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At least 1,531 records · Page 85Linked to original sources
PubMed1994

Reconstitution of 5'-nucleotidase of bull seminal plasma in spin-labeled liposomes.

Seminal plasma separated from freshly ejaculated bull semen contains vesicles with a 5'-nucleotidase activity incorporated as an ectoenzyme anchored by glycosyl phosphatidylinositol (GPI). After its extraction from bull seminal plasma vesicles, the protein was purified and reconstituted into hen egg yolk lecithin liposomes obtained through prolonged dialysis of buffered n-octylglucoside detergent solutions of lipid, protein and various effectors against detergent-free solutions. Gel filtration experiments showed that the enzyme incorporated into liposomes in a dimeric form with its two subunits linked by disulfide bridges. In the presence of reduced glutathione, the protein dissociated into monomers and failed to incorporate into liposomes. Electron spin resonance (ESR) experiments, performed with liposomes containing electron spin labels localized at the hydrophilic lipid headgroups (5-doxyl stearic acid) or in the hydrophobic lipid hydrocarbon chains (16-doxyl stearic acid), demonstrated that the incorporation of 5'-nucleotidase resulted in the immobilization of the spin probes. Furthermore, the spectral parameters obtained before and after treatment of 5'-nucleotidase-containing liposomes with phosphatidylinositol-specific phospholipase C (PI-PLC) indicated that the liposome membrane bilayer did not contain protein segments. This supports the well-known ecto-localization of 5'-nucleotidase and rules out a previously reported possibility of a proteic transmembrane anchoring of the enzyme.

5'-Nucleotidase↗
PubMed1987

Intracellular binding of spin-labeled amiloride: an alternative explanation for amiloride's effects at high concentration.

Amiloride, an important inhibitor of Na+ transport and Na+/H+ exchange, has been used in nontransporting tissues to investigate the relationship between ionic fluxes or intracellular pH change and proliferative or synthetic events. Reports that amiloride is permeant and had direct effects on intracellular processes have led us to investigate the possibility that amiloride binds intracellularly to nuclei, mitochondria, and to purified nucleic acids. Using a nitroxide spin-labeled derivative of amiloride (ASp) and electron paramagnetic resonance (EPR) spectroscopy, we have demonstrated that nuclei and mitochondria isolated from trout liver bind significant amounts of ASp especially at the high amiloride concentrations (approximately mM) commonly used to inhibit proliferative events. While the chemical component responsible for ASp binding in these organelles was not identified, native DNA binds significant amounts of ASp whereas single stranded DNA and RNA bind much less. When these observations are taken together with reports of amiloride's direct action on cellular processes, they support the possibility that some of the effects attributed to inhibition of a transport event are caused by amiloride directly.

Amiloride↗
PubMed2004

Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling.

MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang, and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein. The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques. SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface. The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined. Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.

ATP-Binding Cassette Transporters↗
PubMed1972

Fatty acid desaturase mutants of yeast: growth requirements and electron spin resonance spin-label distribution.

Two respiratory-sufficient and one respiratory-deficient (nuclear petite) strains of yeast Delta(9)-desaturase mutants were analyzed to determine which fatty acids would serve as replacements for the naturally occurring fatty acids, 16:1 Delta(9)cis and 18:1 Delta(9)cis. The requirement can be satisfied by several fatty acids differing in double-bond position, steric configuration, chain length, and degree of unsaturation. The features common to growth-supporting fatty acids are presented and the effects of varying the carbon source and temperature are considered. In addition, we illustrate several pitfalls encountered in membrane studies which exploit lipid-requiring organisms. Since the membrane fatty acid composition of these mutants can be modified readily, electron spin resonance spectroscopy is used to compare membranes of mutant strains enriched for different fatty acids. The lipid distribution pattern of the most commonly employed electron spin resonance spin-label, 12-nitroxide stearate, was ascertained and compared to that of 18:1 Delta(9)cis.

Carbon Isotopes↗
PubMed1999

Determining the occurrence of a 3(10)-helix and an alpha-helix in two different segments of a lipopeptaibol antibiotic using TOAC, a nitroxide spin-labeled C(alpha)-tetrasubstituted alpha-aminoacid.

