A Microassay for Gill Sodium, Potassium-Activated ATPase in Juvenile Pacific Salmonids

Abstract

A microassay well‐plate method is described for determining Na+,K+‐ATPase activities of small gill sections from juvenile Pacific salmon Oncorhynchus spp. The method differs from the established macromethod by detecting inorganic phosphate in nanomole rather than micromole concentrations. This permits the use of much smaller tissue samples, which makes it possible to release fish after sampling. Use of sonication during enzyme extraction and elimination of the need to deproteinize samples before ATPase analysis further simplify the assay. Application of the microwell‐plate technique for both Na+,K+‐ATPase activity and protein analysis permits rapid processing of many samples. It also produces results equivalent to those of the macroassay; no significant differences occurred between sample duplicates run by the two methods with the same enzyme extract (P > 0.05). The coefficient of variation (100·SD/mean) for microassay samples containing enzyme activities of at least 10 umol inorganic phosphate per milligram protein per hour was 12% or less for between‐plate comparisons and 5% or less for same‐plate comparisons. Monitoring of gill‐clipped fish during migration indicated that small gill clips did not cause mortality or alter migration behavior of juvenile salmonids tagged with passive integrated transponders. These are important considerations in programs for monitoring species listed under the U.S. Endangered Species Act.

The Effect of Hatchery Release Strategy on Marine Migratory Behaviour and Apparent Survival of Seymour River Steelhead Smolts (Oncorhynchus mykiss)

“…During sampling, the left gill arches from each fish were removed, placed in foil and frozen in liquid nitrogen. The gill samples were stored at −80°C and later analyzed to determine Na/K-ATPase enzyme activity [28], [29]. Livers were removed, weighed, and with whole body weights used to calculate the hepatosomatic index (HSI, liver weight/whole body weight ×100).…”

Migration performance of wild and hatchery sea trout (Salmo trutta L.) smolts—Implications for compensatory hatchery programs

“…Gill samples were taken from hatchery and wild smolts of the same batches as the tagged fish. A gill biopsy (four to five tips of filaments) was taken from the first gill arch of 10 hatchery and 10 wild smolts and frozen in SEI buffer (300 mm sucrose, 20 mm Na 2 EDTA, 50 mm imidazole, pH 7.3), using the gill biopsy method described by Schrock et al (1994). Gill samples were stored in a −80 • C freezer until Na + ,K + -ATPase activity was analysed (Schrock et al, 1994).…”

Impact of a short‐term exposure to tributyl phosphate on morphology, physiology and migratory behaviour of European eels during the transition from freshwater to the marine environment

“…Gill and kidney Na + /K + ATPase activity was assayed according to the method described by Schrock et al (1994) with the enzyme activity shown as lmolÁPiÁmg À1 proteinÁh À1 .…”