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Catabolism of hemocyanin in the American lobster, Homarus americanus
Comp. Bim'hem. Physiol. Vol. 69B, pp. 781 to 790, 1981 Printed in Great Britain. All rights reserved
0305-0491/81/080781-10502.00/0 Copyright © 1981 Pergamon Press Ltd
CATABOLISM OF HEMOCYANIN IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS EDWARD G. SENKBEIL* and JOHN C. WRISTONJR Department of Chemistry, University of Delaware, Newark, DE 19711, U.S.A. (Received 24 November 1980)
Abstract--1. Cross-reactivity studies by double immunodiffusion suggest no immunological similarity between Homarus americanus hemocyanin and Panulirus argus hemocyanin. 2. Slow hemolymph clearance rates (tin of 15-30 days) for ~25I-Homarus americanus and Panulirus argus hemocyanins were determined after injection in both species, suggesting similar structure of the two hemocyanins relative to clearance recognition sites. 3. No single tissue emerged as the site of clearance and degradation of endogenous ~25I-labelled hemocyanin in Homarus americanus, possibly due to its slow clearance rate. 4. 125I-Limulus polyphemus hemocyanin was rapidly cleared from the hemolymph of Homarus americanus (tl/2 = 45 min) with the epidermis indicated as the initial site of uptake and degradation.
INTRODUCTION A great deal is known about the structure and chemical properties of hemocyanin (for a review see Lontie & Vanquickenborne, 1974) but little about its metabolism. The hepatopancreas has been determined as the site of synthesis of hemocyanin in the American lobster (Senkbeil & Wriston, 1980). The hemolymph clearance of labelled endogenous hemocyanin in lobsters (Stewart & Foley, 1969), crayfish (Sloan et al., 1975) and blue crabs (McCumber & Clem, 1977) is slow, but no further studies on degradation were performed. McCumber & Clem (1977) demonstrated in the blue crab that 125I-labelled hemocyanin from closely related species had much slower hemolymph clearance rates than hemocyanins from more distant species. It has been theorized that hemocyanin is degraded in the hepatopancreas (Van Weel, 1974) of crustaceans primarily because that organ has a central role in all metabolic processes, similar to the liver in vertebrates, but no direct evidence for its importance in hemocyanin degradation has been given. The hemolymph clearance and degradation of foreign soluble proteins in crustaceans has been studied in more detail. Stewart & Foley (1969) demonstrated a fast hemolymph clearance of fluorescentlabelled bovine serum albumin (BSA) and suggested that nonspecific proteolytic degradation might be involved in the clearance. More recent studies (McCumber & Clem, 1977) showed rapid hemolymph clearance of ~25I-BSA from blue crabs and crayfish; since 60% of the radioactivity accumulated in the gills, this organ was proposed as the initial site of hemocyanin degradation. Specificity of clearance was demonstrated since unlabelled BSA, human serum albumin, and rabbit serum albumin retarded clearance of 125I-BSA, whereas bovine gamma globulin and keyhole limpet hemocyanin had no effect. In preliminary studies of purification and characterization * Present address: Physical Science Department, Salisbury State College, Salisbury, MD 21801, U.S.A.
of specific soluble receptors from blue crab hemolymph, McCumber et al. (1979) noted the ability of this factor to neutralize T2 bacteriphage. No studies demonstrating the ability of this factor to bind to soluble proteins were performed. Invertebrates in general have the nonspecific hemolymph defense factors of bactericidins, agglutinins, and phagocytic capacities. These defense mechanisms have been well demonstrated with foreign particles, but the relevance of these systems or the soluble factor of McCumber et al. (1979) to the understanding of soluble protein clearance and degradation is unknown. The present work was undertaken to elucidate the metabolism of hemocyanin in decapod crustaceans. lzsI-labelled hemocyanin was injected into the hemolymph of lobsters and the rate and site of clearance were studied.
MATERIALS AND METHODS Animals
American lobsters (Homarus americanus) were caught off the coast of Lewes, Delaware, and maintained in running seawater, 16°C. Spiny lobsters (Panulirus ar#us), generously donated by Dr William Herrinkind, Florida State University, were maintained in running seawater, 20°C. All experiments were conducted with intermolt lobsters maintained for at least one week in this way, and fed twice weekly on frozen clams, Mercenaria mercenaria. Injected animals were transferred to 5 gallon aquaria and maintained in aerated seawater, 17-18°C. The seawater was changed every 72 hr. Immunological techniques
Antisera were obtained by immunization of white New Zealand male rabbits by modification of the standard procedure of Campbell et al. (1964). Purified hemocyanin was dialyzed against 3.0 mM phosphate buffer, pH 7.0, containing 3.0 mM CaCI2, and 1.0 ml aliquots (10 mg protein/ml) were injected into the outer ear vein every other day for a total of 5 injections. Blood samples were withdrawn every 2 weeks and subcutaneous booster shots of hemocyanin were given every month. Blood samples were allowed to clot, then centrifuged, and the serum frozen with no further
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EDWARD G . SENKBEIL a n d JOHN C. WRISTON JR
purification. The serum samples were used for all immunochemical techniques. Double immunodiffusion using l"J/o Ionoagar no. 2 (Colab Laboratories, Inc.) in 0.07 M Tris, 0.01 M borate, 0.005 M CaC12, pH 8.2, with 0.02°Jo sodium azide was performed according to the methods of Clausen (1969). Immunoelectrophoresis was performed in gels consisting of 1°,o Ionoagar no. 2 in the above buffer. Gels were prepared on microscope slides and samples were run 3 hr at 2 ma/ slide in a Buchler flat-bed electrophoresis apparatus. Gels were stained for protein according to the method of Clausen (1969) with Amido black.
Preparation of hemocyanin Hemocyanin from the 3 species studied (Homarus americanus, Panulirus argus, and Limulus polyphemus) was purified by gel filtration of hemolymph serum on a Sepharose 6B column (2 x 80cm). Purified hemocyanin concentrations were determined spectrophotometrically using the extinction coefficients E~'~ at 280nm (Nickerson & Van Holde, 1971). A solid phase form of the original lactoperoxidase labelling technique (Marchalonis, 1969) was used for iodination of hemocyanin with an Enzymobead Radioiodination kit (Bio-Rad Catalog no. 170-6002) and a modified version of the procedure described in Product Information 1060, copyright 1978 Bio-Rad Laboratories. Protein and Na 1z5I (Amersham/Searle IMS 30) were added on a 1:1 molar ratio so that there would theoretically be a maximum of one label per hemocyanin molecule. Similarity of the iodinated hemocyanin to the native molecule was established by gel filtration studies, immunoprecipitation tests, and immunoelectrophoresis.
Clearance studies of labelled hemocyanin ~25I-labelled hemocyanin (1.5-3.0 microcuries) was injected into the pericardial sinus of the lobster. In clearance studies, samples of the hemolymph were withdrawn periodically from the pericardial sinus. Aliquots (5(~150/~1) were placed in 8 × 75 mm test tubes and refrigerated. Triplicate samples were taken at each time interval. Hemocytes were periodically separated from hemolymph samples (Senkbeil & Wriston, 1980). At the end of the clearance experiment, all tubes were counted directly in a Nuclear Chicago 1185 Series automated gamma counter. Integrity of circulating hemocyanin was determined by gel filtration,
trichloroacetic acid (TCA) precipitation, and immunoprecipitation studies. In other experiments lobsters were sacrificed by freezing at various time intervals after 1251-hemocyanin injection. Tissues were extracted, minced, washed three times in lobster physiological saline (Cole, 1941), blotted dry, and weighed into individual test tubes. Triplicate samples of each tissue were prepared. Aliquots of the tank water were also periodically examined for excreted radioactivity. The molecular size range of the radioactive species in the various tissues was also studied. Washed tissue samples were homogenized (in 0.05 M Tris HCI buffer, pH 7.5, containing 5.0 mM CaCI~ and 0.25 mM phenylmethylsulfonyl fluoride), centrifuged at 15,000rpm for 30rain, and the supernatants applied to either a Sephadex G-25 column (2.5 × 45cm) or a Sepharose 6B column (2.2 × 55cm). Both columns were equilibrated with 0.05 M Tris HCI buffer, pH 7.5, containing 5.0 mM CaC12.
