| The role of ornithine transcarbamylase (OTCase) in arginine metabolism in B. subtilis was examined. A purification procedure for OTCase was developed. Purified OTCase was characterized and used to elicit monospecific antibodies. Immunoprecipitation of OTCase from crude extracts of cells grown in the presence or absence of arginine, showed that the same enzyme protein with the same specific activity was present under both culture conditions. An arginine auxotroph (BR-85), which lacked OTCase activity, produced no OTCase or OTCase CRM under any growth conditions. When BR-85 was grown on arginine as the sole source of carbon and nitrogen, OTCase activity did not appear at any time during the cultivation. Under these conditions, the mutant grew as well as its parent (JH-862), the latter showing OTCase once again only at the end of exponential growth. OTCase was therefore not required for growth on arginine. Since spores of BR-85 contained the same amount of arginine as those of its parent, the physiological role of OTCase induction at the end of exponential growth in the presence of arginine remains obscure.;Studies of the inactivation of OTCase in protease-deficient mutants of B. subtilis showed that it was inactivated 4-fold more slowly in a mutant (S-87) lacking the intracellular serine protease (ISP). Immunoprecipitation of OTCase from crude extracts of JH-168 (the parent organism) showed that the loss of CRM paralleled the loss of activity, and there was no build-up of inactive or fragmented OTCase during inactivation in vivo. Thus, inactivation of OTCase was either a result of, or was rapidly followed by, proteolytic degradation. ISP was purified and reacted with purified OTCase in the presence of a variety of effectors; no loss of activity or fragmentation of OTCase protein could be found. These data indicated that ISP did not inactivate OTCase directly in vivo. This conclusion was confirmed by the demonstration that the specific rate of OTCase synthesis in S-87 continued at a high level even after inactivation had begun. This was not so in JH-168, where OTCase synthesis was shut off shortly after the start of inactivation. The observed extension in OTCase lifetime in S-87 was thus not a result of slowed inactivation, but of its continued synthesis. |
