Biomedical subjects

C C Curtain

Publications and source records attributed to C C Curtain.

At least 19 recordsLinked to original sources

PubMed2001

Selective, high-affinity binding of ferric ions by glycine-extended gastrin(17).

Uptake of dietary iron is essential for replenishment of body stores. A role for the hormone gastrin in iron uptake as a chelator of ferric ions in the gastric lumen has been proposed previously [Baldwin, G. S. (1992) Med. Hypotheses 38, 70-74]. Here, spectroscopic evidence of selective, high-affinity binding of ferric ions to progastrin-derived peptides in aqueous solution at low pH is provided. The maximum at 281 nm in the absorption spectrum of glycine-extended gastrin(17) at pH 4.0 increased (2.07 +/- 0.30)-fold in the presence of > or =2 equiv of ferric ions. Titration of glycine-extended gastrin(17) with ferric ions under stoichiometric conditions indicated that the stoichiometry of binding was 2.00 +/- 0.28 mol of Fe(3+)/mol of peptide. Fluorescence quenching experiments yielded values for the stoichiometry and apparent dissociation constant of the ferric ion-glycine-extended gastrin(17) complex at pH 4.0 of 2.39 +/- 0.17 mol of Fe(3+)/mol and 0.62 +/- 0.19 microM, respectively. No interaction was detected with Co(2+), Cu(2+), Mn(2+), or Cr(3+). Electron paramagnetic resonance spectroscopy suggested that the iron ligands were either oxygen or sulfur atoms. Fluorescence quenching experiments with peptides derived from the glycine-extended gastrin(17) sequence indicated that one or more of the five glutamic acid residues were necessary for iron binding. The binding of ferric ions by glycine-extended gastrin(17) at low pH is consistent with a role for progastrin-derived peptides in iron uptake from the lumen of the gastrointestinal tract.

Binding Sites↗

PubMed2001

Alzheimer's disease amyloid-beta binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits.

Amyloid beta peptide (Abeta) is the major constituent of extracellular plaques and perivascular amyloid deposits, the pathognomonic neuropathological lesions of Alzheimer's disease. Cu(2+) and Zn(2+) bind Abeta, inducing aggregation and giving rise to reactive oxygen species. These reactions may play a deleterious role in the disease state, because high concentrations of iron, copper, and zinc have been located in amyloid in diseased brains. Here we show that coordination of metal ions to Abeta is the same in both aqueous solution and lipid environments, with His(6), His(13), and His(14) all involved. At Cu(2+)/peptide molar ratios >0.3, Abeta coordinated a second Cu(2+) atom in a highly cooperative manner. This effect was abolished if the histidine residues were methylated at N(epsilon)2, indicating the presence of bridging histidine residues, as found in the active site of superoxide dismutase. Addition of Cu(2+) or Zn(2+) to Abeta in a negatively charged lipid environment caused a conformational change from beta-sheet to alpha-helix, accompanied by peptide oligomerization and membrane penetration. These results suggest that metal binding to Abeta generated an allosterically ordered membrane-penetrating oligomer linked by superoxide dismutase-like bridging histidine residues.

Allosteric Regulation↗

PubMed1999

Residues within the HFRIGC sequence of HIV-1 vpr involved in growth arrest activities.

HIV-1 Vpr is a virion-associated protein that can cause growth arrest when produced inside the cell but when added externally it can cause cell death. Employing the yeast model system, the C-terminal domain, in particular the sequence HFRIGCRHSRIG (Vpr(71-82)), is essential for both the growth arrest and cytocidal activities. Conservation of this sequence in HIV-2 and SIV suggests that these residues may be functionally important. Using site-directed mutagenesis we show that the most highly conserved aa residues, His71 and Gly75, were important for the cell cycle inhibitory effects. In contrast, we show that the wild-type Vpr(71-82) peptide and three variants of this peptide with Gly75 changed to Ser, Ala, and Ile all exhibited the same cytocidal activity suggesting that the intracellular and extracellular effects are unrelated.

Amino Acid Sequence↗

PubMed1998

Structural requirements for the cytotoxicity of the N-terminal region of HIV type 1 Nef.

