Determination of the oligomeric states of human and rat monoamine oxidases in the outer mitochondrial membrane and octyl beta-D-glucopyranoside micelles using pulsed dipolar electron spin resonance spectroscopy

Human monoamine oxidase A (hMAOA) is considered to be unique among mammalian MAOs in having a non-conservative Glu-X-Lys mutation (X being 151 in MAOAs and 142 in MAOB's), which is suggested to be the reason for its monomeric structure. This hypothesis has been tested in this work. A pargyline based nitroxide spin labeled irreversible inhibitor (ParSL) was used as a MAO active site specific spin probe to measure intersubunit distances in detergent (octyl beta-d-glucopyranoside, OGP) purified and OMM bound forms by a pulsed dipolar ESR spectroscopic (PDS) technique. In a parallel approach, the covalent flavin cofactor present in the MAO active sites was reduced to its respective anionic flavin semiquinone and used for measuring inter-flavin distances in detergent purified samples. The measured interspin distances are within 0.1-0.3 nm of those estimated from the available dimeric crystal structures of human MAOB and rat MAOA and show that all human and rat MAOs exist as dimers in the OMM. In the OGP micelle, however, human and rat MAOAs exist only partially (相似文献   

Comparative study on the active sites of ficin and papain by the spin labeling method

Ficin was alkylated with a series of haloacetamide spin labels with various distances between the spin probes and reactive groups. From the relation of these distances to the tau c values of the labels incorporated into protein, it was estimated that the depth of the active site hole of ficin is ca. 8 A. The results are somewhat different from those reported previously for papain (S. Nakayama et al. (1981) Biochem. Biophys. Res. Commun. 98, 471-475). Examination of the pH dependence of the ESR spectra for ficin and papain alkylated with an iodoacetamide or a maleimide spin label suggested that these enzymes have an amino acid residue of pKa 4 (probably a histidine residue) around the active site cysteine and that the active site conformations change at around pH 5.     

Structure of rat monoamine oxidase A and its specific recognitions for substrates and inhibitors

Monoamine oxidase (MAO), a mitochondrial outer membrane enzyme, catalyzes the degradation of neurotransmitters in the central nervous system and is the target for anti-depression drug design. Two subtypes of MAO, MAOA and MAOB, are similar in primary sequences but have unique substrate and inhibitor specificities. The structures of human MAOB complexed with various inhibitors were reported early. To understand the mechanisms of specific substrate and inhibitor recognitions of MAOA and MAOB, we have determined the crystal structure of rat MAOA complexed with the specific inhibitor, clorgyline, at 3.2A resolution. The comparison of the structures between MAOA and MAOB clearly explains the specificity of clorgyline for MAOA inhibition. The fitting of serotonin into the binding pockets of MAOs demonstrates that MAOB Tyr326 would block access of the 5-hydroxy group of serotonin into the enzyme. These results will lead to further understanding of the MAOA function and to new anti-depression drug design. This study also presents that MAOA has a transmembrane helix at the C-terminal region. This is the first crystal structure of membrane protein with an isolated transmembrane helix.     

Spin-labelled AMP - an activator of phosphorylase.

1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.     

Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1

The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity to position themselves correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin-labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a DeltaDeltaG of -190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin-labeled at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a K(i) of 9 muM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty-acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction.     

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.     

The 'gating' residues Ile199 and Tyr326 in human monoamine oxidase B function in substrate and inhibitor recognition

The major structural difference between human monoamine oxidases A (MAO A) and B (MAO B) is that MAO A has a monopartite substrate cavity of ~550 ?(3) volume and MAO B contains a dipartite cavity structure with volumes of ~290 ?(3) (entrance cavity) and ~400 ?(3) (substrate cavity). Ile199 and Tyr326 side chains separate these two cavities in MAO B. To probe the function of these gating residues, Ile199Ala and Ile199Ala-Tyr326Ala mutant forms of MAO B were investigated. Structural data on the Ile199Ala MAO B mutant show no alterations in active site geometries compared with wild-type enzyme while the Ile199Ala-Tyr326Ala MAO B mutant exhibits alterations in residues 100-103 which are part of the loop gating the entrance to the active site. Both mutant enzymes exhibit catalytic properties with increased amine K(M) but unaltered k(cat) values. The altered K(M) values on mutation are attributed to the influence of the cavity structure in the binding and subsequent deprotonation of the amine substrate. Both mutant enzymes exhibit weaker binding affinities relative to wild-type enzyme for small reversible inhibitors. Ile199Ala MAO B exhibits an increase in binding affinity for reversible MAO B specific inhibitors which bridge both cavities. The Ile199Ala-Tyr326Ala double mutant exhibits inhibitor binding properties more similar to those of MAO A than to MAO B. These results demonstrate that the bipartite cavity structure in MAO B plays an important role in substrate and inhibitor recognition to distinguish its specificities from those of MAO A and provide insights into specific reversible inhibitor design for these membrane-bound enzymes.     

