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Determination of ceruloplasmin and other copper transport ligands in the blood sera of the nurse shark and clearnose skate
Comp. Biochem. Physiol. Vol. 77B, No. 4, pp. 779-783, 1984 Printed in Great Britain
0305-0491/84 $3.00+ 0.00 © 1984 Pergamon Press Ltd
DETERMINATION OF CERULOPLASMIN A N D OTHER COPPER TRANSPORT LIGANDS IN THE BLOOD SERA OF THE NURSE SHARK A N D CLEARNOSE SKATE A. B. BODINE*, C. A. LUERt and S. GANGJEE* *Department of Dairy Science, Clemson University, Clemson, SC 29631, USA and tMote Marine Laboratory, Sarasota, FL 33577, USA (Received 9 August 1983)
Abstract--l. Sera of the elasmobranchs Ginglymostoma cirratum (nurse shark) and Raja eglanteria (clearnose skate) were examined for the concentration of typical copper transport ligands. 2. The major transport ligands histidine, albumin and ceruloplasmin were quite low in elasmobranch sera although production of melanin by liver cells was quite high. 3. Serum copper content was comparable to certain mammals (rabbit, ox, horse) but considerably lower than reported values for human, rat and swine.
INTRODUCTION It has been recognized for some time that the serum proteins in elasmobranchs are less complex than in higher vertebrates (Lepkovsky, 1929; Irisawa and Irisawa, 1954; Engle et al., 1958). Even so, investigations into the serum proteins that do exist indicate that many components are common to the blood of higher vertebrates. Urist (1961) has established that while the total protein concentration of elasmobranch serum was very low, the majority of the protein had the chemical, electrophoretic and ultracentrifugal properties of globulin. Subsequently, certain of the globulins have been isolated and partially characterized. These include the immunoglobulin IgM (Clem et al., 1967; Voss et al., 1969; Rudikoff et al., 1970) and the various density classes of serum lipoproteins (Lauter et al., 1968; Mills and Taylaur, 1971; Mills et al., 1977). Recently, the complement system in elasmobranch serum has been investigated, with the result that six functionally pure components have been isolated from the nurse shark (Jensen et al., 1981). In addition, the blood coagulation proteins prothrombin and fibrinogen have been demonstrated to be present in the plasma of this subclass of fish (Lewis, 1972). Perhaps the most characteristic feature of elasmobranch serum is the total absence or exceedingly small amount of albumin or an albumin-like protein. Depending upon the species, serum samples have been shown to possess either trace amounts (Rasmussen and Rasmussen, 1967) or a complete lack (Irisawa and Irisawa, 1954; Gunter et aL, 1961; Sulya et al., 1961) of any components having the same electrophoretic mobility as mammalian albumin. With little or no serum albumin, elasmobranchs must rely on alternate means to accomplish the important functions that this protein is known to play in higher vertebrates. The role of albumin in osmotic regulation, for example, is accounted for in elas-
mobranchs by high circulating levels of urea (300-450mM) (Maren, 1967) and trimethylamine oxide (approximately 70 mM) (Groninger, 1959). Substitution for serum albumin's important function as a transport protein, however, has yet to be fully explored. Only a limited number of proteins specific for the binding and transport of elasmobranch serum components have been reported in the literature. Among these are retinol-binding proteins (Idler and Freeman, 1969; Freeman and Idler, 1969; Martin, 1975) and the various lipoproteins involved in lipid transport (Mills et al., 1977). While the binding and transport of the serum calcium and inorganic phosphorus have been attributed to some component of the globulin fraction (Urist, 1961), no studies are available in the literature that specifically explore the existence of metal binding proteins. Metal analyses of elasmobranch tissues indicate that the highest levels of copper are localized in liver and brain (Windom et al., 1973). The existence of a serum copper transport protein, such as ceruloplasmin, would be interesting to establish, especially due to the lack of albumin, which has been shown to be a major transport ligand for copper in vertebrates (Frieden, 1981). In addition, elasmobranch liver is known to possess pigmented cells resembling melanocytes (Boyer and Wade, 1979). If the pigment in these cells is indeed melanin, its biosynthesis would be dependent upon the coppercontaining enzyme tyrosinase (EC 1.10.3.1). The regulation of copper in elasmobranchs therefore might rely on a specific binding and transport mechanism. For this reason, sera from two species of elasmobranch (the nurse shark, Ginglymostoma cirratum, and the clearnose skate, Raja eglanteria) have been examined in order to determine whether cerulopulasmin activity is detectable and to determine the relative distribution of the other known copper transport ligands.
