Materials and Methods

Protein Isolation and Characterization. MTP was isolated as described previously (Wetterau et al., 1990), except as where noted below. Bovine liver was cut into smallcubes and rinsed in 250 mM sucrose. One part bovine liver was mixed with two parts 50 mM Tris, pH 7.4, and 250 mM sucrose and homogenized in a blender. The homogenate was centrifuged at lOOOOg for 30 min, and the supernatant was retained. The yield of MTP appeared to be consistently greater when lOOOOg centrifugation was used as opposed to the previously reported 13000g centrifugation. Microsomes, isolated from the lOOOOg supernatant as previously described, were suspended in 1 mM Tris, pH 8.6, to release the soluble proteinsfrom the lumen of the microsomes.

MTP was further purified by ammonium sulfate precipitation and sequential column chromatography on DEAE-Se-phacel, Sephadex G-200, DEAE-cellulose, Sephacryl S-300, and hydroxylapatite columns. The columns were run as described previously except that 10 mM sodium phosphate, pH 7.4, 0.4 M guanidine hydrochloride, and 0.02% NaN3 was used to elute MTP fromthe Sephacryl S-300 column and the hydroxylapatite matrix was acquired from Calbiochem. Tri-glyceride transfer activity was monitored by measuring the