Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy.

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice-embedded proliferating cells of Escherichia coli K-12 (thymidine-requiring mutant, strain N4316) via correlative light-electron microscopy. For that purpose, erythrosine-11-dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice-embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high-resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.

PEG-coated vesicles from Pluronic/lipid mixtures for the carrying of photoactive erythrosine derivatives.

Liposomes are very attractive membrane models and excellent drug delivery systems. Concerning their drug delivery aspects, the mixing liposomes with biocompatible copolymers allows for stability and the incorporation of several drugs. We developed PEG coated vesicles from the mixture of DPPC and F127 Pluronic copolymer to obtain long-circulating nanoparticles (mixed vesicles). We employed an innovative process previously developed by us: a small amount of F127 mixed in DPPC, thin film preparation, followed by hydration (lipids plus F127) using a bath sonicator cleaner type, forming unilamellar spherical vesicles with diameter ∼100 nm. The formed PEG coated vesicles were incorporated with the xanthene dye Erythrosine B (ERY), and its ester derivatives as photosensitizers (PS) for photodynamic proposes. The F127/DPPC mixed vesicles promoted a higher PS incorporation, and with better thermal and kinetic stability, at least 60 days, when compared to conventional DPPC liposome. The binding constant and quenching analysis revealed that with a higher PS hydrophobicity, PS affinity increases toward the nanoparticle and results in a deeper PS location inside the lipid bilayer. An increment in the fluorescence quantum yield was observed, while the PS singlet oxygen generations remained high. Dialysis studies demonstrated that PS were released based on their hydrophobicity. Permeation analysis showed that all PS can reach the deeper regions of the skin. The Decyl Ester derivative/nanoparticle exhibited high photoactivity against Caco-2 cancer cells (in vitro studies). The PEG coated from F127/DPPC mixed vesicles are very promising nanocarriers for erythrosine and its derivatives.

Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

The morphology, ontogeny and tissue distribution of mast cells were studied in common wolffish(Anarhichas lupus L.) at the larval, juvenile and adult life stages using light and electron-microscopy and immunohistochemistry. Fish were sampled at 1 day, 1, 2, 3, 4, 8 and 12 weeks post-hatching in addition to 6 and 9 months and 2 years and older. From 8 weeks post-hatching, mast cells in common wolffish mainly appeared as oval or rounded cells 8-15 mm in diameter with an eccentrically placed, ovoid nucleus and filled with cytoplasmic granules up to 1.2 mm in diameter. Granules were refractile and eosinophilic to slightly basophilic in H&E and stained bright red with Martius-scarlet-blue and purple with pinacyanol erythrosinate in formalin-fixed tissues. Mast cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 1 day to 4 weeks post-hatching, immature mast cell containing only a few irregularly sized cytoplasmic granules were observed by light and electron-microscopy in loose connective tissue of cranial areas. From 1 day post-hatching, these cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 12 weeks post-hatching, mast cells showed a primarily perivascular distribution and were particularly closely associated with lymphatic vessels and sinuses. Mast cells were mainly located at the peripheral border of the adventitia of arteries and veins, while they were in intimate contact with the endothelium of the lymphatic vessels. Numerous mast cells were observed in the intestine. A stratum compactum, as described in salmonids, was not observed in wolffish intestine,nor were mast cells confined to a separate layer, a stratum granulosum. Lymphatic vessels consisting of endothelium, intimal connective tissue and a poorly developed basal lamina were observed in the intestine. Scanning electron microscopy was used to compare the structure and localization of intestinal mast cells of common wolffish and rainbow trout. Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

Cofilin increases the torsional flexibility and dynamics of actin filaments.

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.

SERCA structural dynamics induced by ATP and calcium.

We have used time-resolved phosphorescence anisotropy (TPA) to probe rotational dynamics of the rabbit skeletal sarcoplasmic reticulum Ca-ATPase (SERCA), to test the hypothesis, generated from X-ray crystallography, that large-scale structural changes are induced by Ca in this system. Previous TPA studies on SERCA used primarily erythrosin 5'-isothiocyanate (ErITC), which binds to the nucleotide-binding domain and inactivates the enzyme. To investigate rotational dynamics of the active enzyme, we labeled SERCA with erythrosin 5'-iodoacetamide, which binds to the phosphorylation domain and has a minimal effect on the calcium-dependent ATPase activity. In the absence of nucleotide and the presence of calcium, TPA results were similar to those observed previously with ErITC, consistent with the global uniaxial rotation of SERCA monomers and oligomers and small amplitude internal protein dynamics. The removal of Ca had only a slight effect, while the addition of adenosine 5'-triphosphate (ATP) increased the amplitude of internal dynamics and changed the probe's orientation, corresponding to tilting of the phosphorylation domain by at least 20 degrees . Ca partially reversed the ATP effects. A nonhydrolyzable ATP analogue had the same effects as ATP, showing that the observed changes were not dependent on active ion transport. Computational analysis indicates that these ligands affect primarily the internal dynamics of the enzyme, with negligible effects on global dynamics and enzyme association. Melittin, which has been shown to aggregate and inhibit SERCA, eliminated the nucleotide-induced internal dynamics and increased the final anisotropy. We propose that (i) the large Ca-dependent structural changes suggested by SERCA crystallography are more dependent on ATP than on Ca and (ii) aggregation-induced inhibition of SERCA is due to the functional coupling between global and internal protein dynamics.

