Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin–horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.     

Partial purification and characterization of a polymorphic protein (Pa) in human parotid saliva.

A polymorphic acidic protein (Pa) has been isolated from human parotid saliva by the use of ion-exchange and gel filtration chromatography. Following these purification procedures, analytical anionic polyacrylamide disc gel electrophoresis revealed a single stainable band. Amino acid analysis demonstrated a protein particularly rich in proline, glutamic acid, and glycine, but with reduced amounts of threonine and no tyrosine. Only a very small percentage of carbohydrate was detected. Isoelectric focusing at pH 3-10 verified the acidic character of this protein with an isoelectric point in the range pH 3.9-4.5. Other salivary proteins called Pa-II, possibly related physiologically and genetically to the Pr system, were also partially purified and studied. Differences were noted between Pa and Pa-II proteins in molecular size and amino acid composition.     

Isolation and partial characterization of an amphiphilic 56-kDa fatty acid binding protein from rat renal basolateral membrane

We have identified a 56-kDa fatty acid binding protein in rat renal basolateral membrane and purified it by extraction in nonionic detergent (Triton X-100), followed by gel filtration, DEAE-cellulose chromatography, and affinity chromatography. The purified protein was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration, polyacrylamide gel electrophoresis, and oleate-Sepharose 4B chromatography. Its molecular mass was estimated to be 56 kDa by SDS-polyacrylamide gel electrophoresis. The protein showed optimal binding activity at pH 7.5 and 37 degrees C. The apparent Kd for palmitic acid was 0.79 microM. It was immunologically clearly distinct from renal cytosolic fatty acid binding protein.     

Purification and characterization of glutathione reductase from bovine erythrocytes

Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.     

The purification and biological activity of a macrophage-derived factor with interleukin 1-like activities from guinea pigs

A macrophage-derived factor with interleukin 1-like activity was purified from culture supernatant of muramyl dipeptide-stimulated peritoneal exudate macrophages of guinea pigs. Starting with serum-free culture supernatant, the purification was carried out by gel permeation chromatography, affinity chromatography on procion red agarose, removal of carry-over serum proteins by Sepharose-coupled antibodies against bovine serum proteins, anion exchange chromatography and hydrophobic chromatography. The purified sample potentiated the phytohemagglutinin-induced thymidine uptake of thymocytes with a 50% effective concentration of 9.6 X 10(-11) M. The sample showed a single band in polyacrylamide gel electrophoresis, and a 65 kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by silver staining. A single peak of activity was detected by thymocyte assay at the position corresponding to the stained band in both of the electrophoretic analyses. The purified factor had activities to potentiate the antigenic activation of sensitized T cells for the production of a lymphokine, macrophage migration inhibitory factor, and also the proliferative response of sensitized T cells to antigen. Thus, the 65 kDa factor has activities to modulate various T cell responses in guinea pigs such as interleukin 1 does in other species. The molecular relationship of the 65 kDa macrophage factor to interleukin 1 remains to be determined.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.     

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Co-purification of the aminoacyl-tRNA synthetase complex with the elongation factor eEF1

A multi-enzyme complex of mammalian aminoacyl-tRNA synthetases was isolated from rabbit reticulocytes, and purified by polyethylene glycol fractionation and gel filtration on Biogel A15m and affinity chromatography on tRNA-Sepharose. The synthetase complex contains nine synthetase activities, and the corresponding proteins as analyzed by SDS polyacrylamide gel electrophoresis. Three of the proteins showed the identical subunit molecular weights to those of the reticulocyte's elongation factor eEF1H. The eEF1 alpha protein could not be removed by second tRNA-Sepharose column chromatography, or gel filtration on Biogel A5m or Biogel A15m. Antibodies against eEF1 alpha react with the purified synthetase complex on the basis of dot blot analysis. This finding should provide new clues for elucidating the structural organization of the mammalian protein biosynthetic machinery.     

Isolation,purification and some properties of a lectin from the winged bean (Psophocarpus tetragonolobus)

We have isolated by affinity chromatography a lectin from the seeds of the winged bean (Psophocrapus tetragonolobus) which agglutinated human (group A, B and O), sheep and rabbit, but not mouse erythrocytes. A molecular weight of 41,000 was obtained from gel filtration, and on sodium dodecyl sulphate polyacrylamide gel electrophoresis a single polypeptide chain of molecular weight 35,000 was seen both before and after reduction. Isoelectric focussing of the lectin on polyacrylamide gel gave a single band with a calculated isoelectric point of 4.0. The lectin was found to be rich in acidic amino acids; cysteine was not detected. Carbohydrate analysis revealed no covalently bound sugars.Abbreviations PBS phosphate-buffered saline - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - WBL winged bean lectin - HPLC high performance liquid chromatography     

Purification, characterization and induction of L-phenylalanine ammonia-lyase in Phaseolus vulgaris

