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G S Bild

Publications and source records attributed to G S Bild.

15 recordsLinked to original sources
PubMed1994

Renaturation and purification of human tissue factor pathway inhibitor expressed in recombinant E. coli.

Tissue factor pathway inhibitor is an inhibitor of the extrinsic coagulation pathway. Evaluation of the pharmacological effects of tissue factor pathway inhibitor in animal models has been limited by the high cost and low availability of mammalian tissue culture produced protein. In order to circumvent this obstacle, a 277-amino-acid nonglycosylated tissue factor pathway inhibitor variant possessing an N-terminal alanine was expressed in recombinant E. coli using the tac promoter expression system. High-level expression in recombinant E. coli resulted in the accumulation of ala-tissue factor pathway inhibitor in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8 M urea, purification of the full-length molecule by cation exchange chromatography, and renaturation in 6 M urea. Fractionation of crude refold mixtures using cation exchange chromatography yielded a purified nonglycosylated tissue factor pathway inhibitor possessing in vitro prothrombin time activity comparable to inhibitor purified from mammalian cell lines.

Amino Acid Sequence↗
PubMed1994

Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock.

Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.

Animals↗
PubMed1994

Refold and characterization of recombinant tissue factor pathway inhibitor expressed in Escherichia coli.

Human tissue factor pathway inhibitor (TFPI) was expressed in E. coli as a non-glycosylated protein with an additional alanine attached to the aminoterminus of the wild type molecule. High-level expression was obtained with pMON6875, a plasmid containing a tac promoter, Gene 10 leader from bacteriophage T7, methionine-alanine-TFPI coding sequence, and the p22 transcriptional terminator. In this system, TFPI accounted for about 5-10% of the total cell protein. The inclusion bodies containing. TFPI were sulfitolyzed, purified by anion-exchange chromatography, refolded through a disulfide interchange reaction, and further fractionated by Mono S cation exchange chromatography. The Mono S resin resolved a peak of highly active TFPI from relatively inactive and possibly misfolded molecules. The E. coli TFPI was shown to be about two-fold more active, on a molar basis, than full-length human SK hepatoma TFPI in a tissue factor-induced clotting assay in human plasma.

Base Sequence↗
PubMed1993

High-level expression and purification of human leukotriene A4 hydrolase from insect cells infected with a baculovirus vector.

Leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation. Leukotriene A4 hydrolase (LTA4H) is an epoxide hydrolase, catalyzing the hydration of LTA4 to LTB4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline. The cDNA for LTA4H was cloned using oligonucleotide-directed amplification of the cDNA sequence by polymerase chain reaction and by oligonucleotide-based screening of a bacteriophage lambda gt11 cDNA library derived from human placental tissue. High levels of biologically active LTA4H were expressed in cultured Spodoptera frugiperda insect cells infected with a baculovirus expression vector containing the LTA4H cDNA. Expression levels were approximately 100 mg per liter of cell-free culture media. LTA4H was recovered from the medium and purified to > 95% purity by ion-exchange and gel-filtration chromatography, with an overall yield of 76%. LTA4H produced by insect cells exhibits both hydrolase and aminopeptidase activities and has kinetic properties similar to those reported for enzyme isolated from human lung. Two major isoforms, with pI's of 5.3 and 5.1, were isolated by preparative chromatofocusing chromatography. NH2-terminal sequence analysis revealed that the two different by an NH2-terminal blocking group. Electrospray ionization mass spectrometry indicates that the two isoforms differ by a molecular mass of 42, indicating that the blocking group is an acetyl group.

Acetylation↗
PubMed1988

Site-directed mutagenesis of Petunia hybrida 5-enolpyruvylshikimate-3-phosphate synthase: Lys-23 is essential for substrate binding.

