ATP稳定肌浆网膜蛋白色氨酸相关构象的机理的探讨

STUDY OF ATP EFFECTS ON CONFORMATION STABILITY OF THE SARCOPLASMIC RETICULUM MEMBRANE PROTEIN

在无ATP存在时,带相同正电荷、但饱和度不同的两种胺类两亲物,即C_(18)饱和的硬脂胺与单不饱和的油胺引起肌浆网蛋白内源荧光强度降低.当ATP或一些阴离子化合物(如磷酸盐或草酸盐)先与肌浆网作用,再加入硬脂胺或油胺,则肌浆网蛋白内源荧光下降幅度明显减小,即存在拮抗作用.腺苷对硬脂胺或油胺均无此拮抗作用.肌浆网与硬脂胺先保温,再加入ATP或阴离子化合物,它们对硬脂胺的拮抗作用消失.与硬脂胺不同,肌浆网与油胺先保温,再加入ATP或阴离子化合物,ATP仍有拮抗、阴离子化合物对油胺则无拮抗作用.然而在肌浆网钙泵蛋白(即Ca^(2+),Mg^(2+)-ATPase)上存在结合位点的ADP和GTP可显著拮抗油胺引起的肌浆网内源荧光强度的降低,而AMP无此拮抗作用.以上提示,ATP有稳定肌浆网膜蛋白构象的作用.它对硬脂胺的拮抗主要依赖于ATP分子内部的负电性基因,而对油胺的拮抗则可能是通过其作为底物引起钙泵蛋白构象变化所产生的一种保护作用.

Stearylamine and oleylamine. C18 saturated and cis -monounsaturated amines, with positive charges. were found to decline the intensity of the intrinsic protein fluorescence of skeletal muscle sarcoplasmic reticulum (SR) in the absence of ATP. When ATP or anion compounds, such as phosphate and oxalate. were added into reaction solution prior to the addition of these amines. the descended extents of fluorescence intensity induced by Stearylamine or oleylamine were decreased. which is called antagonism in this study, and the antagonism to stearylamine was stronger than that to oleylamine. Adenosine had no effect on the falls of fluorescence intensity caused by both the amines. After incubation of SR with Stearylamine. addition of ATP or anion compounds did not antagonize the fall of fluorescence intensity induced by Stearylamine. Inconsistent with Stearylamine. after incubation of SR with oleylamine, the fall of fluorescence intensity induced by oleylamine was antagonized by ATP.but not by anion compounds. The incubation of SR with ADP and GTP.which have their corresponding binding sites on SR Ca2+,Mg2+ -ATPase. respectively. antagonized the fall of fluorescence intensity induced by oleylamine significantly. While in case of .AMP. the antagonism was not observed. These results suggested that ATP may stabilize the membrane protein conformation in sarcoplasmic reticulum and the antagonism of ATP to Stearylamine may mainly depend on the negative charges in the molecule, whereas that to oleylamine may be due to substrate protection through the conformational change of calcium pump protein.