ISSN 10630740, Russian Journal of Marine Biology, 2010, Vol. 36, No. 6, pp. 469–472. © Pleiades Publishing, Ltd., 2010. Original Russian Text © A. M. Andreeva and R. A. Fedorov, 2010, published in Biologiya Morya.
BRIEF NOTESE Biochemistry
Features of the Organization of LowMolecular Weight Proteins from the Blood and Tissue Fluid of the Common Stingray Dasyatis pastinaca (Chondroichthyes: Trygonidae) A. M. Andreeva and R. A. Fedorov Institute for the Biology of Inland Waters, Russian Academy of Sciences, Borok 152742 Russia email:
[email protected] Received January 28, 2010
Abstract—Lowmolecular weight proteins were revealed in the blood serum and tissue fluid from the muscles of the common stingray Dasyatis pastinaca. In the absence of urea in the reaction medium in vitro, these pro teins aggregated into supramolecular complexes; 8 M urea caused the disintegration of these complexes into individual proteins. The role of urea as a component of an organism’s fluids in the formation of the structural arrangements of blood plasma proteins and other biological fluids of cartilaginous fishes is discussed. It is shown that the extracellular fluids of the stingray also contain lowmolecular weight proteins as osmotically active compounds, along with urea, trimetilaminoxid, and salts. Keywords: cartilaginous fishes, common stingray, albuminlike proteins, tissue fluid. DOI: 10.1134/S1063074010060106
A high concentration of urea (0.19–0.6 M) and tri metilaminoxid (0.07–0.1 M and higher) in the blood and other extracellular fluids occurs in cartilaginous fishes [6, 19, 22]. These compounds, together with salts, provide osmotic pressure in the fluids of cartilag inous fishes to a hypertonic level relative to sea water [15, 19, 22]. In contrast to the considered compounds, osmotically active albumins were not revealed in the blood of sharks and rays for many years [10, 12, 16]; they were successfully found only by using modern approaches [13, 14]. In a similar manner to albumins, lowmolecular weight proteins were described in sev eral cartilaginous fishes, the sharks Scyliorhinus canic ula [9], Sphyrna tiburo [11], Mustelus canis [17], Car charhinus plumbeus [20], Squalus acanthias [3] and the bluespotted stingray Dasyatis kuhlii [18]. The objective of this work is a study of the structural organization of lowmolecular weight albuminlike proteins of the blood and tissue fluids from the muscles of the common stingray Dasyatis pastinaca. MATERIAL AND METHODS Stingrays were caught in the Karantinnaya Bay of the Black Sea in November of 2008. Their blood was sampled by techniques that were described previously and the interstitial fluid was obtained by soaking strips of 3MM Whatman paper with muscle tissue fluid [5]. The proteins were determined to be monomers (pro teins of one polypeptide chain)/oligomers (proteins of several polypeptide chains) by comparing the molecu
lar weights of native molecules and those denaturated with 8 M urea [4]. The proteins were fractionated using concentration gradient PAAG (5–40%), PAAG with 8 M urea, and 2D PAAG [4]. The molecular weights of native and denaturated molecules were determined by using proteins of known molecular weights in the polymeric forms of human serum albu min (HSA) or bull serum albumin (BSA) of 67, 134, 201, 268, 335 kDa and ovalbumin (OA) of 45, 90, 135 kDa [2]. The results were processed using OneD scan software. RESULTS AND DISCUSSION A heterogeneous lowmolecular weight fraction (HLF) of eight proteins, which included two macro components with molecular weights of about 67 and 47 kDa and a subfraction of six proteins with molecu lar weights from 13 to 30 kDa, marked as the fastfrac tion (see figure), was revealed in the blood serum of stingrays. The relative content of lowmolecular weight proteins in the serum did not exceed 2.9% of the total protein content; however it reached 28.4% in the tissue fluid of the muscles, mostly due to the 47 kDa molecular weight component; its share was up to 20.5% of the total protein of the muscle fluid (see figure) The addition of 8 M urea to the serum caused, first, disintegration of the 120 and 200 kDa proteins to proteins of about 34 kDa and enrichment of the low molecularweight proteins at their expense, and sec
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Protein electrophoregram of stingray blood serum and tissue fluid. a, electrophoresis in a PAAG concentration gradient (5–40%) of the blood serum of the roach (1) and round goby (2) (given for comparison), tissue fluid from stingray muscles (3) and blood serum (4) (the brackets distinguish proteins of the lowmolecular weight fraction, LMF, the dotted arrows indicate proteins of 47 and 67 kDa), M, polymeric forms of proteins of known molecular masses, human serum albumin (HSA) and ovalbumin (OA); the vertical arrow indicates the direction of electrophoresis. b, 2Delectrophoresis in a PAAG concentration gradient (5–40%) of the lowmolecular weight fraction of proteins from the tissue fluid of stingray muscles (the dotted arrows indicate 47 kDa pro teins and the fastfraction), M, polymeric forms of HSA and OA; the horizontal arrow indicates the direction of disk electro phoresis and the vertical one indicates gradient electrophoresis.
