X-ray Structure Analysis and Characterization of AFUEI,an Elastase Inhibitor from Aspergillus fumigatus

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.     

The determination of myeloperoxidase activity in liver

Myeloperoxidase (MPO) is an enzyme found in granulocytes of neutrophils, but not in mammalian tissues. Previous studies have directly correlated MPO activity with neutrophil accumulation in tissues. This study presents a method for determining MPO activity in liver. Neutrophil accumulation in rat liver was provoked by creating partial ischemia followed by reperfusion. Liver homogenates prepared by a standard procedure showed no MPO activity. The homogenate was applied to Sephadex G100 and DEAE Sepharose CL6B columns which separated MPO activity from inhibitory activity. The inhibitor was identified as catalase based upon its elution from the columns and removal with 3-amino- 1,2,4-triazole (AT), a catalase inhibitor. Based upon these findings, it was determined that full MPO activity can be assayed in unfractionated liver homogenates by first inactivating catalase with AT.     

Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor

Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.     

眼镜蛇毒纤维蛋白原溶解因子的纯化及理化性质的研究

经Heparin-SepharoseCL-6B亲和层析和SephadexG-150分子筛层析,从中国眼镜蛇毒(Najanajaatra)中纯化出一种可水解纤维蛋白原Aα链的蛋白酶——组分F.SDS-PAGE测得分子量为47100,为一含2%中性己糖的单肽链糖蛋白;它对纤维蛋白、酪蛋白和苯甲酰-L-精氨酸乙酯(BAEE)均无水解作用,也无激活纤溶酶原的作用;100μg皮下或皮内注射,未见出血反应。组分F对酸和热均不稳定。EDTA,EGTA,PMSF和肝素等可抑制其纤维蛋白原溶解活性,benzami-dine,aprotinin和大豆胰蛋白酶抑制因子(SBTI)则无抑制作用。     

百合巯基蛋白酶抑制剂的纯化及部分性质研究

百合的鳞茎中含有一种对木瓜蛋白酶有强抑制作用的巯基蛋白酶抑制剂．百合的鳞茎经浸取加热处理,木瓜蛋白酶偶联的Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和层析和ＳｅｐｈａｄｅｘＧ－１００分子筛层析,可获得在ＰＡＧＥ和ＳＤＳ－ＰＡＧＥ均为单一蛋白带的百合巯基蛋白酶抑制剂（ＣＰＩ）．此ＣＰＩ为单链蛋白,含有０．３０７％的中性糖;Ｎ端氨基酸为Ｉｌｅ;ＳＤＳ－ＰＡＧＥ测得亚基分子量为１２０００;ＳｅｐｈａｄｅｘＧ－１００测得分子量为１２５００．百合ＣＰＩ在１００℃内和ｐＨ２～１２范围内非常稳定;对木瓜蛋白酶的抑制属竞争性抑制类型,其Ｋｉ值为１．１５×１０~（－９）ｍｏｌ／Ｌ,对木瓜蛋白酶的抑制摩尔比为８．５：１．     

Inhibition of Histamine Synthesis in Brain by α-Fluoromethylhistidine, a New Irreversible Inhibitor: In Vitro and In Vivo Studies

a-Fluoromethylhistidine (α-FMH), a new potent inhibitor of histidine decarboxylase (HD), has been used for in vitro and in vivo studies of brain HD. Following a preincubation with (+)-α-FMH, brain HD activity was inhibited in a time-dependent and concentration-dependent manner. The enzyme activity was not restored by overnight dialysis against standard buffer. The (–) antimer of α-FMH was ineffective. When injected intraperitoneally in a single dose of 20 mg/kg, (±)-α-FMH induced a complete loss in HD activity in cerebral cortex and hypothalamus as well as in peripheral tissues, such as stomach. At a dosage of 100 mg/kg (±)-α-FMH did not alter histamine-N-methyltransferase, DOPA decarboxylase, and glutamate decarboxylase activities. The maximal decrease of HD activity occurred after 2 h in both cerebral cortex and hypothalamus, but the time course of the recovery of enzyme activity was slower in the cerebral cortex. The enzyme activity reached control value within 3 days in hypothalamus and was not fully restored after 4 days in cerebral cortex. Contrasting with the diminished HD activity, a substantial concentration of histamine remained present in five regions of mouse brain. Thus, α-FMH is a highly specific irreversible inhibitor of brain HD activity and its efficacy makes it useful to study the physiological role of brain histamine.     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Putative apolipoprotein B-100 in the freshwater turtle Chrysemys picta: effects of estrogen and progesterone.

