scholarly journals Binding of NAD+ by cholera toxin

1987 ◽  
Vol 244 (1) ◽  
pp. 225-230 ◽  
Author(s):  
T S Galloway ◽  
S van Heyningen
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Cholera Toxin ◽  
Equilibrium Dialysis ◽  
Accurate Determination ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
Competitive Inhibitors ◽  
Dissociation Constant Kd

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

Free cytosolic Ca2+ measured with Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries

1993 ◽  
Vol 265 (2) ◽  
pp. H741-H746 ◽  
Author(s):  
P. E. Jensen ◽  
M. J. Mulvany ◽  
C. Aalkjaer ◽  
H. Nilsson ◽  
H. Yamaguchi
Keyword(s):  
Dissociation Constant ◽  
Isometric Force ◽  
Force Measurements ◽  
Resistance Arteries ◽  
Fura 2 ◽  
Good Accordance ◽  
Dissociation Constant Kd

Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

scholarly journals Detection of surface-bound ligands by freeze-fracture autoradiography.

1985 ◽  
Vol 101 (3) ◽  
pp. 887-890 ◽  
Author(s):  
J L Carpentier ◽  
D Brown ◽  
B Iacopetta ◽  
L Orci
Keyword(s):  
Cholera Toxin ◽  
Accurate Determination ◽  
Freeze Fracture ◽  
Standard Technique ◽  
Coated Pits ◽  
Experimental Conditions ◽  
Surface Distribution ◽  
Surface Localization ◽  
Autoradiographic Technique

This article describes a new freeze-fracture autoradiographic technique for the detection of radioactive ligands associated with the surface of cells in monolayer or suspension culture. Since freeze-fracture replicas are produced in the conventional way, all membrane features normally seen in freeze-fracture are retained, and autoradiographic grains produced by the labeled ligands are seen superimposed on unaltered exoplasmic membrane fracture faces. To assess the feasibility and resolution of this technique, we compared the surface distribution of alpha 2-macroglobulin and cholera toxin, labeled either with 125I or with colloidal gold, on 3T3-L1 fibroblasts. Both by autoradiography and cytochemical gold labeling, alpha 2-macroglobulin was associated specifically with coated pits, whereas cholera toxin was preferentially found over smaller, apparently non-coated membrane invaginations. Together with data on the surface localization of 125I-transferrin on HL-60 myelomonocytic cells, these results demonstrate the application of this technique for the accurate determination of ligand distribution over large areas of plasma membrane. The simplicity and reproducibility of the method should now allow freeze-fracture autoradiography to become a standard technique for investigating the distribution of both endogenous and exogenous cell surface-associated molecules, as well as the redistribution of such molecules under different experimental conditions.

scholarly journals The adenylate cyclase-activating activity of cholera toxin is not associated with a nicotinamide—adenine dinucleotide glycohydrolase activity

1978 ◽  
Vol 174 (3) ◽  
pp. 1059-1062 ◽  
Author(s):  
R M Tait ◽  
S van Heyningen
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Sodium Dodecyl Sulphate ◽  
Nicotinamide Adenine Dinucleotide ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
True Property

The NAD+ glycohydrolase activity of cholera-toxin samples can be separated from their adenylate cyclase-activating activity by polyacrylamide-gel electrophoresis and is inhibited by sodium dodecyl sulphate (which does not inhibit the action of toxin on cells), but not by antibodies to pure toxin. It is therefore probably not a true property of the toxin.

scholarly journals Equilibrium-binding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions

1980 ◽  
Vol 185 (1) ◽  
pp. 107-114 ◽  
Author(s):  
I A Nieduszynski ◽  
J K Sheehan ◽  
C F Phelps ◽  
T E Hardingham ◽  
H Muir
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Dialysis ◽  
Guanidine Hydrochloride ◽  
Dilute Solution ◽  
Binding Studies ◽  
Laryngeal Cartilage ◽  
Equilibrium Binding ◽  
Cartilage Proteoglycans ◽  
Dissociation Constant Kd

