On the use of the spin labeling technique in the study of erythrocyte membranes

ESR spectra and scanning electron micrographs of human erythrocytes spin labeled with the conventional stearic acid nitroxide substituted at the 5-position have been obtained over a range of label-to-lipid ratios. While morphological changes as previously reported (Bieri V.G.; Wallach D.F.H.; Lin P.S. (1974) Proc. Natl. Acad. Sci. U. S. 71, 4797–4801) are reproduced, it is shown that at label-to-lipid ratios of 1 : 10 or less the basic ESR spectrum is not significantly affected. At low label concentrations the spin labeling technique is a viable one and can be used to investigate membrane properties.     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Interaction of antitumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study.

The cytotoxic and mutagenic properties of antitumor drugs such as adriamycin, acridines, diacridine, actinomycin D and Pt compounds are related to their interaction with nucleic acids and inhibition of protein synthesis. We have examined their interaction with human erythrocyte ghost membranes and murine mastocytoma cells using spin labeling techniques. These drugs induce changes in electron spin resonance of the spin labeled ghost membranes and in the mastocytoma cells. These alterations suggest that these drugs induce changes in protein conformation of the membranes. The membrane binding properties of these drugs may be important in their mechanism of action.     

Localization of immunoreactive testosterone and 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase in cynomolgus monkey (Macaca fascicularis) testes during postnatal development.

The age-related expression of testosterone and 3beta-HSD in the testes of cynomolgus monkeys was detected using light-microscopic immunocytochemistry. Intense deposits of immunoreactive testosterone were labeled in parts of Leydig cells in neonatal, late infantile, pubertal, and adult testes, and only a few Leydig cells in early infantile testes. The immunoreactive 3beta-HSD was labeled in parts of Leydig cells and in all Sertoli cells in neonatal, late infantile, pubertal, and adult testes, whereas only a few Leydig cells, but no Sertoli cells, were labeled in early infantile testes. The fluctuations of testosterone and 3beta-HSD expression in testes correlated well with those already observed plasma testosterone levels during postnatal development in cynomolgus monkeys.     

Electron paramagnetic resonance probing of macromolecules: a comparison of structure-function relationships in chymotrypsinogen, -chymotrypsin and anhydrochymotrypsin

Chymotrypsinogen, chymotrypsin and anhydrochymotrypsin have been covalently spin-labeled by an analog of bromoacetamide, and the latter two proteins have been labeled by an analog of 1-chloro-3-tosylamido-4-phenyl butanone. The electron paramagnetic resonance spectra of the labeled proteins indicate protein conformational changes accompanying (1) activation of the zymogen and (2) the binding of protons and substrates by the native and anhydro enzymes, and tertiary structural differences between these protein forms which are at once informative and predictable. A spin-label linked to the thioether side-chain of methionine 192 in Chymotrypsinogen may be in contact with a hydrophobic surface. This interaction is lost upon zymogen activation with little change in the isotropic rotational freedom of the nitroxide group. The rotational freedom of the group increases sigmoidally with pH; a spectral dependence upon an ionizing group (pK_a = 8.9) is demonstrated. The binding of indole to the labeled enzyme raises the pK_a of the ionizing group to 10.2. A spin-label linked to histidine 57 in chymotrypsin senses both indole binding and pH changes directly; the same label in anhydrochymotrypsin responds directly only to changes in pH. Neither histidine-labeled derivative exhibits enzymic activity. The electron paramagnetic resonance spectra of these two labeled proteins at high pH indicate a decrease in the motional freedom of the spin label. The spectral data show that the conformational state of the labeled zymogen is not similar to the high-pH conformational state of the labeled enzyme. Furthermore, the pH-dependent conformational transition of labeled chymotrypsin requires neither the serine 195 hydroxyl nor the histidine 57 imidazole, since the transition occurs normally in derivatized and chemically modified protein forms. The chemical reactivity of histidine 57 in anhydrochymotrypsin is evaluated and the catalytic activities of two histidine alkylated enzymes are compared.     

Orientation and dynamics of melittin in membranes of varying composition utilizing NBD fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of approximately 8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.     

A ganglioside spin label: ganglioside head group interactions.

A general procedure has been developed for covalent attachment of a nitroxide spin label in the head group region of gangliosides. Gangliosides so labeled and incorporated into lipid bilayer vesicles give a sharp, 3-line spectrum characteristic of a highly mobile structure. The molecular basis of apparent ganglioside-ganglioside head group interaction is briefly discussed.     

Photoaffinity Labeling of Adenylate Cyclase-Linked Serotonin Receptors in Aplysia Neurons

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 microM. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 microM. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact greater than 10(-4) M). Irradiation of the membranes in the presence of 100 microM 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 microM serotonin. Under the same conditions, 100 microM 4-amino, 3-nitrophenylazido-serotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 microM) was irradiated with membranes for 5 min at 4 degrees C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.     