779
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A . B . BODINE et al.
Table 1. Serum ceruloplasmin (Cp) activity and copper concentration in nurse sharks and clearnose skates Species Nurse shark n=9 ~n=5 Skate £ n- 8 9 n=4
Mean Cp activity IUs* ± S.E.t
Range
14.0 -+ 3.2 9.8±2.3
4.2-29.7 5.1 14.3
6.9 ± 0.6 4.2±0.5
4.5 9.9 3.6 11.7
Table 2. Free amino acids and albumin in nurse shark and clearnose skate sera
Amino acid
Mean serum copper # g/m~-+ S2E. Nurse shark n=5 0.43 ± 0.06 0.36-0.51 4' n = 5 0.52 -+ 0.07 0A3-0.59 Skate .3 n = 5 0.40 ± 0.07 0.29-0.46 '~ n = 5 0.50 ± 0.24 0,16-0.72 *IU = #mol of Bandroswki's base formed/l of serum/min (Rice, 1962). tStandard error of the mean.
MATERIALS AND METHODS Blood was obtained from nurse sharks and clearnose skates by cardiac puncture and serum prepared by centrifugation of the clotted blood. The serum so obtained was quick frozen in acetone/dry ice and stored at - 8 0 ' C until analyzed. Ceruloplasmin analysis was conducted by the general copper oxidase method of Houchin (1958) as modified by Rice (1962). Because preliminary results indicated low activity of elasmobranch serum for pphenylenediamine (Aldrich Chemical Co., Milwaukee, WI) as substrate, the methodology was modified by a procedure outlined by Starcher and Hill (1965), which involved incubating 0.2 ml of serum with 0.1% p-phenylenediamine for 30rain at 37°C. Amino acid analyses were conducted on serum {teproteinated with sulfosalicylic acid (Block et al., 1966) using an amino acid analyzer. Serum copper concentration was determined using an atomic absorption spectrophotometer equipped with graphite furnace. Serum albumin was quantitated using a Bromcresol green albumin kit (Sigma Chemical Co., St. Louis, MO). Polyacrylamide gel electrophoresis was conducted in 7% acrylamide run gel and 4% acrylamide stacking gel at 5 mA/tube in pH 8.9 Tris/glycine buffer. Protein bands were visualized with Coomassie G-250. Liver tissue from freshly killed animals was prepared for histology by fixation in buffered 10~ formalin. Fixed liver tissue was then embedded in paraffin, sectioned at 6 ~tm and stained appropriately. Histology procedures included the Fontana-Masson stain to enhance melanin (AFIP 1968a), treatment with 10~, hydrogen peroxide to remove melanin (Humason, 1979) and Gomori's method to stain for iron (AFIP, 1968b).
Alanine Arginine Aspartic acid Cystine Glycine Glutamic acid Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Shark if/? (n - 8) Skate 3/9 (n = 8)
Shark 3'/~: Skate ~/%' (n 13) {n : 8} mg/100 ml -+ S.E. 2.70 ± 0.94 3.30 _+0.51 0.40 -+ 0.09 1.70 ± 0.44 1.17 -+ 0.08 0.23 ± 0.12 0.48 + 0.10 356 + 0.52 5.35 ± 0.99 5.32 _+0.40 2.36 ± 0.48 0.91 +0.15 Trace 1.78 ± 0.65 2.22_+0.37 1.25 -+ 0.09 1.09-+ 0.15 7.08 + 0.92
2.25 -+ 0.88 3.77 ~- 0.62 0.47 + 0.17 2.21 -+ 0.60 1.02 + 0.1 [ 0.19 -+ 0.07 0.44 -+ 0.21 3.14 ± 0.50 6.07 + 1./)5 4.06 + 0.52 2.26 ± 0.39 0.84_+0.13 Trace 1.60-- 0.48 3.11 ±0.28 1.66 -2-_0.17 0.96 ± 0.15 6.69 ± 1.08
Albumin % -+ S.E.