We have found that the hemolytic and cytotoxic activities of myristoylated Nef N-terminal peptides require a net positive charge in the first seven amino residues of the sequence. The activities are considerably less dependent on the secondary structure of the peptides. Film balance studies showed that both active and inactive peptides interacted with neutral phospholipid monolayers, suggesting that binding to neutral lipids was not a sufficient condition for lytic activity. It was also found that nonmyristoylated N-terminal peptide did not interact to the same extent with the monolayer, indicating that myristoylation was essential for lipid interaction. It is considered that the positively charged residues of the proximate N terminus of Nef interact with acidic lipids of biological membranes, reinforcing the weak membrane-targeting properties of the myristyl chain. Parallels are drawn between this mode of interaction with membranes and that of members of the Src family of proteins, which are also myristoylated and have positively charged residues in their proximate N termini. In particular, these proteins and Nef also have serine residues in their proximal N-terminal regions, which when phosphorylated could neutralize the positive charge and thus provide a mechanism for modulating membrane interaction.

Amino Acid Sequence↗

PubMed1998

Efficacy of fusion peptide homologs in blocking cell lysis and HIV-induced fusion.

Contrary to earlier reports, we have found that tri- and hexapeptides analogous or homologous with segments of the 23-residue N-terminal fusion sequence (FS) of the viral transmembrane glycoprotein gp41 (residues 517-539) did not significantly inhibit HIV-1-induced syncytium formation, using an uninfected cell-infected cell fusion assay. In contrast, we found that the high molecular weight apolipoprotein A-1 and a 23-residue analog of the FS, with the phenylalanine residues at positions 524 and 527 replaced with alanine residues, were effective inhibitors. Although the tripeptides were ineffective as inhibitors of syncytium formation, we found a number of them inhibited red cell lysis induced by the synthetic peptide AVGIGALFLGFLGAAGSTMGARS (based on the HIV-1 gp41 FS). This effect was also seen with apolipoprotein A-1. The Ala524,527 analog of the fusion sequence could not be tested in this system because it was hemolytic. We concluded that the smaller peptides were effective inhibitors of hemolysis because they interfered with pore formation by the fusion sequence peptide, either by disrupting the pores or by preventing the peptide from adopting the alpha-helical conformation found in the pores. On the other hand, membrane fusion, which is a prelude to syncytium formation, has been shown to require the fusion sequence in the beta-strand conformation. We argue that small peptides would be unable to block interaction between such strands, although larger molecules, such as apolipoprotein A-1 and the Ala524,527 analog, would be able to do so and thus inhibit fusion. It seems, therefore, that a successful drug directed against the FS-cell membrane interaction stage of syncytium formation would need to be of relatively high molecular weight and complexity.

Giant Cells↗

PubMed1997

Cytotoxic activity of the amino-terminal region of HIV type 1 Nef protein.

Myristoylated 21- and 25-residue N-terminal peptides of the Nef protein of HIV-1 lysed human erythrocytes and were cytotoxic toward a human CD4+ T cell line, CEM, and primary human peripheral blood mononuclear cells (PBMCs). The corresponding nonmyristoylated N-terminal peptides were only very weakly hemolytic and cytotoxic. A myristoylated peptide consisting of residues 31-50 of Nef was neither hemolytic nor cytotoxic. Alteration of the tryptophan residue at position 13 to a serine did not change the hemolytic and cytotoxic activity. Studies of the ultraviolet fluorescence of the tryptophan at position 5 in the peptide, using an artificial membrane system and fluorescence-quenching agents that inserted into the bilayer at different levels, suggested that myristoylation results in this residue being brought into contact with the upper hydrocarbon region of the lipid bilayer of the cell membrane. This tryptophan is flanked by a number of polar residues that would maintain it in this position, resulting in a considerable increase in disorder in the upper regions of the lipid bilayer, leading to its destabilization and to lysis. The cytotoxic activity of the myristoylated Nef fragments may, in part, explain the killing and deletion of cells, especially in lymphoid tissues, during HIV infection.

Amino Acid Sequence↗

PubMed1997

Cytotoxicity resulting from addition of HIV-1 Nef N-terminal peptides to yeast and bacterial cells.

The Nef protein of human immunodeficiency type 1 (HIV-1) has been implicated in diverse intracellular functions; however, extracellular functions have been less studied. Nef and the N-terminus of Nef possess membrane-perturbing and fusogenic activities in artificial membranes that also cause cytotoxicity to human cells, including lymphocytes. The present study investigates the toxicity of HIV-1 Nef peptides employing yeast and bacterial cells. The N-terminal portion of Nef was found to cause cell killing in Escherichia coli and in a variety of yeast cells. This activity was enhanced by myristylation of the Nef N-terminus, a modification that did not lead to toxicity in a control peptide. Cell death in yeast was due to permeabilization of the cell membrane as determined by the propidium iodide uptake of peptide-treated cells. Extracellular Nef, or its breakdown products, may have effects similar to the Nef peptides described here and could be responsible, at least in part, for the death of cells in lymphoid tissues during AIDS. Assays using yeast or bacteria are convenient, inexpensive, and robust and should be useful in further analysis and screening of inhibitors of this activity associated with HIV-1 Nef.

