CONFORMATION AND ION-CHANNEL ACTIVITY OF LYMPHOTOXIN AT NEUTRAL AND LOW PH

Lymphotoxin (LT or TNF-beta), a T cell-derived lymphokine with partial homology to TNF-ar, was found to bind to dimyristoylphosphatidylcholi ne vesicles in a pH-dependent manner: binding increased with decreasin g pH. Binding was not limited to surface association with phospholipid head groups because studies with a photoreactive membrane-restricted probe revealed protein penetration of the hydrocarbon core of the bila yer. Intramembranous photolabeling of the trimeric form of LT demonstr ated maintenance of quaternary structure upon bilayer insertion. The e fficiency of insertion was greatly enhanced with gel-phase bilayers co mpared with fluid phase bilayers even though the binding efficiency wa s much lower. Hence, binding and insertion represent two distinct phys ical processes. Intrinsic fluorescence and dye binding assays showed t hat the acid-facilitated membrane interactions stemmed from acid-induc ed changes in protein conformation. The acquisition of hydrophobic cha racteristics through these conformational changes supplies a physical explanation for LT conversion from a water-soluble form to a membrane- embedded structure. Moreover, the use of vesicle-embedded LT to prepar e planar bilayers vs the addition of soluble LT subsequent to bilayer formation demonstrated that LT exhibits channel activity and that low pH-induced membrane insertion precedes and is distinct from expression of voltage-dependent ion gating. LT's ability to associate intimately with lipid vesicles and form ion channels mirrors the behavior of TNF -alpha. Thus, although LT and TNF-alpha are secreted by different cell types, the conservation of membrane binding, insertion, and channel-f orming activities suggests a functional role in response induction.