Introduction
Several isoforms are known of the a and b subunits of the membrane-bound sodium pump or (Na++K+)-activated ATPase. Individual combinations of isoforms of the two subunits have different affinities to the substrate ATP, to the translocated ions Na+ and K+, and to inhibitors like Ca2+. In most mammalian species all a isoforms have high and nearly equal affinities to the specific inhibitor ouabain, with exception of the rodent a1 isoform that confers extremely low affinity to cardiac glycosides. The total pool of Na,K-ATPase is thus easily determined in tissue biopsies as the (3H)ouabain binding capacity except in rodents if the a1 isoform contributes to the total pool. Since the rat is the preferred experimental model, also in studies aiming at controlling the essential pump function of Na,K-ATPase by cardiac glycosides or Ca2+, a method for quantitative determination of the a1 isoform was developed.
Methods
Homogenates of soleus and EDL were run on SDS gels. In parallel lanes was run a well-characterized preparation of Na,K-ATPase isolated from rat kidney that is known to contain only the a1 isoform. After electroblotting to PVDF membranes blots were incubated with the a1 specific monoclonal antibody 3B, then with an 125I-coupled secondary antibody, and finally the specific labelling of a spots was analysed by means of an electronic autoradiography system (Packard InstantImager). Since the a1 content of reference Na,K-ATPase was known from the specific Na+-dependent 32P-phosphorylation capacity, the a1 content of adjacent a spots in homogenates from soleus and EDL could be calculated.
Results
Two important principles are taken into consideration. First, total homogenates have to be used since the recovery of plasma membrane components like Na,K-ATPase even after sparse purification may be extremely low (Hansen & Clausen, 1988), and second, the Na,K-ATPase used as reference has to be isolated from the very same species since the epitope of the antibody may differ between species. Since a2 and, if present, a3 are the components preferentially determined in (3H)ouabain binding, a combination of this method and the present one will account for the total pool af Na,K-ATPase in rat muscles. In soleus and EDL from 4 wk rats an a1 concentration (in pmol/g tissue) of 160-265 and 140-160, respectively, and in 10-11 wk rats 50-90 and 20-55, respectively, was found. A similar age-dependent decrease in (3H)ouabain binding from 700-800 to about 300 pmol*(g tissue)-1 has been found in rat soleus muscles.
Conclusions
By using specific antibodies to the a1 subunit of Na,K-ATPase and a purified kidney enzyme from the same species it seems possible to determine a ouabain-insensitive a1 component from blots of rat skeletal muscles. The a1 isoform represents 15-25% of the total pool of Na,K-ATPase in EDL and soleus.
References
Hansen, O. & Clausen, T. 1988. Am J Physiol 254, C1-C7