Effects of anoxia exposure and aerobic recovery on metabolic enzyme activities in the freshwater turtle Trachemys scripta elegans

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1 1822 Effects of noxi exposure nd erobic recovery on metbolic enzyme ctivities in the freshwter turtle Trchemys script elegns Willim G. Willmore, Kyr J. Cown, nd Kenneth B. Storey Abstrct: The effects of noxic submergence (20 h t 7 C in nitrogen-bubbled wter) nd subsequent erobic recovery (24 h t 7 C) on the mximl ctivities of 21 metbolic enzymes were ssessed in liver, kidney, hert, brin, nd red nd white skeletl muscle of n noxi-tolernt freshwter turtle, the red-ered slider, Trchemys script elegns. Anoxi exposure ffected the ctivities of only few enzymes; for exmple, it reduced the ctivity of phosphofructokinse in liver nd brin, hexokinse in kidney, glycerol-3-phosphte dehydrogense nd glutmte-oxlocette trnsminse in hert, glutmte dehydrogense nd serine dehydrtse in brin, nd 3-hydroxycyl-CoA dehydrogense in red muscle. During erobic recovery, ctivities of most of these enzymes rebounded nd ctivities of 10 others tht were not ffected by noxi rose during recovery. Anoxi-induced chnges in selected enzymes pper to meet very specific needs such s glycolytic-rte depression, regultion of glycolytic versus gluconeogenic flux in liver, or ltertions in mino cid neurotrnsmitter levels in brin. Overll, the dt demonstrte tht the enzymtic mke-up of turtle orgns undergoes very few chnges during noxi exposure nd recovery, which shows tht the constitutive ctivities of enzymes re well designed to meet the metbolic demnds of noxic excursions. Résumé : Nous vons évlué les effets d une submersion noxique (20 h à 7 C, dns de l eu où brbotit de l zote) et de l récupértion érobie subséquente (24 h à 7 C) sur l ctivité mximle de 21 enzymes métboliques dns le foie, les reins, le coeur, le cerveu, insi que dns les muscles squelettiques blnc et rouge d une tortue d eu douce tolérnt l noxie, Trchemys script elegns. L exposition ux conditions noxiques ffecte l ctivité de seulement quelques enzymes : pr exemple, celle de l phosphofructokinse est réduite dns le foie et dns le cerveu, celle de l hexokinse dns le rein, celle de l glycérol-3-phosphte déshydrogénse et de l glutmte-oxlocétte trnsminse dns le coeur, celle de l glutmte-déshydrogénse et de l sérine déshydrtse dns le cerveu, enfin celle de l 3-hydroxycyl-CoA déshydrogénse dns le muscle rouge. Durnt l récupértion en conditions érobies, l ctivité de l pluprt de ces enzymes reprend et l ctivité de 10 utres enzymes non ffectées pr l noxie ugmente. Les chngements dns l ctivité de certines enzymes prticulières générés pr l exposition à l noxie semblent répondre à des besoins spécifiques comme, pr exemple, l diminution du tux de glycolyse, l régultion du flux glycolytique pr rpport u flux gluconéogénique dns le foie, ou des modifictions des concentrtions des cides minés neurotrnsmetteurs dns le cerveu. Dns l ensemble, ces données démontrent que le complexe enzymtique des orgnes de l tortue subit peu de chngements u cours d une exposition à l noxie et de l période de récupértion subséquente, ce qui veut dire que les ctivités constitutives des enzymes sont ptes à répondre ux exigences métboliques pendnt les excursions noxiques. [Trduit pr l Rédction] Willmore et l Introduction Tolernce of oxygen deprivtion vries widely mong vertebrte species, rnging from the mere minutes tht the brins of most mmmls cn endure without injury to the extrordinry bility of some turtles to survive s long s 3 months when submerged in oxygen-depleted cold wter (Ultsch 1985). Freshwter turtles of the gener Trchemys nd Chrysemys re frequently cited s the best fculttive nerobes mong vertebrtes. Their tolernce serves both breth-hold diving nd winter hiberntion under wter (Ultsch 1989). Although some extrpulmonry gs exchnge Received April 6, Accepted August 21, Published on the NRC Reserch Press Web site t on October 19, W.G. Willmore, 1 K.J. Cown, 2 nd K.B. Storey. 