共查询到20条相似文献,搜索用时 62 毫秒
1.
A new protein component of the Z-line of striated muscles was isolated from chicken breast muscle. This protein has been designated as eu-actinin because of its close similarity in polypeptide molecular weight to actin. Eu-actinin was extracted from myosin-removed myofibrils at low ionic strength at pH 6.5 and purified by column chromatography on Sepharose 4B and DEAE-cellulose. Although the polypeptide molecular weight of eu-actinin measured by SDS-polyacrylamide gel electrophoresis is similar to that of actin, other physico-chemical properties of eu-actinin definitely differ from those of actin. The isoelectric point of eu-actinin was more acidic than that of actin. The amino acid composition of eu-actinin was found to be different from that of actin or those of other muscle structural proteins. The results of analytical gel filtration on Sepharose 4B indicated that eu-actinin forms dimers through non-covalent bonding under aqueous conditions. Eu-actinin has a low axial asymmetry under low-salt conditions, as judged from its intrinsic viscosity ([eta] = 6.4 ml/g for the dimer state) and exhibits a tendency to undergo self-association with increasing ionic strength. Interactions of eu-actinin with other muscle proteins were examined by the affinity column technique. It was shown that eu-actinin binds to actin and alpha-actinin. Eu-actinin exhibited strong seeding ability for the polymerization of actin. Antibody to eu-actinin was raised in a goat and purified by affinity chromatography. The specific antibody against eu-actinin did not form precipitine lines with actin or alpha-actinin. Immunofluorescence studies revealed that eu-actinin is localized at the Z-line of myofibrils. The FITC-conjugated antibody to eu-actinin also stained the Z-lines of rabbit skeletal muscle and chicken cardiac muscle. Therefore, it was concluded that eu-actinin is a new, ubiquitous constituent of Z-lines of striated muscles. 相似文献
2.
Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose. 相似文献
3.
The native dimeric form of enolase from pig muscle was immobilized on Sepharose 4B activated with cyanogen bromide. The amount of matrix-bound enolase, its specific activity and kinetic properties depend on the extent of gel activation with CNBr. Only on the Sepharose activated with small quantities of CNBr the amount of protein which remained after treatment with Gdn.HCl was about 50% of the initially bound enolase, indicating that the enzyme was bound covalently to the matrix through a single subunit. The matrix-bound monomers obtained in this way were inactive and were unable to reassociate to dimers on addition of free subunits. The matrix-bound monomers obtained after KBr treatment were inactive but retained the ability to reassociate into active dimers after addition of free subunits. The results indicate that single matrix-bound subunits of pig muscle enolase are enzymatically inactive and dimeric structure is essential for catalytic activity. 相似文献
4.
N G Gimmel're?kh L G Kravets G N Popova M D Kurski? 《Biokhimii?a (Moscow, Russia)》1978,43(3):481-487
Ca2+-ATPase of skeletal muscle sarcolemma has been isolated and purified. It is prepared from salt extract of sarcolemma by ammonium sulfate fractionation and further purified by gel chromatography on Sepharose 4B. The purity of preparations was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has been shown that Ca2+-ATPase possesses the same mobility as skeletal muscle myosin under gel chromatography on Sepharose 4B and the same mobility as myosin heavy chains in sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Membrane protein binds to rabbit skeletal muscle actin, and this complex dissociates by ATP. Interaction with actin does not change Ca2+- or Mg2+-stimulated ATPase activity. Enzyme has only one pH optimum at 7,0-7,6. Membrane protein is highly specified to calcium--ATPase activity in the presence of Mn2+ is 10% and in the presence of Sr2+, Mg2+ or Co2+ are 3-5% of the activity in the presence of Ca2+. Other nucleoside triphosphate (UTP and ITP) are hydrolyzed at lower rates than is ATP. 相似文献
5.
