'Integrin alpha IIb beta 3 or glycoprotein IIb/IIIa (GPIIb/IIIa, 228 kDa) is a Ca(2+)-dependent, noncovalent heterodimer of glycoproteins IIb (GPIIb or alpha IIb, 136 kDa) and IIIa (GPIIIa or beta 3, 92 kDa), which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. ... More
Effects of membrane thickness on the molecular dynamics and enzymatic activity of reconstituted Ca-ATPase.
AuthorsCornea RL, Thomas DD
JournalBiochemistry
PubMed ID8130205
'We have studied the effect of phospholipid chain length on the activity and molecular dynamics of reconstituted Ca-ATPase from skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence anisotropy (TPA) and electron paramagnetic resonance (EPR). We used reconstituted Ca-ATPase in exogenous phosphatidylcholines with monounsaturated chains 14-24 carbons long, to determine their effects ... More
Mechanism of Ca-ATPase inhibition by melittin in skeletal sarcoplasmic reticulum.
AuthorsVoss JC, Mahaney JE, Thomas DD
JournalBiochemistry
PubMed ID7827051
'We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and ... More
Rotational diffusion of human lipoproteins and their receptors as determined by time-resolved phosphorescence anisotropy.
AuthorsTilley L, Sawyer WH, Morrison JR, Fidge NH
JournalJ Biol Chem
PubMed ID2846567
'Time-resolved phosphorescence anisotropy has been used to assess the rotational dynamics of human serum lipoproteins labeled with phosphorescent probes of high triplet yield. Labeling the lipid phase of low density, very low density, and high density lipoproteins with an eosinyl fatty acid revealed the existence of two motions. The shorter ... More
Estimation of the distance change between cysteine-457 and the nucleotide binding site when sodium pump changes conformation from E1 to E2 by fluorescence energy transfer measurements.
AuthorsLin SH, Faller LD
JournalBiochemistry
PubMed ID8679600
'The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the alpha-subunit. The donor (5''-(iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2''(or 3'')-O-(trinitrophenyl)adenosine 5''-triphosphate) ... More
Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy.
AuthorsVoss J, Birmachu W, Hussey DM, Thomas DD
JournalBiochemistry
PubMed ID1649630
'We have studied the effect of melittin, a basic membrane-binding peptide, on Ca-ATPase activity and on protein and lipid dynamics in skeletal sarcoplasmic reticulum (SR), using time-resolved phosphorescence and fluorescence spectroscopy. Melittin completely inhibits Ca-ATPase activity, with half-maximal inhibition at 9 +/- 1 mol of melittin bound to the membrane ... More
Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.
AuthorsNegash S, Chen LT, Bigelow DJ, Squier TC
JournalBiochemistry
PubMed ID8784178
'We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the ... More
Thermotropic and dynamic characterization of interactions of acylated alpha-bungarotoxin with phospholipid bilayer membranes.
AuthorsBabbitt B, Huang L, Freire E
JournalBiochemistry
PubMed ID6091732
'The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the ... More
Anesthetics alter the physical and functional properties of the Ca-ATPase in cardiac sarcoplasmic reticulum.
AuthorsKaron BS, Geddis LM, Kutchai H, Thomas DD
JournalBiophys J
PubMed ID7756557
'We have studied the effects of the local anesthetic lidocaine, and the general anesthetic halothane, on the function and oligomeric state of the CA-ATPase in cardiac sarcoplasmic reticulum (SR). Oligomeric changes were detected by time-resolved phosphorescence anisotropy (TPA). Lidocaine inhibited and aggregated the Ca-ATPase in cardiac SR. Micromolar calcium or ... More
Phospholamban-dependent effects of C12E8 on calcium transport and molecular dynamics in cardiac sarcoplasmic reticulum.
