New nitrosoureas and their spin-labeled derivatives influence dopa-oxidase activity of tyrosinase.

Tyrosinase is a key enzyme in melanine biosynthesis. The modulating effect of cytostatic agents on DOPA-oxidase activity of tyrosinase could be linked with the drug treatment of melanoma tumors. Two groups of nitrosoureas which influence DOPA-oxidase activity of tyrosinase were studied: new nitrosoureas and their spin-labeled derivatives synthesized in our laboratory. Using Burnett's spectrophotometric method (Burnett et al., 1967) the following effects were established: inhibition by CCNU, inhibition and the activating effects of the other investigated nitrosoureas depend on their physicochemical half-life. The predominant activating effect of the spin-labeled derivatives is due to the nitroxyl radical present in these compounds.     

Superoxide Scavenging Activity of Spin-Labeled Nitrosourea and Triazene Derivatives

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.     

Spectroscopic and biological studies of spin-labeled tetracycline.

A new nitroxyl labeled tetracycline is synthesized. Proton NMR experiments of tetracycline, spin-labeled tetracycline, and the diamagnetic reduced form in DMSO-d6 are reported. The signals observed in the NMR spectra are all assigned. The NMR data revealed that the spin label is attached to the C-2 amide group on ring A of tetracycline. The spin-labeled tetracycline is also tested in vitro for antitumor activity and is found to be active against leukemia P338/ADR cell line and in melanoma LOX cell line.     

Interaction of intestinal brush border membrane vesicles with small unilamellar phospholipid vesicles. Exchange of lipids between membranes is mediated by collisional contact

The kinetics of lipid transfer from small unilamellar vesicles as the donor to brush border vesicles as the acceptor have been investigated by following the transfer of radiolabeled or spin-labeled lipid molecules in the absence of exchange protein. The labeled lipid molecules studied were various radiolabeled and spin-labeled phosphatidylcholines, radiolabeled cholesteryl oleate, and a spin-labeled cholestane. At a given temperature and brush border vesicle concentration similar pseudo-first-order rate constants (half-lifetimes) were observed for different lipid labels used. The lipid transfer is shown to be an exchange reaction leading to an equal distribution of label in donor and acceptor vesicles at equilibrium (time t----infinity). The lipid exchange is a second-order reaction with rate constants being directly proportional to the brush border vesicle concentration. The results are only consistent with a collision-induced exchange of lipid molecules between small unilamellar phospholipid vesicles and brush border vesicles. Other mechanisms such as collision-induced fusion or diffusion of lipid monomers through the aqueous phase are negligible at least under our experimental conditions.     

Increased degradation rate of nitrososureas in media containing carbonate

The stability of two nitrosoureas, tauromustine and lomustine, has been investigated in different media and buffers. All media tested, except Leibovitz's L-15 medium, significantly increased the degradation rate of the investigated nitrosoureas at pH 7.4. Sodium bicarbonate seems to be the cause of the observed increase of the degradation rate, since it provides the main buffering capacity of all the media except for Leibovitz's L-15 medium, which is based on phosphate buffer. Other ingredients in the media, such as amino acids, vitamins, and inorganic salts, or the ionic strength of a buffer, did not have any major effect on the degradation rate of the nitrosoureas. These results suggest that media containing carbonated buffer should be avoided when the anti-tumor effect of nitrosoureas is to be investigated in different cell cultures.     

PDN-1: a spin-labeled analog of cis-diamminedichloroplatinum(II). Biophysical studies in aqueous, lipophilic, and biological media

Biophysical and biochemical studies on a biradical-labeled analog of Cisplatin, PDN-1, in aqueous, biological, and lipophilic media are presented. The ESR spectra from PDN-1 in aqueous meida show five-line spectra typical of biradicals in which intramolecular spin exchange occurs. The spectrum of PDN-1 in 1-octanol is characterized by three broad lines overlying a fourth line that is approximately three times as wide as the others. Although the characteristics of the aqueous spectra differ substantially from those of the octanol spectra, both the midfield peak height and the integral of the absorption spectrum can be used as a linear measure of PDN-1 concentration in either media. The smallest concentration used in these linear calibrations was equivalent to the detection of 60 femptomoles of PDN-1 at a signal-to-noise ratio of 30. PDN-1 is shown to "decay" in tissue culture medium which involves either the reduction of a nitroxyl moiety, the labilization of an amino bond, or a combination of both. Spectra of intracellular PDN-1 show that both a mobile ("free") fraction and slowly tumbling ("bound") fraction of the compound can be detected within CHO cells. The spin-labeled cisplatin method is at least as sensitive as conventional chromatographic and spectroscopic methods, yet has the added advantage of offering information about the molecular environment of the complex.     