Study of the structure of mammalian serum albumin using fluorescent probes

Some peculiarities of hydrophobic structure of serum albumine of some mammals were studied by NMR-spectroscopy, solubilization and fluorescent probes. It has been shown that the FNA probe is bound to the most hydrophobic cavities in the protein molecules, and the sizes of these regions in mammalian albumines are very close. The data obtained by ANS probe show that there exists a proportional relationship between the fluorescence intensity, the total volume of hydrophobic cavities and the quantity of "bound water". When using the ANS--Mg1/2 probe in all cases an increase of fluorescence intensity was obtained. It was concerned with the stabilizing effect of magnesium ions on the protein molecule.     

Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity

Eugenol (1) is an active principle of Rhizoma acori graminei, a medicinal herb used in Asia for the treatment of symptoms reminiscent of Alzheimer's disease (AD). It has been shown to protect neuronal cells from the cytotoxic effect of amyloid beta peptides (Abetas) in cell cultures and exhibit antidepressant-like activity in mice. Results from this study show that eugenol inhibits monoamine oxidase A (MAOA) preferentially with a K(i)=26 microM. It also inhibits MAOB but at much higher concentrations (K(i)=211 microM). In both cases, inhibition is competitive with respect to the monoamine substrate. Survey of compounds structurally related to eugenol has identified a few that inhibit MAOs more potently. Structure activity relationship reveals structural features important for MAOA and MAOB inhibition. Molecular docking experiments were performed to help explain the SAR outcomes. Four of these compounds, two (1, 24) inhibiting MAOA selectively and the other two (19, 21) inhibiting neither MAOA nor MAOB, were tested for antidepressant-like activity using the forced swim test in mice. Results suggest a potential link between the antidepressant activity of eugenol and its MAOA inhibitory activity.     

Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. I. Specificity of thrombin labeling.

In a new strategy for labeling the active sites of serine proteinases with fluorescence probes (Bock, P. E. (1988) Biochemistry 27, 6633-6639), a thioester peptide chloromethyl ketone inhibitor is incorporated into the enzyme active center and used to produce a unique thiol group which provides a site for selective chemical modification with any one of many thiol-reactive fluorescence probes. This approach was developed to increase the opportunities for identifying fluorescent proteinase derivatives that act as reporters of binding interactions by allowing a large number of derivatives, representing a broad range of probe spectral properties, to be readily prepared. In the studies described here, the specificity of the labeling approach was evaluated quantitatively for the labeling of human alpha and beta/gamma-thrombin with the thioester peptide chloromethyl ketones, N alpha-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl and N alpha-[(acetylthio)acetyl]-D-Phe-Phe-Arg-CH2Cl, and the thiol-reactive fluorescence probe, 5-(iodoacetamido)fluorescein. Irreversible inactivation of thrombin by the inhibitors was accompanied by incorporation of 0.98 +/- 0.06 mol/mol of the thioester group into the active site, independent of a 470-fold difference between the thioester peptide chloromethyl ketones in the bimolecular rate constants of alpha-thrombin affinity labeling. Subsequent mild treatment of the covalent thrombin-inhibitor complexes with NH2OH in the presence of 5-(iodoacetamido)fluorescein resulted in generation of the thiol group together with its selective modification and incorporation of 0.96 +/- 0.07 mol of probe/mol of active sites. The incorporated label was localized to a 9000 molecular weight region of alpha and beta/gamma-thrombin containing the catalytic-site histidine residue. Evaluation of competing, side reactions showed that they did not significantly compromise the active site specificity of labeling. These results demonstrated equivalent, active-site-selective fluorescence probe labeling of alpha and beta/gamma-thrombin by use of either of the thioester peptide chloromethyl ketones, with a site specificity of greater than or equal to 94%.     

Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli.