Range
0.86 ± 0.07
0.675 1.20
0.33 ± 0.02
0.25 0.42
*Standard error of the mean.
No significant differences in serum copper concentrations were detected between sexes or species. The values for copper concentration in elasmobranch sera are quite similar to literature values for the ox, horse and rabbit but are much less than the concentrations copper observed in man swine and rat (Airman and Dittmer, 1961). In Table 2 the data for serum free amino acids and albumin are presented. Of interest are the quite low concentrations of aromatic amino acids, acidic amino acids and histidine and the very high concentrations of aliphatic amino acids, particularly valine and leucine. The low concentrations of histidine coupled with the minimal levels of serum albumin, particularly in the skate, would seem to impose a rather severe constraint on copper transport
m
mum
I
l
I I
l [3 l
RESULTS AND DISCUSSION T h e c e r u l o p l a s m i n (Cp) activity a n d s e r u m c o p p e r levels are s u m m a r i z e d in T a b l e 1. Significant differences (P < 0.05) were detected b e t w e e n C p activities in s e r u m o f m a l e n u r s e s h a r k s c o m p a r e d to male or female c l e a r n o s e skates. N o differences (P > 0.05) were detected b e t w e e n m a l e a n d female s h a r k s n o r b e t w e e n the t w o sexes o f skate. T h e s e C p values for b o t h e l a s m o b r a n c h sera are c o n s i d e r a b l y b e l o w literature values for h u m a n s a n d certain o t h e r m a m m a l s (Rice, 1962; G a r a t t i n i , 1960) b u t m a n y fold h i g h e r t h a n the C p activity in chick sera ( S t a r c h e r a n d Hill, 1965).
m
I
l\
/
m
+ Fig. 1. Schematics of the polyacrylamide gel electrophoretograms of clearnose skate serum (A) and nurse shark serum (B). The arrows indicate the albumin bands.
Ceruloplasmin in elasmobranch blood
Fig. 2. Normal nurse shark liver, stained for enhancement of melanin using the Fontana-Masson stain. The dark staining cells are consisted with a positive reaction for the presence of malanin ( x 352).
Fig. 3. Normal nurse shark liver, bleached with 10~ hydrogen peroxide and stained with hematoxylin and eosin. The arrows indicate cells from which melanin has been specifically removed. The nuclei of these cells are observed to be pushed to the periphery. Such distortion of the nuclei might be expected to arise as a consequence of melanin engorgement ( × 352).
781
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A.B. Bor)irqE et al.
