PMID- 19426869
OWN - NLM
STAT- MEDLINE
DCOM- 20090713
LR  - 20131121
IS  - 1557-7988 (Electronic)
IS  - 0076-6879 (Linking)
VI  - 457
DP  - 2009
TI  - Isolation of regulatory-competent, phosphorylated cytochrome C oxidase.
PG  - 193-210
LID - 10.1016/S0076-6879(09)05011-3 [doi]
AB  - The role of posttranslational modifications, specifically reversible 
      phosphorylation as a regulatory mechanism operating in the mitochondria, is a 
      novel research direction. The mitochondrial oxidative phosphorylation system is a 
      particularly interesting unit because it is responsible for the production of the 
      vast majority of cellular energy in addition to free radicals, two factors that 
      are aberrant in numerous human diseases and that may be influenced by reversible 
      phosphorylation of the oxidative phosphorylation complexes. We here describe a 
      detailed protocol for the isolation of mammalian liver and heart mitochondria and 
      subsequently cytochrome c oxidase (CcO) under conditions maintaining the 
      physiological phosphorylation state. The protocol employs the use of activated 
      vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific 
      serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent 
      the activation of calcium-dependent protein phosphatases. CcO purified without 
      manipulation of signaling pathways shows strong tyrosine phosphorylation on 
      subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced 
      by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow 
      liver tissue we further show the identification of a new phosphorylation site on 
      CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of 
      the precursor peptide) via immobilized metal affinity chromatography/nano-liquid 
      chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). 
      This phosphorylation site is located close to the ATP and ADP binding site, which 
      adjusts CcO activity to cellular energy demand, and we propose that 
      phosphorylation of tyrosine 11 enables allosteric regulation.
FAU - Lee, Icksoo
AU  - Lee I
AD  - Center for Molecular Medicine and Genetics, Wayne State University School of 
      Medicine, Detroit, Michigan, USA.
FAU - Salomon, Arthur R
AU  - Salomon AR
FAU - Yu, Kebing
AU  - Yu K
FAU - Samavati, Lobelia
AU  - Samavati L
FAU - Pecina, Petr
AU  - Pecina P
FAU - Pecinova, Alena
AU  - Pecinova A
FAU - Huttemann, Maik
AU  - Huttemann M
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 0 (Protein Subunits)
RN  - 3WHH0066W5 (Vanadates)
RN  - 42HK56048U (Tyrosine)
RN  - EC 1.9.3.1 (Electron Transport Complex IV)
SB  - IM
MH  - Animals
MH  - Cattle
MH  - Chromatography, Liquid
MH  - Electron Transport Complex IV/analysis/*isolation & purification/*metabolism
MH  - Mass Spectrometry
MH  - Mitochondria, Heart/*enzymology
MH  - Mitochondria, Liver/*enzymology
MH  - Phosphorylation
MH  - Protein Subunits/analysis/metabolism
MH  - Spectrometry, Mass, Electrospray Ionization
MH  - Tyrosine/analysis/metabolism
MH  - Vanadates/metabolism
EDAT- 2009/05/12 09:00
MHDA- 2009/07/14 09:00
CRDT- 2009/05/12 09:00
PHST- 2009/05/12 09:00 [entrez]
PHST- 2009/05/12 09:00 [pubmed]
PHST- 2009/07/14 09:00 [medline]
AID - S0076-6879(09)05011-3 [pii]
AID - 10.1016/S0076-6879(09)05011-3 [doi]
PST - ppublish
SO  - Methods Enzymol. 2009;457:193-210. doi: 10.1016/S0076-6879(09)05011-3.