Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability.

Lipoxygenases are key enzymes in the metabolism of unsaturated fatty acids. Soybean lipoxygenase-1 (LOX-1), a paradigm for lipoxygenases isolated from different sources, is composed of two domains: a approximately 30 kDa N-terminal domain and a approximately 60 kDa C-terminal domain. We used limited proteolysis and gel-filtration chromatography to generate and isolate a approximately 60 kDa fragment of LOX-1 ("mini-LOX"), produced by trypsin cleavage between lysine 277 and serine 278. Mini-LOX was subjected to N-terminal sequencing and to electrophoretic, chromatographic, and spectroscopic analysis. Mini-LOX was found to be more acidic and more hydrophobic than LOX-1, and with a higher content of alpha-helix. Kinetic analysis showed that mini-LOX dioxygenates linoleic acid with a catalytic efficiency approximately 3-fold higher than that of LOX-1 (33.3 x 10(6) and 10.9 x 10(6) M(-1) x s(-1), respectively), the activation energy of the reaction being 4.5 +/- 0.5 and 8.3 +/- 0.9 kJ x mol(-1) for mini-LOX and LOX-1, respectively. Substrate preference, tested with linoleic, alpha-linolenic, and arachidonic acids, and with linoleate methyl ester, was the same for LOX-1 and mini-LOX, and also identical was the regio- and stereospecificity of the products generated thereof, analyzed by reversed-phase and chiral high-performance liquid chromatography, and by gas chromatography/mass spectrometry. Mini-LOX was able to bind artificial vesicles with higher affinity than LOX-1, but the binding was less affected by calcium ions than was that of LOX-1. Taken together, these results suggest that the N-terminal domain of soybean lipoxygenase-1 might be a built-in inhibitor of catalytic activity and membrane binding ability of the enzyme, with a possible role in physio(patho)logical conditions.

Activation of different lipoxygenase isozymes induces apoptosis in human erythroleukemia and neuroblastoma cells.

We investigated the ability of different hydroperoxides generated by lipoxygenase isozymes to induce programmed cell death (PCD) in human cells. Erythroleukemia K562 and neuroblastoma CHP100 cells were used, because they showed high basal activity of lipoxygenase. The hydroperoxides generated by 5-, 12-, or 15-lipoxygenases from linoleate, linolenate, or arachidonate, and the corresponding hydroxides, were able to induce PCD in both cell types, in a concentration- and time-dependent manner. After 24 h, K562 and CHP100 cells showed 2.5- to 3.5-fold more apoptotic bodies than the untreated controls. PCD elicited by lipoxygenase products was independent of intracellular glutathione concentration, and did not require mRNA transcription or protein synthesis. On the other hand, lipoxygenase products evoked an immediate and sustained rise in cytoplasmic calcium (within seconds), followed by mitochondrial uncoupling (within hours). Unlike the hydro(pero)xides, the terminal products of the arachidonate cascade (i.e., leukotrienes, prostaglandins and thromboxane) were not cytotoxic.

The early phase of apoptosis in human neuroblastoma CHP100 cells is characterized by lipoxygenase-dependent ultraweak light emission.

Human neuroblastoma CHP100 cells were forced into apoptosis (programmed cell death, PCD) or necrosis by treatment with calcium chloride or sodium nitroprusside (a nitric oxide donor), respectively. Cellular luminescence, a marker of membrane lipid peroxidation, was increased by calcium but not by nitroprusside, and reached a maximum of 4-fold the control value 2 hours after treatment. The increase in luminescence was paralleled by increased 5-lipoxygenase (up to 250% of the control value) and decreased catalase (down to 50%) activity within the same time window. Consistently, incubation of CHP100 cells with inhibitors of 5-lipoxygenase (5,8,11,14-eicosatetraynoic acid and MK886) reduced light emission and PCD, whereas inhibition of catalase by 3-amino-1, 2,4-triazole enhanced both processes. Treatment of CHP100 cells with retinoic acid or cisplatin, unrelated PCD inducers reported to activate the lipoxygenase pathway, also gave enhanced light emission parallel to PCD increase. Altogether, these results suggest that cellular luminescence is an early marker of apoptotic, but not necrotic, program(s) involving generation of hydrogen peroxide and activation of 5-lipoxygenase.

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells.

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 microg ml(-1) blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 microg ml(-1) tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 microg ml(-1). After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 microg ml(-1) tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.

Binding and iron delivering of ovotransferrin to cholesterol-depleted chick-embryo red blood cells.

