Maximum levels of alpha-bungarotoxin binding to human foetal brain membranes remained constant between gestational ages of 10-24 weeks at 30-50 gmol/mg protein (1.1-1.5 p mol/g wet weight in whole brain). Subcellular fractionation of whole brain homogenate showed an enrichment of alpha-bungarotoxin binding in the crude mitochondrial fraction and the subsequent nerve ending sub-fraction. The high proportion of occluded LDH in the nerve ending fraction was indicative of the presence of synaptosomes. Analysis of the fraction at the electron microscope level revealed the existence of synaptosome-like profiles containing mitochondria and very few synaptic vesicles. The specific binding of alpha-bungarotoxin to membranes and crude detergent extracts was shown to be saturable with KD (app) of 3.5 x 10-9 and 2.4 x 10-9M respectively. Scatchard and Hill analysis revealed the presence of a single class of sites exhibiting no cooperativity. Association rate constants, determined by measuring the binding of alpha-bungarotoxin with time to membranes and crude detergent extracts were 2.3 x 105M-1 sec-1 and 2.6 x 105M-1 sec-1 respectively. Dissociation of alpha-bungarotoxin from membranes and crude detergent extract was rapid with a T1/2 in the order of 12 min. and off rate constants of 9.2 x 10-4 sec-1 and 9.8 x 10-4 sec -1 respectively. The intrinsic dissociation constant (KD) was calculated from the on and off rate constants to be 4.0 x 10-9M and 3.3 x 10-9M for membranes and crude detergent extracts respectively. Competition studies with several cholinergic ligands indicated that the alpha-bungarotoxin binding sites displayed a predominantly nicotinic pharmacology. Unlabelled a-BgTx, nicotine and d-tubocurarine were found to be the most effective inhibitors of [125I]-alpha-bungarotoxin binding. A histological study employing autoradiographic techniques showed that [125I] -alpha-bungarotoxin binding was concentrated primarily in the dorsal horn of the spinal cord of a 14 weekold foetus. Clusters of silver grains above the background density were also observed in the ventral horn. Silver grain densities above background were abolished by pre-treatment of the sections with unlabelled alpha-bungarotoxin.A sample of myasthenic plasma and the subsequently prepared immunoglobulin fraction containing antibodies directed against the nAChR from human skeletal muscle were able to precipitate a low but measurable amount of the alpha-bungarotoxin binding sites in human foetal brain. Samples of serum in which an anti-(muscle nAChR) titre was absent failed to precipitate the brain component.
|Date of Award||1985|