高比活力大肠杆菌磷酸转乙酰酶的纯化

从国内大肠杆菌菌株1.505提取磷酸转乙酰酶(PTA),经数步纯化荻得了高比活力酶制品。该酶制品比粗酶纯化了61倍,比活力达1040单位/mg,经聚丙烯酰胺凝胶电泳检查,仅有两条蛋白质区带。用经过纯化的酶测定辅酶A,其结果6.75单位/10微升酶液在光径1cm时,233nm的吸收值△E_(PTA)<0.009。     

Studies on prephenoloxidase-activating enzyme from cuticle of the silkworm Bombyx mori. II. Purification and characterization of the enzyme

Prephenoloxidase-activating enzyme has been purified approximately 4800-fold from cuticular extract of the silkworm, and the preparation seems to be homogeneous as judged by disc- and dodecylsulfate-polyacrylamide gel electrophoresis. By means of gel filtration through Sephadex G-100, it has been supposed that the enzyme exists as mono- and dimeric forms at slightly acidic pH, while a monomeric form is predominant under slightly alkaline condition. The molecular weight of the monomer was estimated to be 33,000–35,000 by dodecylsulfate-polyacrylamide gel electrophoresis and gel filtration.It has been demonstrated that ester substrates for trypsin, benzoyl-l-arginine ethyl ester and tosyl-l-arginine methyl ester, can be hydrolyzed by the purified enzyme. Several lines of evidence indicating that a single protein is involved in both activation and esterolytic reactions have been presented. Some enzymatic properties of the purified preparation as esterase have also been described.In connection to esterase activity of the purified enzyme, a mechanism of prephenoloxidase activation in the silkworm system has briefly been discussed.     

Lipoxygenase in dark-grown Pisum sativum

Lipid oxidizing activity has been detected in acetone powders from both dark- and light-grown dwarf pea seedlings. This activity has been shown by several methods to be due to lipoxygenase. The enzyme from dark-grown seedlings has been purified 5·7-fold by ammonium sulphate precipitation and gel filtration. CM-cel-lulose chromatography of the purified enzyme yielded four active fractions. The properties of the four lipoxy-genase isoenzymes are described.     

Biochemical and immunological characterization of a glycosylated alpha-fucosidase from the invertebrate Unio: interaction of the enzyme with its in vivo binding partners

Mammalian alpha-fucosidase (EC 3.2.1.51) is a lysosomal enzyme that catalyzes the removal of fucose residues from glycosphingolipids and its absence in humans results in a rare metabolic disorder called fucosidosis. Among the invertebrates in the molluscs (Unio) two forms of the enzyme have been reported, a 68 kDa non-glycosylated form and a 56 kDa glycosylated form. The glycosylated form has been purified from the seminal fluid of Unio [Biochem. Biophys. Res. Commun. 234 (1997) 54]. In the present study, the 56 kDa glycosylated form has been purified to homogeneity from the whole body tissue of Unio using a series of chromatographic steps. The purified enzyme migrated as a single protein species in 10% SDS-PAGE. Antibodies to the purified enzyme were raised in a rabbit in order to study its biochemical and immunological properties. The purified enzyme is a glycoprotein that exhibits strong binding to Con A-Sepharose gel and can be deglycosylated by PNGase F enzyme suggesting it to be N-glycosylated. The enzyme has been shown to specifically interact with the mannose 6-phosphate receptor protein (MPR 300) purified from goat and Unio. This specific interaction is discussed in view of its possible in vivo binding partners.     

Isolation of Plasmodium berghei by hemolysin lysis of infected erythrocytes and evidence for a parasite hexokinase.

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.     

Sphingolipid base metabolism. Partial purification and properties of sphinganine kinase of brain.

