共查询到20条相似文献,搜索用时 0 毫秒
1.
Chromatography of Microbial Lipids by Centrifugation Through Microparticulate Gel 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Edgar Ribi Charles Filz Karin Ribi Granville Goode William Brown Makoto Niwa Robert Smith 《Journal of bacteriology》1970,102(1):250-260
An improved apparatus and a procedure are described by which the migration of sample components in column chromatography is accelerated by centrifugal force, thereby making it possible to use beds of densely packed gel prepared with ultrafine silica. This technique was used to resolve components of certain lipid mixtures where other methods have failed, and it has been found generally useful as an adjunct to other methods for the fractionation of lipids. Biologically active phosphoglycolipids from Mycobacterium tuberculosis and a phosphatidylglycerol-like substance from Mycoplasma pneumoniae which formed single spots on thin-layer chromatographic plates were each found to contain a major and several minor components by centrifugal chromatography. The method enabled us to isolate individual components of Wax D from M. tuberculosis rather than a spectrum of components. Minor components were resolved which, although present in insufficient quantity to influence results of chemical analyses, may be responsible for biological activity. The apparatus provides an essentially closed system which reduces highly volatile solvents to minimal evaporation during the chromatographic process. Samples are applied in solution and are not allowed to dry on the columns until after separation has been achieved. Consequently, polar, labile microbial lipids can be resolved without the use of harsh reagents which destroy some of their properties. Single components may be harvested by cutting and removing appropriate segments of the larger chromatograms or by eluting them from the columns. 相似文献
2.
Vu Huu Thanh Kazuyoshi Okubo Kazuo Shibasaki 《Bioscience, biotechnology, and biochemistry》2013,77(7):1501-1503
Two kinds of N-acetylmuramidase, M-1 and M-2 enzymes, that were isolated from the cultural broth of Stm. globisporus 1829, were remarkably different in amino acid composition, immunological properties and modes of lytic action from each other. The M-1 enzyme was composed of 186 amino acid residues of which two moles were of half cystine, while the M-2 enzyme was composed of 99 amino acid residues with no cysteine. The hydrolyzing action of the M-2 enzyme was suppressed by the presence of an O-acetyl group on muramic acid residues in the peptidoglycan moiety, while that of the M-l enzyme was independent of the presence of O-acetyl groups. However, the hydrolyzing activity of both enzymes was enhanced when some muramic acid residues were substituted with stem peptides containing alanine, isoglutamine and lysine. 相似文献
3.
4.
Dolores Bobb 《Preparative biochemistry & biotechnology》2013,43(4):347-354
Abstract Pineapple stem bromelain has been successfully isolated from an acetone powder of a crude plant extract by a single passage through a column of ?-aminocaproyl-D-tryptophan methyl ester coupled to Sepharose 4B. If organic mercury is present during all steps of the procedure, a single band with an apparent molecular weight of 23, 000 daltons is obtained after gel electrophoresis in 1% sodium dodecyl sulfate. Upon reduction with mercaptoethanol, this single species is converted into two chains of about 15, 000 and 8, 500 daltons. 相似文献
5.
E. Ribi R. F. Smith S. M. Strain D. G. Petcoff R. Parker G. Goode 《Preparative biochemistry & biotechnology》2013,43(3):209-220
A technique is described by which Δ9 -tetrahydrocannabinol, the principal psychotomimetic constituent of marihuana and hashish, can be isolated in a chromatographically pure state from synthetic or natural products by pressure-accelerated chromatography through columns of ultrafine silica gel packed by centrifugal force. 相似文献
6.
Kazuhiro Nakanishi Shuichi Yamamoto Ryuichi Matsuno Tadashi Kamikubo 《Bioscience, biotechnology, and biochemistry》2013,77(8):1465-1473
The mechanism of dispersion of solute in gel chromatography using various Sephadex gels was quantitatively studied. In order to simplify the mathematical treatment, non-ad- sorptive low-molecular weight substances such as NaCl and glucose were chosen as samples. A pulse response experiment was carried out in a column. The longitudinal dispersion coefficient and the diffusion coefficient in gel phase were determined separately by applying the moment method to the elution curve. Then, their contribution to the column efficiency characterized by HETP was studied. Particularly, the effect of gel phase diffusion was examined in detail. The gel phase diffusion coefficient was apparently much smaller than the molecular diffusion coefficient. Consequently, it was revealed that gel phase diffusion played a much more important role in gel chromatography than what was expected by other investigators. 相似文献
7.
