Probing The Active And Allosteric Binding Sites Of Soybean Lipoxygenase-1

Soybean lipoxygenase-1 is a model for lipoxygenase activity. While the mechanism of oxygenation is understood, the substrate binding mechanism has not yet been elucidated. Two putative binding mechanisms are the ¿head-first¿ and ¿tail-first¿ models, in which the carboxy-terminus or the methyl terminus of the fatty acid substrate is inserted into the active site while the remainder of the molecule protrudes from the surface, respectively. Previous work has demonstrated that derivatization of fatty acid substrates with D-tryptophan increases active site affinity. It has also been shown that while polyunsaturated fatty acids are the natural substrates of lipoxygenases, monounsaturated fatty acids can be oxygenated at a much slower rate. Starting with a monounsaturated fatty acid, oleic acid, as a platform, the molecule N-oleoyl-D-tryptophan (ODT) was synthesized with the anticipation of it being a potent competitive substrate-analogue inhibitor that could be used to discern the substrate binding mechanism. Inhibition kinetics demonstrated that this molecule functions as a partially competitive inhibitor, through an unknown mechanism. The implication behind partially competitive inhibition is that substrate and inhibitor molecules can bind simultaneously to the enzyme, which alludes to the presence of an allosteric binding domain. To investigate the possibility of an inhibitor binding site on the non-catalytic subunit, limited proteolysis was used to cleave the subunits apart which should have eliminated inhibition. Interestingly, it was observed that at high substrate concentrations the inhibitor was completely ineffective, but at low substrate concentrations the inhibitor maintained its standard efficacy. A satisfactory explanation for these results has not yet been determined.

Crosslinking Studies To Probe Substrate Binding By Soybean Lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO-1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix-2 and helix-11 located on the exterior of the molecule in front of the active site iron. A second hypothesis involves a conformational change in the side chains of residues leucine 541 and threonine 259. To test these hypotheses, site directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C,S545C mutant was found to be unfavorable, but later shown to be present at higher pH values using SDS-PAGE. Treatment of the T259C,S545C with the crosslinker 2,3-dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant, S545C, or the wild type was treated with DBM. Single mutants T259C and L541C both showed approximately 20% reduction in the rate after addition of DBM. Double mutants T259C,L541C and S263C,S545C showed approximately 30% reduction in the rate after addition of DBM. Single mutants T259C and L541C showed an increase in activity after incubation with NEM. Double mutants T259C,S545C and T259C,L541C showed an increase in activity after incubation with NEM. The S263C,S545C double mutant showed a slight decrease in activity in the presence of NEM. It is unclear how the NEM and DBM are interacting with the molecule, but this can easily be determined through mass spectrometry experiments.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Study Of The Binding Of Substrate By The Phe557Val Mutant Soybean Lipoxygenase-1

Lipoxygenases are a class of enzymes which consist of non-heme iron dioxygenases that are produced by fungi, plants, and mammals and catalyze the oxygenation of polyunsaturated fatty acid substrates to unsaturated fatty acid hydroperoxide products. The unsaturated fatty acid hydroperoxide products are stereo- and regiospecific. One such lipoxygenase, soybean lipoxygenase-1 (SBLO-1), catalyzes the conversion of linoleate to 13-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9-hydroperoxy-10(E),12(Z)-octadecadienoate (9-HPOD). Although the structure of SBLO-1 is known and it is the most widely studied lipoxygenase, how it binds to substrate is still poorly understood. Two competing binding hypotheses that have been used to understand and explain the binding are the head first binding model and the tail first binding model. The head first binding model predicts linoleate binds with its polar carboxylate group in the binding pocket and the methyl terminus at the surface of the binding pocket. The tail first binding model predicts that linoleate binds with its methyl terminus end in the binding pocket and the polar carboxylate group at the surface of the binding pocket. Both binding models have been used in the explanation of previous work. In previous work the replacement of phenylalanine with valine has been performed to produce the phe557val mutant SBLO-1. The mutant SBLO-1 was then used in the enzymatic oxygenation of linoleate. With this mutant, the amount of 9-HPOD that is formed increases. This result has been interpreted using the head-first binding model in which the smaller valine residue allows linoleate to bind with the polar carboxylate group of linoleate interacting with arginine-707. The work presented in this thesis confirms the regiochemical results of the previous work and further tests the head-first binding model. If head-first binding occurs, the 9-HPOD is expected to have primarily S configuration. Utilizing chiral-phase HPLC, it was found that the 9-HPOD produced by the phe557val mutant SBLO-1 is primarily S, consistent with head-first binding. The head-first binding model was also tested using linoleyl dimethylamine (LDMA), which has been shown to be a good substrate for SBLO-1 at pH 7.0, where LDMA is thought to be positively charged. This model predicts that less of the 9-peroxide should be produced with this substrate. Through the use of gas chromatography/mass spectrometry, it was found that the conversion of LDMA by the phe557val mutant SBLO-1 resulted in the formation of a 46:54 mixture of the 13-peroxide:9-peroxide. The higher amount of 9-peroxide is the opposite of what is expected for the currently proposed model suggesting that the proposed model may not be entirely correct. The results thus far have been consistent with reverse binding but not with the proposed interaction of the polar end of the substrate with arginine-707.

