The metabolism of proline and arginine was investigated in
kidney, gill, and heart of the pelagic squid, Symplectoteuthis. The rates of CO2
release from 14C-proline exceeded the rates from 14C-arginine. The metabolic
rate of arginine and proline was assessed by monitoring the incorporation of
arginine-derived carbon into various intermediates. Arginine was metabolized,
through ornithine, to proline as well as to glutamate and various subsequent
derivatives (alanine, octopine, aspartate, and carboxylic acids). The same components
became labeled using 14C-proline as the starting substrate, but only the
gill was capable of converting proline to arginine via the urea cycle. In addition,
14C-proline oxidation rates were high enough to exceed those of 14C-glucose in
at least three tissues, kidney, heart, and inner mantle muscle

Emmett, B.

French, C.J.

Hochachka, P.W.

Mommsen, T.P.

Pacific Science

ScholarSpace at University of Hawai'i at Manoa

Pacific Science (1982), vol. 36, no. 3© 1983 by the University of Hawaii Press. All rights reservedThe Fate of Arginine and Proline Carbon in Squid Tissues lT. P. MOMMSEN,2 C. J. FRENCH,2 B. EMMETI,2 and P. W. HOCHACHKA2ABSTRACT: The metabolism of proline and arginine was investigated inkidney, gill, and heart of the pelagic squid, Symplectoteuthis. The rates of CO2release from 14C-proline exceeded the rates from 14C-arginine. The metabolicrate of arginine and proline was assessed by monitoring the incorporation ofarginine-derived carbon into various intermediates. Arginine was metabolized,through ornithine, to proline as well as to glutamate and various subsequentderivatives (alanine, octopine, aspartate, and carboxylic acids). The same com-ponents became labeled using 14C-proline as the starting substrate, but only thegill was capable of converting proline to arginine via the urea cycle. In addition,14C-proline oxidation rates were high enough to exceed those of 14C-glucose inat least three tissues, kidney, heart, and inner mantle muscle.MATERIALS AND METHODSTissue Slice ExperimentsAnimals and ChemicalsPelagic squid of the species Symplecto-teuthis oualaniensis were jigged at nightlightsoff the leeward side of Oahu, Hawaii, duringthe Cruise FIDO XVII of the RjV Kana Keokiin June 1980. Biochemicals were purchasedfrom Sigma Chemical Co., St. Louis, Mis-souri; radiochemicals came from NewEngland Nuclear, Boston, Massachusetts.data for heart, gill, and kidney from the squid,Symplectoteuthis, showing the capacity for ar-ginine conversion to proline. The conversionof proline to arginine was measurable only inthe gill. Although qualitatively similar to re-sults obtained with other species, these dataalso show some important, tissue-specific dif-ferences (Mommsen et al. 1983; Mommsenand Hochachka 1981). Nevertheless, theavailable pathways of proline and argininemetabolism appear to be conserved amongthe cephalopod groups thus far examined,with main differences arising from the wayarginine and proline metabolic potentials areexpressed in different tissues.Heart, kidney, and gills from freshly killedanimals were blotted dry of blood, cut into343AT LEAST IN PART because of the large pool sizeof free amino acids in cephalopod muscles(e.g., see Hochachka, French, and Meredith1978), interest recently has been focusingon their possible roles in energy metabolism.During metabolic studies on the 1979 AlphaHelix Cephalopod Expedition, relatively highrates of CO2 release from arginine andproline were observed in tissues of theoctopus, Octopus macropus (Hochachka andFields, this issue), the squid, Nototodarus(Hochachka, unpublished data), and thechambered nautilus, Nautilus pompilius(Fields and Hochachka, this issue). In mam-mals and bacteria (Adams and Frank 1980),proline and arginine are metabolized throughcommon intermediates, such as pyrroline-5-carboxylate, glutamate, and ornithine, so itwas of interest to assess these potentials incephalopod tissues. In