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At least 163 records · Page 9Linked to original sources
PubMed1991

Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.

Amino Acid Oxidoreductases↗
PubMed1996

No .NO from NO synthase.

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of L-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form L-citrulline (Cit) and nitric oxide (.NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify .NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable .NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal .NO standards were only partially recovered and the dismutation of superoxide (O2-.), which otherwise scavenges. .NO to yield ONOO-, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO- resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8-1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to .NO or that .NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal .NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO-) to .NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from .NO decomposition, was consistent with formation of an .NO precursor molecule such as NO-. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that .NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Electron Transport↗
PubMed1995

Characterization of active and inactive forms of the JAK2 protein-tyrosine kinase produced via the baculovirus expression vector system.

Three forms of rat JAK2 (type 2 Janus tyrosine kinase) were produced via the baculovirus expression vector system. Recombinant baculoviruses encoded either the full-length rat jak2 cloned from the Nb2-SP cell line (rJAK2), a carboxyl-terminal deletion mutant lacking the putative catalytic domain (rJAK2(C delta 795)), or an amino-terminal deletion mutant containing the putative catalytic domain ((N delta 661)rJAK2). The proteins produced in infected Sf21 cells were assayed for phosphotyrosine content and autophosphorylating activity. Tyrosine phosphorylation of rJAK2 was not observed 1 day postinfection when rJAK2 was initially produced but was apparent 2 or more days postinfection when the rJAK2 level had significantly increased. Tyrosine phosphorylation of rJAK2(C delta 795) was not observed; further, coproduction of rJAK2(C delta 795) with rJAK2 blocked tyrosine phosphorylation of rJAK2, consistent with previously published results (Zhuang, H., Patel, S. V., He, T-C., Sonsteby, S. K., Niu, Z., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 21411-21414). Mutant (N delta 661)rJAK2 exhibited a robust tyrosine phosphorylation signal. A second 62-kDa tyrosine phosphoprotein co-immunoprecipitated with (N delta 661)rJAK2 but not with rJAK2 or rJAK2(C delta 795). Both rJAK2 and (N delta 661)rJAK2 incorporated phosphate under in vitro kinase assay conditions, but rJAK2(C delta 795) did not. A JAK2 oligomer with interacting catalytic sites and/or inhibitory sites would provide a simple model to describe these results.

Animals↗
PubMed1997

Argininosuccinate synthetase overexpression in vascular smooth muscle cells potentiates immunostimulant-induced NO production.

Immunostimulants trigger vascular smooth muscle cells (VSMC) to express both the inducible isoform of NO synthase (iNOS) and argininosuccinate synthetase (AS). With constitutively expressed argininosuccinate lyase (AL), AS confers cells with an Arg/Cit cycle that can sustain NO production via continuous regeneration of the NOS substrate, L-arginine (Arg), from the NOS coproduct, L-citrulline (Cit). To assess whether NO synthesis can be rate-limited by Arg recycling, we tested whether AS-overexpressing cells have an enhanced capacity for immununostimulant-induced NO synthesis. Rat VSMC were stably transfected with human AS cDNA in a eukaryotic cell expression vector, driven by a strong viral promoter. AS activity in transfected VSMC exceeded that induced in untransfected cells treated for 24 h with a combination of bacterial lipopolysaccharide and interferon-gamma (LPS/IFN). AS activity was predominantly associated with membranes but was also found in cytosol. Recombinant AS was purified from cytosol and possessed a specific activity exceeding that reported for native AS. Western blotting verified the basal expression of AS antigen in membranes from untreated AS-transfected VSMC and from untransfected VSMC after 24 h exposure to LPS/IFN. Epifluorescence histochemistry revealed a punctate distribution of AS antigen in transfected cells, consistent with a predominant membrane localization. Remarkably, on a per cell basis, LPS/IFN-induced NO production was 3-4-fold greater in AS-transfected cells than untransfected VSMC. In untransfected VSMC, maximal NO production during 48 h required millimolar Arg; notably, Cit was needed at approximately 3-fold higher concentrations than Arg for a comparable NO synthesis rate. In contrast, AS-transfected VSMC utilized Arg and Cit equi-effectively and at much lower concentrations; 100 microM of either precursor supported a maximal rate of NO synthesis for 48 h. The enhanced ability of AS-transfected cells to produce NO, compared with untransfected cells, could not be ascribed to differences in iNOS protein content or LPS/IFN potency for immunoactivation. We conclude that transfection with AS provides a continuous flux of Arg which drives NO synthesis in immunoactivated VSMC. Arg regeneration by AS is rate-limiting to NO synthesis and apparently provides iNOS with a preferred cellular source of Arg. In accord with the reported "channeling" of substrates by urea cycle enzymes, we hypothesize that the Arg/Cit cycle sequesters a discrete pool of recyclable substrate that sustains high-output NO synthesis.

