Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.
 ...
PMID:Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance. 164 24

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 per mole of ATPase (0.1 mol of melittin per mole of lipid). The time-resolved phosphorescence anisotropy (TPA) decay of the Ca-ATPase labeled with erythrosin isothiocyanate (ERITC) shows that melittin restricts microsecond protein rotational motion. At 25 degrees C in the absence of melittin, the TPA is characterized by three decay components, corresponding to a rapid segmental motion (correlation time phi 1 = 2-3 microseconds), the uniaxial rotation of monomers or dimers (phi 2 = 16-22 microseconds), and the uniaxial rotation of larger oligomers (phi 3 = 90-140 microseconds). The effect of melittin is primarily to decrease the fraction of the more mobile monomer/dimer species (A2) while increasing the fractions of the larger oligomer (A3) and very large aggregates (A infinity). Time-resolved fluorescence anisotropy of the lipid-soluble probe diphenylhexatriene (DPH) shows only a slight increase in the lipid hydrocarbon chain effective order parameter, corresponding to an increase in lipid viscosity that is too small to account for the large decrease in protein mobility or inhibition of Ca-ATPase activity. Thus the inhibitory effect of melittin correlates with its capacity to aggregate the Ca-ATPase and is consistent with previously reported inhibition of this enzyme under conditions that increase protein-protein interactions.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy. 164 30

An alanine racemase, encoded by the alr (dal) gene and believed to be the biosynthetic source of D-alanine for cell wall formation, was purified to homogeneity from an overproducing strain of Salmonella typhimurium (dadB), and the enzymological properties of this enzyme were compared with those of the dadB alanine racemase that functions in the catabolism of L-alanine [Wasserman, S. A., Daub, E., Grisafi, P., Botstein, D., & Walsh, C. T. (1984) Biochemistry 23, 5182]. The alr-encoded enzyme has a monomeric structure with a molecular weight of about 40 000. One mole of pyridoxal 5'-phosphate is bound per mole of enzyme, which is essential for catalytic activity of the enzyme. After the internal Schiff base with pyridoxal 5'-phosphate was reduced with NaB3H4, followed by carboxamidomethylation and tryptic digestion of the enzyme, the amino acid sequence of the pyridoxal 5'-phosphate binding peptide was determined. The sequence of 10 amino acid residues around the lysine residue, to which pyridoxal 5'-phosphate is bound, was identical with that of the dadB racemase. No homology was found in the amino-terminal amino acid sequence between the two enzymes. The enzyme was inactivated with D- and L-beta-fluoroalanine, D- and L-beta-chloroalanine, and D-O-acetylserine in a mechanism-based fashion with a common partition ratio of about 150. The enzyme was labeled with an equimolar amount of [14C]-D-beta-chloroalanine. The inactivator-pyridoxal 5'-phosphate adduct was isolated and shown to be the same structure formed in the dadB racemase inactivation [Roise, D., Soda, K., Yagi, T., & Walsh, C. (1984) Biochemistry 23, 5195].
 ...
PMID:Biosynthetic alanine racemase of Salmonella typhimurium: purification and characterization of the enzyme encoded by the alr gene. 352 77

The conditions of nucleotide binding to native, though partly purified, Ca(2+)-ATPase from SR as well as the stoichiometry of nucleotide and strontium binding and the phosphorylation capacity was reevaluated. Binding of MgADP appeared to be aberrant whereas even high-affinity binding of [14C]-ADP took place in the absence of Mg2+. Also low-affinity ATP binding was possible in the absence of divalent cations. A heterogeneity in ADP binding compatible with a two-component model in the absence of thapsigargin was changed to an apparent homogeneity of low-affinity receptors following a mole:mole interaction of enzyme and thapsigargin. Since the affinity of both components was reduced by thapsigargin, high- as well as low-affinity ADP binding seem to be specific and probably to the substrate receptor proper. Analysis of ADP binding isotherms in the absence of Mg2+ according to a model of two independent populations of sites was compatible with a binding capacity of 8.49 +/- 0.43 nmoles/mg protein corresponding to a molecular mass of 118 +/- 6 kD per ADP site. The same total binding capacity was found for ATP. The phosphorylation capacity corresponded to more than one and less than two approximately P per two 110-kD peptides (formally one approximately P per 154 kD protein). Specific binding of Ca2+ and the congener Sr2+ to SR Ca(2+)-ATPase was compatible with their interaction with a single population of sites. The binding capacity was equal to one divalent cation per nucleotide binding peptide. The binding of one nucleotide and one divalent cation per approximately 110 kD peptide and the absence of cooperativity in divalent cation binding might imply that Ca(2+)-ATPase works as a monomer.
 ...
PMID:Binding of ADP to sarcoplasmic reticulum Ca(2+)-ATPase in the absence of Mg2+ is specifically inhibited by thapsigargin: observations on the ligand stoichiometry. 874 53

