Santos-Buch C A, Hall H R, Farfan F, Orlow I, Firpo A, von Kreuter B F, Becker C G
Department of Pathology, Cornell University Medical College, New York, New York, USA.
Arch Toxicol. 1995;69(3):149-59. doi: 10.1007/s002040050151.
Monolayers of L6 rat skeletal myoblast cells formed surface binding isotherms with the purified tobacco leaf glycoprotein TGP1 and the enriched cigarette tar glycoprotein TGP2. Scatchard analysis showed that the binding in the range of the limited concentrations tested was to a single class molecule and the calculated affinity constant (Kd) for TGP1 and TGP2 showed similar values (9.78 x 10(-13) M and 3.09 x 10(-13) M, respectively). The bound TGPs were almost totally displaced by excess nonradiolabeled molecules. The calculated Bmax of the L6 myoblast monolayer was 2.93 fmol for TGP1 and 0.217 fmol for TGP2 per 32.2 mm2. Guinea pig heart sarcolemma binding isotherms were also formed with radiolabeled TGP1 and TGP2. The interaction of tobacco leaf TGP1 with the heart cell membranes was irreversible because only 15-20% of the bound TGP1 was displaced by 100-fold, non-labeled molecules but the interaction of tar TGP2 with heart sarcolemma was reversible and probably saturable. The heart sarcolemma TGP2 affinity constant (Kd) was 5.88 x 10(-7) M and the Bmax, 2.45 x 10(-8) M per 12.5 micrograms sarcolemma. Pretreatment of heart sarcolemma with increasing concentrations of leaf TGP1 did not displace tar TGP2 binding but its absorption on the membrane resulted in increased TGP2 sarcolemma attachment by a complex and unexplained mechanism. Increasing concentrations of the sera of 10 of 15 guinea pigs (67%) that received mainstream emissions of tobacco smoke from a University of Kentucky cigarette smoking machine for 152 days, displaced cigarette tar TGP2 heart cell sarcolemma attachment and this inhibition was significantly different from that produced by the sera of sham smoked and of non-exposed animals (Mann-Whitney test, p = 0.0082). Staphylococcus protein A inhibited the displacement of TGP2 produced by the sera of cigarette smoke exposed guinea pigs and this observation indicated that this action was mediated by IgG molecules. The specific immunoprecipitation of a radiolabeled surface epitope of the L6 myoblast monolayers pretreated with TGP1 or TGP2 by immune IgG against TGP2 and by the IgG of an antiserum against standard TGP showed that the tobacco glycoproteins attached to a unit polypeptide of the plasma membrane of the muscle cells of approximately 76 kDa. These data support the notion that TGP molecules in cigarette smoke are absorbed systemically on smoking and may have a direct toxic effect when they attach to the surface TGP binding proteins of heart and skeletal muscle cells.
L6大鼠骨骼肌成肌细胞单层与纯化的烟草叶糖蛋白TGP1和富集的香烟焦油糖蛋白TGP2形成表面结合等温线。Scatchard分析表明，在所测试的有限浓度范围内，结合是针对单一类分子的，计算得出的TGP1和TGP2的亲和常数（Kd）显示出相似的值（分别为9.78×10⁻¹³ M和3.09×10⁻¹³ M）。结合的TGPs几乎完全被过量的未标记分子取代。计算得出，每32.2平方毫米的L6成肌细胞单层的TGP1的Bmax为2.93飞摩尔，TGP2的Bmax为0.217飞摩尔。用放射性标记的TGP1和TGP2也形成了豚鼠心脏肌膜结合等温线。烟草叶TGP1与心脏细胞膜的相互作用是不可逆的，因为只有15 - 20%的结合TGP1被100倍的未标记分子取代，但焦油TGP2与心脏肌膜的相互作用是可逆的，且可能是可饱和的。心脏肌膜TGP2的亲和常数（Kd）为5.88×10⁻⁷ M，每12.5微克肌膜的Bmax为2.45×10⁻⁸ M。用浓度递增的烟草叶TGP1预处理心脏肌膜并没有取代焦油TGP2的结合，但它在膜上的吸附通过一种复杂且无法解释的机制导致TGP2在肌膜上的附着增加。15只豚鼠中有10只（67%）接受来自肯塔基大学吸烟机的主流烟草烟雾排放152天，其血清浓度增加时，取代了香烟焦油TGP2与心脏肌膜的附着，这种抑制作用与假吸烟和未暴露动物的血清所产生的抑制作用有显著差异（Mann - Whitney检验，p = 0.0082）。葡萄球菌蛋白A抑制了香烟烟雾暴露豚鼠血清产生的TGP2的取代作用，这一观察结果表明这种作用是由IgG分子介导的。用针对TGP2的免疫IgG和针对标准TGP的抗血清的IgG对用TGP1或TGP2预处理的L6成肌细胞单层的放射性标记表面表位进行特异性免疫沉淀，结果表明烟草糖蛋白附着在约76 kDa的肌肉细胞质膜的一个单位多肽上。这些数据支持这样一种观点，即香烟烟雾中的TGP分子在吸烟时被全身吸收，当它们附着在心脏和骨骼肌细胞的表面TGP结合蛋白上时可能具有直接的毒性作用。
