Mitoxantrone changes spectrin-aminophospholipid interactions

Understanding drug-membrane and drug-membrane protein interactions would be a crucial step towards understanding the action and biological properties of anthracyclines, as the cell membrane with its integral and peripheral proteins is the first barrier encountered by these drugs. In this paper, we briefly describe mitoxantrone-monolayer and mitoxantrone-bilayer interactions, focusing on the effect of mitoxantrone on the interactions between erythroid or nonerythroid spectrin with phosphatidylethanolamine-enriched mono- and bilayers. We found that mitoxantrone markedly modifies the interaction of erythroid and nonerythroid spectrins with phosphatidylethanolamine/phosphatidylcholine (PE/PC) monolayers. The change in delta pi induced by spectrins is several-fold larger in the presence of 72 nM mitoxantrone than in its absence: spectrin/mitoxantrone complexes induced a strong compression of the monolayer. Spin-labelling experiments showed that spectrin/mitoxantrone complexes caused significant changes in the order parameter measured using a 5'-doxyl stearate probe in the bilayer, but they practically did not affect the mobility of 16'-doxyl stearate. These results indicate close-to-surface interactions/penetrations without significant effect on the mid-region of the hydrophobic core of the bilayer. The obtained apparent equilibrium dissociation constants indicated relatively similar mitoxantrone-phospholipid and mitoxantrone-spectrin (erythroid and nonerythroid) binding affinities. These results might in part, explain the effect of mitoxantrone on spectrin distribution in the living cells.     

Beta spectrin in human skeletal muscle. Tissue-specific differential processing of 3' beta spectrin pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus

Spectrin, an important component of the mammalian erythrocyte membrane skeleton, is a heterodimeric protein with alpha and beta subunits of 280 and 246 kDa, respectively. Spectrin-like proteins have also been demonstrated in a wide variety of nonerythroid cells. To examine the hypothesis that nonerythroid beta spectrins may be encoded by the "erythroid" beta spectrin gene, we have isolated cDNA clones from a human fetal skeletal muscle library by hybridization to a previously described red cell beta spectrin cDNA. Detailed comparison of muscle and erythroid beta spectrin cDNAs has revealed sequence identity over the majority of their lengths, confirming that they are the product of the same gene. However, there is a sharp divergence in sequence at their 3' ends. A consequence of this divergence is the replacement of the carboxyl terminus of erythroid beta spectrin with a different, longer carboxyl-terminal domain in skeletal muscle. We hypothesize that tissue-specific differential polyadenylation leads to the selective activation of a donor splice site within the beta spectrin coding sequence, splicing downstream nonerythroid exons into the mature muscle beta spectrin mRNA. We predict that replacement, in nonerythroid cells, of the beta spectrin carboxyl terminus, known to participate in spectrin self-association and phosphorylation, has significant functional consequences. These data may explain previously reported nonerythroid beta spectrin isoforms that resemble red cell beta spectrin by immunochemical analysis.     

Cell type-specific association between two types of spectrin and two types of intermediate filaments

We have demonstrated a differential association between two types of spectrin, from erythrocytes and brain, with two types of intermediate filaments, vimentin filaments and neurofilaments. Electron microscopy showed that erythrocyte spectrin promoted the binding of vimentin filaments to red cell inside-out vesicles via lateral associations with the filaments. In vitro binding studies showed that the association of spectrin with vimentin filaments was apparently saturable, increased with temperature, and could be prevented by heat denaturation of the spectrin. Comparisons were made between erythrocyte and brain spectrin binding to both vimentin filaments and neurofilaments. We found that vimentin filaments bound more erythrocyte spectrin than brain spectrin, while neurofilaments bound more brain spectrin than erythrocyte spectrin. Our results show that both erythroid and nonerythroid spectrins are capable of binding to intermediate filaments and that such associations may be characterized by differential affinities of the various types of spectrin with the several classes of intermediate filaments present in cells. Our results also suggest a role for both erythroid and nonerythroid spectrins in mediating the association of intermediate filaments with plasma membranes or other cytoskeletal elements.     

