The molecular heterogeneity of hemocyanin: Structural and functional properties of the 4x6-meric protein of Upogebia pusilla (Crustacea).

The structural properties of the hemocyanin isolated from the Mediterranean mud shrimp, Upogebia pusilla (Decapoda: Thalassinidea), were investigated. Our intent was to make use of the U. pusilla case to perform a structural comparison between crustacean and chelicerate 4x6-meric hemocyanins. The thalassinidean hemocyanin appears similar in size but different in structural organization compared to the chelicerate 4x6-mer. Ultracentrifuge analyses on the purified protein revealed a sedimentation coefficient of 39S, typical of 4x6 hemocyanins. Electron micrographs are in agreement with a model in which four 2x6-meric building blocks are arranged in a tetrahedron-like quaternary structure and not in the quasi-square-planar orientation characteristic of the chelicerate protein. Size-exclusion chromatography-fast protein chromatography analysis showed elevated instability of the protein in absence of divalent ions or at pH values higher than 8.0. This analysis also shows that the dissociation of the U. pusilla 4x6-meric hemocyanin into hexamers occurs without any intermediate 2x6-meric state, in contrast with the dissociation profile of the chelicerate protein exhibiting several dissociation intermediates. The oxygen-binding properties of U. pusilla hemocyanin were studied to disclose possible effects by the typical allosteric effectors that modulate the functional properties of crustacean hemocyanin. A marked Bohr and lactate effect, but no significant influence of urate, on the oxygen affinity of U. pusilla hemocyanin were found.

Prepartum antibiotic treatment of heifers: milk production, milk quality and economic benefit.

Prepartum intramammary antibiotic infusion of heifer mammary glands at 7 or 14 d before expected parturition is an effective procedure for eliminating many infections in heifers during late gestation and for reducing the prevalence of mastitis in heifers during early lactation and throughout lactation. Mastitis pathogens were isolated from 76% of samples obtained from untreated control quarters 7 d before expected calving, from 47% of samples obtained 3 d after calving, and from 29% of samples obtained 10 d postpartum. Mastitis pathogens were isolated from about 30% of control quarters through 240 d of lactation. A similar percentage of samples (70%) was positive for mastitis pathogens at C-7 before antibiotic treatment. However, only 8% of samples obtained at 3 d after calving and 4% of samples obtained at 10 d postpartum from quarters of antibiotic-treated heifers contained mastitis pathogens. Throughout the remainder of lactation, mastitis pathogens were isolated from an average of about 11% of quarters. The percentage of samples with mastitis pathogens was higher in untreated controls than in antibiotic-treated quarters at all sampling intervals during lactation. A similar response was observed in heifers that were treated with antibiotics at 14 d before expected parturition. Prepartum antibiotic-treated heifers produced significantly more milk than control heifers and had significantly lower somatic cell count scores than untreated control heifers. These observations are likely associated with or due to the lower prevalence of mastitis pathogen isolation in prepartum antibiotic-treated heifers throughout lactation. Prepartum antibiotic-treated heifers produced 531 kg more milk than heifers in the untreated control group. Multiplying this increase by a milk price of 0.407 dollars/kg yielded a 216.24 dollars per-heifer increase in gross revenue. The cost of treatment, including the cost of testing for antibiotic residues, was estimated at 15.60 dollars for a net revenue of 200.64 dollars per heifer. Prepartum antibiotic treatment to reduce the rate of mastitis in heifers during lactation was highly effective and economically beneficial.

Two types of urate binding sites on hemocyanin from the crayfish Astacus leptodactylus: an ITC study.

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and L-lactate. We investigated the binding characteristics of urate and the related compound caffeine to the 2 x 6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions employing isothermal titration calorimetry (ITC). An analysis of urate and caffeine binding based on a model of n identical binding sites resulted in approximately four binding sites for caffeine and eight for urate. This result suggests that the binding process for these effectors is more complex than this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. Based on these experiments, at least two different types of binding sites for urate and caffeine exist on the 2 x 6-meric hemocyanin of A. leptodactylus. The two binding sites differ strongly in their specificity towards the two analogues. It can be hypothesized that two different subunit types (beta and gamma) are responsible for the two types of binding sites.

SDS-induced phenoloxidase activity of hemocyanins from Limulus polyphemus, Eurypelma californicum, and Cancer 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 tarantula Eurypelma californicum, exhibit O-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 x 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.

Spider hemocyanin binds ecdysone and 20-OH-ecdysone.

Fluorescence quenching studies and binding experiments with [(3)H]ecdysone reveal that the respiratory protein, hemocyanin, of the tarantula Eurypelma californicum binds ecdysone. The binding constant for ecdysone ranges between 0.5 and 5 mM, indicating a low affinity binding. However, it is comparable with those found for the ecdysone binding to hexamerins from insects. Based on a comparison of sequences and x-ray structures of arthropodan hemocyanins, we propose an evolutionary conserved hydrophobic pocket in domain 1 of the hemocyanin subunit that may bind ecdysone.

Identification, structure, and properties of hemocyanins from Diplopod myriapoda.

Hemocyanins are copper-containing, respiratory proteins that occur in the hemolymph of many arthropod species. Here we report for the first time the presence of hemocyanins in the diplopod Myriapoda, demonstrating that these proteins are more widespread among the Arthropoda than previously thought. The hemocyanin of Spirostreptus sp. (Diplopoda: Spirostreptidae) is composed of two immunologically distinct subunits in the 75-kDa range that are most likely arranged in a 36-mer (6 x 6) native molecule. It has a high oxygen affinity (P(50) = 4.7 torr) but low cooperativity (h = 1.3 +/- 0.2). Spirostreptus hemocyanin is structurally similar to the single known hemocyanin from the myriapod taxon, Scutigera coleoptrata (Chilopoda), indicating a rather conservative architecture of the myriapod hemocyanins. Western blotting demonstrates shared epitopes of Spirostreptus hemocyanin with both chelicerate and crustacean hemocyanins, confirming its identity as an arthropod hemocyanin.