Binding and iron delivering of ovotransferrin (OTf) were evaluated using 14-day old chick-embryo red blood cells (CERBC) and cholesterol-depleted by treatment with chicken egg phosphatidyl choline (E-PC) liposomes. Liposome-treated CERBC assayed for their cholesterol content showed a cholesterol depletion depending on the incubation time, being 25% (w/w) of the maximum cellular removal of cholesterol seen after 22 h incubation at 37 degrees C. Total phosphorus content did not change either for the various samples or during the different incubation times, indicating that specific cholesterol removal occurred, as confirmed also by the increased membrane fluidity revealed through fluorescence anisotropy measurements. The apparent dissociation constant (Kd) of control and treated CERBC was almost of the same value at the same incubation time, ranging from 0.30 microM after 0.25 h incubation to 0.19 microM after 14 or 22 h incubation. In all experiments, the maximum value of bound OTf molecules per cell (Bmax) notably decreased as incubation time increased. But, in cholesterol partly depleted CERBC, the decrease of the Bmax values was less pronounced as the incubation time increased. As far as binding experiments were concerned, iron uptake studies showed that uptaking capacities decreased as incubation time increased. Considering both binding and iron uptake, at the same incubation time, liposome-treated CERBC were slightly more efficient with respect to untreated samples. In any case a passive iron delivering could be evidenced after 22 h incubation. It is suggested that cholesterol may tune binding and iron uptake by either regulating or affecting the expression or mobility of the OTf receptor.

Primary structure of the major glycan from human seminal transferrin.

Human seminal transferrin (HSmT) is an iron-containing glycoprotein whose structural properties have not been adequately investigated. The carbohydrate content of the purified glycoprotein amount to 6.1%, and monosaccharide analysis revealed the major oligosaccharide moiety to be of the N-glycoside type. The carbohydrate chains were released from the iron-free form by digestion with peptide N-glycosidase F (PNGase F) in the presence of detergents such as SDS and beta-octylglucoside. After ethanol precipitation and fractionation on Bio-Gel P-6 and Bio-Gel P-2, the oligosaccharide was further purified on Mono-Q and desalted on Bio-Gel P-2. By 600-MHz 1H-NMR spectroscopy, the primary structure of the major N-linked oligosaccharide component was established to be: [formula: see text]

Exhaustive removal of N-glycans from ascorbate oxidase: effect on the enzymatic activity and immunoreactivity.

Purified ascorbate oxidase from Cucurbita pepo medullosa has been subjected to enzymatic deglycosylation using peptide N-glycosidase F. Experimental conditions were chosen to obtain efficiently deglycosylated and active ascorbate oxidase: in particular, three different detergent solutions were added separately to the incubation mixtures prior to the peptide N-glycosidase F. The detergent solution made of 0.1% (w/v) sodium dodecyl sulphate + 0.5% (v/v) Nonidet P-40 proved to be the only one effective for our purpose. Our results indicate that: (i) the presence of detergents did not affect the enzymatic activity; (ii) fully deglycosylated enzyme retained its activity compared with the native form. Moreover, anti-native ascorbate oxidase antibodies scarcely recognized deglycosylated protein.

Structural analysis of seminal and serum human transferrin by second derivative spectrometry and fluorescence measurements.

Denaturation of human seminal transferrin (HSmT) compared with human serum transferrin (HSrT) was followed to check structural differences between these two proteins. Second derivative UV spectroscopy indicated that treatment with 6 M guanidine hydrochloride (Gnd.HCl) induced greater structural changes in HSrT than in HSmT and, in particular; (i) the exposure value of tyrosinyl residues was almost 2.5-fold higher in native HSmT than in native HSrT; and (ii) a much more pronounced movement of tryptophanyl residues toward a higher polar environment could be noticed in HSrT after incubation with denaturing agent. Fluorescence measurements showed that: (i) a shift of the maximum emission wavelength of HSmT occurred (maximum emission was centered at 333 nm instead of 323 nm as for HSrT; excitation = 280 nm); (ii) the intrinsic tryptophan fluorescence intensity of HSmT increased after 36 hr in the range of 1.5-4.0 M of denaturant, whereas an opposite behavior was found for HSrT in the range 0.0-2.0 M; and (iii) the wavelength maximum of fluorescence emission changed in a biphasic manner for HSrT and, conversely, under the same experimental conditions, HSmT gave a linear and parallel increase of fluorescence emission after 1 and 36 hr. We can conclude that this different behavior of HSmT with respect to HSrT might be due mainly to the fact that both the number and the exposure of tyrosinyl and tryptophanyl residues are different. Lately, these effects are discussed in relationship with the fact that HSmT contains less than half disulphide bridges than HSrT.