HLA genotyping using the Illumina HLA TruSight next-generation sequencing kits: A comparison.

Illumina first introduced their TruSight human leucocyte antigen (HLA) next-generation sequencing (NGS) typing kit in 2015 and subsequently followed up with a new version in 2016. Here we report on our experience comparing the two versions of the Illumina HLA NGS kits.

Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Long-term hazards of neonatal blue-light phototherapy.

Blue-light phototherapy has been an essential therapeutic tool in the management of neonatal jaundice for decades. Rarely, it is accompanied by acute dermatological and systemic side-effects, but fortunately these are reversible and can be adequately and promptly treated in routine neonatal practice. In contrast, much less is known about the potential long-term side-effects of neonatal blue-light phototherapy (NBLP). Many of the data that are currently available on how NBLP influences melanocytic naevus (MN) development are controversial. The results of recent well-designed epidemiological surveys suggest that NBLP could well be a risk factor for MN formation, and highlight the need for additional in vivo and in vitro studies. NBLP is at present the mainstay of treatment for neonatal jaundice, but in the future greater consideration should be given to its long-term side-effects when phototherapy is indicated. It is relevant to emphasize the importance of appropriately restricted and adequate clinical guidelines, and strict monitoring of the management of hyperbilirubinaemia, in order to avoid the unnecessary overtreatment of newborn infants.

Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells.

AIMS/HYPOTHESIS: This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out. METHODS: Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells. RESULTS: TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death. CONCLUSIONS/INTERPRETATION: Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.

Influence of shear viscosity of quark-gluon plasma on elliptic flow in ultrarelativistic heavy-ion collisions.

We investigate the influence of a temperature-dependent shear viscosity over entropy density ratio η/s on the transverse momentum spectra and elliptic flow of hadrons in ultrarelativistic heavy-ion collisions. We find that the elliptic flow in √S(NN)=200 GeV Au+Au collisions at RHIC is dominated by the viscosity in the hadronic phase and in the phase transition region, but largely insensitive to the viscosity of the quark-gluon plasma (QGP). At the highest LHC energy, the elliptic flow becomes sensitive to the QGP viscosity and insensitive to the hadronic viscosity.

Removal of nonspecific binding proteins from cell and tissue extracts using 2-aminobenzimidazole-tethered affinity resin.

Cellular drug target identification through affinity chromatography is often hindered by the quantity of nonspecific binders, such as cytoskeletal and heat shock proteins. Thus, we prepared a 2-aminobenzimidazole-tethered depletion resin designed for removal of these proteins, and tested it on human lung carcinoma cell and rat tissue extracts. Column-bound proteins were identified by two-dimensional gel electrophoresis and MS. Among others, tubulins, actins and heat shock proteins were successfully depleted. Due to the reduction of these highly abundant proteins detection of potential drug targets is considerably facilitated in the pre-purified samples.

Modification of ionotropic glutamate receptor-mediated processes in the rat hippocampus following repeated, brief seizures.

The seizure-induced molecular and functional alterations of glutamatergic transmission in the hippocampus have been investigated. Daily repeated epileptic seizures were induced for 12 days by intraperitoneal administration of 4-aminopyridine (4-AP; 4.5 mg/kg) in adult Wistar rats. The seizure symptoms were evaluated on the Racine's scale. One day after the last injection, the brains were removed for in vitro electrophysiological experiments and immunohistochemical analysis. The glutamate receptor subunits NR1, NR2A, NR2B, GluR1, GluR1(flop), GluR2, and KA-2 were studied using the histoblotting method. The semi-quantitative analysis of subunit immunoreactivities in hippocampal layers was performed with densitometry. In the hippocampus, increase of GluR1, GluR1(flop) and NR2B immunostaining was observed in most of the areas and layers. The significant decrease of GluR2 staining intensity was observed in the CA1 and dentate gyrus. Calcium permeability of hippocampal neurons was tested by a cobalt uptake assay in hippocampal slices. The uptake of cobalt increased in the CA1 area and dentate gyrus, but not in the CA3 region following 4-AP treatment. Effects of AMPA and NMDA (N-methyl-d-aspartate) glutamate receptor antagonists (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) and D-APV respectively) were measured in hippocampal slices using extracellular recording. Analysis of the population spikes revealed the reduced effectiveness of the AMPA receptor antagonist GYKI 52466, while the effect of the NMDA receptor antagonist d-(2R)-amino-5-phosphonovaleric acid was similar to controls. The results demonstrated that repeated convulsions induced structural and functional changes in AMPA receptor-mediated transmission, while NMDA and kainate receptor systems displayed only alterations in receptor subunit composition.

Leflunomide and its metabolite A771726 are high affinity substrates of BCRP: implications for drug resistance.