AuthorsShi Y, Karon BS, Kutchai H, Thomas DD
JournalBiochemistry
PubMed ID8873607
'We have studied the effects of the nonionic detergent C12E8 on Ca-ATPase enzymatic activity and oligomeric state (detected by time-resolved phosphorescence anisotropy, TPA) in skeletal and cardiac sarcoplasmic reticulum (SR). In skeletal, SR, C12E8 inhibits the CA-ATPase, both at high (micromolar and above) and low (submicromolar) Ca. In cardiac SR, ... More
Protein rotation, enzyme activity and photooxidation of SH groups in sarcoplasmic reticulum Ca2+-ATPase.
AuthorsRestall CJ, Arrondo JL, Elliot DA, Jaskowska A, Weber WV, Chapman D
JournalBiochim Biophys Acta
PubMed ID6457648
'The binding of probe molecules such as fluorescein isothiocyanate, eosin isothiocyanate and erythrosin isothiocyanate to the Ca2+-ATPase of sarcoplasmic reticulum followed by illumination of the labelled protein causes substantial reductions of ATPase activity over a 1-h period. The degree of light-sensitivity induced by these probes is related to the triplet ... More
Rotational diffusion of epidermal growth factor complexed to cell surface receptors reflects rapid microaggregation and endocytosis of occupied receptors.
AuthorsZidovetzki R, Yarden Y, Schlessinger J, Jovin TM
JournalProc Natl Acad Sci U S A
PubMed ID6273899
'The rotational diffusion of epidermal growth factor (EGF)--receptor complexes on living human epidermoid carcinoma cells (A-431) has been measured by phosphorescence emission and anisotropy in the mu s time domain. A biologically active phosphorescent conjugate of EGF, erythrosin-EGF, was applied to living cells. The hormone--receptor complexes were mobile with rotational ... More
On the relationship between the mitochondrial inner membrane anion channel and the adenine nucleotide translocase.
AuthorsPowers MF, Smith LL, Beavis AD
JournalJ Biol Chem
PubMed ID7511601
'The mitochondrial inner membrane anion channel (IMAC) is a transport pathway which is believed to be involved in mitochondrial volume homeostasis. The protein, however, has not been identified. In this paper, we examine the relationship between IMAC and the adenine nucleotide translocator. Many inhibitors of the adenine nucleotide translocase are ... More
Rotational diffusion of biological macromolecules by time-resolved delayed luminescence (phosphorescence, fluorescence) anisotropy.
AuthorsJovin TM, Bartholdi M, Vaz WL, Austin RH
JournalAnn N Y Acad Sci
PubMed ID6942745
Multiple spectral parameter imaging.
AuthorsWaggoner A, DeBiasio R, Conrad P, Bright GR, Ernst L, Ryan K, Nederlof M, Taylor D
JournalMethods Cell Biol
PubMed ID2648118
Rotational mobility of the membrane-bound acetylcholine receptor of Torpedo electric organ measured by phosphorescence depolarisation.
AuthorsLo MM, Garland PB, Lamprecht J, Barnard EA
JournalFEBS Lett
PubMed ID7358182
Regulatory volume decrease in HL-60 cells: importance of rapid changes in permeability of Cl- and organic solutes.
AuthorsHallows KR, Knauf PA
JournalAm J Physiol
PubMed ID7943267
'Results obtained through the use of inhibitors and isotope flux and equilibration techniques indicate that the regulatory volume decrease (RVD) response of human promyelocytic leukemic HL-60 cells occurs largely through the efflux of K+ and Cl- through separate conductive membrane pathways. These "channels" differ pharmacologically and in their modes of ... More
Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility.
AuthorsGarland PB, Moore CH
JournalBiochem J
PubMed ID94265
'We used a pulsed dye laser working at 540 nm to excite triplet-state formation of eosin and erythrosin, either bound or unbound to bovine serum albumin, in aqueous solution anaerobically at pH 8 and 20-22 degrees C. Delayed emission from radiative transitions of the triplet state was readily detectable, both ... More
Erythrosin isothiocyanate selectively labels lysine464 within an ATP-protectable binding site on the Ca-ATPase in skeletal sarcoplasmic reticulum membranes.
