Olate J, Allende C C, Allende J E, Sekura R D, Birnbaumer L
FEBS Lett. 1984 Sep 17;175(1):25-30. doi: 10.1016/0014-5793(84)80562-1.
Membranes were obtained from Xenopus laevis oocytes after removal of follicular cells by collagenase treatment. [32P]ADP-ribosylation with pertussis toxin showed them to contain a single Mr = 40000 substrate for this toxin that co-migrates on sodium dodecylsufate-polyacrylamide gel electrophoresis with pure human erythrocyte Ni, the inhibitory regulatory component of adenylyl cyclase. [32P]ADP-ribosylation of oocyte membranes with cholera toxin also showed presence of a single substrate but of Mr = 42000. These results indicate, that the adenylyl cyclase system of oocytes, like that of somatic cells and unlike that of spermatozoids, contains the catalytic unit C and both of the known regulatory N components. The possible susceptibility to pertussis toxin of the guanine nucleotide-dependent inhibition of oocyte adenylyl cyclase by progesterone was investigated. This action of progesterone is mediated by a membrane bound receptor as opposed to a receptor of cytosolic or nuclear localization. However, the inhibitory effect of progesterone was unaffected by pertussis toxin, even though the oocyte membrane Ni was fully ADP-ribosylated with pertussis toxin, as revealed by lack of further [32P]ADP-ribosylation on subsequent re-incubation with pertussis toxin. These results indicate that the action of progesterone, in spite of being nucleotide-dependent, is either not mediated by Ni, suggesting the existence of an additional nucleotide regulatory component, or if mediated by Ni, involves a mode of regulation of this coupling protein that is different from that by which all other inhibitory hormones act on adenylyl cyclase.
通过胶原酶处理去除卵泡细胞后,从非洲爪蟾卵母细胞中获取细胞膜。用百日咳毒素进行的[32P]ADP-核糖基化显示,它们含有一种单一的、分子量为40000的该毒素底物,在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上与纯人红细胞Ni(腺苷酸环化酶的抑制性调节成分)共迁移。用霍乱毒素对卵母细胞膜进行的[32P]ADP-核糖基化也显示存在一种单一底物,但分子量为42000。这些结果表明,卵母细胞的腺苷酸环化酶系统与体细胞的类似,与精子的不同,包含催化单位C和两种已知的调节性N成分。研究了孕酮对卵母细胞腺苷酸环化酶鸟嘌呤核苷酸依赖性抑制作用可能对百日咳毒素的敏感性。孕酮的这种作用是由膜结合受体介导的,与胞质或核定位的受体不同。然而,尽管卵母细胞膜Ni已被百日咳毒素完全ADP-核糖基化(后续再与百日咳毒素孵育时缺乏进一步的[32P]ADP-核糖基化表明),但孕酮的抑制作用不受百日咳毒素影响。这些结果表明,孕酮的作用尽管依赖核苷酸,但要么不是由Ni介导的,提示存在一种额外的核苷酸调节成分,要么如果由Ni介导,则涉及该偶联蛋白的一种调节模式,该模式不同于所有其他抑制性激素作用于腺苷酸环化酶的模式。