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. 1966 Jun;91(6):2286–2290. doi: 10.1128/jb.91.6.2286-2290.1966

Carbamyl Phosphate and Acetyl Phosphate Synthesis in Escherichia coli: Analysis of Associated Enzyme Activities by an Antibody to Acetokinase

Thomas H Brzozowski 1, Sumner M Kalman 1
PMCID: PMC316208  PMID: 5329287

Abstract

Brzozowski, Thomas H. (Stanford University School of Medicine, Palo Alto, Calif.), and Sumner M. Kalman. Carbamyl phosphate and acetyl phosphate synthesis in Escherichia coli: analysis of associated enzyme activities by an antibody to acetokinase. J. Bacteriol. 91:2286–2290. 1966.—Earlier studies have shown that the carbamyl phosphate synthesis from ammonia in cell extracts of wild-type Escherichia coli is due to at least two enzymes, acetokinase and the glutamine-dependent carbamyl phosphate synthetase. Partial purification of the glutamine-dependent carbamyl phosphate synthetase and acetokinase fails to separate from these enzymes this ammonia-dependent activity. An antibody to the partially purified acetokinase was prepared and used to determine the distribution of the ammonia-dependent activity in wild-type organisms and single-step arginine-uracil-requiring mutants with respect to the two enzymes. Such a study was possible because the antibody inhibits acetokinase but not the glutamine-utilizing carbamyl phosphate synthetase. Enzyme inhibition obtained by the stepwise addition of the antibody to cell extracts indicates that all of the ammonia-dependent carbamyl phosphate synthesis observed in the arginine-uracil-requiring mutants is due to a protein in the acetokinase fraction, presumably acetokinase itself, since acetyl phosphate and carbamyl phosphate synthesis were inhibited in a parallel fashion. In wild-type organisms, there is only partial inhibition of the ammonia-dependent activity, even when enough antibody is added to produce maximal inhibition of acetokinase. It is suggested that this residue is due to the glutamine-dependent carbamyl phosphate synthetase, for the ratio of the antibody insensitive to antibody sensitive ammonia-dependent activity present in cell extracts of the two wild-type organisms reported is qualitatively proportional to the level of carbamyl phosphate synthetase present relative to acetokinase.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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