决明子胰蛋白酶抑制剂COTI对菜青虫生长抑制作用的研究

从决明子中分离得到胰蛋白酶抑制剂COTI,利用其缓冲液浸泡油菜叶片饲喂菜青虫(Pieris rapae L)幼虫后,幼虫体重明显下降,分析显示菜青虫中肠提取物中蛋白酶活性受到COTI的抑制,表明决明子胰蛋白酶抑制剂对菜青虫的生长具有明显的抑制作用。     

Circadian Rhythmicity in the 'Basal' Pancreatic Secretion of the Domestic Fowl

The interdigestive pancreatic exocrine secretion of the domestic fowl has been studied following the cosine-vector analysis. For the flow of pancreatic juice the crest phase was at 1040 and the sample amplitude was 2.26μl/min.For the amylase output the crest phase was at 1128 and the sample amplitude was 0.297 U.A.A./min. Both parameters are characterized by a phase-and frequency-synchronized rhythm whose period must be close to 24 hr.     

干预脂毒性改善糖尿病大鼠胰岛素分泌及氧化应激的损害

目的探讨干预脂毒性改善糖尿病大鼠胰岛分泌功能及氧化应激损害的机制。方法将大鼠分为4组①正常组（NC）,全程普通饲料喂养;②高脂组（HF）,全程高脂饲料喂养。糖尿病组,高脂饲料喂养8周后腹腔注射低剂量STZ（30mg/kg）,48h后行OGTT试验判断成模情况后分组。③糖尿病对照组（DM）,不给予药物干预;④血脂干预组（SIM）,灌胃辛伐他汀5mg/（kg.d）4周干预脂毒性。通过免疫组化染色观察胰岛B、A细胞形态学特点,RT-PCR测定胰腺内胰岛素原mRNA表达水平,DHE荧光染色检测胰岛中活性氧化产物ROS水平。结果与糖尿病对照组相比,干预脂毒性4周后血清胆固醇（TC）和甘油三酯（TG）水平分别下降了22.9%（P〈0.01）和57.0%（P〈0.05）。OGTT血糖水平均显著下降（P〈0.01）。胰岛中B细胞相对量是对照组的2.6倍（P〈0.01）,B细胞胞质内胰岛素水平增加了26.5%（P〈0.05）,胰岛素原mRNA表达升高18.3%（P〈0.01）;A细胞相对量减少了50%（P〈0.01）。血清丙二醛（MDA）水平和胰腺中ROS表达显著下降。结论辛伐他汀干预脂毒性4周可以显著改善糖尿病大鼠胰岛分泌功能和氧化应激损害。     

Antifungal Activity of a Bowman–Birk-type Trypsin Inhibitor from Wheat Kernel

A trypsin inhibitor from wheat kernel (WTI) was found to have a strong antifungal activity against a number of pathogenic fungi and to inhibit fungal trypsin-like activity. WTI inhibited in vitro spore germination and hyphal growth of pathogens, with protein concentration required for 50% growth inhibition (IC₅₀) values ranging from 111.7 to above 500 μg/ml. As observed by electron microscopy, WTI determined morphological alterations represented by hyphal growth inhibition and branching. One of the fungal species tested, Botrytis cinerea produced a trypsin-like protease, which was inhibited by the trypsin inhibitor. WTI, as well as other seed defence proteins, appear to be an important resistance factor in wheat kernels during rest and early germination when plants are particularly exposed to attack by potential soil-borne pathogens.     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.     

固定化胰蛋白酶亲和层析法制备低STI残存大豆分离蛋白质

聚苯乙烯阴离子交换树脂(GM201)经预处理除去杂质后先与戊二醛(2—6％)反应,再与胰蛋白酶(5000u/mg,8—10mg/mL,pH 8.0)反应即制得固定化胰蛋白酶。此法得到的固定化胰蛋白酶具有良好的热稳定性,贮藏稳定性和操作稳定性,可用于工业化目的。脱脂豆粉经萃取(PH9.0)后,稀释4倍,在pH5.0下沉淀分离出大豆球蛋白,然后用酸性水(pH5.0)洗涤两次,并进行碱溶与酸沉淀两次,即可将大豆分离蛋白质的STI残留降低到1.85％,比活性降到1u/mg以下。最后再用固定化胰蛋白酶亲和层析,就可以除去大豆分离蛋白质中残留的STI。     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Vegetative Storage Protein with Trypsin Inhibitor Activity Occurs in Sapindus mukorassi,a Sapindaceae Deciduous Tree