Takao Takahashi Toshiharu Yagi Toyohiro Oda Irvin E. Liener 《Bioscience, biotechnology, and biochemistry》2013,77(4):865-867
(+)-Biotin was synthesized from D-glucosamine in a sequence of 13 reactions. 相似文献
8.
采用硅胶柱层析法从中药山栀子中分离制备主要药效成分京尼平甙,色谱条件:常规柱(2.5×30cm),固定相:柱层析硅胶;流动相∶乙醇-石油醚(1∶5,1∶3)。样品通过薄层层析以及600~190nm波长扫描定性鉴定后,用高效液相色谱法(HPLC)检测纯度,结果表明纯度为97.6%。 相似文献
9.
张志群 《现代生物医学进展》2008,8(2):356-357
目的:探讨高效凝胶色谱法测定聚乙烯亚胺(PEI)及其衍生物的分子量及其分布.方法:采用Ultrahydrogel 250(300mmx7.8mm)色谱柱;以已知相对分子量的PEI为标样;醋酸-醋酸盐缓冲液(0.2mol/l醋酸-0.1mol/l醋酸钠)为流动相;柱温40℃;流速lml/min;示差折光检测器.结果:测得自制PEI衍生物的重均分子量(Mw)为5093,数均分子量(Mn)为2090、Z均分子量(Mz)为11031,分布宽度(Mw/Mn)为2.44.结论:高效凝胶色谱法操作简单、灵敏度高,适合于快速、简便地测定PEI及其衍生聚合物的分子量及其分布. 相似文献
10.
The reaction of H2O2 with resting metmyoglobin (MetMb), methaemoglobin (MetHb) and cytochrome-c (Cyt-c) was studied in the Soret and visible regions. The differences between the original and the final peak heights of the native haemproteins at 408 nm was found to be directly proportional to the loss of iron from the molecule. The release of iron from haemproteins was studied in a system generating H2O2 continuously at a low rate by an enzymic system, or by addition of large amounts of H2O2. Cytochrome-c, methaemoglobin and metmyoglobin during interaction with H2O2 at a concentration of 200 μM release 40%, 20% and 3%, respectively, of molecular iron after l0min. The inhibition of haem degradation and iron release by enzymatically-generated H2O2 was determined using several hydroxyl radical scavengers, reducing agents and antioxienzymes, such as superoxide dismutase, catalase and caeruloplasmin. 相似文献
11.