Purification of larvicidal protein from Bacillus sphaericus 1593 总被引:4,自引:0,他引:4
Coat proteins from the spores of Bacillus sphaericus 1593 were separated by preparative polyacrylamide gel electrophoresis. Neutralising antibodies were raised against a single protein band exhibiting toxicity to mosquito larvae. IgG was purified and coupled to CNBr-activated Sepharose 4B to be used as an immunoaffinity matrix. The larvicidal protein was purified to electrophoretic homogeneity using this immunoaffinity column. The single protein species resolved into four peptides of molecular weights 42.6, 44.1, 50.7 and 51.3 KDa on polyacrylamide gel electrophoresis under denaturing conditions. This protein contained 12% carbohydrates. The purified protein exhibited an LC50 value of 8.3 +/- 1.6 ng/ml when tested against early third instar larvae of the mosquito Culex pipiens var quinquefasciatus. 相似文献
6.
Sachiko Okuno Hitoshi Fujisawa 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,799(3):260-269
Dopamine β-monooxygenase was extensively purified from rat adrenal. The specific activity of the final preparation was approx. 1500 nmol/min per mg protein, which was much higher than the highest yet reported. As judged by gel filtration on Ultrogel AcA22, SDS-polyacrylamide gel electrophoresis, and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 88 000. The isoelectric point of the enzyme was estimated to be pH 6.6 in the presence of 8 M urea. Spleen cells from BALB/c mice immunized with rat dopamine β-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine β-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful to isolate rat dopamine β-monooxygenase from crude preparations. 相似文献
7.
The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption. 相似文献
8.
I. Zemanová J. Turková M. Čapka L.A. Nakhapetyan F. Švec J. Kálal 《Enzyme and microbial technology》1981,3(3):229-232
Quantities of serum albumin, papain, chymotrypsin, trypsin and polyvalent natural trypsin inhibitor antilysin coupled to 3-(2′,3′-epoxypropoxy)propyl-glass, 3-(2′,3′-epoxypropoxy)propyl-silica, epoxyactivated Sepharose 6B, glycidyl methacrylate copolymer and oxirane-acrylic beads (Röhm Pharma) were determined as a function of pH of the reaction mixture. Optimal coupling pH and the amounts of attached individual proteins were considerably affected by both the nature of the coupled protein and the nature of the solid matrix. In some cases the effect of increased ionic strength was studied. Differences in plots of the dependence of the amount of the coupled protein on pH and ionic strength are discussed in respect to the differences of isoelectric points, hydrophobicity and charge distribution of proteins and supports. 相似文献
9.
We have investigated methods of stabilizing prolidase by chemical modification and covalent coupling to various supports, for use in protein hydrolysis and possible use in enzyme replacement therapy. Purified acetone powder of calf brain prolidase was further purified by gel filtration on Sephadex G-200 and chromatography on DEAE-Sephadex A25. Polyacrylamide gel electrophoresis showed that the number of bands was reduced from 11 to 2. Since yields were low, the purified (NH4)2SO4 fraction was used in all experiments. Thiolation of the enzyme reduced the amount of protein coupled to AH-or CH-Sepharose 4B. Activities were highest when the protein was linked through its carboxyl groups. The coupled enzyme showed much greater thermal stability than its free counterpart. Of the bound preparations, the thiolated was less stable than the untreated. Untreated and thiolated enzymes bound to either matrix showed higher activity at low pH and less at high pH than the free material. Thiolation shifted the pH maximum from 6.8 to 7.5. The free thiolated enzyme and that bound to activated SH-Sepharose 4B showed greater thermal stability and a broader pH range of optimal activity than the bound untreated enzyme. These results show that prolidase can be immobilized by coupling to an insoluble matrix through various types of covalent bonds with retention of activity and increased stability. 相似文献
10.
Eguchi R Ishihara A Yamauchi K 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2008,147(3):345-350
The association of suspected endocrine-disrupting chemicals (EDCs), diethylstilbestrol (DES), ioxynil and pentachlorophenol (PCP), with chicken serum proteins was investigated in relation to thyroid system disruption. All of these chemicals strongly inhibited l-[(125)I]thyroxine ([(125)I]T(4)) binding to purified transthyretin (TTR) whereas PCP was less potent inhibitor than DES and ioxynil of [(125)I]T(4) binding to diluted whole chicken serum. This result suggested that PCP interacted with serum proteins other than TTR in whole chicken serum. Following the incubation of chicken serum with each chemical (final concentrations 0.25-1.0 microM), serum proteins were fractionated by gel filtration chromatography (Cellulofine GCL-1000) and affinity chromatography (human retinol-binding protein coupled to Sepharose 4B). Although all chemicals were detected in the gel filtration chromatography 50-100 kDa fractions, DES and ioxynil, but not PCP, were co-eluted with TTR during affinity chromatography. Our results indicated that a significant proportion of DES and ioxynil, but a low proportion of PCP, interacted with TTR in whole chicken serum. 相似文献
11.
