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N D Boyd

Publications and source records attributed to N D Boyd.

52 records · Page 3Linked to original sources
PubMed1983

Analysis of receptor-ligand interactions using nitrocellulose gel transfer: application to Torpedo acetylcholine receptor and alpha-bungarotoxin.

A nitrocellulose-gel transfer technique has been adapted to study the interaction of a polypeptide ligand with individual receptor subunits. The acetylcholine receptor isolated from Torpedo californica has been separated into its subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred in a renaturing environment to nitrocellulose sheets. The sheets were incubated with 125I-alpha-bungarotoxin and autoradiographed. A single receptor polypeptide, the alpha subunit (40K) bound the labeled toxin. This binding was demonstrated to be both saturable and specific, although the affinity of 125I-alpha-bungarotoxin (KD, 165 nM) and the potency of d-tubocurarine to displace this binding (IC50, 1 mM) were both reduced by several orders of magnitude when compared to the native receptor.

Animals↗
PubMed1981

Isolation of peptides from uremic plasma that inhibit phenytoin binding to normal plasma proteins.

The binding of many drugs to plasma proteins is altered in renal disease. Explanations include hypoproteinemia, alterations in the native structure of the binding protein, and competitive or noncompetitive inhibition. The binding of phenytoin to proteins was studied in plasma from patients with chronic renal failure by equilibrium dialysis at 37 degrees. Charcoal adsorption was used to normalize the binding. Substances that appeared to be peptides were isolated; they inhibited the binding of phenytoin to normal plasma proteins. The data suggest that the defect in phenytoin-protein binding in chronic renal failure may be due to competitive or noncompetitive inhibition by peptides.

Adsorption↗
PubMed1981

Phenytoin binding to partially purified albumin in renal disease.

Drug binding to protein is known to be altered in renal disease. Explanations include hypoproteinemia, competitive or noncompetitive inhibition, and basic functional defects in the binding protein. Albumin, the primary binding protein for phenytoin (DPH), was isolated from the plasma of patients with severe renal failure as well as from normal controls. DPH binding was not different between the albumin preparations isolated from the two sources, although some differences were detected in the apparent affinity constant and the number of binding sites. It would appear that the significant defect in DPH binding in renal disease cannot be attributed to a basic functional defect in the drug-binding protein albumin.

Humans↗
PubMed1980

Kinetics of binding of [3H]acetylcholine and [3H]carbamoylcholine to Torpedo postsynaptic membranes: slow conformational transitions of the cholinergic receptor.

The kinetics of binding of [3H]acetylcholine (AcCh) and [3H]carbamoylcholine (Carb) to membrane-bound nicotinic receptor from Torpedo electric tissue have been measured on the second time scale by rapid mixing and ultrafiltration. The concentration dependence of the association kinetics of agonist binding and the kinetics of ligand dissociation and receptor reisomerization following the removal of agonist are analyzed in terms of a model in which the observed binding is by a single population of receptors that exists in the absence of agonist in two interconvertible conformations, one binding agonist weakly (R1) and the other binding with high affinity (R2). A computer simulation has been used to determine values of rate and equilibrium constants characterizing the ligand interactions with the two conformations and for the conformational equilibrium in the presence and absence of agonist. At 4 degrees C, R1/R2 - 4.5, and the half-time for isomerization for low to high affinity of unliganded receptor is equal to 200 s, while for receptors occupied by either AcCh of Carb the half-time is reduced to approximately 4 s. For AcCh the apparent dissociation constants of the low- and high-affinity conformations re 800 nM and 2 nM, respectively (Keq = 8 nM), and for Carb the values are 30 microM and 25 nM (Keq = 100 nM). The dissociation rate constant of [3H]AcCh from R2 is equal to 0.04 s-1. The results are further discussed in terms of alternate less satisfactory reaction models and are compared with the receptor conformational equilibria deduced by the use of other kinetic techniques.

Acetylcholine↗
PubMed1980

Kinetics of binding of [3H]acetylcholine to Torpedo postsynaptic membranes: association and dissociation rate constants by rapid mixing and ultrafiltration.