ond, the elimination of the heterogeneity of the fast fraction from six components to one of about 13 kDa. These transformations of lowmolecularweight proteins were obviously related to the aggregation of small proteins in complexes of 120 and 200 kDa in the absence of urea and to disintegration of these com plexes under the effect of urea on individual proteins due to the rupturing of hydrogen bonds. In the pres ence of urea it is possible to explain the elimination of the heterogeneity of the fastfraction by the fact that all the components of this fraction were formed by the oligomerization or covalent modification of one pro tein. Models of the structural arrangements of the serum albumin of vertebrates are based on the model of mammalian albumin, a protein monomer with a low molecular weight (67 kDa), which is, however, large enough for it not to be filtered with ease through the capillary wall [7] and for it to carry out osmotic, plas tic, and transport functions. The structural variety of the albumins of fishes is beyond the given criterion jus tifying use of the term “albuminlike” [3]. The discovery of lowmolecularweight proteins in the blood and tissue fluids of stingrays that are able to aggregate or dissociate depending on the availability of urea in the medium enabled us to expand our ideas on the arrangement of lowmolecular albuminlike pro teins in fish in general. The basic differences between these proteins in rays and other fishes, and also in mammals, are related to their arrangement pattern, molecular weight, and the surface structure of pro teins. The lowmolecular weight proteins of the ray in the presence of urea were found as several protein
monomers that aggregated in the absence of a urea environment in the complexes. In contrast, the albu mins of mammals are monomers that are not inclined to aggregation. The albumins of freshwater bony fishes are organized by their type of oligomeric pro teins; similar to the albumin units of rays they also break down to individual lowmolecular proteins in the presence of urea. The disintegration of oligomers has been observed during gonad maturation and the adaptation of fishes to higher salinities [4]. In sturgeon the albumins were arranged like the monomeric pro teins of mammals [3]. The lowmolecular weight fraction of the stingray serum in the absence of urea contained eight proteins with molecular weights varying from 13 to 67 kDa; with urea only two kinds of molecules were found, with molecular weights of 34 and 13 kDa. In other car tilaginous fishes lowmolecular weight proteins of the serum have other molecular weights, viz., in the shark Mustelus canis, 50 and 25 kDa [17]; in the spurdog Squalus acanthias, 12 proteins with molecular weights from 58 to 70 kDa have been described, which decom pose in SDSPAAG and leave one protein with a molecular weight of about 45 kDa [3]. Oligomeric pro teins of the common bream Abramis brama and roach Rutilus rutilus break down in SDS–PAAG into 10– 13 proteins with molecular weights from 18.5 to 73 kDa [4]. In marine bony fishes ten lowmolecular weight proteins have been described in the arctic flounder Liopsetta glacialis, seven proteins in the shorthorn sculpin Myoxocephalus scorpius with molecular weights from 30 to 90 kDa, and four or five proteins in the Atlantic cod Gadus morhua with
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molecular weights from 45 to 80 kDa [4]. The denatur ated albumin molecules of sturgeon and mammals have identical molecular weights of about 67 kDa [3]. These data show the high level of variability of the molecular weights of the lowmolecular weight pro teins of fishes. The surface structure features of the proteins found in stingrays, other fishes, and vertebrates lead to differ ent behaviors of the proteins in the presence/absence of urea, which can induce dissociation and aggrega tion of proteins [1]. The lowmolecular weight pro teins of stingrays aggregate due to hydrogen bonds in the absence of urea, which distinguishes them from the albumins of humans and bulls, which form poly meric forms due to the effects of covalent S–Sbonds [4]. Therefore, urea in the reaction medium causes disintegration of the albumin complexes in stingrays, whereas the disintegration of polymeric forms of HSA and BSA to monomers also requires a reducing envi ronment. Some sharks also have proteins that are sta bilized by S–Sbridges, viz., in Carcharhinus plumbeus a protein with a molecular weight of 70 