Nora B. Terwilliger

SDS-induced Phenoloxidase Activity of Hemocyanins fromLimulus polyphemus, Eurypelma californicum, andCancer magister

Phenoloxidase, widely distributed among animals, plants, and fungi, is involved in many biologically essential functions including sclerotization and host defense. In chelicerates, the oxygen carrier hemocyanin seems to function as the phenoloxidase. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab Limulus polyphemus and the tarantulaEurypelma californicum, exhibitO-diphenoloxidase activity induced by submicellar concentrations of SDS, a reagent frequently used to identify phenoloxidase activity. The enzymatic activity seems to be restricted to only a few of the heterogeneous subunits. These active subunit types share similar topological positions in the quaternary structures as linkers of the two tightly connected 2 × 6-mers. Because no other phenoloxidase activity was found in the hemolymph of these animals, their hemocyanins may act as a phenoloxidase and thus be involved in the primary immune response and sclerotization of the cuticle. In contrast, hemolymph of a more recent arthropod, the crab Cancer magister, contains both hemocyanin with weak phenoloxidase activity and another hemolymph protein with relatively strong phenoloxidase activity. The chelicerate hemocyanin subunits showing phenoloxidase activity may have evolved into a separate phenoloxidase in crustaceans.

The effect of organic acids on oxygen binding of hemocyanin from the crab Cancer magister

Abstract 1. 1. l -lactate raises the oxygen affinity of both stripped and unstripped hemocyanin (Hc) of Cancer magister . The effect of lactate is independent of pH in the range 6.8-8.3. 2. 2. d -lactate, glycolate and 2-methyl lactate also increase the Hc oxygen affinity, but to a lesser extent than l -lactate. Other structural analogues such as acetate, alanine, α-hydroxybutyrate, pyruvate and propionate have no effect on oxygen binding. 3. 3. These results suggest that C. magister Hc has an allosteric site with high specificity for L-lactate. 4. 4. The amount of lactate and H + in the blood of an exercised C. magister explains the change in Hc oxygen affinity brought about by high levels of locomotor activity. 5. 5. However, the amount of lactate in the blood of a resting crab does not explain the difference in Hc oxygen affinity between stripped and unstripped hemocyanin. 6. 6. End-products of anaerobic metabolism found in other taxa do not affect C. magister Hc oxygen binding. 7. 7. Thus, selection for an oxygen carrier with properties that respond to a particular form of anaerobic metabolism appears to have occurred. 8. 8. These findings confirm an earlier suggestion that an unidentified, low molecular weight factor is also present in crab hemolymph. 9. 9. The respiratory importance of the lactate effect following maximal locomotor activity is its enhancement of O 2 uptake at the gill rather than conservation of venous reserve at the tissues.

Functional and Phylogenetic Analyses of Phenoloxidases from Brachyuran (Cancer magister) and Branchiopod (Artemia franciscana, Triops longicaudatus) Crustaceans

Arthropod phenoloxidases catalyze the melanization and sclerotization of the new postmolt exoskeleton, and they function in the immune response. Hemocyanin, phylogenetically related to phenoloxidase, can function as a phenoloxidase under certain conditions. We investigated the relative contributions of hemocyte phenoloxidase and hemocyanin in the brachyuran crab Cancer magister, using the physiological ratio at which they occur in the hemolymph, and found that hemocyte phenoloxidase has higher activity. They both convert diphenols to o-quinones, but only the hemocyte phenoloxidase is able to catalyze the conversion of monophenols to diphenols. The quaternary structure of hemocyanin affects its reactivity as phenoloxidase. We suggest that prophenoloxidase is released from hemocytes and moves across epidermis into new exoskeleton during premolt and is activated in early postmolt. In addition to functional studies, we have determined the complete cDNA sequence of C. magister hemocyte prophenoloxidase and partial sequences from the branchiopods Artemia franciscana and Triops longicaudatus. We also sequenced C. magister cryptocyanin 2 and a hemocyanin from the amphipod Cyamus scammoni and used these and other members of the arthropod hemocyanin superfamily for phylogenetic analyses. The phylogenies presented here are consistent with the possibility that a common ancestral molecule had both phenoloxidase and reversible oxygen-binding capabilities.

