Biochemical adaptation of deep-sea fishes : susceptibility of dehydrogenases to pressure-inactivation and proteolysis

approved / áoseph F. Siebenaller Muscle-type lactate dehydrogenase (M4-LDH; EC 1.1.1.27), cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) from several shallowand deep-living marine teleost fishes were examined for susceptibility to inactivation by hydrostatic pressure and proteolysis. The molecular mass and subunit aggregation state of these enzymes are identical to those of other vertebrate homologues. The pressures required to half-inactivate the LDH homologues of six confamilial macround fishes are related to the pressures which the enzymes are exposed to in viva. LDH homologues of deeper-living species tend to require greater pressures for inactivation than do shallow-occurring species. Fish MDH and GAPDH homologues are more resistant to inactivation by pressure than are LDH homologues from the same species. Incubation of fish enzymes for 1 h at 10°C and 1750 atm pressure results in 65 to 100% loss of LDH activity, 6 to 14% loss of MDH activity, and no loss of GAPDH activity. Pressures required to inactivate dehydrogenases in vitro are greater than pressures normally encountered in the habitat of the species. 1xnong the homologues of M4-LDH, MDH and GAPDH, resistance to inactivation by hydrostatic pressure appears to be determined by the subunit aggregation state of Redacted for privacy the enzyme and the number of subunit interactions. Enzymes with more subunits and more subunit interactions tend to be less stable at high pressures. This is consistent with the model of Paladini and Weber (Biochemistry, 20:2587-2593, 1981) which predicts that susceptibility to dissociation of oligomeric proteins is controlled by the small "free volumes" at the intersubunit boundaries. Hydrostatic pressure, by affecting protein aggregation state and conformation, may increase susceptibility to proteolysis. At 100C and atmospheric pressure, the rates of inactivation by trypsin and subtilisin are three to four times greater for LDH homologues from shallow-living fishes than for homologues from deep-living fishes. Hydrostatic pressure (as low as 200 atm) has a greater influence on proteolytic inactivation of LDH homologues of shallow-living fishes than for homologues of deep-living fishes. Fish MDII homologues are less susceptible to proteolytic inactivation than LDH homologues from the same species. MDH homologues of shallowand deep-occurring fishes are inactivated to a similar extent by proteases both at atmospheric pressure and at 1750 atm pressure. Under the same conditions fish GAPDH homologues are not inactivated by trypsin or subtilisin. Enzymes that are less susceptible to inactivation by pressure appear to be less susceptible to inactivation by proteases. Conformational perturbations in the structure of shallow-occurring LDH homologues that increase their susceptibility to inactivation by proteases at elevated pressure are not apparent in MDH and GAPDH homologues of the same species. The structural integrity of MDH and GAPDH homologues of marine fishes appear to be pre-adapted to the hydrostatic pressures of the deep-sea. Biochemical Adaptation of Deep-Sea Fishes: Susceptibility of Dehydrogenases to Pressure-Inactivation and Proteolysis