ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINO ACIDS: γ-AMINOBUTYRIC ACID RECEPTOR OF THE SHRIMP MUSCLE

Abstract— The total lipid extract of shrimp muscle ( Artemisia longinaris ) was precipitated with ether. The supernatant containing 95 per cent of the phospholipids and 50 per cent of the protein showed binding for L-[¹⁴C]glutamatc in the first peak of protein. The sediment, redissolved in chloroform-methanol was chromatographed on a Sephadex LH-20 column. A single peak was eluted in the chloroform (20-40 ml) having no lipid phosphorous and high affinity binding for [¹⁴C]GABA. The saturation was achieved at 1 mole per 80.000 g protein and the curve revealed a single type of binding site. The purification achieved was of about 4000-fold. There was no binding of L-[¹⁴C]glulamate to the ether precipitate. The specificity of the binding of [¹⁴C]GABA was further supported by competition experiments with bicuculline. picro-toxin and muscimol. It is suggested that the hydrophobic protein isolated by us represents the GABA receptor. The findings presented in the two papers of the series suggest that the excitatory and inhibitory receptor from crustacean muscle can be separated as two different proteins.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: γ-AMINOBUTYRIC ACID BINDING IN THE RAT CEREBRAL CORTEX

—The binding of [¹⁴C]GABA to nerve-ending membranes isolated from rat cerebral cortex follows a hyperbolic curve saturating at 0·4pmol/μg protein. This binding is about 60% inhibited by chloropromazine, and about 40%, inhibited by bicuculline. A hydrophobic protein fraction binding [¹⁴C]GABA was separated from the total. lipid extract of nerve-ending membranes. The binding follows a hyperbolic curve that saturates at 10·5 pmol of [¹⁴C]GABA/μg of protein, with an apparent K_d= 30 μm . The binding is competitively inhibited by bicuculline with a K_i= 273 μm . These results are compared with those previously obtained on a GABA binding protein from crustacean muscle.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS: l-ASPARTIC ACID-BINDING PROTEIN FROM THE RAT CEREBRAL CORTEX

Abstract— Lyophilized rat cerebral cortex was treated with chloroform-methanol (2:1, v/v), and the extracted hydrophobic proteins (i.e. proteolipids) were separated by column chromatography on Sephadex LH-20. The first peak of protein, eluting with chloroform in the void volume, had high affinity binding for l -[¹⁴C]aspartic acid. The saturation of the binding showed three saturable sites with apparent dissociation constants of 0.2 μ m , 10 μ m and 50 μ m . The binding capacities of the three sites were 2.8, 132 and 617 nmol/mg of protein, respectively. There were 8.0 nmol of high affinity binding sites for l -aspartic acid and 1.53 nmol for l -glutamic acid per g of fresh tissue in the cerebral cortex of the rat. Differentiation between binding of l -aspartic and l -glutamic acid was clearly established by cross-binding and competition experiments with agonists and antagonists.
It is suggested that the isolated protein fraction may correspond to a synaptic receptor and not to the transport system. It is concluded that in the cerebral cortex there is a separate receptor for l -aspartic acid. This is further support to the possible role of this amino acid as a central excitatory transmitter.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS. STEREOSELECTIVITY OF THE BINDING OF L-[14C]GLUTAMIC ACID IN CEREBRAL CORTEX

From the total lipid extract of ncrve-ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L-[¹⁴C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [¹⁴C]GABA-binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd₁= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L-enantiomer. The binding of L-[¹⁴C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL-a-methylglutamic acid, L-nuciferine, L-aspartic acid and L-glutamic acid diethyl ester. These results are compared with those previously obtained with the L-glutamic acid-binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.     

