Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: Phosphorylation studies of nucleoside diphosphate kinase

Ricardo M. Biondi, Katherina Walz, Olaf Georg Issinger, Matthias Engel, Susana Passeron

Research output: Contribution to journalArticle

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Abstract

We have critically analyzed current methodologies for distinguishing histidine and serine phosphorylated residues in proteins and report a simple technique that assures a reliable discrimination. Electrotransfer of a phosphorylated enzyme to Immobilon membranes and its treatment at pH 1 and 14 in buffers containing 5% methanol allows unambiguous distinction between serine/threonine and histidine phosphorylation (O-phosphomonoesters and phosphoramide, respectively) since under these conditions only one type of residue is dephosphorylated. The addition of 5% methanol to all buffers was indispensable to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP kinase cellular function, signaling the suppression of metastasis in the case of human NDP kinase A. Using this improved method, we show that human, Escherichia coli and Candida albicans NDP kinases are only autophosphorylated on histidine residues. In addition, we present evidence that the presence of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe that the technique will be generally useful in histidine phosphorylation screenings.

Original languageEnglish
Pages (from-to)165-171
Number of pages7
JournalAnalytical Biochemistry
Volume242
Issue number2
DOIs
StatePublished - Nov 15 1996
Externally publishedYes

Fingerprint

Nucleoside-Diphosphate Kinase
Phosphorylation
Alkalies
Histidine
Acids
Candida albicans
Serine
Methanol
Buffers
Enzymes
Phosphates
Membranes
Phosphoserine
Candida
Threonine
Escherichia coli
Immobilon
Hydrolysis
Catalytic Domain
Screening

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Discrimination between acid and alkali-labile phosphorylated residues on Immobilon : Phosphorylation studies of nucleoside diphosphate kinase. / Biondi, Ricardo M.; Walz, Katherina; Issinger, Olaf Georg; Engel, Matthias; Passeron, Susana.

In: Analytical Biochemistry, Vol. 242, No. 2, 15.11.1996, p. 165-171.

Research output: Contribution to journalArticle

Biondi, Ricardo M. ; Walz, Katherina ; Issinger, Olaf Georg ; Engel, Matthias ; Passeron, Susana. / Discrimination between acid and alkali-labile phosphorylated residues on Immobilon : Phosphorylation studies of nucleoside diphosphate kinase. In: Analytical Biochemistry. 1996 ; Vol. 242, No. 2. pp. 165-171.
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