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Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第43期275卷 33464-33470页

作者： Silva, PI Daffre, S Bulet, P Univ Sao Paulo Inst Ciencias Biomed Dept Parasitol BR-05508900 Sao Paulo Brazil Inst Butantan Lab Artropodes BR-05503900 Sao Paulo Brazil Inst Biol Mol & Cellulaire CNRS UPR Reponse Immunitaire & Dev Insectes 9022 F-67084 Strasbourg France

We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana, Gomesin has a molecular mass of 2270.4 Ha, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine Lu-amide, and four cysteine residues forming two disulfide bridges, This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions, Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes, Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.

关键词：氨基酸序列抗菌药抗感染药/化学抗感染药/分离和提纯抗感染药/药理学抗微生物阳离子肽类细菌/药物作用血蛋白质类/化学血蛋白质类/分离和提纯血蛋白质类/药理学 DNA结合蛋白质类/化学真菌/药物作用血细胞(无脊椎动物)/化学分子序列数据肽类/化学肽类/分离和提纯肽类/药理学肽类环/化学蜘蛛/化学动物

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Homologous human blood protein separation using immobilized metal affinity chromatography: Protein C separation from prothrombin with application to the separation of factor IX and prothrombin

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BIOTECHNOLOGY PROGRESS 1999年第5期15卷 928-931页

作者： Wu, HP Bruley, DF Univ Maryland Baltimore Cty Dept Chem & Biochem Engn Baltimore MD 21250 USA

Protein C (PC) is a natural anticoagulant and antithrombotic present in human blood at a concentration of 4 mu g/mL. Its deficiency can result in excessive clotting and thrombosis. Protein C can be obtained from human blood plasma;however, there are other coagulant proteins in blood, including prothrombin (factor II), which is present in relatively large amounts and is one of the most active components. Protein C and prothrombin are homologous proteins with similar biochemical features;therefore, immunoaffinity chromatography is used for their separation. However, this technology is very expensive, protein C recovery and activity is low, and contamination problems with mouse antibody are likely. Immobilized metal affinity chromatography (IMAC) utilizes the protein metal-binding properties for protein separation. Protein C has twelve surface-accessible histidines, which are the major metal-binding groups for IMAC separation. After investigating metal ion-binding properties of protein C, we used an IDA-Cu column to separate protein C and prothrombin. Following protein adsorption to the column, prothrombin was washed out using a sodium phosphate buffer containing 2 mM imidazole and protein C was recovered with 15 mM imidazole in the buffer. The mild elution condition allows a high protein C activity and a high recovery. Also, this technology introduces no immunoglobulins, and it is relatively inexpensive. IMAC could replace the immunoaffinity technology for the large-scale separation of protein C from blood plasma Cohn Fraction IV-1. In addition, this work demonstrates a significant application of this technology for the separation of factor IX from prothrombin. Prothrombin has proven to be a harmful contaminant in factor TX cocktails that have been administered to humans in the treatment of hemophilia B.

关键词：血蛋白质类/分离和提纯色谱法亲和/方法铜因子Ⅸ/分离和提纯蛋白质C/分离和提纯凝血酶原/分离和提纯人类

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PROTEINS OF CAMEL ERYTHROCYTE-MEMBRANE

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BIOCHIMICA ET BIOPHYSICA ACTA 1975年第1期401卷 83-94页

作者： RALSTON, GB UNIV SYDNEY DEPT BIOCHEMSYDNEY 2006NEW S WALESAUSTRALIA

Electrophoresis on polyacrylamide gels containing dodecyl sulphate has revealed that the major proteins of the camel erythrocyte membrane are similar to those of the human and bovine species in both electrophoretic mobility and relative abundance. The major difference lies in the major intrinsic membrane protein of molecular weight approx. 100 000. In the camel, this protein has a higher apparent molecular weight than in the human and bovine species. The very high molecular weight water-soluble protein “spectrin” appears to be very tightly bound to the camel erythrocyte membrane, and is only partially extracted after prolonged low ionic strength dialysis. Total release of spectrin is only achieved by means of more drastic treatment such as incubation with urea, guanidine hydrochloride, sodium hydroxide or p -chloromercuribenzoate. Concurrent with the total release of spectrin, the camel cells undergo a shape change from flat ellipsoids to spheres, suggesting an important shape-maintaining role for spectrin in the erythrocytes of this species.

