The nature of polysomes isolated from spleen cells of rats stimulated by antigen.

Kern an(d co-workers1 were the first to examine the molecular mechanism of imnmunoglobulin synthesis and concluded that this process is similar to the synthesis of other proteins. Recently, lymphoid tissues have been studied by more refined electron microscopic2' and biochemical techniques4-6 in an effort to examine details of the mechanism of synthesis of antibody. The data presented in many of these studies are difficult to reconcile with present models of polypeptide synthesis on polysomes. The current hypothesis is that the size of a fully functional polysome should be compatible with the size of the product of synthesis.7 Thus for L and H chains one would expect polysomes in the range of 7 and 15 ribosomes, respectively. The examination of polysomal units of immunoglobulin synthesis isolated from lymph node and spleen homogenates has been complicated by the presence of highactivity ribonucleases (RNases). These tissues seem to lack the RNase inhibitors present in liver cells.8 The contradictory results of experiments in this field might be due to endogenous RNases. On the other hand, synthesis of an antibody molecule might, proceed differently from the model proposed for other polypeptides. The work reported here is the result of efforts to develop a system in which biochemical and morphological studies could be made of polysomes of lymphoid tissue cells during response to antigen injection. The first objective was to devise a means to reduce the degradative action of endonucleases on the preparations being studied. The initial effort to develop such a system was partially successful.9 The experiments presented here indicate success in devising a method to block RNase action completely in homogenates of spleen and lymph nodes. Interpretation of the results of these experiments suggests that some discrepancies may exist between accepted models of protein synthesis and the types of polysomes involved in the assembly of L and H chains of antibody globulins. Materials and Methods.-Animals and immunization: Sprague-Dawley male albino rats weighing between 150 and 300 gm were used in all experiments. They were immunized in two ways: primary immunization consisted of an intravenous injection of 1.0 ml of Salmonella typhi formalinkilled vaccine;10 hyperimmunized animals were given three intravenous injections of 1.0 mnl of the vaccine on 3 consecutive days and 30 days later received a fourth intravenous injection to serve as a booster. No differences were noted in the results of experiments using rats given either immunizing treatment, and therefore no distinction will be made between hyperimmunized and nonhyperimmunized animals. Isotopes: Reconstituted, C'4-labeled Chlorella hydrolysate prepared by Schwarz BioResearch was used in all experiments. Cell preparation, incubation, and centrifugation: Rats were killed by exsanguination under ether anesthesia; the spleen was rapidly removed, placed in ice-cold buffered special saline (BSS, 0.03 M tris, 0.14 M NaCl, 0.005 M KCI, 0.0015 M MgC92), and finely minced. Splenic fragments were then forced gently through an 80-mesh stainless-steel screen by means of a syringe plunger, with frequent BSS washings. Collected cells were washed twice by centrifugation at 250 X g in cold BSS with e-thylenediaminetetraacetate (EDTA) and then in cold BSS without EDTA. The cell suspension from one spleen was then incubated in a Dounce homogenizer vessel in 2.0 ml of Eagle's