Secretion of both partially unfolded and folded apoproteins of dimethyl sulfoxide reductase by spheroplasts from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans.

Spheroplasts prepared from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans secreted dimethyl sulfoxide (DMSO) reductase which had no molybdenum cofactor and therefore no activity, whereas those from wild-type cells secreted the active reductase. The inactive DMSO reductase proteins were separated by nondenaturing electrophoresis into two forms: form I, with the same mobility as the native enzyme, and form II, with slower mobility. Both forms had the same mobility on denaturing gel. Form I and active DMSO reductase had the same profile on gel filtration chromatography. Form II was eluted a little faster than the native enzyme, suggesting that DMSO reductase form II was not an aggregated form but a compactly folded form very similar to the native enzyme. Form II was digested by trypsin and denatured with urea, whereas form I was unaffected, like native DMSO reductase. These results suggested that form II was a partially unfolded but compactly folded apoprotein of DMSO reductase.     

Site-specific mutagenesis of the human interleukin-1 beta gene: the role of arginine residue at the N-terminal region

Using the expression system for site-specific mutagenesis in Escherichia coli, we have made deletion mutants at the N-terminal or C-terminal region of human interleukin-1 beta (IL-1 beta) consisting of 153 amino acids. The truncated mutants showed that at least 147 amino acids (numbers 4-150) in IL-1 beta are necessary for the exertion of biological activity. When we changed the arginine at the 4th position (Arg4) in IL-1 beta to other specific amino acids, there was a marked difference in the relative extent of biological and receptor binding activities among the mutants. The order of the mutants was Arg4 = Lys4 greater than Gln4 greater than Gly4 = des-Arg4 greater than Asp4. Our results demonstrate that the arginine residue at the 4th position in IL-1 beta is important, but not essential, for IL-1 beta to exhibit its biological and receptor binding activities, and the positive charge at this site plays a key role for IL-1 beta to exert the activities.     

Effects of Ca2+ ions on the formation of metaphase chromosomes and sperm pronuclei in cell-free preparations from unactivated Rana pipiens eggs

Nuclei transplanted into unactivated amphibian eggs are known to condense into metaphase chromosomes whereas those transplanted into activated eggs decondense and enlarge. We have made cell-free cytoplasmic preparations from Rana pipiens eggs which can induce demembranated Xenopus laevis sperm to undergo changes similar to those seen in intact eggs. Sperm chromatin which is incubated for 3 hr in unactivated egg preparations made using a buffer containing 3 mM EGTA is induced to form metaphase chromosomes. However, decondensed interphase nuclei are formed when chromatin is incubated in unactivated egg preparations made without EGTA as well as in activated egg preparations. When Ca2+ ions are added to unactivated egg preparations made with EGTA, the preparations lose the ability to induce metaphase chromosome formation and become capable of decondensing sperm chromatin. Once the ability to decondense chromatin has developed, either in unactivated or activated egg preparations, it cannot be suppressed by the addition of EGTA. However, decondensation of sperm chromatin in activated egg preparations can be suppressed by the addition of unactivated egg preparations made with EGTA. In this case, the incubated sperm chromatin is induced to form metaphase chromosomes. These results may indicate that the chromosome condensation activity of unactivated egg cytoplasm can be sustained in cell-free preparations when Ca2+ ion levels are kept low, but when Ca2+ ion levels increase this activity is lost and replaced by a new activity which can decondense chromatin. Since this change in cytoplasmic activities is comparable to that occurring in the intact egg following fertilization, these results suggest that Ca2+ ions play a crucial role during activation in altering the cytoplasmic activities which control nuclear behavior.     

β-xylosidase from Selenomonas ruminantium: Immobilization,stabilization, and application for xylooligosaccharide hydrolysis

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50?°C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55?°C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were V_max 0.20?μmol.min^?1?mg?prot.^?1, K_m 0.45?mM, K_cat 0.82?s^?1, and K_cat/K_m 1.82?s^?1?mM^?1. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.     

Sex and reproductive stage differences in the growth,metabolism, feed,fecal production,excretion and energy budget of the Amazon River prawn (Macrobrachium amazonicum)

The aim of this study was to evaluate differences in various physiological measures (growth, fecal production, feed intake, nitrogenous excretion, oxygen consumption, energy substrate used, and energy budget) among males, ovigerous females and non-ovigerous females of the freshwater prawn Macrobrachium amazonicum. This species exhibits pronounced sexual dimorphism and different male morphotypes and has the potential for use in aquaculture. Males and non-ovigerous females were studied for 30?days. Ovigerous females were studied for 10?days. Prawns were fed commercial prawn food, and all males were of the Translucent Claw (TC) morphotype. The results demonstrate physiological differences both between males and females and between females of different reproductive stages. Males had higher rates of ingestion, growth and oxygen consumption and less fecal loss than females. We postulate that in the absence of other morphotypes, TC males may exhibit increased growth rates. Males and females used protein as an energy substrate. Males channeled approximately 9% of their energy budget into growth, whereas non-ovigerous and ovigerous females channeled only 1.4?±?0.4 and 0.07?±?0.07%, respectively. Whereas males and non-ovigerous females channeled 9.0?±?9.74 and 61.8?±?3.0%, respectively, of the energy ingested into metabolism, ovigerous females channeled 97.7?±?4.7% into metabolism, likely due to the frequent beating of their pleopods, which oxygenates and cleans the eggs. As reported for marine prawns, males and non-ovigerous females of M. amazonicum lost approximately 5% of their ingested energy in exuviae. The physiological differences observed between the sexes and between females of different reproductive stages might reflect corresponding differences in patterns of activity, growth, and reproduction.     

Inhibition of the Functional Interplay between Endoplasmic Reticulum (ER) Oxidoreduclin-1α (Ero1α) and Protein-disulfide Isomerase (PDI) by the Endocrine Disruptor Bisphenol A

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b′ domain, preventing PDI from binding to unfolded proteins. The b′ domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b′ domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding β-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.     

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na⁺ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na⁺ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.     

Crystal structures of human Ero1�� reveal the mechanisms of regulated and targeted oxidation of PDI

In the endoplasmic reticulum (ER) of eukaryotic cells, Ero1 flavoenzymes promote oxidative protein folding through protein disulphide isomerase (PDI), generating reactive oxygen species (hydrogen peroxide) as byproducts. Therefore, Ero1 activity must be strictly regulated to avoid futile oxidation cycles in the ER. Although regulatory mechanisms restraining Ero1α activity ensure that not all PDIs are oxidized, its specificity towards PDI could allow other resident oxidoreductases to remain reduced and competent to carry out isomerization and reduction of protein substrates. In this study, crystal structures of human Ero1α were solved in its hyperactive and inactive forms. Our findings reveal that human Ero1α modulates its oxidative activity by properly positioning regulatory cysteines within an intrinsically flexible loop, and by fine‐tuning the electron shuttle ability of the loop through disulphide rearrangements. Specific PDI targeting is guaranteed by electrostatic and hydrophobic interactions of Ero1α with the PDI b′‐domain through its substrate‐binding pocket. These results reveal the molecular basis of the regulation and specificity of protein disulphide formation in human cells.     

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.     

Structural and biochemical correlates of Na+,K+-ATPase driven ion uptake across the posterior gill epithelium of the true freshwater crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+),K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (≈14?μm2 membrane per μm3cytoplasm), deep invaginations that house the Na(+),K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5?μm2?μm?2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6?μm2?μm?2), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+),K(+)-ATPase specific activity resembles marine crabs but is ≈5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two α-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4) (+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water.