[Blue blood: structure and evolution of hemocyanin]. / Blaues Blut: Struktur und Evolution der Hämocyanine.

Hemocyanins are the oxygen-transporting proteins in arthropods and molluscs, the oxygen is bound by two copper atoms. Spectroscopic studies on the active site show similarities to the active site of a further group of copper-containing proteins, the tyrosinases. Arthropodan and molluscan hemocyanins form high-molecular aggregates which are markedly different in size and quaternary structure. There is only one tertiary structure of an arthropodan hemocyanin available, but from comparison of all amino acid sequences known so far from arthropodan hemocyanins, a common tertiary structure for all arthropodan hemocyanins can be deduced. Again, sequence comparison allows the construction of an evolutionary tree for some oxygen-binding copper proteins.

Hemocyanins in spiders. XXII. Range of allosteric interaction in a four-hexamer hemocyanin. Co-operativity and Bohr effect in dissociation intermediates.

The range of allosteric interaction in the 24-meric hemocyanin from the tarantula Eurypelma californicum was studied by measuring the oxygen-binding properties of defined oligomeric fragments. Dissociation intermediates comprising 19, 12, 7 or 6 subunits were obtained by incubation of native hemocyanin with 10 mM-cysteine at pH 4.4, with 40 mM-dithiothreitol at pH 7 or 8, by short-term alkaline (pH 9.6) treatment or by treatment with 4 M-urea. These could be stabilized by returning to neutral buffer conditions and, in the case of the dodecamer, by carboxymethylation. Conditions were chosen so that part of the starting material remained intact to serve as control in the oxygen-binding measurements. Oxygen equilibrium curves were obtained by a very sensitive fluorimetric/polarographic method. Oxygen affinity and the magnitude of the Bohr effect remain constant from the hexamer up to the control four-hexamer. Co-operativity, in contrast, increases with aggregate size in two steps: n (hexamer) = n (heptamer) less than n (dodecamer) = n (19-mer) less than n (4-hexamer). The characteristic pH-dependence of nH is absent in the hexa- and heptamer but is weakly indicated in the dodecamer, and fully established in the four-hexamer. Results from different preparations are highly consistent, if nH is expressed as a percentage of the control values. Full co-operativity is reached only in the four-hexamer, disproving the dodecameric half-molecule (the smallest repeating unit) as the allosteric unit. The stepwise increase in co-operativity appears to be correlated with higher levels of symmetry in the hierarchy of quaternary structure.

Complete amino-acid sequence of a functional unit from a molluscan hemocyanin (Helix pomatia).

From the beta c-hemocyanin (beta c-Hc) of the vineyard snail, Helix pomatia, the functional unit d (Mr approximately equal to 50,000-55,000) was isolated by limited proteolysis and gel chromatography. A small quantity of functional unit d was obtained intact, but the major part in the form of two peptides (Mr approximately equal to 43,000 and 10,000, respectively) connected by a disulfide bridge. After reduction and carboxymethylation, these were separated from each other and cleaved by conventional methods. The peptides were isolated by gel chromatography and HPLC, and sequenced manually or automatically. The complete sequence of Helix beta c-Hc d comprises 410 residues plus 3 residues at the N-terminus seemingly resulting from incomplete cleavage. There is apparently only one carbohydrate side-chain. Comparison of this gastropodan hemocyanin sequence to the partial sequence of a cephalopodan Hc C-terminal unit revealed sufficient identities to state that the functional units of molluscan hemocyanins have arisen by a series of gene duplications. On the other hand, there is practically no homology with arthropodan hemocyanins except for one section of 42 residues which is clearly homologous. This section corresponds to the "Copper B" site of Panulirus interruptus hemocyanin. It is also found in tyrosinases from Neurospora crassa, Streptomyces glaucescens, and mouse. In the N-terminal half of Helix beta c-Hc d there are other sections clearly homologous to the tyrosinases, but overall homology is limited. The second copper-binding site was not identified but must be completely distinct from the "Copper A" binding site of arthropodan hemocyanins. It is suggested that molluscan and arthropodan hemocyanins have evolved independently from a common ancestral mononuclear copper protein.

The structure of arthropod hemocyanins.

