The alpha1 isoform of Na+,K+-ATPase in rat soleus and extensor digitorum longus

A novel immunochemical method was used for determination of the concentration of Na,K-adenosine triphosphatase (ATPase) containing the ouabain-insensitive alpha1 peptide in rat m. soleus and extensor digitorum longus (EDL). Homogenates of soleus and EDL from 4-week or 10-11-week rats were run on sodium dodecyl sulphate (SDS) gels and in parallel lanes was run a well-characterized preparation of Na,K-ATPase isolated from rat kidney that is known to contain only the alpha1 isoform. After electroblotting to PVDF membranes blots were incubated with the alpha1 specific monoclonal antibody 3B, then with an 125I-coupled secondary antibody, and finally the specific labelling of adjacent alpha spots was analysed by means of an electronic autoradiography system (Packard InstantImager). As the alpha1 content of reference Na,K-ATPase was known from the specific Na+-dependent 32P-phosphorylation capacity, the alpha1 content of adjacent alpha spots in homogenates from soleus and EDL could be calculated. In soleus and EDL from 4-week rats an alpha1 concentration of 135-220 pmol (g tissue)(-1) was found, dependent on the conditions of the experiments but without significant differences between the two types of muscle. In 10-11-week rats a significantly lower concentration of 70-80 and 40-60 pmol (g tissue)(-1) in soleus and EDL, respectively, was found. Ouabain-insensitive Na,K-ATPase containing the alpha1 peptide may thus represent 15-25% of the total number of pumps in skeletal muscle if another 20-30% has to be added to the pool known from (3H)ouabain binding.