Purification and characterization of a lipase from Staphylococcus aureus

An extracellular lipase from Staphylococcus aureus (strain FN 37) was purified to homogeneity. A cell-free culture broth was subjected to ammonium sulphate precipitation, and the lipase was isolated from the resuspended pellet by adsorption chromatography on octyl-Sepharose. The purification was 957-fold, and the recovery of the octyl-Sepharose chromatography was about 100%. The specific activity of the purified lipase was 546 mU of lipase activity per micrograms protein. The purity of the final product was documented by SDS-polyacrylamide gel electrophoresis in which a homogeneous protein band of 43 kDa was found. In gel chromatography on Sephadex G-200 the lipase eluted as a homogeneous peak with an apparent molecular mass of 110 kDa, suggesting that the lipase may exist as an oligomer in physiological media. Analysis of the amino-acid composition revealed a predominance of polar, non-charged amino acids, with serine accounting for 24 mol% of the amino-acid residues.     

Characterization of multiple forms of folate-binding protein from human leukemia cells

Folate-binding proteins were isolated from the particulate fraction (44,000 X g pellet) and the soluble fraction (44,000 X g supernate) of the homogenate of a spleen obtained from a patient who had an acute leukemic (blast) transformation of chronic myelogenous leukemia. The folate-binding activity which was obtained from the particulate fraction by solubilization with 1% Triton X-100 could be resolved into two binding proteins (Mr 310,000 and 28,000) by gel filtration through Sephadex G-200 after incubation with excess [3H]pteroylglutamic acid (PteGlu). The folate-binding protein in the solubilized particulate fraction and the soluble folate-binding protein in the 44,000 X g supernatant cytoplasm were purified by affinity chromatography. Only a 32 kDa protein was identified by SDS-polyacrylamide gel electrophoresis in the final preparation of the purified folate-binding protein from the particulate, whereas two protein bands (Mr 42,000 and 32,000) were identified by SDS-polyacrylamide gel electrophoresis in the purified preparation of the soluble folate-binding protein. Both of these species were immunologically crossreacting. Both the purified folate-binding protein from the particulate fraction and the purified soluble form had higher affinity for oxidized folate than for the reduced folate cofactors, and both proteins had very low affinity for the antifolate compound, methotrexate. The amino-acid composition of the soluble folate-binding protein was similar with regard to the content of apolar amino acids to that reported for the membrane-derived folate-binding protein purified from milk and human placenta.     

Characterization of the Escherichia coli X-ray endonuclease, endonuclease III

The X-ray endonuclease endonuclease III of Escherichia coli has been purified to apparent homogeneity by using the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The most purified fraction shows endonucleolytic activity against apurinic and apyrimidinic (AP) sites and a dose-dependent response to DNA that has been X irradiated, UV irradiated, or treated with OsO4. The endonuclease also nicks OsO4-treated DNA that has been subsequently treated with alkali to produce fragmented thymine residues and DNA treated with potassium permanganate. The enzyme does not incise the alkali-labile sites present in DNA X irradiated in vitro in the presence of hydroxyl radical scavengers. The most purified fractions exhibit two distinct activities, an AP endonuclease that cleaves on the 3' side of the damage leaving a 3'-OH and a 5'-PO4 and a DNA N-glycosylase that recognizes at least two substrates, thymine glycol residues and urea residues. The glycosylase activity is sensitive to N-ethylmaleimide while the AP endonuclease is not.     

The predominant form of mammalian DNA topoisomerase Iin vivo has a molecular mass of 100 kDa

DNA topoisomerase I was purified to apparent homogeneity from human HeLa cells as a single polypeptide with a molecular mass of 100 kDa, as assayed by both gel filtration column chromatography and SDS-polyacrylamide gel electrophoresis. No smaller forms of the enzyme were detected in the purified fraction. Therefore, smaller forms, which have been observed by other investigators, are likely to be the result of proteolysis during isolation and are not relevant to thein vivo activity of DNA topoisomerase I.Abbreviations 2-ME 2-Mercaptoethanol - DTT Dithiothreitol - PMSF Phenylmethylsulfonyl Fluoride - SDS Sodium Dodecyl Sulfate     

dNTP pool imbalance induced endonuclease: 5-fluorodeoxyuridine induced DNA double strand break in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleotide triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. When the cells were treated with 1 microM 5-fluorodeoxyuridine (FdUrd), the imbalance of the cellular dNTP pool was induced. The imbalance was followed by DNA double stranded breaks and subsequent cell death. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. SDS-polyacrylamide gel electrophoresis showed a major protein species of approximate 45 kDa. The endonuclease was revealed, using electrophoretic separation in SDS-polyacrylamide gels containing DNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzyme activity.     

