Membrane reconstitution in chl-r mutants of Escherichia coli K 12. VIII. Purification and properties of the FA factor, the product of the chl B gene.
The isolation and purification of the product of the chl B gene of Escherichia coli K 12 from the chl A mutant have been attempted. The purified protein gives a single band in 10% sodium dodecylsulfate/polyacrylamide gel electrophoresis. The molecular weight is estimated to be 35 000. This protein, that we have named "FA factor", does not contain any lipid, has a strong tendency to lose its activity by polymerizing but can be kept in an active state when stored in buffer containing NaCl. The addition of purified FA protein to a soluble extract from the chl B mutant strain grown under anaerobiosis in the presence of nitrate initiates the "complementation reaction", i.e. the reconstitution of the nitrate reductase activity and the formation of particulate material similar to the native membrane with respect to the structure and enzymatic function. FA protein acts both on the rate of reconstitution and on the total amount of reconstituted enzyme. The complementation leads to the reconstitution of nonsedimentable nitrate reductase and to the formation of three types of particles of different buoyant densities (1.10, 1.18 and 1.23) the two lightest of which contain nitrate reductase. It is shown that FA factor is incorporated only into the particles of intermediate density. In vivo, this factor is located in the native membranes of chl A, chl C, chl D and wild-type strains, whatever the growth conditions, aerobiosis or anaerobiosis, and in the presence or absence of nitrate. Protein FA can be released from either of these membranes (native or reconstituted) by removing Mg-2+ or by subjecting Kaback's vesicles to mechanical treatments; in the case of 1.18-reconstituted particles and wild-type membranes, the release of FA protein does not exert any effect on the level of the nitrate reductase activity.
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000032:analysis* / Q000378:metabolism / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis* / Q000378:metabolism; D005796:Genes; D005816:Genetic Complementation Test; D008970:Molecular Weight; D009154:Mutation; D009565:Nitrate Reductases / Q000378:metabolism
D001426:Bacterial Proteins; D009565:Nitrate Reductases