In vitro incorporation of molybdate into demolybdoproteins in Escherichia coli.
When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.
D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis* / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002474:Cell-Free System; D002701:Chloramphenicol / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D008667:Metalloproteins / Q000096:biosynthesis*; D008982:Molybdenum / Q000378:metabolism*; D009565:Nitrate Reductases / Q000096:biosynthesis* / Q000378:metabolism; D014414:Tungsten / Q000378:metabolism
D001426:Bacterial Proteins; D008667:Metalloproteins; D002701:Chloramphenicol; D008982:Molybdenum; D000445:Aldehyde Oxidoreductases; D009565:Nitrate Reductases; D014414:Tungsten