RESULTS
Preparation of hemocyanin Hemocyanin purified from Homarus americanus, Panulirus argus, and Limulus polyphemus was iodinated by the Enzymobead-lactoperoxidase method. A typical iodination in this study resulted in 15-25°{, of total radioactive iodine being protein incorporated, with an average of less than one atom of radioiodine per hemocyanin molecule. Three methods were used to study the physical and chemical properties of iodinated hemocyanin, and the results strongly suggest a high degree of similarity between labelled hemocyanin and the natural molecule. 1. Gel filtration. Hemocyanins are large aggregates whose dissociation is affected by parameters such as p H a n d calcium concentration (Picket et al., 1966). All iodinated hemocyanins coeluted with native hemocyanin from a Sepharose 6B column, suggesting that iodination had not caused disaggregation of the macromolecules (see Fig. 1). 2. lmmunoprecipitation Studies. Immunoprecipitation studies with 125I-Homarus americanus hemocya-
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Fig. 1. Gel filtration of 125I-labelled Homarus americanus hemocyanin on a Sepharose 6B column. O) 125I-Homarus americanus hemocyanin radioactivity; ( 0 - - 0 ) native Homarus americanus hemocyanin by absorbance at 280 nm. Sepharose 6B was equilibrated with 50 mM Tris-HCl, 5.0 mM CaCI2, pH 7.5; and the same buffer used for elution at 10 ml/hr. Column size was 2.2 x 55 cm. Fraction size was 2.0 ml.
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Catabolism of hemocyanin in the American lobster
(a)
783
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Fig. 2. Immunological comparisons of Homarus americanus hemocyanin and Panulirus argus hemocyanin. Original protein concentrations of Homarus americanus hemocyanin and Panulirus argus hemocyanin are 9.6 mg/ml and 7.7mg/ml, respectively. Plate 2A has the anti-Homarus americanus hemocyanin serum in center well with outer wells 1 through 6 containing Homarus americanus hemo-
wells 1 through 6 containing Panulirus argus hemocyanin at the original, 1/10, 1/50, 1/100, 1/500, and buffer concentration, respectively. Plate 2C has anti-Homarus americanus hemocyanin serum in center well, with original Panulirus argus hemocyanin in 1, 3 and 5 outer wells and 1/10 original Homarus americanus hemocyanin in 2, 4 and 6 outer wells. Plate 2D has anti-Panulirus argus hemocyanin serum in center well with original Panulirus argus hemocyanin in 1, 3 and 5 outer wells and original Homarus americanus hemocyanin in 2, 4 and 6 outer wells.
cyanin at the original, 1/10, 1/100, 1/1000, 1/10,000, and buffer concentration, respectively. Plate 2B has anti-Panulirus argus hemocyanin serum in center well with outer nin showed that greater than 95~o of radioactivity was precipitated with rabbit anti-H, americanus hemocyanin serum. 3. Immunoelectrophoresis. Immunoelectrophoresis of 125I-Homarus americanus hemocyanin followed by autoradiography showed that the radioactivity was present in the same single precipitin band that native H. americanus hemocyanin produces. Immunological properties o f hemocyanin
The immunological cross-reactivity of Panulirus argus and n o m a r u s americanus hemocyanin was determined first. Antisera prepared against these hemocyanins showed only one precipitin band by double immunodiffusion against the respective antigen (Figs 2A & 2B). Both antisera showed a precipitin band at 1/100 the respective original hemocyanin concentration. The results indicate the antisera are specific. Figures 2C and 2D compare the cross-reactivity of H. americanus hemocyanin and P. argus hemocyanin against the two antisera. It may be seen that at c.B.P. 69/4B I
original hemocyanin concentrations in the outerwells, no cross-reactivity is indicated with either antiserum. The two different lobsters are members of the same section, Macrura, but are in different superfamilies. The antigenic determinant sites have apparently evolved independently since divergence from their original ancestor. Although immunological studies of Limulus polyphemus hemocyanin were not done, previous studies indicate that this hemocyanin is unique and does not give an immunological cross-reaction with other species (Ghiretti et al., 1966). An earlier study using quantitative immunoprecipitation tests specifically showed L. polyphemus hemocyanin had no cross-reactivity with Homarus americanus hemocyanin (Boyd, 1937). Clearance rates
(a) Hemocyanin Clearance in Homarus Americanus. ~25I-Homarus americanus hemocyanin shows a very slow clearance rate (Fig. 3A) from H. americanus
784
EDWARD
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those lobsters injected with older labelled hemocyanin (approx 30°,o c o m p a r e d to 20~o for animals injected one week earlier). There was no change in the slope of the second phase of the biphasic plot, thus similar half-lives were determined for all animals. No radioactivity was found in separated hemocytes. Gel filtration of h e m o l y m p h serum samples withdrawn 2, 9 and 15 hours after injection indicated that all radioactivity coeluted with H. americanus hemocyanin. Trichloroacetic acid (TCA) precipitation studies indicated that the radioactivity in the h e m o l y m p h was protein-bound t h r o u g h o u t the determinations. Lastly, immunoprecipitation tests of h e m o l y m p h samples
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Fig. 3. Clearance of ~25I-Hornarus americanus hemocyanin and 125I-Panulirus argus hemocyanin from the hemolymph of Homarus americanus. For injection and sampling procedures, see "Materials and Methods". Each curve represents an average of data from 4 animals. (A) the first sample withdrawn (15 min) was defined as 100°~ radioactivity and the data plotted as mean percentage of radioactivity. The vertical bars represent the limits of +_ l SD. (B) is a semilog plot of the same data. From the fit of a least squares line, coefficients of determination of 0.992 and 0.990 were calculated for ~2-SI-Homarus americanus hemocyanin and 125I-Panulirus argus hemocyanin clearance, respectively. hemolymph, as expected since it is the endogenous hemocyanin. At tx/2 of 25.5 days was calculated from the semilogarithmic plot of the data (Fig. 3B), using the second phase of the apparently biphasic plot. The initial phase of the biphasic plot varies between animals, a n d appeared to be related to the age of the labelled hemoeyanin injected. The total radioactivity cleared in the first 24 hr after injection was higher for
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Fig. 4. Clearance of 1251-Limulus polyphemus hemocyanin from the hemolymph of Homarus americanus. For injection and sampling procedures see "Materials and Methods." The results shown are an average of the results obtained in 4 lobsters. (A) the first sample withdrawn (5 min) was defined as 100.% radioactivity and the data plotted as mean percentage of radioactivity with _+1 SD. (B) is a semilog plot of the same data. From the fit of a least squares line, a coefficient of determination of 0.982 was calculated for the clearance study.
785
Catabolism of hemocyanin in the American lobster showed that greater than 95% of radioactivity was bound to hemocyanin. 125I-Panulirus argus hemocyanin was also cleared slowly from Homarus amer~canus hemolymph (Fig. 3A), although with a slightly faster q/2 (19.5 days) compared to that of the endogenous hemocyanin (25.5 days). The tl/2 was determined from a semilogarithmic plot of the data (Fig. 3B) using the second phase of the apparently biphasic plot for similar reasons as discussed above. Gel filtration of hemolymph serum samples withdrawn one and 10 days after injection showed that all the radioactivity coeluted with purified P. argus hemocyanin. The results of these experiments show that ~25I-P. argus hemocyanin has a similar although somewhat faster clearance rate than endogenous hemocyanin in H. americanus; and suggest that they may have similar recognition sites and clearance mechanisms. In other words, the American lobster does not clear P. argus hemocyanin from its hemolymph rapidly in spite of the fact that the two hemocyanins are immunologically non-cross-reactive, as shown earlier. In contrast, 125I-Limulus polyphemus hemocyanin was rapidly cleared (Fig. 4A) from the hemolymph of Homarus americanus. A semilogarithmic plot of the data indicated an apparent single straight line (Fig. 4B) with a calculated tt/2 of 45 min. Again, hemocytes showed no radioactivity. TCA precipitation studies throughout the clearance period indicated that all the radioactivity was protein bound. Gel filtration studies suggested the circulating radioactivity was bound to L. polyphemus hemocyanin and to slightly lower molecular weight proteins, probably due to degradation. Thus L. polyphemus hemocyanin behaves as a foreign protein in H. americanus, and is rapidly cleared from the hemolymph and taken up by other tissues. (b) Hemocyanin clearance in Panulirus Argus. The clearance rate of ~25i_Panulirus argus and 125I-Homarus americanus hemocyanin from the hemolymph of the spiny lobster, P. argus was also determined (Fig. 5). Each curve represents data from one animal, the data points being plotted as the average of triplicate determinations. The t~/2 values for P. argus and H. americanus hemocyanin are approximately 36 and 14 days, respectively, as determined by a semilogarithmic plot of the clearance data and least square analyses (not shown). Gel filtration of hemolymph serum samples at different time intervals indicated that all the radioactivity coeluted with the original radioactive hemocyanin. The relatively slow clearance rate of the ~25I-Homarius americanus hemocyanin in Panulirus argus suggests that the spiny lobster does not recognize H. americanus hemocyanin as a completely foreign protein. These results are similar to the reciprocal experiments in the previous section where both H. americanus and P. argus hemocyanins exhibited slow clearance rates in H. americanus. Although the exogenous hemocyanin in each study showed a slow clearance rate, it should be noted that the rate was significantly faster than that of the endogenous hemocyanin. The data suggest that the hemocyanins from the two species of lobsters may be similar with respect to their recognition sites for clearance, and also the clearance mechanisms of the two species may
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5 6 7 8 9 I0 Time ( doys ) Fig. 5. Clearance of 125I-Panulirus argus hemocyanin and 125I-Homarus americanus hemocyanin from the hemolymph of Panulirus argus. For injection and sampling procedures, see "Materials and Methods." Each curve represents the average data from one animal. TSe first sample withdrawn was defined as 100% radioactivity and the data plotted as mean percentage of radioactivity.
