Birnbaumer L, Swartz T L, Abramowitz J, Mintz P W, Iyengar R
J Biol Chem. 1980 Apr 25;255(8):3542-51.
Transient and steady state kinetics of the interaction of liver plasma membrane adenylyl cyclase with GTP and the GTP analogues guanyl-5'-yl imidodiphosphate (GMP-P(NH)P), guanyl-5'-yl diphosphonate (GMP-P(CH2)P), and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) was studied before and after treatment of membranes with preactivated cholera toxin. (a) In control experiments, GTP stimulated the enzyme partially and without a noticeable lag, while GTP analogues stimulated to varying degrees (GTP gamma S approximately GMP-P(NH)P greater than GMP-P(CH2)P greater than or equal to GTP) and with lag periods that varied with the nucleotide (GMP-P(NH)P approximately GMP-P(CH2)P greater than GTP gamma S). (b) In toxin-treated membranes, transient kinetics and degree of activation by GTP analogues were unaltered, while activation by GTP remained rapid and became as effective as the most effective of the analogues. (c) Toxin treatment had no effect on the concentration-effect curves for GTP analogues but resulted in a 5- to 9-fold lowering of the apparent K alpha with which GTP stimulates the system. These results indicate that the degree of stimulation of the liver adenylyl cyclase by GTP and its analogues is independent of the susceptibility of the beta-gamma bond of the guanosine triphosphate to be hydrolyzed and that lags in progress curves are not due to slow dissociation of "resident" GDP molecules. The time transients of the interaction of the rat liver adenylyl cyclase with combinations of GTP and GMP-P(NH)P were studied and found to be of the competitive type regardless of the time and sequence of addition of the two nucleotides. The results are consistent with GMP-P(NH)P interacting with and dissociating from the system very slowly and not leading to the formation of an irreversibly activated state of the enzyme. The data obtained on regulation of the basic (no hormone added) liver adenylyl cyclase by guanyl nucleotides are interpreted in terms of a two-state enzyme model (Birnbaumer, L., Bearer, C.F., and Iyengar, R. (1980) J. Biol. Chem. 255, 3552-3557) and the results are discussed in the light of current views of regulation of adenylyl cyclases and involvement of a GTPase. Our experiments are consistent with, but not proof for, presence of a cholera toxin-inhibited GTPase in intimate association with the liver adenylyl cyclase.
在用预激活的霍乱毒素处理肝细胞膜之前和之后，研究了肝细胞膜腺苷酸环化酶与GTP及GTP类似物鸟苷-5'-亚氨二磷酸（GMP-P(NH)P）、鸟苷-5'-亚甲基二磷酸（GMP-P(CH2)P）和鸟苷5'-(3-O-硫代)三磷酸（GTPγS）相互作用的瞬态和稳态动力学。(a) 在对照实验中，GTP对该酶有部分刺激作用且无明显延迟，而GTP类似物的刺激程度各不相同（GTPγS≈GMP-P(NH)P＞GMP-P(CH2)P≥GTP），且延迟期随核苷酸而异（GMP-P(NH)P≈GMP-P(CH2)P＞GTPγS）。(b) 在毒素处理的膜中，GTP类似物的瞬态动力学和激活程度未改变，而GTP的激活仍然迅速，并且变得与最有效的类似物一样有效。(c) 毒素处理对GTP类似物的浓度-效应曲线没有影响，但导致GTP刺激该系统的表观Kα降低了5至9倍。这些结果表明，GTP及其类似物对肝腺苷酸环化酶的刺激程度与鸟苷三磷酸β-γ键被水解的敏感性无关，并且进程曲线中的延迟不是由于“常驻”GDP分子的缓慢解离。研究了大鼠肝腺苷酸环化酶与GTP和GMP-P(NH)P组合相互作用的时间瞬态，发现无论两种核苷酸添加的时间和顺序如何均为竞争型。结果与GMP-P(NH)P与该系统相互作用和解离非常缓慢且不会导致酶形成不可逆激活状态一致。关于鸟苷核苷酸对基础（未添加激素）肝腺苷酸环化酶调节所获得的数据根据双态酶模型（Birnbaumer, L., Bearer, C.F., and Iyengar, R.(1980) J. Biol. Chem. 255, 3552 - 3557）进行解释，并根据目前关于腺苷酸环化酶调节和GTP酶参与的观点进行讨论。我们的实验与肝腺苷酸环化酶紧密相关的霍乱毒素抑制性GTP酶的存在一致，但不能证明其存在。
