1-H and 13-C nuclear magnetic resonance spectra of the lipids in normal and SV 40 virus-transformed hamster embryo fibroblast membranes.

Well resolved 1-H and 13-C NMR spectra were obtained with normal and SV 40-transformed cell membranes. Estimation of the ratio of 13-CT2 values of the normal to transformed cell membranes showed an increased intermolecular motion in the transformed cell membranes. The temperature dependence of the (CH2) line in the 1-H spectra in the temperature range 298-343 degrees K shows an activation energy for the lateral diffusion of the fluid phospholipid regions in the normal cell membranes while the transformed ones show practically no temperature dependence in this temperature range. The fluidity of the phospholipid region in the transformed cell membrane seems to be significantly higher than that observed in the normal cell material. These data support and extend the findings concerning the mobility of the concanavalin A binding/agglutinating sites on the surface of normal and virus-transformed cells and suggest further approaches to the study of the membrane alterations in tumor cells.     

Hormone (3-indoleacetic acid)-phospholipid interaction: 1H, 13C and 31P nuclear magnetic resonance studies

The interaction between the plant hormone, 3-indoleacetic acid (IAA), and some phospholipids in CDCL₃ has been studied by ¹H, ¹³C and ³¹P nuclear magnetic resonance (NMR) spectroscopy. Upon interaction with IAA, significant changes occurred in resonance positions of the phospholipid head group nuclei. Alteration of the fatty acid composition influenced the effects of IAA on these nuclei. These effects were observed in the ethanolamine and phosphate groups of the phosphatidylethanolamines, and in the choline, phosphate and glycerol groups of the phosphatidylcholines. Changes in resonance positions of the phospholipid head group nuclei were used for the determination of dissociation constants (K_d). In all cases, K_d values were approx. 10^?2 molal for 1 : 1 interaction. The NMR results suggest an interaction orientation in which the aromatic ring system of IAA interacts with the quaternary nitrogen function of the head group, and the phosphate group becomes hydrogen-bonded to the NH or carboxyl proton of 1AA.     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

¹H-, ¹³C-and ³¹P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr³⁺, Eu³⁺, Gd³⁺ and Mn²⁺ ions.The differences in chemical shift values. °_n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(²H₂O)³⁺_n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH₂CH₂ group, respectively but relatively far from the N(CH₃)₃ group of the polar head group of the lipid.The integral analysis of the¹ H-NMR spectra obtained from dispersions containing Pr³⁺ and Mn²⁺ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p²H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled ³¹ P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.     

Binding of [3H]cytochalasin B and [3H]colchicine to isolated liver plasma membranes

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([³H]cytochalasin B and [³H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [³H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB³H₄. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insentive to changes of pH or ionic strength. At 10^?6 M [³H]cytochalasin B, glucose or p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 Å) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10^?5 M [³H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [³H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes.[³H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [³H]cytochalasin B binding and cytochalasin B stimulated ³H colchicine binding. [³H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Ergosterol in POPC membranes: physical properties and comparison with structurally similar sterols

The physical properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ergosterol bilayers in the liquid-crystalline phase were determined using deuterium nuclear magnetic resonance (²H NMR) and vesicle extrusion. For the ²H NMR experiments, the sn-1 chain of POPC was perdeuterated, and spectra were taken as a function of ergosterol concentration and temperature. Analysis of the liquid-crystalline spectra provides clear evidence that two types of liquid-crystalline domains, neither of which is a liquid-ordered phase, having distinct average chain conformations coexist in 80:20 and 75:25 POPC/ergosterol membranes over a wide temperature range (from −2 to at least 31°C). Adding ergosterol to a concentration of 25 mol % increases POPC-d₃₁ chain ordering as measured by the NMR spectral first moment M₁ and also increases the membrane lysis tension, obtained from vesicle extrusion. Further addition of ergosterol had no effect on either chain order or lysis tension. This behavior is in marked contrast to the effect of cholesterol on POPC membranes: POPC/cholesterol membranes have a linear dependence of chain order on sterol concentration to at least 40 mol %. To investigate further we compared the dependence on sterol structure and concentration of the NMR spectra and lysis tension for several POPC/sterol membranes at 25°C. For all POPC/sterol membranes investigated in this study, we observed a universal linear relation between lysis tension and M₁. This suggests that changes in acyl chain ordering directly affect the tensile properties of the membrane.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

13C NMR spectral analysis of some isoquinoline alkaloids

The ¹³C NMR spectra of some isoquinoline and tetrahydroisoquinoline alkaloids and their corresponding N-methosalts and of the bisbenzylisoquinoline alkaloid isochondodendrine were recorded and the signals assigned. The substituent shielding effects and the ¹³C¹H long range couplings were analysed and utilized in the spectral interpretation.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

Preparation and comparison of model bilayer systems from chloroplasts thylakoid membrane lipids for 13C-NMR studies

Model bilayer systems from individual purified chloroplast thylakoid membrane lipids, from reconstituted mixtures of these purified lipids, and from leaf total polar lipid extracts have been prepared in water, and the longitudinal relaxation times (T's₁) of the individual carbon atoms of the fatty acyl chains measured by ¹³C-NMR spectroscopy. The T's₁ increasing distance of the carbon atoms from the polar headgroups in all cases, and as the results from each of the preparations are similar, all can be used as models of chloroplast membrane bilayers. Relaxation time measurements on intact chloroplast thylakoid membranes indicate the presence of chlorophyll resonances in the ¹³C-NMR spectrum of the membrane.     

