A novel 35 kDa frog liver acid metallophosphatase.
(17/4151)
The lower molecular weight (35 kDa) acid phosphatase from the frog (Rana esculenta) liver is a glycometalloenzyme susceptible to activation by reducing agents and displaying tartrate and fluoride resistance. Metal chelators (EDTA, 1,10-phenanthroline) inactivate the enzyme reversibly in a time- and temperature-dependent manner. The apoenzyme is reactivated by divalent transition metal cations, i. e. cobalt, zinc, ferrous, manganese, cadmium and nickel to 130%, 75%, 63%, 62%, 55% and 34% of the original activity, respectively. Magnesium, calcium, cupric and ferric ions were shown to be ineffective in this process. Metal analysis by the emission spectrometry method (inductively coupled plasma-atomic emission spectrometry) revealed the presence of zinc, iron and magnesium. The time course of the apoenzyme reactivation, the stabilization effect and the relatively high resistance to oxidizing conditions indicate that the zinc ion is crucial for the enzyme activity. The presence of iron was additionally confirmed by the visible absorption spectrum of the enzyme with a shoulder at 417 nm and by the electron paramagnetic resonance line of high spin iron(III) with geff of 2.4. The active center containing only zinc or both zinc and iron ions is proposed. The frog liver lower molecular weight acid phosphatase is a novel metallophosphatase of lower vertebrate origin, distinct from the mammalian tartrate-resistant, purple acid phosphatases. (+info)
Purification, characterization and cDNA cloning of an endo-exonuclease from the basidiomycete fungus Armillaria mellea.
(18/4151)
We have purified an endo-exonuclease from the fruiting body of the basidiomycete fungus Armillaria mellea by using an ethanol fractionation step, followed by two rounds of column chromatography. The enzyme had an apparent molecular mass of 17500 Da and was shown to exist as a monomer by gel-filtration analysis. The nuclease was active on both double-stranded and single-stranded DNA but not on RNA. It was optimally active at pH8.5 and also exhibited a significant degree of thermostability. Three bivalent metal ions, Mg2+, Co2+ and Mn2+, acted as cofactors in the catalysis. It was also inhibited by high salt concentrations: activity was completely abolished at 150 mM NaCl. The nuclease possessed both endonuclease activity on supercoiled DNA and a 3'-5' (but not a 5'-3') exonuclease activity. It generated 5'-phosphomonoesters on its products that, after a prolonged incubation, were hydrolysed to a mixture of free mononucleotides and small oligonucleotides ranging in size from two to eight bases. Elucidation of its N-terminal amino acid sequence permitted the cDNA cloning of the A. mellea nuclease via a PCR-based approach. Peptide mapping of the purified enzyme generated patterns consistent with the amino acid sequence coded for by the cloned cDNA. A BLAST search of the SwissProt database revealed that A. mellea nuclease shared significant amino acid similarity with two nucleases from Bacillus subtilis, suggesting that the three might constitute a distinct class of nucleolytic enzymes. (+info)
Thermostable aminopeptidase from Pyrococcus horikoshii.
(19/4151)
From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N-terminal amino acid from a variety of substrates including both N-blocked and non-blocked peptides. The enzyme was stable at 90 degrees C, with the optimum temperature over 90 degrees C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N(alpha)-acylated residues in short peptide sequence analysis at high temperatures. (+info)
Mycobacterium sp. as a possible cause of hypersensitivity pneumonitis in machine workers.
(20/4151)
Hypersensitivity pneumonitis (HP) in workers exposed to metal removal fluids (MRFs) is increasing. This study supports the hypothesis that aerosolized mycobacteria colonizing the MRFs likely cause the disease. Three case studies of HP outbreaks among metal workers showed potentially high exposures to a rare and newly proposed Mycobacterium species. Retrospective review of samples submitted to our laboratory showed an association between presence of mycobacteria and HP. (+info)
Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme.
(21/4151)
The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry. (+info)
Zinc is required in pyrrolidine dithiocarbamate inhibition of NF-kappaB activation.
(22/4151)
Pyrrolidine dithiocarbamate (PDTC) is a potent inhibitor of nuclear factor kappa B (NF-kappaB) activation. PDTC inhibited basal NF-kappaB activity of endothelial cells. PDTC, however, failed to inhibit basal NF-kappaB activity after withdrawal of serum in the media, and the inhibitory effect of PDTC could be restored by addition of zinc. When various preparations of metal ion-EDTA were tested with PDTC in serum-containing media, only Zn-EDTA failed to block the inhibitory effect of PDTC. The dependence on zinc was also noted in PDTC inhibition of NF-kappaB stimulated by TNF alpha. These facts suggest that zinc is required for PDTC inhibition of NF-kappaB activation. (+info)
Isolation and primary characterization of an amidase from Rhodococcus rhodochrous.
(23/4151)
Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312. (+info)
Reduction of aneurysm clip artifacts on CT angiograms: a technical note.
(24/4151)
We describe a head tilt technique for use with CT angiography that reduces beam-hardening artifacts in patients with aneurysm clips. This simple maneuver directs the artifacts away from pertinent anatomy, thus increasing the chances for diagnostic accuracy. No significant changes in the CT angiographic protocol are required, and the maneuver can easily be combined with other artifact-minimizing strategies. (+info)