Pressure‐adaptive differences in proteolytic inactivation of M4‐lactate dehydrogenase homologues from marine fishes

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Abstract
The inactivation by hydrostatic pressure of muscle‐type lactate dehydrogenase (M4‐LDH, EC 1.1.1.27; L‐lactate: NAD+ oxidoreductase) homologues from five shallow‐living and six deep‐living marine teleost fishes was compared. The pressures which inactivate these enzymes are much higher than the pressures experienced by any of the species. To determine whether hydrostatic pressure effects on protein aggregation state and conformation might influence proteolysis, the inactivation of LDH by the proteases, trypsin (EC 3.4.21.4) and subtilisin (EC 3.4.4.16) was determined at atmospheric pressure and 1,000 atm pressure. At 10°C and atmospheric pressure, the enzymes of the shallow‐living fishes are inactivated four times faster by trypsin and three times faster by subtilisin than are the homologues of the deep‐living species. At 1,000 atm pressure, the homologues of shallow‐occurring fishes were inactivated 28 to 64% more than predicted from the summed effects of denaturation by 1,000 atm pressure and tryptic inactivation at atmospheric pressure. In contrast, the homologues of the deep‐sea species were inactivated by trypsin 0 to 21% more than expected. At 1,000 atm, inactivation by subtilisin increased to a similar degree for enzymes from both deep‐ and shallow‐living species. However, at 1,000 atm, the M4‐LDH homologues of the deep‐sea species lost less activity (55.3%) than did the homologues of the shallow species (86.4%). In comparisons made at 200 atm, a pressure typical of the habitat of the deep‐occurring species, tryptic inactivation of the LDH of the shallow‐living Sebastes melanops was increased 14%. No pressure inactivation of the enzyme is evident at 200 atm. Two hundred atm pressure does not increase the tryptic inactivation of the enzymes of the two deep‐living macrourids. Increased structural stability of the enzymes of deep‐sea species may be an adaptation which prevents too rapid protein turnover, which would be energetically costly in the food‐poor deep sea.