this paper, we report1 This research was conducted as part of the AlphaHelix Cephalopod Expedition to the Republic of thePhilippines, supported by National Science Foundationgrant PCM 77-16269 to J. Arnold. This study was alsosupported in part by grants from the DeutscheForschungsgemeinschaft to T. P. Mommsen and fromthe Natural Sciences and Engineering Research Councilof Canada to P. W. Hochachka.2 University of British Columbia, Department ofZoology, Vancouver, British Columbia, Canada V6T2A9. Present address of T. P. Mommsen: DalhousieUniversity, Department of Biology, Halifax, NovaScotia, Canada B3H 411.'- , \ ' , . ~, ~ _ " ••• , .. '", ,.. , ,,~r ff' - I' ,.,_ • "'- _, •• ,.-- '"344 PACIFIC SCIENCE, Volume 36, July 1982TABLE 1CO2 PRODUCTION FROM PROLINE AND ARGININE INSymplectoteuthis TISSUESNOTE: Label recovered in the respective compound in percent of label(DPM) loaded onto the amino acid analyzer. For conditions of incubationssee the text. .3.93.90.74.11.70.61.30.80.55.476.697.9KIDNEY0.71 ± 0.40 (4)0.075 ± 0.026 (4)0.55 ± 0.21 (4)[U- 14C]ARGININE4.90.220.41.20.50.20.091.00.50.1193.0102HEARTGILL4.30.840.41.24.20.140.080.60.41.885.999.84.37 ± 0.65 (4)4.04 ± 0.77 (4)1.74 ± 0.53 (8)TISSUEKidneyHeartGillindicate that arginine is metabolized at fastrates in both kidney and gill, with low rates ofturnover in heart, which is consistent with theactivity distribution of arginase in tissues ofsquid (Mommsen and Hochachka 1981) andOctopus (Gaston and Campbell 1966). Prolineis metabolized at high rates, 5-6 times higherthan arginine in both heart and kidney.TABLE 2DISTRIBUTION OF LABEL FROM [U- 14C]ARGININEPRECURSOR IN TISSUE SLICES OF SymplectoteuthisMETABOLITENOTE: Data expressed in micromoles of CO2 produced from the respec-tive L-amino acid per gram offresh tissue in I hrat 23°C ± S. D. The numberof experiments is given in parentheses.Carboxylic acidsUreaAspartateGlutamate/GlutamineProlineAlanineCitrullineOctopiney-Aminobutyric acidOrnithineArgi'riineRecoveryArginine MetabolismThe percent distribution of radiolabel thatwas extractable from tissue slices after incu-bation with uniformly labeled 14C-arginine isshown for gill, kidney, and heart in Table 2.14C-Arginine-derived carbon could be foundin urea, ornithine, glutamate/glutamine, pro-line, citrulline, alanine, octopine, and aspar-tate. Furthermore, a small percentage of labelsmall blocks, and carefully sliced with razorblades into 0.5-I-mm-thick slices. The slices(about 100-200 mg) were taken up in 2.10 mlof medium A (0.35 M NaCI, 8 mM KCI,31 mM MgClz, 8 mM CaClz, 16.2 mMMgS04, 20 mM Tris, adjusted to pH 7.50at 23°C) and preincubated for 10 min withunlabeled substrate (final concentration2.0 mM), using 25-ml flasks in a metabolicshaker. The flasks were capped, and 0.2 ml ofmedium A containing 0.5 ~Ci of 14C-labeledsubstrate was injected through each cap. After30 min at 23°C, the reaction was stopped bythe injection of 0.25 ml trichloroacetic acid(20%), and the 14COZ was collected on glassfiber filters, suspended above the reactionmedium, and soaked with 0.15 ml ofphenyleth-ylamine (Iverson, Bittaker, and Myers 1977).The flasks were shaken for an additional 90min, and then the filters were removed andcounted in toluene-based scintillation fluid.Amino Acid AnalysisThe contents of the flasks were homoge-nized with a Polytron tissue grinder,centri-fuged, and the supernatant adjusted to pH1.95 with crystalline lithium hydroxide. Twoaliquots of the supernatant were separated ona Beckman 118C amino acid analyzer: thefirst run was reacted with ninhydrin; in thesecond run, the column eluate was collectedin 2.0-ml samples and 1.0-ml aliquots werecounted. Amino acid analyses of tissues wereperformed as above, after homogenizing thetissue in 10 vol ice-cold sulfosalicylic acid(3.75%, pH 1.95). Specific activities were cal-culated for the start of the experiments only.We showed that for heart, fin, and gill, theproduction of 14COZ from 14C-labeled