in the serum. The low to very low c o n c e n t r a t i o n s o1 serum a l b u m i n in these e l a s m o b r a n c h s can also be d e m o n s t r a t e d by the acrylamide electrophoresis patterns s h o w n in Fig. 1. Histologic e x a m i n a t i o n of liver tissue from b o t h nurse sharks a n d clearnose skates reveals the characteristic presence of n u m e r o u s pigmented cells scattered t h r o u g h o u t the p a r e n c h y m a . While these pigm e n t e d cells have been described as resembling melanocytes (Boyer a n d Wade, 1979), the actual n a t u r e of the pigment has n o t been documented. A positive reaction following the staining procedure of F o n t a n a - M a s s o n (Fig. 2) c o m b i n e d with a negative response to the G o m o r i ' s stain for iron (data not shown) are consistent with melanin. In addition, t r e a t m e n t o f the liver tissue for the specific removal o f melanin with 10% H202 for 24 hr results in disappearance of the pigment (Fig. 3). This d e m o n s t r a t i o n of the existence of melanin provides a n o t h e r basis for the need to regulate copper t r a n s p o r t in these elasm o b r a n c h s since the tyrosinase enzyme responsible for the biosynthesis of melanin from tyrosine is a copper-containing protein (Jolley et al., 1974). A l t h o u g h the copper c o n t e n t of e l a s m o b r a n c h s ' sera is similar to t h a t in some m a m m a l s (ox, horse, rabbit), the ceruloplasmin, histidine a n d a l b u m i n c o n c e n t r a t i o n s are quite low. It is thus difficult to rationalize these ligands as being chiefly responsible for the control of copper transport. Research is continuing in attempts to identify a coppert r a n s p o r t i n g protein or other ligand t h a t can contribute significantly to copper regulation in these elasmobranchs. Acknowledgements--The authors wish to thank Ms Kellie Dixon, Mote Marine Laboratory for the atomic absorption analyses and Dr. W. H. Luer, Department of Pathology Tulane Medical School, for conducting the histologic examinations. REFERENCES
Altman P. L. and Dittmer D. S. (1961) Blood and Other Body Fluids. Federation of the American Society of Experimental Biology, Bethesda, MD. Armed Forces Institute of Pathology (Edited by Luna L.) (1968a) Manual of Histologic Staining Methods', 3rd Edn, p. 104. McGraw Hill, NY. Armed Forces Institute of Pathology (Edited by Luna, L.) (1968b) Manual of Histologic Staining Methods, 3rd Edn, p. 174. McGraw Hill, NY. Block W. E., Markous M. E. and Steele B. F. (1966) Comparison between amino acid levels in plasma deproteinized with picric acid and with sulfosalicylic acid. Proc. Soc. exp. Biol. Med. 122, 1089-1091. Boyer J. L. and Wade J. B. (1979) Pigmented sinusoidal lining cells in elasmobranch liver. Bull. Mt. Desert Is. biol. Lab. 19, 95-96. Clem L. W., Deboutaud F. and Sigel M. M. (1967) Phylogeny of immunoglobulin structure and function. If. Immunoglobulins of the nurse shark. J. Immunol. 99, 1226-1235. Engle R. L., Woods K. R., Paulsen E. C. and Pert J. H. (1958) Plasma cells and serum proteins in marine fish. Proc. Soc. exp. Biol. Med. 98, 905-909. Freeman H. C. and Idler D. R. (1969) Sex hormone binding proteins. II. Isolation from serum of an elasmobranch (Raja radiata). Gen. comp. Endocrinol. 13, 83-91. Frieden E. and Hsich H. S. (1976) Ceruloplasmin: the
copper transport protein with essential oxidase activity. Adv. Enzymol. 44, 187-236. Frieden E. (1981) Ceruloplasmin: a multifunctional metalloprotein of vertebrate plasma. In Metal Ions in Biological Systems, Vol. 13, Copper Proteins. (Edited by Siegel H.), pp. 131-132. Marcel Dekker, N.Y. Garattini S., Giachetti A. and Pieri L. (1960) Oxidation of ceruloplasmin substrates by serum of different animal species. Archs Biochem. Biophys. 91, 83 85. Groninger H. S. (1959) The occurrence and significance of trimethylamine oxide in marine animals. United States Fish and Wildlife Service, Special Scientific Report. Fisheries No. 333: 1-22. Gunter G., Sulya L. L. and Box B. E. (1961) Some evolutionary patterns in fishes" blood. Biol. Bull. 121, 302-306. Houchin O. B. (1958) A rapid colorimetric method for the quantitative determination of copper oxidase activity (ceruloplasmin). Clin. Chem. 4, 519-523. Humason G. (1979) Animal Tissue Techniques, 9th Edn, p. 260. W. H. Freeman, San Francisco, CA. Idler D. R. and Freeman H. C. (1969) Sex hormone binding proteins. I. Binding of steroids by serum of an elasmobranch (Raja radiata). Gen. comp. Endocrinol. 