Hemocyanins are large multi-subunit copper proteins that transport oxygen in many arthropods and molluscs. Comparison of the amino acid sequence data for seven different subunits of arthropod hemocyanins from crustaceans and chelicerates shows many highly conserved residues and extensive regions of near identity. This correspondence can be matched closely with the three domain structure established by x-ray crystallography for spiny lobster hemocyanin. The degree of identity is particularly striking in the second domain of the subunit that contains the six histidines which ligate the two oxygen-binding copper atoms. The polypeptide architecture of spiny lobster hemocyanin appears to be the same in all arthropods. This structure must therefore be at least as old as the estimated time of divergence of crustaceans and chelicerates, about 540 to 600 million years ago.

Carbonate dehydratase (carbonic anhydrase) in a spider. Association with the hemolymph lipoprotein.

Carbonate dehydratase was detected dissolved in the hemolymph of the tarantula, Eurypelma californicum. The enzyme was purified 31-fold by gel filtration, anion-exchange chromatography, a second gel filtration, and finally, preparative polyacrylamide gel electrophoresis. Zinc content increased during purification to up to 2.4 mol Zn/100 000 g of protein (= 1.58 mg Zn/g protein). In the polyacrylamide electrophoresis of tarantula hemolymph under non-denaturing conditions three major protein bands were observed: hemocyanin, a 16 S lipoprotein and the active band which migrated closely behind the 16 S lipoprotein. After treatment with sodium dodecyl sulfate both the carbonate dehydratase-active protein and the lipoprotein revealed bands corresponding to Mr = 95 000 and 110 000, respectively, but the enzymatically active protein revealed an additional third band with Mr = 40 000. The latter band is though to represent the 'true' carbonate dehydratase protein. Upon isoelectric focusing of material containing carbonate dehydratase activity and lipoprotein, bands were obtained at pH 5.45, 5.6 and 5.7. The band at pH 5.6 contained the peak of enzyme activity, and upon dodecyl sulfate-polyacrylamide gel electrophoresis showed the highest proportion of the 40-kDa polypeptide. It is concluded that tarantula carbonate dehydratase, instead of forming a high molecular mass aggregate, is associated with the 16 S lipoprotein, the latter serving as a carrier for the enzyme. The lipoprotein is probably also involved in other transport processes. It is present in great excess and may therefore occur in two forms, charged with carbonate dehydratase or uncharged. Tarantula carbonate dehydratase is inhibited by acetazolamide and by dansylamide, but not by a number of other known inhibitors, most notably not by 4-(aminomethyl)benzenesulfonamide. Treatment with 1M urea does not affect specific enzyme activity, while 2M urea inhibits by 50%. 2-Mercaptoethanol inhibits activity by 50% at 0.1M. Like other carbonate dehydratases, the tarantula enzyme shows esterase activity. The Km for 4-nitrophenyl acetate is 5mM.

Hemocyanins in Spiders, XVIII. Complete amino-acid sequence of subunit e from Eurypelma californicum hemocyanin.

The complete amino-acid sequence of subunit e of the hemocyanin from the tarantula, Eurypelma californicum, was determined by a combination of manual and automated methods. By limited proteolysis with chymotrypsin, two large fragments (e-CHn 29 and e-CHn 42) were obtained. The large peptides were further cleaved with cyanogen bromide, trypsin (with and without prior blocking of lysine residues), chymotrypsin, Staphylococcus aureus proteinase, Astacus fluviatilis proteinase, or 25% formic acid. The complete chain comprises 621 residues. A remarkable feature of the sequence is a hexapeptide -His-His-Trp-His-Trp-His- which is believed to take part in the binding of copper.

Hemocyanins in spiders, XIX. Complete amino-acid sequence of subunit d from Eurypelma californicum hemocyanin, and comparison to chain e.

The complete primary structure of subunit d of the hemocyanin from the tarantula Eurypelma californicum was determined by manual micro sequencing. Subunit d of Mr = 73000 is split about in the middle of the chain during limited trypsinolysis, only one single bond being attacked. The whole chain contains 14 methionine residues and after cyanogen bromide cleavage 15 peptides could be isolated by gel and ion exchange chromatography and high pressure liquid chromatography. The cyanogen bromide peptides and the large (Mr = 34000 and 37000, respectively) fragments resulting from limited trypsinolysis, were further cleaved with trypsin, chymotrypsin, Staphylococcus aureus proteinase, formic acid, and Astacus fluviatilis proteinase, the latter being very useful in obtaining certain overlapping peptides. The total chain length is 627 residues. Carbohydrate side chains were not found. The sequence is discussed with respect to the gross physical properties of the subunit, to homologies with subunit e and the cleavage specifities of the enzymes employed.