BACKGROUND: Earlier publications have suggested a possible role for the efflux transporter breast cancer resistance protein (BCRP) in acquired resistance to disease-modifying antirheumatic drugs (DMARDs) such as leflunomide and its metabolite A771726 (teriflunomide). However, there is no direct evidence that BCRP interacts with these drugs. OBJECTIVES: To characterise the interaction between BCRP transporter and leflunomide and its active metabolite A771726, with emphasis on the nature of the interaction (substrate or inhibitor) and the kinetic characterisation of the interactions. METHODS: Different in vitro membrane-based methods (ATPase and vesicular transport assay) using BCRP-HAM-Sf9 membrane preparations and cellular assays (Hoechst assay and cytotoxicity assay) were performed on PLB985-BCRP and HEK293-BCRP cell lines overexpressing BCRP. RESULTS: In all assays used, an interaction between the investigated drugs and BCRP was detected. In the vesicular transport assay, both leflunomide and its metabolite inhibited BCRP-mediated methotrexate transport. Both compounds are likely substrates of BCRP as shown by the vanadate-sensitive ATPase assay. In line with the membrane assays, leflunomide and A771726 inhibited BCRP-mediated Hoechst efflux from PLB985-BCRP cells. In the cytotoxicity assay, overexpression of BCRP conferred 20.6-fold and 7.5-fold resistance to HEK293 cells against leflunomide and A771726, respectively. The resistance could be reversed by Ko134, a specific inhibitor of BCRP. CONCLUSION: Based on these results, BCRP could play an important role in the resistance to leflunomide and A771726 via interactions with these drugs. BCRP may also mediate drug-drug interactions when leflunomide is administered with other BCRP substrate drugs such as methotrexate.

Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.

It has been suggested that some glutamatergic synapses lack functional AMPA receptors. We used quantitative immunogold localization to determine the number and variability of synaptic AMPA receptors in the rat hippocampus. Three classes of synapses show distinct patterns of AMPA receptor content. Mossy fiber synapses on CA3 pyramidal spines and synapses on GABAergic interneurons are all immunopositive, have less variability, and contain 4 times as many AMPA receptors as synapses made by Schaffer collaterals on CA1 pyramidal spines and by commissural/ associational (C/A) terminals on CA3 pyramidal spines. Up to 17% of synapses in the latter two connections are immunonegative. After calibrating the immunosignal (1 gold = 2.3 functional receptors) at mossy synapses of a 17-day-old rat, we estimate that the AMPA receptor content of C/A synapses on CA3 pyramidal spines ranges from <3 to 140. A similar range is found in adult Schaffer collateral and C/A synapses.

Surface expression of AMPA receptors in hippocampal neurons is regulated by an NSF-dependent mechanism.

Here, we show that disruption of N-ethylmaleimide-sensitive fusion protein- (NSF-) GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) caused a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). N-methyl-D-aspartate (NMDA) receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduced the surface expression of alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate (AMPA) receptors, whereas NMDA receptor surface expression in the same living cells was unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity was unchanged, and a punctate distribution of GluR2 was observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses.

NSF binding to GluR2 regulates synaptic transmission.

Here, we show that N-ethylmaleimide-sensitive fusion protein (NSF) interacts directly and selectively with the intracellular C-terminal domain of the GluR2 subunit of AMPA receptors. The interaction requires all three domains of NSF but occurs between residues Lys-844 and Gln-853 of rat GluR2, with Asn-851 playing a critical role. Loading of decapeptides corresponding to the NSF-binding domain of GluR2 into rat hippocampal CA1 pyramidal neurons results in a marked, progressive decrement of AMPA receptor-mediated synaptic transmission. This reduction in synaptic transmission was also observed when an anti-NSF monoclonal antibody (mAb) was loaded into CA1 neurons. These results demonstrate a previously unsuspected direct interaction in the postsynaptic neuron between two major proteins involved in synaptic transmission and suggest a rapid NSF-dependent modulation of AMPA receptor function.

HLA genotyping in the clinical laboratory: comparison of next-generation sequencing methods.

Implementation of human leukocyte antigen (HLA) genotyping by next-generation sequencing (NGS) in the clinical lab brings new challenges to the laboratories performing this testing. With the advent of commercially available HLA-NGS typing kits, labs must make numerous decisions concerning capital equipment and address labor considerations. Therefore, careful and unbiased evaluation of available methods is imperative. In this report, we compared our in-house developed HLA NGS typing with two commercially available kits from Illumina and Omixon using 10 International Histocompatibility Working Group (IHWG) and 36 clinical samples. Although all three methods employ long range polymerase chain reaction (PCR) and have been developed on the Illumina MiSeq platform, the methodologies for library preparation show significant variations. There was 100% typing concordance between all three methods at the first field when a HLA type could be assigned. Overall, HLA typing by NGS using in-house or commercially available methods is now feasible in clinical laboratories. However, technical variables such as hands-on time and indexing strategies are sufficiently different among these approaches to impact the workflow of the clinical laboratory.

