Feltner D E, Marasco W A
Department of Pathology, University of Michigan Medical School, Ann Arbor 48109.
J Immunol. 1989 Jun 1;142(11):3963-70.
The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.
通过解离技术研究单价阳离子、鸟嘌呤核苷酸和细菌毒素对[3H]FMLP与兔中性粒细胞质膜结合的调节作用，以确定对不同受体状态的调节效应。在低受体占有率（1 nM [3H]FMLP）条件下，以及在Na+和K+缓冲液中，解离是异质性的，呈现出两种不同的、具有统计学意义的解离速率。用其他单价阳离子替代Na+可增强[3H]FMLP结合。特别是，相对于Na+，K+存在时结合增强是由于与快速和缓慢解离受体的额外结合。通过特定的GTP和百日咳毒素（PT）处理确定了三种受体解离速率，其中两种似乎对应于平衡结合研究中检测到的两种亲和力状态。GTP、PT或霍乱毒素（CT）对[3H]FMLP从快速解离形式受体的解离速率均无影响。100 microM GTP和PT处理均增加了快速解离受体的百分比，相应降低了缓慢解离受体的百分比。GTP、PT和CT处理后快速和缓慢解离受体的观察变化是由于与缓慢解离受体结合量的绝对减少。然而，从未观察到GTP、PT或两者对缓慢解离受体结合的完全抑制。GTP和PT处理，但不是CT处理，使1 nM [3H]FMLP从缓慢解离形式受体的解离速率增加了两倍，导致第三种解离速率。因此，缓慢解离受体包括两种不同的受体状态，一种是与G蛋白相关的鸟嘌呤核苷酸和PT敏感状态，另一种是鸟嘌呤核苷酸不敏感状态。
