Volution of Mammalian Endothermic Metabolism: Itochondrial Activity and Cell Composition

volution of mammalian endothermic metabolism: itochondrial activity and cell composition

A. J. HULBERT AND PAUL LEWIS ELSE Department of Biology, University of Wollongong, Wollongong, New South Wales 2500, Australia ulbert A. J., and Paul Lewis Else. Evolution of mam- m endothermic metabolism: mitochondrial activity and cell as the mammal and the central netted dragon (Amphi bolous nuchalis) as the reptile (8, 19); however, later KTo-n< & »?«hysioL 256 (Re^latory Integrative Comp when it became necessary to prepare isolated liver cells -omparedv 1 ^ ?63f6 in Amphibolous Vf ?-B0dy vitticeps C0raP°si^n and Rattus was measurednorvegicus we changed the comparison to that between the lar-er pti e and a mammal with the same weight and body rat {Rattus norvegicus) and the bearded dragon (A. vit erature). Homogenates were prepared from liver, kidney ticeps) (12). In both of these comparisons the reptile is .heart, lung, and skeletal (gastrocnemius) muscle, and the same size as the mammal and has a preferred body :hondria were isolated. Cytochrome oxidase activities of tlssu* h°m ogenates and isolated mitochondria were meas- emperature that is the same as the mammal's body temperature. These two comparisons have given almost nrif v Wa* ?5?tem content. Phospholipids were identical results, and we believe they offer an excellent £r?mletermined. J1™ MJ?The brain,bdnev' liver, and kidney,ê fatty heart, acid compositionand skeletal system for understanding the cellular basis of endoth te were significantly larger in the mammal, whereas the ermy and its associated thermogenesis. reproductive organs, lung, and digestive tract showed no The initial comparison was restricted to four tissues Oiver, kirJxiey heart, and brain) and showed that the inedSd WT™ -o0% m more SiZG-A11 protein mammaIian and phospholipid tissues than examined the re- mammal had larger internal organs, which contained ve reptilian tissue. Although the mammalian phospho- more mitochondria, and that the total mitochondrial contained significantly less total unsaturated fatty acids membrane surface area of these tissues was approxi unsaturated fatty acids were significantly more polyun- ■ mately fourfold greater in the endotherm than the ec ted than in the reptilian tissues. Tissue cytochrome'oxi- totherm (8). This was later shown to be a general differ ictivity was significantly greater in mammals when ex- d on a wet weight basis but not when expressed on a ence between mammals and reptiles and that mitochon protein basis. Mitochondrial cytochrome oxidase activitv drial membrane surface area was allometrically related protein basis) was the same in both species in liver" to body size with a similar exponent to the metabolic .and brain but in heart, lung, and skeletal muscle rate-body size relationship (10,11). More recently, it has been shown that the mammalian liver and kidney are •onona.lllmTK °ndf-iaThe implications W6re twke of these as active differences as rePtiHan in tissue considerably leakier" to sodium and potassium ions =iuon were discussed relative to the evolution of mam- than the corresponding reptilian tissues, and this greater endothermy. leakiness possibly explains in part the increased Oo consumption ofthe mammalian tissues (12). The present consumption; ectothermy; endothermy; reptiles; phos- paper extends the detailed comparison of the two lar-er ds; membrane fatty acids; tissue protein; cytochrome species and is concerned with two main questions: 1) do mitochondria from mammalian tissues have similar enzymic activity to those from reptilian tissues; and 2) was the increase m metabolism during the evolution of mam malian endothermy associated with any major changes ate are its body size, its body temperature, and in the composition of tissues? Tit is an endotherm or an ectotherm. Many have shown that resting endotherms (mammals MATERIALS AND METHODS •as) have a level of metabolism that is approxi- four to fve times that of similar sized ectotherms y\e1her)iaNN wZ$' ^"l ^-bi^-aminoeth- Tther V6'- physiological 1,8)>. ThlS parameters difference such is also as maximal manifest chromechromp c (horserf * fff heart), add lecithin (EGTA)' (type salt-fê IX-E, egg cyto yolk) ac rate, growth rate, and aerobic endurance are ed oetween the two groups (3, 4). Over the last Mcorhcaci^ (BHtT SiH r(HfES)'.^^ hydroxy toW InTk i CIC 3Cld' and reference fatty acid *££ Tn Tk6 0bttned fr0m the Siâ Chemical. f r^^f^?.^?- V; BSA) was obtained

