Protein synthesis in the isolated giant axon of the squid.

The work of Weiss and his associates,1-3 and more recently of a number of other investigators,4has established the occurrence of a flux of materials from the soma of neurons toward the peripheral regions of the axon. It has been postulated that this mechanism would account for the origin of most of the axonal protein, although the time required to cover the distance which separates some axonal tips from their cell bodies would impose severe delays.4 On the other hand, a number of observations7-9 have indicated the occurrence of local mechanisms of synthesis in peripheral axons, as suggested by the kinetics of appearance of individual proteins after axonal transection. In this paper we report the incorporation of radioactive amino acids into the protein fraction of the axoplasm and of the axonal envelope obtained from giant axons of the squid. These axons are isolated essentially free from small fibers and connective tissue, and pure samples of axoplasm may be obtained by extrusion of the axon. Incorporation of amino acids into axonal protein has recently been reported using systems from mammals'0 and fish."I Materials and Methods.-Giant axons of Loligo pealii were dissected and freed from small fibers: they were tied at both ends. Incubations were carried out at 18-20° in sea water previously filtered through Millipore which contained 5 mM Tris pH 7.8 and 10 Muc/ml of a mixture of 15 C'4-labeled amino acids (New England Nuclear Co., Boston, Mass.). At the end of the incubation, the axons were washed with water, blotted on tissue paper, and the axoplasm was extruded after cutting open one end. Homogenization was carried out with 0.5 ml H20 in a glass homogenizer and was repeated after addition of an equal volume of 10% trichloroacetic acid (TCA). After standing in the cold for at least 10 min, the precipitate was collected by centrifugatiQn and washed with 1 ml of 5% TCA. Supernatants were combined, diluted five times with H20, and plated on copper planchets provided with concentric rings. Under these conditions the quenching of the radioactivity was 43%. The precipitate was treated with 1-2 ml of N NaOH at 500 for 5 minl, any undissolved material being discarded by centrifugation. An aliquot was taken for protein determination with the method of Lowry,'2 and the remaining solution was reprecipitated with 0.6-1.2 ml of 50% TCA, heated at 900 for 15 min, left in the cold for at least 60 min, and filtered through Millipore with an excess of 5% TCA containing 1 mg/ml of a mixture of amino acids (Casamino acids, Difco). Radioactivity was measured in a gas-flow counter with a background of 5 cpm. Results and Discussion.-Incubation of isolated giant axons of squid in buffered sea water containing a mixture of C'4-labeled amino acids resulted in uptake of radioactivity in the TCA-soluble and insoluble fractions of the axoplasm and of the axonal envelope. Entrance of radioactive amino acids in the soluble pool of the axoplasm continued for several hours (Fig. 1). The rate of the process was linear during the first period of incubation, but appeared to decrease later. A saturation level was not reached, however, even after six to seven hours. A significant incorporation of radioactivity occurred also in the protein fraction of the axoplasm with a rate which remained approximately linear for several hours.