Production of specific antibodies to choline acetyltransferase purified from pig brain

Choline acetyltransferase was purified from pig brain and a specific antiserum prepared in a rabbit. The final specific activity after a purification factor of 1,180,000 was 135 μmole × min^?1 × mg prot.^?1 with a yield of about 10%. The purity of this preparation was estimated to be 80% by sodium dodecyl sulphate-gel electrophoresis, the apparent molecular weight of the main band was 67,000. This preparation was used for initial immunization of a rabbit, which subsequently was injected with choline acetyltransferase preparations cut out of the electrophoresis gel. After 4 such additional injections, the rabbit antiserum could precipitate more than 95% of the enzymic activity present in a pig-brain homogenate by either direct or indirect immunoprecipitation. Double immunodiffusion showed that although a precipitation line could be seen with a choline acetyltransferase preparation of high specific activity, no such line was seen using a preparation having 4 times the specific activity of the starting homogenate. This result is in accordance with the prediction (Rossier, 1975) that the amount of choline acetyltransferase present in mammalian tissue extracts is too low to be visualized directly by double immunodiffusion tests.Such a specific antiserum against choline acetyltransferase should be useful as a tool to characterize unambiguously cholinergic neurons, in a way that has not been possible so far.     

Nerve growth factor in mouse and rat serum: correlation between bioassay and radioimmunoassay determinations.

High levels of nerve growth factor (NGF) determined by competition radioimmunoassay do not agree with values obtained by bioassay. This discrepancy is illustrated here with rat and mouse serum as examples in which values up to 1000 ng/ml have been found by using competition radioimmunoassays. An explanation for the discordant results is presented: serum components bind NGF with an intermediate affinity (Kd = 10(-7) M) but with a very large capacity (up to 0.5 mg of NGF per ml of rat serum). The binding of 125I-labeled NGF to serum components competes with the binding to the solid-phase antibodies (Kd = 10(-9)M) present in limiting amounts, according to the principle of competition radioimmunoassays. Thus, less radioactivity is recovered bound to the antibodies and this gives the erroneous impression that NGF is present. To overcome this difficulty, a two-site radioimmunoassay has been developed which utilizes nonlimiting numbers of antibody binding sites. This assay provides a reliable determination of NGF levels in serum and it can be shown that in rat and mouse serum (either sex) there is less than 5 ng of NGF per ml, in agreement with the results of the bioassay.     

A novel 7-transmembrane receptor expressed in nerve growth factor-dependent sensory neurons

This study reports on the full-length cDNA cloning of a gene identified on the basis of its preferential expression in nerve growth factor, compared with neurotrophin-3-dependent neurons. It encodes a putative 7-transmembrane polypeptide that is distantly related to other members of the G protein-coupled receptor superfamily. Unique features of this receptor include a very long carboxy-terminal tail of 360 amino acids and a specific expression pattern in the chick peripheral nervous system, including nerve growth factor-dependent sensory and sympathetic neurons, as well as enteric neurons. In the central nervous system, the receptor is strongly developmentally regulated and is expressed at high levels in the external granule cell layer of the cerebellum, as well as in motoneurons of the spinal cord, and in retinal ganglion cells.