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1.
The super-toxic super-complexes (SSC) of botulinic neurotoxins, types A and B, have been isolated. The preparation of type A SSC (SSC/A) consist of the proteolyzed form of type A botulinic neurotoxin (BoNT/A), 50 and 90 kD, nontoxic nonhemagglutinating protein of 140 kD (NTNH140) in the nonproteolyzed form, hemagglutinin of 17 kD (Ha17), hemagglutinin of 34 kD (Ha34) and the proteolyzed form of hemagglutinin of 70 kD (Ha70) (20 and 50 kD). The preparations of type B SSC (SSC/B) consist of the nonproteolyzed form of type B botulinic neurotoxin (BoNT/B) of 150 kD, the proteolyzed form of BoNT/B of 150 kD (45 and 105 kD), the nonproteolyzed form of NTNH140, Ha17, Ha34 and two nonidentified proteins (32 and 40 kD). As shown in this study, toxic complexes both in native toxins and in the preparations of SSC do not dissociate for several weeks at pH 8.0 and for 18 hours in 3% SDS, as well as after treatment with RNAase or 1 M NaCl. Some part of SSC/A (neurotoxin and Ha70) has been found to dissociate in 3% SDS after 1-hour incubation at 22 degrees C after the addition of 2-ME. The preparations of SSC contain nucleic acids (A260 nm/A280 nm = 2.0), supposedly ensuring the stability of the complexes. In contrast to the L-forms of Clostridium botulinum toxins, the preparations of SSC/A and SSC/B have been found to possess increased toxicity. The specific toxicity of SSC/A has proved to be 1-2 x 10(9) DLM per 1 OD280 nm and that of SSC/B, from 5 x 10(8) to 1 x 10(9) DLM per OD280 nm. One minimal lethal oral dose of these SSC preparations for mice was less than 10 DLM, introduces intraperitoneally.  相似文献   

2.
通过DNA体外重组技术,以pET-3b为表达载体,构建了重组表达质粒pET-6R(B)和PET-6R(B)4,分别编码28kD的hIL-6R配基结合区片段及其53kD的二联体蛋白,并为酶切分析和DNA序列分析所证实。SDS-PAGE分析表明,含有重组表达质粒的菌株可分别表达出28kD的蛋白rIL6R-28和53kD的rIL6R-53。重组蛋白分别占菌体总蛋白的45%和29%左右。重组蛋白主要以包涵体形式存在,Western印迹表明重组蛋白具有IL-6R的抗原性。  相似文献   

3.
SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定   总被引:1,自引:0,他引:1  
ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。  相似文献   

4.
杨征  朱英力 《遗传学报》1999,26(5):518-523
对水稻配子体细胞质雄性不育系粤泰A,保持系粤泰B,F1代泰优2号和孢子体细胞质雄性不育系珍汕97A,保持系珍汕97B,F1代汕优63及另一种孢子体细胞质雄性不育系马协A,保持系马协B,F1代马协63的黄化苗线粒体离体翻译产物进行SDS-PAGE分析。结果表明:粤泰A的线粒体比粤泰B,泰优2号少合成2条多肽,B与F1的带型相近,A特异合成40.7kD多肽;  相似文献   

5.
t-Zeatin (t-Z) and isopentenyladenosine (iPA) occur naturally as highly active plant cell division regulators, t-Z-Sepherose-4B and iPA-Sepherose-4B affinity column were constructed to isolate and purify the cytokinin-binding proteins from etiolated hypocotyl of Phaseolus vulgaris. Two kinds of cytokinin-binding proteins were obtained. One was 15.5 kD in molecular weight (named ZBP) with only one peptide. The other (named IBP), 165 kD in molecular weight, contained two different subunits (40 kD and 43 kD respectively). The binding activity of ZBP was tested and the dissociation constant (Kd) was determined to be 3.2 × 10-7 mol/L. There was one binding site for t-Z in each molecule of ZBP.  相似文献   

6.
Characterization of lamin proteins in BHK cells   总被引:5,自引:0,他引:5  
Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.  相似文献   

7.
Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.  相似文献   

8.
Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two-dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ~6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (~130 kD, pI ~5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α- and β-tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (~50 kD, pI ~5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (~45 kD, pI ~4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (~60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.  相似文献   