Amino Acid Sequence↗

PubMed1996

Preparation and characterization of a biologically active spin-labeled sea anemone toxin.

A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin-labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over the pH range 2-9. The ESR data also show evidence for multimetric states of SL-AP-B over the pH range 2-9, with maximum aggregation at pH 4.5-5, and a slow disaggregation when the pH is adjusted to 8-9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.

Animals↗

PubMed1995

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes.

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

Erythrocyte Aggregation↗

PubMed1995

Antivirals that target the amino-terminal domain of HIV type 1 glycoprotein 41.

Functional and structural studies were made to assess whether a class of antiviral agents targets the N-terminal domain of the glycoprotein 41,000 (gp41) of human immunodeficiency virus type 1 (HIV-1). Previous experiments have shown that the amino-terminal peptide (FP-I; 23 amino acids, residues 519-541) of HIV-1 gp41 is cytolytic to both human erythrocytes (non-CD4+ cells) and Hut-78 cells (CD4+ lymphocytes). Accordingly, FP-I-induced hemolysis may be used as a surrogate assay for evaluating the role of the N-terminal gp41 domain in HIV-cell interactions. Here, we studied the blocking of FP-I-induced lysis of erythrocytes by the following anti-HIV agents: (1) IgG [i.e., anti-(518-541) IgG] raised to an immunoconjugate of Arg-FP-I, (2) apolipoprotein A-1 (apo A-1) and a peptide based on apo A-1, (3) dextran sulfate, (4) gp41 peptide (residues 637-666), and (5) anionic human serum albumins. Dose-response curves indicated that their relative potency in inhibiting FP-I-induced hemolysis was approximately correlated with their previously reported anti-HIV activity. Electron spin resonance (ESR) studies showed that FP-I spin labeled at the N-terminal alanine binds to anti-(518-541) IgG, dextran sulfate, and anionic albumins. The high in vitro antiviral activity and low cytotoxicity of these agents suggest that blocking membrane-FP-I interactions offers a novel approach for AIDS therapy or prophylaxis.

Amino Acid Sequence↗

PubMed1994

Fusogenic activity of amino-terminal region of HIV type 1 Nef protein.

We have studied two isoforms of Nef, Nef-27 and Nef-25, which were produced in E. coli. Nef-25 lacked the first 18 N-terminal residues of Nef-27 and both were nonmyristylated. Nef-27 fuses small unilamellar dipalmitoyl phosphatidylcholine vesicles (SUVs), as indicated by enhanced light scattering of SUVs and lipid mixing using concentration-dependent fluorescence dequenching. Nef-27 also causes the appearance of a shifted isotropic peak in the 31P NMR spectra of these vesicles, suggesting that protein interactions induce nonlamellar lipid structures. Recombinant Nef-25, which lacks only the 18 N-terminal residues of Nef-27, does not fuse vesicles and has little effect on the 31P NMR spectra. On the other hand, synthetic peptides consisting of 18 or 21 of the N-terminal residues of Nef-27 are strongly membrane perturbing, causing vesicle fusion and inducing isotropic peaks in the 31P NMR spectrum. Endogenous fluorescence spectra of the N-terminal peptide (21 residues) with SUVs show that the N-terminal sequence of Nef may achieve these perturbing effects by inserting its hydrophobic side into the lipid bilayer. Theoretical calculations using hydrophobic moment plot analysis indicate that short-length stretches (i.e., six amino acid residues) of the N-terminal sequence may insert into the lipid bilayer as multimeric alpha helices or beta sheets. The above-described membrane activities of Nef-27, which principally reside in its N-terminal domain, may play critical role(s) in certain functional properties of the full-length protein. For example, the fusogenic activity of the N-terminal sequence may be involved in the extracellular release of Nef-27, much of which appears to be associated with small membrane vesicles. The fusion activity may also be relevant to the ability of Nef-27 to downregulate CD4 and IL-2 receptors when this protein is electroporated into cultured lymphocytes, an activity not possessed by Nef-25.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1993

The amino-terminal peptide of HIV-1 gp41 interacts with human serum albumin.

