Saturable Ethanol Binding in Rat Liver Microsomes

Channareddy, Sreelatha; Jose, Sherilyn S.; Eryomin, Vladimir A.; Rubin, Emanuel; Taraschi, Theodore F.; Janes, Nathan

doi:10.1074/jbc.271.30.17625

Cited by 14 publications

(13 citation statements)

References 21 publications

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“…1A and 1B). In the absence of peroxide, EtOH spontaneously incorporates to cyt c at a basal level of 0.3 mol of EtOH/mol protein, most probably noncovalently trapped in hydrophobic protein pockets (Channareddy et al, 1996).…”

Section: Adducts Of Etoh Metabolites With Hemeproteinsmechanism Of Fomentioning

confidence: 99%

Characterization of Adducts of Ethanol Metabolites with Cytochrome c

Anni

Israel

1999

Alcoholism Clin & Exp Res

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Cytochrome c (cyt c) is found in the mitochondria of all mammalian cells where hydrogen peroxide is produced as a byproduct of the electron transport chain. In the presence of peroxide cyt c generates a ferryl heme and radicals at Tyr residues (Barr et al., 1996). These radicals can be transferred to Trp residues within the protein or to Tyr- and Trp-containing peptides (Deterding et al., 1998). We report that addition of ethanol to this system of cyt c plus peroxide results in replacement of the Tyr/Trp radicals by 1-hydroxyethyl radicals (HER), and covalent binding of up to 10 mol of ethanol per mol of cyt c. In the absence of exogenously added peroxide, ethanol incorporation to cyt c is attained also with a reconstituted system of the ethanol-inducible cytochrome P-4502E1 isozyme. Comparative studies with myoglobin and apomyoglobin suggest that the heme is necessary for ethanol adduction of the protein to occur. Structural analysis by mass spectrometry of the tryptic digestion fractions of adducted cyt c is consistent with several peptides bearing one-to-three acetaldehyde moieties on Lys residues, and three distinct Tyr/Trp-containing peptides: P[28-53], P[56-73], P[73-91] carrying one-to-two HER. The x-ray crystallographic structure of cyt c shows that the Tyr/Trp residues in the adducted peptides are in close proximity to the heme. In conclusion, our data show that ethanol metabolites alkylate cyt c under oxidative stress and point to HER-Tyr/Trp adducts as plausible markers of alcoholism.

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Section: Adducts Of Etoh Metabolites With Hemeproteinsmechanism Of Fomentioning

confidence: 99%

Characterization of Adducts of Ethanol Metabolites with Cytochrome c

Anni

Israel

1999

Alcoholism Clin & Exp Res

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“…To investigate the role of microsomal proteins in the activity of PITP or its ethanol enhancement, transfer from thermally pretreated microsomes was monitored. We have shown previously that brief thermal pretreatment abolished the saturable binding of ethanol in microsomes [25]. Thermal pretreatment of the donor microsomes did not alter the activity of PITP and did not alter the enhancement by ethanol.…”

Section: Ethanol and Pitpmentioning

confidence: 99%

“…More recently, opinion has shifted from lipid theories to protein theories as evidence has accumulated that clearly implicate hydrophobic regions of proteins as common targets of alcohols and anaesthetics [25,47]. This has led some to question whether any of the functional effects of alcohols and anaesthetics are mediated by membrane lipids [47].…”

Section: Implications For Ethanol Actionmentioning

confidence: 99%

“…The thermal treatment had previously been shown to abolish the ability of microsomes to bind ethanol saturably [25]. The protease treatment decreased the microsomal protein content by 42 %.…”

Section: Influence Of Microsomal Membrane Proteins On Pitp Activitymentioning

confidence: 99%

“…Microsomal PtdIns was radiolabelled by head-group exchange of inositol by using myo-[2-$H]inositol, as described previously [23,24]. Heat-treated microsomes were incubated in boiling water for 5 min under argon [25].…”

Section: Preparation Of Donor Microsomes and Acceptor Liposomesmentioning

confidence: 99%

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Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature

Komatsu

Bouma

Wirtz

et al. 2000

Biochemical Journal

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Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol. The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-α in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles. Acute exposure to pharmacological levels of ethanol enhanced the function of PITP. Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes. Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer. Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces. The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane. Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles. Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids. The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action. In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid.

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