Adjuvants, Immunologic↗
PubMed2001

Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase.

Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.

Antioxidants↗
PubMed1993

Alcoholic hangover and performance: a review.

The study of alcoholic hangover is reviewed, with particular concern given to its effects upon skills performance. Different models of hangover, and the evidence gathered in their support, are surveyed. Several factors appear to contribute to hangover, and individuals may be differentially susceptible to these influences. The severity of hangover is affected by dose, coproducts of manufacture, and a number of psychological variables. Hangover is differentiated from post-intoxication effects on performance, and the results of a variety of studies on these effects are considered. Post-intoxication effects have been found in a variety of tasks, but no tasks have been consistently affected in a number of studies. This may reflect the fact that few studies have used the same tasks to assess performance. Proposed mechanisms of post-intoxication effects are considered, and the most consistent symptom accompanying impaired performance is found to be fatigue. This notion finds some support in electroencephalographic data collected during the period following intoxication.

Journal Article↗
PubMed2006

Uncertainty analysis of integrated gasification combined cycle systems based on Frame 7H versus 7F gas turbines.

Integrated gasification combined cycle (IGCC) technology is a promising alternative for clean generation of power and coproduction of chemicals from coal and other feedstocks. Advanced concepts for IGCC systems that incorporate state-of-the-art gas turbine systems, however, are not commercially demonstrated. Therefore, there is uncertainty regarding the future commercial-scale performance, emissions, and cost of such technologies. The Frame 7F gas turbine represents current state-of-practice, whereas the Frame 7H is the most recently introduced advanced commercial gas turbine. The objective of this study was to evaluate the risks and potential payoffs of IGCC technology based on different gas turbine combined cycle designs. Models of entrained-flow gasifier-based IGCC systems with Frame 7F (IGCC-7F) and 7H gas turbine combined cycles (IGCC-7H) were developed in ASPEN Plus. An uncertainty analysis was conducted. Gasifier carbon conversion and project cost uncertainty are identified as the most important uncertain inputs with respect to system performance and cost. The uncertainties in the difference of the efficiencies and costs for the two systems are characterized. Despite uncertainty, the IGCC-7H system is robustly preferred to the IGCC-7F system. Advances in gas turbine design will improve the performance, emissions, and cost of IGCC systems. The implications of this study for decision-making regarding technology selection, research planning, and plant operation are discussed.

Coal↗
PubMed1995

Heterogeneity of intracellular cytokine synthesis at the single-cell level in polarized T helper 1 and T helper 2 populations.

CD4+ T helper (Th) cells can be classified into different types based on their cytokine profile. Cells with these polarized patterns of cytokine production have been termed Th1 and Th2, and can be distinguished functionally by the production of IFN-gamma and IL-4, respectively. These phenotypes are crucial in determining the type of immune response that develops after antigen priming. There are no surface markers that define them, and cytokine immunoassay or mRNA analysis both have limitations for characterization of single cells. Using immunofluorescent detection of intracellular IFN-gamma and IL-4, we have studied the emergence of Th1 and Th2 cells in response to antigen exposure and the patterns of cytokine synthesis in established T cell clones. IFN-gamma production by Th1 clones was detectable in almost all cells by 4 h, and it continued in most cells for > 24 h. IL-4 production in Th2 cells peaked at 4 h, but declined rapidly. In Th0 cells containing both cytokines, fewer cells produced IFN-gamma, which did not appear until IL-4 synthesis declined. Cocultivation of clones showed no such cross-regulation. Antigen stimulation of transgenic T cells expressing an ovalbumin-specific T cell receptor generated Th2 cells, probably as a result of endogenous IL-4 production. Addition of IL-12 and/or anti-IL-4 caused Th1 cells to develop, while some Th0 cells were seen when IL-12 alone was added. These results show that stimulation in the presence of polarizing stimuli results in cells producing either IFN-gamma or IL-4, but that coproduction can occur in rare cells under defined conditions.