9.
Transforming growth factor beta 1 (TGF-beta 1) is a potent autocrine growth inhibitor of lymphocytes. In this study, the expression of TGF-beta 1 binding proteins was characterized on murine splenic T cells. With an affinity cross-linking method and by neutralizing antibodies to TGF-beta 1, [125I] TGF-beta 1 was found to bind to three cell surface-binding proteins (280-200 kD, 95-85 kD, 65 kD) that were differentially expressed on resting and mitogen-stimulated T cells. Freshly prepared (resting) T cells were found to constitutively express the 95-85-kD form of these binding proteins, whereas mitogenic stimulation by either concanavalin-A (Con-A), interleukin-1 (IL-1), interleukin-2 (IL-2), or 12-tetradecanoyl-phorbol-13-acetate (TPA) for 12-72 h induced the appearance of all forms of the TGF-beta 1 binding proteins (280-200 kD, 95-85 kD, and 65 kD). Furthermore, antibodies that neutralized the biologic action of TGF-beta 1 also blocked the binding of [125I] TGF-beta 1 to all three binding proteins, suggesting that these binding proteins are involved with signal transduction. These results suggest that the expression of the TGF-beta 1 receptor on T cells is regulated by T cell mitogenic signals and that a regulatory relationship may exist between T cell growth-promoting cytokines (IL-1 and IL-2) and the T cell growth inhibitor, TGF-beta 1.  相似文献   

10.
The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain which terminates with cytochrome oxidase, an alternative pathway that terminates with an alternative oxidase. The alternative oxidase of Sauromatum guttatum Schott has recently been identified as a cluster of proteins with apparent Mr of 37, 36, and 35 kilodaltons (kD). Monoclonal antibodies have now been prepared to these proteins and designated as AOA (binding all three proteins of the alternative oxidase cluster), AOU (binding the upper or 37 kD protein), and AOL (binding the lower or 36 and 35 kD proteins). All three antibodies bind to their respective alternative oxidase proteins whether the proteins are in their native or denatured states (as on protein blots). AOA and AOU inhibit alternative oxidase activity around 49%, whereas AOL inhibits activity only 14%. When coupled individually to Sepharose 4B, all three monoclonal resins were capable of retaining the entire cluster of alternative oxidase proteins, suggesting that these proteins are physically associated in some manner. The monoclonals were capable of binding similar mitochondrial proteins in a number of thermogenic and nonthermogenic species, indicating that they will be useful in characterizing and purifying the alternative oxidase of different systems. The ability of the monoclonal-Sepharose 4B resins to retain the cluster of previously identified alternative oxidase proteins, along with the inhibition of alternative oxidase activity by these monoclonals, supports the role of these proteins in constituting the alternative oxidase.  相似文献   

11.
乌龙岭’龙眼胚胎发育时期特异性蛋白质的变化   总被引:11,自引:0,他引:11  
应用IEF-SDS-PAGE技术分析龙眼胚胎分化发育过程中蛋白质组分的变化。结果表明,在各发育阶段大多数蛋白质组分的电泳图谱基本一致,但也有变化。其中花后38d存在TE1(27.1kD、p,7.3),TE2(17.5kD、pI8.2)2个特异蛋白,45d存在TE3(11.4kD、pI7.6),TE4(13.2kD、pI9.9)2个特异蛋白,52d存在TE5(22.6kD、pI7.2),TE6(18.6kD、pI8.3),TE,(23.5kD、pI3.6)3个特异蛋白。31d胚胎电泳图谱中的蛋白质点数相对较多,表明此时蛋白质旺盛合成与积累,这与蛋白含量的变化基本一致。龙眼胚胎发育过程中特异蛋白的出现或消失.对胚胎的分化发育具有重要作用。  相似文献   

12.
菜心和水稻绿叶中不同等电点的乙醇酸氧化酶   总被引:3,自引:0,他引:3  
The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%~20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.  相似文献   

13.
M V Semenov 《Tsitologiia》1990,32(5):489-493
Sera of rheumatology patients with antikinetochore autoantibodies are identified. Immunoblot of human cell proteins showed that all the sera contained autoantibodies to kinetochore proteins A and B. In Chinese hamster cells proteins with m. m. 45 kD, 100 kD and 110 kD are presumably kinetochore proteins, whereas in murine cells these are proteins with m. m. 45 kD 85 kD and 100 kD.  相似文献   

14.
The conventional protein chromatography technique was adopted to purify the antifreeze proteins (AFPs) from the leaves of Ammopiptanthus mongolicus ( Maxim. ) Cheng f. Two bands on native PAGE gel showed thermal hysteresis activity, one was band Bi, whose thermal hysteresis was 0.46 cE at 8 g/L, which showed two bands (67 kD, 21 kD) on SDS-PAGE gel; the other was B3, whose thermal hysteresis was 0.45 cE at 10 g/L, and it contained only a single protein (39.8 kD). Both B1 and B3 are not glycoproteins, because neither do they interact with Shift-reagent, nor show ultraviolet characteristics of a typical glycoprotein.  相似文献   

