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.     

亲和层析法分离腺苷结合蛋白质

本文报告一种新的腺苷亲和层析凝胶的合成方法。利用这种凝胶可从大鼠心脏、肝脏及小牛主动脉平滑肌的水溶部份分离出几种腺苷结合蛋白质,其亚基分子量(据SDS-PAGE)分别为35,000、37,000、46,000、43,000及15,300Dal。现已证明,35,000Dal蛋白质是乳酸脱氢酶及苹果酸脱氢酶,43,000Dal蛋白质是腺苷激酶,46,000Dal蛋白质可能是S-腺苷同型半胱氨酸水解酶。15,000Dal蛋白质前人未有报道。它对腺苷具有高度特导性和亲和力,推测是腺苷的细胞内受体和/或载体。测定了这种低分子量腺苷结合蛋白质的氨基酸组成及某些物理常数:pI=6.5;沉降系数2.42S,微分比容0.727cm~3/g,与腺苷复合物的解离常数K_D=2.3μM。     

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.     

大鼠骨骼肌组织雄激素受体结合容量测定方法

目的 :以3H testosterone(T)为配基 ,测定大鼠骨骼肌胞浆中的雄激素受体结合容量。方法 :测定温度为 4℃ ,同位素配基的饱和浓度为 5 0 pmol/ml;肌组织以 4倍 (重量 /体积 )缓冲液稀释 ,0～ 4℃温度下 10 80 0 0×g离心 1h ;孵育 18～ 2 4h。结果 :骨骼肌中雄激素受体的Kd =2 ,8× 10 9mol/ml。单点法与多点法之间无显著区别。     

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     

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : IV. SULFHYDRYL GROUPS OF THE PROTEINS OF MUSCLE

1. In the denatured proteins of skeletal muscle, the ratio of SH to S-S groups is higher than in the mixed denatured proteins of other tissues, with a single exception—the proteins of the crystalline lens. 2. The number of active SH groups in the proteins of minced muscle or in any of the protein fractions of muscle is only a fraction of the number found after the proteins have been treated with a denaturing agent. 3. The SH groups of the native proteins of muscle are activated by a rise in pH. 4. The relation between pH and number of active SH groups in the proteins of minced muscle and in the various protein fractions of muscle shows that little, if any, denatured protein is present in minced muscle.     

C－反应蛋白与模型受体的特异性结合

Ｃ－反应蛋白是动物体内一种典型的急性期反应蛋白,本文人工合成了两种可以与Ｃ－反应蛋白特异性结合的兼性分子作为Ｃ－反应蛋白的模型受体,以便进一步在脂单层膜表面上组装Ｃ－反应蛋白的二维晶体。作为第一步工作。本文研究了兼性分子的特性以及荧光光谱方法监测兼性分子与Ｃ－反应蛋白之间特异性相互作用。荧光光谱实验结果表明受体与Ｃ－反应的特异结合会引起荧光强度的下降。     

C－反应蛋白与模型受体的特异性结合

Ｃ－反应蛋白是动物体内一种典型的急性期反应蛋白。本文人工合成了两种可以与Ｃ－反应蛋白特异性结合的兼性分子作为Ｃ－反应蛋白的模型受体,以便进一步在脂单层膜表面上组装Ｃ－反应蛋白的二维晶体。作为第一步工作,本文研究了兼性分子的特性以及荧光光谱方法监测兼性分子与Ｃ－反应蛋白之间特异性相互作用。荧光光谱实验结果表明受体与Ｃ－反应的特异结合会引起荧光强度的下降。     

THE CHANGE IN STATE OF THE PROTEINS OF MUSCLE IN RIGOR

1. When myosin is exposed to a typical denaturing agent (acid) it becomes insoluble and its SH groups are activated. 2. The same number of active SH groups is found in the soluble myosin of resting muscle as in the insoluble myosin of muscle in rigor. No activation of SH groups accompanies the formation of insoluble protein in rigor. 3. When the insoluble myosin of muscle in rigor is treated with a denaturing agent its SH groups are activated. 4. Protein coagulation as brought about by denaturing agents (heat, acid, alkali, alcohol, urea, salicylate, surface forces, ultraviolet light) is a distinctly different change from the coagulation of myosin brought about by the unknown agent in muscle.     

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.     

AMPA受体与谷氨酸在大鼠三叉神经尾侧亚核内分布的免疫组织化学研究

本文用免疫组织化学ＡＢＣ法调查了４种ＡＭＰＡ受体亚单位（ＧｌｕＲ１、２／３、４）和谷氨酸（Ｇｌｕ）在大鼠三叉神经尾侧亚核（Ｖｃ）内的分布及其匹配关系。我们发现,ＧｌｕＲ１和２／３免疫阳性胞体和纤维密集分布于Ｖｃ的Ⅱ层和Ⅲ层外侧部,在Ｖｃ的其余各层呈散在性分布。ＧｌｕＲ４免疫阳性胞体和纤维在Ｖｃ各层均无分布。Ｇｌｕ免疫阳性胞体分布于Ｖｃ各层,尤以浅层（Ⅰ、Ⅱ层）密集,Ｇｌｕ免疫阳性纤维及终末结构主要分布于Ｖｃ浅层。结果表明,Ｇｌｕ免疫阳性纤维及终末样结构与ＡＭＰＡ受体免疫阳性胞体和纤维在Ｖｃ浅层的分布总体上是相互匹配的,但ＡＭＰＡ受体各亚单位在Ｖｃ内的分布存在明显的差异。本文提示,Ｖｃ内的Ｇｌｕ可能通过作用于不同的ＡＭＰＡ受体亚单位而发挥其多种生理功能     

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 .     

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。