Separation and Characterisation of Tryptic Fragments from the Adenosine Triphosphatase of Sarcoplasmic Reticulum

Abstract

The two halves of the ATPase, M_r 115000, from sarcoplasmic reticulum produced by limited trypsin treatment have been purified in sodium dodecylsulphate. The fragment of M_r 60000 has been purified by electrophoresis on cellulose acetate slabs and that of M_r 55000 by gel filtration. The two halves of the 60000 M_r fragment (M_r 33000 and 24000) produced by more extensive trypsin treatment have also been purified by gel filtration in sodium dodecylsulphate. The sum of the amino acid analyses of the constituent tryptic fragments is in good agreement with that for the whole ATPase. The amino acid compositions of the two halves of the ATPase were strikingly similar. N-terminal analysis shows that the ATPase and its constituent tryptic polypeptides all possess a single N-terminal alanine implying no further cleavage of the polypeptide by trypsin. Attempts to solubilize selectively the tryptic fragments from the membrane by a variety of denaturing and solubilising agents under a variety of conditions have proved unsuccessful, suggesting that the interaction between the tryptic polypeptides is stronger than between the lipid and the protein. The possibility that the interaction between the tryptic polypeptides includes disulphide bonding has been eliminated.