大鼠脂多糖结合蛋白的分离纯化及对脂多糖的调节作用

经硫酸铵沉淀、Bio Rex70树脂的阳离子交换层析和MonoQ预装柱的阴离子交换层析 ,从大鼠急性期血清中分离纯化了脂多糖结合蛋白 .SDS PAGE为单一条带 ,分子量 60kD ,所纯化的大鼠脂多糖结合蛋白可明显增强脂多糖与单核巨噬细胞的结合 .脂多糖结合蛋白对脂多糖诱导肺泡巨噬细胞中肿瘤坏死因子α(TNF α)mRNA表达的调节作用具有明显的剂量依赖性 .     

Separation of a New Deoxyribonucleic Acid Polymerase from Vaccinia-infected HeLa Cells

HeLa cell deoxyribonucleic acid (DNA) polymerase was purified about 100-fold by sequential column chromatography on phosphocellulose, hydroxylapatite, and Bio Rex 70. A new form of DNA polymerase found in vaccinia virus-infected cells was separated from HeLa DNA polymerase by chromatography on diethylamino-ethyl cellulose. The new form was also purified approximately 100-fold in the same manner as the HeLa DNA polymerase. In addition to chromatographic differences, the two enzymes differed with regard to primer response, relative activity at high pH, inactivation by heat and p-chloromercuribenzoate, and inhibition by vaccinia antiserum.     

Purification of Thermus aquaticus DNA polymerase expressed in Escherichia coli

DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitation of the enzyme with polyethyleneimine and elution from Bio Rex 70 ion exchange resin in a single salt step. The resulting enzyme preparation contains a single, nearly homogeneous protein consistent with the previously established size of the Taq DNA polymerase in a yield of 40-50 mg of protein per liter of cell culture.     

苦瓜籽核糖体失活蛋白的理化性质及生物活性

采用硫酸铵分级分离,假配基亲和层析和ＳｅｐｈａｃｒｙｌＳ－１００分子筛层析等方法,从苦瓜籽中获得核糖体失活蛋白（ＲＩＰ）．经ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ、ＩＥＦ和ＰＡＳ方法分析均表明为单一蛋白着色带或单一糖蛋白着色带．根据ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ Ｇ－１５０分子筛层析结果计算其相对分子质量为３．０×１０４,经ＩＥＦ－ＰＡＧＥ结果计算其ｐＩ为８．９～９．０．对无细胞系统中蛋白质生物合成抑制活性明显,其ＩＣ５０为５．３×１０－ １０ ｍ ｏｌ／Ｌ左右．体外生物活性试验结果表明其对人肝癌细胞、Ｖｅｒｏ、ＳＰ２／０、３Ｔ３、Ｋｂ、Ｎａｖａｎａ 等肿瘤细胞株均表现有不同程度的抑制作用．而对完整细胞人胚肺二倍体细胞却毒性极小．因此,上述实验结果为该ＲＩＰ的进一步深入研究和有可能开发成免疫毒素的高效弹头药物提供了一定的工作基础．     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

Characterization and partial purification of human epithelial transforming growth factor

A polypeptide growth factor has been partially purified from medium conditioned by the human adrenocortical carcinoma cell line SW13. This factor, designated h-TGFe, stimulates anchorage-independent growth of the SW13 cells. Similar activity was observed in human milk, and in conditioned media from seven of 14 epithelial cell lines. The SW13-derived activity is stable to low pH and 8M urea but labile to dithiothreitol and 2% sodium dodecyl sulfate. Human TGFe does not bind to heparin and fails to stimulate growth of endothelial cells in monolayer culture. The apparent molecular weight of h-TGFe is 59k by size exclusion chromatography in the presence of 8M urea and the activity binds strongly to cation exchangers. The activity elutes at 15-30% acetonitrile from a C18 reverse-phase column and has been partially purified by using a four-step chromatographic procedure. TGFe appears to be a novel growth factor produced by many epithelial cells and tissues.     

