Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B4 isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B4) whereas their skeletal muscles express LDH-A4. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.

The tolerance of abyssal pressures likely depends on adaptive modifications of fish proteins. However, structural modifications of proteins which allow functioning at high pressure remain unclear. We compared the activities of lactate dehydrogenase (LDH), an important enzyme in glycolytic reaction, in three hagfishes inhabiting different depths under increased pressure. LDH in Eptatretus okinoseanus, found at a depth of 1,000 m, was highly active at high pressure of 100 MPa maintaining the activity at 70% of that at 0.1 MPa. In contrast, LDH activity in Paramyxine atami, found at 250-400 m, decreased to 55% at 15 MPa, and that in Eptatretus burgeri, found at 45-60 m, was completely absent at 5 MPa. The result suggests that subunit interaction of the LDH-tetramer is more stable in E. okinoseanus than that in P. atami and E. burgeri under high-pressure conditions. We found six amino acid substitutions between the three LDH primary structures. Accordingly, these amino acid residues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions.

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A(4) from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.

The lactate dehydrogenases (LDHs) in hagfish have been estimated to be the prototype of those in higher vertebrates. The effects of high hydrostatic pressure from 0.1 to 100 MPa on LDH activities from three hagfishes were examined. The LDH activities of Eptatretus burgeri, living at 45–60 m, were completely lost at 5 MPa. In contrast, LDH-A and -B in Eptatretus okinoseanus maintained 70% of their activities even at 100 MPa. These results show that the deeper the habitat, the higher the tolerance to pressure. To elucidate the molecular mechanisms for adaptation to high pressure, we compared the amino acid sequences and three-dimensional structures of LDHs in these hagfish. There were differences in six amino acids (6, 10, 20, 156, 269, and 341). These amino acidresidues are likely to contribute to the stability of the E. okinoseanus LDH under high-pressure conditions. The amino acids responsible for the pressure tolerance of hagfish are the same in both human and hagfish LDHs, and one substitution that occurred as an adaptation during evolution is coincident with that observed in a human disease. Mutation of these amino acids can cause anomalies that may be implicated in the development of human diseases.

Amplified Eptatretus okinoseanus cDNA was digested with NdeI and EcoRI, cloned into pCold trigger factor (TF), and transformed with Escherichia coli strain BL 21 in which a csp A promoter was introduced to inhibit the expression of foreign peptides. Recombinant lactate dehydrogenase (LDH) was obtained in the soluble fraction after sonication of the cells. The protein was digested by HRC 3C protease, thrombin, and factor Xa. The specific activity of TF-tagged protein and tagless protein were 0.646 × 10 6 mIU/mg and 3.56 × 10 6 mIU/mg, respectively. The deletion of the TF tag enhanced the activity compared with the native protein to 13.4 × 10 6 mIU/mg, showing that this expression method is effective for the mass production of the protein to allow further study of the structure of LDH.