be similar. In contrast, the earlier immunodiffusion studies suggested no immunological similarity between the two lobster hemocyanins. Site of clearance of hemocyanin (a) Endogenous hemocyanin. Lobsters were killed at various time intervals after 125I-Homarus americanus hemocyanin injection, and tissues were counted for radioactivity. Fig. 6 shows the percentage of total injected radioactivity in different tissues and in tank water at various time intervals. Radioactivity w a s accounted for within 15Yo of that injected, any discrepancy most likely being due to approximation of average tissue weights. Radioactivity was found in almost all tissues, not specifically in one. Due to the open circulatory system of lobsters and the slow clearance rate of the radioactive endogenous hemocyanin (tl/2 = 25.5 days), all the tissues were bathed in radioactive hemolymph, making it difficult to detect accumulation in any one tissue. (Hydroxyl [~4C]methyl) Inulin, a non-membrane diffusible and non-metabolizable oligosaccharide (Dinda et al., 1977), was injected simultaneously with 125I-Homarus americanus hemocyanin into American lobsters. A comparison of ~25I vs ~4C uptake in tissues (compare Figs 6 and 7) should indicate whether any tissue is specifically taking up ~25I-H. americanus hemocyanin. Most tissues showed a somewhat higher accumulation of 1-125 than C-14 except for the green gland, the excretory organ. This is reasonable since our results also show that 14C-inulin is being excreted into the tank water, in agreement with others (Bryan & Ward, 1962), whereas very little 1-125 is being excreted into the tank. The percent of t25I-radioactivity in muscle appears to rise significantly at 6 and 14.5 hr (i.e. in animals killed 6 and 14.5 hr after injection) as shown in Fig. 6. However, these two lobsters were injected with
786
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Time of sacrifice (days) Fig. 6. Percentage o f total radioactivity in tissues of Homarus americanus after 125|-Hon~rus americanus
hemocyanin injection. Each data point represents the average result of triplicate samples from one lobster tissue. Although not well shown in the diagram, the percentage of total radioactivity in the green gland was 0.06, 0.18, 0.10, 0.05, 0.04, and 0.04 for sacrifice times of 3, 6, 14.5 hr, 1, 5 and 14.5 days, respectively. O O Epidermis; I I - - - I I Muscle; A A Exoskeleton; O- ..... O Gills; [] .......[] Hepatopancreas; • . . . . . . . . . . . • Green Gland; t t - - - - - O Tank Water. 1251_Homaru s americanus hemocyanin that was one week older than the rest, and the labelled hemocyanin is apparently degrading slowly during refrigeration. When the clearance rates from these animals were plotted on a semilogarithmic plot, the initial phase of the curve is much steeper than for other studies. This indicates a greater amount of radioactivity (approximately 10~o more of the total) is removed from the hemolymph in the first 24 hr of clearance before the assumed natural hemolymph clearance rate of hemo-
cyanin is established. It is unclear why the muscle should trap this radioactivity, but the uptake does not seem to be due to specific degradation of natural hemocyanin. The muscle does account for the highest percentage of total animal weight and probably the most extracellular space as well. The increase with time of radioactivity in the exoskeleton may be due to the synthesis and sclerotization of cuticle proteins. Halogcnated tyrosines have been found in arthropod cuticle (Welinder, 1972),
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Fig. 7. Percentage of total radioactivity in tissues of Homarus americanus after 14C-inulin injection. Each data point represents the average result of triplicate samples from one lobster tissue. Although not well shown in the diagram, the percentage of total radioactivity in the green gland was 0.14, 0.21, 0.19, and 0.30 for sacrifice times of 3hr, 14.5hr, 1 day, and 5 days, respectively. O O Epidermis; II-----II Muscle; A A Exoskeleton; ( ~ 0 Gills; El-- [] Hepatopancreas; • . . . . . . . . . . . • Green Gland; 0 - - ~ Tank Water.
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Fig. 8. Percentage of total radioactivity in tissues of Homarus americanus after 12SI-Limulus polyphemus hemocyanin injection. (Due to the time required for freezing, death of the lobsters occurred approximately one hour later than the reported time of sacrifice in the diagram.) Each data point represents the average result of triplicate samples from one lobster tissue. Although not shown in the diagram, the percentage of total radioactivity in either the green gland or the hepatopancreas was less than 1% at any sacrifice time and was relatively constant throughout the time study. O O Epidermis; • m Muscle; A A Exoskeleton; O - - - - - O Gills; O--------O Tank Water. where they are utilized in formation of scleroproteins. The gills (Sloan et al., 1975; McCumber & Clem, 1977) and hepatopancreas (Vonk, 1960) have both been suggested as sites of protein degradation. Studies by Sloan et al. (1975) and McCumber & Clem (1977) have pointed to the gills as the primary site of uptake of 125I-BSA in crayfish and blue crabs. However, our results suggest no specific significance of gills or hepatopancreas in the degradation of endogenous hemocyanin. Gills show a much higher radioactivity per gram tissue (both for 1-125 and C-14), but this appears to be due mainly to the gills being highly vascularized. Although the graphs of radioactivity per gram for all tissues are not shown, they were prepared and showed no significant results. No one tissue can be identified as playing the major role in the degradation of endogenous hemocyanin. When extracts of homogenized tissues taken at various times were examined by gel filtration, no trends were evident with respect to a shift in the distribution of radioactivity from high molecular weight material (undegraded hemocyanin) to low. In vitro incubations of 125I-Homarus americanus hemocyanin with fresh gill, muscle, and hepatopancreas homogenates showed no degradation of hemocyanin. Exogenous hemocyanin. Since t2SI-Limulus polyphemus hemocyanin has a fast clearance rate (tl/2 = 45min) from the hemolymph of Homarus americanus, its site of clearance in the American lobster was also studied with the results shown in Fig. 8. Radioactive L. polyphenms hemocyanin is taken up rapidly by the epidermis. The radioactivity in the epidermis of the claws, main body, and tail segment were all determined, each showing a high uptake. Radioactivity in the epidermis decreased from 85% of the total at approximately one hour after
injection to 16~o of total after 7 days, while it progressively accumulated in the exoskeleton and muscle during the same time period. Very little radioactivity was excreted into the tank water. These surprising results suggest that the epidermis is the initial site o}" the uptake of 125I-Limulus polyphemus hemocyanin from the hemolymph of the American lobster. Although the epidermis is known to be important in the metabolism of cuticle proteins (Van Weel, 1970), there are to the best of our knowledge no reports that suggest it as a site of degradation of foreign proteins. Gel filtration results with homogenized epidermis extracts at increasing time intervals (Fig. 9) indicate that the radioactivity is bound to protein of decreasing molecular weight, further implicating the epidermis as a site of degradation of the foreign hemocyanin. Radioactivity progressively accumulated in the muscle. Gel filtration (Sepharose 6B) of homogenized muscle extracts taken 7 days after injection indicated that the radioactivity is bound to low molecular weight species (<10,000 molecular weight) which were not separable on the column. Thin layer chromatography of the muscle extract and standard iodotyrosine, according to the procedure of Chau & Riley (1966), suggested that part of the radioactivity was present as free iodotyrosine. Due to the small sample volume applied and the large amount of extraneous amino acids present, the amount of 12sI-iodotyrosine can only be estimated approximately as 50~o of the total radioiodine. DISCUSSION
The clearance rates of several hemocyanins in the hemolymph of Homarus americanus and Panulirus
788
EDWARD G. SENKBEIL and JOHN C. WRISTON JR I
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Fig. 9. Gel filtration of epidermis tissue extracts at various time intervals after 1251-Limulus polyphemus hemocyanin injection. At various time intervals after ~25I-Limulus polyphemus hemocyanin injection, animals were sacrificed and epidermis tissue prepared for gel filtration on Sepharose 6B as discussed in "Materials and Methods." The Sepharose 6B column (2.2 × 55 cm) was equilibrated with 50.0mM Tris-HC1, 5.0mM CaCI2, pH 7.5; and the same buffer used for elution at 10ml/hr. Fractions (2.0ml) were collected and counted directly in an automated gamma counter. Each elution pattern represents data from one lobster. O - - - - O Sacrificed 28 min after injection; • • Sacrificed 4 hr 40 min after injection; A A Sacrificed 2 days 4 hr after injection.
