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

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1966 Jun 1

PMID 5329287

Authors

T H Brzozowski

S M Kalman

Affiliations

Soon will be listed here.

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.

References

PIERARD A, Wiame J . Regulation and mutation affecting a glutamine dependent formation of carbamyl phosphate in Escherichia coli. Biochem Biophys Res Commun. 1964; 15(1):76-81. DOI: 10.1016/0006-291x(64)90106-8. View

Lacroute F, PIERARD A, GRENSON M, Wiame J . The biosynthesis of carbamoyl phosphate in Saccharomyces cerevisiae. J Gen Microbiol. 1965; 40(1):127-42. DOI: 10.1099/00221287-40-1-127. View

Kalman S, DUFFIELD P, Brzozowski T . Purification and properties of a bacterial carbamyl phosphate synthetase. J Biol Chem. 1966; 241(8):1871-7. View

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View

THURSTON J, Rheins M, BUEHLER E . A rapid method for recovering serologically active globulins by sodium sulfate precipitation. J Lab Clin Med. 1957; 49(4):647-50. View

Grisolia S, Harmon P, RAIJMAN L . Studies on the mechanism of action of carbamate kinase. Biochim Biophys Acta. 1962; 62:293-9. DOI: 10.1016/0006-3002(62)90042-2. View

LEVENBERG B . Role of L-glutamine as donor of carbamyl nitrogen for the enzymatic synthesis of citruline in Agaricus bisporus. J Biol Chem. 1962; 237:2590-8. View

Beckwith J, Pardee A, Austrian R, Jacob F . Coordination of the synthesis of the enzymes in the pyrimidine pathway of E. coli. J Mol Biol. 1962; 5:618-34. DOI: 10.1016/s0022-2836(62)80090-4. View

THORNE K, Jones M . CARBAMYL AND ACETYL PHOSPHOKINASE ACTIVITIES OF STREPTOCOCCUS FAECALIS AND ESCHERICHIA COLI. J Biol Chem. 1963; 238:2992-8. View

10.

YASHPHE J, GORINI L . PHOSPHORYLATION OF CARBAMATE IN VIVO AND IN VITRO. J Biol Chem. 1965; 240:1681-6. View

11.

Davis B, MINGIOLI E . Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol. 1950; 60(1):17-28. PMC: 385836. DOI: 10.1128/jb.60.1.17-28.1950. View

12.

Kalman S, DUFFIELD P, Brzozowski T . IDENTITY IN ESCHERICHIA COLI OF CARBAMYL PHOSPHOKINASE AND AN ACTIVITY WHICH CATALYZES AMINO GROUP TRANSFER FROM GLUTAMINE TO ORNITHINE IN CITRULLINE SYNTHESIS. Biochem Biophys Res Commun. 1965; 18:530-7. DOI: 10.1016/0006-291x(65)90786-2. View

13.

KANAZIR D, Barner H, Flaks J, COHEN S . Some physiological and genetic properties of a strain of Escherichia coli requiring thymine, arginine and uracil. Biochim Biophys Acta. 1959; 34:341-53. DOI: 10.1016/0006-3002(59)90287-2. View