3 Institute of Biochemistry, Deprtments of Biology nd Chemistry, Crleton University, 1125 Colonel By Drive, Ottw, ON K1S 5B6, Cnd. 1 Present ddress: Hemtology Oncology Division, Brighm nd Women s Hospitl, Hrvrd Medicl School, Longwood Medicl Reserch Center, Boston, MA 02115, U.S.A. 2 Present ddress: Surgicl Reserch Lbortory, Deprtment of Surgery, Sn Frncisco Generl Hospitl nd University of Cliforni, Sn Frncisco, CA 94110, U.S.A. 3 Corresponding uthor (e-mil: kenneth_storey@crleton.c). Cn. J. Zool. 79: (2001) DOI: /cjz

2 Willmore et l occurs in submerged turtles, nd turtles survive considerbly longer in oxygented versus deoxygented wter (Ultsch nd Jckson 1982; Ultsch 1985), their cpcity for long-term nerobiosis is remrkble. The moleculr mechnisms supporting tolernce of noxi by turtles hve been well studied. These include high levels of fermenttive fuels (glycogen) in ll orgns, the bility to buffer high lctic cid lods from nerobic glycolysis (nd to store lctte in the shell), nd metbolic-rte depression (Clrk nd Miller 1973; Storey 1996; Lutz nd Storey 1997; Jckson et l. 2000). By lowering the metbolic rte during noxi to vlue only 10 20% of the erobic rte t the sme temperture (Herbert nd Jckson 1985), nimls reduce ATP expenditure to level tht cn be supported over the long term by glycolytic ATP output nd gin 5- to 10- fold extension of the time tht endogenous fuels cn support survivl. Metbolic-rte depression is chieved vi coordinted reduction in the rtes of most or ll metbolic processes. For exmple, chnnel rrest in brin strongly reduces the net rtes of ion movements cross membrnes (vi ion chnnels nd ATP-dependent pumps) while lrgely mintining membrne potentil difference (Bickler et l. 2001), wheres rtes of protein synthesis re reduced by >90% in heptocytes (Lnd nd Hochchk 1994). Biochemicl mechnisms tht contribute to metbolic rrest include posttrnsltionl modifiction of selected enzymes nd functionl proteins, chnges in protein/enzyme ggregtion or binding to subcellulr structures, llosteric controls, nd chnges in the mounts of selected proteins s result of ltered gene expression or ltered rtes of protein biosynthesis or degrdtion (Storey nd Storey 1990; Storey 2000). Studies tht nlyzed protein synthesis ptterns in vivo, mrna trnsltion ptterns in vitro, nd differentil gene expression hve ll indicted tht orgn-specific chnges in the mounts or types of selected proteins occur under noxic conditions (Brooks nd Storey 1993; Dougls et l. 1994; Ci nd Storey 1996; Willmore et l. 2001). Another pproch to identifying metbolic djustments tht support noxi survivl is to ssess chnges in the mximl ctivities of enzymes in response to the stress. By nlyzing enzymes in multiple pthwys of intermediry metbolism, n overll picture cn be developed of the pthwys nd types of cellulr functions tht re enhnced or suppressed in order to provide coordinted metbolic response to low oxygen levels. The present study mesures the effects of 20 h of noxi exposure nd subsequent 24-h erobic recovery on the mximl ctivities of 21 metbolic enzymes in six orgns of the red-ered slider, Trchemys script elegns. These include enzymes of crbohydrte, ftty cid, denylte, nd mino cid metbolism. Anoxiinduced chnges in enzyme ctivities indicte ltered flux potentil in selected pthwys during noxic submergence. Mterils nd methods Chemicls nd nimls Chemicls were purchsed from Sigm Chemicl Compny (St. Louis, Mo., U.S.A.) or Boehringer Mnnheim Corportion (Montrel, Quebec). Adult red-ered sliders were obtined from Wrds Nturl Science, Mississug, Ontrio, in Februry nd mintined in lrge tnks of dechlorinted wter t 7 C for 3 weeks prior to experimenttion. Turtles