glu-cose, proline, and arginine under the aboveconditions was linear with time for at least 60min.RESULTS AND DISCUSSIONAll three tissues under consideration pro-duced significant amounts of 14COZ whensupplied with either [U- 14C]proline or [U-14C]arginine. The results presented in Table IL' • • • ~ "'''' ~., " , I ,... , '.01. ~, _ .,., .". ,,_Squid Metabolism-MoMMsEN ET AL.could be localized in y-aminobutyric acid(GABA), indicating that at least a small por-tion of arginine is degraded by an L-aminoacid oxidase rather than by arginase. Theoccurrence of this pathway and the identifica-tion of intermediates and enzymes involvedhave been described by van Thoai (1965) andA "~UA'b"rgmme rea y- mmo utync aCId345Baret et al. (1965). The observed distributionof labeled carbon in the squid is consistentwith the following reaction scheme, which isalso thought to be operative in other cepha-lopods (Mommsen and Hochachka 1981),mammals, and bacteria (Adams and Frank1980):Ornithine - Pyrroline-5-carboxylate - Glutamate - 2-0xoglutarate~ ~ tCitrulline Proline Malate/ ~Pyruvate Oxaloacetate~. ~Octopine Alamne AspartateAlthough urease has been demonstrated ina variety of mollusks, mainly in terrestrialsnails (Campbell and Speeg 1968; Florkin1966; Hammen, Miller, and Geer 1966), wefailed to detect any urease activity in tissues ofthe short-finned squid, Illex illecebrosus. Ourdata in Table 2 indicate that urease is alsoabsent in the three tissues from Symplecto-teuthis, but the definitive proof has yet to besupplied. It should also be emphasized thatlabel can be distributed into octopine throughtwo different pathways. In addition, occur-rence of label in citrulline, particularly in thekidney and gill (see below), implies the pre-sence of a functional urea cycle; in our experi-ments, the concurrent synthesis of argininefrom endogenous precursors would lead to adrop in specific activity of [U-14C]arginine,which we did not follow up in this paper.Proline MetabolismUsing proline as the 14C-labeled substrate,the same metabolites as already mentioned inTable 2 became labeled during incubation ofsquid tissue slices, with the exception of ureaand y -aminobutyric acid (Table 3). In thesquid gill, the distribution of label is consis-tent with the scheme given below:Prolinepyrroline-5-~arbOXYlate -Glutamate -2-0xoglutaric acid~ tI Ornithine MalateCitrulline Urea \ Urea . J~\ cycle y Alanme ~Pyruvate oxaloa~'--Arginine ---------------0t . Aspartatec opIne, N' • • " ~. ,"~, ., -" , .. ~~ ., ""~".,,,,,,,~... ., .. '"''346TABLE 3DISTRIBUTION OF LABEL FROM [U- 14C]PROLINEPRECURSOR IN TISSUE SLICES OF SymplectoteuthisMETABOLITE GILL HEART KIDNEYCarboxylic acids 1.2 1.7 0.9Aspartate 1.2 0.2 0.7Glutamate/Glutamine 1.7 2.9 1.2Proline 93.7 91.2 95.4Alanine 0.4 0.3 0.25Citrulline 0.05 0.02 0Octopine 0.3 0.2 0.6Ornithine 0.12 0.04 0.04Arginine 0.51 0 0Recovery 98.9 96.6 99.0NOTE: Label recovered in the respective compound in percent of totallabel loaded (DPM) onto the amino acid analyzer. For conditions of incuba-tions see the text.Under our experimental conditions, gillwas found to be the only organ to possess afully operative urea cycle, but our failure todetect it in kidney may be due to the con-siderable dilution of label at the ornithinelevel in this tissue (Table 4), where the con-centration of ornithine is more than an orderof magnitude higher than in gill or heart.Physiologically, some of these reactions arethe reverse of those in arginine metabolism,but they are drawn separately, because theyare catalytically distinct and may be localizedin separate compartments in the cell (Momm-sen and Hochachka 1981, Phang, Yeh, andHagedorn 1981). The fact that the tissueslices produce labeled alanine from both[U-14C]arginine and [U-14C]proline indicatesthat the three tissues possess the potential totruly oxidize these two amino acids and thatat least some of the recovered 14C02 is liber-ated through malic enzyme (malate