Hemocyanins in spiders, XVII. A presumptive active-site sequence of arthropod hemocyanins.

Peptides containing the sequence -His-His-Trp-His-Trp-His- have been isolated from the subunits e and a of tarantula (Eurypelma californicum) hemocyanin and III B of Limulus polyphemus hemocyanin. In view of the published spectroscopic and titration data on the copper-polypeptide complex and the strong quenching effect of oxygenation on hemocyanin fluorescence, it is concluded that this sequence represents the major part of the active site of Eurypelma, Limulus, and possibly other arthropod hemocyanins.

Hemocyanins in spiders, XV. The role of the individual subunits in the assembly of Eurypelma hemocyanin.

The role of the seven different subunits in the quaternary structure of the 24-meric (37 S) hemocyanin of the tarantula, Eurypelma californicum, was studied by reassembly experiments. Individual subunits and combinations of 2, 3, 4, etc. different subunits were incubated in a total concentration of 1-2 mg/ml overnight in Tris buffer, pH 7.5, omitting divalent cations. The reassembly mixtures were then analyzed by thinlayer gel filtration, electron microscopy, analytical ultracentrifugation, and polyacrylamide gel electrophoresis. At least at the low protein concentration employed, none of the isolated monomeric subunits (a, d, e, f, g) is capable of hexamer are likewise ineffective. The minimal number of monomers required for hexamer formation is three, but only about half of the 10 possible combinations was effective. The best results were obtained with a + f + g. Four and five monomers always yielded hexamers, although the composition of the latter is not known. The heterodimer bc is a prerequisite to go beyond the hexameric state. bc alone forms tetramers and large, strand-like aggregates. a shows a high affinity towards bc, trimers and tetramers being formed. Out of all combinations of bc and two monomers, only one (bc + a + g) produced hexamers or heptamers. With three monomers plus bc, dodecamers and larger structures (but no 24-mers) were obtained, but only if also a was present (bc + a + f + g being the best combination). If all subunits minus one monomer were combined, the hexamer/heptamer level was not exceeded if a was omitted. 24-mers were formed in appreciable yield only in one case, namely if e was absent. However, this reassembly product turned out to be unstable. To obtain stable 24-meric hemocyanin, the complete set of subunits had to be present. The roles of the different subunits are defined as follows: bc is a "core" subunit which forms the inter-hexamer link within each dodecameric half. It serves also in inter-dodecamer bonding. The artificial homodimers bb and cc are ineffective in this respect. bc must be supplemented by a which is needed for dodecamer stabilization. f is also required to link two dodecamers, while g serves to stabilize this bridge. d and e are required to finish off and to stabilize the 24-mer. A model is proposed showing some neighbourhood relationships of the subunits within the hemocyanin 24-mer, and a pathway of reassembly is discussed.

Hemocyanins in spiders, XVI[1]. Subunit topography and a model of the quaternary structure of Eurypelma hemocyanin.

Specific antibodies were prepared against the individual subunits of the hemocyanin isolated from the tarantula Eurypelma californicum. From the antibodies, the monovalent antigen-binding fragments (Fab) were made by papain treatment. Native 37S hemocyanin was incubated with individual Fab species and the labelling of the seven subunits (a, b, c, d, e, f and g) analyzed by electron microscopy. Specific labelling patterns were found for each Fab, which allowed the allocation of specific positions to each subunit within the (4 x 6)-subunit oligomer. Along the large cleft which separates the two dodecameric half-molecules, subunits f, b, c, f are located; e forms the four corners of the 37S particle, while a is found on both sides of the small cleft which separates the two hexamers within each dodecamer, d is close to a on the outer long edges, and g close to f at the side of the particle. These results and those obtained previously by means of partial dissociation and reassembly experiments, confirm and support each other, allowing the construction of a model of the quaternary structure of Eurypelma hemocyanin.