were fed d libitum on diet of trout pellets, lettuce, nd eggshells. For experimenttion, turtles were crried to the lbortory in buckets of pool wter, which were plced in n incubtor t 7 C. Aerobic control turtles were smpled from this condition within 1 h. For noxic submergence, groups of 3 turtles were trnsferred to 40-L continers filled with wter tht hd been previously bubbled with 100% nitrogen gs for 10 h; wire mesh ws fitted bout 10 cm below the wter surfce to prevent turtles from surfcing. Bubbling with nitrogen gs ws continued over the 20-h noxi exposure nd the continer ws held in the 7 C incubtor. After noxi exposure, one group of turtles ws smpled immeditely, while turtles in second group were trnsferred to buckets of erted wter with no obstruction to surfcing nd llowed to recover from noxi exposure for 24 h t 7 C. All nimls were killed by decpittion nd tissues were immeditely removed, frozen in liquid nitrogen, nd trnsferred to 70 C for storge until use. Animls were cred for in ccordnce with the principles nd guidelines of the Cndin Council on Animl Cre nd ll experimentl procedures hd the prior pprovl of the Crleton University Animl Cre Committee. Preprtion of tissue extrcts for enzyme ssy Frozen tissue smples were quickly weighed nd homogenized 1:5 (w/v) in ice-cold buffer contining protein kinse nd protein phosphtse inhibitors (50 mm imidzole, ph 7.5, 10% v/v glycerol, 30 mm 2-mercptoethnol, 100 mm NF, 5 mm EDTA, 5 mm EGTA, few crystls of phenylmethylsulfonyl fluoride) with Dimed Pro 200 homogenizer set to full power. Homogentes were centrifuged in Hereus Biofuge 15 t g for 25 min nd the superntnt ws collected nd stored on ice. Extrcts for the ssy of crnitine plmitoyltrnsferse (CPT) nd crnitine octnoyltrnsferse (COT) were prepred s bove, except tht the ph of the extrction buffer ws 8.1 nd NF nd 2-mercptoethnol were omitted. Determintion of enzyme ctivity All ssys were performed t 22 C using Dyntech MR5000 microplte reder with bsorbnce mesured t 340 nm for most enzymes nd t 412 nm for COT nd CPT in finl volume of 0.25 ml plus the volume of enzyme extrct. Optiml ssy conditions were determined for ech enzyme using extrcts from liver (these conditions were lso used for kidney, brin, nd hert) nd red muscle (lso used for white muscle). Blnks, mesured in the bsence of the most specific substrte, were subtrcted to yield net ctivity vlues. One unit is defined s the mount of enzyme tht produces one micromole of product per minute nd ll ctivities re reported s units per grm wet mss of tissue. All coupling enzymes were deslted before use by centrifugtion through smll column of Sephdex G25 (Helmerhorst nd Stokes 1980). Assy conditions for enzymes were s in Cown et l. (2000) except for selected modifictions to substrte concentrtions s follows: (i) for hexokinse (HK) nd glucokinse (GK): 10 nd 100 mm glucose, respectively; (ii) for phosphofructokinse (PFK): 2.5 mm ATP nd 8 mm fructose-6-phosphte; (iii) for pyruvte kinse (PK): 6 mm phosphoenolpyruvte (PEP) nd 2 mm ADP; (iv) for fructose-1,6-bisphosphtse (FBPse): 1.4 mm fructose-1,6- bisphosphte; (v) for mlte dehydrogense (MDH): 6 mm oxlocette; (vi) for cretine kinse (CK): 20 mm cretine phosphte; nd (vii) for serine dehydrtse (SDH): 200 mm L-serine. Assy conditions for glycerol-3-phosphte dehydrogense (G3PDH) were 50 mm imidzole buffer, ph 8.1, 30 mm glycerol-3-phosphte, nd 0.8mMNAD. Soluble-protein concentrtions were determined using the Coomssie blue G-250 binding method with the BioRd Lbortories prepred regent with bovine serum lbumin s the stndrd. Spectrophotometric quntifiction ws performed t 595 nm using