dehydro-genase: NADP-linked, decarboxylating, EC1.1.1.40).Carbon Dioxide Release from Proline asCompared to GlucoseThe importance of the oxidation of aminoacids-especially proline-for the overallenergy production of the squid can be seenwhen comparing oxidation rates of prolinewith oxidation rates ofuniformly labeled 14C_glucose in a variety of squid tissues (Table 5).Generally, glucose seems to be the preferredPACIFIC SCIENCE, Volume 36, July 1982substrate, but kidney and especially heartclearly prefer proline. The picture presented inTable 5 might change dramatically in favor ofproline if the stationary concentration of thedifferent metabolites is taken into considera-tion. Although cephalopods seem to accumu-late large glycogen stores (Hochachka andFields, this issue), the resting concentrationsof free glucose (Mommsen, unpublished data)are lower than those generally found for pro-line, particularly in kidney, heart, and muscles(Table 4). Thus, the data in Table 5 mayunderestimate actual rates of proline oxi-dation.Finally, an important specialization ofmantle muscle into inner and outer oxidativebands plus a middle, more anaerobic band hasbeen identified by us in at .least five species ofsquid (Mommsen et al. 1981b) and by Bone,Pulsford, and Chubb (1981) in Alloteuthis. InSymplectoteuthis, this specialization is modi-fied into inner oxidative and middle/outeranaerobic muscle bands (Mommsen et al.1981). It is interesting to note that the ratioof CO2 released from glucose/C02 releasedfrom proline is less than unity for the oxi-dative inner mantle, but is about 2.5 forthe middle mantle. Tentacle muscle, whichenzymatically is more similar to the middlemantle (Mommsen, unpublished data), dis-plays a ratio of about 3 (Table 5). These re-sults imply that not only does the oxidativepotential vary between inner and outermantle, but so also do substrate preferencesfor oxidative metabolism.ImplicationsThese experiments clearly establish thepotential for metabolic functions for arginineand proline additional to those usually as-cribed to them (see Hochachka and Fields,this issue). In the case ofarginine, not only is ita potentially important source of metabolicCO2 and waste nitrogen (as urea), it may alsoplaya role in augmenting proline reserves,should they become depleted in times of pro-longed performance. Because the pool size ofproline is typically large, the appearance of14C-arginine-derived proline in tissues such asthe heart is considered particularly significant... " '" , ~, - ~" • ~ ., T • " • "."., ..~ ~. ,Squid Metabolism-MoMMsEN ET AL. 347TABLE 4FREE AMINO ACIDS IN Symplectoteuthis TISSUESAMINO MIDDLE INNER DIGESTNEACID GILL HEART KIDNEY BRAIN TENTACLE MANTLE MANTLE GLANDHis 0.32 0.84 0.20 0.31 0.55 6.95 4.98 0.15Arg 3.56 1.87 2.27 4.35 16.80 37.40 28.84 1.56Om 0.17 0.13 2.22 0.94 0.23 0.83 0.73 0.14Pro 1.60 5.95 15.96 7.35 10.10 14.70 18.88 1.93Gly 2.12 1.14 3.00 3.58 9.20 14.61 12.74 1.35Ala 3.82 23.28 11.26 7.71 7.83 11.66 13.27 4.23NOTE: Data expressed in micromoles per gram of tissue. Amino acid analyses were performed on tissue pooled from four animals. Three independent heartsamples from three or four pooJed animals were analyzed, and the maximum standard deviation found was below 15% of the mean given in the table. Nomeasurable amounts of pyrroline-5-carboxylate or citrulline could be detected in any of the amino acid analyses.TABLESCO2 PRODUCTION FROM PROLINE AND GLUCOSE IN TiSSUES OF THE SQUID Symplectoteuthis oualaniensisGLUCOSE/PROLINETISSUE PROLINE GLUCOSE RATIOKidney 4.37 ± 0.65 (4) 2.10 ± 0.27 (4) 0.5Heart 4.04 ± 0.77 (4) 0.70 ± 0.19 (4) 0.2Gill 1.74 ± 0.53 (8) 2.82 ± 0.84 (4) 1.6Digestive gland 0.092 ± 0.030 (4) 1.22 ± 0.23 (4) 13.3Middle mantle 0.15 ± 0.06 (4) 0.36 ± 0.12 (4) 2.4Inner mantle 0.41 ± 0.17 (4) 0.36 ± 0.19 (4) 0.9Tentacle 0.19 ± 0.D78 (4) 0.57 ± 0.023 (4) 3.0Fin (center) 0.57 ± 0.25 (3) 0.81 ± 0.15 (3) 1.4Brain 2.74 ± 0.90 (4) 8.43 ± 1.51 (4) 3.1NOH: