FIG. 4.

Elution profile of the 2% Triton-solubilized pellet fraction (Δ) and a 2-h HSS (□), eluted from the gel filtration column with M63, pH 8.9, containing 0.1% Triton X-100 and 2 mM dithiothreitol and monitored using the TP assay. The pellet fraction was solubilized at pH 8.9 with 2% Triton X-100, stirring at 4°C for 2 h, and then filtered through a 0.45-μm Millipore membrane filter. An aliquot of 2.5 ml of the solubilized pellet or 2.5 ml of the 2-h HSS was fractionated through the gel filtration column. The resultant fractions were dialyzed against water for 36 h (three changes; three 2-liter volumes; 12 h each) to reduce the detergent and salt concentrations and then assayed as follows: 100 μl from the dialyzed fractions was mixed with an aliquot of assay mix containing both TP substrates (100 μM [¹⁴C]methyl-α-glucoside and 10 mM glucose-6-P). Phosphatidylglycerol was incorporated at 200 μg per reaction mix (1 mg/ml) in the case of the solubilized pellet fractions only. Assay times were 2 and 1.5 h for the solubilized pellet fractions and the HSS fractions, respectively.