The enzyme L-phenylalanine ammonia-lyase was purified from leaves of Phaseolus vulgaris by Sephacryl S-200 gel filtration and Sepharose-4-B--succinyl-aminoethyl-L-phenylalanine affinity chromatography. L-Phenylalanine ammonia-lyase was specifically eluted from the affinity matrix with its substrate L-phenylalanine at 20-25 degrees C. The purified enzyme was shown to be homogeneous by gel electrophoresis both in presence and absence of SDS. Its Mr, determined by gel filtration and non-denaturing gel electrophoresis, was 320,000 +/- 9000 and 330,000 +/- 4000 respectively. After SDS electrophoresis only one band of Mr 83,000 +/- 4000 was detected, indicating that the enzyme is an oligomer containing four subunits. The pH optimum of enzyme activity was 8.8-9.2. Ampholyte isoelectrofocusing in polyacrylamide demonstrated the presence of a single charged species at pH 4.2. The homogeneous enzyme catalyzed the deamination of L-phenylalanine to trans-cinnamate but did not catalyze the transamination of L-phenylalanine to L-phenylpyruvate. The enzyme showed Km 1.25 mM for L-phenylalanine. Antibodies to homogeneous L-phenylalanine ammonia-lyase recognised specific epitopes on L-phenylalanine aminotransferase as demonstrated by immunoaffinity purification and immunoblotting. The induction of L-phenylalanine ammonia-lyase activity during phaseollin biosynthesis in the Phaseolus vulgaris--Colletotrichum lindemuthianum interaction was regulated by an increase in enzyme concentration resulting from an increase in de novo synthesis of L-phenylalanine ammonia-lyase protein.     

铜绿假单胞菌SOS反应阻遏蛋白LexA的复性及活性研究

目的:对LexA蛋白复性方法进行优化,对复性后的LexA蛋白的生物学活性进行分析。方法:采用含有GSH/GSSG的缓冲液,一步稀释法对变性LexA蛋白进行复性,用镍离子亲合柱及阳离子柱层析法对复性后的LexA蛋白进行纯化,再以Sephadex G-25凝胶柱脱盐,采用非变性聚丙烯酰胺凝胶电泳和RP-HPLC法检测复性效果,Western blot法分析复性前后及经DTT处理后的LexA蛋白的免疫反应性,凝胶滞留电泳试验检测复性LexA蛋白与DNA的特异性结合能力。结果:复性后的LexA蛋白出现单体和多聚体的形式,多聚体是由单条肽链聚合而成。LexA单体和多聚体与兔抗LexA多克隆抗体均有较好的反应性。复性后的LexA蛋白能与SOS盒序列发生特异性结合。     

Purification and properties of the enzyme 6-phosphogluconate dehydrogenase from Dicentrarchus labrax L. liver

The enzyme 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP+ oxidoreductase, decarboxylating EC 1.1.1.44) from bass liver has been purified to over 95% of homogeneity by gel filtration, affinity and ion exchange chromatographies. The apparent molecular weight was estimated by gel filtration chromatography to about 100,000. Analysis of the enzyme on sodium dodecyl sulphate polyacrylamide gel electrophoresis showed to be a dimeric protein. The effect of pH and kinetic properties were studied.     

Purification and characterization of an extracellular acid protease from Neurospora crassa

An extracellular acid protease was purified 1420-fold from sulfur-starved protein-induced cultures of Neurospora crassa. The enzyme was homogeneous as determined by polyacrylamide electrophoresis. The purification procedure consisted of an ultrafiltration step, cation-exchange chromatography, and affinity chromatography on Sepharose-linked pepstatin. The enzyme is homologous to aspartyl proteases that are characterized by pepstatin inhibition and trypsinogen activation. It is extremely autolytic, especially under denaturing conditions. The protease is stable between pH 3 and 7, showing optimal activity near pH 4.0 for both trypsinogen activation and hydrolysis of bovine serum albumin. The molecular weight of the enzyme was 34,500 by gel electrophoresis and gel filtration, and 34,975 by amino acid analysis.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Use of fast protein liquid chromatography in the purification of inhibin from bovine follicular fluid

Inhibin from bovine follicular fluid was partly purified using affinity chromatography on immobilized Procion Red 3B, gel filtration on Sephadex G-25 and ion-exchange chromatography on the fast protein liquid chromatography system. Inhibin was subsequently characterized using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. Biological activity was associated with a protein with an apparent molecular weight of approximately 65 kD.     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.     

Purification of prealbumin from human serum

A method is presented by which prealbumin (thyroxine-binding prealbumin; tryptophan-rich prealbumin) may be purified to homogeneity from human serum. The method involves precipitation of contaminating proteins with dilute aqueous phenol, ion-exchange chromatography on DEAE-Sephacel, and gel permeation chromatography on Sephadex G-100. The yield is 25-30%, and the prealbumin is homogeneous by polyacrylamide gel electrophoresis at pH 8.9 and pH 3.6.