The presence of sphinganine kinase in bovine brain has been demonstrated. The product of the action of the brain enzyme on sphinganine has been characterized as sphingamine 1-phosphate by a combination of chemical, enzymatic, and chromatographic techniques. The bovine brain enzyme has been partially purified and appears to exist in multiple forms. The molecular weight of the most highly purified preparation of the enzyme was estimated to be 190,000 by gel filtration. The purified form of the enzyme showed highest activity with ATP but was also active with other purine nucleoside triphosphates. UTP and CTP did not serve as substrates for the enzyme.     

Rat atrial natriuretic factor. Purification and vasorelaxant activity

The atrial natriuretic activity of rat heart has been found to exist in multiple forms. One of these factors has been purified to apparent homogeneity by a combination of gel filtration and high pressure liquid chromatography in two different systems and its amino acid composition determined. The purified active peptide is shown to have a molecular weight of approximately 3800. In addition, the vasorelaxant activity of rat atrium has been purified and found to cochromatograph with the natriuretic activity in all chromatographic systems employed. Thus, the vasorelaxant activity resides in the natriuretic factor. The existence of this new multifunctional peptide implies a higher level of complexity for cardiovascular control of blood volume and pressure.     

Purification of the prophenoloxidase from Hyalophora cecropia and four proteins involved in its activation

Prophenoloxidase (PPO) has been purified to homogeniety from hemolymph of Hyalophora cecropia. There are two forms of the enzyme with identical molecular weights (76 kDa). Four proteins directly involved in the activation of PPO have also been purified from the hemolymph. Active phenoloxidase is elicited by the addition of factor C1 and a serine protease (SPII), which alone cannot activate PPO. Purified SPII contains two proteins with M_r 43 and 53 kDa, the larger molecule may represent the unactivated enzyme. An inhibitor of the SPII catalyzed activation of PPO has been isolated. In addition a protein presumed to be dopa quinone imine conversion factor has been purified.     

草鱼生长激素单抗免疫亲和柱的制备及初步应用

用纯化的草鱼生长激素单克隆抗体偶联到CNBr活化的Sepharose4B凝胶上,制成约10ml的亲和层析柱．用该柱一步纯化了重组鲤鱼生长激素基因的表达产物．偶联有13．65mg单克隆抗体的亲和柱一次可纯化得到约0．7mg重组鲤鱼生长激素．酶联免疫受体测定表明它具有强烈的生物学活性,SDS－PAGE表明它为单一蛋白带,分子量约为22000．     

4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.     

Isolation of an activator of bilirubin glucuronyltransferase from normal and jaundiced Gunn rats

A microsomal activator of the UDP-glucuronyltransferase for bilirubin has been isolated from lubrol solubilized and salt fractionated liver microsomes. The activator has been partially purified by anion exchange and molecular sieving chromatography and found to have a molecular weight of about 60 kDa. The activator is present in liver from normal and bilirubin UDP-glucuronyltransferase deficient Gunn rats. When tested with purified UDP-glucuronyltransferase for bilirubin it accelerated the conjugation rate 10 fold but with the purified UDP-paranitrophenol transferase the rate of conjugation was increased only 1.5 times.     

Human interleukin 1: purification and properties

Human interleukin 1 (IL 1) has been purified to homogeneity by a procedure of molecular weight fractionation, isoelectric focusing, and preparative polyacrylamide gel electrophoresis. The homogeneity of the purified material has been demonstrated by silver staining of analytical polyacrylamide gels. The homogeneous IL 1 retains only a trace of its original biological activity because of the denturing effects of the sodium dodecyl sulfate used in the final step of purification. Very highly purified IL 1, retaining strong biological activity, has been eluted from nondenaturing polyacrylamide gels. This IL 1 has been demonstrated to stimulate human and mouse T and B lymphocytes, fibroblasts, and synovial cells. In addition, in vivo treatment of animals with IL 1 resulted in the immunologically relevant symptoms of fever, increased plasma levels of acute phase proteins, and increased numbers of circulating neutrophils.     