Purified boveine growth hormone labeled enzymatically with iodine-125 was covalently coupled to cyanogen bromide activated Sepharose 4B gel and to diazotized zirconia-clad glass beads. Under the conditions employed, an average of 0.8 and 7.3 mg of hormone were bound per ml of Sepharose and glass, respectively. When the conjugates were incubated in Krebs-Ringer bicarbonate buffer (pH 7.4), three separate radioactive species were detected in the incubation supernatant by chromatography on Sephadex G-75. The elution volumes of two of the species were identical with those of 125-I-labeled growth hormone and Na-125I controls, while the third component eluted as a moleucle of intermediate size. The rate of release of each species from the solid matrix was linear with time over 4 days and increased with temperature from 4 to 37 degrees. Although significantly less growth hormone was released from glass (0.14%/day) than from Sepharose (0.40%/day) at 37 degrees, active hormone in amounts sufficient to be detectable in a biological assay was nevertheless liberated from the former after as little as 4 hr of incubation. By contrast, the rate of release of 125-Iminus- and the intermediate-size compound from glass was significantly greater than from Sepharose, suggesting that protein bound to glass supports is more susceptible to degradation from exposure to ionizing radiation. 相似文献
12.
13.
K Moriya K Tanizawa Y Kanaoka 《Biochemical and biophysical research communications》1989,161(1):52-58
A new method for intermolecular cross-linking or bridging of protein has been proposed. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde, alpha-amino acid residue and copper(II). Reliability of the process as a tool for protein cross-linking was evaluated by chromatographic procedures. Behavior of salicylaldehyde in a column packed with Sepharose attached alpha-amino acid residue showed that salicylaldehyde was bound tightly to the gel in the presence of copper(II) ion and was eluted by the addition of EDTA. The association was shown strong enough to be applied for the purpose of cross-linking of proteins. It was also proved that BSA salicylaldehyde conjugate was immobilized specifically to the column, and the process was reversed by the addition of EDTA as well. The method is proposed to be useful not only for immobilization of enzyme but also for cross-linking of proteins since the method is free from unexpected random coupling products which are unavoidable with bifunctional cross-linking reagents. 相似文献
14.
Purification and characterization of rat dopamine beta-monooxygenase and monoclonal antibodies to the enzyme 总被引:1,自引:0,他引:1
Dopamine beta-monooxygenase was extensively purified from rat adrenal. The specific activity of the final preparation was approx. 1500 nmol/min per mg protein, which was much higher than the highest yet reported. As judged by gel filtration on Ultrogel AcA22, SDS-polyacrylamide gel electrophoresis, and cross-linking studies, the enzyme appeared to be composed of four identical subunits, each possessing a molecular weight of 88 000. The isoelectric point of the enzyme was estimated to be pH 6.6 in the presence of 8 M urea. Spleen cells from BALB/c mice immunized with rat dopamine beta-monooxygenase were fused to P3-X63-Ag8-653 mouse myeloma cells. From 55 hybrid cells, 10 stable clones secreting anti-dopamine beta-monooxygenase antibody were obtained. Antibody from one clone was coupled to CNBr-activated Sepharose 4B and the monoclonal antibody-Sepharose was shown to be very useful to isolate rat dopamine beta-monooxygenase from crude preparations. 相似文献
15.