Microarray-detected changes in gene expression in gills of green crabs (Carcinus maenas) upon dilution of environmental salinity

The interaction between environmental salinity and gene expression was studied in gills of the euryhaline green shore crab Carcinus maenas. A 4462-feature oligonucleotide microarray was used to analyze changes in transcript abundance in posterior ion-transporting gills at 8 time periods following transfer of animals from 32 to 10 or 15 ppt salinity. Transcripts encoding Na(+)/K(+)-ATPase α-subunit and cytoplasmic carbonic anhydrase were upregulated with significant changes between 6 and 24h post-transfer. Other transport proteins showing similar transcriptional upregulation were an organic cation transporter, a sodium/glucose cotransporter, an endomembrane protein associated with regulating plasma membrane protein composition, and a voltage-gated calcium channel. Transport proteins showing little transcriptional response included Na(+)/H(+) exchanger, Na(+)/K(+)/2Cl(-) cotransporter, and V-type H(+)-ATPase B subunit, all of which have been implicated in osmoregulatory ion transport across crustacean gill. Interestingly, there was little affect of salinity dilution on transcriptional expression of stress proteins, suggesting that salinity acclimation is part of normal physiology for C. maenas. Expression of transcripts encoding a variety of mitochondrial proteins was significantly upregulated between 4 days and 7 days post-transfer, consistent with the proliferation of mitochondria-rich cells previously observed at this time.

The quaternary structure of a molluscan (Helisoma trivolvis) extracellular hemoglobin.

The hemoglobin (erythrocruorin) of the planorbid mollusc Helisoma trivolvis has a molecular weight of 1.7-10(6) and a sedimentation coefficient (s0 20, w) of 33.8 S at pH 7.0. At pH 2.0, the pigment consists of 32 S and 13 S material. The hemoglobin exists as a 350 000 molecular weight submultiple in 6 M guanidine and can be further dissociated into a 175-200 000 dalton polypeptide in 6M guanidine, 0.1 M 2-mercaptoethanol or by sodium dodecyl sulfate gel electrophoresis of globin, performic acid oxidized globin or carboxymethylated globin. Electron microscope observations show a ten-membered ring structure measuring 200 A in diameter. It is proposed that Helisoma hemoglobin consists of a 1.7-10(6) dalton circular assembly of ten 175-200 000 dalton polypeptide chains. The amino acid composition of the pigment is reported. The hemoglobin contains one heme per 18-19 000 g protein. Limited proteolysis of the intact pigment shows 60 000, 40 000 and 17 000-18 500 dalton components when analyzed by sodium dodecylsulfate gel electrophoresis. It is likely that the 175-200 000 dalton polypeptide consists of a linear arrangement of 8-12 heme-containing domains, each domain having a molecular weight of 18-19 000.

Thermal Vent Clam (Calyptogena magnifica) Hemoglobin

A heterodont bivalve mollusk Calyptogena magnifica, from the East Pacific Rise and the Gal�pagos Rift hydrothermal vent areas, contains abundant hemoglobin in circulating erythrocytes. No other known heterodont clam contains a circulating intracellular hemoglobin. The hemoglobin is tetrameric and has a relatively high oxygen affinity, which varies only slightly between 2� and 10�C. The presence of hemoglobin in the clam may facilitate the transport of oxygen to be used in chemoautotrophic hydrogen sulfide metabolism.

Evolution of novel functions: cryptocyanin helps build new exoskeleton in Cancer magister.