Developmental changes in synaptic AMPA and NMDA receptor distribution and AMPA receptor subunit composition in living hippocampal neurons.

AMPA and NMDA receptors mediate most excitatory synaptic transmission in the CNS. We have developed antibodies that recognize all AMPA or all NMDA receptor variants on the surface of living neurons. AMPA receptor variants were identified with a polyclonal antibody recognizing the conserved extracellular loop region of all four AMPA receptor subunits (GluR1-4, both flip and flop), whereas NMDA receptors were immunolabeled with a polyclonal antibody that binds to an extracellular N-terminal epitope of the NR1 subunit, common to all splice variants. In non-fixed brain sections these antibodies gave labeling patterns similar to autoradiographic distributions with particularly high levels in the hippocampus. Using these antibodies, in conjunction with GluR2-specific and synaptophysin antibodies, we have directly localized and quantified surface-expressed native AMPA and NMDA receptors on cultured living hippocampal neurons during development. Using a quantitative cell ELISA, a dramatic increase was observed in the surface expression of AMPA receptors, but not NMDA receptors, between 3 and 10 d in culture. Immunocytochemical analysis of hippocampal neurons between 3 and 20 d in vitro shows no change in the proportion of synapses expressing NMDA receptors (approximately 60%) but a dramatic increase (approximately 50%) in the proportion of them that also express AMPA receptors. Furthermore, over this period the proportion of AMPA receptor-positive synapses expressing the GluR2 subunit increased from approximately 67 to approximately 96%. These changes will dramatically alter the functional properties of hippocampal synapses.

Differences in the susceptibility of various cation transport ATPases to vanadate-catalyzed photocleavage.

Illumination of sarcoplasmic reticulum vesicles by ultraviolet light in the presence of 1 mM vanadate causes photocleavage of the Ca(2+)-ATPase into two fragments (Vegh et al. (1990) Biochim. Biophys. Acta 1023, 168-183). In the absence of Ca2+ the photocleavage occurs in the N-terminal half of the molecule near the phosphate acceptor Asp-351. In the presence of 2 mM Ca2+ the photocleavage shifts to the C-terminal half of the ATPase, near the FITC binding site (Lys-515). About half of the Ca(2+)-ATPase was cleaved rapidly, accompanied by nearly complete, irreversible loss of ATPase activity when illuminated in the presence of 2 mM CaCl2; further cleavage of the enzyme was slow and affected primarily the C-terminal fragment produced in the presence of Ca2+. Solubilization of the Ca(2+)-ATPase with C12E8 did not affect the site of photocleavage in either conformation. The vanadate-induced Ca(2+)-ATPase crystals were disrupted during photocleavage, while the binding of anti-ATPase antibodies directed against the phosphorylation site (PR-8) and against the FITC binding region (PR-11) was enhanced. The bovine kidney Na+,K(+)-ATPase was insensitive to photocleavage under conditions where about half the Ca(2+)-ATPase was fragmented. The slight cleavage of the pig gastric H+,K(+)-ATPase after prolonged illumination produced fragments that are distinct from the fragments of the Ca(2+)-ATPase.

Vanadate-catalyzed, conformationally specific photocleavage of the Ca2(+)-ATPase of sarcoplasmic reticulum.

Vanadate-sensitized photocleavage of the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum was observed upon illumination of sarcoplasmic reticulum vesicles or the purified Ca2(+)-ATPase by ultraviolet light in the presence of 1 mM monovanadate or decavanadate. The site of the photocleavage is influenced by the Ca2+ concentration of the medium. When the [Ca2+] is maintained below 10 nM by EGTA, the vanadate-catalyzed photocleavage yields fragments of approximately equal to 87 and approximately equal to 22 kDa, while in the presence of 2-20 mM Ca, polypeptides of 71 and 38 kDa are obtained as the principal cleavage products. These observations indicate that the site of the vanadate-catalyzed photocleavage is altered by changes in the conformation of Ca2(+)-ATPase. Selective tryptic proteolysis, at Arg-505-Ala-506, combined with covalent labeling of Lys-515 by fluorescein 5'-isothiocyanate and with the use of anti-ATPase antibodies of defined specificity, permitted the tentative allocation of the sites of photocleavage to the A fragment near the T2 cleavage site in the absence of Ca2+, and to the B fragment between Lys-515 and Asp-659 in the presence of 2-20 mM Ca2+. The loss of ATPase activity during illumination is accelerated by calcium in the presence of vanadate. The vanadate-catalyzed photocleavage in the presence of Ca2+ is consistent with the existence of an ATPase-Ca2(+)-vanadate complex (Markus et al. (1989) Biochemistry 28, 793-799).