筛选一种高效重组金黄色葡萄球菌蛋白A(SpA)用于制备抗体纯化亲和介质。首先通过基因操作获得金黄色葡萄球菌蛋白A(SpA)的Z结构域单体、二串体、三串体、四串体和五串体基因,将目的基因分别克隆至pET-22b表达载体并转化至大肠杆菌BL21(DE3)感受态细胞,获得不同串联个数的Z结构域基因工程菌,经诱导表达和Ni2+亲和层析纯化得到Z结构域单体和二-五串体蛋白。纯化后的目的蛋白偶联至琼脂糖凝胶作为亲和层析介质,对人免疫球蛋白G(IgG)进行分离纯化。分析比较Z结构域串联体蛋白产量及其偶联的亲和介质对抗体吸附载量的差异。结果表明,构建的Z结构域单体、二串体、三串体、四串体和五串体基因工程菌能有效表达目的蛋白,制备的凝胶亲和介质可特异性吸附人IgG。增加Z结构域串联数,重组蛋白产量和单位摩尔数多聚体蛋白吸附载量获得提高,其中,重组四串体蛋白产量大(160 mg/10 g湿菌体),对抗体的吸附载量高(34.4 mg人IgG/mL胶),更适合作为配基用于亲和层析介质的制备。 相似文献
16.
Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding properties from cartilage. 总被引:15,自引:0,他引:15
A Tengblad 《Biochimica et biophysica acta》1979,578(2):281-289
Partially degraded hyaluronate was coupled to AH-Sepharose 4B using carbodiimide. Approximately 1 mg of hyaluronate was incorporated per ml of wet gel. The derivatized gel was used to purify components of the hyaluronate-proteoglycan complex of cartilage. Two link-proteins were isolated from a crude cartilage extract by affinity binding to the gel and eluted with 4 M guanidinium chloride. By the same procedure one link-protein and the globular portion of the proteoglycan monomer were isolated from a trypsin-treated cartilage extract and were separated from each other by subsequent gel chromatography on Sepharose 6B and Sephacryl S-200. The affinity technique was also used for the preparation of these proteins labelled with dansyl groups. 相似文献
17.
Characterization of alpha-actinin from Acanthamoeba 总被引:5,自引:0,他引:5
T D Pollard P C Tseng D L Rimm D P Bichell R C Williams J Sinard M Sato 《Cell motility and the cytoskeleton》1986,6(6):649-661
Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions. 相似文献
18.
The tail domain of vinculin (V(t)) is an actin binding module containing two regions that interact with F-actin. Although intact V(t) purified from a bacterial expression system is a globular monomer, each actin binding region dimerizes when expressed individually, suggesting the presence of cryptic self-association sites whose exposure is regulated. We show that actin modulates V(t) self-association by inducing or stabilizing a conformational change in V(t) that allows dimerization. Chemical cross-linking studies implicate one of the actin binding regions in mediating dimerization in the presence of actin. Actin-induced V(t) dimers may play a role in the filament cross-linking activity of this protein. The V(t) dimers induced by actin are biochemically distinct from the V(t) dimers and higher oligomers induced by acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate, suggesting structural differences in V(t) bound to these two ligands that may provide a mechanistic basis for inhibition of F-actin binding by phosphatidylinositol 4,5-bisphosphate. The ability of actin to regulate the dimerization state of an actin binding protein suggests that, rather than serving a passive structural role, actin filaments may directly participate in signal transduction and other cellular events that are known to depend on cytoskeletal integrity. 相似文献
19.
This paper reports the properties of human hemoglobin covalently bound to Sepharose 4B both in 'high-affinity' and 'low-affinity' conformations. The results suggest that the coupling reaction is strongly affected by the conformational changes linked to oxygenation of the protein. The rate and the extent of the reaction are different for the oxy and deoxyderivatives, probably due to the change in reactivity of the amino groups in the liganded and unliganded tetramer. The data on the equilibrium which is established between matrix-bound and soluble subunits, measured by the 'subunit-exchange chromatography', indicate that the system displays a minimal heterogeneity when hemoglobin is coupled to the gel in the deoxy state at intermediate protein concentration and pH 8. Maxtrix-bound hemoglobin is characterized by a higher oxygen affinity and by decreased homotropic and heterotropic interactions with respect to hemoglobin in solution, but the changes depend strongly on the conditions used in the coupling procedure. 相似文献
20.
Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin. 相似文献