Cholesterol rotation in phospholipid vesicles as observed by 13C-NMR

¹³C-NMR spectra of cholesterol 90% enriched at C-4 with ¹³C have been obtained in CHCl₃ and in sonicated egg phosphatidylcholine vesicles. ¹³C spin-lattice relaxation times, nuclear Overhauser effects and spin-spin relaxation times have been measured for the C-4 carbon of cholesterol in phosphatidylcholine bilayers as a function of cholesterol content and temperature. All the data are consistent with a correlation time for axial rotation of about 10^?10 s. This rotation is one or two orders of magnitude faster than axial rotation of the phospholipid molecule.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

13C NMR spectroscopy of Pyrrolizidine alkaloids

The ¹³C NMR spectra of nine pyrrolizidine alkaloids of the macrocyclic diester type, seven of the corresponding N-oxides and of the parent base retronecine have been recorded and the signals assigned. The 13C NMR signals were found to be sensitive to structural variation in both the diester moiety and the heterocyclic ring system, providing useful information for structural elucidation, particularly when the ¹H NMR spectra may be difficult to interpret.     

Biophysical studies of the membrane location of the voltage-gated sensors in the HsapBK and KvAP K channels

The membrane location of two fragments in two different K⁺-channels, the KvAP (from Aeropyrum pernix) and the HsapBK (human) corresponding to the putative “paddle” domains, has been investigated by CD, fluorescence and NMR spectroscopy. Both domains interact with q = 0.5 phospholipid bicelles, DHPC micelles and with POPC vesicles. CD spectra demonstrate that both peptides become largely helical in the presence of phospholipid bicelles. Fluorescence quenching studies using soluble acrylamide or lipid-attached doxyl-groups show that the arginine-rich domains are located within the bilayered region in phospholipid bicelles. Nuclear magnetic relaxation parameters, T₁ and ¹³C-¹H NOE, for DMPC in DMPC/DHPC bicelles and for DHPC in micelles showed that the lipid acyl chains in the bicelles become less flexible in the presence of either of the fragments. An even more pronounced effect is seen on the glycerol carbons. ²H NMR spectra of magnetically aligned bicelles showed that the peptide derived from KvAP had no or little effect on bilayer order, while the peptide derived from HsapBK had the effect of lowering the order of the bilayer. The present study demonstrates that the fragments derived from the full-length proteins interact with the bilayered interior of model membranes, and that they affect both the local mobility and lipid order of model membrane systems.     

Binding of peroxiredoxin 6 to substrate determines differential phospholipid hydroperoxide peroxidase and phospholipase A2 activities

Peroxiredoxin 6 (Prdx6) differs from other mammalian peroxiredoxins both in its ability to reduce phospholipid hydroperoxides at neutral pH and in having phospholipase A₂ (PLA₂) activity that is maximal at acidic pH. We previously showed an active site C47 for peroxidase activity and a catalytic triad S32-H26-D140 necessary for binding of phospholipid and PLA₂ activity. This study evaluated binding of reduced and oxidized phospholipid hydroperoxide to Prdx6 at cytosolic pH. Incubation of recombinant Prdx6 with 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine hydroperoxide (PLPCOOH) resulted in peroxidase activity, cys47 oxidation as detected with Prdx6-SO₂₍₃₎ antibody, and a marked shift in the Prdx6 melting temperature by circular dichroism analysis indicating that PLPCOOH is a specific substrate for Prdx6. Preferential Prdx6 binding to oxidized liposomes was detected by changes in DNS-PE or bis-Pyr fluorescence and by ultrafiltration. Site-specific mutation of S32 or H26 in Prdx6 abolished binding while D140 mutation had no effect. Treatment of A549 cells with peroxides led to lipid peroxidation and translocation of Prdx6 from the cytosol to the cell membrane. Thus, the pH specificity for the two enzymatic activities of Prdx6 can be explained by the differential binding kinetics of the protein; Prdx6 binds to reduced phospholipid at acidic pH but at cytosolic pH binds only phospholipid that is oxidized compatible with a role for Prdx6 in the repair of peroxidized cell membranes.     

1H-nmr study on the preferred conformations of chiral pyridine-dinucleotide models

The synthesis of four chiral NAD⁺ models 1 and their 1,4-dihydro analogs 2 is described. From the temperature dependence of the ¹H-nmr spectra it is concluded that for these compounds two preferred conformations I and II, differing slightly in energy, exist. Both conformations are “folded” with the more or less parallel p-anisyl and pyridine groups mutually gauche, but in I the pyridine group is rotated by about 180° as compared with II, thus leading to a conspicuous difference in orientation of the substituent Z (NH₂CO, C₆H₅NHSO₂, (CH₂)₄NSO₂, or (C₄H₈ON)SO₂) in the pyridine ring toward the anisyl group. The most stable conformation (I) has Z closest to the center of the p-anisyl group. In 360-MHz spectra of the dihydropyridines at low temperature (?10°C), slow interconversion of I and II leads to the observation of an XY pattern for the C-4 methylene protons of the 1,4-dihydropyridine system. The anisochronity in this methylene group is caused mainly by the anisotropy of the neighboring p-anisyl group.