Subunit heterogeneity in arthropod hemocyanins: II. Crustacea

Markl, Jürgen; Höfer, Andreas; Bauer, Georg; Markl, A.; Kempter, B.; Brenzinger, Matthias; Linzen, Bernt

doi:10.1007/bf00691462

Cited by 92 publications

(43 citation statements)

References 30 publications

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“…Polyacrylamide gel electrophoresis was carried out in vertical gel slabs according to the methods of Jeffrey et al (1976) and Markl et al (1979) using the Hoeffer Scientific Instruments SE600 electrophoresis unit.…”

Section: Methodsmentioning

confidence: 99%

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Dainese

Muro

Beltramini

et al. 1998

European Journal of Biochemistry

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Carcinus aestuarii hemocyanin (Hc) exists in two aggregation forms at pH 7.5 and 20 mM Ca 2ϩ : 24S accounting for 90% of total hemocyanin and 16S accounting for 10%. Removal of metal cations by EDTA at neutral pH causes the complete dissociation of 24S hemocyanin into two different 16S. At pH 9.2, 24S hemocyanin dissociates into a pH stable 16S and a 5S component. The 5S component consists of three monomeric fractions named CaeSS1 (10%), CaeSS2 (50%) and CaeSS3 (40%); the latter fraction consisting of two isoforms. The fractions CaeSS1, CaeSS2 and CaeSS3 have been studied as far as their reassociation properties to form hexamers are concerned. We investigated the oxygen-binding properties of the native form (24S), the mixture of the two 16S forms, the pH-stable 16S alone and of purified subunit fractions to define the role of each species on the expression of the allosteric behaviour of the 24S aggregate. The analysis of O 2 -binding data reveals that 24S-Hc can be well described by the modified Monod Wyman and Changeaux-model (nested MWC-model), while the half-molecules (16S) bind oxygen according to the simple MWC-model. The two hexameric 16S within the dodecameric 24S hemocyanin can be regarded as nested allosteric units. They behave as being functionally coupled in the T-states (tT and rT). In the R-states (tR and rR) the two half-molecules seem to be functionally uncoupled since they have the same values of oxygen binding constants as deduced for isolated 16S hexamers.Keywords : hemocyanin; subunit; heterogeneity; oxygen binding ; allostery ; nesting.Hemocyanins (Hc) are giant oligomeric copper-containing proteins. They occur freely dissolved in the hemolymph and are responsible for oxygen transport in many arthropods and molluscs (Ghiretti, 1962;Préaux and Gielens, 1984;Salvato and Beltramini, 1990; van Holde and Miller, 1995). Arthropod Hc occur in the hemolymph as aggregates of the basic 16S unit (24S, 37S, 48S, 53S and 62S). The 16S unit is a hexamer of 5S functional subunits. The specific aggregation forms, present in the hemolymph of the various animals, depend on the species (van Holde and van Bruggen, 1971;Markl, 1986;Ellerton et al., 1983). In the hemolymph of most crustacea, hexamers (16S) or dodecamers (24S) or both are found. However, the biological significance for the occurrence of such different aggregation forms is still controversial (Markl and Decker, 1992). Dissociation in functionally intact subunits can be accomplished by removing Ca 2ϩ and increasing the pH above 9.0 (Ellerton et al., 1983; van Holde and Miller, 1995). Various studies elucidate the subunit composition of crustacean Hc. In general, three subunits are found namely a, b and c subunits (Bak and Beintema, 1987;Jekel et al., 1988; Neuteboom et al., 1992;Markl, 1986). They are differently expressed in the various species (Mangum, 1993) and are necessary to guarantee the specific aggregation state (Markl and Decker, 1992). Hc bind oxygen as peroxide in a µϪη 2 :η 2 -coordination mode between the two Cu(II) ions ; while subunit...

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Section: Methodsmentioning

confidence: 99%

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Dainese

Muro

Beltramini

et al. 1998

European Journal of Biochemistry

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“…Analytical electrophoresis was carried out in vertical mini-gel slabs using Bio-Rad Mini Protean Ò 3 and runs were carried out at the constant current of 20 mA per gel. Native PAGE under dissociating conditions, performed as in Markl et al (1979), was carried out in discontinuous gels consisting of a stacking gel (4% pH 9.6) over a resolving gel (8.5% pH 9.6). SDS-PAGE was performed using a discontinuous pH system detailed in Fling and Gregerson (1986), SDS gels consisting in a stacking gel (4%, pH 6.8) over a resolving gel (8.5%, pH 8.6).…”

Section: Chromatographic and Electrophoretic Analysesmentioning

confidence: 99%

Structural and functional heterogeneity of hemocyanin: intra- and inter-specific comparison in four species of portunid crabs (Crustacea: Portunidae)

et al. 2007

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This work is focused on hemocyanin (Hc) heterogeneity at population level and on the biochemical characterization of the different subunit patterns. Two different approaches have been used: we have characterized Hc subunit composition to describe the subunit polymorphism. Then, we have measured the Hc oxygen-binding affinity to disclose the physiological implications of such heterogeneity. In order to evaluate the intra-and inter-specific variability, different populations of Liocarcinus depurator, Liocarcinus marmoreus, Liocarcinus holsatus, Necora puber (Crustacea: Portunidae), from British Isles and Adriatic Sea, have been sampled. Results indicate that Hc polymorphism normally occurs at both intra-and interspecific levels and involves the protein subunit type.These evidences extend to the portunid Hc the concept of molecular heterogeneity within species that have been previously reported for other decapod and amphipod crustaceans. Besides, the results support the view that subunit composition of crustacean Hcs is almost species-specific, but also that closely related species share a common pattern. Furthermore, this heterogeneity corresponds to different stability of the native oligomers quaternary structure and different oxygen affinity. The results are discussed in relation with the environmental regimes that characterize the different sampling areas. In addition to Hc, the hemolymph collected in specimens from different sampling areas contained also a non-respiratory pseudo-Hc. This paper reports for the first time the occurrence of pseudo-Hc at the level of population.

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“…The most common aggregation states are hexamers and dodecamers, particularly amongst the crustaceans (reviewed by Herskovits, 1988 andMarkl eta!., 1979). In a number of cases a single organism contains more than one form of hemocyanin: the hemolymph contains primarily dodecamers with a minor component of hexamers in Cancer magister (Ellerton et at., 1970), Homarus gammarus, Cancer pagurus (Markl et a/.,1979), and Callinectes sapidus .…”

Section: Structure Of Hemocyaninmentioning

confidence: 99%

Developmental changes in the structure and function of lobster hemocyanin

Olson¹

1991

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Subunit heterogeneity in arthropod hemocyanins: II. Crustacea

Cited by 92 publications

References 30 publications

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Subunits composition and allosteric control in Carcinus aestuarii hemocyanin

Structural and functional heterogeneity of hemocyanin: intra- and inter-specific comparison in four species of portunid crabs (Crustacea: Portunidae)

Developmental changes in the structure and function of lobster hemocyanin

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