argus were studied and the results are summarized in Table 1. The half-lives of the proteins were determined from semilogarithmic plots of the hemolymph clearance data. The absolute values are of less significance for our purposes than the comparisons that can be made. The results (Table 1) indicate that 125I-Homarus americanus and 125I-Panulirus argus hemocyanin have slow clearance rates from the hemolymph of either of the two lobster species. H. americanus hemocyanin has a molecular weight of approx 825,000, being composed of 12 subunits (75,000g/mol). P. argus hemocyanin has a molecular weight of 450,000, being composed of 6 subunits (75,000 g/mol). The subunits are similar in the two species with respect to amino acid composition. Based on available structural information and the fact that the two species, H. americanus and P. argus, are morphologically similar, one might expect that the hemocyanin molecules from the two species are structurally similar; however, our immunodiffusion studies showed no cross-reactivity between the two hemocyanins. The morphological similarities of the lobsters are not necessarily related
to biochemical similarities such as protein structure. Our results indicate how important it is to define exactly the meaning of similarity when comparing protein molecules. The immunological studies suggested that the two hemocyanins are dissimilar, but this experimental approach, of course, only demonstrates that the antigenic determinant sites are different. The clearance studies, on the other hand, suggest that the molecules are similar with respect to clearance recognition sites, and that the lobsters may have similar hemolymph protein clearance mechanisms. The two results are not contradictory, but simply result from two different experimental approaches to determining the similarity of the lobster hemocyanins examined. It was not possible to determine conclusively the primary tissue site of clearance of 12SI-labelled endogenous hemocyanin in Homarus americanus because the slow hemolymph clearance rate (tl/2 = 25.5 days) did not allow significant accumulation of radioactivity in any tissue. On the other hand, the catabolism of the rapidly cleared foreign
Table 1. Hemolymph clearance rates of injected proteins Injected protein 125I-Homarus americanus hemocyanin 125I-Panulirus argus hemocyanin 12SI-Limulus polyphemus hemocyanin
t~/2 in Homarus americanus Panulirus argus
25.5 days 19.5 days 45 minutes
14 days 36 days --
Catabolism of hemocyanin in the American lobster protein, 12SI-Limulus polyphemus hemocyanin, in H. americanus led to the accumulation of up to 85~o of the initial radioactivity in the epidermis. Gel filtration studies suggest the epidermis as the site of degradation of this foreign protein. Radioactivity slowly decreased with time in the epidermis and was picked up by the exoskeleton and muscle, with less than 10~o being excreted into the tank water. These results appear contradictory to the published results of the catabolism of the foreign protein, ~25I-BSA, in crayfish (Sloan et al., 1975) and blue crabs (McCumber & Clem, 1977). In both of these studies, the ~25I-BSA was rapidly cleared and approximately 60~o of the radioactivity accumulated in the gills. The blue crab excreted 60~o of the injected radioactivity into the tank water within 3 days. Reasons for these differences in results are unknown. Sloan et al. (1975) have demonstrated some specificity with respect to clearance of various foreign proteins from crayfish, and they postulate that crayfish have receptors or recognition molecules for different foreign proteins. If this is true, lobsters might have different receptor molecules and possibly different sites of degradation. The epidermis is of major importance in degradation and biosynthesis of cuticle proteins (Van Weel, 1970), especially during the molting process, although other investigations have not demonstrated a role for the epidermis with respect to foreign protein degradation. Our studies showed that this tissue has the ability to degrade the foreign protein, 125I-Limulus polyphemus hemocyanin, and that the degraded radioactive products remain in the lobster and are not excreted into the tank water within 7 days after injection. Little is known about the mechanism of protein clearance from the hemolymph of crustaceans, and what protein structural features are involved in determining whether clearance is to be slow (as with endogenous hemocyanin) or fast. The importance of the sugar moiety in the clearance of plasma glycoproteins in mammals has been well studied (for a review, see Ashwell & Morell, 1974). In preliminary studies in this laboratory, the sugar content of the three hemocyanins studied here (Homarus americanus, Panulirus argus and Limulus polyphemus) has been characterized. The lobster hemocyanins apparently contain short chains of glucosamine and mannose, with mannose at the non-reducing end of the carbohydrate moiety. Our results also indicate that L. polyphemus contains no sugars. Preliminary studies of clearance of native and glycosylated asparaginase (N-acetylglucosamine units attached) showed no significant difference in hemolymph clearance rates from H. americanus. These results suggest, but do not prove, that sugars do not have a vital function in clearance mechanisms from the hemolymph. Recent studies by Ashwell & Morgan (1979) have not shown a role for the carbohydrate moiety with respect to clearance in fish, and the authors suggest that this clearance mechanism emerged at an evolutionary stage later than that of fish. Lastly, preliminary results here also indicated that phagocytosis is not involved in the rapid clearance of a foreign protein from lobster hemolymph, since simultaneous injection of ~2SI-L. polyphemus hemocyanin and heat denatured BSA did not alter the hemolymph clearance rate of
789
the labelled hemocyanin. Further studies to elucidate the mechanism of hemolymph protein clearance are needed. Acknowledgements--We thank Dr-Lowell Sick--r his" assistance and support throughout this research project. This work was supported in part by NOAA Seagrant No. 04-6-158-44025 awarded to the Delaware Seagrant College Program. The work was taken from a Ph.D. dissertation (E.S.) presented to the Department of Chemistry, University of Delaware. REFERENCES
ASHWELLG. & MORELLA. G. (1974) Advances in Enzymology, Vol. 41, pp. 99-128. ASHWELLG. & MORGANR. P. (1979) A.C.S. Symposium Series no. 88. Carbohydrate-Protein Interaction, pp. 181-185. American Chemical Society. BovD W. C. (1937) Cross-reactivity of various hemocyanins with special reference to the blood proteins of the black widow spider. Biol. Bull. 73, 181 183. BRYAN G. W. & WARD E. (1962) Potassium metabolism and the accumulation of 137Caesiumby decapod crustacea. d. Mar. Biol. Ass. U.K. 42, 199-241. CAMPBELLD. H., GARVE+J. S., CRIMERN. E. & SUSSDORF D. H. (1964) Methods in Immunology, pp. 69-71. Benjamin, New York. CHAU Y. K. &RtLEY J. P. (1966) The determination of amino acids in seawater. Deep-Sea Res. 13, 111.%1124. CLAUSENJ. (1969) lmmunochemical Techniques for the Identification and Estimation of Macromolecules (Edited by WORK T. S. & WORK E.), pp. 423~;37. Elsevier, New York. COLE W. H. (1941) A perfusing solution for the lobster (Homarus) heart and the effects of its constituent ions on the heart. J. gen. Physiol. 25, 1 6. DINDA P. K., BECK I. T. & BECK M. (1977) Some observations on the determination of extracellular fluid volume of jejunal tissue using [3H]inulin and [14C]inulin. Can. d. Physiol. Pharmac. 55, 389-393. GHmETTIA. N., NUZZOLOC. & GHmEYrl F. (1966) Chemical studies on hemocyanins. I. Amino acid composition. Biochemistry 5, 1943-1951. LONTIE R. & VANQUICKENBORNEL. (1974) Metal Ions in Biological Systems, Vol. 3, pp. 183-200. Marcell Dekker, New York. MARCHALONISJ. J. (1969) An enzymic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113, 299 305. McCUMBER L. & CLEM L. (1977) Recognition of viruses and xenogeneic proteins by the blue crab, Callinectes sapidus. I. Clearance and organ concentration. Devel. comp. Immunology 1, 5-14. McCUMBER L., HOFFMANNE. & CLEM L. (1979) Recognition of viruses and xenogenic proteins by the blue crab, Callinectes sapidus: a humoral receptor for T2 bacteriophage, d. invert. Pathology 33, 1-9. NICKEaSOND. E. & VAN HOLDEK. E. (1971) A comparison of mulluscan and arthropod hemocyanin. 1. Circular dichroism and absorption spectra. Comp. Biochem. Physiol. 39B, 855-872. PICKETTS. M., RIGGSA. F. & LARIMERJ. L. (1966) Lobster hemocyanin: Properties of the minimum functional subunit and of aggregates. Science 151, 1005-1007. SENKBEILE. G. & WRISTONJ. C. (1980) Hemocyanin synthesis in the American lobster, Homarus americanus. Comp, Biochem. Physiol. in press. SLOAN B., YOCUM C. & CLEM L. (1975) Recognition of self and non-self in crustaceans. Nature 258, 521-523. STEWART J. E. & FOLEY D. M. (1969) A precipitin-like reaction of the hemolymph of the lobster Homarus americanus. J. Fish Res. Bd Can. 26, 1392-1397.
