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Proteomic analysis of post-nuclear supernatant fraction and percoll-purified membranes prepared from brain cortex of rats exposed to increasing doses of morphine.


ABSTRACT: BACKGROUND: Proteomic analysis was performed in post-nuclear supernatant (PNS) and Percoll-purified membranes (PM) prepared from fore brain cortex of rats exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. RESULTS: In PNS, the 10 up (?)- or down (?)-regulated proteins exhibiting the largest morphine-induced change were selected, excised manually from the gel and identified by MALDI-TOF MS/MS: 1-(gi|148747414, Guanine deaminase), ?2.5×; 2-(gi|17105370, Vacuolar-type proton ATP subunit B, brain isoform), ?2.6×; 3-(gi|1352384, Protein disulfide-isomerase A3), ?3.4×; 4-(gi|40254595, Dihydropyrimidinase-related protein 2), ?3.6×; 5-(gi|149054470, N-ethylmaleimide sensitive fusion protein, isoform CRAa), ?2.0×; 6-(gi|42476181, Malate dehydrogenase, mitochondrial precursor), ?1.4×; 7-(gi|62653546, Glyceraldehyde-3-phosphate dehydrogenase), ?1.6×; 8-(gi|202837, Aldolase A), ?1.3×; 9-(gi|31542401, Creatine kinase B-type), ?0.86×; 10-(gi|40538860, Aconitate hydratase, mitochondrial precursor), ?1.3×. The identified proteins were of cytoplasmic (1, 4, 5, 7, 9), cell membrane (2), endoplasmic reticulum (3) and mitochondrial (6, 8, 10) origin and 9 of them were significantly increased, 1.3-3.6×. The 4 out of 9 up-regulated proteins (4, 6, 7, 10) were described as functionally related to oxidative stress; the 2 proteins participate in genesis of apoptotic cell death.In PM, the 18 up (?)- or down (?)-regulated proteins were identified by LC-MS/MS and were of plasma membrane [Brain acid soluble protein, ?2.1×; trimeric G? subunit, ?2.0x], myelin membrane [MBP, ?2.5×], cytoplasmic [Internexin, ?5.2×; DPYL2, ?4.9×; Ubiquitin hydrolase, ?2.0×; 60S ribosomal protein, ?2.7×; KCRB, ?2.6×; Sirtuin-2, ?2.5×; Peroxiredoxin-2, ?2.2×; Septin-11, ?2.2×; TERA, ?2.1×; SYUA, ?2.0×; Coronin-1A, ?5.4×] and mitochondrial [Glutamate dehydrogenase 1, ?2.7×; SCOT1, ?2.2×; Prohibitin, ?2.2×; Aspartate aminotransferase, ?2.2×] origin. Surprisingly, the immunoblot analysis of the same PM resolved by 2D-ELFO indicated that the "active", morphine-induced pool of G? subunits represented just a minor fraction of the total signal of G? which was decreased 1.2x only. The dominant signal of G? was unchanged. CONCLUSION: Brain cortex of rats exposed to increasing doses of morphine is far from being adapted. Significant up-regulation of proteins functionally related to oxidative stress and apoptosis suggests a major change of energy metabolism resulting in the state of severe brain cell "discomfort" or even death.

SUBMITTER: Ujcikova H 

PROVIDER: S-EPMC3936806 | BioStudies | 2014-01-01T00:00:00Z

REPOSITORIES: biostudies

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