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G N Somero

Publications and source records attributed to G N Somero.

At least 19 recordsLinked to original sources
PubMed1998

Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes.

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4 (A4-LDH) from nine Antarctic (-1.86 to 1 degree C) and three South American (4 to 10 degree C) notothenioid teleosts. Higher Michaelis-Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in kcat and Km are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting kcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation in Km and kcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase kcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high Km values relative to warm-adapted orthologs.

Adaptation, Physiological↗
PubMed1997

Evolution of lactate dehydrogenase-A homologs of barracuda fishes (genus Sphyraena) from different thermal environments: differences in kinetic properties and thermal stability are due to amino acid substitutions outside the active site.

Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.

Amino Acid Sequence↗
PubMed1996

Differences in protein patterns of gill epithelial cells of the fish Gillichthys mirabilis after osmotic and thermal acclimation.

Different protein patterns in gill epithelium of a euryhaline and eurythermal teleost fish (Gillichthys mirabilis, Family Gobiidae) in response to long-term (2 months) osmotic and thermal acclimation were found for the first time. Gill epithelial cells were isolated to remove extracellular proteins and quantify specialized cell types. Chloride cells were identified on the basis of size (> 10 microns) and bright appearance after [2-(p-dimethylaminostyryl)-1-methyl-pyridinium-iodine] staining. Small mitochondria-rich cells were < 5 microns in diameter and showed intermediate fluorescence. Abundance of chloride cells and small mitochondria-rich cells was significantly influenced by osmotic but not thermal acclimation (dilute seawater/25 degrees C: 1.4 +/- 0.2% chloride cells, 11.9 +/- 4.6% small mitochondria-rich cells; seawater/25 degrees C: 2.4 +/- 0.6% chloride cells, 2.2 +/- 1.3% small mitochondria-rich cells; seawater/10 degrees C: 2.9 +/- 0.3% chloride cells, 1.2 +/- 0.7% small mitochondria-rich cells). Pavement cells, identified by low fluorescence and intermediate size (5-10 microns), largely predominated under all conditions (> 85% of cells). Thus, they represented the major protein source in gill epithelium. Differences in protein patterns were detectable using two-dimensional but not one-dimensional electrophoresis. Of 602 proteins identified by charge and molecular weight properties, only two were induced by high temperature (25 degrees C) and three in response to cold acclimation (10 degrees C). Nine proteins were induced in diluted seawater-acclimated fish, whereas no seawater-induced proteins were found. We hypothesize that proteins induced under dilute seawater conditions are important for the function of pavement cells in gills of hyper-osmoregulating G. mirabilis.

Acclimatization↗
PubMed1996

Activity of lactate dehydrogenase but not its concentration of messenger RNA increases with body size in barred sand bass, Paralabrax nebulifer (Teleostei).

In white skeletal muscle of conspecific pelagic fishes, the activity of enzymes associated with anaerobic glycolysis, e.g., lactate dehydrogenase (LDH), usually scale positively with increasing body size; this pattern is opposite to that found for enzymes of aerobic metabolism, which decrease in mass-specific activity with size (1-3). The higher mass-specific capacities for anaerobic ATP generation in larger conspecifics are thought to facilitate conservation of high-speed ("burst") swimming ability in fishes of different sizes (1). To investigate the mechanisms responsible for scaling of LDH activity, total RNA, and the specific mRNA for LDH-A (the skeletal muscle isoform of the enzyme) in white muscle of paralabrax nebulifer, the barred sand bass. We also measured total protein concentration and the concentration of actin, the major protein of thin filaments, and its specific mRNA. Although LDH activity scaled significantly with body size as predicted (1-4), no other biochemical trait measured showed a significant size-dependent concentration. We conclude that the regulation of LDH activity in white muscle of this species is not governed by LDH-A mRNA concentrations, but rather by one or more other mechanisms, for example rate of translation of LDH message or a reduced rate of degradation of LDH-A in larger fish.

Animals↗
PubMed1995

Ion transport in gills of the euryhaline fish Gillichthys mirabilis is facilitated by a phosphocreatine circuit.