Animals↗
PubMed1999

Human interleukin-12 enhances interferon-gamma-producing influenza-specific memory CD8+ cytotoxic T lymphocytes.

Interferon (IFN)-gamma synthesis of CD45RO+ (memory) and CD45RA+ (naive) CD8+ cytotoxic T lymphocytes (CTLs) and the role of interleukin (IL)-12 in the regulation of human CTL functions in virus-specific immunity were investigated. After culture with influenza virus, CD45RO+ CD8+ T cells from human peripheral blood mononuclear cells increased in frequency and exhibited significant major histocompatibility complex class I-mediated CTL activity, whereas CD45RA+ CD8+ T cells did not. Influenza virus-stimulated CD45RO+ CD8+ T cells contained significantly higher levels of IFN-gamma-producing cells and IFN-gamma-specific mRNA than did CD45RA+ CD8+ T cells. Recombinant human IL-12 further enhanced CTL activity and IFN-gamma production by CD45RO+ CD8+ T cells. These data clearly show that human virus-specific CTL activity and coproduction of IFN-gamma are associated with the CD45RO+ CD8+ T cells that are modulated by the cell-mediated, immunity-inducible cytokine IL-12 in humans.

Adult↗
PubMed2002

Recombinant enzyme therapy for Fabry disease: absence of editing of human alpha-galactosidase A mRNA.

For more than a decade, protein-replacement therapy has been employed successfully for the treatment of Gaucher disease. Recently, a comparable therapy has become available for the related lipid-storage disorder Fabry disease. Two differently produced recombinant alpha-galactosidase A (alpha-gal A) preparations are used independently for this purpose. Agalsidase alpha is obtained from human fibroblasts that have been modified by gene activation; agalsidase beta is obtained from Chinese hamster ovary cells that are transduced with human alpha-gal A cDNA. It has previously been claimed that alpha-gal A mRNA undergoes editing, which may result in coproduction of an edited protein (Phe 396 Tyr) that might have a relevant physiological function. We therefore analyzed the occurrence of alpha-gal A editing, as well as the precise nature, in this respect, of the therapeutic enzymes. No indications were obtained for the existence of editing at the protein or RNA level. Both recombinant enzymes used in therapy are unedited and are capable of functionally correcting cultured fibroblasts from Fabry patients in their excessive globotriaosylceramide accumulation. Although RNA editing is apparently not relevant in the case of alpha-gal A, a thorough analysis of the potential occurrence of editing of transcripts is nevertheless advisable in connection with newly developed protein-replacement therapies.

Amino Acid Sequence↗
PubMed2026

Yeast Strain Development and Process Intensification in High-Gravity Fermentation.

High- and very-high-gravity (HG/VHG) fermentation increases substrate loading and product titers, thereby improving fermenter utilisation and potentially reducing water use and downstream processing requirements. Initially developed for brewing and fuel ethanol production, these approaches are now applied more broadly in food, beverage, and bioproduct manufacturing. This MiniReview summarises operational definitions and industrial drivers of HG/VHG fermentation and examines the associated constraints in rheology, mass and heat transfer, osmotic and ethanol stress, nutrient availability, and oxidative damage. Yeast improvement strategies are reviewed, including adaptive laboratory evolution, mutagenesis, genome shuffling, multiplex genome editing, non-conventional yeasts, and multi-omics-guided selection. Process developments such as no-cook simultaneous liquefaction, saccharification and fermentation (SLSF), enzyme formulation, nutrient management, and in situ product recovery are considered together with applications in alcoholic beverages, organic acids, microbial lipids, and other value-added products. The review also discusses coproduct valorisation and the need to integrate strain development with process design. Current evidence supports HG/VHG fermentation as a useful process-intensification platform, although performance and sustainability depend strongly on feedstock, operating conditions, product requirements, and the basis used to report fermentation outcomes.

circular bioeconomy↗
PubMed1983

Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase.

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.

Alkaline Phosphatase↗
PubMed2001

The effect of ethoxyquin on the quality of ground poultry mortality carcasses preserved by lactic acid fermentation and phosphoric acid stabilization.

