Levels of mRNA encoding biotindependent carboxylases and holocarboxylase synthetase

Probably, cells increase carboxylase activities by increasing the biotinylation of apocarboxylases to produce holocarboxylases. Theoretically, the apo-carboxylases could come from either one of the following two sources: (i) pre-formed apocarboxy-lases from the cellular carboxylase pool; or (ii) new apocarboxylases that have been synthesized specifically in response to cell proliferation. Most probably, the latter would be associated with an increase of mRNA levels encoding biotin-dependent carboxylases. The studies summarized here quantified levels of mRNA encoding

  1. 5.7. Activity of 3-methylcrotonyl-CoA carboxylase (MCC) in mitogen-stimulated human PBMCs. PBMCs were incubated with pokeweed lectin (2.0 mg ml-1) or concanavalin A (20 mg ml-1) for the indicated times. At timed intervals, the activity of MCC was measured. Values are means ± 1 sd; n = 6. ^Significantly different from controls (P < 0.01 vs. time zero) (Stanley et al., 2002).
  2. 5.7. Activity of 3-methylcrotonyl-CoA carboxylase (MCC) in mitogen-stimulated human PBMCs. PBMCs were incubated with pokeweed lectin (2.0 mg ml-1) or concanavalin A (20 mg ml-1) for the indicated times. At timed intervals, the activity of MCC was measured. Values are means ± 1 sd; n = 6. ^Significantly different from controls (P < 0.01 vs. time zero) (Stanley et al., 2002).
  1. 5.8. Activity of propionyl-CoA carboxylase (PCC) in mitogen-stimulated human PBMCs. PBMCs were incubated with pokeweed lectin (2.0 mg ml-1) or concanavalin A (20 mg ml-1) for the indicated times. At timed intervals, the activity of PCC was measured. Values are means ± 1 sd; n = 6. ^Significantly different from controls (P < 0.05 vs. time zero) (Stanley et al., 2002).
  2. 5.8. Activity of propionyl-CoA carboxylase (PCC) in mitogen-stimulated human PBMCs. PBMCs were incubated with pokeweed lectin (2.0 mg ml-1) or concanavalin A (20 mg ml-1) for the indicated times. At timed intervals, the activity of PCC was measured. Values are means ± 1 sd; n = 6. ^Significantly different from controls (P < 0.05 vs. time zero) (Stanley et al., 2002).

biotin-dependent carboxylases in order to determine whether PBMCs respond to cell proliferation with increased synthesis of new apocarboxylases.

Levels of mRNA encoding biotin-dependent carboxylases were greater in proliferating PBMCs than in quiescent controls (Stanley et al., 2002). For example, the level of mRNA encoding propionyl-CoA carboxylase in PBMCs that were stimulated with pokeweed lectin was approximately 4.2 times the mRNA levels in quiescent controls, as judged by quantitative PCR (Fig. 5.9). Likewise, mRNA encoding 3-methylcrotonyl-CoA carboxylase in stimulated PBMCs was 2.3 times that in quiescent controls (data not shown). It remains uncertain whether these increases were due to increased transcription, decreased mRNA degradation or both.

Holocarboxylase synthetase catalyzes the covalent binding of biotin to apocarboxylases to form holocarboxylases. Levels of mRNA encoding holocarboxylase synthetase in mitogen-stimulated, proliferating PBMCs were approximately 2.4 times the level in quiescent controls (Fig. 5.10). This finding is consistent with the hypothesis that PBMCs respond to proliferation with increased expression of the holocarboxylase synthetase gene in order to increase binding of biotin to carboxylases. Levels of mRNA encoding housekeeping genes

(glyceraldehyde-3-phosphate dehydrogenase and histone H4) were the same in proliferating and quiescent PBMCs, suggesting that the effects observed for biotin-dependent carboxylases and holocarboxylase synthetase were not artefacts due to unequal sample loading on the gels.

In summary, the findings presented here provide evidence that PBMCs respond to proliferation with increased de novo synthesis of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and holocarboxylase synthe-tase. Subsequently, holocarboxylase synthetase mediates biotinylation of the newly synthesized 3-methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase.

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