14C2H2- and 14CO2-labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase.
Incubation of cells of the nitrifying bacterium Nitrosomonas europaea with 14C2H2 results in the covalent attachment of 14C label to a membrane-bound polypeptide of an approximate Mr of 28,000 (Hyman, M.R., and Wood, P.M. (1985) Biochem. J. 227, 719-725). A labeling procedure using 14C2H2 generated from Ba14CO3 has been used to investigate the correlation between the extent of covalent modification of this polypeptide by 14C from 14C2H2 and the level of ammonia oxidizing activity in whole cells. The time-dependent inactivation of ammonia monooxygenase by 14C2H2 resulted in a progressive and saturable incorporation of 14C into a 27-kDa polypeptide. In contrast, the specific, time-dependent and complete inactivation of ammonia monooxygenase by light resulted in concomitant decrease in the ability of cells to incorporate 14C from 14C2H2 into this polypeptide. The 14C2H2 labeling procedure was also used to investigate the recovery of ammonia monooxygenase activity after complete inactivation of pre-existing ammonia monooxygenase by either C2H2 or light. The recovery of ammonia monooxygenase activity was closely correlated with a recovery of ability of cells to incorporate 14C label from 14C2H2 into the 27-kDa polypeptide. This recovery process was energy (NH4+)-dependent and was inhibited by chloramphenicol and rifampicin, implying that de novo protein synthesis was required. Additional polypeptides labeled with 14C from 14CO2 were also identified during recovery from C2H2 or light inactivation of ammonia monooxygenase.
Hyman
M R
MR
Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis 97331-2902.
Arp
D J
DJ
D000114:Acetylene / Q000378:metabolism* / Q000494:pharmacology*; D001345:Autoradiography; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D002245:Carbon Dioxide / Q000378:metabolism*; D002250:Carbon Radioisotopes; D007700:Kinetics; D008027:Light; D009606:Nitrosomonas / Q000378:metabolism*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D010101:Oxygen Consumption; D010452:Peptide Biosynthesis
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002245:Carbon Dioxide; D010088:Oxidoreductases; C045582:ammonia monooxygenase; D000114:Acetylene