Hemocyanins in spiders, XIV. Subunit composition of dissociation intermediates and its bearing on quaternary structure of Eurypelma hemocyanin.

The 37 S hemocyanin (24 subunits of 7 types) isolated from the tarantula, Eurypelma californicum, was dissociated partially by various agents and the dissociation intermediates analyzed for their subunit composition by crossed immunoelectrophoresis. The subunit composition of the native hemocyanin was reexamined and the pending problem of the ratio between subunits a and g (= c2) clarified. The subunits are present in the ratio of a:b:c:d:e:f:g = 4:2:2:4:4:4:4. Breakdown products were shown to contain 19 (20?), 16, 12, 8, 7 and 6 polypeptide chains (possibly, there is also a 15-mer). The 19-mer is formed by removal of 5 monomeric subunits from one of the constituent hexamers of the native (4 x 6) hemocyanin, the 16-mer by losing one further copy each of e, f and g. The dodecamer is formed by cleavage with 2-mercaptoethanol and represents a half-molecule. The octamer could not be clearly analyzed but probably contains one copy of chain d more compared to the heptamer. The heptamer represents a one-quarter hemocyanin with one additional polypeptide chain sticking out of the hexameric structure. This can be either subunit b or c of the heterodimer bc. Only one type of hexamer was obtained after cleavage of the 37 S hemocyanin with 4M urea, containing one copy each of chains a, b, d, e, f and g. It is concluded that the 37 S hemocyanin is composed of two identical dodecameric halves linked by dimerization of subunit f, and that each half-molecule is constituted by two non-identical though similar hexamers, both encompassing a complete set of subunits a, d, e, f and g, but differing in their share of the rather stable, the 'c'-hexamer unstable. The relative positions of some of the subunits within the native oligomer are discussed.

On the role of dimeric subunits in the quaternary structure of arthropod hemocyanins.

Partial alkaline dissociation of 24 S (12-meric), 35 S (24-meric) and 60 S (48-meric) hemocyanin from various arthropods was studied by polyacrylamide gradient gel electrophoresis and, in some cases, by electron microscopy. If there are no stable dimers among the subunits, dissociation starts by cleavage of interhexamer bonds, leading to intermediates which are hexamers or multiples of hexamers. Whenever a hemocyanin contained stable dimers, inter-hexamer bonds were also very stable as indicated by the formation of 30 S (19-meric) or 18 S (7-eric) intermediates as primary products. In such cases, inter-hexamer bonds could be cleaved by treatment with reducing agents or with 4M urea; correspondingly, these agents also cleaved the respective dimers into the constituent polypeptide chains. It is concluded that in all cases the dimeric subunits function as bridges between hexamers.

Hemocyanins in spiders, XII. Dissociation and reassociation of Eurypelma hemocyanin.

The dissociation and reassembly of Eurypelma californicum hemocyanin was studied under various conditions of pH, ionic strength and protein, calcium and magnesium concentrations. The hemolymph concentrations of calcium and magnesium were determined to be ca. 4 and 0.9mM, respectively. Eurypelma hemocyanin does not dissociate upon dilution down to 0.04 mg/ml. At physiological pH, phosphate causes partial dissociation. Dissociation at alkaline pH requires two days for completion and is dependent on hemocyanin concentration. Reassociation, starting with the total dissociated subunits, yielded 37 S, 16 S and unreacted 5 S material. The yield of 37 S particles after one day was 75%, but rose slowly to 84% after two weeks. Raising the calcium concentration above 1mM resulted in an increase of 16 S particles (hexamers). High magnesium concentrations interfered with ordered reassembly. However, 37 S, native-like particles are readily formed in the absence of both calcium and magnesium. Reassociation is strongly favoured if ionic strength is increased. The significance of the 16 S reassociation product was studied by repeated dissociation and reassociation and analysis of subunit composition. It was found that the 37 S reassociate contained the whole set of subunits, their quantitative proportions being identical compared to native 37 S hemocyanin. In contrast, the 16 S material was lacking the dimeric subunit bc4 (= 4D). Evidently, this hexameric product arises by incorrect reassociation of monomeric subunits. P50 and nH values were recorded for the reassociated hemocyanins. The 37 S component had a higher affinity and lower cooperativity than native hemocyanin, possibly due to ageing of the subunits.

