Cell cycle control of aspartate transcarbamylase levels in Chlorella sorokiniana

Aspartate transcarbamylase (carbamylphosphate:L-aspartate carbamyltransferase, E. C. 2.1.3.2) from Chlorella sorokiniana was stabilized in vitro by uridine and UMP with 0.04 mM uridine and 0.05 mM UMP giving half maximal stability. Positive cooperative effects on stabilization were observed for UMP but not uridine. The enzyme was stabilized at all temperatures between 2° and 50°, but in the absence of the nucleotide the enzyme was both cold and heat labile and had a temperature stability optimum of 32° for an incubation time of 90 min~ The enzyme was more stable in glycylglycine buffer than in Tris-HCl buffer. The enzyme was inhibited by uridine and UMP, but concentrations of 1.6 mM uridine or 2.6 mM UMP were required for 50% inhibition. Sensitivity to inhibition was diminished by ammonium sulfate fractionation or multiple passages through a French pressure cell. The loss of sensitivity to inhibition may be due to breakdown of the enzyme into subunits or breakdown of a multienzyme complex of pyrimidine enzymes. An assay for activity of carbamyl-P synthetase (E. C. 2.7.2.5), a suspected component of the multienzyme complex, was developed for Chlorella. Neither aspartate transcarbamylase nor carbamyl-P synthetase was sedimented by centrifugation at 100,000 x g for 5.5 hours. Either a multi-enzyme complex does not exist, the complex is of low molecular weight, or the complex was destroyed during preparation of the cellular material.

Synchronous Chlorella cells were used to study the regulation of aspartate transcarbamylase during the cell cycle. Under certain culture conditions (constant light intensity per cell and nitrate as the nitrogen source) the enzyme accumulated in a step pattern with the step increase in enzyme accumulation occurring during DNA replication. This pattern is consistent with two hypotheses: The structural gene is transcribed only during the S-phase, or the structural gene may be transcribed continuously if the enzyme is unstable and either under a constant level of repression or free from repression. In the second case, if the enzyme were synthesized and broken down at the same rate, the enzyme would accumulate only when the gene dosage increased as a result of DNA replication. When culture conditions were altered in such a way as to cause the light intensity per cell to oscillate during the cell cycle, accumulation of the enzyme began before the onset of DNA replication. Therefore, the structural gene for aspartate transcarbamylase is not expressed constitutively, and enzyme accumulation is not restricted to the S-phase as predicted by the first hypothesis. In another experiment, the nitrogen source was changed from nitrate to ammonium, and the effective light intensity was increased but held constant during the cell cycle. The enzyme accumulated in a continuous pattern and DNA in a step pattern, again demonstrating that DNA and enzyme accumulation are not obligately coupled. When DNA synthesis was inhibited by 74%, by the addition of 2'-deoxyadenosine, there was no corresponding effect on enzyme accumulation.