SUMMARY Hemocyanin, the blue blood protein of many arthropods and molluscs, reversibly binds oxygen at its highly conserved copper–oxygen-binding sites and supplies tissues with oxygen. Cryptocyanin, closely related structurally and phylogenetically to arthropod hemocyanin, lacks several of the six critical copper-binding histidines, however, and has lost the ability to bind oxygen. Despite this loss of function, cryptocyanin continues to be synthesized, an indication that it has been exploited to carry out new functions. Here, we show that cryptocyanin is present in extremely high concentrations in the hemolymph of the crab during the premolt portion of the molt cycle. Both proteins are specifically expressed in the same type of cell in the hepatopancreas and secreted into the hemolymph, but cryptocyanin plays a major role in forming the new exoskeleton, while hemocyanin functions in oxygen transport. A cessation in cryptocyanin, but not hemocyanin, synthesis after eyestalk ablation supports our hypothesis that cryptocyanin is closely regulated by molting hormones. The contrasts between the two gene products illustrate how a gene duplication of a copper–oxygen protein and its subsequent mutation may work in concert with the evolution of new regulatory mechanisms, leading to the assumption of new functions.

Developmental changes in oxygen uptake in Cancer magister (Dana) in response to changes in salinity and temperature

Abstract The various life stages of the Dungeness crab, Cancer magister , may experience broad environmental fluctuations in both salinity and temperature, parameters that can affect nearly all aspects of their physiological function. The routine rates of oxygen uptake of megalopa, first and fifth instar juvenile and adult C. magister were measured, using closed vessel respirometry over 8 h during acute exposure to 100, 75 and 50% seawater at 10 and 20°C. At 10°C there is no significant effect of salinity on the rate of oxygen uptake of the megalopa. At 20°C, however, the rate of oxygen uptake rises and is greater at 75 and 50% SW than in 100% SW. The rates of oxygen uptake of the first instar juvenile, fifth instar juvenile and adult are not affected by salinity at either 10 or 20°C. The oxygen uptake of the fifth instar juvenile is less temperature sensitive, at all salinities, than the other stages examined. Weight specific cardiac output is affected by both salinity and temperature. The effect of temperature on cardiac output is especially pronounced for the first instar juvenile. The first instar juvenile is the first benthic stage and therefore is an important step in recruitment of crabs in the estuary. The differences in metabolic response between the stages indicate that the first instar juvenile, in particular, may be very near the limit of its respiratory and circulatory capacities as a result of tidal changes in salinity and temperature in the intertidal estuarine habitat.

The structure of hemoglobin from an unusual deep sea worm (Vestimentifera)

Abstract 1. 1. The extracellular hemoglobin from a species of Vestimentifera, obtained from hydrothermal vents at 2.5 km depths of the ocean, has been studied. 2. 2. The hemoglobin molecules appear as two-tiered hexagonal structures in the electron microscope. The hexagons measure 160 A high and 245 A wide. 3. 3. The pigment, which was purified from frozen specimens, appears to be very unstable and elutes on Sepharose 4B as aggregates of approx 1.7 × 10 6 and 400,000 mol. wt. 4. 4. The subunit molecular weight of purified hemoglobin, determined by sodium dodecyl sulfate gel electrophoresis, shows heterogeneity with molecular weights around 15,000 and 30,000. 5. 5. The purified hemoglobin contains one heme per 23,000 g protein. The amino acid compositions of isolated fractions are presented. 6. 6. This Vestimentifera, which shows characteristics of both the phylum Pogonophora as well as Annelida, has an extracellular hemoglobin whose structure is very similar to that of annelid extracellular hemoglobins.

Comparison of chlorocruorin and annelid hemoglobin quaternary structures

Abstract 1. 1. The structures of Eudistylia vancouveri chlorocruorin and Pista pacifica and Thelepus crispus extracellular hemoglobins have been examined by electron microscopy. The intact pigments display a two-tiered hexagonal array characteristic of other annelid high molecular weight hemoglobins and chlorocruorins. 2. 2. The smallest subunit molecular weight of these pigments determined by SDS gel electrophoresis is 14–15,000; Pista hemoglobin contains some additional 30,000 molecular weight material under a variety of denaturing conditions. Earthworm hemoglobin is also heterogenous upon SDS gel electrophoresis, consisting of at least six components with mol. wt ranging from 13, 000 to 35,000. 3. 3. The amino acid compositions of these high molecular weight pigments are compared with one another and with the intracellular hemoglobins of other invertebrates and vertebrates. It is likely that Eudistylia chlorocruorin and certain high molecular weight annelid hemoglobins form a group of closely related proteins.