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J Markl

Publications and source records attributed to J Markl.

49 records · Page 3Linked to original sources
PubMed1984

Immunological correspondence between arthropod hemocyanin subunits. I. Scorpion (Leiurus, Androctonus) and spider (Eurypelma, Cupiennius) hemocyanin.

The hemocyanins of the scorpions Leiurus quinquestriatus and Androctonus australis, the tarantula Eurypelma californicum (all 24-mers), and the lycosid spider Cupiennius salei (dodecamer) were dissociated into subunits, the subunits isolated and studied by two-dimensional immunoelectrophoresis for interspecific cross-reactivities. Androctonus hemocyanin yielded a pattern of 8 subunit types in agreement with data from Lamy et al. (1979, Arch. Biochem. Biophys. 193, 140-149). Leiurus hemocyanin is also composed of 8 immunologically distinct subunits which could be assigned to the pattern of Androctonus in a subunit-to-subunit correlation. The subunit designations 1 to 6 of Lamy et al. could be adopted for both scorpion hemocyanins; however, in the present communication, Lamy's subunits 3A/3B are designated as 3'/3", because we could not unequivocally decide if 3' = 3A and 3" = 3B or vice versa. The 7 subunit types a to g of Eurypelma hemocyanin could be correlated with the scorpion hemocyanin subunits as follows: a = 3', b = 5B, c = 3C, d = 5A, e = 6, f = 2, g = 4. Additional cross-reactivities were detected between e/4, and f/5A, respectively. No subunit of Eurypelma hemocyanin is homologous to scorpion 3", which could not be precipitated by anti-Eurypelma antiserum. Antiserum against Cupiennius hemocyanin precipitated subunit f of Eurypelma and subunits 2 and 5A of scorpion hemocyanin. The published models of quaternary structure and a possible subunit phylogeny of arachnidan hemocyanins are discussed in view of the present results.

Animals↗
PubMed1982

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.

Animals↗
PubMed1981

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.

Hemocyanins↗
PubMed1981

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.

Animals↗
PubMed1981

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.

Animals↗
PubMed1980

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.

Animals↗
PubMed1980

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.

Animals↗
PubMed1979

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.

Amino Acids↗
PubMed1979

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.

Animals↗
PubMed1977

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.

Animals↗
PubMed1976

Haemocyanins in spiders, III. Chemical and physical properties of the proteins in Dugesiella and Cupiennius blood.

The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid composition is reported. As in the haemocyanins, the N-terminus is blocked. The carbohydrate content is 0.9%, glucose being the only sugar identified.

Amino Acids↗