Fermentation and acidification have been shown to preserve the protein quality of ground poultry coproducts, but the effects of these processes on their lipid stability are unknown, especially in the presence of an antioxidant. To evaluate the effects of these treatments on lipid quality, ground poultry mortality carcasses, with and without an addition of 500 ppm ethoxyquin, were stabilized for 14 and 45 d by lactic acid fermentation or acidification with 2.76, 5.07, 7.35, or 9.65% feed-grade H3PO4. Ethoxyquin treatment significantly (P < 0.001) improved the oxidative stability of lipids from all storage treatments. However, the addition of ethoxyquin increased (P < 0.001) the levels of volatile N (VN) from 2.51 to 3.18% in products stored for 45 d and resulted in an increase (P < 0.001) in free fatty acids in all ensiled products. Ethoxyquin addition had no effect (P > 0.120) on the fatty acid profile of products stored for 14 d but significantly increased (P < 0.001) the levels of stearic (C18:0) and arachidonic acids (C20:4) in products stored for 45 d. In this experiment, the addition of ethoxyquin to preservation systems for the short-term storage of poultry mortality carcasses improved the lipid quality of the ground material without compromising the protein quality or affecting proximate analysis parameters. However, the increased oxidative stability of mortality silage materials that contain ethoxyquin may contribute to enhanced microbial or enzymatic activities that result in proteolytic or lypolytic breakdown products following longer periods of storage.

Animals↗
PubMed1990

Neonatal necrotizing enterocolitis associated with delta toxin-producing methicillin-resistant Staphylococcus aureus.

Delta toxin-producing coagulase-negative staphylococci previously have been associated with necrotizing enterocolitis in neonates. We identified three preterm infants (body weight, 845 +/- 59 g) infected with methicillin-resistant Staphylococcus aureus (MRSA) who had a similar clinical syndrome, characterized by pustular dermatitis, bacteremia and necrotizing enterocolitis accompanied by gastric residua, abdominal distention, hematochezia and pneumatosis intestinalis. MRSA was recovered from all three infants at infected skin sites, blood or venous catheters and from two of three infants in stool specimens. Two infants also had Staphylococcus epidermidis isolated from stool. All MRSA isolates had identical microbiologic profiles: four plasmids with identical molecular weights; coproduction of enterotoxins A and B; and the same antibiotic susceptibilities. Of one skin isolate, two blood isolates and two stool isolates of MRSA that were tested, all had characteristic delta toxin hemolytic activity. All culture supernatants of these isolates evaluated for delta toxin were positive by Western blot analysis. The two strains of S. epidermidis isolated from stool were negative for delta-like toxin by a standardized enzyme-linked immunoassay. The clustering of these cases, the similarity of the clinical syndrome, and the prior association of necrotizing enterocolitis with delta-like toxins produced by S. epidermidis, suggest that delta toxin-producing MRSA (or other S. aureus isolates) also may be etiologic agents in some cases of necrotizing enterocolitis in newborns.

Bacterial Toxins↗
PubMed2006

Contribution of ethylene biosynthesis for resistance to blast fungus infection in young rice plants.

The role of ethylene (ET) in resistance to infection with blast fungus (Magnaporthe grisea) in rice (Oryza sativa) is poorly understood. To study it, we quantified ET levels after inoculation, using young rice plants at the four-leaf stage of rice cv Nipponbare (wild type) and its isogenic plant (IL7), which contains the Pi-i resistance gene to blast fungus race 003. Small necrotic lesions by hypersensitive reaction (HR) were formed at 42 to 72 h postinoculation (hpi) in resistant IL7 leaves, and whitish expanding lesions at 96 hpi in susceptible wild-type leaves. Notable was the enhanced ET emission at 48 hpi accompanied by increased 1-aminocyclopropane-1-carboxylic acid (ACC) levels and highly elevated ACC oxidase (ACO) activity in IL7 leaves, whereas only an enhanced ACC increase at 96 hpi in wild-type leaves. Among six ACC synthase (ACS) and seven ACO genes found in the rice genome, OsACS2 was transiently expressed at 48 hpi in IL7 and at 96 hpi in wild type, and OsACO7 was expressed at 48 hpi in IL7. Treatment with an inhibitor for ACS, aminooxyacetic acid, suppressed enhanced ET emission at 48 hpi in IL7, resulting in expanding lesions instead of HR lesions. Exogenously supplied ACC compromised the aminooxyacetic acid-induced breakdown of resistance in IL7, and treatment with 1-methylcyclopropene and silver thiosulfate, inhibitors of ET action, did not suppress resistance. These findings suggest the importance of ET biosynthesis and, consequently, the coproduct, cyanide, for HR-accompanied resistance to blast fungus in young rice plants and the contribution of induced OsACS2 and OsACO7 gene expression to it.

