Deoxyribonuclease I purification from protease contaminations

Affinity chromatography as a tool in purification of commercial DNA-ase I preparation from protease contaminations by the use of insoluble--protease inhibitors is described.     

Long-chain acyl-CoA hydrolase from rat brain cytosol: purification, characterization, and immunohistochemical localization

To determine whether scavenger receptors are susceptible to regulation by granulocyte macrophage colony-stimulating factor (GM-CSF), a macrophage-specific cytokine, human monocytes were differentiated into macrophages in the absence or presence of 20 U/mL GM-CSF. Binding, uptake, and degradation of acetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) were measured. Treatment with GM-CSF resulted in a significant twofold to threefold decrease in the number of binding sites for Ac-LDL and Ox-LDL on the surface of macrophages without affecting the affinity of the receptor for these ligands. Competition experiments revealed that two binding sites were responsible for the recognition and uptake of Ac-LDL; one specific for Ac-LDL and one that recognized both Ac-LDL and Ox-LDL. No binding site specific for Ox-LDL could be detected in either control or GM-CSF-treated macrophages. Treatment of human monocyte-derived macrophages with GM-CSF resulted in a decrease of the Ac-LDL/Ox-LDL receptor but did not affect the binding site specific for Ac-LDL. Northern blot analysis showed that mRNA levels of both types I and II scavenger receptor were reduced in macrophages differentiated in the presence of GM-CSF. Human macrophages that were differentiated in the presence of GM-CSF accumulated approximately 50% fewer cholesteryl esters. Taken together, these results indicate that GM-CSF can downregulate both types I and II scavenger receptor in human monocyte-derived macrophages, which might have implications for foam cell formation.     

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.     

Biochemical and immunochemical characterization of Brassica juncea glyoxalase I

A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.     

Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.     

Deoxyribonuclease I and its clinical applications

DNases are DNA hydrolyzing enzymes. The well-characterized bovine pancreatic DNase I, the first DNase discovered, is a model DNase for studying the structure-function relationships of the DNase I type enzymes. The Epstein-Barr virus produces a DNase with an unknown biologic function other than degrading DNA, and this viral DNase has been used as an Epstein-Barr viral marker. Human DNase I exhibits polymorphism that can be used for forensic identification and for correlation with certain diseases. Variations in serum DNase activities have been implicated as the result of disease states and measurements of DNase activities are often used for diagnosis and prognosis. Recombinant human DNase I has been administered in cystic fibrosis patients to improve mucociliary clearance and pulmonary function. Thus, although the primary function of DNase is to degrade DNA, there are many reports of its clinical applications.     

Rhodobacter capsulatus DNA topoisomerase I purification and characterization

A 30-kDa DNA topoisomerase has been purified to near homogeneity from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus. The enzyme is recognized by an antibody against a 16-mer peptide sequence from human DNA topoisomerase I. The purified enzyme is a type I topoisomerase. Consistent with the properties of other prokaryotic type I DNA topoisomerases, the isolated enzyme is unable to relax positively supercoiled DNA and absolutely requires divalent cations for its relaxation activity. However, regardless of the Mg+2 concentrations, ATP concentrations above 5 mM completely inhibit the relaxing activity. The enzyme is sensitive to high salt concentrations and the optimal activity occurs at salt concentrations between 3 and 30 mM for monovalent cations. Single-stranded M13 DNA is a strong inhibitor of this relaxing activity. The enzyme is inhibited by ethidium bromide, confirming that this DNA topoisomerase is incapable of relaxing positive supercoils. Topoisomerase I-specific inhibitors like Hoechst 32258 and actinomycin D inhibit the enzymatic activity while the enzyme is resistant to type II topoisomerase inhibitors such as norfloxacin, nalidixic acid, and novobiocin. From these enzymatic characteristics, we conclude that the R. capsulatus DNA topoisomerase is a prokaryotic type I DNA topoisomerase.     

Phosphatidylserine specific binding protein in rat brain: purification and characterization

A calcium-independent phosphatidylserine specific binding protein detected on liposome blotting analysis was purified from rat brain and revealed to be identical to myristoylated, alanine-rich C kinase substrate (MARCKS). MARCKS specifically binds to phosphatidylserine but not phosphatidylcholine. The binding of MARCKS to phosphatidylserine was abolished on protein kinase C-dependent phosphorylation. Since bacterially expressed MARCKS also specifically binds to phosphatidylserine, myristoylation of the N-terminal glycine seems not to be essential for the binding of MARCKS to phosphatidylserine. These data suggest that phosphatidylserine is a membranous target molecule of MARCKS.     

Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase

Mitochondrially bound dihydroorotate-ubiquinone oxidoreductase (dihydroorotate dehydrogenase, EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. Based on the recent functional expression of the complete rat dihydroorotate dehydrogenase by means of the baculovirus expression vector system in Trichoplusia ni cells, a procedure is described that allows the purification of baculovirus expressed enzyme protein fused to a carboxy-terminal tag of eight histidines. Extracts from mitochondria of Spodoptera frugiperda cells infected with the recombinant virus using Triton X-100 were loaded onto Ni2+-nitrilotriacetic acid agarose and histidine-tagged rat protein was selectively eluted with imidazole-containing buffer. In view of our previously published work, the quality of the electrophoretic homogenous rat enzyme was markedly improved; specific activity was 130-150 micromol dihydroorotate/min per milligram; and the stoichiometry of flavin content was 0.8-1.1 mol/mol protein. Efforts to generate mammalian dihydroorotate dehydrogenases with low production costs from bacteria resulted in successful overexpression of the carboxy-terminal-modified rat and human dihydroorotate dehydrogenase in XL-1 Blue cells. By employing the metal chelate affinity chromatography under native conditions, the histidine-tagged human enzyme was purified with a specific activity of 150 micromol/min/mg and the rat enzyme with 83 micromol/min/mg, respectively, at pH 8.0-8.1 optimum. Kinetic constants of the recombinant histidine-tagged rat enzyme from bacteria (dihydroorotate, Km = 14.6 micromol electron acceptor decylubiquinone, Km = 9.5 micromol) were close to those reported for the enzyme from insect cells, with or without the affinity tag. HPLC analyses identified flavin mononucleotide as cofactor of the rat enzyme; UV-vis and fluorometric analyses verified a flavin/protein ratio of 0.8-1.1 mol/mol. By spectral analyses of the functional flavin with the native human enzyme, the interaction of the pharmacological inhibitors Leflunomide and Brequinar with their target could be clarified as interference with the transfer of electrons from the flavin to the quinone. The combination of the bacterial expression system and metal chelate affinity chomatography offers an improved means to purify large quantities of mammalian membrane-bound dihydroorotate dehydrogenases which, by several criteria, possesses the same functional activities as non-histidine-tagged recombinant enzymes.     

Electrotransport purification and some characterization studies of vanadium metal

High-purity vanadium metal having an interstitial impurity content of <5 wt ppm and a resistance ratio,R _{300 K}/R _{4.2 K}, of >1880 has been prepared by an electrotransport technique. The lattice parameter of vanadium of this purity is 3.0269 +- 3Å. An extrapolated value for the room temperature resolved flow stress of vanadium free of C, O, and N was determined to be 0.41 kg/mm² on this material.     

Lipoxygenase activity in rat dermis and epidermis: partial purification and characterization

Lipoxygenase (LOX) activity in epidermis and dermis was distributed among microsomal and cytosolic fractions. The main products of polyunsaturated fatty acid metabolism were 12-hydroperoxy-cis-5,8,14, trans-10-eicosatetraenoic acid (12-HPETE), 15-hydroperoxy-cis-5,8,11, trans-13-eicosatetraenoic acid (15-HPETE) and 13-hydroxy-cis-9, trans-11-octadecadienoic acid (13-HOD). Enzyme activities were isolated from rat dermis and epidermis by ammonium sulphate precipitation, hydrophobic chromatography and gel filtration. In the dermis, activity was found at a molecular mass of 68 kDa, a pI of 4.6 and a Km of 50 microM. This activity was inhibited by known LOX inhibitors. The main reaction products indicated that this was 15-LOX. In the epidermis, activity was found in a fraction with a molecular mass of 68 kDa, a pI of 4.6 and a Km of 80 microM. Activity was inhibited by known LOX inhibitors whereas the reaction products indicated that this was 12-LOX. LOX activity in rat skin may involve one enzyme with dual regional specificities or may comprise two different enzymes.     

Urease from Staphylococcus saprophyticus: purification, characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420,000; it consisted of subunits with molecular weights of 72,400 (alpha), 20,400 (beta), 13,900 (gamma) in an estimated (alpha beta gamma)4 stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420,000 to 100,000. In the native enzyme, 4.09 (+/- 0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.     

Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization

The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.     

Dipeptidyl peptidase IV from human serum: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) in normal human serum was purified 14,400-fold with a 25% yield to homogeneity. The molecular weight of the purified enzyme was approximately 110,000 on SDS-PAGE, almost the same as that of human kidney membrane-bound DPP IV. No difference was found between the two enzymes enzymologically and immunologically, either in substrate specificity, susceptibility to inhibitors, or cross-reactivity with an anti-rat kidney DPP IV antibody, or in their ability to bind adenosine deaminase. However, the N-terminal amino acid sequence of serum DPP IV lacked the transmembrane domain of the membrane-bound enzyme and started at the 39th position, serine, from the N-terminus predicted from the cDNA nucleotide sequence. These results suggest that membrane-bound DPP IV loses its transmembrane domain upon release into the serum, and that its structure on the plasma membrane is not required for its binding to adenosine deaminase.     