The function of creatine kinase (CK) isozymes in energy metabolism and the short-term regulation of active ion transport in gills of the euryhaline teleost Gillichthys mirabilis was investigated. After a transfer of fish from regular seawater [36 parts/thousand (ppt)] to hypersaline water (60 ppt), the plasma osmolality increased significantly from 361.0 +/- 5.2 to 434.2 +/- 20.6 mosmol/kgH2O within 2 h and was regulated down to 391.8 +/- 11.3 mosmol/kgH2O within 12 h. Although the ATP concentration in the gill tissue remained unchanged, the creatine concentration increased significantly from 17.3 +/- 3.2 to 37.6 +/- 5.9 nmol/mg protein within 2 h after the salinity change. CK and Na(+)-K(+)-adenosinetriphosphatase-(Na(+)-K(+)-ATPase) activities were unchanged 48 h after transfer. Independent of salinity, the activities of CK were three to seven times those of the Na(+)-K(+)-ATPase, and the creatine concentration in the gill was at least one order of magnitude higher than the ATP concentration. The occurrence of muscle-type CK (CK-M), brain-type CK, and mitochondrial CK was demonstrated. CK-M was predominant in gills (59 +/- 7.1% of total CK activity). Evidence for a direct functional coupling between CK and Na(+)-K(+)-ATPase was obtained with permeabilized gill cells, by using the CK inhibitor iodoacetamide, which abolishes the competitive channeling of ADP from the external pyruvate kinase reaction to the endogeneous CK reaction in a coupled in situ Na(+)-K(+)-ATPase assay. Our results show the significance and the central regulatory role for energy metabolism and adaptive ionoregulation of a phosphocreatine-CK circuit in situations of high and fluctuating energy demands for euryhaline fishes.

Adenosine Triphosphate↗
PubMed1992

Biochemical ecology of deep-sea animals.

Deep-sea ecosystems contain unique endemic species whose distributions show strong vertical patterning in the case of pelagic animals and sharp horizontal patterning in the case of benthic animals living in or near the deep-sea hydothermal vents. This review discusses the biochemical adaptations that enable deep-sea animals to exploit diverse deep-sea habitats and that help establish biogeographic patterning in the deep-sea. The abilities of deep-sea animals to tolerate the pressure and temperature conditions of deep-sea habitats are due to pervasive adaptations at the biochemical level: enzymes exhibit reduced perturbation of function by pressure, membranes have fluidities adapted to deep-sea pressures and temperatures, and proteins show enhanced structural stability relative to homologous proteins from cold-adapted shallow-living species. Animals from the warmest habitable regions of hydrothermal vent ecosystems have enzymes and mitochondria adapted to high pressure and relatively high temperatures. The low metabolic rates of bathypelagic fishes correlate with greatly reduced capacities for ATP turnover in locomotory muscle. Reduced light and food availability in bathypelagic regions select for low rates of energy expenditure in locomotory activity. Deep-sea animals thus reflect the importance of biochemical adaptations in establishing species distribution patterns and appropriate rates of metabolic turnover in different ecosystems.

Adaptation, Physiological↗
PubMed1992

The threshold induction temperature of the 90-kDa heat shock protein is subject to acclimatization in eurythermal goby fishes (genus Gillichthys).

Two extremely eurythermal goby fishes, Gillichthys mirabilis and Gillichthys seta, which encounter habitat temperature variations of approximately 30 degrees C, showed seasonal acclimatization of endogenous levels and of onset temperatures for enhanced synthesis of a 90-kDa-class heat shock protein (HSP90). Summer-acclimatized fishes had higher levels of HSP90 in brain tissue than winter-acclimatized specimens, as shown by Western blot analysis. For winter-acclimatized fishes, increased synthesis of HSP90 was observed when the temperature was raised from a control temperature (18 degrees C) to 28 degrees C. For summer-acclimatized fish, no significantly increased synthesis of HSP90 occurred until the experimental temperature was raised to 32 degrees C. These data suggest that the threshold temperature at which enhanced expression of HSP-encoding genes occurs is not hard-wired genetically but may be subject to acclimatization. A causal relationship between seasonal changes in steady-state levels of HSP90 and the threshold temperature for enhanced HSP90 synthesis is discussed in terms of existing models for the regulation of HSP gene expression.

