狗肝脏苯甲二氮茸结合抑制因子的cDNA克隆及序列分析

在研究狗抗吗啡活性肽ＰＰＣ过程中,发现它与牛的ＤＢＩ（ｄｉａｚｅｐａｍｂｉｎｄｉｎｇｉｎｈｉｂｉｔｏｒ）的氨基酸序列有很高的同源性,但尚未见到有关狗的ＤＢＩ的文献报道,为了更好的探讨ＰＰＣ和ＤＢＩ相互间的关系,对狗的ＤＢＩ的ｃＤＮＡ进行了克隆和序列分析。本研究利用大鼠ＤＢＩ的基因片段为探针,从狗肝脏ｃＤＮＡ文库中,筛选得到了一阳性克隆,并进行了全自动和手工测序,得到了ＤＢＩ的全长基因。根据ＥＢＭＬｂａｎｋ序列检索,发现狗的ＤＢＩ核酸序列与牛的同源性为８１％,将其核酸序列翻译成氨基酸序列,进行同源序列比较,结果显示：狗的ＤＢＩ的氨基酸序列与猪、牛、人、酵母ＤＢＩ的同源性分别为８８．５％、８７．４％、８３．９％、４６．５％。研究还发现狗的ＤＢＩ序列与抗吗啡活性肽ＰＰＣＮ端６２个氨基酸只有两个不同,Ｃ端１７个氨基酸序列完全相同。只是ＰＰＣ比ＤＢＩ中间多了２３个氨基酸。     

中枢去甲肾上腺素和内阿片肽系统对苯二氮受体反相激动剂DMCM致焦虑作用的影响

在大鼠条件反射性惊恐反应致焦虑模型上,中枢苯二氮(艹卓)(BZD)受体激动剂安定,可使冲突惊恐反应期(PP)的操作数目显著增加,BZD受体反相激动剂DMCM则显著减少PP的操作数目;DMCM的这一作用,可分别为中枢BZD受体拮抗剂Ro 15-1788,GABA A型受体激动剂蝇蕈醇、阿片受体拮抗剂环丙甲羟二氢吗啡酮和中枢去甲肾上腺素能(NA)突触前α_2受体激动剂可乐定所阻断。结果表明,中枢阿片肽和NA系统共同参与BZD受体反相激动剂DMCM的致焦虑作用。     

Isolation and Characterization of a Rat Brain Triakontatetraneuropeptide, a Posttranslational Product of Diazepam Binding Inhibitor: Specific Action at the Ro 5-4864 Recognition Site

This report describes the purification and characterization from rat brain of triakontatetraneuropeptide (TTN, DBI 17-50), a major biologically active processing product of diazepam binding inhibitor (DBI). Brain TTN was purified by immunoaffinity chromatography with polyclonal octadecaneuropeptide, DBI 33-50) antibodies coupled to CNBr-Sepharose 4B followed by two reverse-phase HPLC steps. The amino acid sequence of the purified peptide is: Thr-Gln-Pro-Thr-Asp-Glu-Glu-Met-Leu-Phe-Ile-Tyr-Ser-His-Phe-Lys-Gln-Ala-Thr-Val - Gly-Asp-Val-Asn-Thr-Asp-Arg-Pro-Gly-Leu-Leu-Asp-Leu-Lys. Synthetic TTN injected intracerebroventricularly into rats induces a proconflict activity (IC50 0.8 nmol/rat) that is prevented by the specific "peripheral" benzodiazepine (BZ) receptor antagonist isoquinoline carboxamide, PK 11195, but not by the "central" BZ receptor antagonist imidazobenzodiazepine, flumazenil. TTN displaces [3H]Ro 5-4864 from synaptic membranes of olfactory bulb with a Ki of approximately 5 microM. TTN also enhances picrotoxinin inhibition of gamma-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding. These data suggest that TTN, a natural DBI processing product acting at "Ro 5-4864 preferring" BZ binding site subtypes, might function as a putative neuromodulator of specific GABAA receptor-mediated effects.     