关键词：血蛋白质类/分析血蛋白质类/分离和提纯血蛋白质类/生理学细胞膜/分析细胞膜/药物作用氯汞苯甲酸盐类/药理学色谱法凝胶透析电泳聚丙烯酰氨凝胶红细胞/分析红细胞/细胞学红细胞/超微结构糖蛋白类/分析胍类/药理学显微镜检查相差分子量聚乙烯二醇类/药理学蛋白质类/分析氢氧化钠/药理学溶解度尿素/药理学动物牛女(雌)性人类

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A SPECIFIC STAIN FOR THE DETECTION OF NONHEME IRON PROTEINS IN POLYACRYLAMIDE GELS

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ANALYTICAL BIOCHEMISTRY 1992年第2期207卷 317-320页

作者： LEONG, LM TAN, BH HO, KK NATL UNIV SINGAPORE FAC SCIDEPT BOTSINGAPORE 0511SINGAPORE

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.

关键词：血蛋白质类/分析血蛋白质类/分离和提纯载体蛋白质类/分析载体蛋白质类/分离和提纯生色化合物电泳聚丙烯酰氨凝胶/方法铁氧化还原蛋白类/分析铁氧化还原蛋白类/分离和提纯铁氰化物铁蛋白质类/分析铁蛋白质类/分离和提纯胎血/化学马铁/分析铁结合蛋白质类等电聚焦/方法微量化学/方法肌红蛋白/分析肌红蛋白/分离和提纯品红类染料血清白蛋白牛/分析血清白蛋白牛/分离和提纯染色与标记转铁蛋白/分析转铁蛋白/分离和提纯转铁结合蛋白质类三嗪类动物牛人类

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ISOLEUCINE METABOLISM BY LEUKEMIC AND NORMAL HUMAN LEUKOCYTES IN RELATION TO CELL MATURITY AND TYPE

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BLOOD 1975年第5期45卷 643-651页

作者： BURNS, CP UNIV IOWA COLL MEDDEPT MEDDIV HEMATOL ONCOLIOWA CITYIA 52242

The metabolism of an essential amino acid, isoleucine, by human leukemic and gradient-separated normal human leukocytes of various types and maturity was studied. Blood leukocytes were isolated and incubated with [U-14 C] isoleucine. Separation of metabolic intermediates was accomplished by sequential extraction. The rate of isoleucine incorporation into protein by immature cells from untreated patients with acute leukemia (15.9 ± 2.4 nmoles/hr per 108 leukocytes) was considerably higher than the rates of incorporation by mature neutrophils (3.2 ± 0.5 nmoles/hr per 108 leukocytes), lymphocytes (7.7 ± 1.2 nmoles/hr per 108 leukocytes), and eosinophils (6.2 ± 1.3 nmoles/hr per 108 leukocytes). Those cell preparations with more blast cells had higher rates of protein synthesis. In addition, those cells with greater thymidine incorporation had higher rates of protein synthesis. The leukocytes both oxidized isoleucine and incorporated it into cell lipid. The rates of these metabolic processes were characteristic for various types and maturity of leukocytes. This study demonstrates a relationship of rate of protein synthesis to leukocyte immaturity. This relationship is maintained in neoplastic leukocytes. It suggests that the requirement of the mitotic process for newly synthesized protein is greater than that for the elaboration of the protein products of the mature leukocyte.

