scholarly journals The enzymic degradation of alkyl-substituted gentisates, maleates and malates

1971 ◽  
Vol 122 (1) ◽  
pp. 29-40 ◽  
Author(s):  
D. J. Hopper ◽  
P. J. Chapman ◽  
S. Dagley
Keyword(s):  
Dimethyl Ester ◽  
Fission Products ◽  
Gas Liquid Chromatography ◽  
Benzene Nucleus ◽  
Synthetic Compound ◽  
Intact Cells ◽  
General Sequence ◽  
Fluorescent Pseudomonas ◽  
Cell Extracts ◽  
Ring Fission

1. Cell-free extracts, prepared from a non-fluorescent Pseudomonas grown on m-cresol, oxidized gentisate and certain alkyl-substituted gentisates with the consumption of 1 mol of oxygen and the formation of 1 mol of pyruvate from 1 mol of substrate. 2. In addition to pyruvate, malate was formed from gentisate; citramalate was formed from 3-methylgentisate and 4-methylgentisate; 2,3-dimethylmalate was formed from 3,4-dimethylgentisate. 3. One enantiomer, d-(-)-citramalate, was formed enzymically from 3-methylgentisate, 4-methylgentisate and citraconate. l-(+)-Citramalate was formed from mesaconate by the same extracts. When examined as its dimethyl ester by gas–liquid chromatography, enzymically formed 2,3-dimethylmalate showed the same behaviour as one of the two racemates prepared from the synthetic compound. 4. Maleate, citraconate and 2,3-dimethylmaleate were rapidly hydrated by cell extracts, but ethylfumarate and 2,3-dimethylfumarate were not attacked. 5. Cell extracts oxidized 1,4-dihydroxy-2-naphthoate to give pyruvate and phthalate. 6. Alkylgentisates were oxidized by a gentisate oxygenase (EC 1.13.1.4) present in Pseudomonas 2,5. The ring-fission products were attacked by maleylpyruvase, but not by fumarylpyruvase, and their u.v.-absorption spectra were those expected for alkyl-substituted maleylpyruvates. 7. When supplemented with ATP, CoA, succinate and Mg2+ ions, an enzyme system from cells grown with 2,5-xylenol formed pyruvate from d- but not from l-citramalate. Extracts from cells grown with dl-citramalate or with itaconate attacked both d- and l-citramalate; other alkylmalates were cleaved in similar fashion to give pyruvate or 2-oxobutyrate. 8. These results accord with a general sequence of reactions in which the benzene nucleus of an alkylgentisate is cleaved to give an alkyl-substituted maleylpyruvate. The ring-fission products are hydrolysed to give pyruvate, plus alkylmalic acids which then undergo aldol fissions, probably as their CoA esters. In Pseudomonas 2,5 several homologous sequences of this general type appear to be catalysed by a single battery of enzymes with broad substrate specificities, whereas the metabolic capabilities of the fluorescent Pseudomonas 3,5 are more restricted. 9. Intact cells of both organisms metabolize d-malic acid by reactions that have not been elucidated, but are different from those which degrade alkylmalates.

scholarly journals Bacterial metabolism of 5-aminosalicylic acid. Initial ring cleavage

1992 ◽  
Vol 282 (3) ◽  
pp. 675-680 ◽  
Author(s):  
A Stolz ◽  
B Nörtemann ◽  
H J Knackmuss
Keyword(s):  
Bacterial Metabolism ◽  
Ring Cleavage ◽  
Pseudomonas Sp ◽  
Growth Substrate ◽  
Aminosalicylic Acid ◽  
Intact Cells ◽  
Cell Extracts ◽  
Ring Fission ◽  
Stoichiometric Reaction ◽  
Neutral Conditions

The metabolism of 5-aminosalicylate (5AS) by a bacterial strain, Pseudomonas sp. BN9, was studied. Intact cells of Pseudomonas sp. BN9 grown with 5AS oxidized 5AS and 2,5-dihydroxybenzoate (gentisate), whereas cells grown with gentisate oxidized only the growth substrate of all substituted salicylates tested. Cell extracts from Pseudomonas sp. BN9 catalysed the stoichiometric reaction of 1 mol of oxygen with 1 mol of 5AS to a metabolite with an intense u.v.-absorption maximum at 352 nm (pH 8.0). This metabolite was accumulated under neutral conditions, but was rapidly destroyed at acid pH. It was identified by m.s. and acid-catalysed deamination to fumarylpyruvate (trans-2,4-dioxohept-5-enedioic acid) as cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, thus demonstrating direct cleavage of the monohydroxylated substrate 5AS to a non-aromatic ring-fission product. The enzyme responsible for conversion of 5AS was shown to be Fe(II)-dependent and to be distinct from gentisate 1,2-dioxygenase in strain BN9.

scholarly journals Endothelial cell-derived heparan sulfate binds basic fibroblast growth factor and protects it from proteolytic degradation.

