Polyribosomal synthesis and assembly of the H and L chains of gamma globulin.

Abstract

Polyribosonmes have been shown to be the active uiiit of proteirn synlthesis in gamma globulin-producing cells from both rabbit lymph nodes1 andl mouse plasmna cell tumors.2 7S gamma globulii conisists of two heavy (H) anid two light (L) chains linked by disulfide bonds. The availability of techniques for separating and identifying the two chains affords a unique opportunity to study the synthesis and assembly of the polypeptide chains of a disulfide-linked protein. A/louse plasma cell tumors provide a cell population in which 20-30 per cent of the newly made protein is homogeneous gamma globulin. With these tunmor cells it has been possible to determine (1) whether the two chains are made on polyribosomes of different sizes, (2) whether the two chains are assembled on polyribosomes, (3) the relative rates of synthesis of the two chains, and (4) the approxinmate size of the messenger RNA's. AMaterials and Methods.-Two transplaintable inouise plasmna cell tumnors wer e used. Cells from the MPC-11 tumor (provided by Dr. John Fahey) produce approximately four tirnes as many L as H chains. The B-J tumor (provided by Dr. Elliot Osserman) synthesizes L chains, but few if any H chains. Immunologic an-d electrophoretic analysis indicated that these two polypeptides represented 20-30% of the total protein synthesized by both tumors. Cells were teased from the tumors, filtered through a stainless steel screen, washed four times, and resuspended in Eagle's medium4 containing I/loo the normal amounts of amino acids.3 After adjusting the cell concentration to 1-2 X 107 cells per ml and preincubating at 370 for 15 min, the cells were exposed to various mixtures of C14-amino acids at 10-20 mc/ml. Incorporation of the radioactive precursors was stopped by adding 10 vol of chilled Earle's saline, after which 107 cells were washed three times in cold Earle's saline and resuspended in 1 ml of a cytoplasmic extract freshly prepared from 108 HeLa cells.' The tumor cells were then disrupted by adding desoxycholate to a final conceltration of 0.5%, the cell lysates were immediately layered on 15-30% linear sucrose gradients,6 and centrifuged for 130-150 min at 24,000 rpm in the SW25.1 swingingbucket rotor (Spinco). Gradients were analyzed for UV absorbancy at 26() mn, anid for acid-precipitable radioactivity as previously described.3 Pulse-chase experiments were performed by iticubatiiig cells for 11/2 tllill withb a inxtUil't' of C14-labeled argininie, lysine, threonine, valinie, and leulcine (New Ei-iglaiid Niiclear Corp., 150)30()0 me/mM) at a final coticentratioln of 1l0-20 ,uc/ml an-id chasing'' with a 200-fold excess of tnlabeled amin-o acids. Samples takeii at the time of additioni of the chase, an-d at 15-sec iiitervals thereafter, were analyzed for polyribosome-associated acid-precipitable radioactivity. The addition of the uinlabeled amino acids immediately stopped the incorporation of radioactive label into acid-precipitable material. The identification and quantification of H and L chains (a) in whole-cell lysates, (b) as newly completed and released polypeptide chains from the tops of sucrose gradients, and (c) as nascent polypeptides on polyribosomes, was carried out by a modification of polyacrylamide gel electrophoresis.7 Samples were prepared for electrophoresis by treatment with 10% acetic acid, 1% sodium dodecyl sulfate (SDS), 0.5 M urea, and 1% 2-mercaptoethanol (ME). Under these conditions, aggregated or insoluble proteins are solubilized so that they are neither lost nor trapped at the origin of the gel.7' 8 The sample was thern dialyzed at room temperature for 16 hr against, 0.1% SDS, 0.5 M urea, 0.1.1yo AME, an-d 0.01 M phosphate btiffer at pll 7.1. Five per cent polyacrylamlide gels were plepared inl the samne blLff er btit withl .1 phosphate coticeiitlat ioii of 0.1 AE,