关键词：血蛋白质类/分离和提纯碳放射性同位素细胞分裂 DNA/生物合成嗜酸细胞/代谢异亮氨酸/代谢白血病/代谢白细胞/代谢脂类/分离和提纯淋巴细胞/代谢氚尿苷/代谢人类

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Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure

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ANNALS OF CLINICAL BIOCHEMISTRY 2001年第6期38卷 701-707页

作者： Umeda, M Otsuka, Y Ii, T Matsuura, T Shibati, H Ota, H Sakurabayashi, I Nissui Pharmaceut Co Ltd Div Res Yuki Ibaraki 307 Japan Jichi Med Sch Omiya Med Ctr Dept Clin Labs Omiya Saitama 330 Japan

We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO4) and potassium carbonate (K2CO3), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO4). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO4. After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9.0) containing NAD(+) and D-sorbitol dehydrogenase. After incubation for 30 min at 37 degreesC, the NADH produced was measured fluorimetrically. The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content. The method had an analytical range of 1-180 mu mol/L. The intra- and inter-assay precisions were < 3.3% and < 5.8%, respectively. The detection limit was 0.65 mu mol/ L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO4 was superior to the conventional method using HClO4 and K2CO3.

关键词：血液化学分析/方法血液化学分析/统计学和数值数据血蛋白质类/分离和提纯红细胞/化学氢离子浓度 L-艾杜糖醇-2-脱氢酶 NAD 敏感性与特异性山梨醇/血液光谱法荧光/方法光谱法荧光/统计学和数值数据人类

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Multiple human serum components act as bridging molecules in rosette formation by Plasmodium falciparum-infected erythrocytes

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BLOOD 2000年第2期95卷 674-682页

作者： Somner, EA Black, J Pasvol, G Northwick Pk Hosp Imperial Coll Sch Med Lister Unit Dept Infect & Trop Med Harrow HA1 3UJ Middx England

Resetting, the binding of parasitized erythrocytes to 2 or more uninfected erythrocytes, is an in vitro correlate of disease severity in Plasmodium falciparum malaria. Although cell ligands and receptors have been identified and a role for immunoglobulin M has been suggested, the molecular mechanisms of rosette formation are unknown. The authors demonstrate unequivocally that rosette formation by P falciparum-infected erythrocytes is specifically dependent on human serum, and they propose that serum components act as bridging molecules between the cell populations. Using heparin treatment and Percoll density gradient centrifugation, they have developed an assay in which parasitized erythrocytes grown in serum-containing medium and optimally forming rosettes are stripped of serum components, These infected cells were no longer able to form rosettes when mixed with erythrocytes and incubated in serum-free medium. Rosette formation was re stored by the addition of serum or certain serum fractions obtained by concanavalin A (conA) affinity, anti-IgM affinity, anion exchange, and gel filtration chromatography. The authors clearly demonstrate that multiple serum components-IgM and at least 2 others-are involved in rosette formation. Those others consist of 1 or more acidic components of high-molecular mass that binds to conA (but that is not thrombospondin, fibronectin, or von Willebrand's factor) and of at least 1 more basic, smaller component that does not bind to conA, Data on the size and number of rosettes formed support the authors' hypothesis that multiple bridges are involved in this complex cellular interaction. These findings have important implications for the understanding of pathogenic adhesive interactions of P falciparum and host susceptibility to severe malaria.

关键词：血液血蛋白质类/免疫学血蛋白质类/分离和提纯细胞黏附色谱法亲和色谱法凝胶色谱法离子交换伴刀豆球蛋白A 培养基红细胞/免疫学红细胞/寄生虫学免疫球蛋白M 疟原虫恶性/免疫学疟原虫恶性/生理学玫瑰花结形成动物人类

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RED-CELL PROTEINS .1. 2-DIMENSIONAL MAPPING OF HUMAN-ERYTHROCYTE LYSATE PROTEINS

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BLOOD 1979年第6期53卷 1121-1132页

作者： EDWARDS, JJ ANDERSON, NG NANCE, SL ANDERSON, NL ARGONNE NATL LAB DIV BIOL & MED RES PROGRAM MOLEC & ANAT ARGONNE IL 60439 USA

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by 2-dimensional electrophoresis using isoelectric focusing in the 1st dimension and electrophoresis in the presence of sodium dodecyl sulfate (SDS) in the 2nd. The overwhelming excess of Hb made such analyses difficult in the past. With the ISO-DALT 2-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of Hb. When Hb and several other major proteins were removed by adsorption to DEAE-cellulose, additional minor components were seen, giving a total of over 275. With the use of purified preparations, the map positions of 5 red cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase and carbonic anhydrase. These mapping techniques complement and extent those traditionally used to find human red cell protein variants.