Responsiveness and habituation of soluble icam-1 to acute psychosocial stress in men: determinants and efect of stress-hemoconcentration

We studied the psychophysiology of soluble intercellular adhesion molecule-1 (sICAM-1) in 25 apparently healthy middle-aged men who underwent an acute psychosocial stressor three times with one week apart. Measures of the biological stress response were obtained at week one and three. The magnitude of the sICAM-1 stress response showed no habituation between visits. At week one, cognitive stress appraisal independently predicted integrated sICAM-1 area under the curve (AUC) between rest, immediately post-stress, and 45 min and 105 min post-stress (beta=.67, p=.012, deltaR(2)=.41). Diastolic blood pressure AUC (beta=-.45, p=.048, deltaR(2)=.21) and heart rate (AUC) (beta=.44, p=.055, deltaR(2)=.21) were independent predictors of sICAM-1 (AUC) at week three. Adjustment for hemoconcentration yielded a decrease in sICAM-1 levels from rest to post-stress (p<.001). Stress responsiveness of plasma sICAM-1 was predicted by stress perception and hemodynamic reactivity and affected by stress-hemoconcentration but unrelated to cortisol reactivity and not readily adapting to stress repeats.

Stable transduction of bovine TLR4 and bovine MD-2 into LPS-nonresponsive cells and soluble CD14 promote the ability to respond to LPS

The interaction of bovine cells with lipopolysaccharide (LPS) was explored using human embryo kidney (HEK) 293 cell line stably transduced with bovine toll-like receptor-4 (TLR4) alone or in combination with bovine MD-2. These lines and mock-transduced HEK293 cells were tested by flow cytometry for LPS-fluorescein isothiocyanate (LPS-FITC) binding, nuclear factor kappa B (NFkappaB) activation, interleukin-8 (IL-8) production and interferon-beta mRNA expression/interferon (IFN) type I production. Whereas bovine TLR4 was sufficient to promote binding of high concentrations of LPS-FITC, both bovine TLR4 and MD-2 were required for activation by LPS, as assessed by NFkappaB activation and IL-8 production. Induction of IFN bioactivity was not observed in doubly transduced HEK293 cells, and no evidence for IFN-beta mRNA induction in response to LPS was obtained, although cells responded by IFN-beta mRNA expression to stimulation by Sendai virus and poly-inosinic acid-poly-cytidylic acid (poly(I:C)). Cells stably transduced with both bovine TLR4 and bovine MD-2 responded to LPS by IL-8 production, in decreasing order, in the presence of fetal bovine serum (FCS), of human serum, and of human serum albumin (HSA). The reduced activity in the presence of HSA could be restored by the addition of soluble CD14 (sCD14) but not of LPS binding protein (LBP). This is in contrast to macrophages which show a superior response to LPS in the presence of HSA when compared with macrophages stimulated by LPS in the presence of FCS. This suggests that macrophages but not HEK293 cells express factors rendering LPS stimulation serum-independent. Stably double-transduced cells reacted, in decreasing order, to LPS from Rhodobacter sphaeroides, to LPS from Escherichia coli, to synthetic lipd-IVa (compound 406), to diphosphoryl-lipid-A (S. minnesota) and to monophosphoryl-lipid-A (S. minnesota). They failed to react to the murine MD-2/TLR4 ligand taxol. This resembles the reactivity of bovine macrophages with regard to sensitivity (ED(50)) and order of potency but is distinct from the reactivity pattern of other species. This formally establishes that in order to react to LPS, cattle cells require serum factors (e.g. sCD14) and cell-expressed factors such as MD-2 and TLR4. The cell lines described are the first of a series expressing defined pattern recognition receptors (PRR) of bovine origin. They will be useful in the study of the interaction of the bovine TLR4-MD-2 complex and Gram-negative bovine pathogens, e.g. the agents causing Gram-negative bovine mastitis.