kDa consists of two S–Sbound chains with molecular weights of 36 and 24 kDa [20], in Mustelus canis a Creactive pro tein with a molecular weight of about 250 kDa in media without urea consists of dimers with a molecu lar weight about 50 kDa in which monomers of about 25 kDa are connected by S–Sbonds, and the P com ponent has a molecular weight of 250 kDa and consists of 25 kDa monomers [17]. These examples enable one to assume a certain similarity in the arrangement of lowmolecular weight proteins in stingrays and other cartilaginous fishes: in environments that lack urea, lowmolecular weight proteins aggregate into highmolecular complexes that break down into lowmolecular proteins in the presence of 8 M urea. The ability of proteins to undergo structural transformations was probably also the cause of the difficulty in finding lowmolecular weight proteins in the blood of sharks. Highmolecular weight oligomeric complexes were found in electro phoregrams instead. Whether the reversible structural transitions of proteins in monomers/oligomers have any physiological importance for the regulation of the colloidalosmotic pressure in the blood of sharks is not known. It is not impossible that these complexes can be induced by fluctuation of the urea concentration in the blood related to the variation of the volume of body fluids during salinity fluctuations [8]. Our results showed the presence of lowmolecular weight albuminlike proteins in the blood and tissue fluids of the stingray and enabled us to assume that the pattern of organization of the lowmolecular weight albumin fraction of the blood of stingrays is deter mined by a high concentration of urea in their internal fluids. RUSSIAN JOURNAL OF MARINE BIOLOGY
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ACKNOWLEDGMENTS The work was performed with the support of RFBR Grant no. 100400954a. The authors are grateful to I.I. Rudneva and V.G. Shaide, experts at the Institute of Biology of Southern Seas NAS of Ukraine for samples of blood serum and tissue fluids from the muscles of stingrays. REFERENCES 1. Aleksandrov, V.Ya., Reaktivnost’ kletok i belki (Reactiv ity of Cells and Proteins), Leningrad: Nauka, 1985. 2. Andreeva, A.M., Fizikokhimicheskie svoistva osnov nykh grupp belkov krovi u razlichnykh po ekologii i tak sonomicheskomu polozheniyu predstavitelei khry aschevyh, khryaschevyh ganoidov i kostistykh ryb (Phys ical and Chemical Properties of Basic Groups of Pro teins of Blood in Various by Ecology and Taxonomic Position Representatives of Cartilaginous Fishes, Ganoidomorpha and Bony Bishes): Thes. Diss. … Cand. Biol. Sci., Borok. 1997. 3. Andreeva, A.M., Structurally Functional Organization of Albumin Systems of Fish Blood, Vopr. Ikhthiologii, 1999, vol. 39, no. 6, pp. 825–832. 4. Andreeva, A.M., Strukturnofunkcional’naya organi zatsiya belkov krovi i nekotorykh drugikh vnekletochnykh zhidkostei ryb (Structurally Functional Organization of Proteins of Blood and Some Other Extracellular Fluids of Fishes): Thes. Diss. … Doct. Biol. Sci. Moscow. 2008. 5. Andreeva, A.M., Ryabtseva, I.P., and Bolshakov, V.V., Analysis of Permeability of Capillaries of Different Departments of Microcirculatory System for Plasma Proteins in Some Representatives of Bony fishes, Zhurn. evol. biokhim. i fiziol., 2008, vol. 44, no. 2, pp. 212–214. 6. Stroganov, N.S., Ekologicheskaya fiziologiya ryb (Eco logical Physiology of Fishes), Moscow: MGU, 1962, vol. 1. 7. White, A., Hendler, Ph., Smith, E.L., Hill, R.L. and Lehman, I.R., Osnovy biokhimii. V 3 T., (Principles of Biochemistry. In 3 Vol.), Moscow: Mir, 1981. 8. Anderson, W.G., Taylor, J.R., Good, J.P., et al., Body Fluid Volume Regulation in Elasmobranch Fish, Comp. Biochem. Physiol. A: Mol. Integr. Physiol., 2007, vol. 148, no. 1, pp. 3–13. 9. Cordier, D., Barnoud, R., and Branndonn, A.M., Etude sur la protéinémie de la roussette (Scyllium can icula L.). Influence du jeune, C. R. Soc. Biol. Paris, 1957, vol. 151, pp. 1912–1915. 10. Griffith, R.W., Umminger, B.Z., Grant B.F., et al., Serum Composition of the Coelacanth, Latimeria cha lumnae Smith, J. Exp. Zool., 1974, vol. 187, no. 1, pp. 87–102. 11. Harms, C., Ross, T., and Segars, A., Plasma Biochem istry Reference Values of Wild Bonnethead Sharks, Sphyrna tiburo, Vet. Clin. Pathol., 2002, vol. 31, no. 3, pp. 111–115. 12. Hyodo, S., Bell, J.D., Healy, J.M., et al. Osmoregula tion in Elephant Fish Callorhinchus milii (Holocephali) No. 6
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