运动对大鼠骨骼肌雄激素分布水平及雄激素受体结合容量的影响

本文测定了不同运动条件下的大鼠骨骼肌中的雄激素受体（ａｎｄｒｏｇｅｎｒｅｃｅｐｔｏｒ,ＡＲ）结合容量及雄激素的水平。一次力竭运动可使大鼠股四头肌组织的雄激素结合容量水平上升,但降低了其组织中的雄激素水平。在长期力竭性运动后,股四头肌组织雄激素受体结合容量及雄激素水平均没有变化,而长期的适宜运动则可提高股四头肌的雄激素结合容量的水平,但对其雄激素的水平仍无影响。通过机体注射ＨＣＧ（人促绒毛膜性腺激素）,可提高骨骼肌组织的雄激素水平,但骨骼肌雄激素受体结合容量水平没有变化。连续注射ＨＣＧ４天,其骨骼肌组织雄激素水平显著高于连续注射ＨＣＧ８天的水平。根据上述结果,我们认为,在考虑骨骼肌同化过程时,不但要注意其雄激素的水平,还应注意其雄激素受体的水平。运动对骨骼肌组织的雄激素受体结合容量及骨骼肌雄激素分布的影响是双向的     

γ-AMINOBUTYRIC ACID RECEPTOR BINDING and UPTAKE IN MEMBRANE FRACTIONS OF CRAYFISH MUSCLE

Abstract— The uptake and binding of [³H]GABA and the binding of [³H]muscimol were measured in cell-free fractions of crayfish muscle. The uptake of GABA was saturable, of high affinity ( K _m= 0.5μ m ), and inhibited by low concentrations of compounds believed to block GABA uptake specifically, such as nipecotic acid and 2,4,diaminobutyric acid. The GABA uptake activity was localized to sucrose gradient fractions enriched in sarcolemma as demonstrated by marker enzymes and electron microscopy. The binding of the potent GABAergic agonist muscimol was also localized to the sarcolemma. The binding was saturable, of high affinity (K _D = 9 n m ), and inhibited by GABA (K ₁ = 125 n m ) and by low concentrations of receptor-specific GABA analogues, such as isoguvacine, imidazole acetic acid, and 3-aminopropane sulfonic acid. The rank order for inhibition by GABA analogues of [³H]muscimol binding sites correlated very well with activity on GABA synapses in invertebrates, consistent with specific postsynaptic receptor labeling.     

ISOLATION AND CHARACTERIZATION OF THE SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was studied after isolation of a vesicle fraction and of vesicular subfractions by means of differential and density gradient centrifugations. The different fractions were examined electron microscopically by negative and positive staining; their content in protein and phospholipid and their ability to bind Ca⁺⁺ were determined. After homogenization, differential centrifugation yielded a "sarcovesicular fraction" (SVF) which was mainly composed of numerous vesicles of different types mixed with fibrous proteins and mitochondrial fragments. This SVF contained 2% of the protein and 25% of the phospholipid of the initial tissue extract. It had a high Ca⁺⁺ binding activity that was preserved for several days by storage in the presence of oxalate. After centrifugations of the SVF on sucrose density gradients, two vesicular subfractions were obtained which were characterized by different sedimentation rates, isopycnic banding, morphology, and composition in protein and phospholipid. (a) The low-density subfraction (ρ 1.10–1.12) contained a heterogeneous population of membranous structures: thick- and thin-walled vesicles, tubular formations, triads, and plasma membranes. Its content in protein and phospholipid was very low. (b) The high-density subfraction (ρ 1.13–1.17) was a very pure subfraction composed only of thin-walled vesicles. Its content in phospholipid was high and the ratio of phospholipid-phosphorus to protein was about 20. The calcium-binding activity found in the total SVF was recovered only in this latter homogeneous subfraction. The origin of these two subfractions from the SR is discussed.     