Covalent labeling of the cytoplasmic or luminal domains of the sarcoplasmic reticulum Ca(2+)-ATPase with fluorescent azido dyes.

Sarcoplasmic reticulum (SR) vesicles were incubated with azido derivatives of Cascade blue (ACB), Lucifer yellow (ALY), 2,7-naphthalene-disulfonic acid (ANDS), and fluorescein (AF) for 0.1-24 h at 2 degrees C. All four dyes gave intense reaction with the cytoplasmic domain of the Ca(2+)-ATPase on photoactivation after brief incubation. The penetration of the dyes into the luminal space of the SR was determined after centrifugation through Sephadex microcolumns to remove the external dye, followed by photolabeling and gel electrophoresis of the photolabeled proteins. The reaction of ACB and ANDS with the Ca(2+)-ATPase and with calsequestrin increased progressively during incubation up to 24 h indicating their slow accumulation in the luminal space, while ALY and AF did not show significant penetration into the vesicles. The distribution of the covalently attached ACB in the Ca(2+)-ATPase was tested by tryptic proteolysis after labeling exclusively from the outside (OS), from the inside (IS) or from both sides (BS). In all cases intense ACB fluorescence was seen in the A fragment with inhibition of ATPase activity. In the OS preparations the A1, while in IS the A2 fragment was more intensely labeled. There was no significant incorporation of ACB into the region of B fragment identified by FITC fluorescence. The crystallization of the Ca(2+)-ATPase by EGTA + decavanadate was completely inhibited in the BS samples after labeling either in the Ca2E1 or E2V conformation. There was no inhibition of crystallization in the OS preparations. In the IS preparations labeled in the Ca2E1 state the crystallization was impaired, while in the E2V state there was only slight disorganization of the crystals. The total amount of ACB photoincorporated into SR proteins after incubation for 24 h was 1.75 nmol/mg protein; 2/3 of this labeling occurred from the outside and 1/3 from the inside. Similar level of labeling was obtained in media that stabilize the E1 or the E2 conformation of the Ca(2+)-ATPase.

Characterisation of endoplasmic reticulum and plasma membrane Ca(2+)-ATPases in pancreatic beta-cells and in islets of Langerhans.

We have investigated the plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca(2+)-ATPases involved in active transport of Ca2+ in pancreatic beta-cell lines (MIN6, HIT T15, RINm5F) and in islets of Langerhans. Under selective membrane phosphorylation conditions (at low ATP concentration, in the presence of Ca2+ and La3+ and in the absence of Mg2+ at 4 degrees C) the only labelled proteins are the phosphoenzyme intermediates of the Ca(2+)-ATPases. Under these conditions, beta-cell membranes incorporated 32P from [gamma-32P]ATP into two proteins with molecular mass on acidic SDS-polyacrylamide gels of around 115 and 150 kDa. The 150 kDa band was identified as PMCA (i) by reaction with a monoclonal anti-human erythrocyte plasma membrane Ca(2+)-ATPase antibody; (ii) by its typical tryptic cleavage pattern which generated an 80 kDa band; (iii) by lack of inhibition of its autophosphorylation by SERCA-specific inhibitors. The 115 kDa band was identified as SERCA (i) by reaction with a polyclonal anti-rat fast skeletal muscle Ca(2+)-ATPase antibody; (ii) by the concentration-dependent inhibition of its autophosphorylation by thapsigargin and 2,5-di(t-butyl)-1,4-benzohydroquinone (tBHQ), which are specific inhibitors of SERCA. The 115 kDa band was further characterised as the SERCA-2b isoform by reaction with a polyclonal rabbit antibody against the 12 C-terminal amino acids of SERCA-2b.

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.

Polarized infrared attenuated total reflectance spectroscopy of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca(2+)-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl2, 1-10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing congruent to 7.5 micrograms protein/cm2, and decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH2 vibrations (2923 cm-1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm-1) of the Ca(2+)-ATPase in the Ca2-E1 state and in the EGTA and vanadate stabilized E2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH2 vibrations was not affected by changes in the concentration of KCl (25-100 mM) or Ca2+ (approximately equal to 10(-8)-10(-4) M) and by the addition of vanadate (1 mM) or Pi (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm-1) or CO stretching band (1046 cm-1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of approximately 70 degrees between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca(2+)-ATPase.