| 1. |
- Struglics, André, et al.
(författare)
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Phosphoproteins and protein kinase activities intrinsic to inner membranes of potato tuber mitochondria
- 1999
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Ingår i: Plant and Cell Physiology. - 0032-0781. ; 40:12, s. 1271-1279
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out submitochondrial particles (IO-SMP) were isolated and purified from potato (Solanum tuberosum L. cv.) tubers. When these IO-SMP were incubated with [γ32P]ATP more then 20 proteins became labelled as a result of phosphorylation. The 32P incorporation was stimulated by the oxidizing reagent ferricyanide. Except for a 17 kDa protein which was phosphorylated only in the absence of divalent cations, the protein phosphorylation required Mg2+. The time for half-maximum 32P incorporation was 4 min for the 22 kDa phospho-F1 δ-subunit and 2 min for the 28 kDa phospho-F0 b-subunit of the proton-ATPase. The K(m) for ATP for the detected phosphoproteins was between 65 μM and 110 μM. The pH optimum for protein phosphorylation in inner membranes was between pH 6 and 8, and for the F1 δ-subunit and the F0 b-subunit the pH optima were 6.5-8 and pH 8, respectively. A 37 kDa phosphoprotein was phosphorylated on a histidine residue while the remainder of the inner membrane proteins were phosphorylated on serine or threonine residues. Two autophosphorylated putative kinases were identified: one at 16.5 kDa required divalent cations for autophosphorylation, while another at 30 kDa did not. A 110 kDa protein was labelled only with [α-32P]ATP, suggesting adenylylation.
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| 2. |
- Struglics, André
(författare)
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Protein phosphorylation in plant mitochondria
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein phosphorylation in the subcompartments of plant mitochondria was investigated by labelling with [gamma-32P]ATP and by SDS-PAGE/autoradiography. About 20 proteins in inside-out inner mitochondrial membranes from potato tubers were phosphorylated by endogenous protein kinases when incubated with [gamma-32P]ATP. None of the phosphorylated proteins were labelled when using [alpha-32P]ATP as a phosphate donor. The reversible protein phosphorylation in these membranes is mainly a result of the action of inner membrane serine/threonine protein kinases/phosphatases, although the dephosphorylation of inner membrane proteins appears to be catalysed by protein phosphatases located in the matrix. In addition, a 37 kDa protein contained acid-labile phosphate groups, indicating modification of histidine residues. The phosphorylation of inner membrane proteins requires divalent cations (i.e. Mg2+ or Mn2+) and is stimulated under oxidising conditions. Two autophosphorylated inner membrane proteins with molecular mass of 16.5 and 30 kDa are putative protein kinases. Two inner membrane phosphoproteins with molecular mass of 22 and 28 kDa were identified by N-terminal sequencing as the delta?-subunit and b-subunit, respectively, of the FoF1-ATPase. A 17 kDa phosphoprotein has some characteristics of a nucleoside diphosphate kinase. All other plant inner mitochondrial membrane phosphoproteins remain to be identified. A 39.5 and 41 kDa protein are the most heavily labelled phosphoproteins in the matrix of potato tuber mitochondria. The 41 kDa protein is probably the alpha-subunit of pyruvate dehydrogenase, and the 39.5 kDa protein, which was also labelled with 32Pi, may be the alpha-subunit of succinyl-CoA synthetase. In pea leaf mitochondria, a 17.4 kDa protein, which is probably located in the intermembrane space, was purified and identified as a nucleoside diphosphate kinase. This pea protein is autophosphorylated on both the catalytic histidine and on serine residues. The labelling on serine residues was higher then for the histidine, and kinetic studies demonstrated a faster rate of phosphorylation of serine compared to histidine.
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| 3. |
- Struglics, André, et al.
(författare)
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Purification of a serine and histidine phosphorylated mitochondrial nucleoside diphosphate kinase from Pisum sativum
- 1999
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 262:3, s. 765-773
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Tidskriftsartikel (refereegranskat)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 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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| 4. |
- Struglics, André, et al.
(författare)
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Two subunits of the F0F1-ATPase are phosphorylated in the inner mitochondrial membrane
- 1998
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 0006-291X. ; 243:3, s. 664-668
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Tidskriftsartikel (refereegranskat)abstract
- Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [γ-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the δ'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.
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