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		· Introduction · The Na+,K+ pump in skeletal muscle · Questions · Analysis of the concentration of Na+,K+ pumps in skeletal muscle · Thyroid hormones · Food restriction · Training and immobilisation · Perspectives for future research · Concluding remarks · References
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Analysis of the concentration of Na⁺, K⁺ pumps in skeletal muscle
Quantitative analysis of membrane bound enzymes, such as Na⁺,K⁺-ATPase, is often performed on plasma membrane fractions that have been isolated from cell homogenates using differential centrifugation. However, this procedure requires large amounts of tissue, making it impractical for both medical and veterinary clinical studies, it may result in a loss of > 95% of Na⁺,K⁺ pumps and recovery rates between preparations vary enormously [18]. Thus, the development of techniques to measure the concentration of Na⁺,K⁺ pumps in small samples of intact skeletal muscle has proved invaluable to physiological studies [30].

Cardiac glycosides, such as digoxin and ouabain, bind specifically to the outer surface of the Na⁺,K⁺ pump, a stoichiometric process: one molecule of cardiac glycoside binds to one Na⁺,K⁺ pump molecule. The concentration of Na⁺,K⁺ pumps is measured using radioactively-labelled [³H] ouabain, provided the isozyme of the Na⁺,K⁺ pump in a specific tissue has a high affinity for the molecule. Analysis of mRNA coding for the Na⁺,K⁺ pump in skeletal muscle has revealed that most of it does, indeed, code for an isozyme with a high affinity for ouabain [41].

Fig.5 Schematic representation of the vanadate-facilitated binding of [3H]ouabain to a muscle biopsy. The open ends of the muscle cells allow vanadate ions access to the phosphorylation site of the Na+,K+ pump, on the inner surface of the sarcolemma. This binding results in a configuration of the Na+,K+ pump capable of binding [3H]ouabain to its outer surface (Taken from Reference 6).

Binding of [³H]ouabain to the Na⁺,K⁺ pump is facilitated by the presence of the phosphate analogue vanadate (Figure 5). Using this anion, a simple and rapid assay has been developed for the measurement of Na⁺,K⁺ pump concentration in muscle samples weighing as little as 5 mg [30]. The recovery of such small samples has the considerable advantage of enabling multiple biopsies to be taken from a tissue and therefore duplicate, triplicate or quadruplicate measurements may be made.

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