Deigweiher K, Koschnick N, Pö rtner H, Lucassen M. Acclimation of ion regulatory capacities in gills of marine fish under environmental hypercapnia.

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The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria, and eukaryotes. We have chosen one of the two CPX-ATPases of the thermophile Sulfolobus solfataricus, CopB (= SSO2896) for the investigation of the molecular mechanism of this integral membrane protein. We recombinately expressed three different soluble domains of this protein (named CopB-A, CopB-B, and CopB-C) in Escherichia coli and purified them to homogeneity. 3D crystals of CopB-B, the 29 kDa catalytic ATP binding/phosphorylation domain were produced, which diffracted to a resolution of 2.2 A. CopB-B has heavy metal stimulated phosphatase activity, which was half maximal in the presence of 80 microM Cu2+. The protein forms a phosphorylated intermediate with the substrate gamma-(32P)-ATP. No specific activation of the polypeptide was observed, when CopB-B phosphatase activity was tested in the presence of the purified CopB-C and CopB-A proteins, which provide the cation binding and the phosphatase domains. We conclude that CopB is a putatively copper translocating ATPase, in which structural elements integrally located in the membrane are required for full, coordinated activation of the catalytic ATP binding domain.

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 °C) and high (18 °C) temperatures leads to differential expression of alternative forms of the LDH‐A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH‐A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5′‐UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3′‐UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH‐Aα and LDH‐Aβ subunits. It is expected that the β‐subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH‐A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm‐acclimated fish the relative expression between α and β isoforms of the LDH‐A mRNA is around 5 : 1, whereas in cold‐acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH‐A is more homogeneous in terms of α/β‐subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine‐tuning of the properties of skeletal LDH‐A, which is in line with previously observed kinetic and thermodynamic differences between ‘cold’ and ‘warm’ LDH‐A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.