TIBS - D~c~mher 1977
269 Barents Sea at the edge of the Arctic Sea in the northeastern most tip of the Siberian Russian islands, Novaya Zemlya.
Muscle enzymes of Antarctic fishes
Polar fish proteins Robert
E. Feeney and David T. Osuga
Fishfivm polar murine seas mu,, he exposed to tcn~peratures qf less tflan - 1.8 C. Some of tfwirproteins must tflereffvY~ f1ciI~epr~~pertie.s and,fimctions [email protected]
fj.om tflose of species ,fi-om u’armer environments.
Temperature is one of the most important environmental parameters for living organisms. Consequently, adaptations to temperatures have long interested scientists in many different fields of endeavor. At the molecular level, how enzymes function at different temperatures have been included in many different studies, but, as yet, no general rules on mechanisms of temperature adaptation have been proposed [1,2]. The effects of variations in temperature on the enzymes and proteins of poikilothermic organisms are obvious. The effects of temperature on enzymes and other proteins of homeothermic organisms are also important because of the possible fluctuations in internal temperature due to the extremes in environment or disease as well as by normal physiological changes occurring in some species undergoing cycles such as those involved in hibernation. Biochemists have been using temperature changes as a probe to study enzyme properties for some time . A high degree of sophistication has developed in studies ofenzymes at very low temperatures (e.g., -6O- C). These have recently even involved crystallographic studies of enzyme intermediates that are ‘frozen’ at the enzyme active site at these low temperatures . Obviously, the two parts of the world where very low temperatures exist ‘externally’ are at the poles. The temperatures of surface ocean waters at the poles are approximately - 1.85 C, the equilibrium temperature of the ocean water of normal salinity in equilibrium with ice. Marine species that live near the surface of the water where ice exists are consequently exposed to a temperature of approximately - I .85 C.
During the last few decades there has been a large expansion of research at both poles. In the Antarctic, the main thrust started with the International Geophysical Year in the mid-1950s. The development of the Polar Programs by the U.S. National Science Foundation and the establishment of all-winter bases in Antarctica gave impetus to these studies . The authors began their program in Antarctica in 1964, and they and members of their laboratory visited Antarctica for periods of up to several months in each of seven different years until 197 1. Utilizing the comprehensive support and logistics supplied them, it was possible to do extensive biochemical studies on the proteins and enzymes of fish from these cold waters . An extension in the studies by the authors to the Arctic region was made possible in 1976 by a sea voyage of 2 1 days, in the
The majority of the work on the enzymes of Antarctic fishes have their origin in the extensive physiological studies of Wohlschlag and his students . He studied a variety of different types of Antarctic fishes living in the Ross Sea and concluded that they are all truly coldadapted. Three species, Trematomus horchgrcvinki, T. bernucchii, and Dissostithus ma#sorzi have a very low upper lethal temperature of approximately 7 or 8” C. In fact, they do very poorly in temperatures above 4 C. Metabolic studies on one of the enzyme systems in gill tissue homogenates of T. hernacchii revealed that the enzymatic activity had an unusually low activation energy . Such a low value could be an advantage for cold adaptation. Later studies by Rakusa-Suszczewski and McWhinnie  have expanded this work in a general physiological sense. Studies by our laboratory have also found low activation energies for some enzymes but very different adaptations or effects with others. The heart mitochondrial cytochrome oxidase system of T. horchgrevinkidiffered from the enzymes of warm-blooded homeothermic mammalian organisms in that the homeothermic enzyme system had a temperature induced transition whereas the cold fish system did not  (Fig. 1). Muscle aldolases and dehydrogenases Muscle aldolases from T. borchgrevinki and D. martssoni were purified by conventional procedures and their properties compared with one purified from the rabbit [IO]. The fish aldolases had certain properties similar to the rabbit aldolase, such as similar molecular weights, electrophoretic properties, K, values, and activation energies, although their amino acid compositions differed. The fish aldolases also differed significantly from the rabbit aldolase in being much more labile to temperatures, (Fig. 2) and to sulfhydryl reagents. The glyceraldehyde-3-phosphate dehydrogenase was prepared from the D. muiclsoni muscle and rabbit muscle [I I]. Most of the properties of the rabbit and fish enzymes were very similar, such as molecular weights, specific activities, K, values, amino acid compositions, and temperature-stability curves. There was, however, a significant difference in the activation energies. The values were approximately 14,500 cal/mole for the D. mawsoni enzyme and 18,000 cal/mole for the rabbit enzyme. The lower activation energy of the
[ 121. Three some important differences salient differences of biochemical interest are (a) the absence of hemoglobin and red blood cells in the so-called ‘bloodless’ fish in Antarctica [ l3], (b) a capacity for rapid clotting of blood near 0 C in polar fish and the inability of clotting at higher temperatures [ 141, and (c) the presence of a unique protein which lowers the freezing temperature without affecting the melting TEMPERATURE / ‘0 / temperature in order to allow the fish to exist in equilibrium mixtures of ice and salt near - 1.85 C without undue osmotic stress [ 14- 181. Since the early studies of Rudd [ 131 there has been much interest in the respiratory system of the ‘bloodless’ fish. One of the simplest explanations, although not universally supported, is that these species have a rapid turnover of blood in the tissues and a higher solubility af oxygen at fish enzyme gave it a slightly higher specia very low temperature, which would fic activity at 0 C than the activity of the clearly aid the oxygen supply. In at least rabbit enzyme and a relatively lower specione subspecies, Chaenochepolis aceratus. fic activity at higher temperatures, becomits scaleless skin did not appear to be a ing as small as only half of the activity