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Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

ABSTRACT: The subunit structure of erythrocruorin from the cladoceran Daphnia magna was studied. The native protein was found to have a sedimentation coefficient (S2(20), w) of 17.9 +/- 0.2 S and a molecular weight, as determined by sedimentation equilibrium, of 494 000 +/- 33 000. Iron and haem determinations gave 0.312 +/- 0.011% and 3.84 +/- 0.04%, corresponding to minimal molecular weights of 17900 +/- 600 and 16 100 +/- 200 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis gave one band with mobility corresponding to a molecular weight of 31 000 +/- 1 500. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M-guanidinium chloride and 0.1 M-2-mercaptoethanol is 31 100 +/- 1300. On a molecular-weight basis, Daphnia erythrocruorin is composed of 16 identical polypeptide chains carrying two haem groups each. The native structure is stable between pH5 and 8.5. At alkaline and acidic pH, a gradual decrease in the sedimentation coefficient down to 9.8S occurs. Above pH 10 and below pH4, a slow component with S20, w between 2.7S and 4.0S is observed. The 2.7S, 4.0S and 9.8S species are identified as single-chain subunits, subunit dimers and half-molecules respectively. We propose a model for the molecule composed of 16 2.7S subunits grouped in two layers stacked in an eclipsed orientation, the eight subunits of each layer occupying the vertices of a regular eight-sided polygon. Support for this arrangement is provided from electron microscopy and from analysis of the pH-dissociation pattern.

SUBMITTER: Ilan E

PROVIDER: S-EPMC1153860 | biostudies-other | 1982 Nov

REPOSITORIES: biostudies-other

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Project description:Microarray analyses were used to evaluate patterns of gene transcription following exposure of the waterflea Daphnia magna to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed the water flea Daphnia magna to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Most gene fragments were downregulated under stress (82.4% downregulation compared to 17.6% upregulation) irrespective of the treatment. In approximately 5% of the cases up- or downregulation depended on stressor identity. Based on a PCA, we could identify two different groups within our exposure treatments: small and big gene expression differences compared to the control condition. The treatments parasite 96h exposure, carbaryl 144h of exposure and fish 144h of exposure are combined in the high stress group. 26 samples x 2 biological replicates were analysed in a carriage wheel design (Khanin & Wit, 2005; Knapen et al., 2009) with a reference sample composed of a clutch of non-exposed D. magna that were collected at different time points ranging from 0-24h until three weeks old. The 26 samples included: 4 treatments (control, carbaryl, fish and parasite exposure) x three time points (48h, 96h and 144h) x 2 replicates + 2 start samples (non-exposed individuals younger than 24h old). This led to 52 different microarray hybridizations: 26 hybridizations for the inner part of the wheel, where every sample is hybridized against the reference, and another 26 for the outer part. Color flip experiments were incorporated per sample to fulfill the conditions of technical replicates on the level of labelling; every stressor-time combination was included at least once in red and once in green. One hybridization in the outer part of the wheel had technical problems, and is not included here.

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Erythrocruorin from the water-flea Daphnia magna. Quaternary structure and arrangement of subunits.

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