Carbon and hydrogen isotope systematics of bacterial formation and oxidation of methane

Biogenic methane formation in marine and freshwater environments: CO2 reduction vs. acetate fermentation—Isotope evidence

Biogas production from co-digestion of dairy manure and food waste

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

[This corrects the article on p. 517 in vol. 41.].

The biology of methanogenic bacteria.

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-(14)C 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 alpha-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.

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

the superscripts denoting separate isotopic labels. Since Pine and Barker (1956) established that the methyl group of acetate is transferred intact during its incorporation into methane, we wished to determine whether methanol could be metabolized analogously. The deuterium incorporation procedure previously employed for the study of the acetate fermentation was used to establish that all three hydrogen atoms attached to the carbon of methanol remained attached during methane formation. In addition, we have investigated the relationship between methanol and acetate utilization, and discuss a possible common intermediary pathway.

The methane fermentations of acetate and methanol.

Kinetics of maturation of the amino termini of the cell proteins of Escherichia coli

Individual nitrogenous metabolites have been examined as regulating agents for the breakdown of intracellular proteins in Escherichia coli . Generally, NH 4 + is the most effective regulator. Its depletion progressively increases the basal proteolytic rate to maximum in most strains when the doubling time is increased to 2 h. In E. coli 9723, the rate is further increased at longer doubling times. Amino acids have individual effects on intracellular proteolysis. The basal rate in amino acid-requiring auxotrophs of E. coli 9723 is stimulated weakly by starvation for histidine, tryptophan, or tyrosine, moderately by four other amino acid depletions, and more strongly by eight others. The degree of stimulation roughly correlates with the frequency of the amino acid in the cell proteins. Amino acid analogues that incorporate extensively into protein generally slightly inhibit intracellular proteolysis, except for selenomethionine, which is slightly stimulatory. Metabolic inhibitors were studied at graded concentrations. Chloramphenicol inhibits the basal level of intracellular proteolysis when protein synthesis is slightly or moderately inhibited, and stimulates proteolysis slightly at higher levels. Graded inhibition of ribonucleic acid synthesis with rifampin progressively stimulates intracellular proteolysis. Uracil depletion is also stimulatory. Inhibition of deoxyribonucleic acid synthesis with mitomycin C or by thymine starvation slightly inhibits intracellular proteolysis. Intracellular proteolysis is postulated to be regulated primarily by active ribosomal function. At 43 to 45 C, intracellular proteolysis becomes maximally induced and unresponsive to normal regulatory control by metabolites. Most regulation is directed towards the breakdown of the more stable cell proteins. Total proteolysis in all cell proteins is no more than doubled by the most effective conditions of starvation.

Regulation of Intracellular Proteolysis in Escherichia coli

Pine, Martin J (Roswell Park Memorial Institute, Buffalo, NY) Metabolic control of intracellular proteolysis in growing and resting cells of Escherichia coli J Bacteriol 92:847–850 1966—Protein breakdown was examined in Escherichia coli under varying conditions of growth and during nutritional deprivation Optimal breakdown rates, estimated over short time periods of protein synthesis and decay, are invariant and unrepressible by any condition of growth or starvation A more important aspect of metabolic control is manifest in the selection imposed by the physiological state of the cell for breakdown of limited populations of proteins During the progress of growth, one-half to three-quarters of the protein susceptible in the resting state is progressively spared, and breakdown is continued in populations that are more selected and more frequently regenerated

Martin J. Pine

Papers

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

The methane fermentations of acetate and methanol.

Kinetics of maturation of the amino termini of the cell proteins of Escherichia coli

Regulation of Intracellular Proteolysis in Escherichia coli

Metabolic Control of Intracellular Proteolysis in Growing and Resting Cells of Escherichia coli