Hemocyanins in spiders, X. Limited proteolysis of chain e of Eurypelma hemocyanin and partial sequence of two large fragments.

The polypeptide chain e of the homocyanin from the spider Eurypelma californicum was isolated by ion exchange chromatography. Incubation of the undenatured protein with chymotrypsin, subtilisin, or trypsin resulted in a small number of large fragments which were easily isolated after denaturation. Of the chymotryptic peptides e-Chn-29 was found to be N-terminal, and e-Chn-42 C-terminal. These peptides were characterized by their N-terminal amino acid sequences. The N-terminal sequence of subunit e shows homologies with other arthropod hemocyanins.

Hemocyanins in spiders, IX. Homogeneity, subunit composition and the basic oligomeric structure of Eurypelma californicum hemocyanin.

37S hemocyanin isolated from the tarantula Eurypelma californicum was subjected to a variety of fractionation procedures: Electrophoresis in polyacrylamide gels, isoelectrofocusing, and ion exchange chromatography. Single fractions were dissociated at alkaline pH and/or by sodium dodecyl sulfate and the resulting subunits separated by polyacrylamide gel electrophoresis in gradient slab gels followed, in many cases, by quantitative estimation of the subunit bands. In addition, crossed immunoelectrophoresis was employed to analyze the subunit composition. Separation of the native hemocyanin into fractions with different subunit composition was not observed. Also, hemocyanin samples taken from individual spiders showed a strikingly uniform composition. It is concluded that Eurypelma hemocyanin is homogeneous for all practical means. This implies that the seven different polypeptide chains described previously for Eurypelma hemocyanin all take part in the formation of the 24 subunits, 37S oligomeric molecule. By monitoring chromatographic effluents at 280 nm, scanning stained electrophoresis gels and determining peak areas in these experiments and in crossed immunoelectrophoresis, the relative quantities of the seven different polypeptide chains in the whole molecule were determined as follows (number per 37S molecule): 6a, 2b, 2c2, 2c4, 4d, 4e, 4f. The ratio between a and c2 (6:2) is the least well established. Since 7 different polypeptide chains are incorporated, the hexameric molecule cannot represent the basic oligomeric structure in tarantula hemocyanin, but probably the dodekamer. If Eurypelma hemocyanin is incubated with sucrose, partial dissociation occurs, yielding a fragment sedimenting with ca. 28S. This process is reversible. The 28S fragment still contains all subunit species; it is thought to represent a (3 x 6) or closely related structure. The implications of this finding with respect to the symmetry of the native hemocyanin molecule are discussed.

Hemocyanins in spiders, VII. Immunological comparison of the subunits of Eurypelma californicum hemocyanin.

The isolated subunites of Eurypelma californicum hemocyanin were studied by aid of antibodies raised against whole, dissociated hemocyanin. The proportion of impurities was found to be low in almost all subunits. There was no cross reaction between the individual chains, and the total number of antigenically different subunits was found to be seven, confirming results obtained by different methods. If an artificial mixture prepared from purified subunits is compared to whole, dissociated hemocyanin, an overall very similar pattern is obtained but differences appear which are due to specific interaction.--The dimeric subunit 4D was shown to be a heterodimer (asymmetric dimer) composed of chains b and c4.

Hemocyanins in spiders, VI[1]. Comparison of the polypeptide chains of Eurypelma californicum hemocyanin.

The subunits of the hemocyanin from the tarantula, Eurypelma californicum, were isolated, following dissociation at pH 9.6, by a sequence of chromatographic and electrophoretic steps. Fraction 2 (containing two chains, a and c2) and the constituent polypeptide chains of the dimeric subunit 4D (b and c4) were resolved by anion exchange chromatography at pH 8.9 and 6.5, respectively. Since c2 and c4 have different electrophoretic mobilities in polyacrylamide gradient gels, the total number of different polypeptide chains is seven. The amino acid compositions of the seven chains are reported. There are major differences for at least half of the amino acids, while more consistent proportions become evident, if the amino acids are grouped by types of side chains. The N-terminal amino acid is proline in the case of chains b and e,, while no end group called be detected in any of the other chains by different methods. The C-terminal end group was found to be valine in both chains d and e. Cleavage by 70% formic acid, and by cyanogen bromide in formic acid results in fragmentation patterns distinct for each chain. After cyanogen bromide cleavage, the two largest peptides of each chain are of molecular weight near 2400. Tryptic fingerprints also reveal significant differences between all chains. Subunit heterogeneity of Eurypelma hemocyanin is clearly not the consequence of secondary modifications, but resides in major differences of the amino acid sequences.

