ABSTRACT
When Astacus leptodactylus were kept at various temperatures for several weeks, different ratios between di-hexameric and hexameric hemocyanins were observed in their hemolymph. The higher the temperature the more hexamers were present. This long-term adaptation to different temperatures or/and to temperature-induced pH-shifts as observed in the hemolymph has different effects on the expression of subunit types building up hexamers and those which covalently link two hexamers within the di-hexamers. The oxygen binding behaviour of di-hexameric hemocyanins from cold and warm adapted animals do not show differences with respect to affinity, Bohr effect and cooperativity.
Subject(s)
Adaptation, Physiological , Crustacea/physiology , Hemocyanins/metabolism , Animals , Crustacea/metabolism , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , TemperatureABSTRACT
Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20 degrees C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20 degrees C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10 degrees C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.
Subject(s)
3-Hydroxyacyl CoA Dehydrogenases/metabolism , Adaptation, Physiological , Caenorhabditis elegans/physiology , Citric Acid Cycle/physiology , Cold Temperature , Isocitrate Dehydrogenase/metabolism , L-Lactate Dehydrogenase/metabolism , Oxygen/metabolism , Animals , Hypoxia/metabolism , NAD/metabolism , NADP/metabolism , Oxidation-Reduction , Spectrometry, Fluorescence , Tissue ExtractsABSTRACT
Fluorescence quenching studies and binding experiments with [(3)H]ecdysone reveal that the respiratory protein, hemocyanin, of the tarantula Eurypelma californicum binds ecdysone. The binding constant for ecdysone ranges between 0.5 and 5 mM, indicating a low affinity binding. However, it is comparable with those found for the ecdysone binding to hexamerins from insects. Based on a comparison of sequences and x-ray structures of arthropodan hemocyanins, we propose an evolutionary conserved hydrophobic pocket in domain 1 of the hemocyanin subunit that may bind ecdysone.
Subject(s)
Ecdysone/metabolism , Ecdysterone/metabolism , Hemocyanins/metabolism , Spiders/metabolism , Amino Acid Sequence , Animals , Binding Sites , Glycoproteins/genetics , Hemocyanins/chemistry , Hemocyanins/genetics , Insect Proteins/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , TritiumABSTRACT
In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.
Subject(s)
Caenorhabditis elegans/metabolism , Acetates/metabolism , Adaptation, Physiological , Anaerobiosis/physiology , Animals , Breeding , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Carbohydrate Metabolism , Escherichia coli/metabolism , Lactic Acid/metabolism , Propionates/metabolism , Succinic Acid/metabolism , Survival Rate , TemperatureABSTRACT
Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24-meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90 degrees C. Our results demonstrate that the cation-mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.