13, 75 82. Irisawa H. and Irisawa A. F. (1954) Blood serum protein of the marine elasmobranchii. Science 120, 849-850. Jensen J. A., Festa E., Smith D. S. and Cayer M. (1981) The complement system of the nurse shark: hemolytic and comparative characteristics. Science 214, 566-569. Jolley R, L., Evans L. H, Makino N. and Mason H. S. (1974) Oxytyrosinase J. biol. Chem. 249, 335-339. Lauter C. J., Brandenburger-Brown E. A. and Trams E. G. (1968) Composition of plasma lipoproteins of the spiny dogfish Squalus acanthias. Comp. Biochem. Physiol. 24, 243 247. Lepkovsky S. (1929) The distribution of serum and plasma proteins in fish. J. biol. Chem. 85, 667 673. Lewis J. H. (1972) Comparative homeostasis: studies on elasmobranchs. Comp. Biochem. Physiol. 42A, 223-240. Maren T. H. (1967) Special body fluids of the elasmobranch. In Sharks, Skates and Rays (Edited by Gilbert P. W., Mathewson R. F. and Rall D. P.), pp. 287 292. John Hopkins, Baltimore, MD. Martin B. (1975) Steroid-protein interactions in nonmammalian vertebrates. I. Elasmobranch steroidsbinding protein (E. SBP) in dogfish serum (Scyliorhinus canicula). Gen. comp. Endocrinol. 25, 42 51. Mills G. L. and Taylaur C. E. (1971) The distribution and composition of serum lipoproteins in 18 animals. Comp. Biochem. Physiol. 40B, 489 501. Mills G. L., Taylaur C. E., Chapman M. J. and Forster G. R. (1977) Characterization of serum lipoproteins of the shark Centrophorus squamosus. Biochem. J. 163, 455-465. Rasmussen L. E. and Rasmussen R. A. (1967) Comparative protein and enzyme profiles of the cerebrospinal fluid, extradural fluid, nervous tissue, and sera of elasmobranchs. In Sharks, Skates and Rays (Edited by Gilbert P. W., Mathewson R. G. and Rail D. P.), pp. 360-379. John Hopkins, Baltimore, MD. Rice E. W. (1962) Standardization of ceruloplasmin in terms of international enzyme units. Analyt. Bioehem. 3, 452-456. Rudikoff S., Voss E. W. and Sigel M. M. (1970) Biological and chemical properties of natural antibodies in the nurse shark. J. lmmunol. 105, 1344-1352. Shidoji Y. and Yasutoshi M. (1977) Vitamin A transport in plasma of the non-mammalian vertebrates: isolation and partial characterization of piscine retinol-binding protein. J. Lipid Res. 18, 679 691. Starcher B. and Hill C. H. (1965) Hormonal induction of ceruloplasmin in chicken serum. Comp. Biochem. Physiol. 15, 429434.
Ceruloplasmin in elasmobranch blood Sulya L. L., Box B. E. and Gunter G. (1961) Plasma proteins in the blood of fishes from the Gulf of Mexico. Am. J. Physiol. 200, 152-154. Urist M. R. (1961) Calcium and phosphorous in the blood and skeleton of the elasmobranchii. Endocrinology 69, 778-801. Voss E. W., Russell W. J. and Sigel M. M. (1969)
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Purification and binding properties of nurse shark antibody. Biochemistry 8, 4866-4872. Windom H., Stickney R., Smith R., White D. and Taylor F. (1973) Arsenic, cadmium, copper, mercury, and zinc in some species of North Atlantic finfish. J. Fish. Res. Board Can. 30, 275-279.
0305-0491/84 $3.00+ 0.00 © 1984 Pergamon Press Ltd
DETERMINATION OF CERULOPLASMIN A N D OTHER COPPER TRANSPORT LIGANDS IN THE BLOOD SERA OF THE NURSE SHARK A N D CLEARNOSE SKATE A. B. BODINE*, C. A. LUERt and S. GANGJEE* *Department of Dairy Science, Clemson University, Clemson, SC 29631, USA and tMote Marine Laboratory, Sarasota, FL 33577, USA (Received 9 August 1983)
Abstract--l. Sera of the elasmobranchs Ginglymostoma cirratum (nurse shark) and Raja eglanteria (clearnose skate) were examined for the concentration of typical copper transport ligands. 2. The major transport ligands histidine, albumin and ceruloplasmin were quite low in elasmobranch sera although production of melanin by liver cells was quite high. 3. Serum copper content was comparable to certain mammals (rabbit, ox, horse) but considerably lower than reported values for human, rat and swine.
