Structural stabilities of recombinant scombridae fish myoglobins

An expression system of recombinant myoglobins (Mb) of 3 scombridae fish species was constructed. The stability of these Mbs was compared with native Mbs purified from slow skeletal muscle. The addition of hemin during the cultivation of an Escherichia coli strain harboring a pGEX-2T expression vector was found to be necessary to prevent recombinant Mb from degrading and to attain its proper folding. The stabilities of recombinant Mbs were generally lower than those of native Mbs, partly due to the absence of post-translational modification. The alpha-Helical content of bullet tuna recombinant Mb at 10 degrees C was the lowest (29.0%) among the recombinant Mbs examined (the values for bluefin tuna and bigeye tuna Mbs being 34.8 and 35.5%, respectively). On the other hand, the stabilities of recombinant Mbs of bluefin tuna and bigeye tuna against denaturants (urea and guanidine hydrochloride) were found to be similar, whereas bullet tuna recombinant Mb exhibited the lowest stability among these Mbs. The pattern of temperature-dependent decrease in the alpha-helical content supported these results.     

The protein synthesis machinery operates at the same expense in eurythermal and cold stenothermal pectinids

Translationally active cell-free systems from gills of the Antarctic scallop Adamussium colbecki and the European scallop Aequipecten opercularis were developed, characterised, and optimised for an analysis of translational capacity. The aim was to determine the energetic cost of protein synthesis in the in vitro cell-free system by directly measuring the required energy equivalents in the lysates. Protein synthesis rate in assays conducted with lysates of A. colbecki (1.029+/-0.061 micromol Phe min(-1) at 15 degrees C; Phe=phenylalanine) were higher compared with lysates of A. opercularis (0.087+/-0.013 micromol Phe min(-1) at 15 degrees C and 0.156+/-0.023 micromol Phe min(-1) at 25 degrees C). This can in part be attributed to the naturally occurring higher RNA content in lysates of A. colbecki (0.883+/-0.037 mg RNA mL(-1) lysate) compared with A. opercularis (0.468+/-0.013 mg RNA mL(-1) lysate). There was no significant difference in the energetic costs of protein synthesis in cell-free systems of gill lysates of the cold stenothermal A. colbecki with 4.3+/-0.7 energy equivalents per peptide bond formed and the eurythermal A. opercularis with 5.6+/-0.6 energy equivalents, indicating that there are no differences in the efficiency of the translation machinery. The energetic costs specified for protein synthesis correspond with the generally accepted theoretical value of four energy equivalents per peptide bond formed, especially in gill lysates of A. colbecki, whereas the value for gill lysates of A. opercularis was slightly higher.     

In vitro protein synthesis capacities in a cold stenothermal and a temperate eurythermal pectinid

The translational system was isolated from the gills of the Antarctic scallop Adamussium colbecki (Smith) and the European scallop Aequipecten opercularis (Linnaeus) for in vitro protein synthesis capacities (g protein mg FW^–1 day^–1) and the translational capacities of RNA (k_{RNA in vitro} mg protein mg RNA^–1 day^–1). In vitro protein synthesis capacity in the cold-adapted pectinid at 0 °C was similar to the one found in the temperate scallop at 25 °C. These findings might reflect cold compensated rates in Adamussium colbecki, partly explainable by high tissue levels of RNA. Cold-compensated in vitro protein synthesis capacities may further result from increments in the translational capacity of RNA. The thermal sensitivity of the translation machinery was slightly different in the two species, with significantly lower levels of Arrhenius activation energies E_a and Q₁₀ in Adamussium colbecki in the temperature range 0–15 °C. Reduced protein synthesis and translational capacities were found in vitro in gills of long-term aquarium-maintained Adamussium colbecki and were accounted for by a loss of protein synthesis machinery, i.e. a reduction in RNA levels, as well as a decrease in the amount of protein synthesized per milligram of RNA (RNA translational capacity, k_{RNA in vitro}). Such changes may involve food uptake or mirror metabolic depression strategies, like those occurring during winter. Consequences of high in vitro RNA translational capacities found in the permanently cold-adapted species are discussed in the context of seasonal food availability and growth rates at high latitudes.Abbreviations DPM disintegrations per minute - DTT dithiothreitol - E_a Arrhenius activation energy - k_s fractional protein synthesis rate - k_{RNA in vivo} translational efficiency - k_{RNA in vitro} translational capacity - PCA perchloric acid - Phe phenylalanine - PLA phospho-L-arginine - PSU practical salinity units - RNAse ribonuclease - TCA trichloroacetic acidCommunicated by G. Heldmaier     

Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

Temperature-dependent pH regulation in stenothermal Antarctic and eurythermal temperate eelpout (Zoarcidae): an in-vivo NMR study

