MATERIALS AND METHODS Bacterial Strains and Their Cultivation-P. aeruginosa PA01 and K799 were used (3); they were usually grown in L broth (1% Difco Tryptone, 1%) Difco yeast extract, and 0.5% NaCI) at 37 “C with aeration by shaking. Cells were harvested at the late exponential phase by centrifugation at room temperature. Outer membranes were prepared as described previously (9). Purification of Porin in Cholate-Outer membrane fraction obtained from 4 liters of a late exponential culture of P. aeruginosa was suspended in 20 rnl of 2% sodium cholate, 0.2 M NaCl, 50 mM Tris-CI, pH 8.0. After 20 min at room temperature, the suspension was centrifuged at 125,000 X g for 30 min. The pellet was then extracted with 5 ml of 2% sodium cholate, 1 M NaCl, 50 mM Tris-CI, pH 8.0, 10 rn~ EDTA with brief sonication, and the mixture was centrifuged as above. Ammonium sulfate was added to the supernatant up to 354 saturation, and the salted out proteins were removed by centrifugation. The supernatant solution was applied to a column of Sephacryl S-200 (1.5 X 91 cm) equilibrated with 1% sodium cholate, 1 M NaCI, 10 mM Tris-C1, pH 8.0, and the column was eluted with the same buffer. Porins were eluted as a broad peak soon after the void volume. Porin-containing fractions were combined and concentrated tu 1.7 ml by dialysis against Ficoll 400, and the sample was fractionated again on the same column. The fractions wit. h the highest degree of purity were pooled and dialyzed at 4 “C against 10 mM Tris-C1, pH 8.0, to remove most of the NaCl and cholate. Purification of Porin in Dodecyl Sulfate-Cells from 4 liters of late exponential culture were washed once and were suspended in 40 ml of 15 mM Tris-HCI buffer, pH 8.0, 0.1 mM phenylmethylsulfonyl fluoride, containing about 1 mg each of pancreatic deoxyribonuclease and ribonuclease, and the suspension was passed through a French pressure cell at 15,000 psi three times. After removing large debris by centrifuging at 3,000 rpm for 10 min, the crude envelope fraction was obtained by centrifugation at 100,000 X g for 45 min. The envelope fraction was extracted three times with 45 ml of 2% LDS’, 15 mM Tris-HC1, pH 8.0, at 0 “C, each time followed by centrifugation