Diazepam Binding Inhibitor (DBI) processing: Immunohistochemical studies in the rat brain

Diazepam Binding Inhibitor (DBI) is an endogenous 11-kDa peptide originally isolated from rat brain. In rat brain DBI coexists with at least three different processing products and the members of this peptide family have been shown to displace benzodiazepines and beta carbolines from recognition sites located on the allosteric modulatory centers of GABA_A receptors. Immunocytochemical methods were used to study the location of DBI and two of the processing products, octadecaneuropeptide (ODN) DBI 33-50 and triakontatetraneuropeptide (TTN) DBI 17-50, in rat brain. DBI-LI was found in selected neuronal perikarya and in many glia and glial-like cells. All circumventricular organs displayed a strong DBI like immunoreactivity (LI). The distribution and cellular location of the ODN-LI and TTN-LI differed from that of DBI because they were preferentially associated with DBI in neurons, but not in glia or glial-like cells. The presence of DBI, but not of its processing products, in glial cells, circumventricular organs, and cells of peripheral tissues suggests that the function of this peptide may extend to other yet unknown function in addition to an action on the allosteric modulatory center of GABA_A receptors located in neurons.     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

Subcellular Location and Neuronal Release of Diazepam Binding Inhibitor

Diazepam binding inhibitor (DBI), a peptide located in CNS neurons, blocks the binding of benzodiazepines and beta-carbolines to the allosteric modulatory sites of gamma-aminobutyric acid (GABAA) receptors. Subcellular fractionation studies of rat brain indicate that DBI is compartmentalized. DBI-like immunoreactivity is highly enriched in synaptosomes obtained by differential centrifugation in isotonic sucrose followed by a Percoll gradient. In synaptosomal lysate, DBI-like immunoreactivity is primarily associated with synaptic vesicles partially purified by differential centrifugation and continuous sucrose gradient. Depolarization induced by high K+ levels (50 mM) or veratridine (50 microM) released DBI stored in neurons of superfused slices of hypothalamus, hippocampus, striatum, and cerebral cortex. The high K+ level-induced release is Ca2+ dependent, and the release induced by veratridine is blocked by 1.7 microM tetrodotoxin. Depolarization released GABA and Met5-enkephalin-Arg6-Phe7 together with DBI. DBI is also released by veratridine depolarization, in a tetrodotoxin-sensitive fashion, from primary cultures of cerebral cortical neurons, but not from cortical astrocytes. Depolarization fails to release DBI from slices of liver and other peripheral organs. These data support the view that DBI may be released as a putative neuromodulatory substance from rat brain neurons.     

Stimulation of Brain Pregnenolone Synthesis by Mitochondrial Diazepam Binding Inhibitor Receptor Ligands In Vivo

