共查询到20条相似文献,搜索用时 15 毫秒
1.
A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography. 相似文献
2.
A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver. 相似文献
3.
A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase. 相似文献
4.
Hjertén M Rezeli M Kilár F Hjertén S 《Journal of biochemical and biophysical methods》2008,70(6):1188-1191
A novel approach is described for the synthesis of beds for enzyme reactors. The method is based on the use of artificial antibodies in the form of polyacrylamide gel particles with diameters around 0.1–0.3 mm. These gel particles mimic protein antibodies, raised in experimental animals, in the sense that they selectively recognize and adsorb only the protein present during the preparation of the “antibodies”. The gel antibodies have several advantages over conventional protein antibodies, which can be taken advantage of in the design of enzyme reactors; for instance, if upon prolonged use the immobilized enzyme loses its activity it can easily be replaced by an active enzyme, which is not possible when the enzyme is immobilized via a conventional protein antibody (a new bed with immobilized protein antibodies must be prepared); and equally or more remarkable: the enzyme can be applied in the form of a non-purified extract since the selectivity of the artificial gel antibodies is so high that they will “fish-out” the enzyme, but no other proteins in the extract. In addition, no preconcentration of the enzyme solution is required prior to the immobilization, since the enzyme is enriched at the top of the column upon the application. These unique properties make enzyme reactors based on artificial gel antibodies very attractive, also in process chromatography. The potential application range of the artificial gel antibodies is enormous since the same method for their synthesis can be used independent of the structure and the size of the “antigen”; for instance, renewable biosensors based on gel antibodies for the selective detection of protein biomarkers, as well as pathogenic viruses, bacteria, and spores (for instance Anthrax) should not be difficult to design. 相似文献
5.
Two molecular forms of adenosine deaminase have been found in human tissues. The column gel filtration method has been used for the separation of the two enzyme forms. Routine separation and analysis of the enzyme forms based on the molecular size difference can be achieved by thin-layer gel filtration on Sephadex G-200 superfine gel. The thin-layer method has been found to be more rapid and efficient than the column method. Enzymes in crude preparations can be studied effectively with the thin-layer method. 相似文献
6.
Gordon S. Rule 《Analytical biochemistry》1984,138(1):99-106
A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given. 相似文献
7.
K Moriya K Tanizawa Y Kanaoka 《Biochemical and biophysical research communications》1989,162(1):408-414
A new method for cross-linking of protein was proposed in our previous work. The method is based on the spontaneous chelate formation process involving three components, salicylaldehyde moiety, alpha-amino acid residue and copper(II). In this paper versatility of the method as a purpose of immobilization of enzyme was described. Chymotrypsin-salicylaldehyde conjugate was immobilized to the agarose gel attached alpha-amino acid residue in the presence of copper(II) ion The enzyme was not eluted from the gel by washing with a copper free buffer though it was exclusively eluted by a medium containing EDTA. Catalytic activity of the chymotrypsin salicylaldehyde conjugate was not changed upon the immobilization. The method was proposed as a new tool for reversible immobilization of enzyme. 相似文献
8.
《Experimental mycology》1987,11(1):36-48
A method was developed to assay glucose-1-phosphate uridylyltransferase and 2-acetamido-2-deoxyglucose-1-phosphate uridylyltransferase by separation and quantitation of the corresponding sugar nucleotides by HPLC. Glucose-1-phosphate uridylyltransferase (GPUT) fromNeurospora crassa was purified by a method involving ion-exchange, gel filtration, adsorption, and affinity chromatographic procedures. The enzyme was stable until the last step of purification, after which it became extremely labile, apparently due to disaggregation. With the purified enzyme, kinetic properties of GPUT were determined. Polyacrylamide gel electrophoresis (PAGE) of the purified enzyme under nondenaturing conditions showed a single band which contained all the enzymatic activity. Denaturation of the enzyme with sodium dodecyl sulfate followed by PAGE resolved the single band into four polypeptides of different molecular masses. The minimal molecular mass of the enzyme was calculated to be 537,000 Da. This value was similar to that calculated by sucrose density sedimentation, 580,000 Da, but different from that estimated by gel filtration, 1,600,000 Da. It is proposed that the native enzyme is a trimer which may be disaggregated. By electron microscopy of negatively stained samples, the enzyme appeared in the form of rosettes 10 nm in diameter. 相似文献
9.