The binding of hyaluronate oligosaccharide fractions to proteoglycans from pig laryngeal cartilage has been studied by equilibrium dialysis in dilute solution. It has been shown that: (1) each proteoglycan monomer binds only one hyaluronate oligosaccharide molecule [containing about eighteen saccharide residues (HA approximately 18) and of number-average molecule weight (Mn) 37501]; (2) the dissociation constant, Kd, for interaction between proteoglycan monomer and oligosaccharide HA approximately 18 is 3 × 10(-8) M at 6 degrees C at I 0.15-0.5, pH 7.4; (3) the dissociation constant has little dependence on temperature, so that Kd at 54 degrees C is 3 × 10(-7) M under the same conditions; (4) the aggregatability is high at 6 degrees C, falls significantly at 54 degrees C, but much of it can be recovered on cooling to 6 degrees C again, demonstrating reversible denaturation; (5) a method for determining the proportion of the proteoglycan molecules capable of binding to hyaluronate by equilibrium dialysis was compared with gel-chromatographic and ultracentrifugal methods; (6) a hyaluronate oligosaccharide, HA approximately 56 (Mn 11 000), could bind more than one proteoglycan molecule; (7) consideration of ultracentrifugal data shows that when proteoglycans bind to a hyaluronate of larger size (mol.wt. 670 000), an average Kd of 12 × 10(7) M fits the data in 0.5 M-guanidine hydrochloride at 20 degrees C.

A quantitative approach to inner shell losses

1978 ◽  
Vol 36 (1) ◽  
pp. 526-527 ◽  
Author(s):  
R.D. Leapman ◽  
P. Rez ◽  
D.F. Mayers
Keyword(s):  
Energy Transfer ◽  
Cross Section ◽  
Cross Sections ◽  
Accurate Determination ◽  
Background Intensity ◽  
Core Excitation ◽  
Quantitative Basis ◽  
Cross Section Differential ◽  
Bound Electrons

Microanalysis by EELS has been developing rapidly and though the general form of the spectrum is now understood there is a need to put the technique on a more quantitative basis (1,2). Certain aspects important for microanalysis include: (i) accurate determination of the partial cross sections, σx(α,ΔE) for core excitation when scattering lies inside collection angle a and energy range ΔE above the edge, (ii) behavior of the background intensity due to excitation of less strongly bound electrons, necessary for extrapolation beneath the signal of interest, (iii) departures from the simple hydrogenic K-edge seen in L and M losses, effecting σx and complicating microanalysis. Such problems might be approached empirically but here we describe how computation can elucidate the spectrum shape.The inelastic cross section differential with respect to energy transfer E and momentum transfer q for electrons of energy E0 and velocity v can be written as

Fast calibration of CBED patterns for quantitative analysis

1993 ◽  
Vol 51 ◽  
pp. 674-675
Author(s):  
M.A. Gribelyuk ◽  
M. Rühle
Keyword(s):  
Reciprocal Lattice ◽  
Accurate Determination ◽  
Incident Beam ◽  
Crystal Thickness ◽  
Structure Model ◽  
Beam Direction ◽  
Transmitted Beam ◽  
Reciprocal Vector ◽  
On Line

A new method is suggested for the accurate determination of the incident beam direction K, crystal thickness t and the coordinates of the basic reciprocal lattice vectors V1 and V2 (Fig. 1) of the ZOLZ plans in pixels of the digitized 2-D CBED pattern. For a given structure model and some estimated values Vest and Kest of some point O in the CBED pattern a set of line scans AkBk is chosen so that all the scans are located within CBED disks.The points on line scans AkBk are conjugate to those on A0B0 since they are shifted by the reciprocal vector gk with respect to each other. As many conjugate scans are considered as CBED disks fall into the energy filtered region of the experimental pattern. Electron intensities of the transmitted beam I0 and diffracted beams Igk for all points on conjugate scans are found as a function of crystal thickness t on the basis of the full dynamical calculation.

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