1. The isolation and purification of a putative apolipoprotein B-100 in the plasma of the freshwater turtle Chrysemys picta is described. 2. The protein was purified through differential ultracentrifugation and subsequent Sepharose 6B column chromatography. 3. The molecular weight of the protein determined by electrophoresis was approximately 350 kDa. 4. An antibody to chicken apolipoprotein B-100 specifically recognizes this 350 kDa protein in Western blots, suggesting its identity with apolipoprotein B-100. 5. An antibody to the putative Chrysemys apolipoprotein B-100-like protein was developed and used in an ELISA to quantitate protein levels in plasma. 6. Acute estrogen treatment increased levels of apolipoprotein B-100 (7.64 +/- 0.79 mg/ml plasma) over that of control animals (5.07 +/- 1.74 mg/ml plasma). 7. In contrast, chronic estrogen treatment reduced apolipoprotein B-100 significantly to 2.94 +/- 0.53 mg/ml plasma (P < 0.05).     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

Isolation and partial characterization of an antigen shared between Schistosoma mansoni, Fasciola hepatica, and Biomphalaria glabrata

An antigen was isolated and partially characterized from adult Schistosoma mansoni which immunologically cross-reacted with Fasciola hepatica and Biomphalaria glabrata, a schistosome intermediate snail host. The antigen was isolated from a solubilized freeze-thaw preparation containing 0.5% Triton X-100 by passage through a CNBr-activated Sepharose 4B column coupled with rabbit IgG prepared against a homogenate of B. glabrata hepatopancreas. The eluted antigen (designated as SMw-53P) stained with Coomassie Brilliant Blue R250, but not with Nile Blue A or periodic acid-Schiff's stains. The antigen did not bind to a Concanavalin A affinity column. SMw-53P was determined to have a molecular weight of 53,000 daltons by SDS-polyacrylamide gel electrophoresis. Western blotting, using sera from mice infected with S. mansoni, revealed the presence of the antigen in whole worm preparations of both S. mansoni and F. hepatica. The serodiagnostic potential of the antigen was evaluated by ELISA utilizing sera from S. mansoni-infected mice, or rabbits injected with homogenates of F. hepatica or S. mansoni whole worm, or B. glabrata hepatopancreas. SMw-53P was shown to strongly react with all antisera, but not normal mouse or rabbit sera. These data suggest a limited value for the antigen for the specific immunodiagnosis of schistosomiasis mansoni, but do suggest a possible potential as a general screening tool for detecting trematode infections. Further studies regarding the antigen's potential as a vaccine are indicated.     

眼镜蛇毒中降压因子的提取及其降血压作用的研究

目的：从广西眼镜蛇蛇毒中分离纯化血管紧张素转换酶抑制剂(Angiotensin Converting Enzyme Inhibitor,ACEI),命名为降压因子（Hypotensive Factor,HF）,并测定其生物活性。方法：采用Sephacryl S-100凝胶过滤,CM Sepharose F.F.离子交换层析分离纯化HF,高效液相鉴定纯度,SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定其分子量,紫外分光光度法测定HF对血管紧张素转换酶（ACE）的抑制活性。用离体兔子十二指肠平滑肌测定HF增强缓激肽(Bradykinin,BK)的效应。结果：纯化的广西眼镜蛇蛇毒HF经SDS-PAGE检测显示单一条带,测得其相对分子量约为8.2kD,由十二种氨基酸组成,蛋白回收率为5.70%。HF对ACE有明显的抑制作用,其抑制作用与剂量呈正相关。IC50为1.02?g/ml。HF能增强BK对离体兔子十二指肠平滑肌的收缩效应。结论：本方法成功地从广西眼镜蛇蛇毒中纯化出降血压成分。该成分与血管紧张素转换酶抑制剂作用相似,对血管紧张素转换酶有明显的抑制作用。     

用聚焦层析法纯化人巯基蛋白酶抑制肽C-hCRI_c

以肾衰病人尿为材料,通过碱变性、酸变性和热变性除去部分杂质和尿蛋白。然后用羧甲基-木瓜蛋白酶-琼脂糖4B亲和层析,最后用多缓冲剂聚焦层析,把人的巯基蛋白酶抑制肽C(human cysteine proteinase inhibitor C,简称 hCPI_C)和其他的hCPI_C分开,制备得高纯度、高活性的hCPI_C。在SDS-PAGE中为均一条带,每mg蛋白可抑制50单位木瓜蛋白酶活性的50-63％。     

A neutral subtilopeptidase inhibitor from porcine serum some evidence for alpha2-macroglobulin.

