Phylogenetic analysis of glycolytic enzyme expression.

Although differences among species in enzyme maximal activity or concentration are often interpreted as adaptive and important for regulating metabolism, these differences may simply reflect phylogenetic divergence. Phylogenetic analysis of the expression of the glycolytic enzymes among 15 taxa of a North American fish genus (Fundulus) indicated that most variation in enzyme concentration is due to evolutionary distance and may be nonadaptive. However, three enzymes' maximal activities covary with environmental temperature and have adaptive value. Additionally, two pairs of enzymes covary, indicating coevolution. Thus, metabolic flux may be modulated by many different enzymes rather than by a single rate-limiting enzyme.

The effect of urea exposure on isoaspartyl content and protein L-isoaspartate methyltransferase activity in Drosophila melanogaster.

Urea is a protein unfolding agent that can accumulate to locally high concentrations in tissues of many organisms. We used Drosophila melanogaster to test the hypothesis that urea loading would promote formation of isoaspartate (beta-carboxyl-linked aspartate), a common form of protein damage that occurs most readily in unstructured polypeptides and flexible regions of folded proteins. Ten populations of flies were tested; five control populations of urea-sensitive flies and five previously selected urea-tolerant populations. We measured the effects of urea consumption on levels of both isoaspartate and protein L-isoaspartate methyltransferase (PIMT), an enzyme believed to function in the repair or removal of isoaspartyl proteins. For both sets of populations, urea feeding for 6 days increased isoaspartyl levels by approximately 60%, supporting the idea that disruption of protein secondary and tertiary structures can accelerate the formation of isoaspartate in vivo. Urea feeding tended to increase PIMT activity in both control and urea-tolerant populations. There were no significant differences in PIMT activities or isoaspartyl levels between the control and urea-tolerant flies raised on normal or urea food. The latter findings indicate that urea tolerance evolved in the selected populations without any significant change in PIMT expression or activity.

Phylogenetic analysis of thermal acclimation of the glycolytic enzymes in the genus Fundulus.

Physiological acclimation that alters enzyme activity can compensate for the effect of temperature on function and may be achieved by altering enzyme concentration. This study uses phylogenetic analyses to investigate the evolutionary history of and to test several hypotheses about acclimation responses among all the glycolytic enzymes. These hypotheses are that (1) acclimation increases enzyme concentration at lower temperatures to compensate for reduced activity; (2) equilibrium enzymes tend to show acclimation responses; and (3) acclimation responses are more common in species whose populations experience either large temporal or geographical temperature variations. Using maximal activities as indices of enzyme concentration, the presence of acclimation responses in all the glycolytic enzymes in the heart ventricle was determined for five species in the teleost genus Fundulus. Three of these species are distributed along the steep thermal cline of the North American Atlantic coast, and thus these species experience both seasonal and geographical variation in temperature. The other two species are found in the Gulf of Mexico and experience seasonal variation similar to the Atlantic species but no geographical variation in temperature. Two Atlantic coast species, Fundulus heteroclitus and Fundulus majalis, have unique derived acclimation responses. No derived acclimation responses occur in the Gulf species. A conserved response in hexokinase was observed within one subgenus comprising both Atlantic and Gulf species. In F. heteroclitus, enolase responded to acclimation, and in F majalis, aldolase, triphosphate isomerase, and lactate dehydrogenase had acclimation responses. These enzymes are equilibrium enzymes, and the concentrations of all of them increase at lower temperatures, which would compensate for the effect of temperature on enzyme activity. The compensatory changes all occur in the Atlantic species and may be a mechanism for species to expand their ranges. These data suggest that physiological acclimation is evolutionarily labile.

Rapid enzyme assays investigating the variation in the glycolytic pathway in field-caught populations of Fundulus heteroclitus.

Variation in enzyme expression may be important in evolutionary adaptation, yet is seldom studied. Furthermore, no studies have examined the expression of all enzymes in a defined metabolic pathway. Enzyme concentration is a measure of enzyme expression and was ascertained by assaying maximal activity. Presented here is an analysis of variation of maximal enzyme activity for all the enzymes in a single metabolic pathway, glycolysis, from three clinically distributed populations of the fish, Fundulus heteroclitus. Techniques for rapidly analyzing maximal enzyme activity for all the enzymes of an entire metabolic pathway from many individuals are described. The high degree of repeatability (mean coefficient of variation for replicates, 4.4%) and sensitivity (less than 3 mg of tissue is required to measure all 10 enzymes) of these assays demonstrate the utility of such an approach for analyzing variation among populations for a large numbers of enzymes. Results from these studies indicate that (1) the average coefficient of variation for all enzyme determinations within a population is 45.3% and (2) between populations, the activity of 5 of the 10 glycolytic enzymes are significantly different. This considerable variation occurs even in populations where there is little allelic variation. These data demonstrating substantial variation in enzyme expression support the idea that changes in gene regulation may be as important as, or even more important than, changes in biochemical kinetic parameters in evolutionary processes.

Osmoregulation in Drosophila melanogaster selected for urea tolerance.

Animals may adapt to hyperosmolar environments by either osmoregulating or osmoconforming. Osmoconforming animals generally accumulate organic osmolytes including sugars, amino acids or, in a few cases, urea. In the latter case, they also accumulate 'urea-counteracting' solutes to mitigate the toxic effects of urea. We examined the osmoregulatory adaptation of Drosophila melanogaster larvae selected to live in 300 mmol l(-)(1) urea. Larvae are strong osmoregulators in environments with high NaCl or sucrose levels, but have increased hemolymph osmolarity on urea food. The increase in osmolarity on urea food is smaller in the selected larvae relative to unselected control larvae, and their respective hemolymph urea concentrations can account for the observed increases in total osmolarity. No other hemolymph components appear to act as urea-counteractants. Urea is calculated to be in equilibrium across body compartments in both selected and control larvae, indicating that the selected larvae are not sequestering it to lower their hemolymph osmolarity. The major physiological adaptation to urea does not appear to involve increased tolerance or improved osmoregulation per se, but rather mechanisms (e.g. metabolism, decreased uptake or increased excretion) that reduce overall urea levels and the consequent toxicity.