Structural and functional studies were made to assess interactions between human serum albumin (HSA) and the amino-terminal peptide (FP-I; 23-residue peptide 519-541) of glycoprotein 41,000 (gp41) of human immunodeficiency virus type-1 (HIV-1). Circular dichroism (CD) spectroscopy indicated that the peptide binds to albumin with dominant alpha-helical character. Peptide binding to albumin was also examined using FP-I spin labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). Electron spin resonance (ESR) spectra of FP-II bound to HSA at 38 degrees C indicated that the spin label at the amino-terminal residue (Ala-519) was motionally restricted. The ESR spectrum of 12-nitroxide stearate (12-NS)-labeled HSA was identical to that obtained with FP-II, indicating that the reporter groups for the 12-NS and FP-II probes are similarly bound to albumin. Contrarily, ESR spectra of HSA labeled with FP-III indicated high mobility for the reporter group (Met-537) at the aqueous-protein interface. This suggests that the N-terminal gp41 peptide binds as an alpha helix (residues 519-536) to fatty acid sites on HSA, such that Ala-519 of the peptide residues in the interior of the protein while Met-537 lies outside the protein in aqueous solution. It is also of interest that addition of HSA to human red blood cells dramatically reduced the ability of FP-I to induce hemolysis, presumably through peptide-albumin binding that inhibited FP-I interactions with red cell membranes. The significance of these results focuses on the following three points. The first is that high serum levels of albumin may limit the efficacy of anti-HIV therapies using peptides based on the N-terminal gp41 domain. The second is that the elucidation of FP-I and HSA interactions with physical techniques may provide clues on the molecular features underlying viral FP-I combination with receptors on the target cell surface. Last, the affinity of albumin for the N-terminal gp41 peptide may play a subordinate role in the blocking of HIV infectivity in vitro that has been reported for chemically modified albumins.

Amino Acid Sequence↗

PubMed1992

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).

Amino Acid Sequence↗

PubMed1992

The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation.

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.

Amino Acid Sequence↗

PubMed1989

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts.

A model for the binding of 5-nitroxide stearate, I(12.3), to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L less than 1/2500), I(12.3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L = 1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (greater than 1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12.3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both 'dilute' and 'clustered' I(12.3) are due to membrane-incorporated probe.

Binding Sites↗

PubMed1988

Thermotropic lipid phase separation in the human immunodeficiency virus.

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Lipid composition and fluidity of the human immunodeficiency virus.

Lipid analyses of the human immunodeficiency virus (HIV) propagated in Hut 78 cells indicated a low total lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine; comparable lipid profiles were noted for human erythrocytes and other RNA viruses. Electron spin resonance (ESR) studies of HIV labeled with 5-nitroxide stearate (N-oxy-4',4'-dimethyloxazolidine derivative of ketostearate) showed a low "fluidity" at 37 degrees C, similar to other enveloped RNA viruses and erythrocytes and probably due to the high cholesterol/phospholipid ratio. Ethanol (50%) completely disrupts the envelope, contributing to the rapid inactivation of HIV by ethanol. Contrarily, heating to 57 degrees C causes much less fluidization, and this heating may play a role in the slower viral inactivation at high temperatures. Should a critical minimum ordering in the HIV envelope be necessary for viral stability and infectivity, manipulating the lipid composition or fluidizing the HIV membrane, or both, may provide an untried therapeutic approach.

Cholesterol↗

PubMed1987

Estimation of spin probe clustering in biological membranes.

An iterative spectral subtraction technique has been developed which accurately estimates the proportion of 'dilute' and 'clustered' I(12, 3) (i.e., 5-nitroxide stearate) in human erythrocyte ghosts at 37 degrees C, even if subtractant spectra free from probe-probe interactions cannot be measured due to technical limitations. Gordon et al. ((1985) J. Membrane Biol. 84, 81-95) earlier showed that I(12, 3) occupies a class of high-affinity sites in ghosts at probe/total lipid ratios (P/L) less than 1/2250. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. Although this model allows determination of the dilute/clustered probe ratio, it requires subtraction of experimental spectra with a 'magnetically dilute' spectrum obtained using P/L less than 1/4600. The new methodology accurately profiles the % probe clustering in human erythrocyte ghosts over the entire P/L range, even if the lowest P/L for the subtractant spectrum contains substantial probe-probe interactions (i.e., P/L of 1/604 or 1/303). Application of either the subtraction technique in Gordon et al. (1985) or the iterative subtraction protocol described here should allow determination of probe clustering in a wide range of I(12, 3)-labeled biological membranes.

Cyclic N-Oxides↗