Abstract: Evidence that neurosteroids are potent modulators of the action of GABA at GABA_A receptors has prompted the investigation of the mechanism that controls brain neurosteroid synthesis by glial cell mitochondria in vivo. In vitro studies suggest that the interaction of the diazepam binding inhibitor (DBI)—a polypeptide that is abundant in steroidogenic cells—with glial mitochondrial DBI receptors (MDRs) is a crucial step in the physiological regulation of neurosteroid biosynthesis. MDRs bind 4-chlorodiazepam (4′-CD), N,N-di-n-hexyl-2-(4-fluorophenyl)-indol-3-acetamide (FGIN-1–27), and the isoquinoline carboxamide PK 11195 with high affinity, and these ligands have been used to investigate whether the stimulation of glial MDRs increases brain pregnenolone production in vivo. Adrenalectomized and castrated (A-C) male rats (to eliminate peripheral sources of pregnenolone) were pretreated with trilostane (to prevent pregnenolone metabolism to progesterone), and the pregnenolone content in brain regions dissected after fixation with a 0.8-s exposure to microwave irradiation focused to the head was determined by HPLC followed by specific radioimmunoassay. The forebrain and cerebellum of A-C rats contained 4–7 ng of pregnenolone/g of tissue, and the olfactory bulb contained 10–14 ng/g. These concentrations of brain pregnenolone are only 30–40% lower than those of shamoperated rats. In contrast, the plasma pregnenolone content of sham-operated rats was 2–3 ng/ml, but it was only 0.15–0.20 ng/ml in the plasma of A-C rats. In A-C rats, treatment with the MDR ligands 4-CD and FGIN-1–27 increased the pregnenolone content in the brain but failed to change the plasma or peripheral tissue content of this steroid. The effect of 4′-CD on brain pregnenolone content was maximal (70–100% increase) at the dose of 18 μmol/kg, 5–10 min after intravenous injection. The effect of oral administration of FGIN-1–27 on brain pregnenolone content was maximal (80–150% increase) at doses of 400–800 μmollkg and peaked at ～ 1 h. That this effect of FGIN-1–27 was mediated by the MDR was documented by pre-treatment with the MDR partial agonist PK 11195 (100 μmol/kg, i.p.). PK 11195 did not affect basal brain pregnenolone content but prevented the accumulation of brain pregnenolone induced by FGIN-1–27. FGIN-1–27 and 4-CD failed to increase the brain concentration of dehydre epiandrosterone in A-C rats. These data suggest that glial cell MDRs play a role in neurosteroid biosynthesis in vivo.     

Biochemical Analysis of the Yeast Proteinase Inhibitor (IC) Homolog ICh and Its Comparison with IC

Carboxypeptidase Y (CPY) inhibitor (I^C) and its homologous protein (I^Ch) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I^C and its inhibition mode toward CPY were recently reported, but I^Ch has not been characterized. The molecular mass of I^Ch was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I^Ch is folded as a monomeric β-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I^Ch was exclusively expressed in the early log phase, whereas I^C was expressed in the logarithmic and stationary phase. The intracellular localization of I^Ch was different from that of I^C. These findings provide insights into the physiological functions of I^Ch.     

Identification, Purification, and Characterization of Two Forms of Serotonin Binding Protein from Rat Brain

Serotonin binding protein (SBP) is found in synaptic vesicles of mammalian central and peripheral serotonergic neurons. 5-Hydroxytryptamine (5-HT, serotonin) is physiologically stored as a complex with SBP in vivo. Two forms of SBP have been detected with apparent molecular weights of 45,000 and 56,000 (45K and 56K). To understand the relationship between the two forms more fully, we purified the two proteins to homogeneity and partially characterized them. Purification steps included (NH4)2SO4 fractionation and chromatography on Sepharose 4-B, Affi-Gel-Blue, hydroxylapatite, and phosphocellulose. The 45K from of SBP was obtained pure, whereas the 56K form of SBP was obtained about 90% pure by these methods. To isolate pure 56K SBP for induction of antibodies, the protein was further purified by sodium dodecyl sulfate-gel electrophoresis followed by electroelution. The 56K form of SBP was thus isolated, but in a denatured state; its purity was established by two-dimensional gel electrophoresis. The two forms of SBP (pure 45K and 90% pure undenatured 56K SBP) were similar in their 5-HT binding capacity; the enhancement of 5-HT binding by Fe2+; and inhibition by--SH reagents, chelators, and sodium salts. Antibodies raised against the pure 56K form of SBP cross-reacted with the 45K SBP. The two forms of SBP differed in the following properties: (1) dissociation constants--56K form showed higher affinity for 5-HT (KD1 = 0.4 nM; KD2 = 32 nM), whereas the 45K form showed lower affinity (KD1 = 9.7 nM; KD2 = 120 nM); (2) ratio of number of 5-HT binding sites with low affinity to those with high affinity--56K (19:1), 45K (10:1); (3) isoelectric point--the 56K form of SBP is more acidic (5.6 and 5.9) than the 45K form (6.1); (4) binding enhancement by gangliosides and bicarbonate. To establish whether the 45K form of SBP is found in vivo or is produced by proteolysis during isolation, two additional experiments were carried out. (1) We added a mixture of proteolytic enzyme inhibitors to our homogenization buffer; this addition did not change the ratio of the two forms of SBP. (2) We mixed regions of the CNS enriched in the 45K form of SBP (spinal cord) with regions rich in the 56K form of SBP (raphe nuclei) and homogenized them together. Again, this procedure failed to change the ratio of the two forms of SBP as judged by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 400 WORDS)     