Temperature-dependent adjustments of intracellular pH are thought to play a major role in the maintenance of protein function. Comparative studies were carried out in two species from the same fish family (Zoarcidae), the stenothermal Antarctic eelpout (Pachycara brachycephalum) and the eurythermal eelpout (Zoarces viviparus), to find out whether pH regulation is modified by temperature in the closely related species and to what extent the respective pattern differs between eurytherms and stenotherms. Previous invasive studies had compared individual animals sampled at various temperatures and suggested that a decrease in intracellular pH (pHi) values occurs at rising temperatures, as predicted by the alpha-stat hypothesis of acid-base regulation. The present study used non-invasive in vivo ³¹P-NMR spectroscopy in non-anaesthetized, unrestrained fish for long-term online recordings in individual specimens. Control spectra obtained at T=0°C for P. brachycephalum and at 12°C for Z. viviparus indicated low stress conditions, as well as a high stability of energy and acid-base status over time periods longer than 1 week. Temperature changes had no influence on the concentration of high-energy phosphates like phosphocreatine or ATP. Temperature-induced pH changes were monitored continuously in a range between 0 and 6°C for polar, and 12 and 18°C for temperate eelpout. A pHi change of around -0.015 pH units/°C was observed within both species, in accordance with the alpha-stat hypothesis; however, extrapolation to the same temperature revealed different set points of pH regulation in the two species. These findings confirm that an alpha-stat pattern of pH regulation can be found in stenothermal Antarctic animals, at set points deviating from an alpha-stat pattern, however, in a between-species comparison.     

Primary structure of myoglobins from 31 species of birds

Primary structure of myoglobins (Mbs) from 31 avian species of 15 orders were reported, although portions of the structures in the 2 species could not be determined. At least 68 of the total 153 amino acid sites were invariant all through the avian, reptilian and human Mbs, and 20 of these sites were "internal", forming the internal hydrophobic cavities in which the heme group remains wrapped. Furthermore, at 27 sites, if replaced, the replacements were mostly conservative, and 13 of the conservative sites were "internal". Thus the all 33 "internal" sites, important for structural and functional stability of the protein, have been well preserved, either invariant or conserved, during evolution from reptiles to birds and mammals. The residue 71 (E14) in 4 penguin species was not deleted as previously reported in emperor penguin Mb but occupied by Gln. The residue 121 (GH3) was deleted in all 3 species studied of Falconiformes. Out of 9 anseriforms, 5 species of different genera showed the identical structure. Secondary structures as viewed by hydropathy profiles were highly similar throughout the reptilian, avian and mammalian Mbs.     

Comparative oxygen affinity of fish and mammalian myoglobins

Summary Myoglobins from rat, coho salmon (Oncorhynchus kisutch), buffalo sculpin (Enophrys bison) hearts, and yellowfin tuna (Thunnus albacares) red skeletal muscle were partially purified and their O₂ binding affinities determined. Commercially prepared sperm whale myoglobin was employed as an internal standard. Tested at 20°C, myoglobins from salmon and sculpin bound O₂ with lower affinity than myoglobins from the rat or sperm whale. Oxygen binding studies at 12°C and 37°C suggest that this difference is adaptive, permitting myoglobins from cold-adapted fish to function at physiologically relevant temperatures. Taken together, purification and O₂ binding data obtained in this study reveal a previously unrecognized diversity of myoglobin structure and function.     

Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish

Myoglobin (Mb) buffers intracellular O2 and facilitates diffusion of O2 through the cell. These functions of Mb will be most effective when intracellular PO2 is near the partial pressure of oxygen at which Mb is half saturated (P50) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P50s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P50s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20 degrees C were 0.62 +/- 0.02, 0.59 +/- 0.01, 0.58 +/- 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 +/- 0.07 mmHg) and mackerel (1.39 +/- 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20 degrees C for the endothermic species ranged from 34.1 to 49.3 s(-1), whereas those for mackerel and bonito were 102 and 62 s(-1), respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Comparison of the biochemical and molecular properties of myoglobins from three Biomphalaria species

Myoglobin is a globin with heme as prosthetic group whose main known biological role is to bind to O2 reversibly. On account of their large diversity, globins from mollusks have contributed to the study of this protein class. The cDNA of the myoglobins from Biomphalaria straminea and Biomphalaria tenagophila, which have a glutamine as distal residue (E7), were constructed and analyzed by bioinformatic tools. Native (not recombinant) myoglobins of these two Biomphalaria species were purified and their experimental molecular mass (about 16 kDa) and pI (about (8.0) were provided. Data analysis showed that these proteins are monomers with the signature for the classic myoglobin fold and they are blocked in amino terminus probably by an acetyl group. Values of the autoxidation rates showed that these myoglobins oxidized slowly. About the primary sequences of the myoglobins, they turned out to be satisfactory to group mollusks in phylogenetic class.     

cDNA-derived amino acid sequences of myoglobins from nine species of whales and dolphins