An automated rapid-mixing ultrafiltration apparatus was constructed to measure at subsecond times the kinetics of binding of radiolabeled cholinergic ligands to nicotinic postsynaptic membranes isolated from Torpedo electric tissue. The dissociation of [3H]AcCh-receptor complexes at 7 and 23 degrees C was characterized by dissociation rate constants (kdissoc) of 0.05 s-1 and 0.15S-1, respectively. The association kinetics at low concentrations of [3H]AcCh were determined at 23 degrees C and found to be consistent with a model in which 20% of the AcCh binding sites preexist in a receptor conformation that binds AcCh with high affinity (KD = 3 nM) and with a bimolecular association constant, k+ = 5.7 x 10(7) M-1 SD-1. Initial studies of the association kinetics at higher AcCh concentrations revealed a transient low-affinity binding step that occurred relatively slowly. In the presence of 0.8 microM [3H]AcCh, this component of the association reaction was characterized by an experimental rate constant of 2 s-1. The observed binding kinetics are discussed with reference to the processes of channel activation and receptor desensitization.

Acetylcholine↗
PubMed1980

Irreversible attachment of immunoglobulins and F(ab')2 fragments to their specific cell membrane antigens.

Antiserum was raised in sheep against rat kidney glomerular basement membrane, and the antibodies and their F(ab')2 fragments were prepared. These were reacted with one arm of the bifunctional reagent toluene diisocyanate at pH 7.5 and injected into the tail vein of rats. Kidney sections were taken from the rats and, after washing, incubated at pH 8.5 to permit the other arm of the toluene diisocyanate to become reactive. This procedure resulted in the specific irreversible attachment of the antibodies and their F(ab')2 fragments to their corresponding glomerular basement membrane antigens.

Animals↗
PubMed1979

Determination of plasma octopamine and its level in renal disease.

A radioenzymatic assay method for the estimation of octopamine levels in plasma was developed. Preparation of the enzyme, phenylethanolamine-N-methyl transferase, dilution of the plasma sample, preparation of a suitable blank, and the assay conditions were found to have a significant effect on the sensitivity of the assay. Plasma octopamine levels were measured in a population of 33 normal individuals ranging in age from 19 to 94 years. Significantly higher plasma octopamine levels were found in the age group 70-90 years. Excluding those individuals over the age of 70 years, no significant differences in plasma octopamine levels were found for males or females, the range of values was 0 to 0.68 ng per ml, with a mean value of 0.23 ng per ml (n = 25). Examination of plasma octopamine levels in patients with severe renal disease requiring hemoperfusion dialysis, revealed a significantly higher level of plasma octopamine in renal disease (1.9 ng per ml), and an increase in plasma octopamine during dialysis; the mean level post dialysis being 2.7 ng per ml.

Adult↗
PubMed1979

Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides.

After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides of apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally intact, as demonstrated by the following criteria. The kinetics of binding of [3H]acetylcholine in the presence and absence of 30 micron carbamoylcholine to occupy acetylcholine binding sites, [14C]-meproadifen [2-(diethylmethylaminoethyl)-2,2-diphenylvalerate iodide ] was bound with a dissociation constant, KD, of 0.3 +/- 0.1 micron to 0.3 +/- 0.1 site per [3H]alpha-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholine-stimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.

Acetylcholine↗
PubMed1976

Influence of age and sex on plasma zinc levels in normal and diabetic individuals.

Plasma zinc levels were measured in both healthy and diabetic individuals having an age range of 10-93 years. No significant differences in plasma zinc concentrations were found between males and females in either healthy or diabetic individuals. Up to the age of 50 years, the mean plasma zinc of normal individuals remained relatively constant at 70 +/- 32 mug/100 ml (+/- 2 SE) after which the levels decreased. This decreasing pattern was absent in diabetics, whose plasma zinc of 65 +/- 32 mug/100 ml remained constant over the entire age range. For women on oral contraceptive agents, the mean plasma zinc was 59 +/- 18 mug/100 ml, which was significantly lower than that of controls.

Adolescent↗
PubMed1975

Effect of carbon cup aging on plasma zinc determination by flameless atomic adosorption spectrometry.

Determination of zinc in blood plasma by flameless atomic absorption spectrometry is discussed, with particular reference to the protocol required for the successful use of the Varian-Techtron Carbon Rod Atomizer. Cup aging is shown to be an important factor in limiting the precision of this analytical technique and ways of minimizing the problem are described. Matrix problems have also been encountered, which precluded the use of aqueous standard curves and the method of standard additions. We propose the use of plasma in preparing standard curves, the values for which are corrected for inherent plasma zinc, as a possible solution to the problem.

Carbon↗