Association of spectrin with desmin intermediate filaments

The association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament-erythrocyte inside-out vesicle preparations. Desmin filaments bound to erythrocyte inside-out vesicles in a spectrin-dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration-dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.     

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (K_d) of .50 and .17?μM, for prodan, and .04 and .02?μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain?>?SH3 domain?>?Self-association domain?>?N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.     

Functional diversity among spectrin isoforms

The purpose of this review on spectrin is to examine the functional properties of this ubiquitous family of membrane skeletal proteins. Major topics include spectrin-membrane linkages, spectrin-filament linkages, the subcellular localization of spectrins in various cell types and a discussion of major functional differences between erythroid and nonerythroid spectrins. This includes a summary of studies from our own laboratories on the functional and structural comparison of avian spectrin isoforms which are comprised of a common alpha subunit and a tissue-specific beta subunit. Consequently, the observed differences among these spectrins can be assigned to differences in the properties of the beta subunits.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Comparison of nonerythroid alpha-spectrin genes reveals strict homology among diverse species.

The spectrins are a family of widely distributed filamentous proteins. In association with actin, spectrins form a supporting and organizing scaffold for cell membranes. Using antibodies specific for human brain alpha-spectrin (alpha-fodrin), we have cloned a rat brain alpha-spectrin cDNA from an expression library. Several closely related human clones were also isolated by hybridization. Comparison of sequences of these and other overlapping nonerythroid and erythroid alpha-spectrin genes demonstrated that the nonerythroid genes are strictly conserved across species, while the mammalian erythroid genes have diverged rapidly. Peptide sequences deduced from these cDNAs revealed that the nonerythroid alpha-spectrin chain, like the erythroid spectrin, is composed of multiple 106-amino-acid repeating units, with the characteristic invariant tryptophan as well as other charged and hydrophobic residues in conserved locations. However, the carboxy-terminal sequence varies markedly from this internal repeat pattern and may represent a specialized functional site. The nonerythroid alpha-spectrin gene was mapped to human chromosome 9, in contrast to the erythroid alpha-spectrin gene, which has previously been assigned to a locus on chromosome 1.     

Human cardiac and skeletal muscle spectrins: differential expression and localization.

We describe multiple human cardiac and skeletal muscle spectrin isoforms. Cardiac muscle expresses five erythroid alpha,beta spectrin-reactive isoforms with estimated MR's of 280, 274, 270, 255, and 246 kD, respectively. At least one nonerythroid alpha-spectrin of MR 284 kD is expressed in heart. While skeletal muscle shares the 280, 270, and 246 kD erythroid spectrins, it expresses an immunologically distinct 284 kD nonerythroid alpha-spectrin isoform. The 255 kD erythroid beta-spectrin isoform is specific for cardiac tissue. By immunocytochemistry, both erythroid beta- and nonerythroid alpha-spectrins are localized to costameres, the plasma membrane, and the neuromuscular junctional region.     

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.     

Effects of GM1 on brain spectrin-aminophospholipid interactions

Spectrin, a major component of the membrane skeletal meshwork of metazoan cells, is implicated to associate with membrane domains and is known to act as a scaffold for stabilization and activation of different signalling modules. We have studied the effect of GM1 (monosialotetrahexosyl ganglioside), a well-known model ganglioside and a signalling moiety, on the interaction of non-erythroid brain spectrin with both saturated and unsaturated aminophospholipids by spectroscopic methods. We observe that GM1 modulates brain spectrin-aminophospholipid interaction to the greatest degree whereas its effect on erythroid spectrin is not as pronounced. Fluorescence quenching studies show that brain spectrin interacts with DMPC/DMPE-based vesicles with a 10-fold increased affinity in presence of very low amounts of 2% and 5% GM1, and the extent of quenching decreases progressively in presence of increasing amounts of GM1. Interaction of brain spectrin with unsaturated membrane systems of DOPC/DOPE weakens in presence GM1. Increase in the mean lifetime of the Trp residues of brain spectrin in presence of GM1 indicates change in the microenvironment of spectrin, without affecting the secondary structure of the protein significantly. Studies on pressure – area isotherm of Langmuir-Blodgett monolayer and Brewster's angle microscopy show that GM1 has an expanding effect on the aminophospholipid monolayers, and ordered regions in DMPC/DMPE mixed monolayers are formed and are stabilized at higher pressure. GM1-induced fluidization of the phospholipid membranes and probable physical contact between bulky sugar head group of GM1 and spectrin, may explain the modulatory role of GM1 on aminophospholipid interactions with nonerythroid brain spectrin.     

