PURIFICATION, CHARACTERIZATION, AND CONFORMATIONAL-ANALYSIS OF RABBITPLASMA-LIPID TRANSFER PROTEIN

A procedure for rapid isolation of lipid transfer protein (LTP) from c ommercially available rabbit plasma is described. Use of protease inhi bitors was important for obtaining intact, stable LTP. After lipoprote ins were precipitated from the plasma by dextran sulfate, column chrom atographies through Butyl-Toyopearl 650M, CM-Toyopearl 650M, and Butyl -Toyopearl 650M were employed. Overall purification from plasma was (3 830 +/- 710)-fold with a yield of 3-5%. The isolated LTP migrated as a single band during sodium dodecyl sulfate-polyacrylamide gel electrop boresis with M(r) = 74K and had an NH2-terminal amino acid sequence an d amino acid composition closely matching those predicted by its cDNA. This band was recognized by immunoblotting with an anti-human LTP mon oclonal antibody, TP2. Gel permeation chromatography revealed that LTP behaved as a globular protein of M(r) = 83K. Isoelectric focusing of the isolated LTP demonstrated a ladder of bands with pI's of 5.7-5.9. The specific activity of rabbit LTP was similar to that of human LTP. Monoclonal antibody TP2, that blocked human plasma LTP activity almost completely, only partially inhibited purified rabbit LTP, and rabbit plasma LTP activity to a similar extent. By a centrifugation binding a ssay, rabbit LTP was shown to predominantly associate with lipid micro emulsion in its presence. Circular dichroism spectroscopy indicated a high content of beta structure, and Provencher and Glockner analysis g ave estimated fractional values of 0.30, 0.39, 0.12, and 0.19 for alph a-helix, beta-sheet, beta-turn, and remainder content, respectively. U pon lipid binding, the helical content did not change drastically, alt hough there was some disordering of beta structure.