116 research outputs found
Structure-to-function relationship of mini-lipoxygenase, a 60-kDa fragment of soybean lipoxygenase-1 with lower stability but higher enzymatic activity
No full textLipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agro, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 M). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable
Further structural and functional properties of mini-lipoxygenase, an active fragment of soybean lipoxygenase-1
No full textLipoxygenases (Loxs) form a homologous family of non-heme, non-sulfur iron containing lipid-peroxidizing enzymes, which catalyze the dioxygenation of polyunsaturated fatty acids to the corresponding hydroperoxy derivatives. Soybean lipoxygenase-1 (Lox-1) is widely used as a prototype for studying the structural and functional properties of lipoxygenases. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox", which shows enhanced catalytic efficiency and higher membrane binding ability than the native enzyme (M. Maccarrone, M.L. Salucci, G. van Zadelhoff, F. Malatesta, G. Veldink, J.F.G. Vliegenthart and A. Finazzi-Agro, Biochemistry 40 (2001), 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride (GdHCl) and under high pressure by fluorescence and circular dichroism spectroscopy. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition. Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-8 anilinonaphtalene sulphonic acid binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by fluorescence techniques. In particular, eicosatetraynoic acid (ETYA), an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of ETYA. All these findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible and therefore less stable, that on the other hand is probably causing its higher catalytic efficiency
Structure-to-function relationship of mini-lipoxygenase, a 60kDa fragment of soybean lipoxygenase-1 with lower stability but higher enzymatic activity
No full textLipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membrane-binding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò, A. (2001) Biochemistry 40, 6819-6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0-4.5 m). Both chemical- and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable
Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system
No full textEarly activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death
No full textOxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2-induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti-lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2-induced apoptosis. On the other hand, lentil lipoxygenase products 9- and 13-hydroperoxy-octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells
Methylation and acetylation of 15-hydroxyanandamide modulate its interaction with the endocannabinoid system.
No full textLine emission from circumstellar disks around A stars
No full textThe nature of the tenuous disks around A stars has raised considerable controversy in the literature during the past decade. The debate whether or not the disk around beta Pictoris contains gaseous molecular hydrogen is only the most recent example. Since CO is in general a poor tracer for the gas content of these low mass disks, we discuss here detailed emission line calculations for alternative tracers like C and C+, based on recent optically thin disk models by Kamp & van Zadelhoff (2001). The [C II] 157.7 mum line was searched toward Vega and beta Pictoris - the most prominent A stars with disks - using ISO LWS data, and a tentative detection is reported toward the latter object. From a comparison with emission line observations as well as absorption line studies of both stars, the gas-to-dust ratio is constrained to lie between 0.5 and 9 for beta Pictoris. For Vega the [C II] observations indicate an upper limit of 0.2 M+ for the disk gas mass. Predicted line intensities of C+ and C are presented for a range of models and appear promising species to trace the gas content in the disks around A stars with future instrumental capabilities (SOFIA, Herschel, APEX and ALMA). Searches for CO emission should focus on the J = 3-2 line
Boekaankondigingen
No full textT.W.M. Bakker, Nederlandse kustduinen, geohydrologie, Pudoc, Wageningen, 1981, dissertatie, 189 pag. + bijlage, ƒ 50. Een van de vier publikaties in de reeks Nederlandse kustduinen als resultaat van het in 1976 gestarte TNO-Duinvalleienonderzoek.
T.W.M. Bakker, J.A. Klijns en F.J. van Zadelhoff, Nederlandse kustduinen, landschapsecologie, Pudoc, Wageningen, 1981, 144 pag. + bijlagen, ƒ 37,50. De gezamenlijke publikatie in de reeks Nederlandse kustduinen als resultaat van het in 1976 gestarte TNO-Duinvalleienonderzoek
MODULATION OF THE ENZYMATIC ACTIVITY AND MEMBRANE BINDING PROPERTIES OF SOYBEAN LIPOXYGENASE-1 THROUGH LIMITED PROTEOLYSIS AND METAL SUBSTITUTION
No full textLipoxygenases are non-heme, non-sulfur iron containing enzymes that catalyze the dioxygenation of polyunsatured fatty acids containing one or more pentadiene systems to the corresponding hydroperoxy derivatives. Structural studies in solution of the mammalian and plant enzyme revealed that the latter has a more stable and compact conformation1. As yet, metal atom extraction, reconstitution and substitution with vicariate metals have not been successfully applied to soybean lipoxygenase-1, because of the highly buried position of the iron atom within the active site. Tryptic digestion of lipoxygenase-1 and the subsequent isolation of the 60 kDa C-terminal region allowed to generate a “mini-lipoxygenase-1 (miniLOX)” that retains the catalytically active iron, but in a more accessible position2. In this study, we investigated by near-UV-circular dichroism and fluorescence spectroscopies the structural and functional effects of iron removal, reconstitution and vicariation in miniLOX. Moreover, we report the kinetic analysis and the membrane binding ability of the apo- and metal-substituted forms of miniLOX, using fluorescence resonance energy transfer and monolamellar vesicles. Taken together, these data demonstrate an unprecedented structural role of iron, which is involved not only in the catalytic activity but also in the membrane binding ability of lipoxygenase-1.1. Dainese E. Sabatucci A. van Zadelhoff G. Angelucci C. B. Vachette P., Veldink G. Finazzi Agrò A. and Maccarrone M. (2005). J. Mol. Biol. 349, 143-152.2. Maccarrone M. Salucci M. L. van Zadelhoff G. Malatesta F. Veldink, G. Vliegenthart J. F. and Finazzi Agrò A. (2001). Biochemistry 40, 6819-6827.[...
Structural stability of soybean lipoxygenase-1 in solution as probed by small angle X-ray scattering
No full textSoybean lipoxygenase-1 (LOX-1) is used widely as a model for Studying the structural and functional properties of the homologous family of lipoxygenases. The crystallographic structure revealed that LOX-1. is organized in a beta-sheet N-terminal domain and a larger, mostly helical, C-terminal domain. Here, we describe the overall structural characterization of native unliganded LOX-1 in solution, using small angle X-ray scattering (SAXS). We show that the scattering pattern of the unliganded enzyme in solution does not display any significant difference compared with that calculated from the crystal structure, and that models of the overall shape of the protein calculated ab initio from the SAXS pattern provide a close envelope to the crystal structure. These data, demonstrating that LOX-1 has a compact structure also in solution, rule out any major motional flexibility of the LOX-1 molecule in aqueous solutions. In addition we show that eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase used to mimic the effect Of Substrate binding, does not alter the overall conformation of LOX-1 nor its ability to bind to membranes. In contrast, the addition of glycerol (to 5%, v/v) causes an increase in the binding of the enzyme to membranes without altering its catalytic efficiency towards linoleic acid nor its SAXS pattern, suggesting that the global conformation of the enzyme is unaffected. Therefore, the compact structure determined in the crystal appears to be essentially preserved in these various solution conditions. During the preparation of this article, a paper by M. Hammel and co-workers showed instead a sharp difference between crystal and solution conformations of rabbit 15-LOX-1. The possible cause of this difference might be the presence of oligomers in the rabbit lipoxygenase preparations. (c) 2005 Elsevier Ltd. All rights reserved
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