Brain CCK receptors are structurally distinct from pancreas CCK receptors

Brain and pancreas cholecystokinin (CCK) receptors differ markedly in their selectivity for CCK analogs. To determine the size and subunit structure of the brain CCK receptor and compare it to that of the pancreas, 125I-CCK33 was covalently cross-linked with ultraviolet light to its receptor on mouse brain particles and purified pancreatic plasma membranes. When CCK was crosslinked to brain membranes, a single consistent major labeled protein band of Mr = 55,000 was observed in both the presence and the absence of DTT. These data with brain receptors contrast to results with pancreatic receptors where two bands of Mr = 120,000 and 80,000 are labeled in the absence and presence of DTT, respectively. These studies indicate, therefore, that the brain and pancreas CCK receptors are structurally and functionally distinct.     

Identification of the proteins exposed on the cytoplasmic surface of the pancreatic zymogen granule

Lactoperoxidase-catalyzed ¹²⁵I-iodination was used to label pancreatic zymogen granules. Membrane proteins facing the cytoplasmic surface were specifically labeled. Two low molecular weight proteins of 17000 and 15000 were intensely labeled at 0°C. Another small 13 kDa protein was strongly iodinated at 25°C along with some others, including the 29 kDa subunit of the ATP diphosphohydrolase. The major glycoprotein of the granule membrane was not iodinated but the presence of an iodinated 80 kDa protein suggests that proteolytic fragments of the 92 kDa glycoprotein were accessible to iodination on the intact granule. These proteins localized on the cytoplasmic surface of the granule are believed to play a major role in the exocytotic phenomenon of the exocrine pancreas.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Density of NMDA-Coupled and Uncoupled 1-[1-(2-[3H]Thienyl)cyclohexyl]piperidine Recognition Sites in the Brain and Spinal Cord: Differential Effects of NMDA Agonists and Antagonists

Abstract: Binding of 1-[1-(2-[³H]thienyl)cyclohexyl]piperidine ([³H]TCP) to mouse brain and spinal cord membranes was studied using compounds selective for the NMDA-coupled 1-(1-phenylcyclohexyl)piperidine (PCP) and/or σ recognition sites. In both tissues, [³H]TCP labeled two populations of binding sites. Density of the low-affinity sites was approximately the same in both tissues, but the population of the high-affinity [³H]TCP sites was three times bigger in the brain than in the spinal cord. Self- and cross-displacement studies showed that the high-affinity [³H]TCP binding sites could be identical with NMDA receptor-coupled PCP sites, whereas the low-affinity [³H]TCP sites may be associated with σ binding sites in both tissues. The NMDA-coupled PCP sites labeled in the presence of 6.25 n M [³H]TCP constituted a much higher percentage of the total binding in the brain (75%) than in the spinal cord (44%). Consistent with this, reintroduction of glycine and glutamate significantly increased, but DA antagonists significantly inhibited [³H]TCP binding in the brain but not in the spinal cord. Together, these data suggest that a large component of [³H]TCP-labeled binding sites in the spinal cord may be associated with σ but not the NMDA receptor-coupled PCP sites.     

Binding of the new morphiceptin analogs to human MCF-7 breast cancer cells and their effect on growth

In the present study, we reported on the synthesis of two new mu-opioid peptide analogs, [D-1-Nal3]morphiceptin and [D-1-Nal4]-morphiceptin [1-Nal=3-(1-naphthyl)-alanine] which expressed receptor binding affinities at least at the level of the primary opioid ligands. The new analogs also labeled mu-opioid receptors on the cells of human breast cancer MCF-7 cell line with affinity much higher than that of endomorphins and morphiceptin, the well-known mu-selective opioid peptides. However, none of the tested peptides significantly decreased cell proliferation of MCF-7 cells.     

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. This resistance (> 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture or in suspension or whether choleragen was bound at 0 or 37°C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside G_M1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [³H]G_M1, up to 70% of the cell-associated G_M1 was extracted under the mild conditions. When the G_M1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of G_M1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [³H]galactose by immunoadsorption, only labeled G_M1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.     

