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1.
Lactoferrin (LF), one of the most important and polyfunctional factors of nonspecific cell defense against bacteria, viruses, and cancer diseases, is the main iron-transferring protein of human biological fluids, blood, and milk. Electrophoretically homogeneous preparations of LF from human milk were obtained and studied. Using chromatography on Blue Sepharose, LF preparations were separated into several subfractions differing in their affinity for the sorbent, two of which exhibited nucleoside 5-triphosphate-hydrolyzing activity. Using different methods, including in-gel ATPase activity assay, it was demonstrated that hydrolysis of ATP and other nucleotides is an intrinsic property of LF and that LF is the major ATPase of human milk. It is shown that the ATP-hydrolyzing center is located at the C-terminal domain of the LF molecule and that interaction between ATP and this center increases the oligonucleotide-hydrolyzing activity of the DNA-binding domain of this protein.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 513–520.Original Russian Text Copyright © 2005 by Babina, Semenov, Buneva, Nevinsky. 相似文献
2.
《Archives of biochemistry and biophysics》1986,250(1):19-22
Adenylate kinase from human erythrocytes and skeletal muscle can be purified to homogeneity by a new procedure based on DEAE-Sepharose and Blue Sepharose affinity chromatography and Sephadex G-75 fractionation. For the enzyme purified from erythrocytes the specific activity is 3000 U/mg of protein, and the overall yield is 70%. For the enzyme purified from skeletal muscle the specific activity is 2075 U/mg of protein, and the overall yield is 44%. The sequence of steps takes advantage of the high isoelectric point, the high affinity for Blue Sepharose, and the low molecular weight of the isoenzyme from these two human tissues. 相似文献
3.
Chi-Sun Wang 《Analytical biochemistry》1980,105(1):398-402
Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity. 相似文献
4.
Purification of camel milk lysozyme and its lytic effect on Escherichia coli and Micrococcus lysodeikticus 总被引:1,自引:0,他引:1
A S Duhaiman 《Comparative biochemistry and physiology. B, Comparative biochemistry》1988,91(4):793-796
1. Camel milk lysozyme was purified using heparin-Sepharose 4B, Sephadex G-75 and hydroxyapatite chromatography. By this procedure lysozyme was separated from lactoferrin and a low molecular weight protein. 2. The lytic effect of camel milk lysozyme was assayed using Escherichia coli and Micrococcus lysodeikticus and its activity was compared with that of lysozyme from human milk and egg white. 3. The specific activity of camel milk lysozyme was found to be lower than that of lysozyme from human milk or from egg white. 4. Camel milk lactoferrin did not show a lytic effect on bacteria, while the low molecular weight protein showed lytic activity. 相似文献
5.
Although lactoferrin is known as a basic soluble glycoprotein, the presence of the membrane-bound form of this protein has also been demonstrated in human milk. Membrane-bound lactoferrin was extracted from the human milk fat globule membrane with a detergent mixture of 1% Tween-20, 0.5% C12E8, and 0.5 M KCl in 20 mM Tris-HCl (pH 7.4). Lactoferrin in the detergent-soluble fraction was purified by affinity chromatography with Concanavalin A and by hydrophobic chromatography with phenyl-Superose. The purified protein gave a single band of 80 kDa by SDS-PAGE. Its N-terminal amino acid sequence was consistent with that of human lactoferrin. 相似文献
6.
Human Deoxyribonucleases 总被引:4,自引:0,他引:4
Although mammalian deoxyribonucleases were discovered more than 60 years ago, interest in these enzymes is not weakening. During the last decade, intensive studies of human DNases culminated in discovery of several novel enzymes exhibiting DNase activity. These include an unusual DNase, lactoferrin. For some enzymes, their three-dimensional structure and molecular mechanisms underlying their functioning have been elucidated. In patients with some autoimmune and viral diseases, catalytic antibodies also contribute to alternative pathways of DNA hydrolysis. Some enzymes exhibiting DNase activity play an important role in pathogenesis of various diseases and also in programmed cell death (apoptosis). This review highlights recent achievement in human deoxyribonuclease research. It also considers mechanisms of DNA hydrolysis. The review also summarizes modern data on the biological role of these enzymes in functioning of the human organism, realization of its protective mechanisms, and possible applications of DNases in medicine. 相似文献
7.
Sorrentino S D'Alessandro AM Maras B Di Ciccio L D'Andrea G De Prisco R Bossa F Libonati M Oratore A 《Biochimica et biophysica acta》1999,1430(1):103-110
A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm. 相似文献
8.