A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues. 相似文献
10.
L. Chaiet A. J. Kempf R. Harman E. Kaczka R. Weston K. Nollstadt F. J. Wolf 《Applied microbiology》1970,20(3):421-426
A dextranase, produced by Penicillium funiculosum, was purified 1,000-fold to yield the enzyme which was demonstrated by gel electrophoresis and electrofocusing to be a homogeneous protein. The purification method included acetone partition, ammonium sulfate fractionation, gel filtration, iron defecation and precipitation, and diethylaminoethyl-cellulose chromatography. The pure enzyme was also obtained by preparative gel electrophoresis. Gel-permeation chromatography indicates a molecular weight of 41,000. An isoelectric pH of 4.6 was established by electrofocusing. A 1-mg amount of the enzyme hydrolyzes a dextran substrate to yield 27,000 isomaltose reducing units in 2 hr. 相似文献
11.
IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis and a promising target for cancer chemotherapy, was purified 4860-fold to homogeneity from rat hepatoma 3924A by a method including affinity chromatography in which IMP is bound to epoxy-activated Sepharose 6B. This affinity gel provided a specific elution of the enzyme with 0.5 mM IMP. The final enzyme preparation gave a single band with a molecular weight of 60,000 +/- 1000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. 相似文献
12.
Nicholas J. Hoogenraad Teresa M. Sutherland G.J. Howlett 《Analytical biochemistry》1980,101(1):97-102
Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min?1 mg?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods. 相似文献
13.
A Zsindely M Szabolcs J Aradi M Schablik A Kiss G Szabó 《Acta biologica Academiae Scientiarum Hungaricae》1977,28(3):281-290
The inositol-dependent mutant of Neurospora crassa lacks inositol-1-phosphate synthetase activity. This defect can be revorted by the addition of high-molecular DNA isolated from the wild type. To elucidate the biochemical background of inositol dependence, inositol-1-phosphate synthetase was studied. A method has been developed fro the isolation of the enzyme from the wild type strain in 10 mg scale by salt fractionation, gel filtration and ion-exchange chromatography. The specific activity of the purified enzyme is 4750 U/mg protein and its purity has increased about 100-fold. Polyacrylamide gel electrophoresis indicated that, in addition to the main enzymatically active band, several accompanying proteins occur in very small amount. The molecular weight of the enzyme is 225,000 daltons. Probably it consists of four subunits, two with a molecular weight of 64,000 daltons and another two of 50,000 daltons. An enzymatically inactive protein has been isolated from the mutant with the same procedure as that of the enzyme; it migrated at gel electrophoreis similarly to the enzyme. It may be supposed that the isolated protein is the defective enzyme molecule. 相似文献
14.
An efficient method for isolation of acid alpha-glucosidase from human spleen is developed. The method involves chromatography of the enzyme on rho-aminophenyl-alpha-D-glucopyranoside covalently bound to CH-Sepharose 4B, with subsequent gel filtration on Sephadex G-200. The enzyme was homogeneous by the polyacrylamide gel electrophoresis data; it was purified about 1500-fold, as compared with the crude extract (the total yield 12.5%). Besides acid alpha-glucosidase, the preparations of alpha-L-fucosidase, alpha-D-galactosidase and beta-N-acetylglucosaminidase were isolated and purified 200-, 130- and 280-fold, respectively. The nature of interaction between acid alpha-glucosidase and immobilized rho-aminophenyl-alpha-glucopyranoside is discussed. 相似文献
15.
磷蛋白磷酸酶是磷酸化/脱磷酸化作用中重要的调节酶。本文建立了小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶(PrP)的纯化方法。用~(32)P-酪蛋白为底物测定活力。经纯化的酶纯度提高1380倍,聚丙烯酰胺梯度凝胶电泳检查,只有一条泳带。用凝胶过滤法和聚丙烯酰胺梯度凝胶电泳法测得该酶分子量为200,000。该酶催化~(32)P-酪蛋白脱磷酸化反应的最适pH7.2,对热不稳定。 相似文献
16.
