Evolution of a gene. Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation

The effects of temperature on enzyme structure and function have long been of interest to comparative physiologists and biochemists, and the findings of the past ten years have shown convincingly that temperature stresses on enzymes, and the adaptive responses of enzymes to compensate for these stresses, have played an important role in biological evolution. Many aspects of enzymatic adaptation to temperature have been treated thoroughly in recent monographs (3) and reviews (48, 56, 104), and these sources provide extensive bibliographies of the hundreds of papers dealing with temperature-enzyme interactions. This review attempts to com­ plement existing reviews and to provide an overview of the important structure­ function interrelationships that characterize enzymatic adaptation to temperature. This review also attempts to show how the effects of temperature on enzyme struc­ ture and function may be instrumental in establishing the thermal optima and tolerance limits of metabolic function and, thereby, of the organism itself. Hope­ fully, this discussion will help to provide a bridge between the interesting new discoveries of enzyme chemists and interests of ecologists, systematists, and zoo­ geographers. Attention is first directed toward the sources of thermal stress on enzyme systems; this analysis reveals that most aspects of enzyme structure and function are highly sensitive to temperature variation, whether this variation occurs rapidly or over long evolutionary time. Next the so-called "goals" of temperature adaptation are qut­ lined, namely the set of properties that must be conserved in all organisms, under all thermal regimes. The molecular mechanisms instrumental in effecting these critical conservative adaptations are then discussed. Several distinct classes of mech-

Temperature Adaptation of Enzymes: Biological Optimization Through Structure-Function Compromises

Spectrophotometric and electrophoretic techniques were used to monitor qualitative and quantitative aspects of changes in 12 enzymes from five different tissues of green sunfish after thermal acclimation. Changes in dissolved oxygen concentrations over the range 5–25 ppm had no significant effect on qualitative or quantitative aspects of enzyme distributions.



In response to differences in environmental temperature, however, significant changes in the levels of activity of fructose-biphosphate aldolase, pyruvate kinase, lactate dehydrogenase, malate dehydrogenase, succinate dehydrogenase, cytochrome oxidase, cytochrome c, and glucose-6-phosphate dehydrogenase were observed in one or more tissues. Enzymes in a single metabolic pathway (e.g., glycolysis) usually exhibited parallel changes in a given tissue. Enzymes in different pathways, on the other hand, frequently exhibited changes in opposite directions, one group of enzymes increased in the cold while the other group decreased, indicating that major metabolic reorganizations were occurring. The patterns of change among tissues were distinctly different, therefore changes observed in one tissue cannot readily be generalized to other tissues.



No changes in isozyme patterns were seen for lactate dehydrogenase, creatine kinase, fructose-biphosphate aldolase, malate dehydrogenase, glucosephosphate isomerase, phosphoglucomutase, glycerol-3-phosphate dehydrogenase, or alcohol dehydrogenase in any of the tissues examined. However, some changes in the isozyme patterns of esterases in both liver and eye tissue were observed. These observations indicate that major changes in isozyme repertories do not necessarily accompany the thermoacclimatory responses of all fishes.

Molecular aspects of temperature acclimation in fish: Contributions of changes in enzyme activities and isozyme patterns to metabolic reorganization in the green sunfish

Electrophoretic and immunochemical analysis of the lactate dehydrogenase isozyme patterns of fishes indicates that these isozymes are the tetrameric products of three LDH loci — A, B, and C — which are present in virtually all teleost fishes The A and B genes of fishes and their corresponding isozymes have previously been shown to be homologous to the A and B genes of other vertebrates The eye-band LDH characteristic of many groups of teleosts, and the liver-band LDH of gadoid and cyprinid fishes are shown in this report to be encoded in the same LDH locus, designated C In different groups of fishes this third LDH gene (C) is extremely varied both with regard to its tissue-specific regulation and with regard to the physical properties of its corresponding subunit The electrophoretic mobility of the C4 isozyme ranges from highly anodal in some species to slightly cathodal in others while the mobilities of the A4 and B4 tetramers remain relatively constant Most fish species express the A and B genes for LDH in nearly all tissues In contrast, the C gene is highly restricted in tissue expression in most advanced teleosts where its isozymic products are found predominantly in the eye of some species or in the liver of others

Specialized lactate dehydrogenase isozymes: The molecular and genetic basis for the unique eye and liver LDHS of teleost fishes

1.