1. Comparison of body composition of reptile TABLE 2. Comparison of protein content of tissues wlurus vitticeps and mammal Rattus norvegicus from reptile Amphibolous vitticeps and mammal Rattus norvegicus Significance A. vitticeps R. norvegicus of Differenc Significance %. A. vitticeps R. norvegicus of Difference 9 10 weight, g 304±34 310±24 NS n 6 6 dy weight Body weight, g 340±43 321±33 NS an 0.13±0.01 0.69±0.04 P < 0.01 Protein content, er 2.84±0.42 4.21±0.11 P < 0.01 mg protein/g tissue Iney 0.41±0.04 0.89±0.04 P < 0.01 90±10 165±5 P < 0.01 art 0.29±0.01 0.40±0.01 P < 0.01 Kidney 91±5 126±7 P < 0.01 >mach 1.12±0.01 0.49±0.03 P < 0.01 53±3 105±1 P < 0.01 estines 1.51±0.10 2.04±0.15 P < 0.02 Heart 85±4 114±5 P < 0.01 0.81±0.04 0.68±0.05 NS Lung 60±5 90±7 P < 0.01 ng P < 0.02 productive 0.66±0.13 1.37±0.31 NS Skeletal muscle 81±6 120±12 in + fur 21.21±1.28 20.00±0.55 NS tissue, n, no. of 42.82±0.90 P < 0.01 Values are means ± SE measured as mg protein/g eletal muscle 34.58±1.94 animals. tier + skeleton 36.42±1.43 25.94±1.25 P < 0.01 s are means ± SE measured as percent of body weight (minus stomach contents); n, No. of animals. NS, not significant. TABLE 3. Comparison of phospholipid fatty acid composition of liver from reptile Amphibolous Kile and 6.2% in the mammal. The mammal also vitticeps and mammal Rattus norvegicus jjnificantly more skeletal muscle than the reptile, Significance ere was no significant difference between the two A. vitticeps R. norvegicus of Difference 3 in the proportion of body mass devoted to body n 5 5 rig, the lungs, and the reproductive system. The P < 0.05 Dtal of these differences (10.9% of body weight) Phospholipid content, 0.53±0.09 0.86±0.07 mg/g tissue •mpensated by a significantly greater "other" com- Fatty acid composition, t. This other component is primarily the skeleton, mol % is a much larger part of the reptile than the X, 1.8±0.2 0.8±0.3 P < 0.05 lal. 16:0 14.8±1.6 16.5±2.3 NS 16:1 1.3±0.5 0.3±0.1 NS lough the intestines were significantly larger in the 17:0 0.6±0.1 0.6±0.1 NS lal, the stomach was significantly smaller than in Xa 0.7±0.1 0.5±0.3 NS ptile, and the net result was that there was no 18:0 20.2±1.3 27.1±3.0 NS mce in the portion of body mass devoted to the 18:1" 12.6±2.7 5.3±1.4 NS ive system between the mammal and the reptile. 18:lt 3.0±0.9 2.2±0.2 NS 8.2±0.2 NS of these differences are compatible with the much 18:2 20.7±5.8 ■ level of energy metabolism in the endothermic 18:3 2.2±0.9 0.1±0.1 NS 20:4 13.7±2.0 30.6±1.0 P < 0.001 ial compared with the ectothermic reptile. The lack 20:5 2.2±0.4 0.3±0.1 P < 0.01 erence in the digestive system at first seems un- 22:4 1.0±0.2 0.7±0.1 NS The mammalian system is the more rapid digestor 22:5 2.1±0.1 1.3±0.2 P < 0.01 22:6 1.5±0.3 4.3±0.9 P < 0.05 )sorber of nutrients, and the relatively small stom- Ûnsaturated fatty acids 61.1±1.2 53.7±1.1 P < 0.005 the mammal is probably correlated with its more Unsaturation index 155±4 185±8 P < 0.01 nt mastication of food in the mouth before swal- Average chain length 18.0±0.1 18.4±0.1 P < 0.05 '. The physiological and structural digestive adap- 20:4/18:2 1.02±0.35 3.76±0.17 P < 0.001 s associated with the evolution of endothermy in Values are means ± SE; n, no. of animals. Only fatty acids that lals are excellently covered in a series of papers by constituted >0.5% of total are shown. First no. represents no. of carbon ov and co-workers (20, 21). atoms, whereas second represents no. of double bonds. Xi and X2 are unidentified fatty acids. 18:1* is oleic acid, whereas 18:1+ is cis vacenic the mammalian tissues examined had significantly acid. Unsaturation index is sum of (mol % x no. of double bonds) for protein than the corresponding reptilian tissues each fatty acid. Average chain length is sum of (mol % X no. of carbons/ j 2). The difference was remarkably consistent 100) for each fatty acid. en tissues, with the mammalian tissue having on >e 58% more protein than the same amount of lipid content presumably represents a greater amount of an tissue. As well as having a greater protein membranes in the mammalian tissue compared with the it, mammalian tissues also contained a signifi- reptilian tissues. The difference in protein content, while • greater amount of phospholipids (Tables 3 and 4). including the effect of more membranes (and thus more )holipid content was compared in liver and kidney membrane proteins) in mammals, would seem to be out in both these tissues there was on average 54% predominantly a reflection of the larger amount of non- phospholipid in the tissue from the mammal than membrane protein in the mammal compared with the : tissue from the reptile. This value is very similar reptile. i protein difference, and both are related to the Tables 3 and 4 also contain an analysis of the fatty r metabolic rate of the endothermic mammal com- acids that make up the phospholipids in liver and kidney with the ectothermic reptile. The higher phospho- tissues from the mammal and reptile. One interesting ENDOTHERMY AND CELL COMPOSITION