3 1824 Cn. J. Zool. Vol. 79, 2001 Tble 1. Mximl ctivities (units/grm wet mss) of enzymes in six orgns of turtles (Trchemys script elegns). Liver Kidney Hert Brin Red muscle White muscle Crbohydrte metbolism HK (GK in liver) 0.14 ± ± 0.02* 1.50 ± ± ± ± PFK 0.68 ± 0.044* 0.64 ± ± ± 0.20* 3.32 ± ± 0.50 ALD 0.84 ± ± 0.33* 7.49 ± ± 1.27* 25.4 ± ± 1.05 PK 19.4 ± ± ± ± ± ± 3.73 LDH 267 ± ± ± ± ± ± 5.14 G6PDH 1.58 ± ± ± 0.07* 0.99 ± ± ± PGDH 0.62 ± ± ± 0.033* 0.44 ± nd nd FBPse 0.77 ± 0.060* 0.71 ± ± ± nd nd PEPCK 0.20 ± ± ± ± nd nd G3PDH 0.15 ± ± ± 0.04* 0.17 ± ± ± 0.01 MDH 37.9 ± ± ± ± ± ± 0.98 Ftty cid metbolism HOAD 1.34 ± ± ± ± ± 0.11* 0.30 ± 0.06 CPT 5.16 ± ± 1.79* 0.58 ± 0.47* 3.10 ± 1.66* 1.23 ± ± 0.07 COT 1.01 ± ± ± ± ± ± 0.21 Adenylte metbolism AK 6.22 ± ± ± ± ± ± 3.4 CK 18.1 ± ± ± ± ± ± 60.8 Amino cid metbolism GOT 19.8 ± 0.82* 15.3 ± ± 3.74* 14.8 ± ± 1.23* 2.71 ± 0.25 GPT 0.29 ± ± 0.03 nd ± ± ± 0.76* GDH 23.0 ± ± ± ± 1.26* 0.59 ± 0.15* nd SDH 0.65 ± ± ± 0.025* 0.55 ± 0.05* ± ± 0.16* Note: Vlues re given s the men ± SEM, n = 12 for combined vlues from control, 20 h noxic, nd 24 h recovered turtles or, s indicted by n sterisk, n = 4 for control turtles in those cses where vlues for noxi or recovery differed significntly from control vlues. Glucokinse (GK) ws detected only in liver; hexokinse (HK) ws in ll other orgns. nd, not detected. Other bbrevitions re s follows: AK, denylte kinse; ALD, ldolse; COT, crnitine octnoyltrnsferse; CPT, crnitine plmitoyltrnsferse; CK, cretine kinse; FBPse, fructose-1,6-bisphosphtse; G6PDH, glucose-6-phosphte dehydrogense; GDH, glutmte dehydrogense; GOT, glutmteoxlocette trnsminse; GPT, glutmte-pyruvte trnsminse; G3PDH, glycerol-3-phosphte dehydrogense; HOAD, 3-hydroxycyl- CoA dehydrogense; LDH, lctte dehydrogense; MDH, mlte dehydrogense; PEPCK, phosphoenolpyruvte crboxykinse; PFK, phosphofructokinse; 6PGDH, 6-phosphogluconte dehydrogense; PK, pyruvte kinse; SDH, serine dehydrtse. Dyntech MR-5000 microplte reder to finl well volume of 310 ml. Enzyme rtes provided from the microplte reder were interpreted with the use of the microplte nlysis progrm (Brooks 1994). All vlues were compred by one-wy ANOVA followed by two-tiled Dunnett s or Student Newmn Keuls tests. Results Enzyme mximl ctivities nd soluble-protein content in orgns Activities of mny enzymes were unchnged during noxi exposure (20 h t 7 C) or erobic recovery (24 h t 7 C) nd the overll men mximl ctivities of these, combining dt from control, noxic, nd recovered smples (n = 12), re shown in Tble 1 for the six orgns nlyzed. Tble 1 lso shows control vlues (n = 4) for those enzymes whose ctivities did chnge significntly s the result of noxi or erobic recovery. Soluble-protein content in tissue extrcts did not chnge significntly during noxi or erobic recovery in ny tissue. Men concentrtions were 118 ± 19.8, 111 ± 16.3, 69.3 ± 7.46, 91.0 ± 5.33, 73.1 ± 16.9, nd 58.7 ± 14.4 mg/g wet mss (men ± SEM) in liver, kidney, brin, hert, red muscle, nd white muscle, respectively (vlues re pooled for ll three conditions; n = 12). Effects of noxi nd recovery Mximl ctivities of selected enzymes chnged during noxi exposure nd erobic recovery in turtle orgns. Effects on enzymes in liver nd kidney re shown in Fig. 1. Anoxic submergence ffected the ctivity of only one enzyme in liver. The ctivity of PFK, mjor regultory enzyme of glycolysis, dropped by 33% in liver during noxi nd remined low during erobic recovery. Activities of liver FBPse nd glutmte-oxlocette trnsminse (GOT) were unchnged during noxi exposure but rose during erobic recovery, incresing by 60 nd 28%, respectively, compred with controls. In kidney, the ctivities of only three enzymes chnged under the experimentl conditions (Fig. 1). HK ctivity decresed by 40% during noxi exposure, wheres CPT ctivity in liver incresed by nerly 3-fold during noxi, to 9.59 ± 0.68 units/g wet mss, but hd decresed to 6.12 ± 0.43 units/g wet mss fter 24 h of erobic recovery. Aldolse (ALD) ctivity doubled in kidney of recovered turtles compred with noxic nimls.