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EDWARD G. SENKBEILand JOHN C. WRISTON JR
VAN WEEL P. B. (1970) Chemical Zoology (Edited by FLORKIN M. & SCrmER B. T.), Vol. 5, Part A, pp. 97-115. Academic Press, New York. VAN WEEL P. B. (1974) Hepatopancreas? Comp. Biochem. Physiol. 47A, 1-9.
VONK H. J. (1960) The Physiology ~ Crustacea, Vol. L p. 291. Academic Press, New York. WEUNDER B. S. (1972) Halogenated tyrosines from the cuticle of Limulus polyphemus. Biochim. Biophy. ,4eta 279, 491 497.
0305-0491/81/080781-10502.00/0 Copyright © 1981 Pergamon Press Ltd
CATABOLISM OF HEMOCYANIN IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS EDWARD G. SENKBEIL* and JOHN C. WRISTONJR Department of Chemistry, University of Delaware, Newark, DE 19711, U.S.A. (Received 24 November 1980)
Abstract--1. Cross-reactivity studies by double immunodiffusion suggest no immunological similarity between Homarus americanus hemocyanin and Panulirus argus hemocyanin. 2. Slow hemolymph clearance rates (tin of 15-30 days) for ~25I-Homarus americanus and Panulirus argus hemocyanins were determined after injection in both species, suggesting similar structure of the two hemocyanins relative to clearance recognition sites. 3. No single tissue emerged as the site of clearance and degradation of endogenous ~25I-labelled hemocyanin in Homarus americanus, possibly due to its slow clearance rate. 4. 125I-Limulus polyphemus hemocyanin was rapidly cleared from the hemolymph of Homarus americanus (tl/2 = 45 min) with the epidermis indicated as the initial site of uptake and degradation.
INTRODUCTION A great deal is known about the structure and chemical properties of hemocyanin (for a review see Lontie & Vanquickenborne, 1974) but little about its metabolism. The hepatopancreas has been determined as the site of synthesis of hemocyanin in the American lobster (Senkbeil & Wriston, 1980). The hemolymph clearance of labelled endogenous hemocyanin in lobsters (Stewart & Foley, 1969), crayfish (Sloan et al., 1975) and blue crabs (McCumber & Clem, 1977) is slow, but no further studies on degradation were performed. McCumber & Clem (1977) demonstrated in the blue crab that 125I-labelled hemocyanin from closely related species had much slower hemolymph clearance rates than hemocyanins from more distant species. It has been theorized that hemocyanin is degraded in the hepatopancreas (Van Weel, 1974) of crustaceans primarily because that organ has a central role in all metabolic processes, similar to the liver in vertebrates, but no direct evidence for its importance in hemocyanin degradation has been given. The hemolymph clearance and degradation of foreign soluble proteins in crustaceans has been studied in more detail. Stewart & Foley (1969) demonstrated a fast hemolymph clearance of fluorescentlabelled bovine serum albumin (BSA) and suggested that nonspecific proteolytic degradation might be involved in the clearance. More recent studies (McCumber & Clem, 1977) showed rapid hemolymph clearance of ~25I-BSA from blue crabs and crayfish; since 60% of the radioactivity accumulated in the gills, this organ was proposed as the initial site of hemocyanin degradation. Specificity of clearance was demonstrated since unlabelled BSA, human serum albumin, and rabbit serum albumin retarded clearance of 125I-BSA, whereas bovine gamma globulin and keyhole limpet hemocyanin had no effect. In preliminary studies of purification and characterization * Present address: Physical Science Department, Salisbury State College, Salisbury, MD 21801, U.S.A.
of specific soluble receptors from blue crab hemolymph, McCumber et al. (1979) noted the ability of this factor to neutralize T2 bacteriphage. No studies demonstrating the ability of this factor to bind to soluble proteins were performed. Invertebrates in general have the nonspecific hemolymph defense factors of bactericidins, agglutinins, and phagocytic capacities. These defense mechanisms have been well demonstrated with foreign particles, but the relevance of these systems or the soluble factor of McCumber et al. (1979) to the understanding of soluble protein clearance and degradation is unknown. The present work was undertaken to elucidate the metabolism of hemocyanin in decapod crustaceans. lzsI-labelled hemocyanin was injected into the hemolymph of lobsters and the rate and site of clearance were studied.
MATERIALS AND METHODS Animals
American lobsters (Homarus americanus) were caught off the coast of Lewes, Delaware, and maintained in running seawater, 16°C. Spiny lobsters (Panulirus ar#us), generously donated by Dr William Herrinkind, Florida State University, were maintained in running seawater, 20°C. All experiments were conducted with intermolt lobsters maintained for at least one week in this way, and fed twice weekly on frozen clams, Mercenaria mercenaria. Injected animals were transferred to 5 gallon aquaria and maintained in aerated seawater, 17-18°C. The seawater was changed every 72 hr. Immunological techniques
Antisera were obtained by immunization of white New Zealand male rabbits by modification of the standard procedure of Campbell et al. (1964). Purified hemocyanin was dialyzed against 3.0 mM phosphate buffer, pH 7.0, containing 3.0 mM CaCI2, and 1.0 ml aliquots (10 mg protein/ml) were injected into the outer ear vein every other day for a total of 5 injections. Blood samples were withdrawn every 2 weeks and subcutaneous booster shots of hemocyanin were given every month. Blood samples were allowed to clot, then centrifuged, and the serum frozen with no further
781
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EDWARD G . SENKBEIL a n d JOHN C. WRISTON JR
purification. The serum samples were used for all immunochemical techniques. Double immunodiffusion using l"J/o Ionoagar no. 2 (Colab Laboratories, Inc.) in 0.07 M Tris, 0.01 M borate, 0.005 M CaC12, pH 8.2, with 0.02°Jo sodium azide was performed according to the methods of Clausen (1969). Immunoelectrophoresis was performed in gels consisting of 1°,o Ionoagar no. 2 in the above buffer. Gels were prepared on microscope slides and samples were run 3 hr at 2 ma/ slide in a Buchler flat-bed electrophoresis apparatus. Gels were stained for protein according to the method of Clausen (1969) with Amido black.
Preparation of hemocyanin Hemocyanin from the 3 species studied (Homarus americanus, Panulirus argus, and Limulus polyphemus) was purified by gel filtration of hemolymph serum on a Sepharose 6B column (2 x 80cm). Purified hemocyanin concentrations were determined spectrophotometrically using the extinction coefficients E~'~ at 280nm (Nickerson & Van Holde, 1971). A solid phase form of the original lactoperoxidase labelling technique (Marchalonis, 1969) was used for iodination of hemocyanin with an Enzymobead Radioiodination kit (Bio-Rad Catalog no. 170-6002) and a modified version of the procedure described in Product Information 1060, copyright 1978 Bio-Rad Laboratories. Protein and Na 1z5I (Amersham/Searle IMS 30) were added on a 1:1 molar ratio so that there would theoretically be a maximum of one label per hemocyanin molecule. Similarity of the iodinated hemocyanin to the native molecule was established by gel filtration studies, immunoprecipitation tests, and immunoelectrophoresis.