TABLE 4. Comparison of phospholipid fatty acid Table 5 shows a comparison ofthe cytochrome oxic activity of six tissues from the mammal and the rep composition of kidney from reptile Amphibolous When compared on a wet weight basis, the cytochn vitticeps and mammal Rattus norvegicus oxidase activity of all tissues was significantly greate Significance the mammal than in the reptile, however, when comp, A. vitticeps R. norvegicus of Difference on a tissue protein basis, only one tissue (lung) he o significantly greater cytochrome oxidase activity in Phospholipid content, 0.54±0.02 0.79±0.06 P < 0.005 mammal, one tissue (brain) showed a significantly mg/g tissue duced enzyme activity in the mammal, and all the o Fatty acid composition, tissues showed no significant difference between mol% mammal and the reptile. This change is consistent i X, 2.4±0.6 1.2±0.4 NS 16:0 11.5±0.7 16.2±1.7 P < 0.05 the higher protein content of the mammalian tis; 1.6±0.6 0.5±0.1 NS compared with the equivalent reptilian tissue, espec: 0.4±0.1 0.6±0.1 NS the brain (see Table 2). When isolated mitochoE 0.9±0.3 NS X, 1.0±0.4 from these tissues are compared (Table 5), reptilian 1 18:0 20.5±0.4 25.3±3.2 NS 18:1* 14.0±3.5 6.3±1.5 NS kidney, and brain mitochondria are not significa 18:1+ 3.6±1.0 2.2±0.2 NS different from the equivalent mammalian mitochon 18:2 16.5±3.4 7.7±0.3 NS Both skeletal muscle and cardiac muscle mitochor. NS 18:3 3.2±1.6 0.1±0.1 from the mammal had very high cytochrome oxi 20:4 13.0±1.3 30.7±0.5 P< 0.001 20:5 2.4±0.5 0.5±0.1 P < 0.01 activities (on a per mg protein basis) that were a 22:4 1.1±0.3 0.9±0.1 NS twice those of the equivalent reptilian mitochon 22:5 2.5±0.6 1.2+0.2 P < 0.01 Although the cytochrome oxidase activity of lung r 22:6 1.8±0.5 3.5±0.7 P < 0.05 chondria was not as great as mitochondria from mi; 53.9±0.8 P < 0.005 %Unsaturated fatty acids 61.6±1.7 the total activity of the mammalian mitochondria Unsaturation index 159±5 183±3 P < 0.005 Average chain length 18.0±0.2 18.4±0.1 NS twice that of reptilian mitochondria. 20:4/18:2 1.02±0.28 4.04±0.24 P< 0.001 Mammalian mitochondria tended to have a gr Values are means ± SE; n, no. of animals. Only fatty acids that protein content relative to mitochondrial lipid comp constituted >0.5*rc of total are shown. First no. represents no. of carbon with reptilian mitochondria, although this was onh atoms, whereas second represents no. of double bonds. Xi and X> are unidentified fatty acids. 18:1* is oleic acid, whereas 18:1+ is CIS vacenic acid. Unsaturation index is sum of (mol % x no. of double bonds) for table 5. Comparison of tissue and mitochondrial each fatty acid.. Average chain length is sum of (mol % x no. of carbons/ cytochrome oxidase activity in tissues from reptile 100) for each fatty acid. Amphibolous vitticeps and mammal Rattus norvegi^ Signific finding (in both species) is the remarkable similarity A. vitticeps amount of linoleic acid (18 : 2) in mammalian phospho Liver Kidney 895±117 649±68 r- lipids and an increased content of arachidonic acid Brain 464±73 441±54 > (20: 4). The differences were statistically significant in Heart 891±172 1,782±152 P< both tissues in the case of 20 : 4 but were not statistically Lung 263±37 531±55 P< 751±127 1,532±274 P< significant in the case of 18: 2. This is probably due to Skeletal muscle the high variability of 18:2 content in the reptilian Values are means ± SE; n, no. of animals. All cytochrome phospholipids. activities were measured at 37°C. NS, not significant. ENDOTHERMY AND CELL COMPOSITION R67 iP*k ;ne Siîf!cant CytOChrnrnp in **?er nvinaco and brain opfuM+Jnn (ê Table ;+ 6). :.- -.«—:ui-gastrocnemius *.- •_ muscle - .1 » was t . . the only skeletal muscle ex- involved and^he accuracy of the measurements, reptiles (11). >arison of these values should not be too rigorous, major assumptions are that cytochrome oxidase ity is not affected by the isolation of mitochondria DISCUSSION hat the protein content of the mitochondrial prep- Previously, it has been shown that endothermic mam 3n is both solely mitochondrial protein and contains mals have a greater amount of (metabolically active) ally all ofthe protein originally in the mitochondria. tissues than ectothermic reptiles, and, via the techniques ming these to be true, we can see from Table 7 that, of quantitative electron microscopy, it was shown that the exception of kidney tissue, there are no signifi- these mammalian tissues contain a greater amount of differences in the proportion of cellular