4 Willmore et l Fig. 1. Chnges in mximl ctivities of enzymes in turtle (Trchemys script elegns) liver nd kidney during 20-h noxic submergence t 7 C nd subsequent 24-h erobic recovery t 7 C. Light-shded brs represent the control, drk-shded brs represent noxi exposure, nd solid brs represent erobic recovery. Vlues re given s the men ± SEM (n = 4)., significntly different from the corresponding control vlue (one-wy ANOVA with Student Newmn Keuls test), P < 0.05; b, significntly different from the corresponding vlue in noxi, P < Activity ( units/g wet mss ) Liver,b Kidney Control Anoxi Recovery b 0 PFK FBPse GOT ALD HK CPT Fig. 2. Chnges in mximl ctivities of enzymes in turtle hert over the course of noxi nd recovery. For detils see Fig. 1. Fig. 3. Effects of noxi exposure nd recovery on enzyme ctivities in turtle brin. For detils see Fig. 1. Activity (units/g wet mss) PFK Brin ALD CPT GDH Control Anoxi Recovery SDH Figure 2 shows the effects of noxi nd erobic recovery on enzyme mximl ctivities in turtle hert. Four enzymes showed significnt chnges in ctivity during noxi. The ctivity of glucose-6-phosphte dehydrogense (G6PDH) rose by 52% (nd remined high during recovery) nd CPT ctivity rose 3-fold in noxi nd then to 13.5-fold higher thn controls during recovery. G3PDH nd GOT ctivities decresed during noxi by 78 nd 32%, respectively, nd both rebounded during erobic recovery. Other enzymes chnged only during recovery fter noxi exposure. SDH incresed 2-fold compred with controls, wheres 6-phosphogluconte dehydrogense (6PGDH) ctivity incresed by 33% over the vlue in noxi. The effects of noxi nd erobic recovery on enzyme ctivities in brin re shown in Fig. 3. Anoxi exposure reduced the ctivity of two glycolytic enzymes, PFK nd ALD, by 30 nd 24%, respectively. Glutmte dehydrogense (GDH) nd SDH lso decresed significntly during noxi to ctivities 43 nd 60% of controls. PFK, ALD, nd SDH ctivities remined low fter 24 h of erobic recovery nd GDH ctivity rebounded, wheres CPT ctivity rose by 31% during recovery only. Chnges in enzyme ctivities in red nd white skeletl muscle re shown in Fig. 4. Anoxi exposure resulted in significnt decrese in 3-hydroxycyl-CoA dehydrogense (HOAD) nd GOT ctivities in red muscle, which fell to 35 nd 64% of control vlues, respectively. Activities of both enzymes rebounded during erobic recovery. The ctivity of glutmte dehydrogense (GDH) in red muscle lso rose during recovery by 3.5-fold. In white muscle, SDH nd

5 1826 Cn. J. Zool. Vol. 79, 2001 Fig. 4. Effect of noxi exposure nd recovery on enzyme ctivities in turtle red nd white muscle. For detils see Fig. 1. Activity (units/g wet mss) glutmte-pyruvte trnsminse (GPT) ctivities lso incresed during erobic recovery, by 2- nd 2.5-fold, respectively. Discussion Red muscle HOAD b GDH,b GOT White muscle b SDH Control Anoxi Recovery,b GPT,b Overll, the present study illustrtes tht lrge-scle chnges to the ctivities of enzymes of intermediry metbolism do not occur during noxi exposure or erobic recovery in noxi-tolernt turtles. This indictes tht the metbolic mke-up of turtle orgns is generlly well designed, with constitutive ctivities of enzymes tht cn meet the metbolic demnds of noxic excursions whenever they occur. This is not unexpected, since turtles need to be prepred for submergence t ny time nd the energy-restricted noxic stte is not time for lrge-scle protein synthesis in support of metbolic reorgniztion. Indeed, rtes of protein synthesis by turtle heptocytes were reduced by >90% under noxic conditions, wheres rtes in noxic hert were only bout one-third of vlues under erobic conditions (Lnd nd Hochchk 1994; Biley nd Driedzic 1996). Nevertheless, studies using 35 S to lbel new proteins synthesized in vivo or proteins produced in vitro vi trnsltion of mrna isolted from noxic nimls showed tht synthesis of selected proteins does occur during noxi in turtle orgns (Brooks nd Storey 1993; Dougls et l. 1994). Furthermore, the noxi-induced up-regultion of mitochondrilly encoded genes ssocited with the electron trnsport system occurs in both turtle hert nd brin in response to noxic submergence t 5 7 C, suggesting incresed synthesis of the protein products of these genes in noxi (Ci nd Storey 1996; Willmore et l. 2001). In the present study the mximl ctivities of 21 enzymes in six orgns of dult turtles were surveyed. The dt show only 13 instnces where enzyme ctivities chnged significntly during the 20-h noxic submergence nd, of these, only 3 involved significnt increses in ctivity. CPT ctivity rose in kidney nd hert by 3-fold in ech cse nd G6PDH ctivity lso incresed in hert under