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J Bacteriol. 1970 July; 103(1): 207-215
Copyright © 1970 American Society for Microbiology. All Rights Reserved.

Steady-State Measurement of the Turnover of Amino Acid in the Cellular Proteins of Growing Escherichia coli: Existence of Two Kinetically Distinct Reactions

¹ Department of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York 14203

ABSTRACT

Turnover of cellular protein has been estimated in Escherichia coli during continuous exponential growth and in the absence of extensive experimental manipulation. Estimation is based upon the cumulative release into carrier pools of free leucine-1-¹⁴C over a number of time intervals after its pulsed incorporation into protein. Breakdown rates obtained with other labeled amino acids are similar to those obtained with leucine. Two kinetically separate processes have been shown. First, a very rapid turnover of 5% of the amino acid label occurs within 45 sec after its incorporation, most likely indicating maturative cleavages within the proteins after their assembly. A slower heterogeneous rate of true protein turnover follows, falling by 39% in the remaining proteins for each doubling of turnover time. At 36 C, the total breakdown rate of cellular protein is 2.5 and 3.0% per hr over a threefold range of growth rate in glucose and acetate medium, respectively. This relatively constant breakdown rate is maintained during slower growth by more extensive protein replacement, one fifth of the protein synthesized at any time in the acetate medium being replaced after 4.6 doubling times. Intracellular proteolysis thus appears to be a normal and integral reaction of the growing cell. The total rate equals minimal estimates obtained by others for arrested or decelerated growth but is kinetically more heterogeneous. Quantitatively proteolysis is not directly affected by growth arrestment per se as caused by -methylhistidine, chloramphenicol, or uncouplers of oxidative phosphorylation, but qualitatively it can gradually become more homogeneous kinetically as a secondary event of starvation. Under more extreme conditions as with extensive washing, prolonged phosphorylative uncoupling, or acidification of the growth medium, the proteolytic rate can increase severalfold.

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