Frequency-domain lifetime fluorometry of double-labeled creatine kinase.

Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.

Degranulation, density, and distribution of mast cells in the rat thalamus: a light and electron microscopic study in basal conditions and after intracerebroventricular administration of nerve growth factor.

In the adult rat brain mast cells reside selectively in the thalamus. We investigated thalamic mast cells stained by acidic toluidine blue or pinacyanol, and with histamine immunocytochemistry, focusing on their state of activity revealed by degranulation. Mast cells exhibited perivascular prevalence and high quantitative variability, between cases and in different sections, with no asymmetry or topographical selectivity in thalamic nuclei. Pinacyanol, alone or with erythrosine, stained mast cells with higher sensitivity than toluidine blue. However, toluidine blue was highly predictive of pinacyanol staining and provided the best resolution of mast cell cytoplasmic features. Histamine immunocytochemistry labeled 61% of pinacyanol-stained mast cells. Intensely toluidine blue-stained granulated cells, as well as cells exhibiting different degrees of degranulation that paralleled lighter staining, were observed. The response of thalamic mast cells to intracerebroventricular administration of nerve growth factor (NGF) and control cytochrome-c injections was evaluated after 2, 24, and 72 hours. No obvious changes in mast cell number or distribution were found after treatment, but massive degranulation was frequently observed after NGF administration. Significant decrease of staining intensity of mast cells, supporting enhanced degranulation, was documented in NGF-treated animals by quantitative image analysis. Ultrastructural features of mast cell degranulation, with granule coalescence and matrix dissolution, were detected in untreated and NGF-treated cases. The findings point out that mast cells are active in the thalamus in basal conditions and that NGF has the potential to elicit long-lasting degranulation of thalamic mast cells in vivo, exerting a direct effect and/or priming these cells to react to endogenous stimuli.

Effects of fluorescent pseudo-ATP and ATP-metal analogs on secondary structure of Na(+)/K(+)-ATPase.

The secondary structure of Na(+)/K(+)-ATPase after modification of the ATP-binding sites was analyzed. Consistently with recent reports, we found in trypsin-treated Na(+)/K(+)-ATPase additionally to alpha-helix also beta-sheet structures in the transmembrane segments. However, binding of fluorescein 5'-isothiocyanate (FITC), the pseudo-ATP analog, to the ATP-binding site did not affect the secondary structure of undigested Na(+)/K(+)-ATPase. Consequently, fluorescence intensity changes of FITC-labeled Na(+)/K(+)-ATPase commonly used to observe conformational transitions of the enzyme reflect physiological changes of the native structure. The metal complex analogues of ATP, Cr(H(2)O)(4)ATP and Co(NH(3))(4)ATP, on the other hand, affected the secondary structure of Na(+)/K(+)-ATPase. We propose that these changes in the secondary structure are responsible for inhibition of backdoor phosphorylation.

Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.

Rotational dynamics of the regulatory light chain in scallop muscle detected by time-resolved phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy (TPA) to study the rotational dynamics of chicken gizzard regulatory light chain (RLC) bound to scallop adductor muscle myofibrils in key physiological states. Native RLC from scallop myofibrils was extracted and replaced completely with gizzard RLC labeled specifically at Cys 108 with erythrosin iodoacetamide (ErIA). The calcium sensitivity of the ATPase activity of the labeled myofibril preparation was quite similar to that of the native sample, indicating that the ErIA-labeled RLC is functionally bound to the myosin head. In rigor (in the absence of ATP, when all the myosin heads are rigidly bound to the thin filament), a slight decay was observed in the first few microseconds, followed by no change in the anisotropy. This indicates small-amplitude restricted motions of the RLC or the entire LC domain of myosin. Addition of calcium to rigor restricts these motions further. Relaxation with ATP (no Ca) causes a large decay in the anisotropy, indicating large-amplitude rotational motion with correlation times of 5-50 micros. Further addition of calcium, to induce contraction, resulted in a decrease in the rate and amplitude of anisotropy decay. In particular, there is clear evidence for a slow rotational motion with a correlation time of approximately 300 micros, which is not present either in rigor or relaxation. This indicates rotational motion that specifically correlates with force generation. The changes in the rotational dynamics of the light-chain domain in rigor, relaxation, and contraction support earlier work based on probes of the catalytic domain that muscle contraction is accompanied by a disorder-to-order transition of the myosin head. However, the motions of the LC domain are different from those of the catalytic domain, which indicates rotation of the two domains relative to each other.