Clearance studies of labelled hemocyanin ~25I-labelled hemocyanin (1.5-3.0 microcuries) was injected into the pericardial sinus of the lobster. In clearance studies, samples of the hemolymph were withdrawn periodically from the pericardial sinus. Aliquots (5(~150/~1) were placed in 8 × 75 mm test tubes and refrigerated. Triplicate samples were taken at each time interval. Hemocytes were periodically separated from hemolymph samples (Senkbeil & Wriston, 1980). At the end of the clearance experiment, all tubes were counted directly in a Nuclear Chicago 1185 Series automated gamma counter. Integrity of circulating hemocyanin was determined by gel filtration,
trichloroacetic acid (TCA) precipitation, and immunoprecipitation studies. In other experiments lobsters were sacrificed by freezing at various time intervals after 1251-hemocyanin injection. Tissues were extracted, minced, washed three times in lobster physiological saline (Cole, 1941), blotted dry, and weighed into individual test tubes. Triplicate samples of each tissue were prepared. Aliquots of the tank water were also periodically examined for excreted radioactivity. The molecular size range of the radioactive species in the various tissues was also studied. Washed tissue samples were homogenized (in 0.05 M Tris HCI buffer, pH 7.5, containing 5.0 mM CaCI~ and 0.25 mM phenylmethylsulfonyl fluoride), centrifuged at 15,000rpm for 30rain, and the supernatants applied to either a Sephadex G-25 column (2.5 × 45cm) or a Sepharose 6B column (2.2 × 55cm). Both columns were equilibrated with 0.05 M Tris HCI buffer, pH 7.5, containing 5.0 mM CaC12.
RESULTS
Preparation of hemocyanin Hemocyanin purified from Homarus americanus, Panulirus argus, and Limulus polyphemus was iodinated by the Enzymobead-lactoperoxidase method. A typical iodination in this study resulted in 15-25°{, of total radioactive iodine being protein incorporated, with an average of less than one atom of radioiodine per hemocyanin molecule. Three methods were used to study the physical and chemical properties of iodinated hemocyanin, and the results strongly suggest a high degree of similarity between labelled hemocyanin and the natural molecule. 1. Gel filtration. Hemocyanins are large aggregates whose dissociation is affected by parameters such as p H a n d calcium concentration (Picket et al., 1966). All iodinated hemocyanins coeluted with native hemocyanin from a Sepharose 6B column, suggesting that iodination had not caused disaggregation of the macromolecules (see Fig. 1). 2. lmmunoprecipitation Studies. Immunoprecipitation studies with 125I-Homarus americanus hemocya-
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Fig. 1. Gel filtration of 125I-labelled Homarus americanus hemocyanin on a Sepharose 6B column. O) 125I-Homarus americanus hemocyanin radioactivity; ( 0 - - 0 ) native Homarus americanus hemocyanin by absorbance at 280 nm. Sepharose 6B was equilibrated with 50 mM Tris-HCl, 5.0 mM CaCI2, pH 7.5; and the same buffer used for elution at 10 ml/hr. Column size was 2.2 x 55 cm. Fraction size was 2.0 ml.
(0
Catabolism of hemocyanin in the American lobster
(a)
783
(c)
(b)
(d)
Fig. 2. Immunological comparisons of Homarus americanus hemocyanin and Panulirus argus hemocyanin. Original protein concentrations of Homarus americanus hemocyanin and Panulirus argus hemocyanin are 9.6 mg/ml and 7.7mg/ml, respectively. Plate 2A has the anti-Homarus americanus hemocyanin serum in center well with outer wells 1 through 6 containing Homarus americanus hemo-
wells 1 through 6 containing Panulirus argus hemocyanin at the original, 1/10, 1/50, 1/100, 1/500, and buffer concentration, respectively. Plate 2C has anti-Homarus americanus hemocyanin serum in center well, with original Panulirus argus hemocyanin in 1, 3 and 5 outer wells and 1/10 original Homarus americanus hemocyanin in 2, 4 and 6 outer wells. Plate 2D has anti-Panulirus argus hemocyanin serum in center well with original Panulirus argus hemocyanin in 1, 3 and 5 outer wells and original Homarus americanus hemocyanin in 2, 4 and 6 outer wells.
cyanin at the original, 1/10, 1/100, 1/1000, 1/10,000, and buffer concentration, respectively. Plate 2B has anti-Panulirus argus hemocyanin serum in center well with outer nin showed that greater than 95~o of radioactivity was precipitated with rabbit anti-H, americanus hemocyanin serum. 3. Immunoelectrophoresis. Immunoelectrophoresis of 125I-Homarus americanus hemocyanin followed by autoradiography showed that the radioactivity was present in the same single precipitin band that native H. americanus hemocyanin produces. Immunological properties o f hemocyanin
The immunological cross-reactivity of Panulirus argus and n o m a r u s americanus hemocyanin was determined first. Antisera prepared against these hemocyanins showed only one precipitin band by double immunodiffusion against the respective antigen (Figs 2A & 2B). Both antisera showed a precipitin band at 1/100 the respective original hemocyanin concentration. The results indicate the antisera are specific. Figures 2C and 2D compare the cross-reactivity of H. americanus hemocyanin and P. argus hemocyanin against the two antisera. It may be seen that at c.B.P. 69/4B I
original hemocyanin concentrations in the outerwells, no cross-reactivity is indicated with either antiserum. The two different lobsters are members of the same section, Macrura, but are in different superfamilies. The antigenic determinant sites have apparently evolved independently since divergence from their original ancestor. Although immunological studies of Limulus polyphemus hemocyanin were not done, previous studies indicate that this hemocyanin is unique and does not give an immunological cross-reaction with other species (Ghiretti et al., 1966). An earlier study using quantitative immunoprecipitation tests specifically showed L. polyphemus hemocyanin had no cross-reactivity with Homarus americanus hemocyanin (Boyd, 1937). Clearance rates
(a) Hemocyanin Clearance in Homarus Americanus. ~25I-Homarus americanus hemocyanin shows a very slow clearance rate (Fig. 3A) from H. americanus
784
EDWARD
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SENKBEIL
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those lobsters injected with older labelled hemocyanin (approx 30°,o c o m p a r e d to 20~o for animals injected one week earlier). There was no change in the slope of the second phase of the biphasic plot, thus similar half-lives were determined for all animals. No radioactivity was found in separated hemocytes. Gel filtration of h e m o l y m p h serum samples withdrawn 2, 9 and 15 hours after injection indicated that all radioactivity coeluted with H. americanus hemocyanin. Trichloroacetic acid (TCA) precipitation studies indicated that the radioactivity in the h e m o l y m p h was protein-bound t h r o u g h o u t the determinations. Lastly, immunoprecipitation tests of h e m o l y m p h samples
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Fig. 3. Clearance of ~25I-Hornarus americanus hemocyanin and 125I-Panulirus argus hemocyanin from the hemolymph of Homarus americanus. For injection and sampling procedures, see "Materials and Methods". Each curve represents an average of data from 4 animals. (A) the first sample withdrawn (15 min) was defined as 100°~ radioactivity and the data plotted as mean percentage of radioactivity. The vertical bars represent the limits of +_ l SD. (B) is a semilog plot of the same data. From the fit of a least squares line, coefficients of determination of 0.992 and 0.990 were calculated for ~2-SI-Homarus americanus hemocyanin and 125I-Panulirus argus hemocyanin clearance, respectively. hemolymph, as expected since it is the endogenous hemocyanin. At tx/2 of 25.5 days was calculated from the semilogarithmic plot of the data (Fig. 3B), using the second phase of the apparently biphasic plot. The initial phase of the biphasic plot varies between animals, a n d appeared to be related to the age of the labelled hemoeyanin injected. The total radioactivity cleared in the first 24 hr after injection was higher for
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Fig. 4. Clearance of 1251-Limulus polyphemus hemocyanin from the hemolymph of Homarus americanus. For injection and sampling procedures see "Materials and Methods." The results shown are an average of the results obtained in 4 lobsters. (A) the first sample withdrawn (5 min) was defined as 100.% radioactivity and the data plotted as mean percentage of radioactivity with _+1 SD. (B) is a semilog plot of the same data. From the fit of a least squares line, a coefficient of determination of 0.982 was calculated for the clearance study.