noxi. Notbly, this contrsts with the responses of mmmlin tissues to hypoxic/noxic conditions, which include rpid upregultion of the genes for the most glycolytic enzymes in n ttempt to meet energy demnd by incresing glycolytic ATP production (Semenz et l. 1994; Firth et l. 1994). In turtles, by contrst, glycolytic enzymes re not induced under noxi for two resons: (1) constitutive ctivities of these enzymes re lredy high nd (2) metbolic-rte depression reduces the demnd for ATP produced by glycolysis. In the other 10 instnces, in five orgns (not white muscle) enzyme ctivities significntly decresed during noxi exposure. This included decreses in ctivities of enzymes of glycolysis (HK in kidney, PFK in liver nd brin, ALD in brin) nd mino cid metbolism (GOT in hert nd red muscle, GDH nd SDH in brin), s well s G3PDH in hert nd HOAD in red muscle. Other enzymes responded to erobic recovery from noxi nd, in most cses, ctivities incresed during recovery. In 5 cses these represented reversls of ctivities tht were reduced during noxi, wheres 10 involved new increses in enzyme ctivities. Five enzymes tht were reduced during noxi remined low over the 24-h erobic recovery. These ptterns of reduced enzyme ctivities during noxi nd incresed ctivities during erobic recovery re consistent with lower biosynthetic cpcity in the noxic stte nd renewl of protein synthesis when oxygen returns. However, the mny instnces of unchnged enzyme ctivities in noxi suggest tht, in generl, rtes of protein synthesis nd protein degrdtion re suppressed in coordinted fshion s prt of the overll metbolic-rte depression induced by noxi exposure. Aginst bckground of lrgely unchnged enzyme ctivities, the specific enzymes ffected by noxi exposure gin more significnce. In vitro determintion of enzyme mximl ctivities hs long been employed to provide insight into metbolic flux potentil in vivo nd to suggest possible rtelimiting nd regultory loci (Newsholme nd Crbtree 1986). The utility of enzyme-ctivity mesurements is vlid for two resons: (1) chnges in enzyme levels cn significntly ffect flux through metbolic pthwys nd (2) enzyme (protein) synthesis is ATP-expensive, so those chnges tht do occur re likely of importnce to orgnisml survivl of stress. In liver, noxi exposure suppressed the mximl ctivity of PFK, one of the regultory enzymes of glycolysis (Fig. 1). Such control of PFK in liver cn hve two functions: (1) when glycogenolysis is ctivted, PFK inhibition helps to divert hexose phosphtes into the production of glucose for export, nd (2) under gluconeogenic conditions, PFK inhibition fcilittes the synthesis of hexose phosphtes from substrtes such s lctte, lnine, or glycerol-3- phosphte. The first function is undoubtedly the cuse of the noxi-induced effect on liver PFK, s liver glycogen is the primry source of the blood glucose tht is used s n nerobic fuel by other tissues. Anoxic inhibition t the PFK locus in turtle liver ws lso indicted from the pttern of chnges in substrte nd product levels of the enzyme (Kelly nd Storey 1988). However, it is the second function tht probbly keeps PFK low during erobic recovery, wheres the ctivity of FBPse, which reverses the PFK rection, rises significntly in liver during recovery to fcilitte gluconeogenesis from lctte. Such oppositely directed re-

6 Willmore et l sponses by PFK nd FBPse re well known in vertebrte liver metbolism s one of the min controls on glycolysis versus gluconeogenesis nd chnges in the rtio of PFK:FBPse ctivities in turtle liver (0.88 in control, 0.48 in noxi, nd 0.33 in erobic recovery) support this interprettion. However, the mechnism of PFK- nd FBPse-ctivity chnges over noxi/recovery in liver my not be chnge in enzyme mount but chnge in protein phosphoryltion stte. Both enzymes in vertebrte liver re controlled by reversible phosphoryltion tht hs opposite effects on their ctivities (inhibiting PFK, ctivting FBPse). A previous study of PFK in turtle orgns noted noxi-induced chnges in the kinetic properties nd mximl ctivity ( 40% reduction) of PFK in liver tht were consistent with less ctive enzyme in noxi, probbly resulting from noxi-induced phosphoryltion (Brooks nd Storey 1989). The only other significnt chnge in turtle liver enzyme ctivities during noxi/recovery ws 28% rise in GOT during erobic recovery tht my be relted to the enzyme s role in mino cid metbolism or the mlte sprtte shuttle; both would be suppressed in noxi nd rectivted during recovery. In kidney, HK ctivity ws reduced during noxi by 40% (Fig. 1); this could contribute to metbolic-rte depression by suppressing the entry of exogenous glucose into