Periyasamy S M, Huang W H, Askari A
J Biol Chem. 1983 Aug 25;258(16):9878-85.
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.
研究了纯化的膜结合酶的α亚基和β亚基与几种试剂的交联反应。在1,5-二氟-2,4-二硝基苯存在下，交联的α,β-二聚体的形成受到K⁺ + ATP或酶磷酸化的特异性影响。在4,4'-二氟-3,3'-二硝基二苯砜或邻菲罗啉-Cu²⁺存在下，相同条件影响交联的α,α-二聚体的形成。由于已经确定了非共价的α,β缔合，数据表明K⁺ + ATP诱导或磷酸化诱导的α,β-二聚体寡聚体的α,β结构域和α,α结构域发生变化。当磷酸化诱导交联的α,β-二聚体或α,α-二聚体形成时，只有一半的亚基发生交联，表明存在α,β-二聚体的协同四聚体。当微粒体或红细胞膜在磷酸化诱导的交联条件下暴露于³²Pi时，唯一的产物是α,β-二聚体和α,α-二聚体，表明粗膜中存在α,β-二聚体的寡聚体。用八甘醇十二烷基醚溶解的酶亚基，通过已被建议产生未缔合的α,β-二聚体的方法，经历了不受酶稀释影响的自发交联。由于最大的产物是α₂β₂，溶解的酶至少是α,β-二聚体的二聚体。这些发现证实膜结合酶是α,β-二聚体的寡聚体。单个α,β-二聚体是否能够发挥催化和转运功能尚未确定。