Caprine (Capra hircus) luteinizing hormone: purification and chromatographic investigation of its different isoforms

Luteinizing Hormone (LH) from goat pituitary glands has been purified and characterized with respect to its size and subunit nature. The purification at each step was monitored by protein estimation, SDS-PAGE, and direct binding ELISA. The final product was found to be over 90 fold purified as compared to the starting pituitary extract, and the yield of the final purified LH was found to be 65.3 mg/kilogram of wet pituitary glands. Fractionation of the cLH into different charge isoforms by SP-Sephadex ion exchanger has been observed. Chromatography on immobilized Con A lectin resulted in fractionation of the purified cLH into unbound (2%), loosely bound (85%), and firmly bound (13%) fractions indicating oligosaccharide heterogeneity. The purified hormone was capable of stimulating weight increase in the seminal vesicles in immature male rats, with a biopotency equivalent to the 2200 I.U. of hCG per mg of purified cLH. The FSH content of the purified cLH was found to be less than 0.0165% as indicated by in vivo Steelman-Pohley assay.     

Overproduction and purification of lysyl-tRNA synthetase encoded by the herC gene of E coli.

Lysyl-tRNA synthetases are synthesized from two distinct genes in E coli, lysS and lysU, but neither gene product has been purified distinctively by using overproducing systems. The lysS gene has been identified by a herC mutation which restores maintenance of the mutant ColE1 replicon. The herC gene product was overproduced by using a tac promoter fusion and purified to homogeneity. The purified HerC protein possesses a lysyl-tRNA synthetase activity as predicted by the sequence identity of herC to lysS. The procedure is useful for rapid mass-scale purification of lysyl-tRNA synthetase.     

Antibody against purified human hexosaminidase B cross-reacting with human hexosaminidase A

Human hexosaminidase B has been purified to virtually homogeneous state from placenta. An anti-serum has been prepared in rabbits against the purified preparation. The serum reacted equally with human hexosaminidase B (free of hexosaminidase A) and with human hexosaminidase A (free of hexosaminidase B) as shown by immunodiffusion and by precipitation of enzyme activity from solution.     

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.     

Purification and reconstitution into liposomes of an integral membrane protein conferring immunity to colicin A

Abstract The immunity protein to colicin A protects producing cells from the action of this pore-forming toxin. It is located into the cytoplasmic membrane. This protein has been 'tagged' with an epitope from the colicin A protein for which a monoclonal antibody is available. The fusion protein (named VL1) has been purified after extraction from the membrane in two steps using a chromatofocusing and an immunoadsorbant chromatography. The purified protein has then been reconstituted into lipid vesicles.     

Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties

The gene for pneumolysin, the thiol-activated toxin from Streptococcus pneumoniae, has been expressed in Escherichia coli. The recombinant protein has been purified using a rapid, high yield, purification procedure and has been shown to be identical with respect to N-terminal amino-acid sequence, specific activity, effect on human polymorphonuclear phagocytes and effect on human complement to the native toxin purified from the pneumococcus. This provides a large enough source of material to begin investigation of pneumolysin as a candidate for inclusion in a pneumococcal vaccine.     

Characterization of purified avian 90,000-Da heat shock protein

The so-called 90,000-Da heat shock protein (hsp90) from chicken liver has been purified and physically characterized in the presence of high levels of the serine phosphatase inhibitor fluoride. The protein is an elongated dimer with a molecular weight of 160,000 and a frictional ratio of 1.6. On two-dimensional electrophoresis it exhibits several isoelectric forms lying between pH 5.1 and 5.8. It contains an average of 5.8 mol of covalently bound phosphate per dimer and is thus extensively phosphorylated. Analysis of the ultraviolet spectrum showed the purified protein to be free of nucleotide-containing components. Molybdate has been shown to stabilize complexes between the 90,000-Da heat shock protein and steroid receptors. However, molybdate has no effect on the sedimentation of the purified heat shock protein. Proteins structurally related to hsp90 have been reported to penetrate the endoplasmic reticulum. However, when purified hsp90 was tested using the partition method of Bordier, which distinguishes hydrophilic and lipophilic proteins, it partitioned totally into the aqueous phase.