glycolysis. ALD lso showed trend towrds reduced ctivity during noxi, with substntil rebound during erobic recovery. Although not regultory enzyme, ALD is one of three low-ctivity glycolytic enzymes in kidney (HK, PFK, ALD), nd chnges in ALD lso suggest pttern of metbolic-rte suppression in noxi nd rectivtion in recovery. CPT ctivity rose 3-fold during noxi in kidney, lthough the functionl significnce of this increse is not pprent, since the role of CPT is to trnslocte ftty cids into the mitochondri for use in bet-oxidtion. CPT ctivity lso incresed in turtle hert during noxi (by 3-fold) nd recovery (by 13-fold) nd in brin during recovery (by 33%) (Figs. 2, 3). Elevted CPT ctivity during erobic recovery could support renewed relince on lipid oxidtion to fuel energy metbolism nd biosynthesis when erobic conditions return. Enzyme chnges in hert during noxi included reduced ctivities of both G3PDH nd GOT (Fig. 2). Both enzymes hve multiple roles in metbolism but function tht they shre is redox regultion. Both re components of shuttle systems tht move cytoplsmic reducing equivlents into the mitochondri. Cytoplsmic G3PDH uses NADH to synthesize glycerol-3-phosphte, which moves into the mitochondri, where it is oxidized by FAD-linked glycerol-3-oxidse, with relese of electrons into the electron trnsport system. The mlte sprtte shuttle, which is composed of cytosolic nd mitochondril isoforms of MDH nd GOT, lso crries out this function. Both shuttles would be hlted by the interruption of the electron trnsport system when oxygen is depleted nd reduced ctivities of these enzymes in noxi my contribute to reorgniztion of redox regultion for nerobic survivl. Sephton nd Driedzic (1996) reported no chnge in brin HK, PK, lctte dehydrogense (LDH), nd HOAD over the course of 16 weeks of submergence in normoxic wter t 3 C, which concurs with our findings for the shorter noxi stress. Anoxi exposure in brin reduced the ctivities of two glycolytic enzymes (PFK nd ALD) nd two enzymes of mino cid metbolism (GDH nd SDH) (Fig. 3). The response by glycolytic enzymes my contribute to glycolyticrte suppression, wheres chnges in GDH nd SDH my be linked with neurotrnsmitter metbolism. One negtive effect of oxygen deprivtion on mmmlin brin is the relese of high levels of the excittory mino cids glutmte nd sprtte into the extrcellulr fluid (Young et l. 1993). Turtles do not show this; in fct, both extrcellulr glutmte (Young et l. 1993) nd totl brin glutmte (Nilsson et l. 1990) decresed significntly during noxi, wheres levels of inhibitory mino cids (g-minobutyric cid, glycine, turine, lnine) incresed in noxic turtle brin (Nilsson et l. 1990). Although GDH prticiptes in both glutmte synthesis nd ctbolism, the 57% reduction in GDH ctivity in turtle brin in noxi could reduce the rte of glutmte production, leding to lower glutmte levels. Elevted levels of glycine during noxi my be the result of de novo synthesis but would lso be fvoured by blocking the two routes of glycine ctbolism. In one route, glycine is first converted to serine nd then SDH ctlyzes conversion to pyruvte, nd this is followed by pyruvte oxidtion. In the other, glycine is directly cleved (producing CO 2,NH 4 +, nd methylene group) by the mitochondril NAD-dependent glycine-clevge enzyme. Both routes would be suppressed in noxic turtle brin (SDH ctivity is reduced by 40%; mitochondril NAD vilbility is low) nd this could promote sustined or elevted glycine levels. Anoxi/recovery hd few effects on enzyme ctivities in skeletl muscles nd, except for HOAD, ll enzymes ffected were involved with mino cid metbolism (Fig. 4). The ctivity of HOAD ws strongly reduced under noxi in red muscle, flling to just 35% of the control vlue but rebounding gin during erobic recovery. Under erobic conditions, red muscle relies on ftty cid oxidtion for high percentge of its energy needs. The inbility to oxidize lipids during noxi could ccount for suppressed ctivity of HOAD nd perhps other enzymes of ftty cid ctbolism during nerobiosis. Anoxic suppression of GOT in red muscle might hve the sme function s postulted bove for hert or, tken together with the increses in other enzymes (GDH in red muscle nd SDH nd GPT in white muscle) during recovery, these might imply n overll increse in mino cid ctbolism during recovery from noxi. Acknowledgements The uthors thnk J.M. Storey for editoril commentry on the mnuscript. This study ws supported by reserch grnt from the Nturl Sciences nd Engineering Reserch Council of Cnd (NSERC) to K.B.S. nd NSERC postgrdute scholrships to W.G.W. nd K.J.C. References Biley, J.R., nd Driedzic, W.R Decresed totl ventriculr nd mitochondril protein synthesis during extended noxi in turtle hert. Am. J. Physiol. 271: R1660 R1667. Bickler, P.E., Donohoe, P.H., Buck, L.T The hypoxic brin: suppressing energy-expensive membrne functions by regultion

