Subcellular localization of nerve growth factor receptors. Thirteen-day chick embryo brain.

Abstract

The subcellular location of the specific binding sites of nerve growth factor (NGF) as judged by binding of the 125I-labeled protein in 13-day chick embryo brain, has been examined. The homogenized tissue was separated into four fractions, P1, P2, P3, and S, by differential centrifugation. Fraction P2, which contained the majority of the specific 125I-NGF binding, was further separated by discontinuous sucrose density gradient centrifugation into three fractions. Fraction B contains many synaptosome-like structures, which, when derived from adult brain, result from the shearing off and resealing of synaptic terminals. This fraction contained 65% of the specific 125I-NGF binding of P2. Following hypoosmotic lysis by water, Fraction B was separated into seven fractions, O, D, E, F, G, H, and I, by discontinuous sucrose density gradient centrifugation. The specific 125I-NGF binding was localized with the denser fractions, G, H, and I, with about a 10-fold purification as compared to the original homogenate. However, only 65% of the binding of Fraction B was found in the sum of the tertiary fractions, indicating that some loss of specific binding accompanied the lysis. By means of marker enzymes and macromolecules, as well as electron microscopy, it was determined that the distribution of cellular components of embryonic tissue in this fractionation technique is very similar to that observed for adult brain tissue. Thus, the properties of the NGF receptors determined in whole brain, which are remarkably similar to those found in peripheral neurons, are the properties of the receptors that appear to be located in the developing synaptosomal structures.