Lactoferrin (LF) is a main iron-transfering glycoprotein of human barrier body fluids, blood and milk. LF, a protein of the acute phase, is responsible for nonspecific cells defense against microbial and viral infection and cancer diseases. LF is an important component of the passive immunity of newborns system. LF, an extremely polyfunctional protein, is the object of intensive investigations. In this work electrophoretically homogeneous LF from human milk was prepared. Affinity chromatography of LF on Blue Sepharose separated the protein into several distinct isoforms with different affinities to this resin. Two of this isoforms possess nucleoside-5'-triphosphate-hydrolyzing activity. Using several methods including in-gel ATPase activity assays, we show that ATP (and others NTP) hydrolysis is an intrinsic property of LF, and that LF is the major ATPase of human milk. It was shown that ATP-hydrolyzing site is located in C-lobe of LF. 相似文献
9.
We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin. 相似文献
10.
Comparative studies of the binding of some ligands to human serum albumin non-covalently attached to immobilized Cibacron Blue, or covalently immobilized on Sepharose, by column affinity chromatography.
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A comparative study of the ligand-binding properties of human serum albumin was performed by the technique of affinity chromatography with the protein attached to immobilized Cibacron Blue F3GA (Blue Sepharose), or covalently immobilized on Sepharose. The binding strength of octanoate, decanoate and dodecanoate is much weaker when human serum albumin is attached to immobilized Cibacron Blue, indicating that the binding sites for fatty acids are involved in the attachment of human serum albumin to immobilized Cibacron Blue. The results revealed additional alterations of the ligand binding when human serum albumin was attached to immobilized Cibacron Blue, involving sites outside of the binding domains of fatty acids. Thus the stereoselective binding of L-tryptophan was abolished, and the resolution of the warfarin enantiomers was impaired. However, the binding strength of warfarin and salicylic acid was rather close to the values observed with human serum albumin covalently immobilized on Sepharose. It is suggested that the availability of the binding sites for L-tryptophan, warfarin and salicylic acid is partially blocked by the complex between albumin and the dye without direct participation in the complex-formation. An alternative interpretation involves an allosteric mechanism brought about by complex-formation between serum albumin and the immobilized Cibacron Blue. 相似文献
11.
Recombinant human deoxyribonuclease I (DNase I) is an important clinical agent that is inhaled into the airways where it degrades DNA to lower molecular weight fragments, thus reducing the viscoelasticity of sputum and improving the lung function of cystic fibrosis patients. To investigate DNases with potentially improved properties, we constructed a molecular fusion of human DNase I with the hinge and Fc region of human IgG1 heavy chain, creating a DNase I-Fc fusion protein. Infection of Sf9 insect cells with recombinant baculovirus resulted in the expression and secretion of the DNase I-Fc fusion protein. The fusion protein was purified from the culture medium using protein A affinity chromatography followed by desalting by gel filtration and was characterized by amino-terminal sequence, amino acid composition, and a variety of enzyme-linked immunosorbent assays (ELISA) and activity assays. The purified fusion contains DNase I, as determined by a DNase I ELISA and an actin-binding ELISA, and an intact antibody Fc region, which was quantified by an Fc ELISA, in a 2:1 stoichiometric ratio, respectively. The dimeric DNase I-Fc fusion was functionally active in enzymatic DNA digestion assays, albeit about 10-fold less than monomeric DNase I. Cleavage of the DNase I-Fc fusion by papain resulted in a specific activity comparable to the monomeric enzyme. Salt was inhibitory for wild type monomeric DNase I but actually enhanced the activity of the dimeric DNase I-Fc fusion. The DNase I-Fc fusion protein was also less Ca2+-dependent than DNase I itself. These results are consistent with a higher affinity of the dimeric fusion protein to DNA than monomeric DNase I. The engineered DNase I-Fc fusion protein described herein has properties that may have clinical benefits. 相似文献
12.
