Elisa M. Valenzuela-Soto

Purification and Properties of Betaine Aldehyde Dehydrogenase Extracted from Detached Leaves of Amaranthus hypochondriacus L. Subjected to Water Deficit

Summary The activity of the enzyme betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from leaves of Amaranthus hypochondriacus L. rises from undetectable levels to 10-3 units/ mg protein after 4 h of treatment with 17% (w/v) polyethyleneglycol to produce a water deficit. This enzyme was purified to apparent homogeneity in only three consecutive steps: fractional precipitation with ammonium sulfate, ion exchange, affinity chromatography on 5′-AMP Sepharose. A specific activity of 2.6 mol/min kg (protein) was obtained. The enzyme has a native molecular mass of 125 kDa, estimated by gel filtration chromatography, a subunit molecular mass of 63 kDa, determined by SDS-PAGE. The reaction is highly specific for betaine aldehyde, which is an inhibitor at high concentrations, but can use NAD+ or NADP+ as nucleotide. The estimated Km values at pH 8.0 and 30 °C for NAD+, NADP+, betaine aldehyde were 80 μM, 2.5 mM, 69 μM respectively. The reaction could not be reversed even at very high glycine betaine concentrations. The optimum pH and temperature were 8.0 and 50 °C, respectively. The pH dependence of the velocity indicated the existence of two ionizable groups of macroscopic pK values of 6.78 ± 0.02 and 9.38 ± 0.01 involved in catalysis and/or binding of the substrates. Chemical modification studies suggested the presence of essential cisteine(s), histidine(s), arginine(s) residues. The enzyme was activated by relatively low concentrations of K+, sucrose, proline, while it was inhibited by NH+4, Na+, high concentrations of glycine betaine. Mg++ up to 150 mM and Ca++ up to 50 mM did not have any effect on the activity.

Monovalent cations requirements for the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, porcine kidney and amaranth leaves

Betaine aldehyde dehydrogenase from the human pathogen Pseudomonas aeruginosa requires K(+) ions for maintenance of its active conformation. In order to explore if this property is shared by other BADHs of different origins and to further understand the mechanism underlying the effects of these ions, we carried out a comparative study on the stability and quaternary structure of P. aeruginosa, porcine kidney and amaranth leaves BADHs in the absence of K(+) ions. At low enzyme concentrations, the bacterial and porcine enzymes were totally inactivated upon removal of K(+) following biphasic and monophasic kinetics, respectively, whereas the amaranth enzyme retained its activity. Inactivation of P. aeruginosa BADH was much faster than that of the porcine enzyme. The oxidized coenzyme protected both enzymes against inactivation by the absence of K(+), whereas betaine aldehyde afforded partial protection to the bacterial BADH and increased the inactivation rate of the porcine. Reactivation of the inactive enzymes, by adding back to the incubation medium K(+) ions, was dependent on enzyme concentration, suggesting that enzyme dissociation takes place in the absence of K(+). In the bacterial enzyme, NH(4)(+) but not Na(+) ions could mimic the effects of K(+), whereas the three cations tested reactivated porcine BADH, indicating a requirement of this enzyme for high ionic strength rather than for a specific monovalent cation. Size exclusion chromatography of the inactivated enzymes confirmed that K(+) ions or other monovalent cations are required for the maintenance of the quaternary structure of these two BADHs. At pH 7.0, in the absence of K(+) in a buffer of low ionic strength, the active tetrameric form of P. aeruginosa BADH dissociated into inactive monomers and that of porcine kidney BADH into inactive dimers. Once reactivated, both enzymes reassociated into active tetramers.

Inhibition by Cu2+ and Cd2+ of a mu-class glutathione S-transferase from shrimp Litopenaeus vannamei

Glutathione S‐transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of xenobiotics and aiding its excretion from the cell. The present work presents the inhibition of a mu‐class GST of the marine shrimp Litopenaeus vannamei by copper (Cu2+) and cadmium (Cd2+). The protein was overexpressed in bacteria and its enzymatic activity measured using 1‐chloro‐2,4‐dinitrobenzene. The mean inhibitory concentration (IC50) for shrimp GST against Cu2+ was 4.77 μM and for Cd2+ was 0.39 μM. A molecular model of the protein based on the crystal structure of a maize GST bound to cadmium showed that the metal binds in the GSH‐binding site by coordination with Asp and Gln residues. These results are consistent with the experimental data and suggest that sublethal concentration of metals may affect the capacity of the organism to detoxify pesticides or xenobiotics.