protein that mitochondria than the same reptilian tissue (8, 11). and in the mitochondria of the reptilian tissues When all factors are taken into account, endothermic ared with the mammalian tissues. In the mamma- mammals average a total mitochondrial membrane sur ddney, 37% of cellular protein is calculated to be face area that is four to five times that for ectothermic :hondrial, which is significantly greater than the reptiles, and this difference is similar to that for their calculated for the reptilian kidney. In both species, respective standard metabolic rates (11). Such a corre Dal muscle is the tissue with the smallest proportion lation assumes that mitochondria from endotherms have ltllnr nrntoin OO m^nnl.n-J.:.l j. _• T-v * i • — — — - v * — " " " " w w w u n u o c H u m e ^ L u u i e r m s . i n s because ofthe large amounts of contractile protein the present study we have examined this assumption by iscle. In both species, cardiac muscle has twice the measuring the enzymic activity of isolated reptilian and hondrial protein (relative to total cellular protein) mammalian mitochondria. Cytochrome oxidase activity does skeletal muscle. In the present study, the was chosen because it is relatively easy to measure and has been previously used in a number of comparisons of •: 6. Comparison of relative protein to lipid content tissues between ectotherms and endotherms. It is the ochondria from tissues of reptile Amphibolous terminal respiratory enzyme and is responsible for the ps and mammal Rattus norvegicus consumption of 02. The answer to the question posed in the introduction A. vitticeps R. norvegicus Sjî£lcance is both yes and no. Mitochondria isolated from three of Difference tissues (liver, kidney, and brain) showed similar enzymic activity in both the endotherm and the ectotherm, eight, g 340±43 321±33 NS ondrial protein-to-lipid whereas mitochondria from muscle (both skeletal and io, mg protein/mg lipid cardiac) and lung showed twice the cytochrome oxidase 1.0±0.2* 3.6±0.3 P < 0.02 activity (on a per mg mitochondrial protein basis) in the sy 1.9±0.2 2.2±0.5 NS endotherm compared with the ectotherm (see Table 5). 0.5±0.1 0.9±0.1 P < 0.01 An analysis of previous studies from the literature gives 2.0±0.6 1.5±0.9 NS 1.0±0.5 1.5±0.4 a similar dichotomy between tissues. Mitochondria iso tal muscle NS 0.6±0.1 1.2±0.5* NS lated from the liver of a wide range of ectotherms are 3s are means ± SE; n, no. of animals. NS, not significant. * No very similar in their properties to those isolated from als = o. mammalian liver (5, 28). A similar finding was made in a comparison of brain mitochondria from a number of 7. Comparison of relative mitochondrial protein vertebrates (30). In muscle, although the cytochrome c(as % of total cellular protein) of tissues of reptile oxidase content of fish heart mitochondria is about one- bolurus vitticeps and mammal Rattus norvegicus third of that of beef heart mitochondria, the 02 con sumption per mole of cytochrome oxidase is similar in A. vitticeps R. norvegicus Si£J»ficance both groups (32). of Difference Because cytochrome oxidase activity was measured as 02 consumption (when provided with excess substrate) ight, g 340±43 321±33 ndrial protein, and the size ofthe tissues was also measured, it is possible f total to calculate the total 02 consumption by cytochrome ilar protein oxidase for the summed tissues for both animals. When 32±6<"=:b 41±o NS this is done, the total 02 consumption by cytochrome • 41±5 V 2 5 ± 2 25±2 3 7 ± 437±4 P < 0 . 0 5 oxidase is 22 ml 02/min for the reptile and 63 ml 02/min 55±8 39±5 39±5 NS for the mammal. In both species, skeletal muscle is 33±6 15±1 15±1 NS 20±6 21±3 21±3 NS responsible for 76-80% of the summed 02 consumption "il muscle muscle 13±3 13±3 7+1* 7±1* NS by cytochrome oxidase, and both of these values are very similar to the maximal metabolic rates expected for the \is *** - 5. Tex?S Mitochondrial *_** "' n0' protein 0f animals- was calculated NS, not significant. as tissue *cyto- No. two species (11). ^dase activity x 100/mitochondrial cytochrome oxidase ac- Akhmerov (1) has recently reported that the mito chondria of endothermic animals are qualitatively differ- R68 ENDOTHERMY AND CELL COMPOSITION ent from those frommic ectothermic animais. *punw«*« .animals. *"*.*;— Quantitative . / rr>„ui„„ o n„A ^^^J^PA a\ TViptp ftAXX are lsis difficult rl 1Cv2__STS. however ♦ since " JLlreptilian unsaturated tissue (see fatty Tables acids 3 but and proportionate!} 4). lhere are