牦牛DBI基因的分子特征及其在乳腺组织中的表达与定位

地西泮结合抑制因子(Diazepam binding inhibitor,DBI)与酰基辅酶A具有高亲和力,在动物组织中广泛存在,与脂肪酸代谢、类固醇激素合成密切相关。为研究DBI基因的分子特征及该基因在乳腺发育中的作用,对牦牛DBI基因编码区进行克隆,进行生物信息学分析;采用实时荧光定量PCR (Quantitative real-time PCR,qPCR)、蛋白免疫印迹技术(Western blotting,WB)和免疫组织化学(Immunohistochemistry,IHC)方法对牦牛泌乳前期、泌乳期和干乳期的乳腺组织中DBI的相对表达量和表达部位进行研究。DBI序列分析显示：牦牛DBI基因编码区序列长264 bp,编码87个氨基酸残基,与牛的同源性高达99.62％;qPCR数据表明：牦牛泌乳前期乳腺组织中DBI基因的相对表达量显著高于泌乳期和干乳期(P< 0.05);WB结果显示：牦牛泌乳前期乳腺组织中DBI蛋白的表达量最高,干乳期次之,泌乳期最低(P< 0.05);IHC结果表明：不同发育时期的牦牛乳腺组织中DBI的表达部位并无明显差异,主要表达于乳腺腺泡上皮细胞、导管上皮细胞及小叶间质细胞。DBI在不同发育时期牦牛乳腺组织中的相对表达量具有明显差异(P< 0.05),揭示DBI可能参与牦牛乳腺发育的过程,这为进一步探究DBI基因在生物体中的作用提供相应的理论参考。     

猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。     

Binding of the Recombinant Proteinase Inhibitor Eglin c,of the Soybean Bowman-Birk Proteinase Inhibitor and of its Chymotrypsin and Trypsin Inhibiting Fragments to Leu-Proteinase,the Leucine Specific Serine Proteinase from Spinach (Spinacia oleracea L.) Leaves: Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respetively) to Leuproteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of K_a (at 21°C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from ～6.9, in the free Leu-proteinase, to ～5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c - K_a = 2.2 × 10¹¹ M^-1, δG°= - 64kJ/mol, δH° = + 5.9kJ/mol, and δS° = + 240J/molK; Leu-proteinase:BBI - K_a = 3.2 × 10¹⁰ M^-1, δG° = - 59kJ/mol, δH°= + 8.8kJ/mol, and δS° = + 230J/molK; and Leu-proteinase:F-C - K_a = 1.1 × 10⁶ M^-1, δG°= - 34kJ/mol, δH° = + 18J/mol, and δS° = + 180J/molK (values of K_a, δG° and δS° were obtained at 21.0°C; values of δH° were temperature-independent over the range explored, i.e. between 10.0°C and 40.0°C). F-T does not inhibit Leu-proteinase up to an inhibitor concentration of 1.0 × 10^-3 M, suggesting that the upper limit of K_a is 1 × 10² M^-1. Considering the known molecular models, the observed binding behaviour of eglin c, BBI, F-C and F-T to Leu-proteinase has been related to the inferred stereochemistry of the enzyme/inhibitor contact region