Identification and extraction of Pasteurella haemolytica membrane proteins.

Abstract

The inner and outer membranes of Pasteurella haemolytica were separated by sucrose density gradient centrifugation after plasmolysis of the cells in 20% sucrose and fragmentation in a French pressure cell. Assays of the two membrane fractions for 2-keto-3-deoxyoctonate, succinate dehydrogenase, and NADH dehydrogenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that each of the two membrane fractions was purified fivefold relative to the other. The outer membrane fraction contained two major proteins of molecular weights 30,000 and 42,000 (30K and 42K proteins), and the inner membrane fraction contained five proteins in approximately equal amounts. Intact bacteria as well as membrane fractions were extracted by procedures used by others for vaccine preparation to determine whether the outer membrane proteins were released. Extraction of the isolated membranes with 0.5 M potassium thiocyanate in 0.425 M NaCl with or without EDTA or with M sodium salicylate failed to release more than traces of the outer membrane proteins. Sodium dodecyl sulfate extracted essentially all of the proteins of both membranes, but the products of this procedure were of low solubility and presumably denatured. The inner membrane proteins were extracted with 0.5% Sarkosyl in 0.02 M sodium phosphate buffer (pH 7.5). The 42K outer membrane protein, most of the lipopolysaccharide, and some of the 30K outer membrane protein were extracted with 1% Zwittergent 3-16 in 0.25 M NaCl (pH 8), and the remaining 30K outer membrane protein was extracted with 1% deoxycholate in 0.25% NaCl (pH 8). Extraction of membranes in this sequence yielded partially purified membrane proteins that were soluble in dilute buffers.