Chemical modification of Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase, a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine), with pyridoxal 5'-phosphate suggested that Lys-22 (equivalent to Lys-23 of the Petunia hybrida enzyme) is a potential active site residue (Huynh, Q. K., Kishore, G. M., and Bild, G. S. (1988) J. Biol. Chem. 263, 735-739). To investigate the possible role of this residue in the reaction mechanism, we have used site-directed mutagenesis to replace Lys-23 of the P. hybrida enzyme with 3 other amino acid residues: Ala, Glu, and Arg. Analysis of these mutant enzymes indicates that of these only the Lys-23 to Arg mutant enzyme is active; the other two replacements (Ala and Glu) result in inactivation of the enzyme. Two of the mutant enzymes (Lys-23 to Arg and Ala) were purified to homogeneity and characterized. The purified Lys-23 to Arg mutant enzyme is less sensitive than the wild type enzyme to pyridoxal 5'-phosphate. It showed identical Km values for substrates and a 5-fold higher I50 value for glyphosate in comparison with those from the wild type enzyme. Binding studies using fluorescence measurements revealed that the substrate shikimate 3-phosphate and glyphosate were able to bind the purified Lys-23 to Arg mutant enzyme but not to the purified catalytically inactive Lys-23 to Ala mutant enzyme. The above results suggest that the cationic group at position 23 of the enzyme may play an important role in substrate binding.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1988

5-Enolpyruvyl shikimate 3-phosphate synthase from Escherichia coli. Identification of Lys-22 as a potential active site residue.

5-Enolpyruvyl shikimate 3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enolpyruvyl shikimate 3-phosphate and inorganic phosphate. The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine). In order to determine the role of lysine residues in the mechanism of action of this enzyme as well as in its inhibition by glyphosate, chemical modification studies with pyridoxal 5'-phosphate were undertaken. Incubation of the enzyme with the reagent in the absence of light resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo first-order and saturation kinetics with Kinact of 45 microM and a maximum rate constant of 1.1 min-1. The inactivation rate increased with increase in pH, with a titratable pK of 7.6. Activity of the inactive enzyme was restored by addition of amino thiol compounds. Reaction of enzyme with pyridoxal 5'-phosphate was prevented in the presence of substrates or substrate plus glyphosate, an inhibitor of the enzyme. Upon 90% inactivation, approximately 1 mol of pyridoxal 5'-phosphate was incorporated per mol of enzyme. The azomethine linkage between pyridoxal 5'-phosphate and the enzyme was reduced by NaB3H4. Tryptic digestion followed by reverse phase chromatographic separation resulted in the isolation of a peptide which contained the pyridoxal 5'-phosphate moiety as well as 3H label. By amino acid sequencing of this peptide, the modified residue was identified as Lys-22. The amino acid sequence around Lys-22 is conserved in bacterial, fungal, as well as plant enzymes suggesting that this region may constitute a part of the enzyme's active site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1984

Detection of beta-carbanion formation during kynurenine hydrolysis catalyzed by Pseudomonas marginalis kynureninase.

2-Amino-4-hydroxy-4-phenylbutyric acid has been shown to be formed during the Pseudomonas marginalis kynureninase-catalyzed hydrolysis of kynurenine in the presence of benzaldehyde and pyridoxal phosphate. The formation of 2-amino-4-hydroxy-4-phenylbutyric acid is the first demonstration, to our knowledge, of the controlled trapping of an amino acid beta-carbanion generated either chemically or enzymatically, and is perhaps the best empirical evidence to date that enzyme mechanisms can proceed through a beta-carbanionic intermediate. The lifetime of the beta-carbanionic alanyl intermediate generated by kynureninase is of sufficient duration to allow reaction with benzaldehyde. Other aromatic, but no aliphatic, aldehydes will undergo electrophilic addition with kynureninase-generated beta-carbanionic alanyl intermediates to form the corresponding amino acid.

Benzaldehydes↗
PubMed1983

Facile oxygen exchanges of phosphoenolpyruvate and preparation of [18O]phosphoenolpyruvate.

