Molecular Organization in Bacterial Cell Membranes

Streptomyces albus cytoplasmic membranes revealed a relatively complex pattern in dodecylsul‐phate gel electrophoresis with approximately 25 polypeptide chains; most of them could be grouped into four to five clusters, all showing Schiff‐positive stain. The groups were identified by their relative mobilities in electrophoresis (0.30–0.40; 0.64 ± 0.03; 0.70–0.80; 0.80–0.90 and 1.0). We studied the topological distribution by various probes. These techniques, applied to protoplasts and membranes, included trypsin digestion, introduction of tritium label in the carbohydrate moieties by reduction with sodium boro[ 3 H]hydride, protein labelling by iodination catalyzed by iodide peroxidase, and subsequent examination of the trypsin sensitivity of the labelled components. The results can be summarized as follows: (a) The protein and Schiff‐positive materials of relative mobility 0.30–0.40 gave controversial results with the different probes. Therefore, their distribution in S. albus membranes is not yet clearly established. (b) The predominant protein and Schiff‐positive components of relative mobility 0.64 were slightly more sensitive to trypsin digestion in isolated membranes than in intact protoplasts (about 15% of the Schiff and 0.3% of the protein stains remained in this last case). The incorporation of tritium label in this group confirmed its glycoprotein nature. These glycoproteins were labelled two or three times more in isolated membranes than in protoplasts but were not iodinated by the iodide peroxidase probe. Trypsin treatment degraded the 3 H label in a similar manner to the Schiff‐positive material but did not increase the labelling with 125 I. We propose an asymmetrical location for the glycoproteins of relative mobility 0.64 with a minor proportion richer in carbohydrate residues per mole of protein oriented towards the interior. (c) The glycoprotein group of relative mobility 0.70–0.90 was apparently more resistant to trypsin in protoplasts than in membrane but was equally labelled with sodium boro[ 3 H]hydride in both systems. Some of its component(s) was (were) labelled with 125 I to the same extent in both protoplasts and membranes. The distribution of this glycoprotein group is not yet clearly established but the experimental results suggest a predominant external orientation. (d) The components of relative mobility 1.0 were seemingly composed of glycoproteins and proteins. The glycoproteins were resistant to trypsin. They incorporated about the same amount of tritium label in protoplasts and membranes suggesting a preferential location towards the external surface of the cell. This location was partly confirmed by the iodide peroxidase probe. (e) The proteins ( R x 1.0) were highly labelled with iodine in isolated membranes but not in protoplasts. This label was sensitive to trypsin digestion in membranes. These results point to an asymmetrical distribution towards the interior. (f) n ‐Butanol extraction of 3 H‐labelled membranes suggested that the glycoproteins extracted into the soluble phase were mostly those externally located. (g) The iodide peroxidase probe revealed two bands (molecular weights 80000 and 75000), extensively iodinated as compared with their protein stain, that can not be ascribed to any of the glycoprotein clusters. The 80000 molecular weight component appears to span the membrane. We propose a structural model of the plasma membrane of S. albus to account for these results.