水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性

在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。     

CALCIUM-DEPENDENT BINDING OF BRAIN GLUTAMATE DECARBOXYLASE TO PHOSPHOLIPID VESICLES

The binding of glutamate decarboxylase (GAD), to phospholipid vesicles (liposomes) in the absence and in the presence of several Ca²⁺ and Mg²⁺ concentrations was studied. Phosphatidylcho-line-phosphatidylserine (4:1) liposomes are capable of binding GAD in a Ca²⁺-dependent manner. The per cent of GAD bound increased from 5 to 65°., in a sigmoid shape with Ca²⁺ concentrations in the 0.2-4 mm range. Mg²⁺ also induces GAD binding but is less effective than Ca²⁺ The Ca²⁺ -dependent binding of GAD is not the result of unspecific association of protein, since Ca²⁺ did not promote any binding of choline acetyltransferase or lactate dehydrogenase. Furthermore, the relative specific activity (^o_o enzyme activity/% protein) of GAD associated to liposomes increases 4-fold from 0 to 2 mm Ca²⁺. The per cent of GAD bound attains a plateau at a ratio phospholipid/protein of about 1.5. and decreases when the pH increases from 6.5 or 6.8 to 7 or 7.25. Na⁺ or K⁺ at a 100mm concentration also induce binding of GAD to liposomes. Phosphatidylcholine liposomes (without phosphatidylserine) practically did not bind GAD at any Ca²⁺ concentration. The Ca²⁺-dependent association of GAD to phosphatidylcholine-phosphatidylserine liposomes is very similar to that previously reported using brain membranes, and it correlates also well with the reported Ca²⁺-dependent aggregation of phosphatidylserine molecules in phospholipid membranes of similar composition. It is concluded that phosphatidylserine is probably involved in the Ca²⁺-dependent binding of GAD to brain membranes. Phospholipid vesicles seem to be a useful experimental model for studying the mechanisms of this GAD association to membranes and the possible physiological implications of the GAD-Ca²⁺-membrane interaction regarding the release of newly synthesized GABA from nerve endings.     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

蚕豆保卫细胞原生质体质膜ABA结合蛋白的理化特性

以蚕豆(Vicia faba L.)保卫细胞原生质体为材料,证明在其质膜外侧存在着对ABA高亲和力的结合蛋白。这种蛋白与ABA作用的最适pH为6.5;在25℃时.其特异结合比高于0℃时的特异结合比;在温育30min时即达到其最大结合。它与ABA作用的解离常数为2.0×10~(-8)mol/L,每个原生质体上大约有3.2×10~6个结合位点。该结合蛋白内部具有维持其功能所必须的二硫键,而且其功能的发挥要求介质中有一定浓度的Ca~(2 )的存在。     

谷氨酸下调培养海马神经元AMPA受体G1uR2亚单位的表达

目的 研究在癫痫发病过程中,谷氨酸对AMPA受体G1uR2亚单位表达变化的影响。方法 用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体G1uR2亚单位mRNA和蛋白的表达变化。结果 在谷氨酸刺激后2h,8h,12h,培养海马神经元G1uR2 mRNA和蛋白表达明显下降,与对照组相比,差异有显著性（P〈0．05）,而非NMDA受体拮抗剂CNQX能阻断此变化。结论 在癫痫等疾病中,谷氨酸能通过激活AMAP／KA受体下调AMPA受体G1uR2亚单位的表达,参与发病过程。     

谷氨酸下调培养海马神经元AMPA 受体GluR2亚单位的表达

目的研究在癫痫发病过程中,谷氨酸对AMPA受体Glu R2亚单位表达变化的影响。方法用RT-PCR和Western Blot方法观察谷氨酸诱导培养大鼠海马神经元AMPA受体Glu R2亚单位mRNA和蛋白的表达变化。结果在谷氨酸刺激后2h,8h,12h,培养海马神经元Glu R2mRNA和蛋白表达明显下降,与对照组相比,差异有显著性(P<0.05),而非NMDA受体拮抗剂CNQX能阻断此变化。结论在癫痫等疾病中,谷氨酸能通过激活AMAP/KA受体下调AMPA受体GluR2亚单位的表达,参与发病过程。     

去卵巢大鼠Ru 486与垂体及子宫内膜孕激素受体结合的比较

经雌激素诱发的去卵巢大鼠在肌肉注射不同量的 Ru486(0.1mg/kg—2mg/kg)之后,垂体及子宫内膜的胞液游离孕激素受体结合位点随着 Ru486注射量的升高而逐步下降。注射 Ru486 2mg/kg 30min 后,此两组织中孕激素受体的结合位点就明显降低,2h 降到最低点,3h 开始恢复。以上实验说明 Ru486进入机体即可分别占领垂体及子宫内膜的孕激素受体的结合位点。然而 Ru486对此二组织的孕激素受体的结合作用并不完全相同,似乎子宫孕激素受体更易为 Ru486所占位,而解离也快。     