Expression and silencing of Selenoprotein M (SelM) from the white shrimp Litopenaeus vannamei: Effect on peroxidase activity and hydrogen peroxide concentration in gills and hepatopancreas

Selenoprotein M (SelM), is a selenocysteine containing protein with redox activity involved in the antioxidant response. In the white shrimp Litopenaeus vannamei, SelM expression in gills is induced transiently during viral infection by the White Spot Syndrome Virus (WSSV). We report that SelM expression was detected in healthy shrimp L. vannamei in gills, muscle, hepatopancreas and pleopods, with more abundance in the hepatopancreas and gills. SelM transcripts were silenced by intramuscular injection with double-stranded RNAs (dsRNAs). In gills and hepatopancreas, all shrimp injected with long dsRNAs had lower SelM transcripts levels compared with controls. Peroxidase activity and hydrogen peroxide concentration were measured to detect effects on antioxidants. Peroxidase activity decreased upon silencing of SelM in gills, but no significant effect was detected in hepatopancreas. In contrast, total cell hydrogen peroxide concentration did not change in gills and hepatopancreas of silenced shrimp. Non-heme peroxidases are new players in the oxidative stress system that need to be addressed in detail, as well as selenium as a critical micronutrient for the antioxidant and innate immune systems in crustaceans.

Trypsin from viscera of vermiculated sailfin catfish, Pterygoplichthys disjunctivus, Weber, 1991: Its purification and characterization

Pterygoplichthys disjunctivus viscera trypsin was purified by fractionation with ammonium sulphate, gel filtration, affinity and ion exchange chromatography (DEAE-Sepharose). Trypsin molecular weight was approximately 27.5kDa according to SDS-PAGE, shown a single band in zymography. It exhibited maximal activity at pH 9.5 and 40°C, using N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate. Enzyme was effectively inhibited by phenyl methyl sulphonyl fluoride (PMSF) (100%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (85.4%), benzamidine (80.2%), and soybean trypsin inhibitor (75.6%) and partially inhibited by N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (10.3%), ethylendiaminetetraacetic acid (EDTA) (8.7%) and pepstatin A (1.2%). Enzyme activity was slightly affected by metal ions (Fe(2+)>Hg(2+)>Mn(2+)>K(+)>Mg(2+)>Li(+)>Cu(2+)). Trypsin activity decreased continuously as NaCl concentration increased (0-30%). Km and kcat values were 0.13mM and 1.46s(-1), respectively. Results suggest the enzyme have a potential application where room processing temperatures (25-35°C) or high salt (30%) concentration are needed, such as in fish sauce production.

Role of HIF-1 on phosphofructokinase and fructose 1, 6-bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei

HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and β subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.

Inactivation of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide.

Concentrated urine formation in the kidney is accompanied by conditions that favor the accumulation of reactive oxygen species (ROS). Under hyperosmotic conditions, medulla cells accumulate glycine betaine, which is an osmolyte synthesized by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). All BADHs identified to date have a highly reactive cysteine residue at the active site, and this cysteine is susceptible to oxidation by hydrogen peroxide. Porcine kidney BADH incubated with H(2)O(2) (0-500 μM) lost 25% of its activity. However, pkBADH inactivation by hydrogen peroxide was limited, even after 120 min of incubation. The presence of coenzyme NAD(+) (10-50 μM) increased the extent of inactivation (60%) at 120 min of reaction, but the ligands betaine aldehyde (50 and 500 μM) and glycine betaine (100 mM) did not change the rate or extent of inactivation as compared to the reaction without ligand. 2-Mercaptoethanol and dithiothreitol, but not reduced glutathione, were able to restore enzyme activity. Mass spectrometry analysis of hydrogen peroxide inactivated BADH revealed oxidation of M278, M243, M241 and H335 in the absence and oxidation of M94, M327 and M278 in the presence of NAD(+). Molecular modeling of BADH revealed that the oxidized methionine and histidine residues are near the NAD(+) binding site. In the presence of the coenzyme, these oxidized residues are proximal to the betaine aldehyde binding site. None of the oxidized amino acid residues participates directly in catalysis. We suggest that pkBADH inactivation by hydrogen peroxide occurs via disulfide bond formation between vicinal catalytic cysteines (C288 and C289).