Nichollas E. Scott Benjamin L. Parker Angela M. Connolly Jana Paulech Alistair V. G. Edwards Ben Crossett Linda Falconer Daniel Kolarich Steven P. Djordjevic Peter H?jrup Nicolle H. Packer Martin R. Larsen Stuart J. Cordwell 《Molecular & cellular proteomics : MCP》2011,10(2)
Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or β-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.Campylobacter jejuni is a Gram-negative, microaerophilic, spiral-shaped, motile bacterium that is the most common cause of food- and water-borne diarrheal illness worldwide (1). Typical infections are acquired via the consumption of undercooked poultry where C. jejuni is found commensally (2). Symptoms in humans range from mild, non-inflammatory diarrhea to severe abdominal cramps, vomiting, and inflammation (3). Prior infection with C. jejuni is a common antecedent of two chronic immune-mediated disorders: Guillain-Barré syndrome (4) and immunoproliferative small intestine disease (5). A unique molecular trait of C. jejuni is the ability to post-translationally modify proteins by the N-linked addition of a 7-residue glycan (GalNAc-α1,4-GalNAc-α1,4-(Glcβ1,3)- GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-Bac-β1 where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucopyranose)) (6) at the consensus sequon (D/E)XNX(S/T) where X is any amino acid except proline (7).The N-linked C. jejuni heptasaccharide is encoded by the pgl (protein glycosylation) gene cluster (8–10), and the glycan is transferred to proteins by the PglB oligosaccharyltransferase (11) at the periplasmic face of the inner membrane (12). Removal of the N-glycosylation gene cluster (or indeed pglB alone) results in C. jejuni that displays poor adherence to and invasion of epithelial cell lines (13) and reduced colonization of the chicken gastrointestinal tract (14). Although this demonstrates a requirement for glycosylation in virulence, the proteins that mediate this are still unknown, and the overall role of glycan attachment remains to be elucidated. Our current understanding of the structural context of glycosylation in C. jejuni suggests that it does not play a role in steric stabilization by conferring structural rigidity as seen in eukaryotes (15) but occurs preferably on flexible loops and unordered regions of proteins (16–18). To investigate the role of glycosylation in protein function, recent studies have utilized mutagenesis to remove the N-linked sequon from three glycoproteins: Cj1496c (19), Cj0143c (20), and VirB10 (21). Removal of glycosylation from Cj1496c and Cj0143c had little effect on protein function; however, glycan attachment was required for correct localization of VirB10. Although the exact role of the glycan remains largely unknown, it appears to be site-specific with a single site, Asn97, influencing localization of VirB10, whereas a second site, Asn32, is dispensable (21). It is clear that a more comprehensive analysis of the C. jejuni glycoproteome is required. A further complication in the elucidation of N-linked glycosylation is the use of the NCTC 11168 strain, which because of laboratory passage (22, 23) may not be the most appropriate model in which to study the virulence properties of glycan attachment. For example, we have recently shown that a surface-exposed virulence factor, JlpA, is glycosylated at two sites (Asn146 and Asn107) in all sequenced C. jejuni strains except NCTC 11168, which contains only Asn146 (24).Glycoproteomics in C. jejuni is also a major technical challenge. Unlike eukaryotic N-linked glycans, the C. jejuni glycan is resistant to removal by protein N-glycosidase F (24) and chemical liberation via β-elimination (6) possibly because of the structure of the unique linking sugar, bacillosamine (25). Analysis therefore requires complementary methodology to elucidate the sites of glycosylation in the presence of the glycan. Preferential fragmentation of the glycan itself during collision-induced dissociation (CID) generally results in poor recovery of peptide fragment ions, and thus identification of the underlying protein and site of attachment remains problematic. MS3 has been attempted for site identification (6, 26); however, the data are limited by the requirement for sufficient ions for two rounds of tandem MS. We have also shown previously that C. jejuni encodes several hydrophobic integral membrane and outer membrane proteins possessing multiple transmembrane-spanning regions that are not amenable to gel-based approaches (27), particularly those using