at respiratory compensation, and the gill area 37 C, the environmental temperature of was similar to that of red blooded polar the rabbit. In addition, they differed fish [ 191. The heart of C. aceratm was two extensively in their dissociation and inactito three times larger than other fish examvation by adenine nucleotides at low ined, and its spleen and- many other parts temperatures. The activity ofthe rabbit enof its body were comparatively large, sugzyme was reduced by 80”,, after incubation of with 2 mM ATP at 0 C, while the I). IHCIH‘- gesting this may be the consequence reduced axial musculature. soni enzyme lost only about 25”,, of its acOur laboratory investigated the concentivity in 18 mM ATP (Fig. 3). The losses tration of the iron transport protein, transof activity by the rabbit were shovvn to be ferrin, in another ‘bloodless’ fish, Chinndaccompanied by a dissociation of the tetraWe considered that the ram karhleenue. mer into dimers. No such affects occurred concentration of the serum transferrin at 37 C. might be lowered because of the reduced Blood proteins from polar species requirement for iron, but the transferrin concentration was found to be the same The blood proteins of teleosts have as in the fish possessingred blood cells . many features in common with proteins Extensive studies have been made on the of other higher vertebrates. but there are proteins lowering the freezing temperature, and these proteins have been named ‘antifreeze glycoproteins’ in the case of most fishes, and just ‘antifreeze proteins’ in others [ l5-18,20-251. Because of the unique physics concerned with this phenomenon. these proteins will be discussed in more detail in the next section. Antifreeze proteins Probably the most significant earliest observation on freezing temperature in polar fish blood was by the eminent physiologist. Per Scholander, and coworkers . They showed that there was a high molecular weight substance that lowered the freezing temperature much more than would be estimated from its concentration and apparent size. This work was done on Arctic fish, and also early studies by Leivestad  off northern Norway extended and confirmed such abnormalities. The next definitive work was started
by DeVries and Wohlschlag [ 161 and continued in our laboratory as part of our work done simultaneously on blood proteins [14,17,18]. The main work of a chemical and physical nature has been done with an antifreeze glycoprotein from Antarctic fish, but more recent studies have been done with other antifreeze glycoproteins from Arctic fish [22,26] and with non-glycoproteins from Arctic fish [22-241. An amazing consistency between the two antifreeze proteins is that they both contain approximately two-thirds of the total amino acids as alanine! The Antarctic antifreeze glycoprotein is unique in many respects. First, it is composed of repeating units of a glycotripeptide (Ala, Ala, Thr), with each Thr glycosiditally linked to a disaccharide (Fig. 4) . Each series of active components varies in molecular weight from approximately
TIBS - December I9 77
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11,000 to 25,000. Secondly, it lowers the freezing temperature of water approximately 500 times greater than it should based on its molecular weight as functioning in a colligative manner, and consequently it lowers the freezing temperature of water more than twice as much as an equal weight of sodium chloride (Fig. 5). In agreement with this abnormal lowering, all molecular weight determinations, including those on the, colligative property of osmotic pressure, are in agreement. Thirdly, antifreeze glycoprotein has no effect on the melting temperature. In other words, the melting temperature is the same as it would be for other large proteins of a similar size. There appears to be no evidence of supercooling in the conventional sense because the antifreeze glycoprotein is active in the presence of varying amounts of ice crystals. Fourthly, it is completely included in the developing ice phase of freezing of the solution (i.e., it is not excluded from the ice as is the case with materials acting in a colligative fashion). The normal melting point of the ice as well as other data indicate that the ice is normal ice and that antifreeze is entrapped in some manner although not part of the crystal structure of the ice lattice . The mechanism of action presently considered to be the most likely explanation is one involving an interference of ice crystal development by surface action. Visual observarions of uneven crystal surfaces support this idea . A recent very interesting observation is that fish‘ caught off the northern end of the Russian Siberian islands, Novaya Zemlya, by the authors in 1976, contained active glycoproteins that appear to have the identical structure of those from Antarctica .
The finding of an antifreeze protein in Arctic fish [21,23,24] which contains the same high amount of alanine as the antifreeze glycoprotein, two out of three residues, but no carbohydrate, places additional emphasis on the importance of the hydrophobic aspects of the molecules. It is too early to note whether or not the functions of these two antifreeze agents work by the same mechanism or whether the one without the carbohydrate is as efficient. The one from the Arctic fish does have periodically placed carboxyl groups which may in some way substitute for carbohydrate residues. Polar marine species should continue to offer many opportunities and challenges for investigators interested in biological systems at low temperatures. Some similar adaptations have also been found in nonmarine species, such as the adaptation of the glyceraldehyde-3-phosphate dehydrogenase in the cold-adapted honeybee  and the adaptation of the heart mitochondrial oxidase systems in plants and some other species . Although antifreeze proteins have been reported to exist in some trees , they have so far only been characterized from species living in the icesalt waters of the seas. Acknowledgements
The financial support (grant no. GA12607) and logistical assistance of the U.S. National Science Foundation was essential for the Antarctic studies of the authors. The Iinancial assistance and supply of logistics by the Norwegian Government and the Norwegian Institute of Marine Biology in Bergen for the Arctic research in 1976 is gratefully acknowledged. NIH Grant HD 00122 has supported the chemical and physical chemical studies.
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