Goldfish acclimated either to 5 °C or to 25 °C were transferred to the opposite temperature and the changes in behavioral resistance to high temperature, the fluidity and fatty acid composition of isolated synaptosomal membranes were followed during acclimation to the new temperature with the purpose of establishing some correlation.




2.

In 25 °C-acclimated goldfish, hyperexcitability was induced at 34.5 °C, loss of equilibrium at 37.6 °C and coma at 39.0 °C. In 5 °C-acclimated goldfish the corresponding temperatures were 29.2 °C, 32.0 °C and 33.0 °C. Time to attain 75% of the final acclimated state after transfer was approximately 4 days at 25 °C and 28 days at 5 °C.




3.

Fluidity of synaptosomal membranes isolated from goldfish brains was estimated by use of the fluorescence polarization technique. Membrane viscosity decreased during acclimation to 5 °C, but increased during acclimation to 25 °C. The early stages of the transitions differed in time course from behavioral resistance acclimation but times to reach the new acclimated state were similar.




4.

Fatty acid composition of synaptosomal phospholipids showed increased unsaturation during cold-acclimation and decreased unsaturation during warm-acclimation.




5.

It is concluded that during acclimation, behavior shows changes in resistance to heat which are related to synaptic block. These are correlated in direction and overall time course with viscosity of synaptosomes as dictated by changes in the saturation of membrane phospholipids.

Correlations between behavioral temperature adaptations of goldfish and the viscosity and fatty acid composition of their synaptic membranes

1

The time course of acclimation from 15 ° C to 5 ° C and from 15 ° C to 25 ° C was measured for two compensating enzymes (cytochrome oxidase and succinic dehydrogenase) from epaxial muscle of goldfish (Carassius auratus L) The time course of acclimation from 15 ° C to 25 ° C was measured for one inversely compensating enzyme (catalase)




2

The transition from one acclimation state to another shows initial oscillations before reaching a new steady state at from 3–4 weeks The time necessary to reach the new steady state is shorter for small than for larger fish The oscillations in activity cannot be attributed to the feeding schedule of the animals

Time course of thermal acclimation in goldfish

1. 1. The lactate dehydrogenase and malate dehydrogenase isozyme patterns from goldfish ( Carassius auratus L.) tissues were examined by starch gel electrophoresis and immunoprecipitation. 2. 2. Electrophoretic analyses reveal no detectable qualitative or quantitative alterations in the isozyme patterns due to the effects of temperature acclimation at 5, 15 and 25°C. 3. 3. The only variations of LDH * isozyme patterns observed were those attributable to genetic polymorphism and to different ratios of red and white skeletal muscle fibres.

Lactate dehydrogenase and malate dehydrogenase isozyme patterns in tissues of temperature-acclimated goldfish (Carassius auratus L.).

. Lactate dehydrogenase and malate dehydrogenase isozyme patterns from goldfish ( Carassius auratus L.) tissues were examined by starch gel electrophoresis. Electrophoretic analyses reveal no detectable qualitative or quantitative alterations in the isozyme patterns due to the effects of temperature acclimation at 5°, 15°, and 25°C. The only variations of LDH isozyme patterns observed were those attributable to genetic polymorphism and to the different isozyme patterns of white and red skeletal muscle fibers. Specific activity of LDH was similar for all acclimation temperatures. Various glycolytic and oxidative isozymes from green sunfish ( Lepomis cyanellus ) tissues were examined by starch gel electrophoresis and spectrophotometric activity analysis. No qualitative alterations were noted in the isozyme patterns due to the effects of temperature acclimation at 5° and 25°C although quantitative alterations were observed. Warm-acclimated sunfish display an increase in glycolytic enzymes (LDH, aldolase, phosphoglucomutase, G-3-PDH) and a decrease in an oxidative enzyme (MDH). Cold-acclimated sunfish display increased oxidative enzyme activity and decreased glycolytic enzyme activity. We conclude that qualitative isozyme alterations of these enzyme systems occur as a direct result of temperature acclimation only in trout, while other groups of fish show either quantitative alterations or no changes in isozyme patterns.

F. Ray Wilson

Papers

Time course of thermal acclimation in goldfish

Lactate dehydrogenase and malate dehydrogenase isozyme patterns in tissues of temperature-acclimated goldfish (Carassius auratus L.).

Isozyme patterns in tissues of temperature-acclimated fish