AuthorsHuang S, Negash S, Squier TC
JournalBiochemistry
PubMed ID9578581
'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 ... More
Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.
'The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different ... More
Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.
AuthorsMcPherson RA, Sawyer WH, Tilley L
JournalBiochemistry
PubMed ID1370629
'Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 ... More
Synthesis of erythrosin isothiocyanate and its use as a phosphorescent depolarization probe for slow rotational mobility of membrane proteins [proceedings]
AuthorsMoore CH, Garland PB
JournalBiochem Soc Trans
PubMed ID510751
Phosphorescence depolarization and the measurement of rotational motion of proteins in membranes.
AuthorsMoore C, Boxer D, Garland P
JournalFEBS Lett
PubMed ID160330
Monitoring membrane protein rotational diffusion using time-averaged phosphorescence.
AuthorsMurray EK, Restall CJ, Chapman D
JournalBiochim Biophys Acta
PubMed ID6871203
Rotational motions of membrane proteins have previously been measured using time-dependent phosphorescence techniques. This paper discusses a method of examining membrane protein mobility at temperatures relevant to biological systems, using a technique similar to steady-state fluorescence. The method is demonstrated using sarcoplasmic reticulum ATPase labelled with erythrosin isothiocyanate, both in ... More
The physical mechanism of calcium pump regulation in the heart.
AuthorsVoss J, Jones LR, Thomas DD
JournalBiophys J
PubMed ID7918987
The Ca-ATPase in the cardiac sarcoplasmic reticulum membrane is regulated by an amphipathic transmembrane protein, phospholamban. We have used time-resolved phosphorescence anisotropy to detect the microsecond rotational dynamics, and thereby the self-association, of the Ca-ATPase as a function of phospholamban phosphorylation and physiologically relevant calcium levels. The phosphorylation of phospholamban ... More
Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes.
AuthorsLi Y, Glazer AN
JournalBioconjug Chem
PubMed ID10077473
A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a ... More
Major histocompatibility complex independent T cell receptor-antigen interaction: functional analysis using fluorescein derivatives.
We have isolated T cell receptor (TCR) cDNAs from fluorescein (FL)-specific human T cell clones (alpha FL beta FL), and transferred them to TCR beta- Jurkat cells in order to study direct FL-binding to the TCR. Using either FL-conjugated polymers (FL-polymer) or FL-substituted Sepharose beads, we are able to demonstrate ... More
Rotational mobility of high-affinity epidermal growth factor receptors on the surface of living A431 cells.
AuthorsZidovetzki R, Johnson DA, Arndt-Jovin DJ, Jovin TM
JournalBiochemistry
PubMed ID1647816
The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated ... More
Preformed oligomeric epidermal growth factor receptors undergo an ectodomain structure change during signaling.
AuthorsMartin-Fernandez M, Clarke DT, Tobin MJ, Jones SV, Jones GR
JournalBiophys J
PubMed ID11964230
Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, ... More
A homogeneous immunofluorescence assay based on dye-sensitized photobleaching.
AuthorsBystryak S, Goldiner I, Niv A, Nasser AM, Goldstein L
JournalAnal Biochem
PubMed ID7778763
A novel homogeneous immunoassay requiring only one incubation step, and applicable in principle to the determination of low- as well as high-molecular-weight substances, has been developed. The method is based on (a) photooxidation by singlet oxygen (1O2) of a fluorescent substrate (1,3-diphenylisobenzofuran, DPBF) embedded in unilamellar vesicles on the surface ... More
Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells.
AuthorsZidovetzki R, Bartholdi M, Arndt-Jovin D, Jovin TM
JournalBiochemistry
PubMed ID2428396
The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not ... More
Halothane and cyclopiazonic acid modulate Ca-ATPase oligomeric state and function in sarcoplasmic reticulum.