The interaction of Mn2+, substrates and initiators with RNA polymerase have been studied by kinetic and magnetic resonance methods. As determined by electron paramagnetic resonance, Mn2+ binds to RNA polymerase at one tight binding site with a dissociation constant less than 10 muM and at 6 +/- 1 weak binding sites with dissociation constants 100-fold greater. The binding of Mn2+ to RNA polymerase at both types of sites causes an order of magnitude enhancement of the paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons, indicating the presence of residual water ligands on the enzyme-bound Mn2+. A kinetic analysis of the Mn2+-activated enzyme with poly(dT) as template indicates the substrate to be MnATP under steady-state conditions in the presence or absence of the initiator ApA. ATP and UTP interact with the tightly bound Mn2+ to form ternary complexes with approximately 50% greater enhancement factors. The dissociation constant of MnATP from the tight Mn2+ site as determined by longitudinal proton relaxation rate (PRR) titration (4.7 muM) is similar to the KM of MnATP in the ApA-initiated RNA polymerase reaction (10 +/- 3 muM) but not in the ATP-initiated reaction (160 +/- 30 muM). Similarly, the dissociation constant of the substrate MnUTP from the tight Mn2+ site (90 muM) is in agreement with the KM of MnUTP (101 +/- 13 muM) when poly[d(A-T)]-poly[d(A-T)] is used as template, indicating the tight Mn2+ site to be the catalytic site for RNA chain elongation. Manganese adenylyl imidodiphosphate (MnAMP-PNP) has been found to be a substrate for RNA polymerase. It has the same affinity as MnATP for the tight site but, unlike the results obtained with MnATP, the enhancement is decreased by 43% in the enzyme Mn-AMP-PNP complex. These results suggest that the enzyme-bound Mn2+ interacts with the leaving pyrophosphate group. The initiators ApA and ApU and the inhibitor rifamycin interact with the enzyme-Mn2+ complex producing small (15-20%) decreases in the enhancement. The dissociation constant of ApA estimated from PRR data (less than or equal to 1.5 muM) agrees with that determined kinetically (1.0 +/- 0.5 muM) as the concentration of ApA required to produce half-maximal change in the KM of MnATP. In the presence of the initiation specific reagents ApA, ApU, or rifamycin, the affinity of the enzyme-Mn complex for ATP or UTP shows little change. However, ATP and UTP no longer increase the enhancement factor of the tightly bound Mn2+ but decrease it by 30-55%, indicating a change in the environment of the Mn2+-substrate complex on the enzyme when the initiation site is either occupied or blocked. Although the role of the six weak Mn2+ binding sites is not clear, the presence of a single tightly bound Mn2+ at the catalytic site for chain elongation which interacts with the substrate reinforces the number of active sites as one per molecule of holoenzyme and provides a paramagnetic reference point for further structural studies.     

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism.     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

A 40-kDa polypeptide from papain digestion of the rabbit intestinal Na+/phosphate cotransporter retains Na+ and phosphate cotransport

We searched for new fluorescent probes of catalytic-site nucleotide binding in F(1)F(0)-ATP synthase by introducing Cys mutations at positions in or close to catalytic sites and then reacting Cys-mutant F(1) with thiol-reactive fluorescent probes. Four suitable mutant/probe combinations were identified. beta F410C labeled by 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F) gave very large signal changes in response to nucleotide, allowing facile measurement of fluorescence and nucleotide-binding parameters, not only in F(1) but also in F(1)F(0). The results are consistent with the presence of three asymmetric catalytic sites of widely different affinities, with similar properties in both enzymes, and revealed a unique probe environment at the high-affinity site 1. beta Y331C F(1) labeled by ABD-F gave a large signal which monitored catalytic site polarity changes that occur along the ATP hydrolysis pathway. Two other mutant/probe combinations with significant nucleotide-responsive signals were beta Y331C labeled by 5-((((2-iodoacetyl)amino)ethyl)amino)naphthaline-1-sulfonic acid and alpha F291C labeled by 2-4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid. The signal of the latter responds differentially to nucleoside diphosphate versus triphosphate bound in catalytic sites.     

trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1

The catalytic domain of the flavin-dependent human histone demethylase lysine-specific demethylase 1 (LSD1) belongs to the family of amine oxidases including polyamine oxidase and monoamine oxidase (MAO). We previously assessed monoamine oxidase inhibitors (MAOIs) for their ability to inhibit the reaction catalyzed by LSD1 [Lee, M. G., et al. (2006) Chem. Biol. 13, 563-567], demonstrating that trans-2-phenylcyclopropylamine (2-PCPA, tranylcypromine, Parnate) was the most potent with respect to LSD1. Here we show that 2-PCPA is a time-dependent, mechanism-based irreversible inhibitor of LSD1 with a KI of 242 microM and a kinact of 0.0106 s-1. 2-PCPA shows limited selectivity for human MAOs versus LSD1, with kinact/KI values only 16-fold and 2.4-fold higher for MAO B and MAO A, respectively. Profiles of LSD1 activity and inactivation by 2-PCPA as a function of pH are consistent with a mechanism of inactivation dependent upon enzyme catalysis. Mass spectrometry supports a role for FAD as the site of covalent modification by 2-PCPA. These results will provide a foundation for the design of cyclopropylamine-based inhibitors that are selective for LSD1 to probe its role in vivo.     

