4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 1-5-difluoro-2-4-dinitrobenzene

Cross-linking reactions of the alpha- and beta-subunits of the purified membrane-bound enzyme with several reagents were studied. In the presence of 1,5-difluoro-2,4-dinitrobenzene, formation of a cross-linked alpha, beta-dimer was affected specifically by K+ + ATP or enzyme phosphorylation. The same conditions affected the formation of cross-linked alpha, alpha-dimer in the presence of 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone or o-phenanthroline-Cu2+. Since noncovalent alpha, beta-association has been established, the data suggest K+ + ATP-induced or phosphorylation-induced changes in alpha, beta-domain and alpha, alpha-domain of an oligomer of alpha, beta-dimer. When the formation of cross-linked alpha, beta-dimer or alpha, alpha-dimer was induced by phosphorylation, only half of the subunits were cross-linked, suggesting the existence of a cooperative tetramer of alpha, beta-dimer. When microsomes or red cell membranes were exposed to 32Pi under phosphorylation-induced cross-linking conditions, the only products were alpha, beta-dimer and alpha, alpha-dimer, indicating the existence of an oligomer of alpha, beta-dimer in crude membranes. Subunits of the enzyme solubilized with octaethylene glycol dodecyl ether, by methods that have been suggested to yield unassociated alpha, beta-dimers, underwent spontaneous cross-linking that was not affected by enzyme dilution. Since the largest product was alpha 2 beta 2, the solubilized enzyme is at least a dimer of alpha, beta-dimer. The findings establish that the membrane-bound enzyme is an oligomer of alpha, beta-dimer. Whether or not a single alpha, beta-dimer is capable of catalytic and transport functions has not been determined.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Cross-Linking Reagents; Dimethyl Suberimidate; Dinitrofluorobenzene; Dogs; Glutaral; Kidney; Macromolecular Substances; Maleimides; Sodium-Potassium-Exchanging ATPase; Thiocyanates