GM_3对重建Gs与AC偶联功能的影响

EFFECT OF GM3 ON THE COUPLING FUNCTION BETWEEN Gs AND AC

将神经节苷脂ＧＭ３（Ｍｏｎｏｓｉａｌｏｇａｎｇｌｉｏｓｉｄｅ－ＧＭ３）通过保温法掺入到含激活型Ｇ蛋白（ＳｔｉｍｕｌａｔｏｒｙＧＴＰ－ｂｉｎｄｉｎｇｐｒｏｔｅｉｎ，Ｇｓ）与腺苷酸环化酶（ＡｄｅｎｙｌｙｌＣｙｃｌａｓｅ，ＡＣ）的脂酶体中，研究了ＧＭ３对Ｇｓ和ＡＣ偶联功能的影响。实验结果表明，在４－１０μｍｏｌ／Ｌ浓度范围的ＧＭ３增加ＡＣ的基础活力；在高于４μｍｏｌ／Ｌ时，ＧＭ３可显著抑制Ｇｓ激活ＡＣ的能力；而在ＧＭ３浓度高于１００μｍｏｌ／Ｌ的条件下，Ｇｓ结合ＧＴＰγＳ（Ｇｕａｎｏｓｉｎｅ５＇－Ｏ－（３－ｔｈｉｏｔｒｉｐｈｏｓｐｈａｔｅ））的活力受到明显抑制。随外源ＧＭ３浓度的增加，ＧＭ３对Ｇｓ激活ＡＣ的能力与对ＡＣ基础活力的影响似乎并不完全一致。这些结果提示，Ｇｓ与ＡＣ的解偶联对较低浓度的ＧＭ３的影响更加敏感。用荧光探剂ＭＣ５４０标记脂酶体，测量其荧光光谱的结果显示，随着ＧＭ３浓度增加，ＭＣ５４０的荧光强度增强，这说明外源性的ＧＭ３的掺入使膜脂质分子头部的堆积变得更加疏松。这可能提示，ＧＭ３介导的膜脂物理状态的变化是调节Ｇｓ与ＡＣ偶联功能的重要因素之一。

The ganglioside GM3 was incorporated by incubation into the proteoliposomes containing Gs(Stimulatory GTP-binding protein) and AC(Adenylyl Cyclase), and its effect on the coupling function of Gs and AC was studied. The results indicated that in low concentrations (4～10μmol/L) of added GM3, the basal activity of AC was stimulated, while the activities of Gs stimulating AC, and of Gs binding to GTPγS were significantly inhibited with the concentration of GM3 higher than 4, and 100μmol/L, respectively. With increase of added GM3, the effect of GM3 on Gs stimulating AC was not in accord with that of GM3 on the basal activity of AC. The results may suggest that the uncoupling between Gs and AC was more sensitive to low concentration of GM3. Fluorescence changes of the proteoliposomes with or without GM3, using MC540 as a fluorescence probe, were measured. The results showed that the fluorescent intensity of the labeled proteoliposomes enhanced with increase of GM3, which indicated that added GM3 would loosen packing of the lipid molecules in the proteoliposomes. In conclusion, it seemed that GM3 regulated the coupling function between Gs and AC probably by mediating the fluidity of lipid bilayer in the membranes.