EAAT2 density at the astrocyte plasma membrane and Ca2 + -regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca²⁺ strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca²⁺-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca²⁺-dependent manner, since in the absence of extracellular Ca²⁺ local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

Specific pathway selection by the early projections of individual peripheral sensory neurons in the embryonic medicinal leech

In leech, the central annulus of each midbody segment possesses seven pairs of sensilla, which are mixed clusters of primary peripheral sensory neurons that extend their axons into the CNS where they segregate into distinct fascicles. Pathway selection by individual afferent growth cones of sensillar neurons was examined by double labeling using intracellular dye-filling with anitobody labeling in early Hirudo medicinalis embryos. The monoclonal antibody Lan3–2 was used because sensillar neuronal tracts are specifically labeled by this antibody. Examining 68 individually filled neurons we found that sensillar neuron growth cones bifurcate within the CNS, that they project long filopodia capable to sampling the local environment, and that all of them appeared to choose a single particular CNS fascicle without apparent retraction or realignment of growth cones. Furthermore, each side of the bifurcating afferent growth cones always chose the same fascicle, implying a specific choice of a distinct labeled pathway. By dye-filling individual central neurons (P-cells), we show that there are centrally projecting axons present at the time sensillar afferents enter the ganglionic primordia and select a particular fascicle, and we confirm that at least the dorsal peripheral nerve is likely to be pioneered by central neurons, not by the peripheral afferent. In the sensillum studied here, we sound examples of sensory neurons extending axons into one of all the avilable fascicles. Thus, an individual embryonic sensillum possesses a heterogeneous population of afferents with respect to the central fascicle chosen. This is consistent with the idea that segregation into distinct axon fascicles may be based upon functional differences between individual afferent neurons. Our findings argue strongly in favor of specific pathway selection by afferents in this system and are consistent with previous suggestions that there exists a hierarchy of cues, including surface glycoconjugates that mediate navigation of the sensillar growth cones and the fasciculation of their axons. 1994 John Wiley & Sons, Inc.     

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) causes Akt phosphorylation and morphological changes in intracellular organellae in cultured rat astrocytes

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is widely used for cell viability and cytotoxicity assays, but cell biological effects of MTT itself have not been investigated. In this paper we show that MTT induces a morphological change in an intracellular membranous compartment labeled with anti-Rab5 antibody, dissociation of early endosomal auto-antigen (EEA1) from the membrane fraction, and phosphorylation of Akt probably through a phosphatidylinositol-3-OH kinase [PI(3)K] pathway in cultured rat astrocytes. These findings suggest that MTT affects cellular functions and conditions to some extent, and such effects of MTT may cause some discrepancies of measurement of cell viability using MTT assay and other assays. That is, the effects of MTT on cells could influence the results of cell viability assay. Moreover, MTT or other tetrazolium salts could be used as interesting activators of Akt to investigate the mechanism by which Akt or PI(3)K is activated.     

Site-specific fluorescent derivatization and liquid chromatographic-mass spectrometric characterization of long R(3) IGF-I for bioanalytical applications

Recombinant Long R(3) IGF-I was derivatized with fluorescein isothiocyanate (FITC) at a single location by careful selection of reaction conditions (i.e. pH, and FITC/protein amino group ratio). High-performance liquid chromatography (LC) and electrospray mass spectrometry (MS) were used to confirm the extent of fluorescein conjugation. The protein conjugate was isolated and subjected to cyanogen bromide (CNBr) cleavage, followed by LC-MS to determine the site of modification. The isolated species of Long R(3) IGF-I-FITC was labeled at the N-terminal Met residue. Recognition of this fluorescent analog by monoclonal anti-IGF-I was preserved, indicating its potential for immunodiagnostic applications.     

Melanoprotein Absence of a direct melanin-protein relationship in chick embryp melanocytes

Short-term synthesis of radioactivity labeled melanin (using dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil) by chick retinal pigment tissues in vitro was not influenced by inhibitors of protein synthesis, puromycin and cyloheximide. Co-ordinate synthesis of protein is, therefore, unnecessary for melanin synthesis, and melanoproteins must represent secondary interactions between melanin and protein. Melanin was isolated from chick embryo feather germs by extracting the proteins with hot dodecyl sulfate/mercaptoethanol. Melanin isolated from tissues incubated previously in l-[U-¹⁴C]valine medium had no associated radioactivity compared to the radioactivity of melanin prepared from tissues incubated in dl-[2-¹⁴C]tyrosine or 2-[2-¹⁴C]thiouracil. If melanoproteins exist at all, they are non-covalently bonded associations of melanin and melanosomal proteins.     

In Vitro Exchange of β-Sitosterol between Erythrocytes and Plasma of Rats

The erythrocytes and plasma of rats were not labeled equally with sterols even after feeding plant sterols for 2 months.

When erythrocytes and plasma were labeled in vivo with radioactive sterols, the in vitro exchange of cholesterol between cells and plasma was considerably greater than that of β-sitosterol. The dependence of the transfer on plasma lecithin : cholesterol acyltransferase was much less with β-sitosterol.

More labeled β-sitosterol existed in high density lipoprotein and less in very-low density and low density lipoproteins than cholesterol, when plasma was labeled in vivo. A similar distribution pattern was observed when plasma was incubated with labeled erythrocytes. These results suggest that an extra ethyl group in the side chain of the molecule substantially influences the metabolic behavior of the sterols.