INTRODUCTION It has been recognized for some time that the serum proteins in elasmobranchs are less complex than in higher vertebrates (Lepkovsky, 1929; Irisawa and Irisawa, 1954; Engle et al., 1958). Even so, investigations into the serum proteins that do exist indicate that many components are common to the blood of higher vertebrates. Urist (1961) has established that while the total protein concentration of elasmobranch serum was very low, the majority of the protein had the chemical, electrophoretic and ultracentrifugal properties of globulin. Subsequently, certain of the globulins have been isolated and partially characterized. These include the immunoglobulin IgM (Clem et al., 1967; Voss et al., 1969; Rudikoff et al., 1970) and the various density classes of serum lipoproteins (Lauter et al., 1968; Mills and Taylaur, 1971; Mills et al., 1977). Recently, the complement system in elasmobranch serum has been investigated, with the result that six functionally pure components have been isolated from the nurse shark (Jensen et al., 1981). In addition, the blood coagulation proteins prothrombin and fibrinogen have been demonstrated to be present in the plasma of this subclass of fish (Lewis, 1972). Perhaps the most characteristic feature of elasmobranch serum is the total absence or exceedingly small amount of albumin or an albumin-like protein. Depending upon the species, serum samples have been shown to possess either trace amounts (Rasmussen and Rasmussen, 1967) or a complete lack (Irisawa and Irisawa, 1954; Gunter et aL, 1961; Sulya et al., 1961) of any components having the same electrophoretic mobility as mammalian albumin. With little or no serum albumin, elasmobranchs must rely on alternate means to accomplish the important functions that this protein is known to play in higher vertebrates. The role of albumin in osmotic regulation, for example, is accounted for in elas-
mobranchs by high circulating levels of urea (300-450mM) (Maren, 1967) and trimethylamine oxide (approximately 70 mM) (Groninger, 1959). Substitution for serum albumin's important function as a transport protein, however, has yet to be fully explored. Only a limited number of proteins specific for the binding and transport of elasmobranch serum components have been reported in the literature. Among these are retinol-binding proteins (Idler and Freeman, 1969; Freeman and Idler, 1969; Martin, 1975) and the various lipoproteins involved in lipid transport (Mills et al., 1977). While the binding and transport of the serum calcium and inorganic phosphorus have been attributed to some component of the globulin fraction (Urist, 1961), no studies are available in the literature that specifically explore the existence of metal binding proteins. Metal analyses of elasmobranch tissues indicate that the highest levels of copper are localized in liver and brain (Windom et al., 1973). The existence of a serum copper transport protein, such as ceruloplasmin, would be interesting to establish, especially due to the lack of albumin, which has been shown to be a major transport ligand for copper in vertebrates (Frieden, 1981). In addition, elasmobranch liver is known to possess pigmented cells resembling melanocytes (Boyer and Wade, 1979). If the pigment in these cells is indeed melanin, its biosynthesis would be dependent upon the coppercontaining enzyme tyrosinase (EC 1.10.3.1). The regulation of copper in elasmobranchs therefore might rely on a specific binding and transport mechanism. For this reason, sera from two species of elasmobranch (the nurse shark, Ginglymostoma cirratum, and the clearnose skate, Raja eglanteria) have been examined in order to determine whether cerulopulasmin activity is detectable and to determine the relative distribution of the other known copper transport ligands.
779
780
A . B . BODINE et al.