Oxidative stress and damage in liver, but not in brain, of Fischer 344 rats subjected to dietary iron supplementation with lipid-soluble [(3,5,5-trimethylhexanoyl)ferrocene]

Accumulation of iron probably predisposes the aging brain to progressive neuronal loss. We examined various markers of oxidative stress and damage in the brain and liver of 3- and 24-month-old rats following supplementation with the lipophilic iron derivative [(3,5,5-trimethylhexanoyl)ferrocene] (TMHF), which is capable of crossing the blood-brain barrier. At both ages, iron concentration increased markedly in the liver but failed to increase in the brain. In the liver of TMHF-treated young rats, levels of alpha- and gamma-tocopherols and glutathione (GSH) were also higher. In contrast, the brain displayed unaltered levels of the tocopherols and GSH. Malondialdehyde (MDA) level was also higher in the cerebrospinal fluid (CSF) and the liver but not in the brain. In old rats, the absence of an increase in iron concentration in the brain was reflected by unaltered concentrations of GSH, tocopherols, and MDA as compared to that in untreated rats. In the aging liver, concentrations of GSH and MDA increased with TMHF treatment. Morphological studies revealed unaltered levels of iron, ferritin, heme oxygenase-1 (HO-1), nitrotyrosine (NT), or MDA in the brains of both young and old rats treated with TMHF. In contrast, TMHF treatment increased the level of HO-1 in Kupffer cells, NT in hepatic endothelial cells, and MDA and ferritin in hepatocytes. Although these results demonstrated an increase in the biochemical markers of oxidative stress and damage in response to increasing concentrations of iron in the liver, they also demonstrated that the brain is well protected against dietary iron overload by using iron in a lipid-soluble formulation.

Preparation of soluble extracts from adenovirus-infected cells for studies of RNA splicing

Here we describe a collection of methods that have been adapted to produce highly efficient nuclear and cytoplasmic extracts from adenovirus-infected HeLa cells. We describe how to produce extracts from virus-infected cells and how to analyze RNA splicing in vitro using T7 RNA polymerase-derived splicing substrate RNAs.

Structural characterization of water-soluble atmospheric organic matter by ultrahigh-resolution mass spectrometry

Isolated water-soluble analytes extracted from fog water collected during a radiation fog event near Fresno, CA were analyzed using collision induced dissociation and ultrahigh-resolution mass spectrometry. Tandem mass analysis was performed on scan ranges between 100-400 u to characterize the structures of nitrogen and/or sulfur containing species. CHNO, CHOS, and CHNOS compounds were targeted specifically because of the high number of oxygen atoms contained in their molecular formulas. The presence of 22 neutral losses corresponding to fragment ions was evaluated for each of the 1308 precursors. Priority neutral losses represent specific polar functional groups (H2O, CO2, CH3OH, HNO3, SO3, etc., and several combinations of these). Additional neutral losses represent non-specific functional groups (CO, CH2O, C3H8, etc.) Five distinct monoterpene derived organonitrates, organosulfates, and nitroxy-organosulfates were observed in this study, including C10H16O7S, C10H17NO7S, C10H17 NO8S, C10H17NO9S, and C10H17NO10S. Nitrophenols and linear alkyl benzene sulfonates were present in high abundance. Liquid chromatography/mass spectrometery methodology was developed to isolate and quantify nitrophenols based on their fragmentation behavior.

Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçet's disease

OBJECTIVE: We analysed the production of soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 at sites of inflammation and measured their plasma concentrations to evaluate them as biological markers of disease activity. METHODS: Plasma samples of 35 patients with Behçet's disease (BD) were collected prospectively at monthly intervals and grouped for inactive disease, active BD without arthritis, and active BD with arthritis. sTNFR1 and sTNFR2 concentrations were measured using immunoassays and compared with other biological disease activity parameters. Plasma sTNFR levels were compared to synovial fluid (SF) levels in seven patients. Sixteen tissue samples of mucocutaneous lesions were stained for TNFR2 expression by immunohistochemistry. RESULTS: sTNFR1 and sTNFR2 were found at increased plasma concentrations in active BD, with the highest concentration in active BD with arthritis (p<0.001). Concentrations of both sTNFRs were at least three times higher in SF of arthritic joints than in the corresponding plasma samples (p = 0.025). A change of more than 1 ng/mL of sTNFR2 plasma concentrations correlated with a concordant change in arthritic activity (96% confidence interval). Sensitivity to change was superior to that of sTNFR1, and other biological disease activity parameters such as erythrocyte sedimentation rate (ESR), immunoglobulin (Ig)G, IgA, and interleukin (IL)-10 plasma concentrations. A strong staining for TNFR2 was found in mucocutaneous lesions, where mast cells were identified as the major source for this receptor. CONCLUSIONS: This longitudinal study demonstrates that sTNFR2 plasma concentrations are closely linked with active BD, and especially with arthritis. Taken together with the expression of TNFR molecules in mast cells of mucocutaneous lesions, our results indicate a fundamental role for the TNF/TNFR pathway in BD.