Uridine--cytidine kinase from foetal and adult rat liver. Purification and study of some properties.

Partial purification of uridine--cytidine kinase (EC 2.7.1.48) from foetal rat liver by chromatography on DEAE-cellulose gives two active fractions. The first in order of elution was identified as a form specific for foetal liver. It was purified 300-fold. The second fraction was common to foetal, and adult rat liver and spleen and was purified 20-fold. The foetal fraction of the enzyme was found to be heat-sensitive and protected against inactivation by PO34- anions. The two isolated forms have different apparent Km for uridine, respectively 410 muM for the foetal form and 52 muM for the adult form.     

DNase II digestion of the nucleosome core: precise locations and relative exposures of sites.

The precise locations and relative exposures of the DNase II-accessible sites in the nucleosome core DNA are determined using techniques previously employed for the enzyme DNase I. It is found that there are a number of similarities between the site exposure patterns for the two enzymes but that in general the DNase II seems to discriminate less among adjacent sites' accessibilities than does DNase I. The two enzymes attack essentially the same positions in the DNA, the average difference between the precise location of the site being less than one-half base for the two enzymes. Such close similarities in the digestion patterns of two enzymes with such different mechanisms of scission show that the patterns reflect the structure of the nucleosome core and not merely the properties of the particular enzyme used.     

Preparation of a cathepsin D sensitive near-infrared fluorescence probe for imaging.

A variety of proteases are overexpressed or activated during pathogenesis and represent important targets for therapeutic drugs. We have previously shown that optical imaging probes sensitive in the near-infrared fluorescence (NIRF) spectrum can be used for in vivo imaging of enzyme activity. In the current study, we show that these probes can be designed with specificity for specific enzymes, for example, cathepsin D which is known to be overexpressed in many tumors. A NIR cyanine fluorochrome served as the optical reporter and was attached to the amino terminal of an 11 amino acid peptide sequence with specificity for cathepsin D. The peptides were subsequently attached to a synthetic graft copolymer for efficient tumoral delivery. The close spatial proximity of the multiple fluorochromes resulted in quenching of fluorescence in the bound state. A 350-fold signal amplification was observed post cleavage during in vitro testing. Cell culture experiments using a rodent tumor cell line stably transfected with human cathepsin D confirmed enzyme specific activation within cells. This sequence but not a scrambled control sequence showed enzyme specificity in vitro. We conclude that activatable NIRF optical probes can be synthesized to potentially probe for specific enzymes in living organisms.     

Phospholipid spin probes measure the effects of ethanol on the molecular order of liver microsomes

Ethanol, in vitro, is known to perturb the molecular order of the phospholipids in biological membranes, while chronic ethanol exposure, in vivo, leads to resistance to disordering. Such changes have usually been measured by electron spin resonance, utilizing fatty acid spin probes. The use of such probes is controversial, since their orientation in the membrane may not accurately represent that of individual phospholipids. We, therefore, compared ethanol-induced structural perturbations in the membranes of rat hepatic microsomes measured with the spin probe 12-doxylstearic acid (SA 12) with those assayed with various phospholipid spin probes. With SA 12, the addition of increasing amounts of ethanol (50-250 mM) in vitro caused a progressive decrease in the membrane molecular order, as measured by electron spin resonance (ESR). By contrast, microsomes obtained from rats chronically fed ethanol were resistant to the disordering effect of ethanol. Microsomes labeled with the phospholipid spin probes 1-palmitoyl-2-(12-doxylstearoyl)phosphatidylcholine, -phosphatidylethanolamine, or -phosphatidic acid also exhibited increased disordering with the addition of increasing amounts of ethanol. However, the effect noted with phospholipid spin probes was less than that observed with the fatty acid probe. Microsomes obtained from the livers of chronically intoxicated animals labeled with the phospholipid probes were also resistant to the disordering effects of ethanol in vitro. These results suggest that fatty acid spin probes are qualitatively valid for measuring membrane perturbations in biological membranes, ethanol affects all microsomal phospholipids, regardless of chemical dissimilarities (e.g., head-group structure), in a qualitatively similar fashion, and the fluidization of fatty acyl chains in microsomal membranes is comparable in different membrane phospholipids.