Trichogin GA IV is a 11-residue lipopeptaibol antibiotic exhibiting membrane modifying properties. We synthesized step-by-step by solution methods three trichogin analogues, each with a double Aib (alpha-aminoisobutyric acid)-->TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) replacement. The strict similarity in the conformational propensities of Aib and TOAC allowed us to exploit these analogues in a detailed investigation of the conformation of this lipopeptaibol in different organic solvents and in a membrane-mimetic environment using in particular the double spin labeling ESR technique. We conclude that the secondary structure in solution remains essentially unchanged if compared to that previously found in the crystal state for trichogin. More specifically, the N-terminal region of the peptide folds in a 3(10)-helix, while the central and C-terminal regions are mainly alpha-helical. An additional, significant proof for the modest plasticity of the trichogin structure was obtained by an X-ray diffraction analysis of the nOct-[TOAC4,8, Leu-OMe11] analogue. For the three analogues permeability measurements revealed membrane-modifying properties comparable to those of natural trichogin.

Aminoisobutyric Acids↗
PubMed1978

Spin-labeled ribosomes.

Explore the source record for details and available documents.

Electron Spin Resonance Spectroscopy↗
PubMed2000

Assessment of 2-chloroadenosine treatment after experimental traumatic brain injury in the rat using arterial spin-labeled MRI: a preliminary report.

Adenosine is a putative endogenous neuroprotectant. Its action at A1 receptors mitigates excitotoxicity while action at A2 receptors increases cerebral blood flow (CBF). We hypothesized that cerebral injection of the adenosine analog, 2-chloroadenosine, would decrease swelling and increase CBF early after experimental traumatic brain injury (TBI). To test this hypothesis, rats were anesthetized and subjected to TBI using a controlled cortical impact (CCI) model (n = 5/group). Immediately after injury, 2-chloroadenosine (0.3 nmole in 2 microliters) or an equal volume of vehicle were stereotactically injected lateral to the area of contusion. Using magnetic resonance imaging (MRI), in vivo spin-lattice relaxation time of tissue water (Tlobs) and CBF (arterial spin labeling) were measured in a 2-mm thick slice in the injured and non-injured hemispheres at 3-4 h after CCI. In a separate, preliminary experiment, the effect of 2-chloroadenosine injection in normal rat brain was studied. Rats (n = 2) were anesthetized and a burr hole was made for injection of 2-chloroadenosine into the same site as in the TBI model. One rat received the standard dose of 0.3 nmole and one rat received a 6 nmole injection. Tlobs and CBF studies were obtained 1.5-3.5 h after injection, using the same MRI methods as in the TBI study. In rats subjected to TBI, treatment with 2-chloroadenosine attenuated the increase in Tlobs after injury (p < 0.05 for treatment vs vehicle) in both hippocampus and cortex ipsilateral to injury. However, treatment with 2-chloroadenosine did not improve post-traumatic hypoperfusion. In normal rats, injection of 0.3 nmole of 2-chloroadenosine did not increase CBF, but the higher dosage of 6 nmole dramatically increased hemispheric CBF by 1.5-2.0-fold. The effect of local injection of 2-chloroadenosine at a dose of 0.3 nmole after experimental TBI on Tlobs presumably represents a reduction in post-traumatic edema. This reduction in edema, along with the augmentation of CBF seen in normal rats at higher dosage (6 nmole), supports a role for adenosine in neuroprotection following TBI.

2-Chloroadenosine↗
PubMed1983

Comparison of human and rat liver microsomes by spin label and biochemical analyses.

Detailed lipid analyses of human and rat liver microsomes revealed interesting differences. It was found that human liver microsomes contain twice as much lipid as those from the rat. This increased lipid content is not associated with an increase in content of a particular lipid class; human liver microsomes contain higher amounts of each of the lipid classes. Human and rat liver microsomes differ especially in the essential fatty acid composition of total lipids and phospholipids: human liver microsomes contain more linoleic acid and less arachidonic acid than those of the rat. Such a pattern of distribution of fatty acids is similar to that previously reported for human liver mitochondria and has not been reported for other species. Although the previously reported for human liver mitochondria and has not been reported for other species. Although the unsaturation of lipids is lower in human than in rat liver microsomes, spin label studies revealed a higher fluidity in human membranes. It is suggested that this might arise from a lesser immobilization of lipids by proteins in human liver subcellular membranes.

