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Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum

Struglics, André LU and Håkansson, Gunilla (1999) In European Journal of Biochemistry 262(3). p.765-773
Abstract

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [γ-32]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino- acid sequencing of two peptides, resulting from a trypsin digestion, revealed... (More)

For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [γ-32]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino- acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [32P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali- stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.

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author
organization
publishing date
type
Contribution to journal
publication status
published
keywords
Mitochondria, Nucleoside diphosphate kinase, Pisum sativum, Protein phosphorylation
in
European Journal of Biochemistry
volume
262
issue
3
pages
765 - 773
publisher
Wiley-Blackwell
external identifiers
  • Scopus:0038351354
ISSN
0014-2956
DOI
10.1046/j.1432-1327.1999.00432.x
language
English
LU publication?
yes
id
2cfafe2a-e860-4e57-b0af-9aa2a0445b78
date added to LUP
2016-10-14 16:01:29
date last changed
2016-11-29 10:25:38
@misc{2cfafe2a-e860-4e57-b0af-9aa2a0445b78,
  abstract     = {<p>For the first time, to our knowledge, a nucleoside diphosphate kinase (NDPK) has been purified from plant mitochondria (Pisum sativum L.). In intact pea leaf mitochondria, a 17.4-kDa soluble protein was phosphorylated in the presence of EDTA when [γ-<sup>32</sup>]ATP was used as the phosphate donor. Cell fractionation demonstrated that the 17.4-kDa protein is a true mitochondrial protein, and the lack of accessibility to EDTA of the matrix compartment in intact mitochondria suggested it may have an intermembrane space localization. The 17.4-kDa protein was purified from mitochondrial soluble proteins using ATP-agarose and anion exchange chromatography. Amino- acid sequencing of two peptides, resulting from a trypsin digestion, revealed high similarity with the conserved catalytic phosphohistidine site and with the C-terminal of NDPKs. Acid and alkali treatments of [<sup>32</sup>P]-labelled pea mitochondrial NDPK indicated the presence of acid-stable as well as alkali- stable phosphogroups. Thin-layer chromatography experiments revealed serine as the acid-stable phosphogroup. The alkali-stable labelling probably reflects phosphorylation of the conserved catalytic histidine residue. In phosphorylation experiments, the purified pea mitochondrial NDPK was labelled more heavily on serine than histidine residues. Furthermore, kinetic studies showed a faster phosphorylation rate for serine compared to histidine. Both ATP and GTP could be used as phosphate donor for histidine as well as serine labelling of the pea mitochondrial NDPK.</p>},
  author       = {Struglics, André and Håkansson, Gunilla},
  issn         = {0014-2956},
  keyword      = {Mitochondria,Nucleoside diphosphate kinase,Pisum sativum,Protein phosphorylation},
  language     = {eng},
  month        = {06},
  number       = {3},
  pages        = {765--773},
  publisher    = {ARRAY(0x803d1f0)},
  series       = {European Journal of Biochemistry},
  title        = {Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum},
  url          = {http://dx.doi.org/10.1046/j.1432-1327.1999.00432.x},
  volume       = {262},
  year         = {1999},
}