out previously any company u «««^-- ^ reported for a more extensive comparison between ectotherms*""^SHS and endotherms should take _&ttsss*_5__ into oeen «P° ectothermic and endothermic verfe QWgﬂtliU

ence between r therms is interesting chondrial membranes of endotherms maj

membrane in the endotherm (12). The ﬁnding by Akh- ».\ ,i .i i. l :j—„U«-.J-;n c-V-i/int o miirh crrpatP

ference between these two tissues found in the present tissues compared with reptiliantissues^ (12, 19). study. Although such a qualitative difference in the mi- s: tochondria of ectotherms and endotherms might be a c partial explanation of the difference in heat production fa1 between the two groups it cannot explain the differenc"

amount of "sodium-potassium pumping" (12). permeaDiiitypermeability oi of irug frog »mskin «.are »-«both =ii The other question posed earlier was wh« inrrpﬂ^p in metabolic rate associated with the - ... tfentoh^ Na+"K--ATPase. activity ot cells grown m c the composition of the tissues? The approximate fourfold aff increase in metabolism of the mammal compared with co: the reptile was associated with a 50-60% increase in th protein content and phospholipid content of all tissu- examined (only liver and kidney were analyzed for pho pholipid). As mentioned previously, body size inﬂuence; metabolic

mammals is also associated with changes in the protein tween the mammalian tissues^ and the reptik.

tionship between weight-speciﬁc metabolic rate and pi

IllUll 111 UUtli Sliuobiuus. *v* wM""r~l -— sight-speciﬁc metabolism that is about four times that is not only