Animals↗
PubMed1983

Spin label studies on synaptosomal membranes of rat brain cortex during aging.

Membrane lipids of brain synaptosomes of 2, 12 and 24 months old rats were labeled by stearic acid spin probes with a doxyl group in the C-5 and C-16 position, respectively. We demonstrated that the hydrophobic region of synaptosomal membranes became more rigid during the life span studied, and the region located nearer the surface of membranes displays a significantly decreased fluidity only in the oldest group. Membrane proteins labeled with a nitroxide derivative of maleimide suggest that conformational changes take place during aging.

Aging↗
PubMed1986

Effects of vanadate on the rotational dynamics of spin-labeled calcium adenosinetriphosphatase in sarcoplasmic reticulum membranes.

We have studied the effects of vanadate on the rotational motion of the calcium adenosine-triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron paramagnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spin-labeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Spin-label electron paramagnetic resonance and differential scanning calorimetry studies of the interaction between mitochondrial succinate-ubiquinone and ubiquinol-cytochrome c reductases.

The interaction between succinate-ubiquinone and ubiquinol-cytochrome c reductases in the purified, dispersed state and in embedded phospholipid vesicles was studied by differential scanning calorimetry and by electron paramagnetic resonance (EPR). When the purified, detergent-dispersed succinate-ubiquinone reductase, ubiquinol-cytochrome c reductase, and cytochrome c oxidase undergo thermodenaturation, they show an endothermic transition. However, when these isolated electron-transfer complexes are embedded in phospholipid vesicles, they undergo exothermodenaturation. The energy released could result from the collapse of the strained interaction between unsaturated fatty acyl groups of phospholipids and an exposed area of the complex formed by removal of interacting proteins. The exothermic enthalpy change of thermodenaturation of a protein-phospholipid vesicle containing both succinate-ubiquinone and ubiquinol-cytochrome c reductases was smaller than that of a mixture of protein-phospholipid vesicles formed from the individual electron-transfer complexes. This suggests specific interaction between succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase in the membrane. This idea is supported by saturation transfer EPR studies showing that the rotational correlation time of spin-labeled ubiquinol-cytochrome c reductase is increased when mixed with succinate-ubiquinone reductase prior to embedding in phospholipid vesicles. These results indicate that succinate-ubiquinone reductase and ubiquinol-cytochrome c reductase are indeed present in the membrane as a supermacromolecular complex. No such supermacromolecular complex is detected between NADH-ubiquinone and ubiquinol-cytochrome c reductases or between succinate-ubiquinone and NADH-uniquinone reductases.

Animals↗
PubMed2002

Magnetic resonance imaging with lateralized arterial spin labeling.

We report the development of a new MRI technique which allows spins from right-sided arteries to be labeled separately from spins from left-sided arteries. This method uses two spatially-selective adiabatic inversion pulses to alternate the labeling of the right carotid and vertebral artery separate from the left carotid and vertebral artery. Normal volunteers were scanned on a clinical 1.5 T system and the resultant brain images correlated with the T2 anatomic images. Arterial anatomy was depicted using the new sequence and corresponded to the labeling scheme employed by the sequence. It was demonstrated that spatially selective inversion pulses permit the encoding of the spins within specific vascular origins and the observation of their run-off territory.

Brain↗
PubMedDate not supplied

[Structural characteristics of plasma lipoproteins studied with spin-labelled fatty acids].

As shown by means of electrone paramagnetic resonance of spin probes, a magnitude of ordering of lipoprotein molecular arrangement both internal and external parts of molecule was decreased in a following sequence: high density lipoproteins greater than low density greater than very low density lipoproteins. Permeability towards a reducing agent - ascorbic acid - was altered similarly in the lipoprotein surface monolayer.

Adult↗
PubMed1979

Progressive muscular dystrophy type Duchenne. I. Spin label studies on the physico-chemical state of erythrocyte membranes.

Electron spin resonance studies of erythrocyte membranes from patients with Duchenne muscular dystrophy exhibit changes in the physical state of lipids and proteins in membranes when compared to membranes from normal subjects. The results suggest that the alterations in membrane lipid-protein organization are present in this disease.

Electron Spin Resonance Spectroscopy↗