15.
Regulation of the steady-state tyrosine phosphorylation of the insulin receptor and its postreceptor substrates are essential determinants of insulin signal transduction. However, little is known regarding the molecular interactions that influence the balance of these processes, especially the phosphorylation state of postinsulin receptor substrates, such as insulin receptor substrate-1 (IRS-1). The specific activity of four candidate protein-tyrosine phosphatases (protein-tyrosine phosphatase 1B (PTP1B), SH2 domain-containing PTPase-2 (SHP-2), leukocyte common antigen-related (LAR), and leukocyte antigen-related phosphatase) (LRP) toward IRS-1 dephosphorylation was studied using recombinant proteins in vitro. PTP1B exhibited the highest specific activity (percentage dephosphorylated per microg per min), and the enzyme activities varied over a range of 5.5 x 10(3). When evaluated as a ratio of activity versus IRS-1 to that versus p-nitrophenyl phosphate, PTP1B remained significantly more active by 3.1-293-fold, respectively. Overlay blots with recombinant Src homology 2 domains of IRS-1 adaptor proteins showed that the loss of IRS-1 binding of Crk, GRB2, SHP-2, and the p85 subunit of phosphatidylinositol 3'-kinase paralleled the rate of overall IRS-1 dephosphorylation. Further studies revealed that the adaptor protein GRB2 strongly promoted the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and catalytically inactive PTP1B, increasing their co-immunoprecipitation from an equimolar solution by 13.5 +/- 3.3-fold (n = 7; p < 0.01). Inclusion of GRB2 in a reaction mixture of IRS-1 and active PTP1B also increased the overall rate of IRS-1 tyrosine dephosphorylation by 2.7-3.9-fold (p < 0.01). These results provide new insight into novel molecular interactions involving PTP1B and GRB2 that may influence the steady-state capacity of IRS-1 to function as a phosphotyrosine scaffold and possibly affect the balance of postreceptor insulin signaling.  相似文献   

16.
Many of the cytosolic proteins of the rat brain appear to have the capacity to incorporate L-[14C]arginine posttranslationally. Scanning of the electrophoretic pattern of the labeled proteins showed two main radioactive peaks: peak A, found in the region of proteins of MW above 200 kD, and peak B, found in the region of 33 kD. The ratio of peaks A/B tends to decrease with the age of the rats. Another zone of radioactivity has an apparent MW similar to that of albumin (approximately 66 kD). No differences were found between the effects of ionic strength and of inhibitors on the arginyl transferase of brain and those described for the transferases of other organs.  相似文献   

17.
为了探讨腺病毒 (adenovirus,Ad)E1B 5 5kD癌蛋白 (AdE1B 5 5kD)打破hDaxx和PML共定位细胞核的作用机制 ,本文利用体内外共免疫沉淀反应研究AdE1B 5 5kD与hDaxx的结合反应 ,并通过酵母双杂交体系测定两种蛋白质的相互作用及其作用的氨基酸残基序列。结果显示 :Ad2E1B 5 5kD通过C端 5 8个氨基酸 (aa)与hDaxx结合并发生相互作用。Ad12E1B 5 5kD与hDaxx结合需全序列aa及其构象。共免疫沉淀反应和Westernblot结果证实Ad2 / 5或Ad12E1B 5 5kD能在体内外与hDaxx直接结合  相似文献   

18.
The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.  相似文献   

19.
The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of ?160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold. Thus, one signaling pathway from the insulin and IGF-I receptors probably proceeds as follows: tyrosine phosphorylation of IRS-1, tight association of IRS-1 with PI 3-kinase with accompanying activation of the kinase, elevation of the PI 3-phosphates. © 1993 Wiley-Liss, Inc.  相似文献   

20.
荔枝胚败育过程中内源激素与蛋白质含量的变化   总被引:4,自引:0,他引:4  
连续3年(1999-2001年)对典型的荔枝焦核品种桂味、糯米糍和大核品种黑叶、怀枝花后10-40d的幼胚和胚乳内源激素、多酚含量及蛋白质动态变化进行研究。结果表明,焦核品种幼胚及胚乳中的IAA、GAs和ABA含量低于大核品种;多酚类物质含量在胚中低于大核品种,胚乳中则高于大核品种;胚和胚乳中的蛋白质含量均低于大核品种。蛋白质电泳结果显示,22.5、28.5和45kD这3类蛋白质在怀枝和黑叶的胚蛋白质代谢过程中表现出较高的稳定性,桂味和糯米糍胚蛋白质中的28.5kD蛋白质也有相似的特性。  相似文献   

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