7 1828 Cn. J. Zool. Vol. 79, 2001 of receptors nd ion chnnels. In Moleculr mechnisms of metbolic rrest. Edited by K.B. Storey, BIOS Scientific Publishers, Oxford. pp Brooks, S.P.J The microplte nlysis progrm. BioTechniques, 17: Brooks, S.P.J., nd Storey, K.B Regultion of glycolytic enzymes during noxi in the turtle Pseudemys script. Am.J. Physiol. 257: R278 R283. Brooks, S.P.J., nd Storey, K.B De novo protein synthesis nd protein phosphoryltion during noxi nd recovery in the red-ered turtle. Am. J. Physiol. 265: R1380 R1386. Ci, Q., nd Storey, K.B Anoxi-induced gene-expression in turtle hert: up-regultion of mitochondril genes for NADHubiquinone oxidoreductse subunit-5 nd cytochrome-c oxidse subunit-1. Eur. J. Biochem. 241: Clrk, V.M., nd Miller, A.T Studies on nerobic metbolism in the freshwter turtle (Pseudemys script elegns). Comp. Biochem. Physiol. A, 44: Cown, K.J., McDonld, J.A., Storey, J.M., nd Storey, K.B Metbolic reorgniztion nd signl trnsduction during estivtion in the spdefoot tod. Exp. Biol. Online, 5: 1 ( / htm). Dougls, D.N., Gibnd, M., Altosr, I., nd Storey, K.B Anoxi induces chnges in trnsltble mrna popultions in turtle orgns: possible dptive strtegy for nerobiosis. J. Comp. Physiol. B, 164: Firth, J.D., Ebert, B.L., Pugh, C.W., nd Rtcliffe, P.J Oxygen-regulted control elements in the phosphoglycerte kinse 1 nd lctte dehydrogense A genes: similrities with the erythropoietin 3 enhncer. Proc. Ntl. Acd. Sci. U.S.A. 91: Helmerhorst, E., nd Stokes, G.B Microcentrifuge deslting: rpid quntittive method for deslting smll mounts of protein. Anl. Biochem. 104: Herbert, C.M., nd Jckson, D.C Temperture effects on the responses to prolonged submergence in the turtle Chrysemys pict bellii. II. Metbolic rte, blood cid bse nd ionic chnges, nd crdiovsculr function in erted nd noxic wter. Physiol. Zool. 58: Jckson, D.C., Crocker, C.E., nd Ultsch, G.R Bone nd shell contribution to lctic cid buffering of submerged turtles Chrysemys pict bellii t 3 C. Am. J. Physiol. 278: R1564 R1571. Kelly, D.A., nd Storey, K.B Orgn-specific control of glycolysis in noxic turtles. Am. J. Physiol. 255: R774 R779. Lnd, S.C., nd Hochchk, P.W Protein turnover during metbolic rrest in turtle heptocytes: role nd energy dependence of proteolysis. Am. J. Physiol. 266: C1028 C1036. Lutz, P.L., nd Storey, K.B Adpttions to vritions in oxygen tension by vertebrtes nd invertebrtes. In Hndbook of physiology. Section 13. Comprtive physiology. Vol. 2. Edited by W.H. Dntzler. Oxford University Press, Oxford. pp Newsholme, E.A., nd Crbtree, B Mximl ctlytic ctivity of some key enzymes in provision of physiologiclly useful informtion bout metbolic fluxes. J. Exp. Zool. 293: Nilsson, G.E., Alfro, A.A., nd Lutz, P.L Chnges in turtle brin neurotrnsmitters nd relted substnces during noxi. Am. J. Physiol. 259: R376 R384. Semenz, G.L., Roth, P.H., Fng, H.M., nd Wng, G.L Trnscriptionl regultion of genes encoding glycolytic enzymes by hypoxi-inducible fctor 1. J. Biol. Chem. 269: Sephton, D.H., nd Driedzic, W.R Mintennce of enzyme ctivity levels during long-term erobic diving in the red-ered turtle Trchemys script elegns. Physiol. Zool. 69: Storey, K.B Metbolic dpttions supporting noxi tolernce in reptiles: recent dvnces. Comp. Biochem. Physiol. B, 113: Storey, K.B Stress-induced gene expression in freezetolernt nd noxi-tolernt vertebrtes. In Environmentl stress nd gene regultion. Edited by K.B. Storey. BIOS Scientific Publishers, Oxford. pp Storey, K.B., nd Storey, J.M Metbolic rte depression nd biochemicl dpttion in nerobiosis, hiberntion nd estivtion. Q. Rev. Biol. 65: Ultsch, G.R The vibility of Nerctic freshwter turtles submerged in noxi nd normoxi t 3 nd 10 C. Comp. Biochem. Physiol. A, 81: Ultsch, G.R Ecology nd physiology of hiberntion nd overwintering mong freshwter fishes, turtles, nd snkes. Biol. Rev. Cmb. Philos. Soc. 64: Ultsch, G.R., nd Jckson, D.C Long-term submergence t 3 C of the turtle Chrysemys pict bellii, in normoxic nd severely hypoxic wter. I. Survivl, gs exchnge nd cid bse sttus. J. Exp. Biol. 96: Willmore, W.G., English, T.E., nd Storey, K.B Mitochondril gene responses to low oxygen stress in turtle orgns. Copei, 101: Young, R.S.K., During, M.J., Donnelly, D.F., Aquil, W.J., Perry, V.L., nd Hddd, G.G Effect of noxi on excittory mino cids in brin slices of rts nd turtles: in vitro microdilysis. Am. J. Physiol. 264: R716 R719.