; 3 C l l l / ^ i i v y n j ^ * » ^ . J \ — » / • — — - - - - — - S AsI well between as there the being docosahexanoic more phospholipid in acid the mam- (22 diac : &***^^^*"£ 6) cont ™^

malian tissue, the fatty acid composition of these phos- from the mouse to the whale, with the more me ENDOTHERMY AND CELL COMPOSITION se species having a greater 22 : 6 content (14). This 14. Gudbjarnason, S., B. Doell, G. Oskarsdottir, and J. Hall- lilar to the findings of the present study in that the GRIMSSON. Modification of cardiac phospholipids and catechol amine stress tolerance. In: Tocopherol, Oxygen and Biomembranes, Dolically more intense mammalian tissues have a edited bv C. de Duve and O. Havaishi. Amsterdam: Elsevier, 1978, sr 22 : 6 content than the less metabolically intense p.297-310. ian tissues (Tables 3 and 4). 15. Harris, W. D., and P. Popat. Determination of phosphorus e compositional differences reported in the present content of lipids. J. Am. Oil Chem. Soc. 31: 124-127, 1954. are based on a detailed comparison of only two 16. Hemmingsen, A. M. Energy metabolism as related to body size and respiratory surfaces and its evolution. Rep. Steno Mem. Hosp. is. They agree with the relatively small number of Nord. Insulinlab. 9: 1-110, 1960. 2S in the literature, but whether they represent 17. Hendriks, T., A. A. Klompmakers, F. J. M. Daemen, and S. L. al differences between endotherms and ectotherms BONTING. Biochemical aspects of the visual process. XXXII. s study of a more diverse range of vertebrate species. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids. Biochim. Biophys. Acta 433: 271-281, 1976. thank Ann-Michelle Whitington, Patric Tap, and Namita Sen 18. Hulbert, A. J. On the evolution of energy metabolism in mam ir technical assistance and also Dr. R. Akhmerov for his com- mals. In: Comparative Physiology: Primitive Mammals, edited by on the manuscript. The New South Wales National Parks and K. Schmidt-Nielsen, L. Bolis, and C. R. Taylor. Cambridge, UK: "e Service gave permission to capture the lizards. Cambridge Univ. Press, 1980, p. 129-139. s work was supported by a Peter Rankin Trust Fund Award (to 19. Hulbert, A. J., and P. L. Else. Comparison of the "mammal Use) and an Australian Research Grants Scheme grant (to A. J. machine" and the "reptile machine": energy use and thyroid activ t). ity. Am. J. Physiol. 241 (Regulator,' Integrative Comp. Phvsiol. 10): lent address of P. L. Else: Dept. of Zoology, University of R350-R356, 1981. lrne, Parkville, Vic 3052, Australia. 20. Karasov, W. H., E. Petrossian, L. Rosenberg, and J. M. ress for reprint requests: A. J. Hulbert, Dept. of Biology, Univ. Diamond. How do passage rate and assimilation differ between longong, Wollongong, NSW 2500, Australia. herbivorous lizards and nonruminant mammals? J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 156: 599-609, 1986. ed 2 May 1988; accepted in final form 3 August 1988. 21. Karasov, W. H., D. H. Solberg, and J. M. Diamond. What transport adaptations enable mammals to absorb sugars and amino acids faster than reptiles? Am. J. Phvsiol. 249 (Gastrointest. Liver Physiol. 12): G271-G283, 1985. :HMEROV, R. N. Qualitative difference in mitochondria of en- 22. Kleiber, M. The Fire of Life: An Introduction to Animal Energetics. thermic and ectothermic animals. FEBS Lett. 198: 251-255 New York: Wiley, 1961. B6. 23. Natochin, Yu. V., V. G. Leont'ev, M. N. Maslova, L. F. IVTHOLOMEW, G. A., AND V. A. Tucker. Control of changes in Pomazanskaya, N. I. Pravdina, and E. M. Kreps. Sodium dy temperature, metabolism and circulation by the agamid lizard. reabsorption and membrane systems of vertebrate kidneys. Zh. Evol. Biokhim. i Fiziol. 