关键词：肌动蛋白类血蛋白电泳血蛋白质类/分离和提纯碳酸酐酶类过氧化氢酶色谱法 DEAE纤维素电泳聚丙烯酰氨凝胶红细胞/分析红细胞/酶学葡糖磷酸脱氢酶血红蛋白类次黄嘌呤磷酸核糖转移酶等电聚焦丙酮酸激酶人类

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Inclusion of S-sepharose beads in the culture medium significantly improves recovery of secreted rBPI₂₁ from transfected CHO-K1 cells

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PROTEIN EXPRESSION AND PURIFICATION 2000年第1期18卷 77-85页

作者： Horwitz, AH Carroll, SF Williams, RE Liu, PS XOMA US LLC Santa Monica CA 90404 USA XOMA US LLC Berkeley CA 94710 USA

rBPI(23) a recombinant N-terminal fragment of human bactericidal/permeability-increasing protein (BPI), kills gram-negative bacteria and binds endotoxin, rBPI(21), a variant, in which cysteine 132 is changed to alanine, retains the activities of rBPI(23). Initial attempts using conventional ion-exchange chromatography to purify rBPI(23) from culture supernatants of transfected CHO-K1 cells resulted in lower than expected yields. Also, ELISA of supernatants from CHO-K1 transfectants expressing rBPI(23) or rBPI(21) yielded variable signals. Results from pulse-chase experiments using [S-35]methionine had indicated that rBPI(23) could not be detected in the culture medium by 7 h of chase, suggesting that these proteins were degraded and/or bound to cells, media components, or vessel surfaces. To address these issues, we developed a novel process whereby sterile S-Sepharose beads were added directly to the cell culture medium, For attached cells, the beads were added to confluent cultures with serum-free medium for the expression phase, while for suspension-adapted cells, beads were added at the beginning of culture growth. The S-Sepharose was then separated from cells and media and washed, and BPI was eluted with high-salt buffer. This approach yielded up to a 50-fold improvement in recovery of rBPI(23) and rBPI(21) from roller bottles, shake flasks, and 2-liter fermenters. It also resulted in improved detection and quantitation of secreted rBPI(23) and rBPI(21) by ELISA. Results of competition binding studies with iodinated rBPI(21) in conjunction with unlabeled rBPI(21) and rBPI(23) or with heparin demonstrated that these proteins bound specifically and with high affinity to heparan-containing sites on the surface of the CHO-K1 cells. We conclude that the S-Sepharose included in the culture medium captures the BPI protein products as they are secreted and protects them from degradation and/or irreversible binding to cell surfaces. This method has been scaled up to a m

关键词：抗微生物阳离子肽类结合竞争性血蛋白质类/生物合成血蛋白质类/遗传学血蛋白质类/分离和提纯 CHO细胞仓鼠亚科培养基发酵离子交换树脂类膜蛋白质类肽碎片/生物合成肽碎片/遗传学肽碎片/分离和提纯重组蛋白质类/生物合成重组蛋白质类/遗传学重组蛋白质类/分离和提纯琼脂糖转染动物人类

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GEL CHROMATOGRAPHY ON SEPHADEX GELS WITH NARROW PARTICLE-SIZE DISTRIBUTION OBTAINED BY DRY ELUTRIATION

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ANALYTICAL BIOCHEMISTRY 1976年第2期70卷 628-631页

作者： EKMAN, R JOHANSSON, BG RAVNSKOV, U UNIV HOSP LUND DEPT CLIN CHEM S-22185 LUND SWEDEN UNIV HOSP LUND NEPHROL DEPT S-22185 LUND SWEDEN

A simple and inexpensive method for increasing the resolution in Sephadex gel chromatography is described. Apart from the gel fraction obtained with the lowest air flow, consisting of particles with diameters of around 10 .mu.m, gel columns with satisfactory flow properties were easily prepared from all batches. Elutriated gel fractions were used for the separation of human plasma and urinary proteins on Sephadex G-75-G-200 but should also prove valuable in the separation of low molecular components with Sephadex gels.

关键词：血蛋白质类/分离和提纯色谱法凝胶/方法右旋糖酐类人类

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