Purification of protein methylase II from human erythrocytes

Protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) which methyl esterifies free carboxyl groups of protein substrate using S-adenosyl-L-methionine as the methyl donor, has been purified from human erythrocytes approximately 13000-fold with a yield of 12%. The purified enzyme was over 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A bulk of hemoglobin present in the erythrocyte lysate, which severely limited the use of affinity chromatography for purification, was effectively removed by ammonium sulfate precipitation and by the subsequent salt washing of the precipitates followed by molecular sieve chromatography on Sephadex G-75. This preparation can be further purified by affinity chromatography, in which S-adenosyl-L-homocysteine is covalently linked to Sepharose-4B, followed by Sephadex G-75 chromatography to yield an enzyme with an activity of 27000 pmol methyl group transferred/mg/min at 37 degrees C.     

Purification and characterization of two new high molecular weight forms of DNA polymerase delta

Two high molecular weight DNA polymerases, which we have designated delta I and delta II, have been purified from calf thymus tissue. Using Bio Rex-70, DEAE-Sephadex A-25, and DNA affinity resin chromatography followed by sucrose gradient sedimentation, we purified DNA polymerase delta I 1400-fold to a specific activity of 10 000 nmol of nucleotide incorporated h-1 mg-1, and DNA polymerase delta II was purified 4100-fold to a final specific activity of 30 000 nmol of nucleotide incorporated h-1 mg-1. The native molecular weights of DNA polymerase delta I and DNA polymerase delta II are 240 000 and 290 000, respectively. Both enzymes have similarities to other purified delta-polymerases previously reported in their ability to degrade single-stranded DNA in a 3' to 5' direction, affinity for an AMP-hexane-agarose matrix, high activity on poly(dA) X oligo(dT) template, and relative resistance to the polymerase alpha inhibitors N2-(p-n-butylphenyl)dATP and N2-(p-n-butylphenyl)dGTP. These two forms of DNA polymerase delta also share several common features with alpha-type DNA polymerases. Both calf DNA polymerase delta I and DNA polymerase delta II are similar to calf DNA polymerase alpha in molecular weight, are inhibited by the alpha-polymerase inhibitors N-ethylmaleimide and aphidicolin, contain an active DNA-dependent RNA polymerase or primase activity, display a similar extent of processive DNA synthesis, and are stimulated by millimolar concentrations of ATP. We propose that calf DNA polymerase delta I, which also has a template specificity essentially identical with that of calf DNA polymerase alpha, could be an exonuclease-containing form of a DNA replicative enzyme.     

Identification of a low molecular weight form of epithelial transforming growth factor

We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.     

蛋白质二硫键异构酶相关蛋白A的表达及性质的研究

克隆了Aspergillus niger T21中的蛋白质二硫键异构酶相关蛋白A（PRPA）基因,并将它插入pET23b表达载体。在E. coli中表达时,PRPA占菌体总蛋白的34%。经过超声破细胞、硫酸铵分级沉淀和离子交换层析获得了纯度大于90%的重组蛋白。PRPA有二硫键异构酶活性。在PRPA存在下,变性和还原的溶菌酶复性率和复性速度降低,电泳结果表明溶菌酶聚集增多。荧光结果表明PRPA表面有较多的疏水基团。     

A new high sensitive analytical micro-scale procedure for chromosomal proteins

Utilizing male rat liver cells we describe a method for isolating and fractionating chromosomal proteins. About 99%of chromosomal proteins was dissociated using a three step dissociation procedure. DNA was removed by sedimentation and the histone fractions were separated from the non-histone chromosomal proteins by Bio Rex 70 chromatography. The nonhistone chromosomal proteins were fractionated by micro-gradient electrophoresis on SDS-polyacrylamide gels, which proved to be superior to the electrophoretic procedures currently in use. The histones were further separated on polyacrylamide-SDS slab gels using a micro-two-dimensional electrophoretic system. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.This work was supported by the Deutsche Forschungs-gemeinschaft     

Purification and some properties of alkaline pullulanase from a strain of bacillus no. 202-1, an alkalophilic microorganism.

Pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) was purified about 290-fold from the culture fluid of Bacillus No. 202-1 by DEAE-cellulose adsorption, acetone fractionation, (NH4) 2SO4 precipitation and DEAE--cellulose column chromatography followed by Sephadex G-200 molecular sieve chromatography. The enzyme gave a single band of protein by disc polyacrylamide gel electrophoresis. The molecular weight was estimated as 92 000 by sodium dodecyl sulfate gel electrophoresis. The isolectric point was lower than pH 2.5. The optimum pH for enzyme action was about 8.5-9.0. The action of the enzyme on amylopectin and glycogen resulted in increase in the iodine coloration of 85% and 70%, respectively. The enzyme completely hydrolyzed 1,6-alpha-glucosidic linkages in amylopectin, glycogen and pullulan.     