Contributions of the beta-subunit to spectrin structure and function

The three avian spectrins that have been characterized consist of a common alpha-subunit (240 kD) paired with an isoform-specific beta-subunit from either erythrocyte (220 or 230 kD), brain (235 kD), or intestinal brush border (260 kD). Analysis of avian spectrins, with their naturally occurring "subunit replacement" has proved useful in assessing the relative contribution of each subunit to spectrin function. In this study we have completed a survey of avian spectrin binding properties and present morphometric analysis of the relative flexibility and linearity of various avian and human spectrin isoforms. Evidence is presented that, like its mammalian counterpart, avian brain spectrin binds human erythroid ankyrin with low affinity. Cosedimentation analysis demonstrates that 1) avian erythroid protein 4.1 stimulates spectrin-actin binding of both mammalian and avian erythrocyte and brain spectrins, but not the TW 260/240 isoform, 2) calpactin I does not potentiate actin binding of either TW 260/240 or brain spectrin, and 3) erythrocyte adducin does not stimulate the interaction of TW 260/240 with actin. In addition, a morphometric analysis of rotary-shadow images of spectrin isoforms, individual subunits, and reconstituted complexes from isolated subunits was performed. This analysis revealed that the overall flexibility and linearity of a given spectrin heterodimer and tetramer is largely determined by the intrinsic rigidity and linearity of its beta-spectrin subunit. No additional rigidity appears to be imparted by noncovalent associations between the subunits. The scaled flexural rigidity of the most rigid spectrin analyzed (human brain) is similar to that reported for F-actin.     

The L49F mutation in alpha erythroid spectrin induces local disorder in the tetramer association region: Fluorescence and molecular dynamics studies of free and bound alpha spectrin

The bundling of the N‐terminal, partial domain helix (Helix C′) of human erythroid α‐spectrin (αI) with the C‐terminal, partial domain helices (Helices A′ and B′) of erythroid β‐spectrin (βI) to give a spectrin pseudo structural domain (triple helical bundle A′B′C′) has long been recognized as a crucial step in forming functional spectrin tetramers in erythrocytes. We have used apparent polarity and Stern–Volmer quenching constants of Helix C′ of αI bound to Helices A′ and B′ of βI, along with previous NMR and EPR results, to propose a model for the triple helical bundle. This model was used as the input structure for molecular dynamics simulations for both wild type (WT) and αI mutant L49F. The simulation output structures show a stable helical bundle for WT, but not for L49F. In WT, four critical interactions were identified: two hydrophobic clusters and two salt bridges. However, in L49F, the region downstream of Helix C′ was unable to assume a helical conformation and one critical hydrophobic cluster was disrupted. Other molecular interactions critical to the WT helical bundle were also weakened in L49F, possibly leading to the lower tetramer levels observed in patients with this mutation‐induced blood disorder.     

The present status of erythrocyte spectrin structure: the 106-residue repetitive structure is a basic feature of an entire class of proteins

