Effect of 3-phenylamino-l-alanine on tryptophan binding to rat hepatic nuclear envelopes

We have determined that the addition of 3-phenylamino-l-alanine (PAA), a recently reported contaminant in l-tryptophan implicated in the eosinophilia-myalgia syndrome, affects tryptophan binding by utilizing an in vitro measurement of 3H-tryptophan binding to hepatic nuclei or nuclear envelopes. PAA (10-10 to 10-4M) diminishes the inhibitory effect of binding due to excess unlabeled l-tryptophan (10-4M). PAA alone has no inhibitory effect on binding. The effect of PAA on in vitro tryptophan binding is in contrast to that of another contaminant, 1,1′-ethylidenebis(tryptophan), which together with excess unlabeled l-tryptophan does not appreciably affect the binding. In vitro addition of PAA and l-tryptophan to nuclei of rat brain or of cultured murine macrophages does not affect [3H]tryptophan binding in comparison to l-tryptophan alone as if the case with hepatic nuclear envelopes. Adding PAA to an in vitro protein synthesis system and measuring [3H]tryptophan or [3H]alanine incorporation into acid-precipitable proteins reveals that it competes similarly, but somewhat less, than does equimolar concentrations of unlabeled l-tryptophan or l-alanine, respectively. This suggests that PAA or a breakdown compound becomes incorporated into proteins. Speculation as to how PAA may affect tissues in experimental animals is presented. © 1994

Studies with 1,1’-ethylidenebis(tryptophan), a contaminant associated with L-tryptophan implicated in the eosinophilia-myalgia syndrome

L-Tryptophan binds to a rat liver nuclear envelope protein, and this binding is saturable, stereospecific, and of high affinity. Utilizing an in vitro assay of [3H]tryptophan binding to rat hepatic nuclear envelopes, we have previously determined that the L-tryptophan obtained from Showa Denko and which was implicated in cases of the eosinophilia-myalgia syndrome (EMS) inhibited [3H]tryptophan binding differently than did control L-tryptophan (not implicated in EMS). Therefore, in this study we investigated whether the addition of 1,1\u27-ethylidenebis(tryptophan) (EBT), a contaminant or impurity in L-tryptophan implicated in EMS, would have an effect. Our results indicate that EBT alone has little inhibitory binding effect compared with that of control L-tryptophan and that when EBT was added to control L-tryptophan the inhibitory binding effort was similar to that of control L-tryptophan alone. On the other hand, in vitro addition of EBT plus L-tryptophan to nuclei of cultured murine macrophages (WLG5) results in less inhibition of [3H]tryptophan binding than does addition of L-tryptophan alone. Similar in vitro additions to nuclei of rat brain reveal little effect on binding, as was also the case for hepatic nuclear envelopes. Adding EBT to an in vitro hepatic protein synthesis system and measuring [3H]tryptophan incorporation into acid-precipitable proteins reveal that it competes similarly to that found with equimolar concentrations of unlabeled L-tryptophan. It does not affect [14C]leucine incorporation into proteins. [14C]EBT becomes incorporated in vitro into proteins (acid-precipitable), and this incorporation is diminished in the presence of equimolar concentrations of unlabeled EBT or L-tryptophan. This suggests that EBT or possibly a breakdown product becomes incorporated into proteins. Speculation as to how EBT may affect tissues in experimental animals is presented. © 1994 Academic Press, Inc