The Effects of Enzyme Synthesis and Stability and of Deoxyribonucleic Acid Replication on the Cellular Levels of Aspartate Transcarbamylase during the Cell Cycle of Eucaryote Chlorella

Abstract

Abstract Homogenates of Chlorella pyrenoidosa (strain 7-11-05) contain an organic, low molecular weight (l 1000), heat stable, acid-soluble, Norit A adsorbable compound required for the in vitro stability of aspartate transcarbamylase. Dilution of these homogenates or removal of the stabilizer by either ultrafiltration, gel filtration, ammonium sulfate precipitation, or sucrose density gradient centrifugation resulted in the rapid first order decay of enzyme activity even at 0–3°. The apparent feedback inhibitor, UMP, was the only nucleotide effective as an in vitro stabilizer within physiological concentrations. Although carbamyl phosphate could stabilize the enzyme in vitro, it was eliminated as the possible endogenous stabilizer because carbamyl phosphate is heat labile. The other substrate, aspartate, was ineffective as a stabilizer. In cell cycle studies with synchronous cells of Chlorella, the pattern of aspartate transcarbamylase was observed to parallel the periodic DNA pattern except for a period of gradual enzyme accumulation immediately prior to the step increase in DNA level. Prior to the period of gradual enzyme accumulation, the enzyme level remained constant with a steady state existing between enzyme synthesis and breakdown. The structural gene of the enzyme appeared to be continuously transcribed and the enzyme continuously synthesized so that the enzyme increased from one steady state level to another during the period of gene replication. The gradual increase in activity prior to the step increase in DNA is proposed to be caused by the accumulation of an endogenous stabilizer (e.g. UMP) which reduces the rate of enzyme degradation. This inference is supported by the accumulation of total nucleotide-P and of an endogenous inhibitor of enzyme activity, and by the decreased rate of breakdown of the enzyme in vivo and also in cell homogenates from this period of the cell cycle.