FIGURE 4.

Biochemical fractionations of HspL. A, equal volumes of total proteins (T), soluble fraction (S), and insoluble membrane fraction (IS) of wild type strain NT1RE(pJK270) grown in I-medium at 25 °C for 16 h with the addition of DMSO or AS were subjected to Western blotting. The quality of fractionation was assessed by monitoring the distribution of GroEL (cytosolic marker) and VirB9 (membrane marker). B, the insoluble membrane fraction was treated in 50 mm Tris-HCl, pH 7.5 (buffer only), in the presence of salts or detergents as indicated. Soluble (S) and insoluble (IS) fractions were separated by ultracentrifugation and subjected to Western blotting. C, the insoluble membrane fractions separated by sucrose density gradient centrifugation at 120,000 × g at 4 °C for 18 h were collected from the top of the gradient and analyzed. The fractions were detected by Western blotting. The fractions containing the outer membranes (OM) and inner membranes (IM) were identified on the basis of the activity of the inner membrane specific enzyme NADH oxidase (open triangle) and the outer membrane specific lipopolysaccharide 2-keto-3-deoxyoctonate (KDO, open square) (57). D, spheroplasts were treated with protease at different concentrations (0, 25, 50, or 100 μg/ml) and subjected to Western blotting. The resistance of cytosolic GroEL to protease digestion indicates the quality of spheroplast preparation.