1. A simple method for purifying transferrins and lactoferrin is described. 2. The method consists of a preliminary step of dye-ligand chromatography using DEAE Affi-Gel Blue as the gel matrix at pH 7.5. In this chromatographic step, the transferrins and lactoferrin were readily separated from the bulk of the other proteins by start buffer elution. 3. Differences in the chromatographic behaviour of the various serum transferrins (monkey, human, rabbit, pig, chicken and duck) and ovotransferrin upon DEAE Affi-Gel Blue chromatography can be attributed to differences in the anionic charge of the transferrins in 0.02 M potassium phosphate buffer, pH 7.5, thus resulting in the differential retardation of these protein molecules by the gel matrix. 4. The result of DEAE Affi-Gel Blue chromatography of human lactoferrin is different from that for the transferrins. This may possibly reflect the differences in the strength of interaction between lactoferrin and transferrin with this gel matrix. 相似文献
13.
Escherichia coli DNA photolyase was expressed as C-terminal 6x histidine-fused protein. Purification of His-tagged E. coli DNA photolyase was developed using immobilized metal affinity chromatography with Chelating Sepharose Fast Flow. By one-step affinity chromatography, approximate 4.6 mg DNA photolyase was obtained from 400 ml E. coli culture. The purified His-tagged enzyme was combined with two chromophors, FADH and MTHF. Using the oligonucleotide containing cyclobutane pyrimidine dimer as substrate, both reversed-phase high-performance liquid chromatography and size-exclusion high-performance liquid chromatography were developed to measure the enzyme activity. The enzyme was found to be able to repair the cyclobutane pyrimidine dimer with the turnover rate of 2.4 dimers/photolyase molecule/min. 相似文献
14.
A 40-kDa lactoferrin-binding protein was identified in a strain of Prevotella nigrescens isolated from a patient with periodontitis. The protein was purified by affinity column chromatography using a Sepharose–lactoferrin column and detergent-solubilized membranes. The N-terminal sequence revealed no apparent similarities with any other sequenced bacterial protein. The native conformation of the 40-kDa protein was a condition to bind either iron-free or iron-saturated lactoferrin. A possible function of this Lf-binding protein could be related with an iron acquisition mechanism in P. nigrescens. 相似文献
15.
The effect of matrix on the binding of albumin to immobilized Cibacron Blue. 总被引:3,自引:3,他引:0
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With Cibacron Blue as a ligand, the suitability of Sepharose, Sephacryl, cellulose and Ultrogel as column supports for the affinity chromatography of human serum albumin was examined. Sepharose and Sephacryl were the most effective matrices. Albumin was readily removed from such columns by using an electrophoretic desorption technique. 相似文献
16.
Purification of human lysozyme from milk and pancreatic juice 总被引:5,自引:0,他引:5
Human milk lysozyme was purified by heparin-Sepharose affinity chromatography and Sepharose 4B gel-permeation chromatography. This procedure was also found applicable to the purification of human pancreatic juice lysozyme. Double-diffusion analyses indicated that human milk lysozyme was immunochemically identical to human saliva and human pancreatic juice lysozyme. Based on the identity of the N-terminal 10-amino-acid-residue sequence analyzed, it was suggested that human milk lysozyme and human pancreatic juice lysozyme are identical molecular entities. 相似文献
17.
The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time. 相似文献
18.
Two methods for the separation of α-fetoprotein (AFB) and albumin using (A) affinity chromatography on Blue Sepharose CL-6B and (B) ampholyte displacement chromatography are described. The heterogeneity of immunoreactive human AFP is demonstrated by ampholyte displacement chromatography; possibly seven variants of AFP can be distinguished by this method. 相似文献
19.
重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析 总被引:1,自引:0,他引:1
李伟 《中国生物化学与分子生物学报》2006,22(12):979-983
为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框(ORF)插入原核表达载体pGEX6P1 GST基因下游的多克隆位点(MCS).将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性. 相似文献
20.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,694(1):49-53
The simultaneous isolation of three enzymes from the southern copperhead snake venom (Agkistrodon contortrix contortrix; ACC) is described. The first step is a chromatography of crude venom on a Mono S cation-exchange column at pH 6.5. A fibrin clot promoting enzyme (fiprozyme) that preferentially releases fibrinopeptide B from fibrinogen is isolated from the fraction not binding to the Mono S by a further three-step process. The procedure involves affinity chromatography on Blue Sepharose, gel chromatography on Sephacryl S-200 and metal–chelate chromatography on Chelating Sepharose. Protein C activator and phospholipase coelute from the Mono S column. They are separated by a gel chromatography on Sephacryl S-200. After this step two enzymes are obtained: a highly purified protein C activator applicable in methods for determination of functional level of protein C (a plasma regulator of hemostasis) and an electrophoretically pure enzyme with the activity of phospholipase A2. 相似文献