View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Electronic Publication Information Center Marine Biology )2001) 139: 113±126 DOI 10.1007/s002270100557 I.M. Sokolova á H.O. PoÈ rtner Temperature effects on key metabolic enzymes in Littorina saxatilis and L. obtusata from different latitudes and shore levels Received: 23 October 2000 / Accepted: 31 January 2001 / Published online: 30 March 2001 Ó Springer-Verlag 2001 Abstract Eects of temperature on activities of key L. obtusata £ low shore L. saxatilis< high shore metabolic enzymes, citrate synthase )CS), NADP- L. saxatilis. Possibly, an increased anaerobic capacity is dependent isocitrate dehydrogenase )NADP-IDH), as- advantageous for snails at high shore levels, where they partate aminotransferase )AAT), pyruvate kinase )PK) may experience limited oxygen access during prolonged and phosphoenolpyruvate carboxykinase )PEPCK), air exposure. In contrast, the activation energies of the were studied in high and low shore Littorina saxatilis respective enzymes did not reveal a consistent pattern of and in low shore L. obtusata from the temperate variation and were similar in animals from dierent North Sea and the sub-arctic White Sea. It was found latitudes and shore levels. Arrhenius breakpoint tem- that adaptation of L. saxatilis and L. obtusata to life peratures )ABT) of the studied enzymes characterising a at dierent latitudes and/or shore levels involves con- change in the thermal properties of the protein were stitutive changes in enzymatic activities, so that ani- found well within the average highs of ambient tem- mals naturally adapted to )micro-) environments with peratures )20±35°C). Denaturation temperatures )Td), lower mean temperatures )i.e. in the White Sea or at indicating heat inactivation of the protein, were close to low shore levels) tend to have higher enzyme activities the environmental extremes experienced by L. saxatilis as compared to their counterparts from warmer hab- during summer low tide. These ®ndings suggest that the itats )e.g. the North Sea or high shore levels, respec- metabolic machinery of this eurythermal species may tively). This suggests metabolic temperature function close to its physiological limits during summer compensation in dierent populations/subpopulational low tide, especially at high shore levels. groups of this eurythermal species. Activities of all ®ve studied enzymes were modulated by adaptations to high shore life in L. saxatilis, whereas only NADP- IDH, PK and PEPCK had dierent activities in ani- Introduction mals from dierent latitudes. Adaptation to high shore life also involved an enhanced potential for anaero- Temperature is an important environmental factor in- bic energy production via the succinate pathway in ¯uencing all life functions of an organism through Littorina spp. )measured as the ratio of PEPCK and changes in the rates of biochemical and physiological PK activities), which increased in the order: low shore processes and in the stability of biomolecules )reviews: Hochachka and Somero 1973; Homann 1983; Prosser 1991). Adaptation to the thermal environment is rec- ognised as one of the evolutionary mainstreams and Communicated by O. Kinne, Oldendorf/Luhe comprises the organism's ability to adjust metabolism I.M. Sokolova )&) á H.O. PoÈ rtner on both short-term and evolutionary time scales Alfred Wegener Institute for Polar and Marine Research, )Hochachka and Somero 1973; Prosser 1991; Clarke Laboratory for Ecophysiology and Ecotoxicology, 1998). Changes in the kinetic characteristics of enzymes Columbusstr. 30, 27568 Bremerhaven, Germany re¯ect dierences in metabolic regulation and are E-mail: [email protected] inevitably involved in adaptation and acclimation to Tel.: +49-471-48311311 Fax: +49-471-48311149 ambient temperature )Somero 1975, 1978, 1995; Lag- erspetz 1987; Vetter and Buchholz 1998). Hence, the I.M. Sokolova analysis of temperature-dependent changes in enzyme White Sea Biological Station, Zoological Institute of Russian Academy of Sciences, structure and function is traditionally used as a powerful Universitetskaya emb. 1, 199034 St. Petersburg, Russia tool in studies of temperature acclimation and adapta- 114 tion of animals, particularly ectotherms, which are es- iccation and oxygen deprivation during low tide, pecially prone to direct temperature eects of their en- whereas environmental conditions are milder and more vironment. stable at low shore levels )Sokolova et al. 2000b). Thus, There is a continuously growing body of data con- comparison of temperature eects on enzyme activities cerning metabolic adaptations on the molecular )in- in high versus low shore and White versus North Sea cluding enzymatic) level in dierent ectotherms, )sub-) populations