lectins for glycoprotein purification (28). We hypothesize that N-linked glycosylation is more widespread than previously demonstrated (6, 7, 26) because these studies examined only soluble proteins (6, 26) or used lectin affinity (6, 7), which limits the amount and type of detergents that can be used. Recent work (26) has demonstrated the potential of exploiting the hydrophilic nature of the C. jejuni glycan to enable glycopeptide enrichment.The ability to generate product ions useful for the identification of a glycosylated peptide is governed by three factors: the peptide backbone, the glycan, and the fragmentation approach. Multiple strategies exist to separately exploit the first two of these parameters (29, 30), but it is only recently that selective fragmentation of modified peptides has been available through electron transfer dissociation (ETD)1 and electron capture dissociation (31, 32). ETD/electron capture dissociation enable the selective cleavage of the peptide while maintaining the carbohydrate structure, and this has been demonstrated using eukaryotic glycopeptides (33, 34) and more recently glycopeptides isolated from the pathogen Neisseria gonorrhoeae (35). A more recent fragmentation approach is higher energy collisional (C-trap) dissociation (HCD), which uses higher fragmentation energies than standard CID and enables identification of modifications, such as phosphotyrosine (36), via diagnostic immonium ions and high mass accuracy over the full mass range in MS/MS. HCD has not previously been applied to glycopeptides.We applied several enrichment and MS fragmentation approaches to the characterization of the glycoproteome of C. jejuni HB93-13. Sequence analysis determined that the HB93-13 genome contains 510 N-linked sequons ((D/E)XNX(S/T)) in 382 proteins of which 261 (with 371 potential N-linked sites) are predicted to pass through the inner membrane and are therefore the subset that may be glycosylated. We examined trypsin digests of whole cell and membrane protein preparations using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and graphite enrichment of gel-separated proteins using several mass spectrometric techniques (CID, HCD, and ETD). This is the first study to demonstrate the potential of using the high energy fragmentation of HCD to overcome the signal disruption caused by labile glycan fragmentation and to provide peptide sequencing within a single step. Manual data analysis was also simplified as the GalNAc fragment ion (204.086 Da) provides a signature that can be used to highlight glycopeptides within a complex mixture. We identified 81 glycosylation sites, including 47 not described previously in the literature and a single site that cannot be unambiguously assigned. The majority of these are present on proteins not amenable to traditional gel-based analyses, such as hydrophobic transmembrane proteins. Our work more than doubles the previously known N-linked C. jejuni glycoproteome and provides a clear rationale for other studies where the peptide and glycan need to remain associated. 相似文献
12.
13.
Takashi Kuge Kimie Kobayashi Hiroshi Tanahashi Tatsuo Igushi Shinichi Kitamura 《Bioscience, biotechnology, and biochemistry》2013,77(9):2375-2376
Changes in the crystallinity and polymorph of chitosan, which may affect its functionality, by heating (up to 200°C) its water suspension were studied by X-ray diffraction measurements, using tendon chitosan prepared by N-deacetylation of a crab tendon chitin, and chitosan powders with various degrees of polymerization (DPv = 1,720–12,600) and N-acetylation (zero to 26%). It was found that the presence of hydrated polymorphs or anhydrous crystals in a chitosan sample could be examined easily by measuring the powder diffraction pattern of a sample. Chitosan with a low molecular weight or low degree of N-acetylation was highly crystallized, especially in the anhydrous form that is considered to spoil chitosan’s functionality, by heating. 相似文献
14.
15.
16.
17.
以魔芋葡甘聚糖(KGM)凝胶作为铜金属螯合亲和层析的载体一步亲和纯化猪血SOD,得到电泳均一,比活为8622U/mg,纯化倍数为77.8倍的SOD,其回收率为85.4%.探讨了魔芋葡甘聚糖凝胶作为亲和载体的可能性及前景. 相似文献
18.
目的:利用硝酸银硅胶填料有效分离出血浆混合脂肪酸中的亚油酸。方法:通过改进的FOLCH法提取血浆总脂后,再采用皂化、酸化水解的方法将总脂转化为混合脂肪酸。用ghosh法将硅胶改性为硝酸银硅胶后,以亚油酸为对象,通过静态吸附试验了解硝酸银硅胶对不饱和脂肪酸的吸附特性,采用柱层析的方法分离血浆混合脂肪酸中的亚油酸。结果:血浆与有机溶剂在1∶5时既能有效萃取血浆总脂,用正己烷∶二氯甲烷∶乙醚=89∶10∶1作为洗脱剂,将洗脱液甲酯化后进行GC和GC-MS检测,硝酸银硅胶柱的洗脱液中亚油酸的纯度60.74%,硅胶柱的为23.65%,不饱和脂肪酸得到了较好的纯化。 相似文献
19.
建立了一种亲和层析纯化肌质网Ca2+-ATP酶的方法.用非离子型去污剂C12E8 溶解肌质网,再通过反应红-120琼脂糖亲和层析柱使肌质网Ca2+-ATP酶纯度从粗品中的65%提高到99%,并具有较高ATP水解活性.经SDS-聚丙烯酰胺凝胶电泳检测,为电泳纯. 相似文献
20.