM and B _max = 567.1 ± 26.5 fmol/mg protein. On the other hand, [³H]ZM241385 binding experiments revealed a single binding site population of receptors with K _D = 16.5 ± 1.3 nM and B _max = 358.9 ± 52.4 fmol/mg protein in intact cells, and K _D = 4.7 ± 0.6 nM and B _max = 74.3 ± 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A_2A receptor in C6 cells. A₁, A_2A, A_2B and A₃ adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Giα and Gsα proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A₁R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A₁ and A₂ receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Meiosis reinitiation of mussel oocytes involves L-type voltage-gated calcium channel

In the present work we assessed the involvement of L-type voltage opening Ca²⁺ channels in KCl-induced meiosis reinitiation of metaphase-arrested blue mussel (Mytilus galloprovincialis) oocytes by performing binding assays with a tritiated dihydropyridine analog (+)PN 200110. Our data reveal the existence of a single class of dihydropyridine receptors in plasma membrane-enriched rough microsome preparations of mussel oocytes. The apparent affinity (K_d) of characterized receptors equals 1.32 ± 0.21 μM while their maximal binding capacity (B_max) is 620 ± 150 pmol/mg protein. The comparison of the rank order of potency of analogs tested to: 1) inhibit [(+)-l³H]PN 200110 specific binding and 2) block KCl-induced meiosis reinitiation pointed to the pharmacological profile similar to but not identical with those previously described for mammalian dihydropyridine receptors. The efficiencies of all antagonists tested are linearly related (r = 0.995) in binding- (inhibition of [(+)-l³H]PN 200110 specific binding) and biological (inhibition of meiosis reinitiation) assays thus arguing for functional involvement of L-type Ca²⁺ channels in oocyte activation. Reversibility of antagonist actions on meiosis reinitiation and dependence of receptor binding characteristics on a membrane polarization state further suggested such a role. J. Cell. Biochem. 64:152–160. © 1997 Wiley-Liss, Inc.     

人血浆HDL对动脉粥样硬化家兔肝细胞膜 LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化（Ａｓ） 模型家兔通过静脉注射人血浆ＨＤＬ 制剂, 观察ＨＤＬ 对Ａｓ家兔肝细胞膜ＬＤＬ受体活性的影响． 结果发现, 摄取高胆固醇饲料的Ａｓ 家兔, 其肝细胞膜ＬＤＬ 受体 Ｋｄ 值虽无明显变化但Ｂｍａｘ 值显著减小（ Ｐ＜ ０－０１ , 与正常对照组比较） ; 注射ＨＤＬ 制剂后, Ａｓ 家兔肝细胞膜ＬＤＬ受体Ｋｄ 值仍无明显改变, 但Ｂｍａｘ 值却显著回升（ Ｐ＜ ０－０１ , 与高脂组比较） ． 表明人血浆ＨＤＬ 具有增加Ａｓ 家兔肝细胞膜ＬＤＬ 受体活性的作用．     

Early decreases in plasma lipid transfer proteins during weight reduction

Objective: To determine the effect of short‐term weight loss in obese women on concentrations of plasma cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), two new risk factors for cardiovascular disease. Research Methods and Procedures: Plasma CETP and PLTP mass concentrations were measured in 38 obese, non‐diabetic women before and after a moderate, 4% weight loss that was obtained by a 1250 kcal/d diet for 4 weeks. Anthropometric and biological parameters were measured before and after weight loss. Results: Plasma CETP concentration decreased substantially after weight loss (2.76 ± 0.79 before and 2.31 ± 0.69 mg/L after; p = 0.000), and the same was true for plasma PLTP concentration (9.01 ± 2.44 mg/L before vs. 8.34 ± 2.57 after; p = 0.043). The HDL profile shifted toward the small‐sized range, with significant decreases in the relative abundance of HDL_2b and HDL_2a at the expense of HDL_3b after weight loss. A significant, positive correlation between CETP and PLTP mass concentrations is reported for the first time in obese patients (r = 0.43, p = 0.004), and weight reduction was accompanied by early, concomitant, and parallel decreases in plasma CETP and PLTP levels (r = 0.47, p = 0.003). The significant relationship between CETP and PLTP levels was lost after the dietary intervention (r = 0.27; p = 0.11). Discussion: CETP and PLTP correlate positively and significantly in obese patients. The hypocaloric dietary manipulation constitutes a relevant intervention to reduce rapidly and simultaneously plasma levels of CETP and PLTP. The impact of reduced PLTP activity on HDL size appeared to be more prominent than the impact of concomitant reduction in CETP activity.     