Acclimatization↗
PubMed1992

Temperature- and concentration-dependence of compatibility of the organic osmolyte beta-dimethylsulfoniopropionate.

The effects of the organic osmolyte beta-dimethylsulfoniopropionate (DMSP) on the structural stability of three model proteins were examined to determine whether DMSP, like the structurally similar solute dimethyl sulfoxide (DMSO), is compatible with native protein structure at low, but not elevated, temperatures. DMSP stabilized phosphofructokinase under conditions of cold-induced denaturation. Thus, DMSP, like DMSO, may be an effective protein cryoprotectant. However, DMSP was not an effective stabilizer of protein structure under conditions of heat denaturation. Whereas low (0.2 M) concentrations of DMSP stabilized lactate dehydrogenase against inactivation at 50 degrees C, higher DMSP concentrations were ineffective. DMSP favored the denaturation of glutamate dehydrogenase at all DMSP concentrations tested. DMSP may be a compatible osmotic solute only under conditions of moderate temperature and low, yet physiological, concentrations. The mechanistic basis of DMSP's temperature- and concentration-dependent effects and the possible roles played by adaptation temperature and severity of osmotic stress in the evolutionary selection of organic osmolytes are discussed.

Dimethyl Sulfoxide↗
PubMed1992

Adaptations to high hydrostatic pressure.

The importance of adaptation to high pressure has long been implicit in the findings of studies in which 1 atm-adapted species were subjected to elevated pressures. Recent comparative studies have shown that pressure sensitivities of enzymes, structural proteins, and membrane-based systems differ markedly between shallow- and deep-living species. These studies allow operational definition of what constitutes high pressures for different biological structures and processes. These are the habitat (adaptation) pressures at which a given type of system first exhibits reduced perturbation by pressure. These threshold pressures vary among physiological systems, but are similar for a given system among different species. Dehydrogenase enzymes and adenylyl cyclases exhibit threshold perturbation pressures of only 50-100 atm; the Na(+)-K(+)-ATPase of teleost gills appears to have a pressure perturbation threshold near 200 atm, and a similar threshold was found for actin self-assembly. Even this limited sample of physiological processes indicates that the terms deep and high pressure begin to apply at depths of only 500 m or less--and processes yet to be examined in comparative analysis may yield even lower pressure thresholds. The differences in sensitivity to pressure of homologous systems in shallow- and deep-living organisms have implications at several levels of biological organization. The vertical distribution patterns of species in aquatic habitats may be established, in part, by interspecific differences in resistance to pressure. High pressures may restrict the depths to which shallow-living species can penetrate, and the obligately barophilic systems found in deep-living organisms may limit their upper distribution limits. The similarities noted among the adaptations of deep-sea species with different shallow-water ancestors reflect a high degree of convergent evolution in pressure adaptation. It will be interesting to learn if the similarities in pressure-resistance of function among diverse deep-sea species are the result of similar or identical changes at the molecular level, e.g. in protein sequence. Acclimation to pressure may be of widespread occurrence among species that undergo large changes in depth, e.g. during ontogeny. Pressure acclimation may require pressure-regulation of gene expression. Lastly, comparisons of species from the cold deep sea with those from hydrothermal vents have shown that adaptations to both temperature and pressure play critical roles in determining the distribution patterns of deep-living species.

Adaptation, Physiological↗
PubMed1989

Properties of the interaction between phosphofructokinase and actin.

