Orr I, Martin C, Ashley R, Shoshan-Barmatz V
Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel.
J Biol Chem. 1993 Jan 15;268(2):1376-82.
Fluorescein 5'-isothiocyanate (FITC) markedly inhibited ryanodine binding to rabbit skeletal muscle junctional sarcoplasmic reticulum. Half-maximal inhibition was obtained with about 20 microM and complete inhibition by 80 microM FITC. Inhibition was enhanced in the presence of high salt and at alkaline pH. The pH dependence of the inactivation of ryanodine binding suggested that FITC bound to a very reactive lysine epsilon-amino group with a pK alpha of about 7.5 or above. Kinetic analysis of the time course of inactivation of ryanodine binding by various concentrations of FITC suggested that the inactivation resulted from the modification of 2 or more amino acid residues. The inhibition of ryanodine binding by FITC was partially prevented by ATP, ADP, adenyl imidodiphosphate, and 3-O-(benzoyl)-benzoyl-ATP (Bz-ATP) but not by AMP. FITC modification of sarcoplasmic reticulum membranes inhibited the photoaffinity labeling by [alpha-32P]Bz-ATP of the 450-kDa protein and the ryanodine receptor with half-maximal inhibition at about 100 microM. The results suggest that the inhibition of ryanodine binding is mainly due to FITC modification of sites which are not involved in ATP binding. The FITC moiety is bound to the 160-, 96-, 76-, and 60-kDa ryanodine receptor tryptic fragments, and the FITC site is apparently on the 21.5-, 18-, and 17-kDa fragments which are formed by the V8 protease. Covalent modification by FITC dramatically affected the activity of single Ca2+ release channels incorporated into planar lipid bilayers. FITC caused a marked increased in channel open probability mainly to a noisy approximately 60% subconductance state. FITC-modified channels were no longer affected by ryanodine but were still abolished by Mg2+ and ruthenium red. We suggest that FITC modifies reactive lysine residues involved in channel activation by transmembrane charge movement in the t-tubular system.
异硫氰酸荧光素（FITC）显著抑制了ryanodine与兔骨骼肌连接肌浆网的结合。约20微摩尔的FITC可产生半数抑制作用，80微摩尔的FITC则可完全抑制。在高盐存在及碱性pH条件下，抑制作用增强。ryanodine结合失活的pH依赖性表明，FITC与一个非常活泼的赖氨酸ε-氨基结合，其pKα约为7.5或更高。对不同浓度FITC使ryanodine结合失活的时间进程进行动力学分析表明，失活是由2个或更多氨基酸残基的修饰所致。ATP、ADP、腺苷亚氨基二磷酸和3 - O -（苯甲酰基）-苯甲酰基-ATP（Bz - ATP）可部分阻止FITC对ryanodine结合的抑制，但AMP则不能。肌浆网膜的FITC修饰抑制了[α-32P]Bz - ATP对450 kDa蛋白和ryanodine受体的光亲和标记，约100微摩尔时产生半数抑制作用。结果表明，ryanodine结合的抑制主要是由于FITC对不参与ATP结合的位点的修饰。FITC部分与160 kDa、96 kDa、76 kDa和60 kDa的ryanodine受体胰蛋白酶片段结合，FITC位点显然位于由V8蛋白酶形成的21.5 kDa、18 kDa和17 kDa片段上。FITC的共价修饰显著影响了整合到平面脂质双分子层中的单个Ca2+释放通道的活性。FITC使通道开放概率显著增加，主要转变为一个嘈杂的约60%亚电导状态。FITC修饰的通道不再受ryanodine影响，但仍可被Mg2+和钌红消除。我们认为，FITC修饰了参与通过横管系统跨膜电荷移动激活通道的活性赖氨酸残基。
