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Measurements were made of the rate of intracellular protein turnover in Chlorella fusca var. vacuolata, using two methods. Incorporation of 18O into protein from H218O-enriched medium occurred at linear rates in both autotrophically growing and nitrogen-starved cells. On the assumption that the only port of 18O incorporation is via the hydrolytic cleavage of peptide bonds followed by resynthesis of the labelled amino acids into new protein, apparent turnover rates were between 1.2 and 2.13% h-1 in growing cells, falling to 0.5% h-1 at the commencement of starvation. After 17.5 h starvation, the rate rose again to the growing cell value. However, analysis of the exchanges between the acid and amides of aspartate and glutamate in the free pool showed that 18O can also be incorporated via side-chain exchange, thus causing overestimation of turnover. Labelling of protein by growth in 3H2O followed by selective measurement of the α-carbon tritium has also been used to avoid the problem of isotope reincorporation. However, in growing cells, incorporation continued after washing the cells free of unincorporated tritium. In nitrogen-starved cells, two stability classes were seen, 85% of protein being degraded at 3.47% h-1, and 15% at 1.11% h-1. It is suggested that this high rate of turnover results from the exposure of the culture to high levels of radioactivity for long periods, and concluded that neither method alone will yield an absolute value for the rate of protein turnover, but differences between classes of protein with different decay rates will be apparent.

microbiology

Protein Turnover in Chlorella fusca var. vacuolata: Measurement of the Overall Rate of Intracellular Protein Degradation using Isotope Exchange with Water