An inhibitor of neutral subtilopeptidase [EC 3.4.24.4] was purified from porcine serum by salting out with (NH4)2SO4, chromatography on anion exchange sephadex, gel filtration with Sepharose 6B, and isoelectric focusing. The preparation was homogeneous by electrophoretic and ultracentrifugal criteria, and was shown to be a glycoprotein with a molecular weight of 740,000. It inhibited the caseinolytic activities of thermolysin, subtilisin, trypsin [EC 3.4.21.4], and alpha-chymotrypsin [EC 3.4.21.1] as well as that of neutral subtilopeptidase by an equimolar binding to those proteolytic enzymes. SDS-polyacrylamide gel electrophoresis after reduction with beta-mercaptoethanol indicated that the inhibitor was made up of four subunit monomers having a molecular weight of 190,000. From comparisons of its physiocochemical and inhibitory properties with those of well-investigated plasma proteins, the inhibitor was identified as alpha2-macroglobulin. On treatment of the inhibitor with neutral subtilopeptidase, a protein with a molecular weight of 95,000 appeared after treatment with SDS and beta-mercaptoethanol, suggesting that a peptide bond susceptible to the enzyme exists near the mid-point of the subunit chains.     

An Endogenous Inhibitor of Ca++-ATPase from Human Placenta

Intracellular free calcium is regulated by Ca⁺⁺-ATPase, one form present on the plasma membrane (PM Ca⁺⁺-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca⁺⁺-ATPase). An endogenous inhibitor of SR Ca⁺⁺-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca⁺⁺-ATPase by this inhibitor was concentration-and time-dependent. The inhibitor neither bound to DEAE-nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15min up to 55°C and completely destroyed at 80°C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca⁺⁺-ATPase in the placenta during pregnancy.     

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition

The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel β-sheet gripping an α-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting β-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S′ side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp³²–Asp²¹⁵ diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

新的端粒酶活性抑制蛋白LPTS-L的制备

LPTS基因是利用定位候选克隆策略克隆的一个新的肝相关候选肿瘤抑制基因。LPTS基因编码一个全长为328氨基酸的蛋白质(LPTS-L),该蛋白具有抑制细胞端粒酶活性的功能。为了进一步研究LPTS-L蛋白的结构与功能,利用DNA重组技术,将LPTS-L的cDNA克隆到表达载体pET-24a中构建重组克隆pET-24-LPTS,并在大肠杆菌BL-21中进行融合表达,获得可溶形式的LPTS-L融合蛋白。采用Ni Sepharose4B柱亲和层析,可以获得纯度较高的蛋白,但不适合大量制备。通过设计引物去掉了pET-24a载体上的6×His tag将LPTS-L基因进行了非融合表达,然后采用磷酸纤维素P11阳离子交换层析纯化LPTS-L蛋白,纯度可达到55%。再经Sephadex G-100凝胶过滤,LPTS-L蛋白的纯度可达到80%。Western blot实验显示经纯化后的LPTS-L蛋白可与兔抗GST-LPTS-L的多抗发生特异性结合。采用TRAP法测定蛋白质活性,结果显示纯化得到的LPTS-L蛋白可抑制端粒酶的活性,与采用Ni Sepharose4B纯化获得的LPTS-L融合蛋白比较,其抑制效率基本一致。因此,所建立的技术可以有效地制备LPTS-L蛋白。     

An Anti-plasmin Inhibitor,Eckol, Isolated from the Brown Alga Ecklonia kurome Okamura

The biological activities of eckol, a novel phlorotannin with a dibenzo-β-dioxine skeleton, were examined. Eckol inhibited the antiplasmin activity of a₂-plasmin inhibitor very efficiently (IC₅₀; 1.6 μg/ml) as well as those of α₂-macroglobulin and -antitrypsin. However, its inhibitory effect on the antithrombin III-heparin complex was very weak. Eckol also showed inhibitory activity on thrombin (IC₅₀; 12 μg/ml), but not on plasmin. Its inhibitory activity was reduced in whole human plasma, but at concentrations of above 200 μg/ml it enhanced urokinase-induced fibrinolysis in human plasma. Studies on the inhibitory spectra of several derivatives of eckol showed that the dibenzo-l,4-dioxane skeleton was necessary for inhibition of plasmin inhibitor. These observations suggest that eckol or its derivatives may be useful clinically for potentiating thrombolytic activity.     

Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA.     

Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity.

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.