Characterization of the tobacco glycoprotein surface binding property of heart and skeletal muscle cells. I. Modulation of the heart cell membrane TGP interaction by anti-TGP IgG.

Abstract

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.