Purification and characterization of a low molecular weight zinc binding protein from human placenta

A low molecular weight, native zinc binding, cytosolic protein (LMZP) has been isolated, purified and characterized from human normal term placenta. Gel filtration of heat treated placental cytosol after sequential acetone precipitation (80% ppt) revealed a major zinc binding protein in the range of low molecular weight. This partially purified zinc binding fraction was further fractionated on DEAE-Sephadex A-25. The zinc was eluted in one of the three peak fractions. Further, the purity of zinc binding protein was confirmed on fast protein liquid chromatography (FPLC). The purified placental LMZP was homogenous on SDS-polyacrylamide gel electrophoresis with a single band. Ultraviolet (UV) spectrum of LMZP showed an absorption maximum at 257 nm which disappeared at pH 2. Molecular weight of LMZP as determined by gel chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis was 6 kDa. It was calculated that 1 g atom of zinc was bound to 1 mole of the LMZP. Unlike in classical metallothionein, the amino acid composition of placental LMZP revealed the presence of aromatic amino acids, lower content of cysteine and higher content of histidine, glutamic acid and aspartic acid (10, 9 and 5 residues/mole, respectively).     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

Purification and characterization of heparinase that degrades both heparin and heparan sulfate from Bacillus circulans

A heparinase that degrades both heparin and heparan sulfate (HS) was purified to homogeneity from the cell-free extract of Bacillus circulans HpT298. The purified enzyme had a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular mass of 111,000. The enzyme showed optimal activity at pH 7.5 and 45 degrees C, and its activity was stimulated in the presence of 5 mM CaCl2, BaCl2, or MgCl2. Analysis of substrate specificity and degraded disaccharides demonstrated that the enzyme acts on both heparin and HS, similar to heparinase II from Flavobacterium heparinum.     

Purification and characterization of a Cu, Zn-superoxide dismutase from adult Paragonimus westermani.

In cytosolic fraction of adult Paragonimus westermani, superoxide dismutase activity was identified (4.3 units/mg of specific activity) using a xanthine-xanthine oxidase system. The enzyme was purified 150 fold in its activity using the ammonium sulfate precipitation, DEAE-Trisacryl M anion-exchange chromatography and Sephadex G-100 molecular sieve chromatography. The enzyme exhibited the enhanced activity at pH 10.0. The enzyme activity totally disappeared in 1.0mM cyanide while it remained 77.8% even in 10 mM azide. These findings indicated that the enzyme was Cu, Zn-SOD type. Molecular mass of the enzyme was estimated to be 34 kDa by gel filtration and 17 kDa on reducing SDS-polyacrylamide gel electrophoresis which indicated a dimer protein.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Isolation and characterization of proteins that stimulate the activity of mammalian DNA methyltransferase

Previously, the purification of DNA methyltransferase from murine P815 mastocytoma cells by immunoaffinity chromatography was described (Pfeifer, G.P., Grünwald, S., Palitti, F., Kaul, S., Boehm, T.L.J., Hirth, H.P. and Drahovsky, D. (1985) J. Biol. Chem. 260, 13787-13793). Proteins that stimulate the enzymatic activity of DNA methyltransferase have been purified from the same cells. These proteins, which partially coelute with DNA methyltransferase from DEAE-cellulose and heparin-agarose, are separated from the enzyme during the immunoaffinity purification step. A further purification of the stimulating proteins was achieved by butanol extraction, DEAE-cellulose chromatography and gel filtration on Superose 12. Two DNA methyltransferase-stimulating protein fractions were obtained. SDS-polyacrylamide gel electrophoresis of one fraction showed a single polypeptide with a molecular mass of 29 kDa. The second fraction consisted of 5 or 6 polypeptides with molecular masses 78-82 and 51-54 kDa. The proteins stimulate both de novo and maintenance activity of DNA methyltransferase about 3-fold. They enhance the methylation of any natural DNA and of poly[(dI-dC).(dI-dC)] but inhibit the methylation of poly[(dG-dC).(dG-dC)]. The purified proteins do not form a tight complex with DNA methyltransferase; however, they bind both to double-stranded and single-stranded DNA. The sequence specificity of DNA methyltransferase is obviously altered in presence of these proteins.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100-6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol-activated cytolysin family.     

Purification and characterization of a cortical secretory vesicle membrane fraction

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.     

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.     

韭菜线粒体锰超氧化物歧化酶纯化及性质研究

经硫酸铵沉淀、ＤＥＡＥ－Ｓｅｐｈａｃｅｌ层析和Ｓｅｐｈａｄｅｘ Ｇ－２００凝胶过滤,将韭菜线粒体ＳＯＤ纯化到均一程度。从６０００ｇ韭菜叶片线粒体中纯化得到２．５ｍｇ酶,酶比活力达１２００Ｕ／ｍｇ蛋白。该酶对ＫＣＮ和Ｈ２Ｏ２都不敏感,热稳定性弱 外光区吸收峰在２８０ｎｍ,凝胶过滤法测得其分子量为８２００Ｄ,ＳＯＳ－ＰＡＧＥ法测定其亚基分子量的２２０００Ｄ,ＤＮＳ法测得其Ｎ－末端氨基酸为缬氨酸。上述结     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.     

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.     

Purification and characterization of alpha-keto amide reductase from Saccharomyces cerevisiae

An NADPH-dependent alpha-keto amide reductase was purified from Saccharomyces cerevisiae. The molecular mass of the native enzyme was estimated to be 33 and 36 kDa by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis, respectively. The purified enzyme showed a reducing activity not only for aromatic alpha-keto amides but also for aliphatic and aromatic alpha-keto esters. The internal sequence of the enzyme was identical with that of a hypothetical protein (ORF YDL 124w) coded by yeast chromosome IV.