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. 1983 Aug;72(4):1133–1135. doi: 10.1104/pp.72.4.1133

Evidence for a β-Aspartyl Phosphate Residue in the Phosphorylated Intermediate of the Red Beet Plasma Membrane ATPase 1

Donald P Briskin 1, Ronald J Poole 1
PMCID: PMC1066388  PMID: 16663134

Abstract

A borohydride reduction method was used to identify the phosphorylated amino acid in the phospho-enzyme of the red beet (Beta vulgaris L.) plasma membrane ATPase. Plasma membrane fractions were phosphorylated with unlabeled ATP in the presence of MgSO4 at pH 6.5 and then treated with sodium [3H]borohydride. The borohydride-treated samples were subjected to hydrolysis in 6 normal HCl at 110°C for 22 hours and then analyzed by high voltage paper electrophoresis and thin layer chromatography. This analysis demonstrated the formation of labeled homoserine as the major reduction product when phosphorylated membrane samples were treated with sodium [3H]borohydride. This suggests that the phosphoryl group in the plasma membrane ATPase of red beet storage tissue is attached to the β-carboxyl side chain of an aspartic acid residue in the active site of the enzyme.

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Selected References

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

  1. Amory A., Gofffeau A. Characterization of the beta-aspartyl phosphate intermediate formed by the H+-translocating ATPase from the yeast Schizosaccharomyces pombe. J Biol Chem. 1982 May 10;257(9):4723–4730. [PubMed] [Google Scholar]
  2. Briskin D. P., Leonard R. T. Partial characterization of a phosphorylated intermediate associated with the plasma membrane ATPase of corn roots. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6922–6926. doi: 10.1073/pnas.79.22.6922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Briskin D. P., Leonard R. T. Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots. Plant Physiol. 1982 Nov;70(5):1459–1464. doi: 10.1104/pp.70.5.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Briskin D. P., Poole R. J. Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet. Plant Physiol. 1983 Feb;71(2):350–355. doi: 10.1104/pp.71.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Briskin D. P., Poole R. J. Plasma membrane ATPase of red beet forms a phosphorylated intermediate. Plant Physiol. 1983 Mar;71(3):507–512. doi: 10.1104/pp.71.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  7. Dame J. B., Scarborough G. A. Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate. J Biol Chem. 1981 Oct 25;256(20):10724–10730. [PubMed] [Google Scholar]
  8. Degani C., Boyer P. D. A borohydride reduction method for characterization of the acyl phosphate linkage in proteins and its application to sarcoplasmic reticulum adenosine triphosphatase. J Biol Chem. 1973 Dec 10;248(23):8222–8226. [PubMed] [Google Scholar]
  9. GAUDRY R. The synthesis of amino acids from 2,3-dihydrofuran DL-ornithine, DL-proline, and DL-alpha-amino-delta-hydroxyvaleric acid. Can J Chem. 1951 Jul;29(7):544–551. doi: 10.1139/v51-063. [DOI] [PubMed] [Google Scholar]
  10. Goffeau A., Slayman C. W. The proton-translocating ATPase of the fungal plasma membrane. Biochim Biophys Acta. 1981 Dec 30;639(3-4):197–223. doi: 10.1016/0304-4173(81)90010-0. [DOI] [PubMed] [Google Scholar]
  11. Hobbs A. S., Albers R. W. The structure of proteins involved in active membrane transport. Annu Rev Biophys Bioeng. 1980;9:259–291. doi: 10.1146/annurev.bb.09.060180.001355. [DOI] [PubMed] [Google Scholar]
  12. Nishigaki I., Chen F. T., Hokin L. E. Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. XV. Direct chemical characterization of the acyl phosphate in the enzyme as an aspartyl beta-phosphate residue. J Biol Chem. 1974 Aug 10;249(15):4911–4916. [PubMed] [Google Scholar]
  13. Paz M. A., Henson E., Rombauer R., Abrash L., Blumenfeld O. O., Gallop P. M. Alpha-amino alcohols as products of a reductive side reaction of denatured collagen with sodium borohydride. Biochemistry. 1970 May 12;9(10):2123–2127. doi: 10.1021/bi00812a014. [DOI] [PubMed] [Google Scholar]

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