Artery wall binding peptide (AWBP; Cys-Gly-Arg-Ala-Leu-Val-Asp-Thr-Leu-Lys-Phe-Val-Thr-Gln-Ala-Glu-Gly-Ala-Lys), a specific targeting peptide, was conjugated to poly(ethylene glycol)-grafted-poly(L-lysine) (PEG-g-PLL) to enhance the gene transfer to artery wall cells. AWBP-PEG-PLL was synthesized by the reaction between the vinylsulfone group of PEG-g-PLL and the thiol group of cysteine in AWBP. 1H-NMR analysis confirmed the composition of the obtained polymer and indicated that four mol of AWBP were reacted to one mole of VS-PEG-PLL. The particles of AWBP-PEG-PLL/pDNA complexes were determined spherical with a size of approximately 100 nm by dynamic light scattering (DLS) and atomic force microscopy (AFM). Agarose gel retardation assay indicated that AWBP-PEG-PLL was able to condense plasmid DNA and reach complete complexation at and above a charge ratio 1/1 (+/-). Transfection efficiency of AWBP-PEG-PLL/pDNA complexes was 150-180 times higher than that of control systems, such as PEG-g-PLL/pDNA and PLL/pDNA, in both bovine aorta endothelial cells and smooth muscle cells. Luciferase activities of AWBP-PEG-PLL depended on the amount of free AWBP, while those of the control carriers such as PLL and PEG-g-PLL were not affected by free AWBP. These results supported that gene transfer of AWBP-PEG-PLL/pDNA complexes to bovine aorta wall cells was mediated by specific artery wall cell receptor-mediated endocytosis.
 ...
PMID:Artery wall binding peptide-poly(ethylene glycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells. 1177 68

We have used the orthogonal carbodiimide condensation and copper-catalyzed azide-alkyne "click" cycloaddition (CuAAC) reactions to prepare self-assembled monolayers that present distinct peptides to stem cells in a bioinert background. The approach involved first forming mixed SAMs with three components: (i) an azide-terminated hexaethylene glycol alkanethiolate (HS-EG6-N3), (ii) a carboxylate-terminated hexaethylene glycol alkanethiolate (HS-EG6-COOH), and (iii) a triethylene glycol alkanethiolate (HS-EG3). An acetylene-bearing peptide and an amine-terminated peptide were then immobilized to these substrates using a "click" CuAAC reaction and a carbodiimide condensation reaction, respectively. Polarization-modulated infrared reflectance-absorbance spectroscopic analysis demonstrated formation of well-ordered, close-packed self-assembled monolayers (SAMs), chemoselective conjugation of amine-terminated peptides to surface carboxylate groups, and subsequent conjugation of acetylene-terminated peptides to the azide groups on SAMs. Varying the mole fraction of HS-EG6-N3, HS-EG6-COOH, and HS-EG3 during SAM formation allowed for control over the densities of each peptide on the substrate. Substrates presenting varying surface densities of RGESP (a nonfunctional peptide), RGDSP (a cell adhesion peptide), or TYRSRKY (a heparin/heparan sulfate-binding peptide) were then used to characterize the relationship between peptide surface density and human mesenchymal stem cell (hMSC) adhesion. Results demonstrate that RGESP does not influence RGDSP-mediated adhesion of hMSCs, which indicates that a second peptide with distinct bioactivity can be immobilized alongside RGDSP to characterize the influence of two peptides on hMSC behavior. Our results also demonstrate that RGDSP and TYRSRKY act synergistically to promote hMSC adhesion in the absence of serum. Interestingly, heparin sequestered by TYRSRKY inhibits cell adhesion on substrates presenting RGDSP = 0.1% and > or = 0.1% TYRSRKY or RGDSP = 1% and > or = 0.5% TYRSRKY. Taken together, these results indicate that two peptides can be controllably presented to stem cells on the same otherwise bioinert SAM substrate, and that multiple, distinct extracellular moieties act in concert to regulate hMSC adhesion.
 ...
PMID:Immobilization of peptides with distinct biological activities onto stem cell culture substrates using orthogonal chemistries. 2035 53