scholarly journals Nonequivalent binding sites in cystathionase. Nanosecond and steady fluorescence studies.

1975 ◽  
Vol 250 (19) ◽  
pp. 7722-7727
Author(s):  
JE Churchich ◽  
T Beeler ◽  
KJ Oh
Keyword(s):  
Binding Sites ◽  
Fluorescence Studies

scholarly journals Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies

1985 ◽  
Vol 229 (3) ◽  
pp. 687-692 ◽  
Author(s):  
F Tabary ◽  
J P Frénoy
Keyword(s):  
Oryza Sativa ◽  
Binding Sites ◽  
Wheat Germ ◽  
Equilibrium Dialysis ◽  
Blue Shift ◽  
Association Constants ◽  
Fluorescence Studies ◽  
Saccharide Binding ◽  
Binding Enthalpy

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M−1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from −12.1 to −20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.

scholarly journals Serological and fluorescence studies of the heat stability of bovine lactoferrin

1979 ◽  
Vol 46 (1) ◽  
pp. 83-93 ◽  
Author(s):  
Augustin Baer ◽  
Marko Oroz ◽  
Bernard Blanc
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Complement Fixation ◽  
Heat Stability ◽  
Heat Denaturation ◽  
Bovine Lactoferrin ◽  
Iron Binding ◽  
Binding Ability ◽  
Fluorescence Studies ◽  
Tryptophan Residues

SUMMARYThe heat denaturation of Fe-saturated lactoferrin (If) and Fe-free lactoferrin (apo-lf) was studied using the methods of micro-complement fixation and fluorescence. It was established that the change in conformation of apo-lf, induced by iron binding, conferred a higher heat stability to the molecule: the changes were observed at temperatures above 40 °C for apo-lf and above 60 °C for If. The Fe-binding ability of the protein was partially independent of the degree of denaturation. Fluorescence analyses indicated that tryptophan residues were probably not directly involved in the metal binding. There was no evidence of antibodies interfering with the binding sites.

scholarly journals Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase

1985 ◽  
Vol 225 (3) ◽  
pp. 573-580 ◽  
Author(s):  
P N B Gibbs ◽  
M G Gore ◽  
P M Jordan
Keyword(s):  
Metal Ions ◽  
Binding Sites ◽  
The Other ◽  
Stopped Flow ◽  
Thiol Groups ◽  
Protein Fluorescence ◽  
Fluorescence Studies ◽  
Nitrobenzoic Acid ◽  
Rate Of Reaction ◽  
Molar Equivalent

The reaction of human 5-aminolaevulinate dehydratase with 5,5′-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.

Fluorescence Studies of Metal-Humic Complexes with the Use of Lanthanide Ion Probe Spectroscopy

1996 ◽  
Vol 50 (3) ◽  
pp. 401-408 ◽  
Author(s):  
J. W. Thomason ◽  
W. Susetyo ◽  
L. A. Carreira
Keyword(s):  
Fluorescence Lifetime ◽  
Metal Binding ◽  
Binding Sites ◽  
Peak Shift ◽  
Binding Energies ◽  
Lanthanide Ion ◽  
Fluorescence Studies ◽  
Ion Probe ◽  
Humic Materials ◽  
Probe Spectroscopy

The acidic functional groups of humic materials are an abundant source of metal binding sites in the natural environment. Studies of metal binding to humics are of great environmental interest because the biological and physicochemical properties of metals are often changed dramatically as a result of complexation with humics. In order to understand how these heterogeneous organic macromolecules bind metals with such a large range of binding energies, lanthanide ion probe spectroscopy (LIPS) has been used to study changes in the fluorescence lifetime of the europium probe metal as it binds to these substances. A method developed by Horrocks and Sudnick for the determination of the number of water molecules bound to Eu3+ was used to calculate the coordination number of humic-bound Eu3+ from the fluorescence data. The peak shift of the Eu3+ hypersensitive emission band (616 nm) was used to calculate the change in charge of the complex. Equations based on Horrocks and Sudnick's method were also developed to calculate the distribution of metal associated with the different types of binding sites on humic substances by computer modeling of the fluorescence lifetime data.

scholarly journals Vertebrate lectins, Comparison of properties of beta-galactoside-binding lectins from tissues of calf and chicken.

1979 ◽  
Vol 81 (3) ◽  
pp. 528-537 ◽  
Author(s):  
E B Briles ◽  
W Gregory ◽  
P Fletcher ◽  
S Kornfeld
Keyword(s):  
Binding Sites ◽  
Gel Electrophoresis ◽  
Cellular Localization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thiol Group ◽  
Cross Reactivity ◽  
Cytoplasmic Localization ◽  
Binding Properties ◽  
Fluorescence Studies ◽  
Species Specific

Beta-galactoside-binding lectins were isolated from various calf tissues and from chicken hearts by affinity chromatography on asialofetuin-Sepharose, and were compared with respect to biochemical characteristics, binding properties, antigenic cross-reactivity, and cellular localization. The lectins are all thiol group-requiring, divalent cation-independent dimers, of apparent monomer mol wt 12,000 (calf lectins) or 13,000 (chicken lectin), and acidic pI. The calf lectins appear essentially identical by dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid composition, and radioimmunoassay, while the chicken lectin is distinctly different by these criteria. However, all of the lectins competed for the same binding sites on rabbit erythrocytes, and could be inhibited by the same saccharide haptens (notably lactose and thiodigalactoside). Immuno-fluorescence studies on several cultured cell lines revealed that the bovine and chicken lectins had primarily an intracellular cytoplasmic localization. The beta-galactoside-binding lectins of vertebrates appear to be species-specific rather than tissue-specific.

scholarly journals Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315

1976 ◽  
Vol 155 (1) ◽  
pp. 37-53 ◽  
Author(s):  
R A Dwek ◽  
D Givol ◽  
R Jones ◽  
A C McLaughlin ◽  
S Wain-Hobson ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Metal Binding ◽  
Spin Label ◽  
Binding Sites ◽  
Binding Constants ◽  
Kinetic Scheme ◽  
Difference Spectrum ◽  
Protein Fluorescence ◽  
Myeloma Protein ◽  
Fluorescence Studies

1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a ‘slow’ conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Electron Probe Analysis of Muscle

1982 ◽  
Vol 40 ◽  
pp. 398-401
Author(s):  
A. V. Somlyo ◽  
H. Shuman ◽  
A. P. Somlyo
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Resting State ◽  
Binding Sites ◽  
Electron Probe ◽  
Frog Skeletal Muscle ◽  
Electron Probe Analysis ◽  
Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

Sign in / Sign up

Export Citation Format

Share Document