Rearrangement of domain elements of the Ca-ATPase in cardiac sarcoplasmic reticulum membranes upon phospholamban phosphorylation.

Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions. Two major Er-ITC labeling sites were identified using electrospray ionization mass spectrometry (ESI-MS), corresponding to Lys464 and Lys650, which are respectively located within the phosphorylation and nucleotide binding domains of the Ca-ATPase. Frequency-domain phosphorescence measurements of the rotational dynamics of Er-ITC bound to these cytoplasmic sequences within the Ca-ATPase permit the resolution of the dynamic structure of individual domain elements relative to the overall rotational motion of the entire Ca-ATPase polypeptide chain. We observe a significant decrease in the rotational dynamics of Er-ITC bound to the Ca-ATPase upon phosphorylation of PLB by PKA, as evidenced by an increase in the residual anisotropy. These results suggest that phosphorylation of PLB results in a structural reorientation of the phosphorylation or nucleotide binding domains with respect to the membrane normal. In contrast, calcium activation of the Ca-ATPase in the presence of dephosphorylated PLB results in no detectable change in the rotational dynamics of Er-ITC, suggesting that calcium binding and PLB phosphorylation have distinct effects on the conformation of the Ca-ATPase. We suggest that PLB functions to alter the efficiency of phosphoenyzme formation following calcium activation of the Ca-ATPase by modulating the spatial arrangement between ATP bound in the nucleotide binding domain and Asp351 in the phosphorylation domain.

Interleukin 1 (IL-1) causes changes in lateral and rotational mobilities of IL-1 type I receptors.

To investigate IL-1-dependent interactions of IL-1 type I (IL-1 RI) receptors on intact cells, lateral and rotational mobilities and detergent insolubility were investigated. Lateral mobility was measured by fluorescence photobleaching recovery, using a Cy3-modified, noncompetitive mAb specific for IL-1RI (M5) bound to wild-type IL-1 RI or mutant IL-1 RI with a truncated cytoplasmic tail. Addition of IL-1 causes significant reduction in the mobile fraction of wild-type IL-1 RI for two different transfected cell lines. For the mutant IL-1 RI, no significant decrease in response to IL-1 is observed, indicating that the missing cytoplasmic segment is involved in IL-1-dependent interactions of IL-1 RI that lead to reduced lateral mobility on the cell surface. The rotational mobility of IL-1 RI was assessed with phosphorescence anisotropy decay measurements using erythrosin-labeled M5. IL-1 decreases the rotational mobility of cell surface IL-1 RI on the microsecond time scale and also increases the initial anisotropy, indicating loss in segmental motion. Measurements of resistance to solubilization by Triton X-100 showed that IL-1 binding increases the fraction of IL-1 RI sedimenting with cytoskeletal residues. The IL-1 receptor antagonist protein (IL-1ra) causes partial effects in reducing rotational mobility and increasing detergent insolubility of M5-lableled IL-1 RI, indicating that this ligand causes structural changes in the presence of the dimerizing M5 mAb. These ligand-dependent physical interactions of IL-1 RI on the cell surface may be related to signal initiation by this receptor.

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits.

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Förster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of Förster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. Förster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).

Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.