Amino Acid Sequence↗
PubMed1993

Mass determination of 15-hydroxyprostaglandin dehydrogenase from human placenta and kinetic studies with (5Z, 8E, 10E, 12S)-12-hydroxy-5,8,10-heptadecatrienoic acid as substrate.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the first step in the metabolism of prostaglandins which is usually associated with physiological inactivation. A highly purified homogenous enzyme preparation from human placenta was used to determine the molecular mass and lack of quaternary structure of the enzyme. Furthermore we have examined enzyme kinetics of the purified enzyme with (5Z,8E,10E,12S)-12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) an equimolar coproduct of thromboxane biosynthesis. Using gel electrophoresis and gel filtration on FPLC, we could estimate a molecular mass of 28 +/- 1 kDa, indicating that the enzyme consists of one single protein chain. The exact molecular mass of the monomer was calculated by matrix-assisted laser desorption/ionization mass spectrometry to 28740 +/- 30 Da. (5Z,8E,10E)-12-oxo-5,8,10-heptadecatrienoic acid (oxo-HT) could be identified as the only product obtained from the enzymatic reaction with HHT. Quantification of this metabolite was achieved by gas chromatography/tandem mass spectrometry. The calculated enzyme kinetic constants for the formation of the metabolic product [Km (HHT) = 9.68 microM, Vi = 12.78 mU/micrograms] were in agreement with those determined for NADH formation (Km = 7.65 microM, Vi = 11.79 mU/micrograms). This demonstrates that HHT shows high affinity to the enzyme which is comparable to prostaglandin E2 (PGE2). As the product oxo-HT is a potent inhibitor of platelet aggregation, dehydrogenation of HHT might represent a biological activation step.

Chromatography, Gel↗
PubMed1997

Mutational analysis of domain II of flavonol 3-sulfotransferase.

The flavonol 3- and 4'-sulfotransferases (ST) from Flaveria chloraefolia catalyze the transfer of the sulfonate group from 3'-phosphoadenosine 5'-phosphosulfate (PAdoPS) to position 3 of flavonol aglycones and position 4' of flavonol 3-sulfates. We identified previously a protein segment, designated domain II, that contains all the determinants responsible for the specificity of these enzymes. Within domain II, at least five amino acids specific to the 4'-ST that could bind the sulfate group of quercetin 3-sulfate were identified. In this study, these amino acid residues were introduced at equivalent positions in the flavonol 3-ST sequence by site-directed mutagenesis of the cloned cDNA. No reversal of the substrate specificity was observed after the individual mutations. However, mutation of Leu95 to Tyr had different effects on the kinetic constants depending on the substitution pattern of the flavonoid B ring, suggesting that the tyrosine side chain may be in direct contact with this part of the molecule. The function of conserved amino acids present in domain II was also investigated. Unconservative mutations at Lys134, Tyr137 and Tyr150 resulted in protein instability in solution, suggesting that these residues might be important for the structural stability of the enzyme. Replacement of Arg140 with Lys or Ser had no effect on protein stability, but resulted in a strong reduction in specific activity. The results of photoaffinity-labeling experiments with PAdoP[35S]S suggest that this residue is required to bind the cosubstrate. In addition, the reduced affinity of [Ser140]ST for 3'-phosphoadenosine 5'-phosphate (PAdoP)-agarose indicates that Arg140 is also involved in binding the coproduct. Replacement of His118 with Glu or Ala resulted in a strong reduction in catalytic activity. However, [Lys118]ST retained a significant amount of catalytic activity. The results of photoaffinity-labeling experiments with PAdoP[35S]S and affinity chromatography on PAdoP-agarose suggest that His118 might be involved in catalysis in the flavonol 3-ST.

Amino Acid Sequence↗
PubMed2004

Comparative analysis of high-affinity ligand binding and G protein coupling of the human CXCR1 chemokine receptor and of a CXCR1-Galpha fusion protein after heterologous production in baculovirus-infected insect cells.

In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.

Animals↗