Structural and kinetic characterization of myoglobins from eurythermal and stenothermal fish species.

Teleost myoglobin (Mb) proteins from four fish species inhabiting different temperature environments were used to investigate the relationship between protein function and thermal stability. Mb was isolated from yellowfin tuna (homeothermal warm), mackerel (eurythermal warm), and the Antarctic teleost Notothenia coriiceps (stenothermal cold). Zebrafish (stenothermal tropical) myoglobin was expressed from cloned cDNA. These proteins differed in oxygen affinity, as measured by O2 dissociation rates and P50 values, and thermal stability as measured by autooxidation rates. Mackerel Mb had the highest P50 value at 25 degrees C (3.7 mmHg), corresponding to the lowest O2 affinity, followed by zebrafish (1.0 mmHg), yellowfin tuna (1.0 mmHg), and N. coriiceps (0.6 mmHg). Oxygen dissociation rates and Arrhenius plots were similar between all teleost species in this study, with the exception of mackerel myoglobin, which was two-fold faster at all temperatures tested. Myoglobin from the Antarctic teleost had the highest autooxidation rate (0.44 h(-1)), followed by mackerel (0.26 h(-1)), zebrafish (0.22 h(-1)), and yellowfin tuna (0.088 h(-1)). Primary structural analysis revealed residue differences distributed throughout the polypeptide sequences, making it difficult to identify, which, if any, residues contribute to structural flexibility. However, analysis of molecular dynamics trajectories indicates that Mb from the eurythermal mackerel is predicted to be the most flexible protein within the D loop and FG turn. At the same time, it has the lowest O2 affinity and the highest O2 dissociation rates when compared to myoglobins from teleosts that appear to be less flexible in our dynamics simulations.

Isolation, characterization and nucleotide sequence of the muscle isoforms of creatine kinase from the Antarctic teleost Chaenocephalus aceratus.

Creatine kinase (CK) was isolated from the white muscle of the Antarctic icefish Chaenocephalus aceratus, which is deficient in glycolytic capacity. C. aceratus white myotomal creatine kinase (MMCK) displayed an apparent K(m) at 0.5 degrees C of 0.06 mM for ADP and 17 mM for Phosphocreatine. These K(m) values are similar to those reported for other vertebrate MMCKs at their physiologically relevant body temperatures. C. aceratus MMCK exhibited optimal activity at pH of 7.6-7.7 at 0.5 degrees C, in contrast to rabbit MMCK which had optimum activity at pH 6.2 at 30 degrees C. The apparent V(max) of C. aceratus MMCK at 0.5 degrees C is 94+/-4 S.D. (n=9) micromol ATP/min/mg (i.e. U/mg), which is comparable to rabbit MMCK assayed at 20 degrees C and 8-fold greater than rabbit MMCK measured at 0.5 degrees C. DEAE chromatography of C. aceratus white muscle CK resolved two distinct activity peaks. Cloning and sequencing of C. aceratus CK cDNAs confirmed that two muscle-specific isoforms of CK were expressed that were distinct from the mitochondrial and brain isoforms. Icefish MMCK was sensitive to transient temperature elevation, and the DEAE-fractionated forms were highly unstable. These results indicate that C. aceratus MMCK displays significant activity at physiological temperature and intracellular pH of icefish muscle that could contribute to sustaining energy charge during burst-swimming.

The myoglobin gene of the Antarctic icefish, Chaenocephalus aceratus, contains a duplicated TATAAAA sequence that interferes with transcription.

Six of the 16 known species of Antarctic icefish (family Channichthyidae) have lost the ability to express cardiac myoglobin (Mb) via at least four independent events during radiation of these species. We report here that the lesion in Chaenocephalus aceratus Mb is a duplicated TATAAAA element that blocks transcription. This lesion is distinct from those of other icefish species that do not express cardiac Mb. The C. aceratus Mb gene is nearly identical to that of Chionodraco rastrospinosus, a closely related Mb-expressing icefish species, with one exception. A 15-bp segment is present in C. aceratus but absent from C. rastrospinosus; this insertion is located 648 bp upstream from the reference transcription start site of C. rastrospinosus and includes the sequence TATAAAA, which bound HeLa cell transcription factor IID (TFIID) and icefish nuclear proteins in gel-retardation assays. Reporter constructs containing the 'full-length' C. aceratus Mb promoter were not expressed in transient expression assays in oxidative skeletal muscle of live icefish. By contrast, constructs employing the nearly identical 'full-length' C. rastrospinosus Mb promoter were efficiently expressed in parallel assays in the same tissue. Truncated constructs of C. aceratus Mb that did not contain the 15-bp duplication were expressed at very low levels. These data confirm a third independent mechanism of Mb loss among channichthyid species, indicate that C. aceratus aerobic muscle is capable of expressing functional Mb genes and demonstrate that duplication of the muscle-specific TATAAAA sequence in an inappropriate context can result in loss of a gene's expression, resulting in significant physiological consequences.

Teleost introns are characterized by a high A+T content.

We previously observed that Antarctic fish genes contain intron sequences of high A+T content (60-70% average A+T) which are in stark contrast with adjacent protein coding-sequences. Here, we report that this disparity in intron/exon base composition is a common feature among teleosts. We analyzed 483 teleost genomic DNA sequences, containing 2583 introns, from 80 teleost genera that populate polar, temperate, or tropical habitats. Eighty-nine percent of teleost introns display an A+T content between 50-84% A+T with a mean of 60% A+T. In contrast, only 37% of teleost exons have an A+T content greater-than 50% with a mean of 48% A+T. A comparison to homologous mammalian genes showed a striking difference; in this case, introns and exons have similar base compositions, averaging 45-47% A+T. This indicates that most teleost genes exhibit a large difference in base composition between their introns and exons. There was no correlation of teleost intron A+T content to intron length or habitat temperature range. Thus, teleost intron sequences tend to show the common feature of being much higher in A+T content then neighboring exons.