AuthorsKaron BS, Mahaney JE, Thomas DD
JournalBiochemistry
PubMed ID7947799
We have studied the effects of cyclopiazonic acid (CPA) and halothane on the enzymatic activity, oligomeric state, and conformational equilibrium of the Ca-ATPase in skeletal muscle sarcoplasmic reticulum (SR). CPA is a potent inhibitor of Ca-ATPase activity, and this inhibition is competitive with respect to ATP concentration. Time-resolved phosphorescence anisotropy ... More
Triplet-state detection of labeled proteins using fluorescence recovery spectroscopy.
AuthorsCorin AF, Blatt E, Jovin TM
JournalBiochemistry
PubMed ID2441744
The experimental procedures for detecting the triplet states of chromophores in solutions (cuvettes) by fluorescence recovery spectroscopy (FRS) are described in detail, together with applications in studies of protein structure and protein-cell interactions in the microsecond to millisecond time domain. The experimental configuration has been characterized by measuring the emission ... More
Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer.
AuthorsAmler E, Abbott A, Ball WJ
JournalBiophys J
PubMed ID1312368
The oligomeric nature of the purified lamb kidney Na+,K(+)-ATPase was investigated by measuring the fluorescence energy transfer between catalytic (alpha) subunits following sequential labeling with fluorescein 5'-isothiocyanate (FITC) and erythrosin 5'-isothiocyanate (ErITC). Although these two probes had different spectral responses upon reaction with the enzyme, our studies suggest that a ... More
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.
AuthorsLinnertz H, Urbanova P, Obsil T, Herman P, Amler E, Schoner W
JournalJ Biol Chem
PubMed ID9786881
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 ... More
An autoinhibitory peptide from the erythrocyte Ca-ATPase aggregates and inhibits both muscle Ca-ATPase isoforms.
AuthorsReddy LG, Shi Y, Kutchai H, Filoteo AG, Penniston JT, Thomas DD
JournalBiophys J
PubMed ID10354431
We have studied the effects of C28R2, a basic peptide derived from the autoinhibitory domain of the plasma membrane Ca-ATPase, on enzyme activity, oligomeric state, and E1-E2 conformational equilibrium of the Ca-ATPase from skeletal and cardiac sarcoplasmic reticulum (SR). Time-resolved phosphorescence anisotropy (TPA) was used to determine changes in the ... More
Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme.
AuthorsMartin DW, Sachs JR
JournalJ Biol Chem
PubMed ID10831595
The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity ... More
Molecular mechanism of Ca-ATPase activation by halothane in sarcoplasmic reticulum.
AuthorsKaron BS, Thomas DD
JournalBiochemistry
PubMed ID8393342
We have studied the molecular mechanism of Ca-ATPase activation in sarcoplasmic reticulum (SR) by the volatile anesthetic halothane. Using time-resolved phosphorescence anisotropy, we determined the rotational correlation times and mole fractions of different oligomeric states of the enzyme, as a function of halothane and temperature. Lipid fluidity was measured independently, ... More
Rotational dynamics of the Fc epsilon receptor on mast cells monitored by specific monoclonal antibodies and IgE.
AuthorsPecht I, Ortega E, Jovin TM
JournalBiochemistry
PubMed ID1849426
The rotational motions of the type I receptor for the Fc epsilon domains (Fc epsilon RI) present on mast cells were investigated by measuring the phosphorescence emission and anisotropy decay kinetics of erythrosin (Er) covalently bound to several Fc epsilon RI-specific macromolecular ligands. The latter consisted of three murine monoclonal ... More
Interleukin 1 (IL-1) causes changes in lateral and rotational mobilities of IL-1 type I receptors.
AuthorsGuo C, Georgiadis KE, Dower SK, Holowka D, Baird BA
JournalBiochemistry
PubMed ID9931029
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 ... More
The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.
