Cloning and expression of the algL gene, encoding the Azotobacter chroococcum alginate lyase: purification and characterization of the enzyme.
(9/6642)
The alginate lyase-encoding gene (algL) of Azotobacter chroococcum was localized to a 3.1-kb EcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme. (+info)
An improved separation procedure for nucleoside monophosphates on polyethyleneimine-(PEI-)cellulose thin layers.
(10/6642)
A procedure is described for the two-dimensional separation of the 4 major and 16 modified nucleoside-(5') monophosphates on anion-exchange thin layers of polyethyleneimine- (PEI-)cellulose. The method, which is simple and less time-consuming than existing partition chromatographic methods, may be used for the identification of 5'-termini of RNA and RNA fragments. (+info)
Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins.
(11/6642)
The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, we have isolated a native Gbeta5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gbeta5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocked the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor. (+info)
Evaluation of a new method for the analysis of free catecholamines in plasma using automated sample trace enrichment with dialysis and HPLC.
(12/6642)
BACKGROUND: Analysis of urinary free catecholamines was automated recently, but analysis of plasma samples posed special difficulties. The present study was undertaken to evaluate a new method for the automated analysis of plasma catecholamines. METHODS: The procedure is based on an improved sample handling system that includes dialysis and sample clean-up on a strong cation trace-enrichment cartridge. The catecholamines norepinephrine, epinephrine, and dopamine are then separated by reversed-phase ion-pair chromatography and quantified by electrochemical detection. RESULTS: Use of a 740- microL sample is required to give the catecholamine detection limit of 0.05 nmol/L and analytical imprecision (CV) between 1.1% and 9.3%. The assay can be run unattended, although >12 h of analysis time is not recommended without cooling of the autosampler rack. Comparison (n = 68) of the automated cation-exchange clean-up with the well-established manual alumina procedure gave excellent agreement (mean, 3.78 +/- 2.76 and 3.8 +/- 2.89 nmol/L for norepinephrine and 0.99 +/- 1.72 and 1.08 +/- 1.78 nmol/L for epinephrine). Hemodialysis had no clear effect on plasma norepinephrine. Epinephrine concentrations were similar (0.05 < P < 0.1) in chronic renal failure patients (0.24 +/- 0.3 nmol/L; n = 15) and healthy controls (0.5 +/- 0.24 nmol/L; n = 31). Dopamine was not quantified, being usually <0.2 nmol/L. CONCLUSION: The availability of such a fully automated procedure should encourage the more widespread use of plasma catecholamine estimation, e.g., after dialysis, exercise, or trauma/surgery and in the investigation of catecholamine-secreting tumors, particularly in the anuric patient. (+info)
Purification and characterization of a mitochondrial thymine glycol endonuclease from rat liver.
(13/6642)
Mitochondrial DNA is exposed to oxygen radicals produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions in mitochondrial DNA may lead to structural genomic alterations, mitochondrial dysfunction, and associated degenerative diseases. The pyrimidine hydrate thymine glycol, one of many oxidative lesions, can block DNA and RNA polymerases and thereby exert negative biological effects. Mitochondrial DNA repair of this lesion is important to ensure normal mitochondrial DNA metabolism. Here, we report the purification of a novel rat liver mitochondrial thymine glycol endonuclease (mtTGendo). By using a radiolabeled oligonucleotide duplex containing a single thymine glycol lesion, damage-specific incision at the modified thymine was observed upon incubation with mitochondrial protein extracts. After purification using cation exchange, hydrophobic interaction, and size exclusion chromatography, the most pure active fractions contained a single band of approximately 37 kDa on a silver-stained gel. MtTGendo is active within a broad KCl concentration range and is EDTA-resistant. Furthermore, mtTGendo has an associated apurinic/apyrimidinic-lyase activity. MtTGendo does not incise 8-oxodeoxyguanosine or uracil-containing duplexes or thymine glycol in single-stranded DNA. Based upon functional similarity, we conclude that mtTGendo may be a rat mitochondrial homolog of the Escherichia coli endonuclease III protein. (+info)
Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning.
(14/6642)
Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys152 and Ile153. The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorso-ventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid. (+info)
Structure and anticoagulant activity of sulfated fucans. Comparison between the regular, repetitive, and linear fucans from echinoderms with the more heterogeneous and branched polymers from brown algae.
(15/6642)
Sulfated fucans are among the most widely studied of all the sulfated polysaccharides of non-mammalian origin that exhibit biological activities in mammalian systems. Examples of these polysaccharides extracted from echinoderms have simple structures, composed of oligosaccharide repeating units within which the residues differ by specific patterns of sulfation among different species. In contrast the algal fucans may have some regular repeating structure but are clearly more heterogeneous when compared with the echinoderm fucans. The structures of the sulfated fucans from brown algae also vary from species to species. We compared the anticoagulant activity of the regular and repetitive fucans from echinoderms with that of the more heterogeneous fucans from three species of brown algae. Our results indicate that different structural features determine not only the anticoagulant potency of the sulfated fucans but also the mechanism by which they exert this activity. Thus, the branched fucans from brown algae are direct inhibitors of thrombin, whereas the linear fucans from echinoderms require the presence of antithrombin or heparin cofactor II for inhibition of thrombin, as reported for mammalian glycosaminoglycans. The linear sulfated fucans from echinoderms have an anticoagulant action resembling that of mammalian dermatan sulfate and a modest action through antithrombin. A single difference of one sulfate ester per tetrasaccharide repeating unit modifies the anticoagulant activity of the polysaccharide markedly. Possibly the spatial arrangements of sulfate esters in the repeating tetrasaccharide unit of the echinoderm fucan mimics the site in dermatan sulfate with high affinity for heparin cofactor II. (+info)
Rapid purification of membrane extrinsic F1-domain of chloroplast ATP synthase in monodisperse form suitable for 3D-crystallization.
(16/6642)
A new chromatographic procedure for purification of the membrane extrinsic F1-domain of chloroplast ATP synthase is presented. The purification is achieved by a single anion exchange chromatography step. Determination of the enzyme-bound nucleotides reveals only 1 mole of ADP per complex. The purified enzyme shows a latent Ca(2+)-dependent ATPase activity of 1.0 mumol.mg-1 min-1 and a Mg(2+)-dependent activity of 4.4 mumol.mg-1 .min-1. Both activities are increased up to 8-10-fold after dithiothreitol activation. Analysis of the purified F1-complex by SDS/PAGE, silver staining and immunoblotting revealed that the preparation is uncontaminated by fragmented subunits or ribulose-1,5-bisphosphate carboxylase/oxygenase. Gel filtration experiments indicate that the preparation is homogenous and monodisperse. In order to determine the solubility minimum of the purified F1-complex the isoelectric point of the preparation was calculated from pH mapping on ion exchange columns. In agreement with calculations based on the amino acid sequence, a slightly acidic pI of 5.7 was found. Using ammonium sulphate as a precipitant the purified CF1-complex could be crystallized by MicroBatch. (+info)