785
Catabolism of hemocyanin in the American lobster showed that greater than 95% of radioactivity was bound to hemocyanin. 125I-Panulirus argus hemocyanin was also cleared slowly from Homarus amer~canus hemolymph (Fig. 3A), although with a slightly faster q/2 (19.5 days) compared to that of the endogenous hemocyanin (25.5 days). The tl/2 was determined from a semilogarithmic plot of the data (Fig. 3B) using the second phase of the apparently biphasic plot for similar reasons as discussed above. Gel filtration of hemolymph serum samples withdrawn one and 10 days after injection showed that all the radioactivity coeluted with purified P. argus hemocyanin. The results of these experiments show that ~25I-P. argus hemocyanin has a similar although somewhat faster clearance rate than endogenous hemocyanin in H. americanus; and suggest that they may have similar recognition sites and clearance mechanisms. In other words, the American lobster does not clear P. argus hemocyanin from its hemolymph rapidly in spite of the fact that the two hemocyanins are immunologically non-cross-reactive, as shown earlier. In contrast, 125I-Limulus polyphemus hemocyanin was rapidly cleared (Fig. 4A) from the hemolymph of Homarus americanus. A semilogarithmic plot of the data indicated an apparent single straight line (Fig. 4B) with a calculated tt/2 of 45 min. Again, hemocytes showed no radioactivity. TCA precipitation studies throughout the clearance period indicated that all the radioactivity was protein bound. Gel filtration studies suggested the circulating radioactivity was bound to L. polyphemus hemocyanin and to slightly lower molecular weight proteins, probably due to degradation. Thus L. polyphemus hemocyanin behaves as a foreign protein in H. americanus, and is rapidly cleared from the hemolymph and taken up by other tissues. (b) Hemocyanin clearance in Panulirus Argus. The clearance rate of ~25i_Panulirus argus and 125I-Homarus americanus hemocyanin from the hemolymph of the spiny lobster, P. argus was also determined (Fig. 5). Each curve represents data from one animal, the data points being plotted as the average of triplicate determinations. The t~/2 values for P. argus and H. americanus hemocyanin are approximately 36 and 14 days, respectively, as determined by a semilogarithmic plot of the clearance data and least square analyses (not shown). Gel filtration of hemolymph serum samples at different time intervals indicated that all the radioactivity coeluted with the original radioactive hemocyanin. The relatively slow clearance rate of the ~25I-Homarius americanus hemocyanin in Panulirus argus suggests that the spiny lobster does not recognize H. americanus hemocyanin as a completely foreign protein. These results are similar to the reciprocal experiments in the previous section where both H. americanus and P. argus hemocyanins exhibited slow clearance rates in H. americanus. Although the exogenous hemocyanin in each study showed a slow clearance rate, it should be noted that the rate was significantly faster than that of the endogenous hemocyanin. The data suggest that the hemocyanins from the two species of lobsters may be similar with respect to their recognition sites for clearance, and also the clearance mechanisms of the two species may
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5 6 7 8 9 I0 Time ( doys ) Fig. 5. Clearance of 125I-Panulirus argus hemocyanin and 125I-Homarus americanus hemocyanin from the hemolymph of Panulirus argus. For injection and sampling procedures, see "Materials and Methods." Each curve represents the average data from one animal. TSe first sample withdrawn was defined as 100% radioactivity and the data plotted as mean percentage of radioactivity.
be similar. In contrast, the earlier immunodiffusion studies suggested no immunological similarity between the two lobster hemocyanins. Site of clearance of hemocyanin (a) Endogenous hemocyanin. Lobsters were killed at various time intervals after 125I-Homarus americanus hemocyanin injection, and tissues were counted for radioactivity. Fig. 6 shows the percentage of total injected radioactivity in different tissues and in tank water at various time intervals. Radioactivity w a s accounted for within 15Yo of that injected, any discrepancy most likely being due to approximation of average tissue weights. Radioactivity was found in almost all tissues, not specifically in one. Due to the open circulatory system of lobsters and the slow clearance rate of the radioactive endogenous hemocyanin (tl/2 = 25.5 days), all the tissues were bathed in radioactive hemolymph, making it difficult to detect accumulation in any one tissue. (Hydroxyl [~4C]methyl) Inulin, a non-membrane diffusible and non-metabolizable oligosaccharide (Dinda et al., 1977), was injected simultaneously with 125I-Homarus americanus hemocyanin into American lobsters. A comparison of ~25I vs ~4C uptake in tissues (compare Figs 6 and 7) should indicate whether any tissue is specifically taking up ~25I-H. americanus hemocyanin. Most tissues showed a somewhat higher accumulation of 1-125 than C-14 except for the green gland, the excretory organ. This is reasonable since our results also show that 14C-inulin is being excreted into the tank water, in agreement with others (Bryan & Ward, 1962), whereas very little 1-125 is being excreted into the tank. The percent of t25I-radioactivity in muscle appears to rise significantly at 6 and 14.5 hr (i.e. in animals killed 6 and 14.5 hr after injection) as shown in Fig. 6. However, these two lobsters were injected with
786
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Time of sacrifice (days) Fig. 6. Percentage o f total radioactivity in tissues of Homarus americanus after 125|-Hon~rus americanus
hemocyanin injection. Each data point represents the average result of triplicate samples from one lobster tissue. Although not well shown in the diagram, the percentage of total radioactivity in the green gland was 0.06, 0.18, 0.10, 0.05, 0.04, and 0.04 for sacrifice times of 3, 6, 14.5 hr, 1, 5 and 14.5 days, respectively. O O Epidermis; I I - - - I I Muscle; A A Exoskeleton; O- ..... O Gills; [] .......[] Hepatopancreas; • . . . . . . . . . . . • Green Gland; t t - - - - - O Tank Water. 1251_Homaru s americanus hemocyanin that was one week older than the rest, and the labelled hemocyanin is apparently degrading slowly during refrigeration. When the clearance rates from these animals were plotted on a semilogarithmic plot, the initial phase of the curve is much steeper than for other studies. This indicates a greater amount of radioactivity (approximately 10~o more of the total) is removed from the hemolymph in the first 24 hr of clearance before the assumed natural hemolymph clearance rate of hemo-
cyanin is established. It is unclear why the muscle should trap this radioactivity, but the uptake does not seem to be due to specific degradation of natural hemocyanin. The muscle does account for the highest percentage of total animal weight and probably the most extracellular space as well. The increase with time of radioactivity in the exoskeleton may be due to the synthesis and sclerotization of cuticle proteins. Halogcnated tyrosines have been found in arthropod cuticle (Welinder, 1972),
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Fig. 7. Percentage of total radioactivity in tissues of Homarus americanus after 14C-inulin injection. Each data point represents the average result of triplicate samples from one lobster tissue. Although not well shown in the diagram, the percentage of total radioactivity in the green gland was 0.14, 0.21, 0.19, and 0.30 for sacrifice times of 3hr, 14.5hr, 1 day, and 5 days, respectively. O O Epidermis; II-----II Muscle; A A Exoskeleton; ( ~ 0 Gills; El-- [] Hepatopancreas; • . . . . . . . . . . . • Green Gland; 0 - - ~ Tank Water.