AuthorsAmler E, Abbott A, Malak H, Lakowicz J, Ball WJ
JournalBiophys J
PubMed ID8770197
The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence ... More
Rotational mobility of Sendai virus glycoproteins in membranes of fused human erythrocytes and in the envelopes of cell-bound virions.
AuthorsAroeti B, Jovin TM, Henis YI
JournalBiochemistry
PubMed ID2176844
The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in ... More
Conformation of human fibrinogen in solution from polarized triplet spectroscopy.
AuthorsMontejo JM, Naqvi KR, Lillo MP, González-Rodríguez J, Acuña AU
JournalBiochemistry
PubMed ID1324718
The rotational motions of human fibrinogen in solution at 20 degrees C have been examined, in the 0.2-12-microseconds time range, by measuring the laser-induced dichroism of the triplet state of an erythrosin probe covalently bonded to the protein. The decay of the anisotropy was multiexponential, and up to three correlation ... More
Dynamics of molecules involved in antigen presentation: effects of fixation.
AuthorsBarisas BG, Wade WF, Jovin TM, Arndt-Jovin D, Roess DA
JournalMol Immunol
PubMed ID10593509
Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen ... More
Differential effects of general anesthetics on the quaternary structure of the Ca-ATPases of cardiac and skeletal sarcoplasmic reticulum.
AuthorsKutchai H, Geddis LM, Jones LR, Thomas DD
JournalBiochemistry
PubMed ID9485389
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 ... More
Rearrangement of domain elements of the Ca-ATPase in cardiac sarcoplasmic reticulum membranes upon phospholamban phosphorylation.
AuthorsNegash S, Huang S, Squier TC
JournalBiochemistry
PubMed ID10387060
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 ... More
Lipid phase separations induced by the association of cholera toxin to phospholipid membranes containing ganglioside GM1.
AuthorsGoins B, Freire E
JournalBiochemistry
PubMed ID3839133
The interactions of cholera toxin and their isolated binding and active subunits with phospholipid bilayers containing the toxin receptor ganglioside GM1 have been studied by using high-sensitivity differential scanning calorimetry and steady-state and time-resolved fluorescence and phosphorescence spectroscopy. The results of this investigation indicate that cholera toxin associates with phospholipid ... More
Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.
AuthorsVoss JC, Mahaney JE, Jones LR, Thomas DD
JournalBiophys J
PubMed ID7612820
We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which ... More
Self-association of class I major histocompatibility complex molecules in liposome and cell surface membranes.
AuthorsChakrabarti A, Matko J, Rahman NA, Barisas BG, Edidin M
JournalBiochemistry
PubMed ID1322696
Fluorescent derivatives of a human MHC class I glycoprotein, HLA-A2, were reconstituted into dimyristoylphosphatidylcholine (DMPC) liposomes. Measurements of lateral diffusion of fluorescein-(Fl-) labeled HLA-A2 by fluorescence photobleaching recovery (FPR), of rotational diffusion of erythrosin-(Er-) labeled HLA-A2 by time-resolved phosphorescence anisotropy (TPA), and of molecular proximity by flow cytometric fluorescence resonance ... More
Enhanced rotational dynamics of the phosphorylation domain of the Ca-ATPase upon calcium activation.
AuthorsHuang S, Squier TC
JournalBiochemistry
PubMed ID9922175
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 ... More
Lysophosphatidylcholine modulates catalytically important motions of the Ca-ATPase phosphorylation domain.
AuthorsHunter GW, Bigelow DJ, Squier TC
JournalBiochemistry
PubMed ID10194382
Catalytically important motions of the Ca-ATPase, modulated by the physical properties of surrounding membrane phospholipids, have been suggested to be rate-limiting under physiological conditions. To identify the nature of the structural coupling between the Ca-ATPase and membrane phospholipids, we have investigated the functional and structural effects resulting from the incorporation ... More