Mechanistic studies of the biosynthesis of paratose: purification and characterization of CDP-paratose synthase

The 3,6-dideoxyhexoses can be found in the cell wall lipopolysaccharide of Gram-negative bacteria, where they have been shown to be the dominant antigenic determinants. All naturally occurring 3,6-dideoxyhexoses, with colitose as the only exception, are biosynthesized via a complex pathway that begins with CDP-d-glucose. Included in this pathway is CDP-paratose synthase, an essential enzyme in the formation of the 3,6-dideoxy sugars, CDP-paratose and CDP-tyvelose. Recently, the gene encoding CDP-paratose synthase in Salmonella typhi, rfbS, has been identified and sequenced [Verma, N., and Reeves, P. (1989) J. Bacteriol. 171, 5694-5701]. On the basis of this information, we have amplified the rfbS gene by polymerase chain reaction (PCR) from S. typhi and cloned this gene into a pET-24(+) vector. Expression and purification of CDP-paratose synthase have allowed us to fully characterize the catalytic properties of this enzyme, which is a homodimeric protein with a preference for NADPH over NADH. It catalyzes the stereospecific hydride transfer of the pro-S hydrogen from the C-4' position of the reduced coenzyme to C-4 of the substrate, CDP-3,6-dideoxy-D-glycero-D-glycero-4-hexulose. The overall equilibrium of this catalysis greatly favors the formation of the reduced sugar product and the oxidized coenzyme. Interestingly, this enzyme also exhibits a high affinity for NADPH with a much smaller dissociation constant (Kia) of 0.005 +/- 0.002 microM compared to the Km of 26 +/- 8 microM for NADPH. While this unusual property complicated the interpretation of the kinetic data, the kinetic mechanism of CDP-paratose synthase as explored by the combination of bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies is most consistent with a Theorell-Chance mechanism. The present study on CDP-paratose synthase, a likely new member of the short-chain dehydrogenase family, represents the first detailed characterization of this type of ketohexose reductase, many of which may share similar properties with CDP-paratose synthase.     

Expression, purification and characterization of GDP-D-mannose 4,6-dehydratase from Escherichia coli

Two separate N-terminal fragments of the 470-amino-acid Escherichia coli DnaB helicase, comprising residues 1-142 and 1-161, were expressed in E. coli. The proteins were extracted in a soluble fraction, purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly alpha-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The 1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.     

Maltose phosphorylase from Lactobacillus brevis: purification, characterization, and application in a biosensor for ortho-phosphate

With the goal to obtain maltose phosphorylase as a tool to determine ortho-phosphate, the enzyme from Lactobacillus brevis was purified to 98% by an expeditious FPLC-aided procedure which included anion exchange chromatography, gel filtration, and hydroxyapatite chromatography. The native maltose phosphorylase had a molecular mass of 196 kDa and consisted of two 88 kDa subunits. In isoelectric focusing two isoforms with pI values of 4.2 and 4.6 were observed. Maximum enzyme activity was obtained at 36 degrees C and pH 6.5 and was independent of pyridoxal 5'-phosphate. The apparent K(m) values with maltose and phosphate as substrates were 0.9 mmol l-1 and 1.8 mmol l-1, respectively. Maltose phosphorylase could be stored in 10 mM phosphate buffer pH 6.5 at 4 degrees C with a loss of activity of only 7% up to 6 months. The stability of the enzyme at high temperatures was enhanced significantly using additives like phosphate, citrate, and imidazole. The purified maltose phosphorylase was used as key enzyme in a phosphate sensor consisting of maltose phosphorylase and glucose oxidase. A detection limit of 0.1 microM phosphate was observed and the sensor response was linear in the range between 0.5 and 10 microM.     

Acid xylanase from yeast Cryptococcus sp. S-2: purification, characterization, cloning, and sequencing

A xylan-degrading enzyme produced by yeast Cryptococcus sp. S-2 was isolated and purified, and characterized as an endoxylanase (1,4-beta-D-xylan xylanohydrolase [EC 3.2.1.8]). We estimated the molecular weight and isoelectric point of purified xylanase (xyn-CS2) to be 22,000 and 7.4, respectively. This low-molecular-weight xylanase had an unusual pH optimum of 2.0, and showed 75% of maximal activity even at pH 1.0. An open reading frame of the cDNA specified 209 amino acids, including a putative signal peptide of 25 amino acids. The deduced amino acid sequence of xyn-CS2 shared significant similarities with the family-G xylanases of B. pumilus, C. acetobutylicum, T. reesei, and A. kawachii. Xyn-CS2 included two unique cysteine residues in a putative catalytic region, raising the possibility that these residues are at least partially responsible for its acidophilic nature.