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G Somero

Publications and source records attributed to G Somero.

7 recordsLinked to original sources
PubMed1997

Amino acid sequence differences cannot fully explain interspecific variation in thermal sensitivities of gobiid fish A4-lactate dehydrogenases (A4-LDHs)

We compared the deduced amino acid sequences, heat stabilities and thermal sensitivities of a kinetic property, the apparent Michaelis­Menten constant (Km) of pyruvate, of A4-lactate dehydrogenase (A4-LDH) in four species of goby fishes (Family Gobiidae), adapted to different temperatures, to examine how changes in primary structure influence the adaptation of enzymes. The effect of temperature on Km of pyruvate reflected each species' environmental temperature. For the most eurythermal species, Gillichthys seta, which is endemic to shallow intertidal regions of the upper Gulf of California and encounters temperatures between approximately 9 and 40 °C, Km of pyruvate was minimally affected by temperature, compared with the A4-LDH orthologues from a less eurythermal congener, G. mirabilis (9­30 °C), a cold temperate goby, Coryphopterus nicholsi (10­18 °C) and a tropical species, C. personatus (25­32 °C). Heat denaturation profiles failed to correlate with habitat temperature; G. mirabilis A4-LDH was most thermally stable, followed by the orthologues of C. nicholsi and G. seta. Complementary DNAs (cDNAs) encoding LDH-As of G. seta, Gulf of California and Pacific coast populations of G. mirabilis and C. nicholsi were isolated and sequenced, and the corresponding amino acid sequences deduced. The nucleotide sequences of LDH-A of the two populations of G. mirabilis were identical. Five nucleotide differences in the coding region and one amino acid substitution (at position 78) distinguished LDH-As of G. mirabilis and C. nicholsi. The substitution of a glycyl residue (C. nicholsi) for an alanyl residue (G. mirabilis) may account for the difference in thermal stability between these two orthologues. Comparisons of the LDH-A cDNAs of G. mirabilis and G. seta revealed four differences in nucleotide sequence in the coding region, but all nucleotide substitutions were synonymous. The identical deduced primary structures of the two enzymes suggested the possibility of different protein conformational variants ('conformers') in the two species. This hypothesis is supported by electrospray ionization mass spectrometry, which indicates that the masses of the A4-LDH orthologues of the two species are the same within the resolution of the technique. To explore the possibility that the two enzymes were different conformers of the same primary structure, we treated purified G. seta and G. mirabilis A4-LDHs with 3.0 mol l-1 urea or 6 mol l-1 guanidine­HCl and, after removing the denaturant, compared their kinetic properties and heat stabilities. Neither treatment had an effect on the A4-LDH of G. mirabilis, but both converted the Km versus temperature profile of the G. seta enzyme to that of the G. mirabilis A4-LDH. The thermal stability of neither enzyme was affected. We propose, as has been suggested in several previous studies of A4-LDH, that this enzyme can fold into a number of conformers with different stabilities and functional properties. The A4-LDH of G. seta furnishes evidence that such conformers may provide an important mechanism for adaptation of proteins to temperature.

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PubMed1996

Seasonal-, tidal-cycle- and microhabitat-related variation in membrane order of phospholipid vesicles from gills of the intertidal mussel Mytilus californianus

Alterations in the order, or fluidity, of cellular membranes in response to variations in environmental temperature are well known. The mussel Mytilus californianus, a common inhabitant of mid-intertidal regions along the Pacific coast of North America, can experience large (20 °C or more) and cyclic (every 6 h) changes in body temperature (Tb ) during tidal cycles. In the present study, we explore membrane order during seasonal and tidal temperature cycles and find that vesicles prepared from gill phospholipids exhibit significant seasonal differences in order that are consistent with homeoviscous adaptation and suggest winter-acclimatization to mean Tb values and summer-acclimatization to upper extreme Tb values or to large cyclic thermal fluctuations, despite repeated resubmergence in sea water at 10 °C during both seasons. Phospholipid vesicles prepared from the gills of mussels acclimated for 6 weeks to constant high or low temperatures in the laboratory fail to exhibit temperature-compensatory differences in order. In addition, during the summer, mussels inhabiting high intertidal sites, but not those from low sites, possess the ability to alter membrane order rapidly (within hours). This alteration of order appears to represent a mechanism designed to offset the thermal variations encountered during the tidal cycle. Thus, M. californianus have the ability to adjust membrane order on seasonal as well as hourly time scales and do so on the basis of their height in the intertidal region.