We determined the myoglobin (Mb) cDNA sequences of nine cetaceans, of which six are the first reports of Mb sequences: sei whale (Balaenoptera borealis), Bryde's whale (Balaenoptera edeni), pygmy sperm whale (Kogia breviceps), Stejneger's beaked whale (Mesoplodon stejnegeri), Longman's beaked whale (Indopacetus pacificus), and melon-headed whale (Peponocephala electra), and three confirm the previously determined chemical amino acid sequences: sperm whale (Physeter macrocephalus), common minke whale (Balaenoptera acutorostrata) and pantropical spotted dolphin (Stenella attenuata). We found two types of Mb in the skeletal muscle of pantropical spotted dolphin: Mb I with the same amino acid sequence as that deposited in the protein database, and Mb II, which differs at two amino acid residues compared with Mb I. Using an alignment of the amino acid or cDNA sequences of cetacean Mb, we constructed a phylogenetic tree by the NJ method. Clustering of cetacean Mb amino acid and cDNA sequences essentially follows the classical taxonomy of cetaceans, suggesting that Mb sequence data is valid for classification of cetaceans at least to the family level.     

Structural and kinetic characterization of early folding events in beta-lactoglobulin

We have defined the structural and dynamic properties of an early folding intermediate of beta-lactoglobulin known to contain non-native alpha-helical structure. The folding of beta-lactoglobulin was monitored over the 100 micros--10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 micros is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising beta-strands F, G and H, and the main alpha-helix, which was thus identified as the folding core of beta-lactoglobulin. At the same time, weak protection (up to approximately 10-fold) of amide protons in a segment spanning residues 12--21 is consistent with formation of marginally stable non-native alpha-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like alpha/beta core domain.     

Structural and kinetic characterization of an archaeal beta-class carbonic anhydrase

The beta-class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer. Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta-class carbonic anhydrase indicate that the secondary structure of the beta-class enzymes is predominantly alpha-helical, unlike that of the alpha- or gamma-class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K(m) for CO(2) hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H(2)O than in D(2)O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K(m) is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta-class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta-class enzymes and firmly establish that the alpha-, beta-, and gamma-classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.     

Immunochemical investigation of four Saprolegnia species with parasitic activity in fish: Serological and kinetic characterization of a chymotrypsin-like activity

The antigenic structure of four aquatic Phycomycetes of the genusSaprolegnia with parasitic activity in fish was analyzed with immunoelectrophoresis and double diffusion techniques. It is shown that these serological tests might represent a valuable criterion for the identification of the fourSaprolegnia species:S. parasitica, S. ferax, S. delica, andS. diclina. A specific chromogenic reaction revealed the presence of a protease with chymotrypsin-like activity among the precipitation arcs obtained after immunoelectrophoresis. Analysis of this proteolytic activity by kinetic assay methods confirmed the results of the serological tests. Moreover estimates of the quantity of enzyme present in the cellular extract and in the culture medium were obtained. A possible relationship between the proteolytic activity and the host-parasite interaction was discussed.     

Molecular characterization of two myoglobins of Paragonimus westermani

Myoglobins (Mbs), globin proteins, are present in high concentrations in trematodes. In Paragonimus westermani, 2 cDNAs were found to encode Mbs. The first clone, Pwmyo1, codes a total of 149 amino acids with a calculated mass of 16.6 kDa. The second, Pwmyo2, encodes a 146-amino acid protein with a calculated mass of 16.2 kDa. The predicted secondary structures showed the presence of 8 helices, which is the basic characteristic of Mbs. Sequence alignment revealed a high homology with the other trematode Mbs. The 2 clones contained the characteristic tyrosyl residues at helical positions B10 and distal E7, which are substitutions that have been previously shown to contribute to the high oxygen affinity of Mbs. Polyclonal antibodies against the recombinant Mbs were raised with no cross-reactivity observed. Immunolocalization revealed the proteins to be distributed generally throughout the parenchymal tissues, but absent from the tegument and reproductive organs. The cell mass of the eggs of the worm stained positive to Pwmyo2 but not Pwmyo1, suggesting the stage-specific expression of these Mbs.     

Multiple aminoacyl-tRNA synthetases (translases) in temperature acclimation of eurythermal fish

The temperature dependency of activity of the entire set of aminoacyl-tRNA synthetases (protein synthetic translases) has been studied in the laboratory rat and in toadfish (Opsanus tau) acclimated to 20 δC or to 10 δC. The complex temperature responses of these enzyme systems reveal the presence of multiple forms for the translases of most amino acids and show adaptive behavior of these systems with respect to body temperature of the animal. The phenylalanine translase system has been studied in detail, and adaptation of this system at low temperatures correlates with adaptation in the elongation factor system. All known protein synthetic components appear to be coordinated in adaptive responses with the exception of ribosomes. Our data indicate no rôle for ribosomes in adaptation of the protein synthetic system and apparently no rôle for ribosomes in protein synthesis at all in rat and fish. This finding may solve some long-standing paradoxes in the protein synthesis field concerning the mechanism by which ribosomes participate in protein synthesis.