Solubilization of High-Affinity,Guanine Nucleotide-Sensitive μ-Opioid Receptors from Rat Brain Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the μ-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [³H][d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin ([³H]DAMGO), a μ-selective opioid agonist, with high affinity (K_D = 1.90 ± 0.93 nM; B_max = 629 ± 162 fmol/mg of protein). Of the membrane-associated [³H]-DAMGO binding sites, 29 ± 7% were recovered in the solubilized fraction. Specific [³H]DAMGO binding was completely abolished in the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate). The solubilized receptor also bound [³H]diprenorphine, a nonselective opioid antagonist, with high affinity (K_D = 1.4 ± 0.39 nM, B_max = 920 ± 154 fmol/mg of protein). Guanosine 5′-O-(3-thiotriphosphate) did not diminish [³H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 µM competed with [³H]diprenorphine for the solubilized binding sites; in contrast, [d -Pen²,d -Pen⁵]-enkephalin, a δ-selective opioid agonist, and U50488H, a κ-selective opioid agonist, failed to compete with [³H]diprenorphine for the solubilized binding sites at concentrations of <1 µM. In the absence of guanine nucleotides, the DAMGO displacement curve for [³H]diprenorphine binding sites better fit a two-site than a one-site model with K_Dhigh = 2.17 ± 1.5 nM, B_max = 648 ± 110 fmol/mg of protein and K_Dlow = 468 ± 63 nM, B_max = 253 ± 84 fmol/mg of protein. In the presence of 10 µM guanosine 5′-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with K_D = 815 ± 33 nM, B_max = 965 ± 124 fmol/mg of protein.     

Solubilization of High-Affinity, Guanine Nucjeotide-Sensitive μ-Opioid Receptors from 7315c Cell Membranes

Abstract: High-affinity μ-opioid receptors have been solubilized from 7315c cell membranes. Occupancy of the membrane-associated receptors with morphine before their solubilization in the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate was critical for stabilization of the receptor. The solubilized opioid receptor bound [³H]-etorphine with high affinity (K_D= 0.304 ± 0.06 nM; B_max= 154 ± 33 fmol/mg of protein). Of the membrane-associated [³H]etorphine binding sites, 40 ± 5% were recovered in the solubilized fraction. Both μ-selective and non-selective enkephalins competed with [³H]etorphine for the solubilized binding sites; in contrast, 5- and K-opioid enkephalins failed to compete with [³H]etorphine for the solubilized binding sites at concentrations of <1 μM.The μ-selective ligand [³H][D-Ala²,A/-Me-Phe⁴,Gly⁵-ol]enkephalin also bound with high affinity (K_D= 0.79 rM; B_max= 108±17 fmol/mg of protein) to the solubilized material. Of the membrane-associated [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin binding sites, 43 ± 3% were recovered in the solubilized material. Guanosine 5′-O-(3-thiotriphosphate), GTP, and guanosine 5′-O-(2-thiodiphosphate), but not adenylylimidodiphosphate, diminished [³H][D-Ala²,N-Me-Phe⁴,Gly⁵-ol]enkephalin binding in a concentration-dependent manner. Finally, μ-opioid receptors from rat brain membranes were also solubilized in a high-affinity, guanine nucleotide-sensitive state if membrane-associated receptors were occupied with morphine before and during their solubilization with the detergent 3-[(3-cholamidopropyl) dimethyl]-1-propane sulfonate.     

Characterization of the Binding of the GABA Agonist [3H]Piperidine-4-Sulphonic Acid to Bovine Brain Synaptic Membranes

Abstract: The binding of radioactive piperidine-4-sulphonic acid ([³H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0°C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: K_D= 17 ± 7 nM (B_max= 0.15 ± 0.07 pmol/mg protein) and K_D= 237 ± 100 nM (B_max= 0.80 ± 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [³H]P4S. The slow binding component (k_on= 5.6 × 10⁷ or 8.8 × 10⁷ M^-1 min^-1, k_Off= 0.83 min^-1, and K_D= 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing potassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (K_D= 11 nM, B_max= 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [³H]P4S appears to involve the same two populations of sites with B_max values similar to those for [³H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [³H]P4S and [³H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.