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PubMed1996

Adaptation to temperature stress and aerial exposure in congeneric species of intertidal porcelain crabs (genus Petrolisthes): correlation of physiology, biochemistry and morphology with vertical distribution

We examined physiological and biochemical responses to temperature and aerial exposure in two species of intertidal porcelain crabs (genus Petrolisthes) that inhabit discrete vertical zones. On the shores of the Northeastern Pacific, P. cinctipes (Randall) occurs under rocks and in mussel beds in the mid to high intertidal zone and P. eriomerus (Stimpson) occurs under rocks in the low intertidal zone and subtidally to 80 m. Because of their different vertical distributions, these two species experience very different levels of abiotic stress. Individuals of P. cinctipes can be emersed during every low tide, but P. eriomerus is only emersed during the lowest spring tides and on most days is not emersed at all. Temperatures measured underneath rocks in the mid intertidal zone were as high as 31 °C, 15 °C higher than maximal temperatures measured under rocks in the low intertidal zone. In air, at 25 °C, large specimens of P. cinctipes were able to maintain a higher respiration rate than similarly sized P. eriomerus. No interspecific differences in the respiratory response to emersion were seen in small specimens. Examination of the response of heart rate to temperature revealed that P. cinctipes has a 5 °C higher Arrhenius break temperature (ABT, the temperature at which there is a discontinuity in the slope of an Arrhenius plot) than its congener (31.5 °C versus 26.6 °C). The heart rate of P. cinctipes recovered fully after exposure to cold (1.5 °C), but the heart rate of P. eriomerus did not recover after exposure to 2 °C or cooler. The ABT of heart rate in P. cinctipes was very close to maximal microhabitat temperatures; thus, individuals of this species may be living at or near their thermal tolerance limits. P. cinctipes were able to maintain aerobic metabolism during emersion, whereas P. eriomerus shifted to anaerobic metabolism. A pronounced accumulation of whole-body lactate was found in specimens of P. eriomerus incubated in air at 25 °C over a 5 h period, but not in P. cinctipes similarly treated. P. cinctipes possesses a membranous structure on the ventral merus of each walking leg, but this structure is not found in P. eriomerus. To test the function of the leg membrane, we measured the aerial respiration rates and the lactate accumulation of P. cinctipes with their leg membranes obscured. These individuals had significantly lower aerial respiration rates at 30 °C than control crabs. Crabs with leg membranes obscured also accumulated a considerable amount of lactate during a 5 h period of emersion at 28 °C, but control crabs showed no accumulation under the same conditions. These data suggest that the leg membrane functions as a respiratory structure. The results of this study illustrate that a suite of morphological, physiological and biochemical features allows P. cinctipes to live higher in the intertidal region than P. eriomerus.

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PubMed1995

Thermal adaptation of cytoplasmic malate dehydrogenases of eastern Pacific barracuda (Sphyraena spp): the role of differential isoenzyme expression

Kinetic properties, electrophoretic patterns and thermal stabilities of cytoplasmic malate dehydrogenases (cMDHs) were compared in Eastern Pacific barracuda (Sphyraena spp) from different latitudes. All tissues of the tropical species S. ensis contained only a single, thermostable form of cMDH. Subtropical (S. lucasana) as well as north (S. argentea) and south (S. idiastes) temperate barracuda contained both thermostable and thermolabile cMDHs, the pattern characteristic of most teleosts. Kinetic studies using unfractioned cMDHs showed that the apparent Michaelis­Menten constant (Km) of cofactor (NADH) increased with temperature, but at the physiological temperatures of the four species, Km of NADH was conserved within a narrow range (20­23 µmol l-1). Thermostable and thermolabile cMDHs were chromatographically separated and compared. Thermolabile cMDHs had higher Km values for NADH at all measurement temperatures than did thermostable cMDHs. Thermolabile cMDHs isolated from congeneric barracuda exhibited similar kinetic properties (Km versus temperature, optimal pH, optimal substrate and cofactor concentrations). Thermostable cMDHs, likewise, were similar among the barracuda. Conservation of Km in the differently thermally adapted barracudas is, therefore, apparently due to adjustments in the ratio of expression of the thermostable and thermolabile isoforms, rather than to temperature-adaptive differences among orthologous homologues, as is commonly found for enzymes encoded by a single gene locus. The effects of temperature on the Km of NADH for isolated thermostable and thermolabile cMDHs of a eurythermal goby, Gillichthys mirabilis, however, were consistent with adaptive change in orthologous homologues of cMDH. The selective basis for the absence of thermolabile cMDH in warm-adapted ectotherms, mammals and birds is discussed.