A vegetative storage protein （VSP） with trypsin inhibitor activity in a deciduous tree, Sapindus mukorassi, was characterized by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot, immuno-histochemical localization, light- and electro-microscopy, together with analysis of proteinase inhibitor activity of the purified VSP in vitro. There were two proteins with molecular masses of about 23 and 27 kDa in a relatively high content in the bark tissues of terminal branches of S. mukorassi in leafless periods. The proteins decreased markedly during young shoot development, indicating their role in seasonal nitrogen storage. Immuno-histochemical localization with the polyclonal antibodies raised against the 23 kDa protein demonstrated that the 23 kDa protein was the major component of protein inclusions in protein-storing cells. The protein inclusions were identified by protein-specific staining and should correspond to the electron-dense materials in different forms in the vacuoles of phloem parenchyma cells and phloem ray parenchyma cells under an electron microscope. So, the 23 kDa protein was a typical VSP in S. mukorassi. The 23 and 27 kDa proteins shared no immuno-relatedness, whereas the 23 kDa protein was immuno-related with the 22 kDa VSP in lychee and possessed trypsin inhibitor activity. The 23 kDa protein may confer dual functions： nitrogen storage and defense.     

Binding of the Bovine and Porcine Pancreatic Secretory Trypsin Inhibitor (Kazal) To Human Leukocyte Elastase: A Thermodynamic Study

Abstract

The effect of pH and temperature on the apparent association equilibrium constant (K_a) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – K_a = 6.3 × 10⁴M^?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 10² entropy units; porcine PSTI –K_a = 7.0 × 10³M^?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 10² entropy units (values of K_a δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of K_a for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s).     

胰蛋白酶处理对质膜H~ -ATPase钒酸钠抑制效应的影响

采用经蔗糖密度梯度法纯化的大豆 (GlycinemaxL .)下胚轴质膜微囊为材料 ,分析了胰蛋白酶处理对质膜H ATPase钒酸钠抑制效应的影响。实验结果显示 ,温和胰蛋白酶处理显著提高H ATPase的ATP水解活力。并且发现酶切处理降低了钒酸钠对ATPase的抑制效应 ,当钒酸钠浓度为 2mmol/L时 ,ATPase活力仅被抑制 5 3.49% ,而未经酶切的对照组则被抑制 6 4.13%。ATP水解动力学分析表明 ,胰蛋白酶酶切处理既不影响ATP水解的Km 值也不影响钒酸钠的抑制类型 ,酶切前后的Km 值都等于 0 .34mmol/L ,并且都属于反竞争抑制。以上结果显示胰蛋白酶酶切处理可能改变了磷酸酶结构域的结构而影响了钒酸钠的抑制效应 ,暗示C_末端调节着磷酸酶结构域的结构和功能     

Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion

Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr^595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion.     

Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.

Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14-38]Abu, at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by alpha-amino-n-butyric acid (Abu). [14-38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D degree -the diffusion coefficient extrapolated to infinite dilution-is determined. From D degree, a value of the hydrodynamic radius. Rh, is computed from the Stokes-Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38]Abu, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions.     

Heterogeneous Packing in the Folding/Unfolding Intermediate State of Bitter Gourd Trypsin Inhibitor

The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.     

Effect of Ethanol on Nuclear Casein Kinase II Activity in Brain

Casein kinase II (CK II) plays an important role in serine/threonine dependent protein phosphorylation. In brain it is associated with long term potentiation besides its involvement in DNA, RNA and protein metabolism. Ethanol has been shown to induce cognitive impairment and affects DNA, RNA and protein metabolism at various steps. Since CK II is central in all these events, which are specifically affected by ethanol, the role of nuclear CK II is investigated in the present study. Total nuclear casein kinase activity was unaffected while heparin sensitive nuclear casein kinase II activity showed a 30% decrease in the brain from chronic alcohol fed rats. Cytosolic CK II activity was also unaffected. Immunological detection by western analysis using CK II antibodies showed no alteration in the quantity of enzyme. The decrease in nuclear casein kinase II might be responsible for ethanol induced cognitive impairment in the brain.     

Effect of phospholipids on conformational structure of bovine pancreatic trypsin inhibitor (BPTI) and its thermolabile mutants

The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (DeltaG(U)) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of DeltaG(U) was negative, only two types of non-native structures were formed: one with high beta structure content at low PS fluidity state, and the other with a high alpha-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies.     

Simultaneous Binding of Basic Peptides at Intracellular Sites on a Large Conductance Ca2+-activated K+ Channel : Equilibrium and Kinetic Basis of Negatively Coupled Ligand Interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca²⁺-activated K⁺ channels (maxi-K_Ca) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-K_Ca channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (K _d, 16.5 nM) and BPTI (K _d, 1,490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (K _d, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (K _d, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (K _d, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (K _d, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (K _d, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand–ligand interactions at distinct sites on the intracellular surface of maxi-K_Ca channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-K_Ca channel.     

