Isolation and Characterization of Hydrophobic Proteins (H Proteins) in the Membrane Fraction of Bacillus subtilis

Abstract

Cytoplasmic membranes of B. subtilis, grown in complex medium containing glucose, were fractionated into 3 membrane subfractions [light band (1.155-1.158 g/cm3); medium band (1.181-1.183 g/cm3); heavy band (1.21-1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions the light and medium bands consisted mainly of membranes, but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5-8 nm in diameter. It was named the H-protein. The H-protein formed trilamellar unit membrane structure when combined with lipid. Pulse-labeling and pulse-chase experiments with radioactive leucine, showed that H-protein consisted of the newest membrane protein synthesized in the cells, and the label incorporated into H-protein was shifted into light and medium bands of the membranes during the chase. Cytochromes were not found in H-protein, but when H-protein was incubated with heme a and protoheme, these compounds were incorporated into the apoproteins of the cytochromes present in H-protein and formed cytochromes a and b. Cytochromes were also formed in H-protein which were isolated from the cells grown in the presence of hemin (hemin-grown H protein). Succinate dehydrogenase [EC 1.3.99.1] activity was increased .apprx. 4-fold by combining H-protein or hemin-grown H protein with lipid. H-protein had no cytochrome oxidase [EC 1.9.3.1] activity, but hemin-grown H protein had some of the activity, and this was increased .apprx. 4-fold by combining the protein with lipid. Hemin-grown H protein also formed succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. Succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase [EC 1.6.99.3] was also found in hemin-grown H protein and was activated .apprx. 9-fold in constituted reaction systems. Vesicles formed by hemin-grown H protein and lipid could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline were also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. The H-protein apparently contains the apoprotein of cytochromes and a carrier involved in the active transport of alanine and proline.