Interactions of spin-labeled calmodulin with trifluoperazine and phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III)

Bovine calmodulin analogues, spin-labeled at methionine and tyrosine residues, have been utilized in electron paramagnetic resonance (EPR) studies designed to investigate calmodulin interactions with the antipsychotic drug trifluoperazine and the calmodulin-binding protein 3',5'-cyclic nucleotide phosphodiesterase. Trifluoperazine titrations of spin-labeled calmodulin analogues were carried out in the presence of Ca(II), Cd(II), and Tb(III). Similar experiments were performed with the phosphodiesterase in the presence of Ca(II), Cd(II), La(III), Tb(III), and Lu(III). EPR signals from the methionine-directed probe proved to be more sensitive to the binding of target molecules than signals from the tyrosine-directed probe, perhaps indicating that the spin-labeled methionine is at a site close to the target molecule binding site. While the binding of TFP, as monitored by EPR spectral changes in the methionine spin-labeled calmodulin, was in evidence with Ca(II), Cd(II), and all the lanthanides examined, no binding of phosphodiesterase to calmodulin could be detected in the presence of the lanthanide ions, perhaps due to inactivation of the phosphodiesterase by lanthanide ion binding. The abilities of the spin-labeled calmodulins to activate phosphodiesterase were also investigated. The spin-labeled tyrosine calmodulin was able to activate phosphodiesterase as well as native calmodulin, while a lower degree of activation was found when the spin-labeled methionine analogue was used.     

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.     

Inhibition of protein A24 lyase by nitrosoureas

The protein A24 content of Ehrlich ascites tumor cells increased several-fold following treatment of cell cultures with nitrosoureas, but did not increase when other alkylating agents not containing carbamoyl moieties were tested. The same nitrosoureas and, in addition, 2-chloroethyl isocyanate inhibited an A24 lyase-containing cytoplasmic extract in cleaving protein A24 into histone H2A and ubiquitin. It appears that carbamoylation of A24 lyase by nitrosoureas inhibits the enzyme and is responsible for the measured increases in cellular protein A24 content due to reduced turnover of this protein.     

Biosynthesis of spin-labeled peptidoglycan: spin-spin interactions.

Membrane preparations from Gaffkya homari catalyzed the in vitro biosynthesis of soluble uncross-linked spin-labeled peptidoglycan, a uniformly labeled polynitroxide, from the spin-labeled nucleotide UDP-MurNAc-Ala-DGlu-Lys(Nepsilon-2,2,5,5-tetramethyl-1-pyrrolin-1-oxyl-3-carbonyl)-DAla-DAla (I) and UDP-GlcNAc. Soluble spin-labeled peptidoglycan was separated from membrane fragments and its spin-labeled precursor by centrifugation and gel filtration. The molecular weight distribution of the polymer was examined by agarose gel filtration. Spin-labeled [14C]peptidoglycan was polydisperse with a peak of radioactivity corresponding to a molecular weight of 5.0 X 10(5). The electron spin resonance spectrum of spin-labeled peptidoglycan was extensively broadened by spin-spin exchange interactions. These interactions were modified by changes in temperature, reduction by ascorbate, hydrolysis by lysozyme, and complexation with the antibiotic, vancomycin. Spin-spin exchange was reduced or eliminated in spin-labeled peptidoglycan by the random reduction of free radicals by ascorbate. A rotational correlation time of 0.37 ns was calculated for the probe in partially reduced spin-labeled peptidoglycan. This compares to a correlation time of 0.13 ns for the substrate (I). Raising the temperature increases spin-spin exchange line broadening. No transition points were observed for spin-labeled peptidoglycan as measured by this method. Degradati on of spin-labeled peptidoglycan by lysozyme eliminated the observed spin-spin exchange and yielded products with a mobility similar to I. Complexation of spin-labeled peptidoglycan with vancomycin resulted in both pronounced free-radical immobilization and a decrease in spin-spin exchange. The exchange effects are consistent with distance measurements in molecular models for peptidoglycan.     

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.     

An ESR study of the hydration steps of lecithin multilayers.

A spin-labeled stearic acid and other spin-labeled molecules have been used for an ESR study of the hydration process of the egg lecithin lamellar phase for water concentrations, CW greater than 10%. Two hydration steps are found for CW approximately 20% and CW congruent to 30%, in good agreement with results of the literature. To explain discontinuities in the behavior of three spin-labeled molecules at CW congruent to 20%, a conformational change of the phosphatidylcholine polar head and a phase transition in the polar interface are proposed.     

