Fung B K, Young J H, Yamane H K, Griswold-Prenner I
Jules Stein Eye Institute, UCLA School of Medicine 90024.
Biochemistry. 1990 Mar 20;29(11):2657-64. doi: 10.1021/bi00463a006.
The cyclic GMP phosphodiesterase of the retinal rod is composed of three distinct types of polypeptides: alpha (90 kDa), beta (86 kDa), and gamma (10 kDa). The gamma subunit has been shown to inhibit phosphodiesterase activity associated with alpha and beta. To investigate the subunit stoichiometry of the retinal phosphodiesterase, we have developed a panel of monoclonal and peptide antibodies that recognize individual phosphodiesterase subunits. By quantitative and immunochemical analysis of the purified subunits, we have shown that each phosphodiesterase molecule contains one copy each of alpha and beta subunit and two copies of gamma subunit. Moreover, gamma can be chemically cross-linked to both alpha and beta, but not to itself, suggesting that alpha and beta may each bind one gamma. The phosphodiesterase is fully activated when both copies of gamma were removed by proteolysis with trypsin. Upon recombination of the purified gamma subunit with the trypsin-activated phosphodiesterase containing alpha beta, the alpha beta gamma 2 stoichiometry is once again restored, with concomitant total inhibition of activity. Our results suggest that at least two activated transducin molecules are required to fully activate one molecule of phosphodiesterase in retinal rods.
α（90 kDa）、β（86 kDa）和γ（10 kDa）。已证明γ亚基可抑制与α和β相关的磷酸二酯酶活性。为了研究视网膜磷酸二酯酶的亚基化学计量，我们开发了一组识别单个磷酸二酯酶亚基的单克隆抗体和肽抗体。通过对纯化亚基的定量和免疫化学分析，我们表明每个磷酸二酯酶分子包含一个α亚基拷贝、一个β亚基拷贝和两个γ亚基拷贝。此外，γ可以化学交联到α和β，但不能交联到其自身，这表明α和β可能各自结合一个γ。当用胰蛋白酶进行蛋白水解去除两个γ拷贝时，磷酸二酯酶被完全激活。将纯化的γ亚基与含有αβ的胰蛋白酶激活的磷酸二酯酶重组后，αβγ2化学计量再次恢复，同时活性被完全抑制。我们的结果表明，在视网膜视杆细胞中，至少需要两个激活的转导素分子才能完全激活一个磷酸二酯酶分子。