Spectrin, the major component of the erythroid membrane skeleton, is a long, asymmetrical rodlike protein that interacts with several other proteins to form a two-dimensional membrane skeleton. Progress in several laboratories over the past few years including substantial partial peptide and nucleotide sequence determination has greatly enhanced our knowledge of the structural properties of this large molecule (heterodimer = 465,000 daltons). The alpha and beta subunits are homologous with approximately 30% identity. They are aligned in an antiparallel side-to-side orientation with the amino- and carboxy-termini near opposite physical ends of the molecule. The predominant structural feature elucidated from sequencing this large molecule is the nearly universal occurrence in both subunits of a single type of repetitive structure. The periodicity of this homologous structure is exactly 106 amino acid residues. As many as 36 homologous, but nonidentical, repeats exist and comprise more than 90% of the mass of the heterodimer. Each of these repetitive units is folded into a triple-stranded structure that is highly helical. Peptide maps, antibody crossreactivity, peptide sequence analysis, and more recently nucleic acid sequences have defined several major properties of the erythroid molecule and related proteins in other tissues. Tissue-specific spectrins have the same 106-residue repetitive structure and show sequence homology to erythroid spectrin.     

Beta spectrin bestows protein 4.1 sensitivity on spectrin-actin interactions

The ability of protein 4.1 to stimulate the binding of spectrin to F-actin has been compared by cosedimentation analysis for three avian (erythrocyte, brain, and brush border) and two mammalian (erythrocyte and brain) spectrin isoforms. Human erythroid protein 4.1 stimulated actin binding of all spectrins except the brush border isoform (TW 260/240). These results suggested that the beta subunit determined the protein 4.1 sensitivity of the heterodimer, since all avian alpha subunits are encoded by a single gene. Tissue-specific posttranslational modification of the alpha subunit was excluded by examining the properties of hybrid spectrins composed of the purified alpha subunit from avian erythrocyte or brush border spectrin and the beta subunit of human erythrocyte spectrin. A hybrid composed of avian brush border alpha and human erythroid beta spectrin ran on nondenaturing gels as a discrete band, migrating near human erythroid spectrin tetramers. The actin-binding activity of this hybrid was stimulated by protein 4.1, while either chain alone was devoid of activity. Therefore, although both subunits were required for actin binding, the sensitivity of the spectrin-actin interaction to protein 4.1 is a property uniquely bestowed on the heterodimer by the beta subunit. The singular insensitivity of brush border spectrin to stimulation by erythroid protein 4.1 was also consistent with the absence of proteins in avian intestinal epithelial cells which were immunoreactive with polyclonal antisera sensitive to all of the known avian and human erythroid 4.1 isoforms.     

Role of spectrin in Amoeba proteus, as studied by microinjection of anti-spectrin monoclonal antibodies.

Spectrin is a major protein accounting for about 5% of whole-cell proteins in Amoeba proteus, and the precipitation of spectrin by intracellular injection of purified anti-spectrin monoclonal antibodies has a profound effect on cell morphology, motility, and movement-related cell activities in amoebae. Thus, amoebae injected with anti-spectrin antibodies show drastic changes in their shape and movement, suggesting that amoeba spectrin plays an important structural role, unlike nonerythroid spectrins in other cells. However, precipitation of spectrin does not affect the distribution of F-actin in amoebae.     

Characterization of the calmodulin-binding site of nonerythroid alpha-spectrin. Recombinant protein and model peptide studies

An important function of the mammalian nonerythroid alpha-spectrin chain (alpha-fodrin) that distinguishes it from the closely related erythroid isoform is its ability to bind calmodulin. By analysis of a series of deleted recombinant spectrin fusion proteins, we have identified a region in the nonerythroid alpha chain involved in calcium-dependent binding of calmodulin. The region is distinctive in that the sequence is absent from the homologous domain of the erythroid alpha chain and diverges from the normal internal repeat structure observed throughout other spectrins. In order to determine limits of this functional site, a synthetic peptide as small as 24 residues was shown to compete with either recombinant or brain alpha-spectrin in binding to calmodulin. The active peptide, which was derived from a segment between repeats 11 and 12, was composed of the following sequence: Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala-Thr-Phe-Asn - Ser-Ile-Lys-Glu. Comparison of this sequence with functional sites in other diverse calcium-dependent calmodulin-binding proteins has revealed a structural motif common to all of these proteins, namely clusters of hydrophobic residues interspersed with basic residues. When folded into alpha-helical conformations, these binding sites are predicted to form amphipathic structures.     

Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B(max)) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in deltapi change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The deltapi change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a "raft-like" mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.