787
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Fig. 8. Percentage of total radioactivity in tissues of Homarus americanus after 12SI-Limulus polyphemus hemocyanin injection. (Due to the time required for freezing, death of the lobsters occurred approximately one hour later than the reported time of sacrifice in the diagram.) Each data point represents the average result of triplicate samples from one lobster tissue. Although not shown in the diagram, the percentage of total radioactivity in either the green gland or the hepatopancreas was less than 1% at any sacrifice time and was relatively constant throughout the time study. O O Epidermis; • m Muscle; A A Exoskeleton; O - - - - - O Gills; O--------O Tank Water. where they are utilized in formation of scleroproteins. The gills (Sloan et al., 1975; McCumber & Clem, 1977) and hepatopancreas (Vonk, 1960) have both been suggested as sites of protein degradation. Studies by Sloan et al. (1975) and McCumber & Clem (1977) have pointed to the gills as the primary site of uptake of 125I-BSA in crayfish and blue crabs. However, our results suggest no specific significance of gills or hepatopancreas in the degradation of endogenous hemocyanin. Gills show a much higher radioactivity per gram tissue (both for 1-125 and C-14), but this appears to be due mainly to the gills being highly vascularized. Although the graphs of radioactivity per gram for all tissues are not shown, they were prepared and showed no significant results. No one tissue can be identified as playing the major role in the degradation of endogenous hemocyanin. When extracts of homogenized tissues taken at various times were examined by gel filtration, no trends were evident with respect to a shift in the distribution of radioactivity from high molecular weight material (undegraded hemocyanin) to low. In vitro incubations of 125I-Homarus americanus hemocyanin with fresh gill, muscle, and hepatopancreas homogenates showed no degradation of hemocyanin. Exogenous hemocyanin. Since t2SI-Limulus polyphemus hemocyanin has a fast clearance rate (tl/2 = 45min) from the hemolymph of Homarus americanus, its site of clearance in the American lobster was also studied with the results shown in Fig. 8. Radioactive L. polyphenms hemocyanin is taken up rapidly by the epidermis. The radioactivity in the epidermis of the claws, main body, and tail segment were all determined, each showing a high uptake. Radioactivity in the epidermis decreased from 85% of the total at approximately one hour after
injection to 16~o of total after 7 days, while it progressively accumulated in the exoskeleton and muscle during the same time period. Very little radioactivity was excreted into the tank water. These surprising results suggest that the epidermis is the initial site o}" the uptake of 125I-Limulus polyphemus hemocyanin from the hemolymph of the American lobster. Although the epidermis is known to be important in the metabolism of cuticle proteins (Van Weel, 1970), there are to the best of our knowledge no reports that suggest it as a site of degradation of foreign proteins. Gel filtration results with homogenized epidermis extracts at increasing time intervals (Fig. 9) indicate that the radioactivity is bound to protein of decreasing molecular weight, further implicating the epidermis as a site of degradation of the foreign hemocyanin. Radioactivity progressively accumulated in the muscle. Gel filtration (Sepharose 6B) of homogenized muscle extracts taken 7 days after injection indicated that the radioactivity is bound to low molecular weight species (<10,000 molecular weight) which were not separable on the column. Thin layer chromatography of the muscle extract and standard iodotyrosine, according to the procedure of Chau & Riley (1966), suggested that part of the radioactivity was present as free iodotyrosine. Due to the small sample volume applied and the large amount of extraneous amino acids present, the amount of 12sI-iodotyrosine can only be estimated approximately as 50~o of the total radioiodine. DISCUSSION
The clearance rates of several hemocyanins in the hemolymph of Homarus americanus and Panulirus
788
EDWARD G. SENKBEIL and JOHN C. WRISTON JR I
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Fig. 9. Gel filtration of epidermis tissue extracts at various time intervals after 1251-Limulus polyphemus hemocyanin injection. At various time intervals after ~25I-Limulus polyphemus hemocyanin injection, animals were sacrificed and epidermis tissue prepared for gel filtration on Sepharose 6B as discussed in "Materials and Methods." The Sepharose 6B column (2.2 × 55 cm) was equilibrated with 50.0mM Tris-HC1, 5.0mM CaCI2, pH 7.5; and the same buffer used for elution at 10ml/hr. Fractions (2.0ml) were collected and counted directly in an automated gamma counter. Each elution pattern represents data from one lobster. O - - - - O Sacrificed 28 min after injection; • • Sacrificed 4 hr 40 min after injection; A A Sacrificed 2 days 4 hr after injection.
argus were studied and the results are summarized in Table 1. The half-lives of the proteins were determined from semilogarithmic plots of the hemolymph clearance data. The absolute values are of less significance for our purposes than the comparisons that can be made. The results (Table 1) indicate that 125I-Homarus americanus and 125I-Panulirus argus hemocyanin have slow clearance rates from the hemolymph of either of the two lobster species. H. americanus hemocyanin has a molecular weight of approx 825,000, being composed of 12 subunits (75,000g/mol). P. argus hemocyanin has a molecular weight of 450,000, being composed of 6 subunits (75,000 g/mol). The subunits are similar in the two species with respect to amino acid composition. Based on available structural information and the fact that the two species, H. americanus and P. argus, are morphologically similar, one might expect that the hemocyanin molecules from the two species are structurally similar; however, our immunodiffusion studies showed no cross-reactivity between the two hemocyanins. The morphological similarities of the lobsters are not necessarily related
to biochemical similarities such as protein structure. Our results indicate how important it is to define exactly the meaning of similarity when comparing protein molecules. The immunological studies suggested that the two hemocyanins are dissimilar, but this experimental approach, of course, only demonstrates that the antigenic determinant sites are different. The clearance studies, on the other hand, suggest that the molecules are similar with respect to clearance recognition sites, and that the lobsters may have similar hemolymph protein clearance mechanisms. The two results are not contradictory, but simply result from two different experimental approaches to determining the similarity of the lobster hemocyanins examined. It was not possible to determine conclusively the primary tissue site of clearance of 12SI-labelled endogenous hemocyanin in Homarus americanus because the slow hemolymph clearance rate (tl/2 = 25.5 days) did not allow significant accumulation of radioactivity in any tissue. On the other hand, the catabolism of the rapidly cleared foreign
Table 1. Hemolymph clearance rates of injected proteins Injected protein 125I-Homarus americanus hemocyanin 125I-Panulirus argus hemocyanin 12SI-Limulus polyphemus hemocyanin
t~/2 in Homarus americanus Panulirus argus
25.5 days 19.5 days 45 minutes
14 days 36 days --
Catabolism of hemocyanin in the American lobster protein, 12SI-Limulus polyphemus hemocyanin, in H. americanus led to the accumulation of up to 85~o of the initial radioactivity in the epidermis. Gel filtration studies suggest the epidermis as the site of degradation of this foreign protein. Radioactivity slowly decreased with time in the epidermis and was picked up by the exoskeleton and muscle, with less than 10~o being excreted into the tank water. These results appear contradictory to the published results of the catabolism of the foreign protein, ~25I-BSA, in crayfish (Sloan et al., 1975) and blue crabs (McCumber & Clem, 1977). In both of these studies, the ~25I-BSA was rapidly cleared and approximately 60~o of the radioactivity accumulated in the gills. The blue crab excreted 60~o of the injected radioactivity into the tank water within 3 days. Reasons for these differences in results are unknown. Sloan et al. (1975) have demonstrated some specificity with respect to clearance of various foreign proteins from crayfish, and they postulate that crayfish have receptors or recognition molecules for different foreign proteins. If this is true, lobsters might have different receptor molecules and possibly different sites of degradation. The epidermis is of major importance in degradation and biosynthesis of cuticle proteins (Van Weel, 1970), especially during the molting process, although other investigations have not demonstrated a role for the epidermis with respect to foreign protein degradation. Our studies showed that this tissue has the ability to degrade the foreign protein, 125I-Limulus polyphemus hemocyanin, and that the degraded radioactive products remain in the lobster and are not excreted into the tank water within 7 days after injection. Little is known about the mechanism of protein clearance from the hemolymph of crustaceans, and what protein structural features are involved in determining whether clearance is to be slow (as with endogenous hemocyanin) or fast. The importance of the sugar moiety in the clearance of plasma glycoproteins in mammals has been well studied (for a review, see Ashwell & Morell, 1974). In preliminary studies in this laboratory, the sugar content of the three hemocyanins studied here (Homarus americanus, Panulirus argus and Limulus polyphemus) has been characterized. The lobster hemocyanins apparently contain short chains of glucosamine and mannose, with mannose at the non-reducing end of the carbohydrate moiety. Our results also indicate that L. polyphemus contains no sugars. Preliminary studies of clearance of native and glycosylated asparaginase (N-acetylglucosamine units attached) showed no significant difference in hemolymph clearance rates from H. americanus. These results suggest, but do not prove, that sugars do not have a vital function in clearance mechanisms from the hemolymph. Recent studies by Ashwell & Morgan (1979) have not shown a role for the carbohydrate moiety with respect to clearance in fish, and the authors suggest that this clearance mechanism emerged at an evolutionary stage later than that of fish. Lastly, preliminary results here also indicated that phagocytosis is not involved in the rapid clearance of a foreign protein from lobster hemolymph, since simultaneous injection of ~2SI-L. polyphemus hemocyanin and heat denatured BSA did not alter the hemolymph clearance rate of
789
the labelled hemocyanin. Further studies to elucidate the mechanism of hemolymph protein clearance are needed. Acknowledgements--We thank Dr-Lowell Sick--r his" assistance and support throughout this research project. This work was supported in part by NOAA Seagrant No. 04-6-158-44025 awarded to the Delaware Seagrant College Program. The work was taken from a Ph.D. dissertation (E.S.) presented to the Department of Chemistry, University of Delaware. REFERENCES
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EDWARD G. SENKBEILand JOHN C. WRISTON JR
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VONK H. J. (1960) The Physiology ~ Crustacea, Vol. L p. 291. Academic Press, New York. WEUNDER B. S. (1972) Halogenated tyrosines from the cuticle of Limulus polyphemus. Biochim. Biophy. ,4eta 279, 491 497.