Table 1. Serum ceruloplasmin (Cp) activity and copper concentration in nurse sharks and clearnose skates Species Nurse shark n=9 ~n=5 Skate £ n- 8 9 n=4
Mean Cp activity IUs* ± S.E.t
Range
14.0 -+ 3.2 9.8±2.3
4.2-29.7 5.1 14.3
6.9 ± 0.6 4.2±0.5
4.5 9.9 3.6 11.7
Table 2. Free amino acids and albumin in nurse shark and clearnose skate sera
Amino acid
Mean serum copper # g/m~-+ S2E. Nurse shark n=5 0.43 ± 0.06 0.36-0.51 4' n = 5 0.52 -+ 0.07 0A3-0.59 Skate .3 n = 5 0.40 ± 0.07 0.29-0.46 '~ n = 5 0.50 ± 0.24 0,16-0.72 *IU = #mol of Bandroswki's base formed/l of serum/min (Rice, 1962). tStandard error of the mean.
MATERIALS AND METHODS Blood was obtained from nurse sharks and clearnose skates by cardiac puncture and serum prepared by centrifugation of the clotted blood. The serum so obtained was quick frozen in acetone/dry ice and stored at - 8 0 ' C until analyzed. Ceruloplasmin analysis was conducted by the general copper oxidase method of Houchin (1958) as modified by Rice (1962). Because preliminary results indicated low activity of elasmobranch serum for pphenylenediamine (Aldrich Chemical Co., Milwaukee, WI) as substrate, the methodology was modified by a procedure outlined by Starcher and Hill (1965), which involved incubating 0.2 ml of serum with 0.1% p-phenylenediamine for 30rain at 37°C. Amino acid analyses were conducted on serum {teproteinated with sulfosalicylic acid (Block et al., 1966) using an amino acid analyzer. Serum copper concentration was determined using an atomic absorption spectrophotometer equipped with graphite furnace. Serum albumin was quantitated using a Bromcresol green albumin kit (Sigma Chemical Co., St. Louis, MO). Polyacrylamide gel electrophoresis was conducted in 7% acrylamide run gel and 4% acrylamide stacking gel at 5 mA/tube in pH 8.9 Tris/glycine buffer. Protein bands were visualized with Coomassie G-250. Liver tissue from freshly killed animals was prepared for histology by fixation in buffered 10~ formalin. Fixed liver tissue was then embedded in paraffin, sectioned at 6 ~tm and stained appropriately. Histology procedures included the Fontana-Masson stain to enhance melanin (AFIP 1968a), treatment with 10~, hydrogen peroxide to remove melanin (Humason, 1979) and Gomori's method to stain for iron (AFIP, 1968b).
Alanine Arginine Aspartic acid Cystine Glycine Glutamic acid Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
Shark if/? (n - 8) Skate 3/9 (n = 8)
Shark 3'/~: Skate ~/%' (n 13) {n : 8} mg/100 ml -+ S.E. 2.70 ± 0.94 3.30 _+0.51 0.40 -+ 0.09 1.70 ± 0.44 1.17 -+ 0.08 0.23 ± 0.12 0.48 + 0.10 356 + 0.52 5.35 ± 0.99 5.32 _+0.40 2.36 ± 0.48 0.91 +0.15 Trace 1.78 ± 0.65 2.22_+0.37 1.25 -+ 0.09 1.09-+ 0.15 7.08 + 0.92
2.25 -+ 0.88 3.77 ~- 0.62 0.47 + 0.17 2.21 -+ 0.60 1.02 + 0.1 [ 0.19 -+ 0.07 0.44 -+ 0.21 3.14 ± 0.50 6.07 + 1./)5 4.06 + 0.52 2.26 ± 0.39 0.84_+0.13 Trace 1.60-- 0.48 3.11 ±0.28 1.66 -2-_0.17 0.96 ± 0.15 6.69 ± 1.08
Albumin % -+ S.E.
Range
0.86 ± 0.07
0.675 1.20
0.33 ± 0.02
0.25 0.42
*Standard error of the mean.