11: 45-52, 1975. iphibolurus barbatus. Physiol. Zool. 36: 199-218, 1963. 24. Lin, M. H., D. R. Romsos, T. Akera, and G. A. Leveille. NNETT, A. F. Activity metabolism of the lower vertebrates. nu. Rev. Physiol 40: 447-469, 1978. Increase in Na*,K*-ATPase enzyme units in liver and kidney from SE, T. J. On the evolution and adaptive significance of postnatal essential fatty acid deficient rats. Experientia Basel 35: 735-736, >wth rates in terrestrial vertebrates. Q. Rev. Biol. 53: 243-282 1979. 78. 25. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran dall. Protein measurement with the Folin phenol reagent. J. Biol. SSUTO, Y. Oxidative activities of liver mitochondria from mam- Chem. 193: 265-275, 1951. Js, birds, reptiles and amphibia as a function of temperature. 26. Moore, J. L., T. Richardson, and H. F. Deluca. Essential fatty mp. Biochem. Physiol. B Comp. Biochem. 39: 919-923, 1971. acids and ionic permeabilitv of lecithin membranes. Chem. Phys. RISTIE, W. W. Lipid Analysis (2nd ed.). Oxford, UK: Pergamon, Lipids 3: 39-58, 1969. 27. MUNRO, H. N., and E. D. Downie. Relationship of liver compo WSON, T. J., and A. J. Hulbert. Standard metabolism, body sition to intensity of protein metabolism in different mammals. aperature, and surface areas of Australian marsupials. Am. j. Nature Lond. 203: 603-604, 1964. ysioL 218:1233-1238, 1970. 28. Smith. C. L. The temperature dependence of oxidative phospho SB, P.^L., AND A. J. HULBERT. Comparison of the "mammal chine" and the "reptile machine": energy production. Am. J. rylation and of the activity of various enzyme systems in liver mitochondria from cold- and warm-blooded animals. Comp. ysioL 240 (Regulatory Integrative Comp. Physiol. 9): R3-R9 Biochem. Physiol. B Comp. Biochem. 46B: 445-461, 1973. 11. 29. Solomonson, L. P., V. A. Liepkalns, and A. A. Spector. se, P. L., and A. J. Hulbert. A comparative study of the Changes in (Na* + K*)-ATPase activity of Ehrlich ascites tumor taboiic capacity of hearts from reptiles and mammals. Comp. cells produced by alteration of membrane fatty acid composition. •chem. PhysioL A Comp. Physiol. 76A: 553-557, 1983. Biochemistry 15: 892-897, 1976. 5E, P. L., and A. J. Hulbert. Mammals: an allometric study 30. Wahbe, V. G., W. M. Balfour, and F. E. Samson. A comparativ. metabolism at tissue and mitochondrial level. Am. J. Physiol. study on vertebrate brain mitochondria. Comp. Biochem. Physiol. • [Regulatory Integrative Comp. Physiol. 17): R415-R421, 1985. 3: 199-205, 1961. 5E, P. L., and A. J. Hulbert. An allometric comparison of the 31. Wharton, D. C, and D. E. Griffiths. Studies on the electron ccr.ondria of mammalian and reptilian tissues: the implications transport system. XXXIX. Assay of cytochrome oxidase. Effects the evolution of endothermy. J. Comp. Physiol. B Biochem. of phospholipids and other factors. Arch. Biochem. Biophys. 96: t Environ. Physiol. 156: 3-11, 1985. 103-114, 1962. ;e. P. L., and A. J. Hulbert. Evolution of mammalian endo- 32. Wilson, M. T., J. Bonaventura, and M. Brunori. Mitochon rmic metabolism: "leaky" membranes as a source of heat. Am. drial cytochrome content and cytochrome oxidase activity of some 'hysiol 253 (Regulatory Integrative Comp. Physiol. 22): R1-R7, Amazonian fish. Comp. Biochem. Physiol. A Comp. Physiol. 62: i. 245-249,1979. .ch, J., M. Lees, and G. H. Sloane-Stanley. A simple 33. YORIO, T., S. TORRES, and N. Tarapoom. Alteration in mem hod for the isolation and purification of total lipids from animal brane permeability by diacylglycerol and phosphatidylcholine con ues. J. Biol. Chem. 226: 497-509, 1957. taining arachidonic acid. Lipids 18: 96-99, 1983.