Binding of spin-labeled galactosides to the lactose permease of Escherichia coli

A series of nitroxide spin-labeled alpha- or beta-galactopyranosides and a nitroxide spin-labeled beta-glucopyranoside have been synthesized and examined for binding to the lactose permease of Escherichia coli. Out of the twelve nitroxide spin-labeled galactopyranosides synthesized, 1-oxyl-2, 5, 5-trimethyl-2-[3-nitro-4-N-(hexyl-1-thio-beta-D-galactopyranosid-1 -yl )]aminophenyl pyrrolidine (NN) exhibits the highest affinity for the permease based on the following observations: (a) the analogue inhibits lactose transport with a K(I) about 7 microM; (b) NN blocks labeling of single-Cys148 permease with 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) with an apparent affinity of about 12 microM; (c) electron paramagnetic resonance demonstrates binding of the spin-labeled sugar by purified wild-type permease in a manner that is reversed by nonspin-labeled ligand. The equilibrium dissociation constant (K(D)) is about 23 microM and binding stoichiometry is approximately unity. In contrast, the nitroxide spin-labeled glucopyranoside does not inhibit active lactose transport or labeling of single-Cys148 permease with MIANS. It is concluded that NN binds specifically to lac permease with an affinity in the low micromolar range. Furthermore, affinity of the permease for the spin-labeled galactopyranosides is directly related to the length, hydrophobicity, and geometry of the linker between the galactoside and the nitroxide spin-label.     

Ganglioside-protein interactions: spin-label electron spin resonance studies with (Na+,K+)-ATPase membranes

Lipid-protein interactions in (Na+,K+)-ATPase-rich membranes from Squalus acanthias have been studied using spin-labeled derivatives of the mono- and disialogangliosides GM1, GM2, GM3, and GD1b, in conjunction with electron spin resonance (ESR) spectroscopy. Ganglioside-protein interactions are revealed by the presence of a second component in the ESR spectra of the membranes in addition to a component that corresponds closely to the ESR spectra obtained from dispersions of the extracted membrane lipids. This second component corresponds to spin-labeled gangliosides whose chain motion is significantly restricted relative to that of the fluid lipids in the membrane or the lipid extract. A small selectively for the motionally restricted component associated with the protein is found in the order GD1b greater than GM1 approximately equal to GM2 approximately equal to GM3. Comparison with previous results from spin-labeled phospholipids in the same system [Esmann, M., Watts, A., & Marsh, D. (1985) Biochemistry 24, 1386-1393] shows that the spin-labeled monosialogangliosides GM1, GM2, and GM3 display little selectivity in the lipid-protein interaction relative to spin-labeled phosphatidylcholine. The spectral characteristics of both the fluid and motionally restricted spin-labeled components differ very significantly, however, between the gangliosides and the phospholipids. The outer hyperfine splitting of the motionally restricted component is smaller for the gangliosides than for the phospholipids, indicating a smaller degree of motional restriction on interaction of the ganglioside lipid chains with the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.     

Melting studies on spin-labeled polyadenylic–polyuridylic complexes

Temperature-dependent conformational transitions of spin-labeled poly rA, spin-labeled poly rU and the two-stranded helical complexes consisting either of spin-labeled rA·poly rU or spin-labeled poly rU·poly rA have been measured by electron spin resonance spectrocopy. The polynucleotides were spin labeled with 4-(2-iodoacetamido)2,2,6,6-tetramethylpiperidinooxyl and the spin label to nucleotide base ratio was approximately 1:600. The relationship between the log of tumbling time τ and the reciprocal absolute temperature for the spin-labeled single and double-stranded polynucleotides is presented. An agreement between T_m^OD (optical density melting) and T_m^sp (spin melting) is found for the complexes, which strongly supports the conclusion that the same temperature-dependent structural changes are monitored with both techniques.     

Spin-labeling studies of rat liver NADPH-cytochrome p450 reductase: conformation and function relationship.

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP+, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP+, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP+ and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W/S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.     

Use of double spin-labeled histones to monitor histone-chromatin integration

Tyrosine-specific nitroxide spin labels have been synthesized that utilize either deuterium or deuterium and [15N] isotopic substitution within the nitroxide ring. These probes have been used to differentially spin label and simultaneously monitor both histones H1 and H5, during the displacement of endogenous spin-labeled H1 from reconstituted chromatin by exogenously added spin-labeled H5.     

Oxygen profiles in membranes

Transmembrane profiles of molecular oxygen in lipid bilayers are not only significant for membrane physiology and pathology, but also are essential to the determination of membrane protein structure by site-directed spin labeling. Oxygen profiles obtained with spin-labeled lipid chains have a Boltzmann sigmoidal dependence on the depth into each lipid leaflet, which represents a two-compartment distribution between outer and inner regions of the membrane, with a transfer free energy that depends linearly on distance from the dividing planes. Transmembrane profiles for intramembrane polarity, and for water penetration into the membrane, have an identical form, but are of the reverse sign. Comparison with recently published oxygen profiles from a site-specifically spin-labeled alpha-helical transmembrane peptide validates the use of spin-labeled lipids for all these profiles and provides the necessary bridge to generate the full bilayer from a single lipid leaflet.     

Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin.

Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.