Data expressed in micromoles ofCO, produced from proline or glucose per gram of fresh tissue in I hr at230C ± S.D. The number ofexperiments isgiven in parentheses.Although different fates of proline are notyet fully quantitated, the spread of labelamong various intermediates implies thepotential for a complex metabolism leadingmainly to alanine, octopine, and aspartate.Tentatively, we assume that the pathways forproline metabolism in these tissues are com-parable to those observed in insects (Weedaet al. 1980), except for the complicatinginteractions with pathways of arginine meta-bolism.The main questions that remain to be moreclosely analyzed are the following: (1) How dothese various metabolic interactions changefollowing arginine-phosphate depletion andoctopine accumulation during anaerobicwork? (2) How do the arginine-proline inter-actions change following exhaustive aerobicwork, when proline reserves may be depleted?(3) How is proline carbon partitioned betweencomplete oxidation and Krebs cycle augmen-tation? (4) How is waste nitrogen partitionedbetween urea and ammonia under differentmetabolic conditions? Further work is re-quired before these problems can be put intoclearer perspective.ACKNOWLEDGMENTSThe cooperation of the crew of the RjVKana Keoki and of N. Rinaldi, radiationofficer at the University of Hawaii, is grate-fully acknowledged.LITERATURE CITEDADAMS, E., and L. FRANK. 1980. Metabolismof proline and hydroxyproline. Ann. Rev.Biochem. 49: 1005-1062., " ., ..... , " ,... ' ... - .~, - .. ~. ,.,348BARET, R., M. MOURGUE, A. BROC, andJ. CHARMOT. 1965. Etudecomparativedeladesamidination de l'acide y-guanidinobu-tyrique et de I'arginine par l'hepato-pancreas ou Ie foie de divers Invertebres.Compt. Rend. Acad. Sci. Soc. BioI. 159:2446-2450.BONE, Q., A. PULSFORD, and A. C. CHUBB.1981. Squid mantle muscle. J. Mar. BioI.Assoc. (U.K.) 61 :327-342.CAMPBELL, J. W., and K. V. SPEEG. 1968.Arginine biosynthesis and metabolism interrestrial snails. Compo Biochem. Physiol.25:3-32.FLORKIN, M. 1966. Nitrogen metabolism.Pages 309-352 in K. M. Wilbur and C. M.Yonge, eds. Physiology ofMollusca. Vol. 2.Academic Press, New York.GASTON, S., and J. W. CAMPBELL. 1966. Dis-tribution of arginase activity in mollusks.Compo Biochem. Physiol. 17: 259-270.HAMMEN, C. S., H. F. MILLER, and W. H.GEER. 1966. Nitrogen excretion of Crasso-strea virginica. Compo Biochem. Physiol.17 :1199-1201.HOCHACHKA, P. W., C. J. FRENCH, andJ. MEREDITH. 1978. Metabolic and ultra-structural organization in Nautilus muscles.J. Exp. ZooI. 205: 51-62.IVERSON, R. c., H. F. BITTAKER, and V. B.PACIFIC SCIENCE, Volume 36, July 1982MYERS. 1977. Loss of radiocarbon in directuse of aquasol for liquid scintillation count-ing of solutions containing 14C-NaHC03 .Limnol. Oceanogr. 21: 756-758.MOMMSEN, T. P., and P. W. HOCHACHKA.1981. Respiratory and enzymatic proper-ties of squid heart mitochondria. Eur. J.Biochem. 120: 345-350.MOMMSEN, T. P., P. W. HOCHACHKA, and C. J.FRENCH. 1983. The intermediary metabo-lism of arginine in squid tissues. Submitted.MOMMSEN, T. P., J. S. BALLANTYNE, D. D.MACDONALD, J. GOSLINE, and P. W.Hochachka. 1981. Analogues of red andwhite muscle in squid mantle. Proc. Nat.Acad. Sci. u.S. 78: 3274-3278.PHANG, J. M., G. C. YEH, and C. H.HAGEDORN. 1981. The intercellular prolinecycle. Life Sci. 28: 53-58.VAN THOAI, N. 1965. New aspects of the meta-bolism of arginine. Pages 3-19 in G. E. W.Wolstenholme and M. P. Cameron, eds.Comparative biochemistry of arginine andderivatives. Ciba Foundation Study GroupNo. 19, Churchill, London.WEEDA, E., A. B. KOOPMANSCHAP, C. A. D. DEKORT, and A. M. T. BEENAKKERS. 1980.Proline synthesis in fat body of Leptino-tarsa decemlineata. Insect Biochem. 10:631-636.

The Fate of Arginine and Proline Carbon in Squid Tissues

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The Fate of Arginine and Proline Carbon in Squid Tissues

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