Enhancement of adsorptive pinocytosis of a polyaspartamide by incorporation of tyramine residues

We have previously shown that poly a$-( N-2-hydroxyethyl)-D,L-aspartamide is captured by rat visceral yolk sacs cultured in vitro by pinocytosis (Duncan et al., 1982) and also that substitution of the parent polymer with tyramine residues (19.2molX) caused a marked increase in its rate of capture. In this study we have investigated this phenomenon further by synthesizing a series of polyaspartamides with a range of tyramine contents (1.2, 4.9, 10.4, 16.9 and 21.9 mol%) and studying the relationship between their tyramine content and their rates of pinocytosis by rat visceral yolk sacs cultured in uitro. Using samples of narrow polydispersity we examined the effect of molecular weight on rate of capture. The effect of serum proteins and substrate concentration on the rate of polymer capture was also investigated. The polyaspartamide derivatives were prepared as previously described (VlsPk et al., 1979; Drobnik et al., 1979) and radiolabelled with lz5I using the chloramine-T method (Duncan et al., 1982). The resultant 1251-labelled preparations were stable during both storage and incubation. To quantify their rates of capture by rat visceral yolk sacs, 1251-labelled polymers were incubated with the tissue in serum-free medium 199 and uptake measured as described previously (Ibbotson & Williams, 1979). It was found that pinocytic capture of the radiolabelled polymers was linear with time over a 5 h incubation period and that the rate of uptake of polyaspartamide increased with increasing tyramine content. Above a tyramine content of 10mol% the rate of uptake of polymer increased dramatically and linearly with rising tyramine content of the polymer (up to 21.9mol%). The effect of increasing tyramine content on the pinocytic uptake of polyaspartamide derivatives was very similar to that already described for tyrosinamidecontaining N-2-hydroxypropyl methacrylamide copolymers (Duncan et al., 1984). The molecular weight of synthetic polymers has been shown previously to influence their rate of pinocytosis by yolk sacs in vitro (Duncan et al., 1982; Cartlidge et al., 1982). Three samples were chosen for this study, each containing 4.9molx tyramine, but of M , 8000, 39000 and 60000. Uptake by yolk sacs was measured over a 3 h incubation period and all three samples were captured at the same rate. Therefore over the molecular weight range studied, polymer size has no effect on rate of capture. The presence of calf serum (10-50%) in the incubation medium severely inhibited capture of all polyaspartamide derivatives. Although this effect was partially attributable to the inhibition of pinocytic vesicle formation in the yolk sac by serum (Forster & Williams, 1984), competition by serum proteins with polyaspartamide molecules for cellsurface binding sites was largely responsible for the decrease in the rate of polymer capture observed. As the serum proteins can effectively compete with the polymeric substrate, the polymer-membrane interaction is likely to be very non-specific. The effect of substrate concentration (5-100jig/ml) on the uptake of the derivatives containing the least (1.2mol%) and the most (21.9molX) tyramine was examined. In this experiment a trace amount of radiolabelled polymer was added to the incubation in addition to the appropriate concentration of non-radioactively labelled polymer. Whereas increasing substrate concentration had no effect on the rate of uptake (measured in pl/mg of protein per h) of the former, it caused a progressive decrease in the rate of capture of the latter. These data indicate that over the substrate concentration range studied the polymer with low tyramine content is captured by fluid-phase pinocytosis whereas the more highly substituted polymer is captured by adsorptive pinocytosis.