1988 ◽  
Vol 107 (2) ◽  
pp. 743-751 ◽  
Author(s):  
O Saksela ◽  
D Moscatelli ◽  
A Sommer ◽  
D B Rifkin
Keyword(s):  
Growth Factor ◽  
Heparan Sulfate ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Deae Cellulose ◽  
Proteolytic Degradation ◽  
Fibroblast Growth ◽  
Intact Cells ◽  
High Affinity ◽  
Cell Extracts

Cultured bovine capillary endothelial (BCE) cells were found to synthesize and secrete high molecular mass heparan sulfate proteoglycans and glycosaminoglycans, which bound basic fibroblast growth factor (bFGF). The secreted heparan sulfate molecules were purified by DEAE cellulose chromatography, followed by Sepharose 4B chromatography and affinity chromatography on immobilized bFGF. Most of the heparinase-sensitive sulfated molecules secreted into the medium by BCE cells bound to immobilized bFGF at low salt concentrations. However, elution from bFGF with increasing salt concentrations demonstrated varying affinities for bFGF among the secreted heparan sulfate molecules, with part of the heparan sulfate requiring NaCl concentrations between 1.0 and 1.5 M for elution. Cell extracts prepared from BCE cells also contained a bFGF-binding heparan sulfate proteoglycan, which could be released from the intact cells by a short proteinase treatment. The purified bFGF-binding heparan sulfate competed with 125I-bFGF for binding to low-affinity binding sites but not to high-affinity sites on the cells. Heparan sulfate did not interfere with bFGF stimulation of plasminogen activator activity in BCE cells in agreement with its lack of effect on binding of 125I-bFGF to high-affinity sites. Soluble bFGF was readily degraded by plasmin, whereas bFGF bound to heparan sulfate was protected from proteolytic degradation. Treatment of the heparan sulfate with heparinase before addition of plasmin abolished the protection and resulted in degradation of bFGF by the added proteinase. The results suggest that heparan sulfate released either directly by cells or through proteolytic degradation of their extracellular milieu may act as carrier for bFGF and facilitate the diffusion of locally produced growth factor by competing with its binding to surrounding matrix structures. Simultaneously, the secreted heparan sulfate glycosaminoglycans protect the growth factor from proteolytic degradation by extracellular proteinases, which are abundant at sites of neovascularization or cell invasion.

Cytoplasmic structure and contractility: the solation-contraction coupling hypothesis

1982 ◽  
Vol 299 (1095) ◽  
pp. 185-197 ◽  
Keyword(s):  
Quantitative Analysis ◽  
Dictyostelium Discoideum ◽  
Calcium Ions ◽  
Soluble Extract ◽  
Intact Cells ◽  
Contraction Coupling ◽  
Cell Extracts ◽  
Cytoplasmic Structure ◽  
Cytological Features ◽  
Amoeboid Cells

We have briefly described our studies of cytoskeletal and contractile elements in intact cells, in cell extracts, and in mixtures of purified proteins. Changes in the concentration of calcium and of protons have been found to modulate both gelation and contraction in all of these preparations. The distribution of calcium, protons and actin has been studied in intact amoeboid cells. Using these results, we have refined our working model of the relation of cytoskeletal and contractile proteins: the solation-contraction coupling hypothesis. The model is also supported by quantitative analysis of the rates of contraction in a soluble extract of Dictyostelium discoideum amoebae allowed to gel in a capillary and stimulated by the addition of calcium ions at one end. A plausible interpretation of the most prominent cytological features of amoeboid locomotion is obtained by application of the principles of our model. In addition, we propose that the solation-contraction coupling hypothesis may be useful in further study of a variety of motile phenomena observed in many types of cells.

scholarly journals Phosphorylation of lymphocyte myosin catalyzed in vitro and in intact cells.

1982 ◽  
Vol 93 (2) ◽  
pp. 261-268 ◽  
Author(s):  
M Fechheimer ◽  
J J Cebra
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Intact Cells ◽  
Surface Immunoglobulin ◽  
Cell Extracts ◽  
Lymphocyte Populations ◽  
Immunologic Reactions