代谢型谷氨酸受体5与白细胞介素1受体Ⅰ型在大鼠大脑皮质及海马神经元的共存

目的 研究代谢型谷氨酸受体5(mGluR5)和白细胞介素1受体I型(IL-1RI)在大鼠大脑皮质和海马的分布及共存状况。方法 采用相邻切片的免疫细胞化学双标法,在两张相邻脑冠状切片上分别显示mGluR5与IL-1RI的免疫组化染色结果,通过显微摄像确定mGluR5／IL-1RI双标神经元。结果 切片上mGluR5阳性产物为蓝黑色,定位于细胞膜上;IL-1RI阳性产物为棕黄色,主要定位于细胞膜,也存在于神经元内。在大鼠大脑皮质及海马锥体细胞层均存在较丰富的mGluR5及IL-1RI阳性神经元。在大脑皮质,有部分神经元内mGluR5与IL-1RI共存;在海马锥体细胞层,mGluR5及n1RI阳性反应细胞密集,分布区重叠,很可能也存在二共存的神经元。结论 大鼠大脑皮质内存在mGluR5及IL-1RI共存神经元。本研究的结果为免疫神经调质在神经元内相互作用提供了形态学依据。     

斑节对虾溶菌酶基因克隆及序列分析

参考对虾溶菌酶基因和类溶菌酶基因及其他多种生物的溶菌酶基因序列 ,设计并合成引物。运用RT PCR技术 ,从斑节对虾血细胞总RNA中扩增获得特异性片段。所获片段回收纯化后克隆到pGEM TEasyVector系统的T载体上。重组子的序列分析表明 ,所克隆的斑节对虾溶菌酶基因片段长 6 5 8bp ,包括溶菌酶基因开放阅读框 (ORF)4 77bp和 3′端非编码区的 181bp。 4 77bpORF共编码 15 8个氨基酸 ,包括溶菌酶成熟肽 14 0个氨基酸残基和信号肽 18个氨基酸残基。斑节对虾溶菌酶成熟肽推测分子量为 16 ,32kd ,等电点为 8 78。与南美白对虾溶菌酶基因的碱基序列及推测氨基酸序列比较 ,同源性分别为 89 5 %和 93 0 % ;与日本对虾类溶菌酶基因的同源性分别为 84 0 %和91 0 %。进一步的序列分析表明 ,斑节对虾溶菌酶氨基酸序列与多种类群生物的c 型溶菌酶氨基酸序列具有较高的同源性 ,并具有与c 型溶菌酶相同的活性位点氨基酸残基Glu51和Asp68,且与活性位点相邻的序列高度保守。斑节对虾溶菌酶氨基酸序列还具有与c 型溶菌酶相同的结构氨基酸——— 8个半胱氨酸残基。因而可认为所克隆的斑节对虾溶菌酶基因属c 型溶菌酶基因。     

鸡视网膜谷氨酸受体和GABA受体在两栖类卵母细胞中的表达

本工作利用两栖类卵母细胞作为功能表达系统,对鸡视网膜中的谷氨酸受体和ＧＡＢＡ受体的类型和基本性质进行了研究。在注射鸡视网膜ｍＲＮＡ的卵母细胞上,谷氨酸受体有明显的表达。Ｌ－Ｇｌｕ及其类似物ＫＡ,ＡＭＰＡ,ＱＡ都毫无例外地能诱导卵母细胞产生快速平滑的去极化电流,而ＮＭＤＡ,Ｌ－ＡＰ４,ＡＣＰＤ以及天冬氨酸不能诱导明显的电流反应。并且ＡＭＰＡ,ＱＡ对ＫＡ反应存在一定的抑制作用,提示ＡＭＰＡ,ＱＡ可能与ＫＡ作用于同一受体。抑制性氨基酸ＧＡＢＡ的受体被证明大部分为ＧＡＢＡＡ亚型,但有小部分的ＧＡＢＡ反应不能为荷包牡丹碱（ｂｉｃｕｃｕｌｌｉｎｅ）所阻断。