Conditions that permit the selective modification of an ATP-protectable site on the Ca-ATPase in skeletal sarcoplasmic reticulum (SR) membranes using erythrosin isothiocyanate (Er-ITC) have been identified. The major labeling site for Er-ITC has been identified using reversed-phase HPLC and positive FAB mass spectrometry after exhaustive tryptic digestion of the Er-ITC-modified Ca-ATPase. An ATP-protectable peptide corresponding to M452NVFNTEVRNLSK464VER467 is modified by Er-ITC, the average mass of which is 2830.1 +/- 0.3 Da. The exclusive modification of lysine residues indicates Lys464 as the site of Er-ITC modification. Derivatization with Er-ITC diminishes the secondary activation of steady-state ATPase activity and the rate of dephosphorylation by millimolar concentrations of ATP. In contrast, in the presence of micromolar ATP concentrations Er-ITC modification of the Ca-ATPase does not affect (i) the apparent affinity of ATP, (ii) the maximal extent of phosphoenzyme formation by ATP, (iii) the rate of steady-state ATP hydrolysis, or (iv) the rate of dephosphorylation of the Ca-ATPase. Furthermore, ATP utilization by the Ca-ATPase is unaffected by detergent solubilization, irrespective of Er-ITC modification, indicating that the secondary activation of ATP hydrolysis involves a single Ca-ATPase polypeptide chain. Therefore, Er-ITC does not interfere with the normal structural transitions associated with phosphoenzyme decay. Rather, these results indicate that Er-ITC bound to Lys464 interferes with either ATP binding to a low-affinity site or the associated structural transitions that modulate the rate of enzyme dephosphorylation.

Differential effects of general anesthetics on the quaternary structure of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum.

The effects of the general anesthetics hexanol, halothane, and diethyl ether on Ca-ATPase activity and on the oligomeric state of the Ca-ATPase of sarcoplasmic reticulum (SR) from cardiac and skeletal muscle were investigated. The effects of these general anesthetics on Ca-ATPase activity were similar in cardiac and skeletal SR and were characterized by stimulation of Ca-ATPase activity at lower concentrations of anesthetics and inhibition at higher concentrations. The distribution of the Ca-ATPase among its oligomeric states was estimated from the time-resolved phosphorescence anisotropy (TPA) decay of SR in which Ca-ATPase was covalently labeled with erythrosin isothiocyanate (ERITC) or with erythrosin iodoacetamide (ERIA). In contrast to the similar responses of Ca-ATPase activity, there were marked differences in the responses to general anesthetics of the TPA decay between cardiac and skeletal SR. In cardiac SR hexanol, halothane, and diethyl ether caused pronounced increases in the limiting anisotropy at very long times (r infinity), which indicate increases in the fraction of oligomers too large to rotate on the millisecond time scale of the experiments. In skeletal SR, by contrast, there were no significant changes in r infinity in response to the three general anesthetics. This difference between cardiac and skeletal SR in response to general anesthetics is not due to the presence of phospholamban in cardiac SR, since SR from AT-1 cells, which have the SERCA2a isoform of Ca-ATPase, but only trace levels of phospholamban, have increases in r infinity in response to the general anesthetics that resemble those in cardiac SR. Experiments with cardiac SR labeled with ERIA give similar results, showing that the results with ERITC are not an artifact of the labeling procedure. Increasing the ionic strength with LiCl diminished the proportion of large immobile oligomers of cardiac Ca-ATPase under control conditions but enhanced the formation of large oligomers in response to hexanol.

Erythrosin 5'-isothiocyanate labels Cys549 as part of the low-affinity ATP binding site of Na+/K+-ATPase.

The high-affinity E1ATP site of Na+/K+-ATPase labeled with fluorescein 5'-isothiocyanate and its E2ATP site labeled with erythrosin 5'-isothiocyanate (ErITC), as was shown recently [Linnertz et al. (1998) J. Biol. Chem. 273, 28813-28821], reside on separate and adjacent catalytic alpha subunits. This paper provides evidence that specific labeling of the E2ATP binding site with ErITC resulted in a modification of the Cys549 residue in the tryptic fragment with the sequence Val545-Leu-Gly-Phe-Cys549-His550. Hence, Cys549 is part of or close to the low-affinity E2ATP binding site of Na+/K+-ATPase.

Enhanced rotational dynamics of the phosphorylation domain of the Ca-ATPase upon calcium activation.

We have used labeling conditions that permit the specific and covalent attachment of erythrosin isothiocyanate (Er-ITC) to Lys464 within the phosphorylation domain of the Ca-ATPase in skeletal sarcoplasmic reticulum membranes. These labeling conditions do not interfere with high-affinity ATP binding, phosphoenzyme formation, or phosphoenzyme hydrolysis [Huang, S., Negash, S., and Squier, T. C. (1998) Biochemistry 37, 6949-6957]. Thus, we can use frequency-domain phosphorescence spectroscopy to measure the rotational dynamics of the Ca-ATPase stabilized in different enzymatic states corresponding to the absence of bound ligands (E), calcium activation (E x Ca2), the presence of bound nucleotide (E x ATP), and formation of phosphoenzyme (E-P). We resolve three rotational correlation times corresponding to (i) a large-amplitude domain motion of the phosphorylation domain (phi1 approximately 5 +/- 1 micros), (ii) overall protein rotational motion with respect to the membrane normal (phi2 approximately 50 +/- 10 micros), and (iii) the rotational motion of the SR vesicles (phi3 approximately 1.1 +/- 0.4 ms). No differences are observed in the rotational dynamics of E, E x ATP, or E-P, indicating that phosphoenzyme formation or nucleotide binding result in no global structural changes involving the phosphorylation domain. In contrast, calcium activation enhances the amplitude of motion of the phosphorylation domain. These observed calcium-dependent changes in rotational dynamics result from structural changes within a single Ca-ATPase polypeptide chain, since protein-protein interactions do not change upon calcium binding. Thus, calcium binding induces concerted domain motions within a single Ca-ATPase polypeptide chain that may play a critical role in facilitating substrate binding and utilization.

