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.