Moffet F J, Kidwai A M, Bär H P
Recent Adv Stud Cardiac Struct Metab. 1976;9:183-91.
The method of Kidwai et al. (1971. Biochem. Biophys. Res. Commun. 45:901) offers a rapid and simple technique for the preparation of membrane fractions from rat heart, using sucrose density centrifugation of a 100,000x g pellet. We have investigated the distribution of adenylate cyclase, cyclic AMP phosphodiesterase, and norepinephrine binding activity in these fractions. Specific activity of adenylate cyclase was high in the plasma membrane (PM) and sarcoplasmic reticulum, as well as the nuclear (N) fractions, but 80-90 percent of the total activity was found in the N fraction. Epinephrine stimulation of adenylate cyclase was present in all fractions but did not exceed 25 percent of basal activity. The activity in the mitochondrial fraction was low and insensitive to epinephrine. About 10 percent of phosphodiesterase activity was found in the 100,000 x g pellet. After density gradient centrifugation, 70 percent of this portion was recovered in the N fraction, with the highest specific activity present in the PM fraction. Binding of [3H] norepinephrine was measured by a membrane filtration technique. Binding activity was found in all fractions, and it paralleled roughly the distribution of adenylate cyclase. However, only about 25-30 percent of the binding was blocked by propranolol, except in the PM fraction where binding was not prevented by this drug. Further, a comparison of adenylate cyclase activities with norepinephrine binding yields a turnover number for the enzyme of the order of 10(-2) sec-1, assuming a 1:1 relationship between beta-adrenergic recptor and adenylate cyclase. Since this value seems unrealistic, we suspect that either the binding activity measured is unrelated to the beta-adrenergic receptor or the receptor to cyclase ratio is considerably larger than unity.
基德瓦伊等人（1971年，《生物化学与生物物理研究通讯》45:901）的方法提供了一种快速简便的技术，用于从大鼠心脏制备膜组分，采用对100,000×g沉淀进行蔗糖密度离心。我们研究了腺苷酸环化酶、环磷酸腺苷磷酸二酯酶和去甲肾上腺素结合活性在这些组分中的分布。腺苷酸环化酶的比活性在质膜（PM）、肌浆网以及核（N）组分中较高，但总活性的80 - 90%存在于N组分中。所有组分中均存在肾上腺素对腺苷酸环化酶的刺激作用，但不超过基础活性的25%。线粒体组分中的活性较低且对肾上腺素不敏感。约10%的磷酸二酯酶活性存在于100,000×g沉淀中。密度梯度离心后，该部分的70%在N组分中回收，PM组分中比活性最高。通过膜过滤技术测量[3H]去甲肾上腺素的结合。所有组分中均发现结合活性，且其大致与腺苷酸环化酶的分布平行。然而，只有约25 - 30%的结合被普萘洛尔阻断，PM组分除外，该药物对其结合无抑制作用。此外，假设β - 肾上腺素能受体与腺苷酸环化酶呈1:1关系，将腺苷酸环化酶活性与去甲肾上腺素结合进行比较，得出该酶的周转数约为10(-2)秒-1。由于该值似乎不切实际，我们怀疑所测量的结合活性与β - 肾上腺素能受体无关，或者受体与环化酶的比例远大于1。
