Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.     

Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum

Lactobacillus plantarum 423, isolated from sorghum beer, produces a bacteriocin (plantaricin 423) which is inhibitory to several food spoilage bacteria and food-borne pathogens, including Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria spp. and Staphylococcus spp. Plantaricin 423 is resistant to treatment at 80 degrees C, but loses 50% of its activity after 60 min at 100 degrees C and 75% of its activity after autoclaving (121 degrees C, 15 min). Plantaricin 423 remains active after incubation at pH 1-10 and is inactivated when treated with pepsin, papain, alpha-chymotrypsin, trypsin and Proteinase K. Plantaricin 423 was partially purified and its size estimated at 3.5 kDa, as determined by tricine-SDS-PAGE. The mechanism of activity of plantaricin 423 is weakly bactericidal, as determined against Oenococcus oeni (previously Leuconostoc oenos). High DNA homology was obtained between the plasmid DNA of strain 423 and the pediocin PA-1 operon of Pediococcus acidilactici PAC 1.0, suggesting that plantaricin 423 is plasmid-encoded and related to the pediocin gene cluster.     

Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1

Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.     

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber

The involvement of mdr1a P-glycoprotein (P-gP) on the tissue distribution of the multidrug resistance-reversing agent SDZ PSC 833 was assessed by use of mdr1a (-/-) mice. The mdr1a (-/-) and wild-type mdr1a (+/+) mice received a 4-h constantrate i.v. infusion (2 micrograms/min) of [14C]SDZ PSC 833. Mice were sacrificed at 0, 0.5, 1, 2 and 4 h during infusion and at 0.5, 1, 3, 8 and 24 h after stopping the infusion. Blood and tissues were analyzed on total (14C) and parental SDZ PSC 833 concentrations. Mdr1a (-/-) mice exhibited increased SDZ PSC 833 accumulation in cerebrum, cerebellum and somewhat in testes and small intestine compared with the wild-type mice. The difference between mdr1a (-/-) and (+/+) brain (cerebrum and cerebellum) penetration depended on SDZ PSC 833 blood concentrations, because this cyclosporin analog apparently governs its own brain penetration by inhibiting the P-glycoprotein pump in mdr1a (+/+) mice. Thus the mdr1a (-/-)/(+/+) ratio of brain concentrations tended to decrease and increase at high and low blood concentrations, respectively. These findings clearly demonstrate the interaction of SDZ PSC 833 with the P-glycoprotein present at the blood-brain barrier. The SDZ PSC 833 distribution in other mdr1a P-glycoprotein-expressed tissues, as well as its metabolism and elimination, was not affected by the mdr1a gene disruption. This suggests that factors other than mdr1a P-gP are involved in the disposition of this multidrug resistance-reversing agent.     

Deoxyribonuclease I from rat urine: affinity purification, characterization, and immunochemical studies

Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-fold to apparent homogeneity with a 14% yield by affinity chromatography utilizing polyguanylic acid-agarose and DNA-cellulose. The purified enzyme preparation was found to contain no other detectable nucleases. Isoelectric focusing electrophoresis revealed that all six isoelectric forms of the enzyme had been purified, and the resulting bands all contained DNase I activity. Quantitative amino acid analysis and N-terminal amino acid sequencing were performed on the purified DNase I. The N-terminal sequence up to the 15th residue of the enzyme was identical to that of rat parotid DNase I. The enzyme was found to be a glycoprotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine, and at least 3 sialic acid residues per molecule. The isoelectric multiplicity of the enzyme was partly due to differences in the sialic acid content of the isoforms. Gel filtration on Superose 12 and electrophoresis on sodium dodecyl sulfate polyacrylamide gels indicated an approximate molecular mass for DNase I of 32 kDa. The enzyme had an optimum pH of 6.5 and required divalent cations such as Ca2+ for its activity. Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin. An antibody against the purified enzyme was found to be monospecific against rat urine and the pure antigen, and completely blocked the activity of the purified enzyme.     

Purification and characterization of trehalose phosphorylase from Catellatospora ferruginea

Trehalose phosphorylase was purified from the cell extracts of Catellatospora ferruginea. The enzyme had an apparent molecular weight of 400,000 by gel filtration and 98,000 by SDS-PAGE, suggesting that the enzyme was a tetramer. The enzyme was specific for trehalose in phosphorolysis and specific for beta-D-glucose 1-phosphate in synthesis. In addition to D-glucose, D-xylose and D-fucose were also possible sugar acceptors during synthesis. Phosphate ions were a key to the activity and stability of the enzyme, controlling the equilibrium of the reversible reaction and the heat stability of the enzyme. The enzyme was strongly inhibited by p-chloromercuribenzoate and pyridoxal phosphate. The enzyme was inactivated by heat or by storage frozen with ammonium chloride and lithium chloride.     

Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization

OBJECTIVE: To study the safety and efficacy of methylphenidate in children with the dual diagnosis of epilepsy and attention deficit hyperactivity disorder (ADHD). STUDY DESIGN: Thirty children, aged 6.4 to 16.4 years, with epilepsy and ADHD were studied during a 4-month period. During the initial 2 months of the study, the children were treated with antiepileptic drugs (AEDs) only, and for the remaining 2 months, methylphenidate was added at a morning dose of 0.3 mg/kg. They underwent neurologic assessment, brain computed tomography, IQ testing, and assessment with the Childhood Behavior Checklist at baseline before methylphenidate therapy. Electroencephalography, AED determinations, and the continuous-performance task (CPT) test were done at baseline and after 2 months of methylphenidate therapy. A double-blind, crossover design was used to compare the effects of methylphenidate versus placebo on an electroencephalogram, AED levels, and the CPT. On the 2 days of testing, the child received AEDs and a capsule containing either placebo or methylphenidate. RESULTS: None of the 25 children of this sample who were seizure free had attacks while taking methylphenidate. Of the 5 children with seizures, 3 had an increase in attacks, whereas the other 2 showed no change or a reduction. There were no significant changes in AED levels or electroencephalographic findings. Methylphenidate benefited 70% of children according to parental report; methylphenidate also enhanced performance on the CPT. Side effects of methylphenidate were mild and transient. CONCLUSION: Methylphenidate is effective in treating children with epilepsy and ADHD and safe in children who are seizure free. Caution is warranted for those still having seizures while receiving AED therapy.     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50,900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42 degrees C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 micrograms/ml) and not inhibited by antipain dihydrochloride (120 micrograms/ml), aprotinin (4 micrograms/ml), bestatin (80 micrograms/ml), chymostatin (50 micrograms/ml), E-64 (20 micrograms/ml), leupeptin (4 micrograms/ml), Pefabloc SC (2000 micrograms/ml), pepstatin (4 micrograms/ml), phosphoramidon (660 micrograms/ml), or phenylmethylsulfonyl fluoride (400 micrograms/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.     

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.     

Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus

The finding of heat-stable enzymes or the engineering of moderately thermostable enzymes into more stable ones by random or site-directed mutagenesis has become a main priority of modern biotechnology. We report here for the first time a heat-stable phytase able to withstand temperatures up to 100 degrees C over a period of 20 min, with a loss of only 10% of the initial enzymatic activity. The gene (phyA) encoding this heat-stable enzyme has been cloned from Aspergillus fumigatus and overexpressed in Aspergillus niger. The enzyme showed high activity with 4-nitrophenyl phosphate at a pH range of 3 to 5 and with phytic acid at a pH range of 2.5 to 7.5.     

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.     

Intercellular signaling in Stigmatella aurantiaca: purification and characterization of stigmolone, a myxobacterial pheromone

The myxobacterium Stigmatella aurantiaca passes through a life cycle that involves formation of a multicellular fruiting body as the most complex stage. An early step in this differentiation process depends on a signal factor secreted by the cells when nutrients become limited. The formation of a fruiting body from a small cell population can be accelerated by addition of this secreted material. The bioactive compound was found to be steam volatile. It was purified to homogeneity by steam distillation followed by reversed-phase and normal-phase HPLC. The pheromone was named stigmolone, in accordance with the structure 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, as determined by NMR and mass spectrometry. Stigmolone represents a structurally unique and highly bioactive prokaryotic pheromone that is effective in the bioassay at 1 nM concentration.     

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.     

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.     

Dipeptidyl peptidase IV from porcine seminal plasma: purification, characterization, and N-terminal amino acid sequence

Dipeptidyl peptidase IV (DPP IV) was purified to homogeneity from porcine seminal plasma by polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was calculated to be approximately 290,000 on PAGE in the absence of sodium dodecyl sulfate (SDS) and 310,000 on Sephacryl S-300 HR column chromatography, and to be 115,000 and 105,000 on SDS-PAGE in the absence and presence of beta-mercaptoethanol. The enzyme is suggested to be composed of three identical subunits. The enzyme rapidly hydrolyzed the substrate Gly-Pro-MCA, and weakly the substrate Lys-Ala-MCA. It was strongly inhibited by diisopropylphosphofluoridate (DFP), and moderately by both phenylmethyl-sulfonyl fluoride (PMSF) and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). It was also strongly inhibited by zinc ion. The amino acid sequence of the first 18 residues of the enzyme was Asn-Lys-Gly-Thr-Asp-Asp-Ala-Ala-Ala-Asp-Ser-Arg-Arg- Thr-Tyr-Thr-Leu-Thr-. This sequence was highly homologous to the sequences in the rear of the transmembrane site of human and rat liver DPP IVs and mouse thymus DPP IV. The native DPP IV is suggested to be released into the seminal plasma after the cleavage of the hydrophobic N-terminal domain by chymotrypsin-like or pepsin-like enzymes. Other properties of DPP IV including kinetic parameters, pH stability and heat stability were characterized.     

Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase

The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D-Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala-D-Ser rather than a D-Ala-D-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent D-Ala-D-Ser ligase activity. The kcat/Km2 (Km2 = Km for D-Ser or C-terminal D-Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. VanC2 also has substantial D-Ala-D-Asn ligase activity (kcat/Km2 = 74 mM-1min-1).