UBC Theses and Dissertations
Changes in the ouabain-sensitive, sodium and potassium-activated adenosine triphosphatase of the gills of coho salmon Oncorhynchus kisutch, during the fry to smolt stages of its life history and upon exposure to sea water Giles, Michael Arthur
Some of the kinetic characteristics of the sodium and potassium-activated adenosine triphosphatase of the fragmented cell membranes of cells from the gills of sea water adapted coho salmon Oncorhynchus kisutch, and changes in this enzyme upon exposure to sea water and during the fry to smolt stages of fresh water reared juvenile coho were investigated. Inhibition with 4 X 10⁻⁴ moles/liter ouabain was used to assay the activity of this enzyme since this ATPase is specifically inhibited by ouabain (Skou, 1957). The following assay conditions were found to result in maximal hydrolysis of ATP in enzyme preparations from sea water adapted coho: pH, 7.4; incubation temperature, 40°C; NaCl and KCl concentrations of 100.0 and 20.0 mmoles/liter, respectively, and Mg²⁺ -ATP, 5.0 mmoles/liter. The Km for ATP was 0.2 mmoles/liter. The enzyme activity recorded with magnesium ions as the only cation present (Mg²⁺-ATPase) was not affected by any concentration of ouabain, although the addition of sodium ions (100 mmoles/liter) appeared to inhibit this activity slightly. The additional hydrolysis of ATP observed when sodium, potassium and magnesium ions were present was inhibited by ouabain. The Ki for ouabain was; 7 X 10⁻⁶ moles/liter when sodium and potassium ion concentrations were 100.0 and 20.0 mmoles liter, respectively. The (Na⁺ + K⁺)- activated ATPase of sea water adapted coho was characterized by its high ouabain-sensitive activity and the large activating effect of potassium ions in the presence of magnesium and sodium ions compared to the activity observed with the latter two ions alone. This enzyme in preparations from the gills of fresh water reared fish was characterized by a high activating effect of sodium ions when present with magnesium ions. This sodium activation often comprised over 60% of the total ouabain-sensitive activity. Considerable increases in the total activity, and activating effects of potassium ions and decreases in the activating effects of sodium ions alone were observed when fresh water reared coho were transferred directly to sea water. The changes in the activating effect of the ions were noticable after 5 days exposure to sea water although no changes in the total activity of the enzyme occurred until after 10 days exposure. On a seasonal basis changes in enzyme activity occurred which were apparently linked to the stage of development of the parr-smolt transformation in fresh water reared juvenile coho. Activities during the period of October 1, 1968 to late November, 1968 were generally quite low. A sharp peak in activity occurred in December, 1968 to late January, 1969 which decreased to a low level by mid-February. Up to and including this last period the activity of enzymes from the gills of both fresh water and sea water reared coho were qualitatively similar although the seawater fish always had a higher enzyme activity. During the period of mid-February to late April,1969 the enzyme from fresh water reared coho changed in total activity and characteristics of sodium activation and potassium activation and became very similar to that of sea water reared fish of the same age.