No significant differences in serum copper concentrations were detected between sexes or species. The values for copper concentration in elasmobranch sera are quite similar to literature values for the ox, horse and rabbit but are much less than the concentrations copper observed in man swine and rat (Airman and Dittmer, 1961). In Table 2 the data for serum free amino acids and albumin are presented. Of interest are the quite low concentrations of aromatic amino acids, acidic amino acids and histidine and the very high concentrations of aliphatic amino acids, particularly valine and leucine. The low concentrations of histidine coupled with the minimal levels of serum albumin, particularly in the skate, would seem to impose a rather severe constraint on copper transport
m
mum
I
l
I I
l [3 l
RESULTS AND DISCUSSION T h e c e r u l o p l a s m i n (Cp) activity a n d s e r u m c o p p e r levels are s u m m a r i z e d in T a b l e 1. Significant differences (P < 0.05) were detected b e t w e e n C p activities in s e r u m o f m a l e n u r s e s h a r k s c o m p a r e d to male or female c l e a r n o s e skates. N o differences (P > 0.05) were detected b e t w e e n m a l e a n d female s h a r k s n o r b e t w e e n the t w o sexes o f skate. T h e s e C p values for b o t h e l a s m o b r a n c h sera are c o n s i d e r a b l y b e l o w literature values for h u m a n s a n d certain o t h e r m a m m a l s (Rice, 1962; G a r a t t i n i , 1960) b u t m a n y fold h i g h e r t h a n the C p activity in chick sera ( S t a r c h e r a n d Hill, 1965).
m
I
l\
/
m
+ Fig. 1. Schematics of the polyacrylamide gel electrophoretograms of clearnose skate serum (A) and nurse shark serum (B). The arrows indicate the albumin bands.
Ceruloplasmin in elasmobranch blood
Fig. 2. Normal nurse shark liver, stained for enhancement of melanin using the Fontana-Masson stain. The dark staining cells are consisted with a positive reaction for the presence of malanin ( x 352).
Fig. 3. Normal nurse shark liver, bleached with 10~ hydrogen peroxide and stained with hematoxylin and eosin. The arrows indicate cells from which melanin has been specifically removed. The nuclei of these cells are observed to be pushed to the periphery. Such distortion of the nuclei might be expected to arise as a consequence of melanin engorgement ( × 352).
781
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A.B. Bor)irqE et al.
in the serum. The low to very low c o n c e n t r a t i o n s o1 serum a l b u m i n in these e l a s m o b r a n c h s can also be d e m o n s t r a t e d by the acrylamide electrophoresis patterns s h o w n in Fig. 1. Histologic e x a m i n a t i o n of liver tissue from b o t h nurse sharks a n d clearnose skates reveals the characteristic presence of n u m e r o u s pigmented cells scattered t h r o u g h o u t the p a r e n c h y m a . While these pigm e n t e d cells have been described as resembling melanocytes (Boyer a n d Wade, 1979), the actual n a t u r e of the pigment has n o t been documented. A positive reaction following the staining procedure of F o n t a n a - M a s s o n (Fig. 2) c o m b i n e d with a negative response to the G o m o r i ' s stain for iron (data not shown) are consistent with melanin. In addition, t r e a t m e n t o f the liver tissue for the specific removal o f melanin with 10% H202 for 24 hr results in disappearance of the pigment (Fig. 3). This d e m o n s t r a t i o n of the existence of melanin provides a n o t h e r basis for the need to regulate copper t r a n s p o r t in these elasm o b r a n c h s since the tyrosinase enzyme responsible for the biosynthesis of melanin from tyrosine is a copper-containing protein (Jolley et al., 1974). A l t h o u g h the copper c o n t e n t of e l a s m o b r a n c h s ' sera is similar to t h a t in some m a m m a l s (ox, horse, rabbit), the ceruloplasmin, histidine a n d a l b u m i n c o n c e n t r a t i o n s are quite low. It is thus difficult to rationalize these ligands as being chiefly responsible for the control of copper transport. Research is continuing in attempts to identify a coppert r a n s p o r t i n g protein or other ligand t h a t can contribute significantly to copper regulation in these elasmobranchs. Acknowledgements--The authors wish to thank Ms Kellie Dixon, Mote Marine Laboratory for the atomic absorption analyses and Dr. W. H. Luer, Department of Pathology Tulane Medical School, for conducting the histologic examinations. REFERENCES
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