of the periwinkles provides a useful especially in those inhabiting extreme environments model allowing to identify potentially adaptive changes )reviews: Clarke 1998; Somero 1998; PoÈ rtner et al. 1999, in enzymatic function in Littorina from dierent thermal in press). However, the frequent absence of closely environments on vertical and latitudinal scales. related species at high and low latitudes )Eastman 1993) For the present study, ®ve enzymes representative of makes comparative work on metabolic adaptations in dierent metabolic pathways were chosen: citrate syn- cold-and warm-adapted organisms very dicult. As a thase )CS), NADP-dependent isocitrate dehydrogenase result, temperature eects per se are often confounded )NADP-IDH), aspartate aminotransferase )AAT), with interspeci®c dierences in other traits, which may pyruvate kinase )PK) and phosphoenolpyruvate carb- play much stronger roles in governing metabolic rates in oxykinase )PEPCK). Citrate synthase is a key regulatory general and enzymatic activities in particular than the enzyme in the tricarbonic acid )TCA) cycle )Krebs and one played by temperature )Pierce and Crawford Johnson 1937) and was chosen as an indicator of aerobic 1997a,b; Somero 1998). To overcome this problem, one capacity of animals. NADP-dependent isocitrate dehy- should ideally choose a group of closely related species drogenase is suggested to adopt a key role in a futile with known phylogeny or a single species with a wide mitochondrial substrate cycle which is likely to be rele- geographical distribution. Such studies have been quite vant to the control of respiration and mitochondrial rare )e.g. Pierce and Crawford 1997a,b; Sommer et al. proton leakage )Sazanov and Jackson 1994; PoÈ rtner 1997; van Dijk et al. 1999) due to diculties with ®nding et al. 1999, in press). Pyruvate kinase and phospho- suitable animal models. The intertidal gastropod enolpyruvate carboxykinase are assumed to compete for Littorina saxatilis chosen for the present study is an a common substrate, phosphoenolpyruvate, channelling example of a uniquely convenient object in this respect. it to the TCA cycle via pyruvate )PK) and acetyl-CoA or Its distribution area covers >50° by latitude, ranging to mitochondrial anaerobic pathways via oxaloacetate from North Africa up to Svalbard )Reid 1996). This )PEPCK) and malate )Saz 1971; Bryant 1975). Thus, a allows comparison of warm-and cold-adapted popula- ratio of speci®c activities of PK and PEPCK is consid- tions within an ecologically, morphologically and ge- ered as a good indicator of the potential anaerobic netically cohesive unit such as a single species. capacity of an animal )de Vooys 1980; Bowen 1984; Moreover, L. saxatilis is characterised by an excep- SimpendoÈ rfer et al. 1995). Aspartate aminotransferase is tionally wide vertical distribution. It occupies the range involved in amino acid metabolism and suggested to from the upper subtidal to the supratidal splash zone play an important role during early anaerobiosis, and thus encounters environments with greatly diering together with alanine aminotransferase, by channelling temperature and humidity regimes even within a single aspartate to the mitochondrial production of succinate population )Sokolova et al. 2000b). )de Zwaan 1991), as well as during post-anaerobic The aim of the present investigation was to study the recovery, by supplying aspartate for the regeneration eects of temperature on the activity of key metabolic of AMP from IMP )Mommsen and Hochachka 1988). enzymes in L. saxatilis from dierent shore levels from Thus, the chosen set of enzymes should give a good the sub-arctic White Sea and the temperate North Sea. overview of aerobic and anaerobic capacities of These two study areas dier greatly with respect to their L. saxatilis. For comparative purposes, PK and PEPCK climatic conditions, especially temperature regime. Sub- activities were measured in a closely related species, arctic White Sea populations of L. saxatilis experience L. obtusata from the North and White Seas, which extreme seasonal variations of temperature, from freez- covers a slightly more restricted latitudinal range and ing )below ±1.5°C in brackish seawater to temperatures occupies only mid-to low shore levels )Reid 1996). of from ±10°Cto+15°C in air) in late autumn and For the ®ve studied enzymes, maximal activities winter to highs of from +15°Cto+20°C and more in )Vmax) were measured at dierent temperatures in summer )Babkov 1998; Sokolova et al. 2000b). In con- L. saxatilis and L. obtusata from dierent latitudes and/ trast, both temperature ¯uctuations and temperature or shore levels. Vmax was chosen because it is a mecha- extremes are much less pronounced in the North Sea and nistically