间羟苯甲酸4－羟化酶纯化及部分性质研究

经超声破碎、硫酸铵分级沉淀、凝胶过滤、磷酸钙胶层析和离子交换层析等步骤, 从Comamonas testosteroni菌株中获得了SDS-PAGE单一条带, 相对分子质量为62×10³的间羟苯甲酸4-羟化酶比活提高21倍, 产率为30%.此酶为FAD加单氧酶, 催化间羟苯甲酸转变为3,4二羟苯甲酸.     

Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.     

Purification of D-beta-hydroxybutyrate dehydrogenase from rat brain

D-beta-Hydroxybutyrate dehydrogenase (BDH), a lipid-requiring enzyme, has been purified to homogeneity from rat brain using a new improved method. The purified rat brain BDH has a subunit molecular mass of 31 kilodaltons on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The apoenzyme, i.e., the enzyme devoid of phospholipid, has no activity, but can be activated by phospholipid to a specific activity of 125 mumol/(min.mg). This is 625-fold greater than the activity in the mitochondrial fraction. The new purification procedure involves chromatography using a quaternary amine Sepharose resin followed by a sulphonate Sepharose resin, and eliminates the need for glass bead adsorption chromatography.     

Partial Purification and Some Properties of Stearyl-Coa: Sn-Glycerol-3-Phosphate Acyltransferase from Liver Microsomes

About fourfold purification of the stearyl-CoA: sn-glycerol-3-phosphate acyltransferases (GPAT) was achieved from liver microsomes by extraction with 1M phosphate buffer (pH 7–4) followed by gel filtration. The partially purified enzyme synthesized mainly diacylgly-cerophosphate and a small amount (5%) of monoacylglycerophosphate. Patty aoyl-CoA synthetase and to some extent stearyl-CoA desaturase were copurified along with the transferases. Data obtained from molecular sieve chromatography, polyacrylamide gel electrophoresis and electron microscopy showed that GPAT activity was tightly bound to a high molecular weight protein fraction with vesicular structure.     

Identification of a membrane-associated receptor for transforming growth factor type E

Abstract

We have identified the receptor for epithelial type transforming growth factor (TGFe). TGFe, a member of the epithelin/granulin family of proteins, is present primarily in tissues of epithelial origin. It is a powerful mitogen for epithelial and fibroblastic cells. TGFe, iodinated using an immobilized glucose oxidase-lactoperoxidase method, was chemically crosslinked to receptors on membranes isolated from SW-13 adrenal carcinoma cells by the crosslinker disuccinimidyl suberate (DSS). The receptor appears to be a protein which migrates at an apparent molecular weight of approximately 170-175 kDa under reducing and nonreducing conditions in SDS-polyacrylamide gels.     

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.     

Purification of human alpha-fetoprotein

By employing complex and highly specialized immunochemical methods, several investigators have achieved purification of human α-fetoprotein (AFP) found in fetal serum and/or sera of patients with hepatoma. The present report describes a simpler method which results in the isolation of homogeneous preparation of AFP from human cord serum. AFP was purified by sequential use of Affi-Gel Blue affinity, DE-52 diethylaminoethyl cellulose ion-exchange, immunoadsorption with anti-albumin covalently coupled to Sepharose 4B, and Sephacryl S-200 molecular sieve chromatographic techniques. The homogeneity of the purified AFP was established by subjecting it to polyacrylamide gel electrophoresis, analytical isoelectric focusing, molecular sieve chromatography and immunological techniques. The purified AFP has a molecular weight of approximately 68,000 as determined by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and molecular sieve chromatography, and upon isoelectric focusing yielded a single band pI = 4.8. In addition, the purified AFP gives a single precipitin line when tested against rabbit antiserum to whole human hepatoma serum proteins, and no line(s) of precipitin when tested against rabbit antiserum to normal serum proteins.