Spin Labeling of Membrane Proteins in Mammalian Brain Synaptic Plasma Membranes: Partial Characterization

Self Cite

Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age-related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N-tert-butyl-a-phenylnitrone (PBN) and 4-hydroxy-2,2, 6,6-tetramethylpiperidin-1 -oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPA) protein-specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of eitherof the two free radical scavengers. After 30 mm, the gerbils were exposed to 90-100% 02 for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol-specific label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1 -oxyl). As in our earlier study, the EPR spectral parameter of MAL-6--labeled membranes, the W/S ratio, decreased with hyperoxia (p < 0.00001). This effect was lessened significantly with administration of PBN (p <0.0003) or Tempol (p < 0.00003). For the GS and protein carbonyl assays, cortical proteins were used. The activity of the GS decreased with hyperoxia (p <0.000005), and this effect likewise was lessened with administration of PBN (p <0.004) orlempol (p < 0.002). The protein carbonyl content increased with hyperoxia (p < 0.0002), and there was a protective effect found with Tempol (p < 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as potential antiaging agents.

Section: Resultsmentioning

confidence: 99%

“…Spin labeling of synaptosomal membrane proteins was performed as previously described (Umhauer et al, 1992;Hensley et al, 1994). Lysed synaptosomal membranes were labeled with the protein-specific spin label MAL-6.…”

Section: Spin Labelingmentioning

confidence: 99%

Prevention of Hyperoxia‐Induced Alterations in Synaptosomal Membrane‐Associated Proteins by N‐tert‐Butyl‐α‐Phenylnitrone and 4‐Hydroxy‐2,2,6,6‐Tetramethylpiperidin‐1‐oxyl (Tempol)

Howard

Yatin

Hensley

et al. 1996

Self Cite

“…Spin labeling of synaptosomal membrane proteins was performed as described (Umhauer et al 1992;Hensley et al 1994). Isolated synaptosomes were suspended in lysing buffer for 30 min.…”

Section: Tbarsmentioning

confidence: 99%

The antioxidants α‐lipoic acid and N‐acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice

Farr

Poon

Doğrukol-Ak

et al. 2003

Self Cite

433

253

Oxidative stress may play a crucial role in age-related neurodegenerative disorders. Here, we examined the ability of two antioxidants, alpha-lipoic acid (LA) and N-acetylcysteine (NAC), to reverse the cognitive deficits found in the SAMP8 mouse. By 12 months of age, this strain develops elevated levels of Abeta and severe deficits in learning and memory. We found that 12-month-old SAMP8 mice, in comparison with 4-month-old mice, had increased levels of protein carbonyls (an index of protein oxidation), increased TBARS (an index of lipid peroxidation) and a decrease in the weakly immobilized/strongly immobilized (W/S) ratio of the protein-specific spin label MAL-6 (an index of oxidation-induced conformational changes in synaptosomal membrane proteins). Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

“…Brain specimens were removed at autopsy and immediately suspended in ice-cold isolation buffer (0 .32 M sucrose, 10 mM HEPES, pH 7.4, and the following protease inhibitors : 0.5 pg/ml leupeptin, 0.5 pg/ml pepstatin, 0.5 pg/ml aprotinin, and 0.2 mM PMSF, and the following calcium and divalent metal chelators: 2 mM EDTA and 2 mM EGTA) . Spin labeling was performed on these fresh autopsy samples as previously described (Umhauer et al ., 1992 ;Hensley et al ., 1994b ;Hall et al ., 1995) . Samples were thoroughly homogenized with a Wheaton motorized Tefloncoated homogenizer with 0.25-mm clearance.…”

Section: Synaptosome Preparation and Membrane Protein Spin Labelingmentioning

confidence: 99%

Brain Regional Correspondence Between Alzheimer's Disease Histopathology and Biomarkers of Protein Oxidation

Hensley¹,

Hall²,

Ramachandran³

et al. 1995

Self Cite

735

421

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL‐6 spin‐labeled synaptosomes, phenylhydrazine‐reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)‐demented and age‐matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation‐sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque‐dense regions of the AD brain may represent environments of elevated oxidative stress.