Role of invariant tyrosines in a crustacean mu-class glutathione S-transferase from shrimp Litopenaeus vannamei: Site-directed mutagenesis of Y7 and Y116

Y6 and Y115 are key amino acids involved in enzyme-substrate interactions in mu-class glutathione S-transferase (GST). They provide electrophilic assistance and stabilize substrates through their hydroxyl groups. Two site-directed mutants (Y7F and Y116F) and the wild-type shrimp GSTs were expressed in Escherichia coli, and the steady-state kinetic parameters were determined using CDNB as the second substrate. The mutants were modeled based on a crystal structure of a mu-class GST to obtain further insights about the changes at the active site. The Y116F mutant had an increase in kcat contrary to Y7F compared to the wild type. Molecular modeling showed that the shrimp GST has a H108 residue that may contribute to compensate and lead to a less deleterious change when conserved tyrosine residues are mutated. This work indicates that shrimp GST is a useful model to understand the catalysis mechanisms in this critical enzyme.

Manganese inactivation of renal betaine aldehyde dehydrogenase from swine

Manganese is an essential micronutrient for mammals, however high manganese concentrations cause adverse health effects. Swine renal betaine aldehyde dehydrogenase catalyzes the synthesis of glycine betaine, which plays an important role in renal cells osmoregulation. In vitro inactivation of BADH was observed by incubating the purified enzyme in the presence of 1 mM MnCl2 under physiological and low ionic strength conditions. Enzyme inactivation followed first order kinetics in a monophasic process with an inactivation constant of 0.126 ± 0.011 min and 0.137 ± 0.017 at physiological and low ionic strength, respectively. Enzyme inactivation was not prevented by physiological ionic strength, nor by the substrates NAD and betaine aldehyde at saturated concentrations. The enzyme was reactivated with DTT and GSH. Native-PAGE of the inactivated enzyme showed no change in the tetrameric conformation. Intrinsic protein fluorescence studies demonstrated an increased exposure of the tryptophan residues to the aqueous solvent when the enzyme was incubated with Mn. These results suggest that BADH inactivation by Mn may result from the oxidation of cysteines, which induces changes in the tertiary structure of the enzyme.

Interrelation of Collagen Chemical Structure and Nanostructure with Firmness of three Body Regions of Jumbo Squid (Dosidicus gigas)

The chemical structure, thermal denaturation and nanostructure of collagen, obtained from a cation-exchange separation of the mantle, fins and tentacles of jumbo squid (Dosidicus gigas), were comparatively studied. The main idea of this work, was to provide an in-depth understanding of the interdependence between pyridinoline (Pyr) content, helix chemical structure and nanostructure of squid collagen with squid tissue firmness. The tentacles required more shear force and its collagen presented the higher temperature and enthalpy of transition, than the mantle and fins. The tentacle firmness may be explained by the relatively higher imino amino acid content, proline and lysine hydroxylation degrees and Pyr content of its collagen. Moreover, among the regions studied, the collagen from the tentacles had a more intense β band chain. Also, the Fourier transform infrared analysis and Raman spectra, implied that the collagen in the tentacles, was more intermolecularly ordered than the mantle and fins. Consistent with these results, a comparative evaluation of the surface morphology of the three regions, with atomic force microscopy, suggested a more ordered collagen structure in the tentacles (lower roughness values). Based on the above, collagen from tentacles has a higher degree of molecular order that sustains a higher muscle firmness compared to that of other anatomical regions.