The polypeptides of the ox heart mitochondrial inner membrane: isolation of the major component.

attempted a reversal of the cross-linking. The stained band was out from the gel and the reversal of the cross-linking by ammonolysis was carried out in the gel slice as described by Hulla & Gratzer (1972). On re-electrophoresis a very faint band which migrated approximately as the a subunit was found. Owing to the small amount of regenerated subunit obtained we could not rule out the presence of other subunits in band A which may not be detectable by Coomassie Blue staining in the quantity likely to be present. To increase the detectability of the regenerated subunits formed on ammonolysis of band A we chemically labelled the band with lZsI in the excised gel slice. Experimental details are given in the legend to Fig. 1 . The products of the reversal were then located by radioautography of the gel. A radioactive band exactly corresponding to the a subunit was obtained (Figs. l e and lf). No other radioactive bands were found, suggesting that band A is derived solely from the a subunit. The estimated molecular weight for band A of 14OOOO indicates that it is probably a dimer of a subunits. This finding demonstrates that a subunits are adjacent to one another in the ATPase molecule. Bragg & Hou (1975) have shown that the predominant species formed after cross-linking the ATPase from Escherichiu coli, which has a similar subunit composition to that of the ox heart enzyme, is a dimer of a and B subunits. Although it is possible that some of the faint bands formed after cross-linking the ox heart ATPase arise from cross-links between the a and subunits, it appears that the arrangements of the subunits in the two molecules is different.