Activation of pigeon erythrocyte membrane adenylate cyclase by guanylnucleotide analogues and separation of a nucleotide binding protein.

作者：Thomas Pfeuffer, ERNST J.M. HELMREICH · 发表于：Journal of Biological Chemistry · 年份：1975 · DOI：10.1016/s0021-9258(19)41866-8 · 被引用次数：341 · 研究领域：Erythrocyte Function and Pathophysiology、Lipid Membrane Structure and Behavior、Cellular transport and secretion

Metabolically stable GTP analogues were 10 to 40 times more potent activators of DL-isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) from pigeon erythrocyte membranes that GTP. The order of effectiveness was guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) greater than guanylyl imidodiphosphate (Gpp(NH)p) greater than guanylyl methylenediphosphonate (Gpp(CH2)p greater than GTP. In contrast to activation with GTP, activation by analogues was independent of ATP concentration. The analogues seem to bind, however, to the same regulatory sites in membrane preparations to which GTP is bound but with higher affinity; Kdiss for (14C)Gpp-(nh)p and (3H)Gpp(CH2)p and membranes was 0.7 and 2.4 x 10-7 M, respectively. DL-Isoproterenol did not increase the amount of guanylnucleotide bound, it merely accelerated and potentiated activation. Bound radioactive GTP analogues were recovered unchanged from the membrane pellet. This and mutual displacement of analogues and GTP ruled out covalent attachment of the whole or of part of the nonphosphorylating GTP analogues. Treatment of the membrane preparation with Gpp(NH)p effectively (greater than 80%) protected DL-isoproterenol-activated adenylate cyclase against the action of Filipin or Lubrol PX. Activation of membrane-bound adenylate cyclase with GTP analogues resulted in a stable enzyme which could be nearly completely resolved from membranes with Lubrol PX and stripped of lipids and detergent without loss of activity. This effect was ...