Myosin has been isolated from guinea pig B-lymphocytic leukemia cells (L2C). The myosin has been enzymatically phosphorylated and dephosphorylated in vitro using both heterologous and lymphocyte-derived enzymes. Both the heavy chain and 20,000-dalton light chain of lymphocyte myosin are phosphorylated in vitro. Phosphorylation of myosin enhances actin-activated ATPase activity. Phosphorylation of myosin in murine lymphocytes was analyzed by use of a novel technique for rapid immunoprecipitation of myosin from cell extracts. Both the heavy chain and 20,000-dalton light chain of myosin are phosphorylated in intact cells. Addition of antibody reactive with cell-surface immunoglobulin to lymphocyte populations enriched for B cells stimulates locomotion of these cells and also increases the quantity of 32P isolated in association with the 20,000-dalton light chain of lymphocyte myosin, when 32Pi was present in the medium. In addition, an unidentified, phosphorylated polypeptides with a molecular mass of 22,000 daltons is co-isolated with myosin from cells by rapid immunoprecipitation. These results are consistent with the hypothesis that phosphorylation of myosin may contribute to regulation of movements performed by lymphocytes which are related to their participation in immunologic reactions.

scholarly journals Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

2004 ◽  
Vol 70 (1) ◽  
pp. 340-345 ◽  
Author(s):  
Joanna D. Moody ◽  
James P. Freeman ◽  
Peter P. Fu ◽  
Carl E. Cerniglia
Keyword(s):  
High Performance ◽  
Culture Media ◽  
Optical Purity ◽  
Fission Products ◽  
Reversed Phase ◽  
Environmental Pollutant ◽  
Ring Fission ◽  
Uv Visible ◽  
Derivatives Of ◽  
Absolute Stereochemistry

ABSTRACT Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

scholarly journals Energetics of Helicobacter pylori and Its Implications for the Mechanism of Urease-Dependent Acid Tolerance at pH 1

2002 ◽  
Vol 184 (11) ◽  
pp. 3053-3060 ◽  
Author(s):  
Kerstin Stingl ◽  
Eva-Maria Uhlemann ◽  
Roland Schmid ◽  
Karlheinz Altendorf ◽  
Evert P. Bakker
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Potential ◽  
Urease Activity ◽  
Cytoplasmic Ph ◽  
Ph Homeostasis ◽  
Intact Cells ◽  
Ph Values ◽  
Cell Extracts ◽  
H Pylori ◽  
External Ph

ABSTRACT In the presence of urea the neutrophilic human pathogen Helicobacter pylori survives for several hours at pH 1 with concomitant cytoplasmic pH homeostasis. To study this effect in detail, the transmembrane proton motive force and cytoplasmic urease activity of H. pylori were determined at various pH values. In the absence of urea, the organism maintained a close-to-neutral cytoplasm and an internally negative membrane potential at external pH values greater than 4 to 5. In the presence of urea, H. pylori accomplished cytoplasmic pH homeostasis down to an external pH of 1.2. At this external pH, the cytoplasmic pH was 4.9 and the membrane potential was slightly negative inside. The latter finding is in contrast to the situation in acidophiles, which develop inside-positive membrane potentials under similar conditions. Measurements of the time course of the membrane potential confirmed that addition of urea to the cells led to hyperpolarization. Most likely, this effect was due to electrogenic export of ammonium cations from the cytoplasm. The urease activity of intact cells increased nearly exponentially with decreasing external pH. This activation was not due to enhanced gene expression at low external pH values. In cell extracts the pH optimum of urease activity was dependent on the buffer system and was about pH 5 in sodium citrate buffer. Since this is the cytoplasmic pH of the cells at pH 1 to 2, we propose that cytoplasmic pH is a factor in the in vivo activation of the urease at low external pH values. The mechanism by which urease activity leads to cytoplasmic pH homeostasis in H. pylori is discussed.

scholarly journals Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity.

1992 ◽  
Vol 119 (5) ◽  
pp. 1297-1307 ◽  
Author(s):  
M J Levis ◽  
H R Bourne
Keyword(s):  
Adenylyl Cyclase ◽  
Cholera Toxin ◽  
Soluble Fraction ◽  
Covalent Modification ◽  
Alpha Subunit ◽  
Wild Type ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Cell Extracts ◽  
Membrane Bound

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.

scholarly journals Molecular Basis for Enhanced Activity of Posaconazole against Absidia corymbifera and Rhizopus oryzae

2006 ◽  
Vol 50 (11) ◽  
pp. 3917-3919 ◽  
Author(s):  
Andrew S. Chau ◽  
Guodong Chen ◽  
Paul M. McNicholas ◽  
Paul A. Mann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Basis ◽  
Rhizopus Oryzae ◽  
Intact Cells ◽  
Absidia Corymbifera ◽  
Cell Extracts ◽  
Enhanced Activity ◽  
Ergosterol Synthesis

ABSTRACT Posaconazole and itraconazole were more potent inhibitors of ergosterol synthesis, in both intact cells and cell extracts from Absidia corymbifera and Rhizopus oryzae, than voriconazole and fluconazole. Similarly, expression of CYP51 from R. oryzae in Saccharomyces cerevisiae significantly increased resistance to fluconazole and voriconazole but not to posaconazole and itraconazole.

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