The interaction of rabbit skeletal muscle phosphofructokinase (PFK) with actin is characterized in terms of the binding of PFK to actin in the presence and absence of tropomyosin and troponin, the effect of PFK on actin polymerization, and the involvement of adenylates in the binding of PFK to actin. The thin filament proteins, tropomyosin and troponin, are associated with skeletal muscle actin and reduce the binding of PFK to actin, thus influencing the probable distribution of PFK in skeletal muscle. The binding of PFK to actin is inhibited by ATP and ADP but not by fructose 6-phosphate or fructose 2,6-bisphosphate. This specific inhibition, plus evidence from fluorescence quenching and photoaffinity labeling, suggests that actin binds at the adenosine activation sites of PFK. Light scattering measurements used to monitor actin polymerization indicate that PFK dramatically increases the level of light scattering produced by the polymerization of actin, indicative of a superaggregate of PFK and actin. PFK inhibits the polymerization of actin when polymerization is induced by low concentrations of added salts. Although PFK binds to actin with high affinity, it seems to have little effect on the high shear viscosity of actin filaments.

Actins↗
PubMed1989

Pressure adaptation of Na+/K+-ATPase in gills of marine teleosts.

The effects of pressure and temperature on an integral membrane protein, Na+/K+-adenosine triphosphatase (Na+/K+-ATPase), were studied in fish gill membrane preparations from shallow- and deep-living marine teleosts. The inhibition by pressure of maximal velocity of the enzyme is nonlinear, increasing at higher pressures. Na+/K+-ATPases from deep-sea fish were less inhibited by pressure than those of shallow-living species. Habitat temperature also affected the pressure response of the enzyme. As a function of physiological pressure and temperature, the order of increasing pressure-sensitivity was cold, deep-sea less than warm, deep-sea (hydrothermal vents) less than polar = shallow and mid-depth, cold less than shallow, warm. Activation volumes in all species were conserved at 30-60 ml mol-1 at physiological pressures, which may reflect a similar membrane physical state at the actual pressure the animal experiences. Arrhenius plots [In(Na+/K+-ATPase activity) vs 1/T] were steeper for warm-water and shallow-living species than for deep-sea species. The depth at which adaptation was first observed was about 2000 m (approximately equal to 200 atm: 1 atm = 101.3 kPa). The data are consistent with a model of increased membrane fluidity resulting in reduced pressure-sensitivity of Na+/K+-ATPase from deep-sea species.

Acclimatization↗
PubMed1988

Regulation of binding of phosphofructokinase to myofibrils in the red and white muscle of the barred sand bass, Paralabrax nebulifer (Serranidae).

The binding of phosphofructokinase (PFK) to myofibrils from the white muscle of the fish Paralabrax nebulifer (Girard, 1854) is sensitive to factors known to be allosteric regulators of PFK activity. PFK in Triton-X-100-extracted muscle remains bound to myofibrils at pH 7.0 and is fully solubilized by increasing the pH to 8.0. The curve describing the pH-dependence of PFK binding to myofibrils is similar in its steepness to pH versus activity curves of PFK at low temperature. Nucleotides are also potent modulators, preventing the association of PFK with myofibrils at concentrations between 20 and 60 mumol l-1 of ATP, ADP, MgATP or GTP, listed in order of effectiveness. PFKs in the red and white muscle extracts differ in their pH-dependence of binding to myofibrils, and their kinetic and regulatory properties (response to citrate, pH and fructose-2,6-bisphosphate). Reversible binding of PFK to myofibrils may be important in the control of glycolysis, especially in the highly glycolytic white muscle fibres.

Animals↗
PubMed1987

Binding of phosphofructokinase to filamentous actin.

Phosphofructokinase (PFK) and filamentous actin from rabbit skeletal muscle form a specific association as demonstrated by electron microscopy of the negatively stained proteins. Actin paracrystals have distinct cross-striations when PFK is present. The periodicity of these striations, 37 +/- 1.0 nm, corresponds to the crossover spacing of the actin helix, 36 +/- 1.0 nm. Assays based on the sedimentation of actin indicate that PFK binds to actin in a concentration-dependent manner with no indication of saturation at a PFK:actin ratio 33 times higher than the ratio in mammalian skeletal muscle. This binding is maintained at physiological ionic strength. Increasing the pH from 6.7 to 7.5 causes a gradual elution of PFK from purified actin filaments. The binding of PFK to actin decreases the rate and extent of activity loss caused by the dissociation of PFK tetramers at low pH and low temperature. The reversible association of PFK with actin may play a role in regulating PFK activity and, therefore, glycolysis during periods of metabolic acidosis.

