ISOLATION OF FACTOR I

THE inhibitory activity of extracts of brain and spinal cord on the generation of impplses by the stretch -tor neuron of the cray6sh has been reported previously (FLORIN, 1954). The agent (or agents) responsible for this action was called Factor I. In a previous communication in this Journal (ELLIOTT and FUIREY, 1956) the results of other published studies on Factor I were summarized, a method for the assay of Factor I activity was described and the effects of a number of known substances on the stretch receptor preparation were noted. In the study now reported the assay method has been applied in the isolation from bed brain of a substance with high Factor I activity. This substance has been identilied as y-aminobutyric acid. EXPERIMENTAL A s 9 and amffs. Following every step in the purification, samples of the products were held for assay. The assay of Factor I activity was carried out as described by ELuarr and F L ~ R ~ Y (1956). Bridy this method consists in the application of the unltnown makid, freed from organic solvent and dissolved in a buffered saline solution, to the crayfish stretch receptor preparation. Various dilutions of the material arc tested until a concentration, the “minimum blocking concentration,” is reached which just blocks the spontaneous discharges of the stretch receptor neuron for 10 sec. Since the sensitivity of test ~ ~ ~ ~ a r a t i o n s varies, the minimum blocking concentration of a reference solution containing Factor Iis determined before and after each unknown. A singk reference solution was used throughout the present work; samples were taken as requ id from the stock reference solution which was stored in the frozen state. The activity of the unknown solution was thus compared with that of the reference solution and the concentration of Factor I was expressed in ‘c.u.r.’ (crayfish units reference) per ml. A concentration of 1 c.u.r. per mi is about the minimum blocking concentration with many stretch receptor preparations. Table 1 summarizeS the results of the steps in the isolation procedure described below. It should be understood that the objective was to obtain increased specific activity (activity per unit weight of material) rather than maximal yields of activity.