Glucosamine metabolism in Drosophila salivary glands. Separation of metabolites and some characteristics of three enzymes involved.

The conversion of fructose 6-phosphate to mucopolysaccharide precursors was studied in extracts of Drosophila virilis salivary glands. 1. Methods for chromatography of sugar phosphates were adapted and modified to allow routine separation and quantitation of radioactivity of the metabolites from milligram amounts of tissue. Anion exchange chromatography was performed on Dowex 1-X8 employing steps of increasing ammonium formate. Final isolation of each compound was achieved by various thin-layer chromatographic systems. 2. Data obtained by isotope incorporation into glucosamine 6-phosphate compare well with results of the Morgan-Elson colorimetric assay for amino-sugars. 3. Glucosaminephosphate isomerase (glutamine-forming) (EC 5.3.1.19) in gland extracts has a Km of 0.35 mM for fructose 6-phosphate, and of 0.25 mM for glutamine. The enzyme is inhibited at glutamine concentrations exceeding 1 mM and by UDP-N-acetylglucosamine (50% at 0.6 mM). Feedback inhibition by UDP-N-acetylglucosamine is enhanced by AMP and by glucose 6-phosphate. 4. Glucosaminephosphate isomerase (EC 5.3.1.10) has a twenty fold lower affinity towards fructose 6-phosphate (Km = 6.0 mM) compared to the glutamine-forming isomerase. Km (NH+4) is 7.4 mM. In the presence of 20 mM glucose 6-phosphate, the pH optimum is shifted from 6.6 to 7.4, and V increased by a factor of 2.5. Furthermore, the affinity is approximately doubled for both substrates. 5. Glucosamine acetyltransferase (EC 2.3.1.3) has a Km of 2 mM for glucoseamine 6-phosphate. Its activity is not rate-limiting in salivary glands. Since N-acetylglucosamine 6-phosphate and 1-phosphate were found near equilibrium concentrations, acetylglucosamine phosphomutase (EC 2.7.5.2) must also be present in the extracts.

Hemocyanins in spiders, IV[1]. Subunit heterogeneity of Eurypelma (Dugesiella) hemocyanin, and separation of polypeptide chains.

The hemocyanin of the North American tarantula Eurypelma californicum (Dugesiella californica) is dissociated at pH 9.6 into monomers (Mr about 70 000) and dimers (Mr about 140 000), which were separated by gel filtration. The monomer peak was resolved by preparative polyacrylamide gel electrophoresis and yielded 4 protein bands, three of which (1, 3 and 4M) are apparently homogeneous. Band 2 contains two sub-fractions (2I and 2II). The dimer peak contains two dimers (bands 4D and 5). Upon treatment with 5mM cysteine the dimer band 5 is dissociated, yielding only one type of monomer identical with band 3. The other dimer, which was only partially dissociated by 10mM EDTA, is most probably a heterodimer, one component being electrophoretically indistinguishable from band 2II. After treatment of the native hemocyanin with sodium dodecylsulfate and analysis in gradient gel slabs, 6 polypeptide chains were observed (labeled a - f). They correspond to the products of alkaline dissociation as follows: band 1 = e, band 2I = a, band 2II = c, band 3 = f, band 4M = d, band 4D = b plus c, band 5 = f. The molecular weights were determined by dodecylsulfate gel electrophoresis in gradient gels, and by sedimentation equilibrium analysis and found to range between 67 000 and 76 000. The sedimentation coefficients are between 4.4 and 4.7 S for the monomers and 6.6 and 6.7 for the dimers. The isoelectric points range from pH 4.5 to pH 5.4. The findings are discussed with respect to the limitations of molecular weight determination by conventional dodecylsulfate gel electrophoresis, to the structure of the hemocyanin oligomers and to possible biological significance.