Biophysical studies on the lipid transfer particle from the hemolymph of the tobacco hornworm, Manduca sexta

Abstract

Hydrodynamic studies conducted in the analytical ultracentrifuge provided evidence for two populations of lipid transfer particle (LTP) when centrifuged in a buffer solution containing 10 mM Tris, pH S.O/lOO mM KCl. The apparent sedimentation coefficients of the two species was 23.3 S and 15.3 S. Upon changing the buffer pH to 7.0 or 5.7, two species of LTP were still present but the ratio of their relative abundance was altered. When the KC1 concentration in the buffer was lowered to 50 mM the sample sedimented as a single species with an apparent s~,~ of 22.9 S. In higher ionic strength buffers (10 mM succinate, pH 5.7/500 mM KCl) LTP sedimented with an apparent s~,~ of 14.8 S. Further experiments revealed that these two forms are interconvertable as a function of buffer ionic strength. Given previous estimates of the molecular size of LTP we concluded that the slower sedimenting peak observed at high ionic strength represents monomeric LTP while the faster sedimenting material observed at low ionic strength is likely to be an aggregated state of LTP. This interpretation is supported by molecular weight determinations made by sedimentation equilibrium experiments conducted in 10 mM succinate, pH 5.7/500 mM KC1 which yielded a particle M,= 887000. Circular dichroism spectra of monomeric LTP sample revealed 6% a-helix, 49% B-sheet, 7% /?-turn and 35% random coil while aggregated LTP contained 13% a-helix, 66% p-sheet and 21% random coil. The transfer activity of the two LTP forms was assayed and found to be the same indicating that either the state of LTP aggregation did not affect transfer activity or that upon exposure to a large excess of lipoprotein substrate disaggregation, without loss of activity, occurs.