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PubMed1995

Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus

We examined the seasonal variation in environmentally induced protein damage in natural populations of the intertidal mussel Mytilus trossulus. In order to compare the state of protein pools during seasonal variations in environmental temperature, we used solid-phase immunochemical analysis to quantify ubiquitin conjugate concentrations and relative levels of the stress protein hsp70. The two biochemical indices were selected for their cellular roles in irreversible and reversible protein denaturation, respectively. Proteins that are ubiquitinated are irreversibly damaged and are degraded by intracellular proteases; stress proteins act as molecular chaperones to re-fold thermally denatured proteins and, thus, indicate degrees of reversible protein damage. Comparisons involved mussels collected in February and August from two study sites: an intertidal site which subjected animals to a wide range of body temperatures (from approximately 10 to 35 C in summer), and a subtidal site where animals remained submerged throughout the tidal cycle. Our results show that quantities of ubiquitin conjugates and hsp70 were greater in gill tissue from summer-collected mussels than in gills of winter-collected specimens. Ubiquitin conjugate and hsp70 levels were also greater in mussels collected from an intertidal location than in mussels from a submerged population. Our results show that the high summer temperatures normally experienced in the field are sufficient to cause increased denaturation of cellular proteins. Despite increases in the concentrations of heat shock proteins in summer-acclimatized mussels, elevated levels of irreversibly denatured, i.e. ubiquitinated, proteins were still observed, which indicates that the heat shock response may not be able to rescue all heat-damaged proteins. The energy costs associated with replacing heat-damaged proteins and with maintaining the concentrations and activities of heat shock proteins may contribute substantially to cellular energy demands. These increased energy demands may have an impact on the ecological energetic relationships of species, e.g. in the allocations of energy for growth and reproduction, and, as a consequence, may contribute to determining their distribution limits.

Journal Article↗
PubMed1995

Osmotic and thermal effects on in situ ATPase activity in permeabilized gill epithelial cells of the fish Gillichthys mirabilis

Long-jawed mudsuckers (Gillichthys mirabilis) were acclimated to sea water (SW) at 7 °C, SW at 26 °C or dilute sea water (DSW) at 26 °C for 5 months. Gill cells were isolated and the proportion of mitochondria-rich (MR) cells was determined. The number of cells harvested amounted to 4.7x10(7)±0.6x10(7) to 10.6x10(7)±1.1x10(7) and the yield was between 7.1x10(8)±0.6x10(8) and 10.7x10(8)±1.4x10(8) cells g-1 gill epithelial mass. Cell viability was 96.8±0.4 to 97.8±0.6 %. The number, size and volume of MR cells decreased significantly during DSW acclimation, but did not change during thermal acclimation. The protein content was not influenced by osmotic or thermal acclimation and ranged between 20.0±1.6 and 22.1±1.5 pg cell-1. Using a new method, which is based on the formation of plasma membrane channels by alamethicin, we were able to permeabilize gill cells. For the first time, the Na+/K+-ATPase and H+-ATPase activities of fish gills were determined in intact cells in situ. The activity of both ATPases was dependent on alamethicin concentration (optimum 100 µg mg-1 protein) and on preincubation time (optimum 10 min). The in situ activity of both ATPases was influenced by osmotic, but not thermal, acclimation. A positive linear correlation was found between in situ Na+/K+-ATPase activity and total MR cell volume. However, we show, for the first time, that a negative linear correlation exists between H+-ATPase activity and total MR cell volume, suggesting a localization of H+-ATPase in pavement cells. In permeabilized cells, the activity of both ATPases was 2.6­3.9 times higher than that of crude homogenates and 1.6­2.1 times higher than that of permeabilized homogenate vesicles. We hypothesize that in crude homogenates three-quarters of Na+/K+-ATPase and two-thirds of H+-ATPase activity are not detectable both because of a mixture of inside-out and right-side-out vesicles and because of the disruption of membrane and enzyme integrity.

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