Antihypertensive Effect of Angiotensin I-Converting Enzyme Inhibitory Peptides from a Sesame Protein Hydrolysate in Spontaneously Hypertensive Rats

Sesame peptide powder (SPP) exhibited angiotensin I-converting enzyme (ACE) inhibitory activity, and significantly and temporarily decreased the systolic blood pressure (SBP) in spontaneously hypertensive rats (SHRs) by a single administration (1 and 10 mg/kg). Six peptide ACE inhibitors were isolated and identified from SPP. The representative peptides, Leu-Val-Tyr, Leu-Gln-Pro and Leu-Lys-Tyr, could competitively inhibit ACE activity at respective Ki values of 0.92 μM, 0.50 μM, and 0.48 μM. A reconstituted sesame peptide mixture of Leu-Ser-Ala, Leu-Gln-Pro, Leu-Lys-Tyr, Ile-Val-Tyr, Val-Ile-Tyr, Leu-Val-Tyr, and Met-Leu-Pro-Ala-Tyr according to their content ratio in SPP showed a strong antihypertensive effect on SHR at doses of 3.63 and 36.3 μg/kg, which accounted for more than 70% of the corresponding dosage for the SPP-induced hypotensive effect. Repeated oral administration of SPP also lowered both SBP and the aortic ACE activity in SHR. These results demonstrate that SPP would be a beneficial ingredient for preventing and providing therapy against hypertension and its related diseases.     

Prediction of a low-frequency mode in bovine pancreatic trypsin inhibitor molecule

One of the frontiers today in molecular biology is to measure, identify and go further to predict the low-frequency internal motion of biological macromolecules, which is crucially important for understanding the dynamic mechanism of various biological functions occurring in such molecules. Based on the theory of continuity model developed recently for dealing with the internal low-frequency motion of a biological macromolecule, it is predicted that the low-frequency phonons with wave number of about 23 cm^?1 might be excited in BPTI molecule.     

截短型rBTI的表达、纯化及其对EC9706细胞生长的抑制作用

依据先前获得的重组荞麦胰蛋白酶抑制剂（rBTI）氨基酸序列及三维分子构像,分别构建了rBTI C末端缺失VVM、TPVVM或VDTPVVM的截短型pExsecI BTI t1,pExsecI BTI t2和pExsecI BTI t3重组质粒.转入大肠杆菌BL21中进行表达,并通过Resource Q阴离子交换层析分离.实验结果显示,3个工程菌均以可溶方式表达,目的蛋白在SDS PAGE图谱中显示单一条带,其纯度达98％以上. 理化性质分析表明,截短型rBTI与野生型rBTI具有相似的胰蛋白酶抑制活性,并具有很好的热稳定性及酸碱稳定性. 将野生型和截短型rBTI分别作用于人食管癌EC9706细胞.MTT检测发现,C末端截短不同数目的氨基酸后,与野生型rBTI相比,在相同浓度下截短型rBTI仍具有一定的抑制肿瘤细胞生长作用,其抑制作用范围是截短前的50%左右. 这些结果提示, rBTI的 C末端氨基酸残基缺失未引起活性区域或功能部位的较大改变,从而保留了其对胰蛋白酶的抑制作用和部分生物学功能.     

Combined Effects of Dietary Protein Type and Fat Level on the Body Fat-Reducing Activity of Conjugated Linoleic Acid (CLA) in Rats

The interaction of dietary protein type and fat level on the body fat-reducing activity of conjugated linoleic acid (CLA) was studied in male rats fed diets containing casein (CAS) or soy protein (SOY) as a protein source with low fat (LF, 6.0% soybean oil) or high fat (HF, 13.0% soybean oil) combinations for 4 weeks. CLA was added at the 1.0% level to all diets. The weight of perirenal adipose tissue tended to be lower in the SOY groups than in the corresponding CAS groups, and the difference between the LF diets was significant. The weight of epididymal adipose tissue showed a similar but insignificant trend. The weight of brown adipose tissue was heaviest on the SOY-HF diet and lowest on two CAS diets, the SOY-LF diet being intermediate. The concentration of serum leptin was lowest on the SOY-LF diet and was significantly lower than that of the corresponding CAS group, but this difference disappeared when the dietary fat level increased. The serum cholesterol-lowering activity of SOY in relation to CAS was reproduced even when CLA was given. Thus the body fat-reducing activity of CLA was most marked when rats were fed the SOY-LF diet. Although the CAS-HF diet increased body fat deposition, the magnitude of the reduction by lowering dietary fat level was more marked than in the case of SOY. These results indicate a complicated interaction of dietary manipulations with the body fat-reducing effect of CLA, but the combination of CLA with the SOY-LF diet appears to be an appropriate approach.