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. 1978 Jul 1;173(1):207–217. doi: 10.1042/bj1730207

Proline biosynthesis by cell-free extracts of Escherichia coli and potential errors arising from the use of a bioradiological assay procedure.

D J Hayzer, V Moses
PMCID: PMC1185764  PMID: 356852

Abstract

1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.

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Selected References

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

  1. Baich A. Proline synthesis in Escherichia coli. A proline-inhibitable glutamic acid kinase. Biochim Biophys Acta. 1969 Dec 30;192(3):462–467. doi: 10.1016/0304-4165(69)90395-x. [DOI] [PubMed] [Google Scholar]
  2. Baich A. The biosynthesis of proline in Escherichia coli: phosphate-dependent glutamate -semialdehyde dehydrogenase (NADP), the second enzyme in the pathway. Biochim Biophys Acta. 1971 Jul 20;244(1):129–134. doi: 10.1016/0304-4165(71)90129-2. [DOI] [PubMed] [Google Scholar]
  3. CROWLEY G. J., MOSES V., ULLRICH J. A VERSATILE SOLVENT TO REPLACE PHENOL FOR THE PAPER CHROMATOGRAPHY OF RADIOACTIVE INTERMEDIARY METABOLITES. J Chromatogr. 1963 Oct;12:219–228. doi: 10.1016/s0021-9673(01)83673-6. [DOI] [PubMed] [Google Scholar]
  4. Dawson D. M., Eppenberger H. M., Kaplan N. O. The comparative enzymology of creatine kinases. II. Physical and chemical properties. J Biol Chem. 1967 Jan 25;242(2):210–217. [PubMed] [Google Scholar]
  5. EDELHOCH H. The properties of thyroglobulin. I. The effects of alkali. J Biol Chem. 1960 May;235:1326–1334. [PubMed] [Google Scholar]
  6. ENDE H., MEYERHOFF G., SCHULZ G. V. Das Fibrinogenmolekül und sein Verhalten. I. Bestimmung des Molekulargewichts, des Achsenverhältnisses und der osmotischen Virialkoeffizienten durch Messung der Sedimentation und Diffusion. Z Naturforsch B. 1958 Nov;13B(11):713–721. [PubMed] [Google Scholar]
  7. FRANK L., RANHAND B. PROLINE METABOLISM IN ESCHERICHIA COLI. 3. THE PROLINE CATABOLIC PATHWAY. Arch Biochem Biophys. 1964 Aug;107:325–331. doi: 10.1016/0003-9861(64)90338-8. [DOI] [PubMed] [Google Scholar]
  8. FRANK L., RYBICKI P. Studies of proline metabolism in Escherichia coli. I. The degradation of proline during growth of a proline-requiring auxotroph. Arch Biochem Biophys. 1961 Dec;95:441–449. doi: 10.1016/0003-9861(61)90174-6. [DOI] [PubMed] [Google Scholar]
  9. Gamper H., Moses V. Enzyme organization in the proline biosynthetic pathway of Escherichia coli. Biochim Biophys Acta. 1974 Jun 20;354(1):75–87. doi: 10.1016/0304-4165(74)90055-5. [DOI] [PubMed] [Google Scholar]
  10. Hayzer D. J., Moses V. The enzymes of proline biosynthesis in Escherichia coli. Their molecular weights and the problem of enzyme aggregation. Biochem J. 1978 Jul 1;173(1):219–228. doi: 10.1042/bj1730219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. KELLER P. J., LOWRY C., TAYLOR J. F. The molecular weights of some crystalline enzymes from muscle and yeast. II. Phosphoglucomutase. Biochim Biophys Acta. 1956 Apr;20(1):115–117. [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. MARGOLIASH E. Amino acid sequence of chymotryptic peptides from horse heart cytochrome c. J Biol Chem. 1962 Jul;237:2161–2174. [PubMed] [Google Scholar]
  14. Moses V., Prevost C. Catabolite repression of beta-galactosidase synthesis in Escherichia coli. Biochem J. 1966 Aug;100(2):336–353. doi: 10.1042/bj1000336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. PARDEE A. B., PRESTIDGE L. S. The initial kinetics of enzyme induction. Biochim Biophys Acta. 1961 Apr 29;49:77–88. doi: 10.1016/0006-3002(61)90871-x. [DOI] [PubMed] [Google Scholar]
  16. Williams I., Frank L. Improved chemical synthesis and enzymatic assay of delta-1-pyrroline-5-carboxylic acid. Anal Biochem. 1975 Mar;64(1):85–97. doi: 10.1016/0003-2697(75)90408-x. [DOI] [PubMed] [Google Scholar]

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