CHARACTERIZATION OF THE TOBACCO GLYCOPROTEIN SURFACE BINDING PROPERTYOF HEART AND SKELETAL-MUSCLE CELLS .1. MODULATION OF THE HEART CELL-MEMBRANE TGP INTERACTION BY ANTI-TGP IGG

Monolayers of L(6) rat skeletal myoblast cells formed surface binding isotherms with the purified tobacco leaf glycoprotein TGP(1) and the e nriched cigarette tar glycoprotein TGP(2). Scatchard analysis showed t hat the binding in the range of the limited concentrations tested was to a single class molecule and the calculated affinity constant (K-d) for TGP(1) and TGP(2) showed similar values (9.78 x 10(-13) M and 3.09 x 10(-13) M, respectively). The bound TGPs were almost totally displa ced by excess non-radiolabeled molecules. The calculated B-max of the L(6) myoblast monolayer was 2.93 fmol for TGP(1) and 0.217 fmol for TG P(2) per 32.2 mm(2). Guinea pig heart sarcolemma binding isotherms wer e also formed with radiolabeled TGP(1) and TGP(2). The interaction of tobacco leaf TGP(1) with the heart cell membranes was irreversible bec ause only 15-20% of the bound TGP(1) was displaced by 100-fold, non-la beled molecules but the interaction of tar TGP(2) with heart sarcolemm a was reversible and probably saturable. The heart sarcolemma TGP(2) a ffinity constant (K-d) was 5.88 x 10(-7) M and the B-max, 2.45 X 10(-8 ) M per 12.5 mu g sarcolemma. Pretreatment of heart sarcolemma with in creasing concentrations of leaf TGP(1) did not displace tar TGP(2) bin ding but its absorption on the membrane resulted in increased TGP(2) s arcolemma attachment by a complex and unexplained mechanism. Increasin g concentrations of the sera of 10 of 15 guinea pigs (67%) that receiv ed mainstream emissions of tobacco smoke from a University of Kentucky cigarette smoking machine for 152 days, displaced cigarette tar TGP(2 ) heart cell sarcolemma attachment and this inhibition was significant ly different from that produced by the sera of sham smoked and of none xposed animals (Mann-Whitney test, p = 0.0082). Staphylococcus protein A inhibited the displacement of TGP(2) produced by the sera of cigare tte smoke exposed guinea pigs and this observation indicated that this action was mediated by Ige molecules. The specific immunoprecipitatio n of a radiolabeled surface epitope of the L(6) myoblast monolayers pr etreated with TGP(1) or TGP(2) by immune IgG against TGP(2) and by the IgG of an antiserum against standard TGP showed that the tobacco glyc oproteins attached to a unit polypeptide of the plasma membrane of the muscle cells of approximately 76 kDa. These data support the notion t hat TGP molecules in cigarette smoke are absorbed systemically on smok ing and may have a direct toxic effect when they attach to the surface TGP binding proteins of heart and skeletal muscle cells.