Phosphoenolpyruvate when heated in acidic solution exchanges its phosphoryl and carboxyl oxygens rapidly and its enolic oxygen much more slowly with oxygens from water. The incorporation of 18O into phosphoenolpyruvate was measured by gas chromatography-mass spectrometry and phosphorus-31 nuclear magnetic resonance after heating in H218O at 98 degrees C. The rates of exchange of all six oxygens of phosphoenolpyruvate with water increase with increasing acidity, and the phosphoryl oxygens exchange more rapidly than the carboxyl oxygens. The rate of exchange of each oxygen of the phosphoryl group is 16-fold greater than the hydrolysis rate at 1 N HCl. This provides a simple and useful method for the synthesis of [18O]phosphoenolpyruvate highly enriched in its phosphoryl-group oxygens. An enrichment of 89% was obtained with a 50% yield. The [18O]-phosphoenolpyruvate showed a binomial distribution of 18O in the phosphoryl-group oxygens. The exchange may be explained by the reversible formation of a transient cyclic phosphate and, for exchange of the enolic oxygen, a transient acyl phosphate. Preparation of [18O]phosphoenolypyruvate from [18O]Pi by a chemical synthesis from beta-chlorolactate was not satisfactory because of drastic loss of 18O during the procedures used. Some loss of 18O also occurred during an enzymic synthesis with KCNO, [18O]Pi, carbamate kinase, and pyruvate kinase.

Gas Chromatography-Mass Spectrometry↗
PubMed1980

Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction.

A new approach for assessing if catalytic cooperativity may occur between subunits has been applied to Escherichia coli glutamine synthetase. The extent of oxygen exchange between bound [18O]glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations. This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine. At 1000 microM NH4+ no detectable reversals occurred, and only one glutamate oxygen appeared in product phosphate as required by the reaction mechanism. However, at 10 microM NH4+ over 15 reversals of bound glutamine formation occurred. Controls showed that under the experimental conditions free glutamine does not become significantly involved in exchange and, therefore, the reversal of the oxygen exchange steps appears to be limited to bound glutamine. In contrast to the effect seen with NH4+, adenosine 5'-triphosphate concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly. These findings are interpreted as showing that NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange. The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at one catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations.

Adenosine Triphosphate↗
PubMed1980

Subunit interaction during catalysis. ATP modulation of catalytic steps in the succinyl-CoA synthetase reaction.

A new approach for assessing of catalytic cooperativity may occur between subunits has been applied to succinyl-CoA synthetase. This is based on the extent of oxygen exchange between medium [18O]Pi and succinate per molecule of ATP cleaved during steady state succinyl-CoA synthesis. Suitable traps are used to remove succinyl-CoA and ADP as soon as they are released to the medium. With the Escherichia coli enzyme, which has an alpha 2 beta 2 structure, a pronounced increase in oxygen exchange per ATP cleaved occurs as ATP concentration is lowered. In contrast, when the CoA concentration is varied, the oxygen exchange per molecule of product formed remains constant. Also, with the pig heart enzyme, which is shown to retain its alpha beta structure during catalysis and thus has only one catalytic site, no modulation of oxygen exchange by ATP concentration is observed. These experimental findings show that the binding of an ATP either promotes the dissociation of bound succinyl-CoA or decreases its participation in exchange. Measurement of the distribution of [18O]Pi species found as exchange occurs shows that only one catalytic sequence is involved in exchange at various ATP concentrations. These observations along with other controls and results eliminate most other explanations of the ATP modulation of the exchange and suggest that binding of ATP at one catalytic site promotes catalytic site promotes catalytic events at an alternate catalytic site.

Adenosine Triphosphate↗
PubMed1978

Biosynthesis of a prostaglandin by a plant enzyme.

Lipoxygenase-2 from soybeans catalyzes the oxygenation of arachidonic acid to form significant amounts of a prostaglandin product. Results obtained with the dithionite-reduced derivatized product upon combined gas chromatography-mass spectroscopy are consistent with structures of the stereoisomers of 9,11,15-trihydroxyprosta-5,13-dienoic acid. A portion of the material formed by reduction of the enzyme product with dithionite reacts positively in a radioimmunoassay against rabbit anti-prostaglandin F2alpha antibody. This appears to be the first report of the synthesis of a prostaglandin by a non-animal enzyme.

Arachidonic Acids↗