Actins↗
PubMed1986

Protons, osmolytes, and fitness of internal milieu for protein function.

The composition of the intracellular milieu shows striking similarities among widely different species. Only certain values of intracellular pH, values that generally reflect alphastat regulation, and only narrow ranges of inorganic ion concentrations are found in the cytoplasm of the cells of most animals, plants, and microorganisms. In water-stressed organisms only a few types of low-molecular-weight organic molecules (osmolytes) are accumulated. These highly conserved characteristics of the intracellular fluids reflect the need to maintain critical features of macromolecules within narrow ranges optimal for life. For proteins these features include maintaining adequate rates of catalysis, a high level of regulatory responsiveness, and a precise balance between stability and lability of structure (tertiary conformation, subunit assembly, and multiprotein complexes). The optimal values for these functional and structural features of proteins often lie near the midrange of possible values for these properties, and only under specific conditions of intracellular pH, ionic strength, and osmolyte composition are these optimal midrange values conserved. In dormant cells the departure of solution conditions from values that are optimal for protein function and structure may be instrumental in reducing or shutting down metabolic functions. Seen from a broad evolutionary perspective, the evolution of the intracellular milieu is an important complement to macromolecular evolution. In certain instances appropriate modifications of the internal milieu may reduce the need for adaptive amino acid replacements in proteins.

Acid-Base Equilibrium↗
PubMed1985

Pressure effects on actin self-assembly: interspecific differences in the equilibrium and kinetics of the G to F transformation.

Purified skeletal muscle actins from species whose ambient pressures range from 1 to greater than 500 atm were examined for the sensitivity to hydrostatic pressure of the globular (G) to filamentous (F) self-assembly reaction. Both the equilibrium position and the kinetics of self-assembly were affected by pressure. Increased pressure shifted the self-assembly equilibrium toward the monomer (G) state and reduced the rate of F-actin assembly. For most of the actins studied, the perturbation by pressure of F-actin formation decreased with increasing measurement of pressure, indicating that F-actin has a higher compressibility than G-actin. The increase in system volume and compressibility concomitant with the assembly of F-actin can be interpreted as reflections of the major role played by hydrophobic effects in stabilizing F-actin and of the existence of "hard" binding sites, in the terminology of Torgerson et al. [Torgerson, P. M., Drickamer, H. G., & Weber, G. (1979) Biochemistry 18, 3079-3083], in the actin subunits. For actin from the deepest occurring species studied, the teleost fish Coryphaenoides armatus, which occurs to depths of approximately 5000 m (equivalent to 501 atm of pressure), there was no difference in compressibility between G-actin and F-actin; that is, the effect of increasing pressure on self-assembly was linear over the entire pressure range examined, 600 atm. The self-assembly reaction of the actin from C. armatus also differed from that of the other actins examined in that the G to F equilibrium was relatively insensitive to increased pressure; i.e., the volume change (delta V) of assembly was small.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1984

Influence of osmolytes, thin filaments, and solubility state on elasmobranch phosphofructokinase in vitro.

Skeletal muscle phosphofructokinase (PFK) purified from the thornback ray is rapidly inactivated by urea concentrations as low as 50 mM at pH values below 7.0. Urea-induced loss of PFK activity is not offset by trimethylamine-N-oxide. Protection against urea-inactivation in vivo, where urea concentration may approach 0.5 M, may be due to two effects. Filamentous (F) actin and muscle thin filaments moderately reduce the urea-induced loss of PFK activity. The binding of PFK to F-actin and to thin filaments is shown by ultracentrifugation experiments. PFK activity in vivo also may be stabilized in this species by the formation of a particulate enzyme form which is totally resistant to inactivation by physiological concentrations of urea.

Animals↗