Self-association accompanies inhibition of Ca-ATPase by thapsigargin.

Recent studies have demonstrated a relationship between the activity of the Ca-ATPase of sarcoplasmic reticulum and its state of self-association. In the present study, the effects of thapsigargin (TG), a toxin that specifically inhibits the Ca-ATPase of rabbit skeletal muscle sarcoplasmic reticulum membrane, were studied by detecting the time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase that had been labeled with the phosphorescent probe erythrosin-isothiocyanate (ErITC). Anisotropy decays were fit to a function that consisted of three exponential decays plus a constant background, as well as to a function describing explicitly the uniaxial rotation of proteins in a membrane. In the absence of TG, the anisotropy was best-fit by a model representing the rotation of three populations, corresponding to different-sized oligomeric species in the membrane. The addition of stoichiometric amounts of TG to the Ca-ATPase promptly decreased the overall apparent rate of decay, indicating decreased rotational mobility. A detailed analysis showed that the principal change was not in the rates of rotation but rather in the population distribution of the Ca-ATPase molecules among the different-sized oligomers. TG decreased the proportion of small oligomers and increased the proportion of large ones. Preincubation of the ErITC-SR in 1 mM Ca2+, which stabilizes the E1 conformation relative to E2, was found to protect partially against the changes in the TPA associated with the presence of the inhibitor. These results are consistent with the hypothesis that TG inhibits the Ca-ATPase by stabilizing it in an E2-like conformation, which promotes the formation of larger aggregates of the enzyme. When combined with the effects of other inhibitors on the Ca-ATPase, these results support a general model for the coupling of enzyme conformation and self-association in this system.

Histaminergic and non-histamine-immunoreactive mast cells within the cat lateral geniculate complex examined with light and electron microscopy.

Mast cells and their location in the cat lateral geniculate complex of the thalamus were examined by means of histamine immunohistochemistry and the mast cell stain pinacyanol erythrosinate. Brain sections from seven normal adult pigmented cats were processed for light or electron microscopy. Histamine-containing and pinacyanol erythrosinate-stained mast cells were widespread throughout the dorsal and ventral lateral geniculate nuclei and the surrounding regions. Mast cells were especially numerous rostrally in the complex and in the geniculate C laminae. The cells were found consistently in association with blood vessels, ranging from capillary size to vessels c. 150 microns diameter, and twice as often with arterioles as with venules. Large clusters of many mast cells associated with single blood vessels were seen. Individual mast cells were typically 8 microns in diameter and somewhat oval, although multipolar and crescent-shaped cells were also seen, up to twice as long. The amount of histamine labeling varied across cells. When histamine-labeled material was secondarily stained with pinacyanol erythrosinate, many mast cells were double labeled. In addition, there was a small population of mast cells that stained only with pinacyanol erythrosinate, but was otherwise identical to the histamine-immunoreactive mast cells. Electron microscopic examination showed that the mast cells lie on the brain side of the blood-brain barrier. Mast cells were found in close proximity to the thalamic neuropil, primarily apposed to the processes of astrocytes, but also apposed to neural elements. The distinctive electron-dense cytoplasmic granules in the fully granulated, mature state were largely amorphous in appearance and as large as 700 nm in diameter. Histamine was dispersed throughout some granules and contained within restricted areas of other granules. In degranulated mast cells, large, irregularly shaped, electron-lucent granules were seen fused with the cell membrane on the neuropil side, as well as the lumen side of the mast cell. More mast cells were observed at the electron microscopic level than were expected from the light level observations, which suggests that, despite the numbers of mast cells labeled, these results may still underestimate the total mast cell population present in this region of the thalamus. Mast cells, by their numbers, their distribution and the potent chemical substances they contain, may significantly influence vascular and neural function, directly and indirectly, in the cat lateral geniculate complex.