Toshio Satoh Yasuo Hoshino Hiroshi Kitamura 《Bioscience, biotechnology, and biochemistry》2013,77(9):1749-1751
An alkaline proteinase produced by Bacillus sp. was purified and crystallyzed through isopropanol or ammonium sulfate precipitation, decolorization with DEAE-cellulose and gel filtration with Sephadex G-100. The optimum pH for caseinhydrolysis was 11.5 and the activity was completely inactivated after incubation with DFP. The specific activity on Hammersten casein was about 4,500 units/mg enzyme protein. The enzyme also hydrolyzed synthetic ester substrate such as Ac-Tyr-OEt, Ac-Phe-OEt or Bz-Met-OMe. The isoelectric point was pH 10.7, and the molecular weight was estimated to be about 17,500 by the sedimentation equilibrium method and 16,000 by gel filtration method. Some physicochemical properties and the amino acid composition of the enzyme were investigated, indicating that the enzyme is distinguishable from alkaline proteinase of Bacillus species so far reported. 相似文献
17.
Myung Jin Shin Jae Yeon Park Kyungmoon Park Seung Hoon Song Young Je Yoo 《Biotechnology and Bioprocess Engineering》2007,12(6):640-645
A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing
enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the
method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made
in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other
immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above
95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other
sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized
using the novel sol-gel method described here was more stable than enzymes immobilized using the other method. 相似文献
18.
A rapid and efficient method for the isolation of glycollate oxidase from pea leaves is described. The method utilizes the unusually high isoelectric point (pH 9·6) which has been determined for the enzyme using isoelectric focusing. The enzyme is apparently homogeneous by polyacrylamide gel electrophoresis and has a MW of ca 100000. Some properties of the enzyme are described. 相似文献
19.
An optimized multilocus enzyme electrophoresis method, which involves polyacrylamide-agarose gel electrophoresis followed by electrophoretic transfers on nitrocellulose sheets, was developed for the analysis of enzyme polymorphism in several aerobic and anaerobic bacterial species including Staphylococcus aureus, Streptococcus pneumoniae, S. agalactiae, Klebsiella pneumoniae and K. oxytoca, Clostridium bifermentans and C. sordellii, and Prevotella bivia. Serial electrophoretic transfers (during 5-15 min each) from a single polyacrylamide gel could be achieved for most enzymes studied, and allowed an increased definition of enzyme bands on nitrocellulose as compared to migration gels. Four enzymes, which could not be blotted in such conditions, could still be stained in gels after blotting. Thus, the method allowed the combined analysis of several enzymes after a single gel electrophoresis separation. The analysis of enzyme polymorphism in the various species studied raised the interest of polymorphic loci such as esterase or glutamic-oxaloacetic transaminase for epidemiologic studies. The method characterized a genetic diversity of enzyme loci of S. pneumoniae higher than previously reported, and is thus convenient for the analysis of genetic relationships between related isolates. Since the present method reduces the tediousness of multilocus enzyme electrophoresis and requires experimental conditions that are not specific for the bacterial population studied, it may be proposed for rapid population genetics analysis of a wide variety of bacteria. 相似文献
20.
J C Shih 《Analytical biochemistry》1978,89(1):103-109
A new, simple, and quantitative method was developed to determine lipoamide dehydrogenase (E,C, 1.6,4.3, NADH:lipoamide oxidoreductase). The principle of this technique is to allow the enzyme or tissue extracts to diffuse in an agarose gel containing lipoate. The enzyme, after 24 hr of diffusion and 2 hr of reaction with NADH, can be determined by the size of a dark or fluorescence-quenching zone in the gel when illuminated with uv light. The diameter of the quenching zone which indicates the enzymatic oxidation of NADH is linearly proportional to the logarithm of enzyme concentration. Since lipoamide dehydrogenase is a FAD-enzyme, the activity was decreased in liver homogenates of riboflavin-deficient chicks, as measured by the new method. This demonstrated the potential importance of this new technique in nutritional and clinical applications. 相似文献