Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1979 Nov 1;183(2):239–245. doi: 10.1042/bj1830239

Wheat-germ aspartate transcarbamoylase. Purification and cold-lability.

J E Grayson, R J Yon, P J Butterworth
PMCID: PMC1161552  PMID: 43131

Abstract

1. Aspartate transcarbamoylase was purified approx. 3000-fold from wheat (Triticum vulgare) germ in 15-20% yield. The product has a specific activity of 14 mumol/min per mg of protein and is approx. 90% pure. The purification scheme includes the use of biospecific "imphilyte" chromatography as described by Yon [Biochem.J.(1977) 161, 233-237]. The enzyme was passed successively through columns of CPAD [N-(3-carboxypropionyl)aminodecyl]-Sepharose in the absence and presence respectively of the ligands UMP and L-aspartate. In the second passage the enzyme was specifically displaced away from impurities with which it co-migrated in the first passage. These two steps contributed a factor of 80 to the overall purification. 2. The enzyme is slowly inactivated on dilution at 0 degrees C and pH 7.0, the inactivation being partially reversible. A detailed investigation of the temperature- and pH-dependence of the cold-inactivation suggested that it was initiated by the perturbation of the pKa values of groups with a moderately high and positive heat of ionization, which were tentatively identified as histidine residues. These findings support a new concept of cold-lability proposed by Bock, Gilbert & Frieden [Biochem. Biophys. Res. Commun. (1975) 66, 564-569].

Full text

PDF
239

Selected References

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

  1. Achar B. S., Savithri H. S., Vaidyanathan C. S., Rao N. A. Studies on plant aspartate transcarbamylase. Purification and properties of the enzyme from mung-bean (Phaseolus aureus) seedlings. Eur J Biochem. 1974 Aug 15;47(1):15–22. doi: 10.1111/j.1432-1033.1974.tb03662.x. [DOI] [PubMed] [Google Scholar]
  2. Bock P. E., Gilbert H. R., Frieden C. Analysis of the cold lability behavior of rabbit muscle phosphofructokinase. Biochem Biophys Res Commun. 1975 Sep 16;66(2):564–569. doi: 10.1016/0006-291x(75)90547-1. [DOI] [PubMed] [Google Scholar]
  3. Grayson J. E., Yon R. J. Wheat-germ aspartate transcarbamylase: reversible ligand-dependent aggregation behaviour in vitro. Biochem Soc Trans. 1978;6(1):197–200. doi: 10.1042/bst0060197. [DOI] [PubMed] [Google Scholar]
  4. Johnson L. B., Niblett C. L., Shively O. D. Asparate transcarbamylase activity in etiolated cowpea hypocotyls treated with 2,4-dichlorophenoxyacetic Acid. Plant Physiol. 1973 Feb;51(2):318–321. doi: 10.1104/pp.51.2.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. NEUMANN J., JONES M. E. END-PRODUCT INHIBITION OF ASPARTATE TRANSCARBAMYLASE IN VARIOUS SPECIES. Arch Biochem Biophys. 1964 Mar;104:438–447. doi: 10.1016/0003-9861(64)90487-4. [DOI] [PubMed] [Google Scholar]
  7. Ong B. L., Jackson J. F. Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties. Biochem J. 1972 Sep;129(3):571–581. doi: 10.1042/bj1290571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. PARDEE A. B., YATES R. A. Control of pyrimidine biosynthesis in Escherichia coli by a feed-back mechanism. J Biol Chem. 1956 Aug;221(2):757–770. [PubMed] [Google Scholar]
  9. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  10. Yon R. J. Biospecific-elution chromatography with 'imphilytes' as stationary phases. Biochem J. 1977 Feb 1;161(2):233–237. doi: 10.1042/bj1610233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Yon R. J. Enzyme purification by hydrophobic chromatography: an alternative approach illustrated in the purification of aspartate transcarbamoylase from wheat germ. Biochem J. 1974 Jan;137(1):127–130. doi: 10.1042/bj1370127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Yon R. J., Simmonds R. J. Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase. Biochem J. 1975 Nov;151(2):281–290. doi: 10.1042/bj1510281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Yon R. J., Simmonds R. J. The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose. Biochem J. 1979 Feb 1;177(2):417–424. doi: 10.1042/bj1770417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Yon R. J. Wheat-germ aspartate transcarbamoylase. Kinetic behaviour suggesting an allosteric mechanism of regulation. Biochem J. 1972 Jun;128(2):311–320. doi: 10.1042/bj1280311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Yon R. J. Wheat-germ aspartate transcarbamoylase. The effects of ligands on the inactivation of the enzyme by trypsin and denaturing agents. Biochem J. 1973 Apr;131(4):699–706. doi: 10.1042/bj1310699. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES