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Immunoglobulin biosynthesis
J. Mol. Biol. (1970) 53, 306-320
Immunoglobulin
Biosynthesis
V.t Light Chain Assembly DAVID SCHUBERTANDMELVIN COHN The Salk Institute for Biological Studies San Diego, Calif., U.S.A. (Received 27 November 1969, and in revised forra 28 July 1970) A protein
one-half the size of and antigenically related to light chain was found in immunoglobulin and light chain producing myelomas. The kinetics of its turnover are not consistent with its being a degradation product of light chain. The possibility that this immunoglobulin fragment is a synthetic intermediate in light chain biosynthesis is analyzed and discussed in the context of other data on light chain biosynthesis.
1. Introduction Many mouse myelomas contain, but do not secrete, a protein one-half the size of a related to it (Schubert & Cohn, 1968; Schubert, light chain and immunologically 1970). The most obvious hypothesis is that this immunoglobulin fragment represents an intracellular breakdown product of the light chain (Cioli & Baglioni, 1967 ; Solomon & McLaughlin,
1969). However,
as the data from a large number
of experiments
were
analyzed, contradictions to this interpretation arose. The goal of this study is to describe the contradictions which indicate that the question is still open as to whether variable and constant regions of immunoglobulin are joined at the peptide, rather than at the DNA or messenger RNA level. It should be recalled that separate structural genes appear to code for the variable and constant regions of both the heavy and light chain subunits. Therefore, a mechanism must exist for joining these genes or their products to form a single subunit polypeptide chain (reviewed by Cohn, 1968; Koshland, Davis & Fujita, 1969).
2. Materials and Methods (a) Cell lines The tissue culture line of mouse myeloma 5194 produces an IgA (~a) immunoglobulin which reacts specifically with nucleic acid bases (Schubert, Jobe & Cohn, 1968) and grows in suspension culture as described previously (Schubert, 1970). Mouse myeloma RPC20, a gift from Dr K. R. McIntire, produces a X-type light chain only. Mouse myeloma MOPC46, a gift froin Dr M. Potter, produces a K-type light chain only. Neither cell line synthesizes nor secretes detectable heavy chain (Schubert & Cohn, 1968). S63 (~a) and its non-immunoglobulin producing derivative, XS63, have been previously described (Schubert & Horibata, 1968; Cohn, Notani & Rice, 1969). Cells from the solid subcutaneous tumors were dispersed for isotopic labeling as described by Schubert,, Munro & Ohno (1968). t Paper IV in this series is Schubert, 1970. 305
306
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SCHUBERT
(b) Pulse labeling
AND
11. COHN
and preparation
of polysomes
Cells were washed twice in modified Eagle’s medium minus loucine and resuspended at about 3 X lo6 cells/ml. in Eagle’s medium minus sodium bicarbonate, pH 6.7, and 1 X 10 -5 M-leuoine. The suspension was gently stirred in a 37°C water bath. After a IO-min preincubation, [3H]leucine (40 c/m-mole) was added to a final concentration of 20 PC/ml. When quantitation of the protein synthesized during a labeling experiment wee required, purified [14C]leucine secreted immunoglobulin or light chain was added before the addition of the [3H]leucine. In this manner, the amount of material recovered during the subsequent purification procedures could be normalized to the input protein. Samples were diluted directly into iced pH 6.7 buffer, 0.025 M-phosphate, 0.075 M-NaCI, 0.01 ivr-Mgcl, (Becker & Rich, 1966), and a final concentration of 1 ye v/v Nonidet P40 detergent (Shell Chemical CO.), and 0.25 M-sucrose. The cell lysate was then layered directly onto 0.3 to 1 M linear sucrose gradients. Centrifugation was carried out for 150 min at 24,000 rev./min in the SW25*1 head of a Beckman model L2; the absorbance at 260 nm was monitored through a Gilford continuous flow spectrophotometer. The determination of labeled protein and the serological precipitation of nascent protein from polysomes and polysome supernatants has previously been described (Schubert & Cohn, 1968). (c) Antisera Rabbit antisera against 5194 and S63 IgA immunoglobulins reacted with monomeric and polymeric IgA, isolated heavy chain, and weakly with light chain dimers. They did not, however, react with light chain monomers. For this reason, all serological precipitations involving 5194 and S63 were performed with a mixture of anti-IgA immunoglobulin and an anti-K-type light chain serum which together precipitated all of the intracellular heavy and light chain. Anti-h type light chain was prepared against RPC20 urinary light chain and anti-K type light chain (a gift from Dr P. Knopf, Salk Institute) was prepared against MOPC46 urinary light chain. The latter serum reacted with IgG and IgA immunoglobulin. (d) Acrylamide
disc electrophoresis
Immune precipitates of intracellular material were washed 3 times in 0.9% N&l and dissolved in 0.3 ml. of a solution containing 9.8 M-urea, 1% sodium lauryl sulfate, 0.5 M-Tris-HCl, pH 8.5 at 37°C. 2-mercaptoethanol was added to 0.25 M, and the mixture incubated at 37°C for 3 hr under nitrogen. Iodoacetamide, dissolved in 2 M-Tris, pH 8.5, was then added to bring the final concentration to 0.30 ivr-iodoacetamide. Incubation was continued for 30 mm, a 10% molar excess of 2-mercaptoethanol was added and the solution was dialyzed against 0.5 M-urea, 0.1% sodium lauryl sulfate, O*2o/o 2-mercaptoethanol, and 0.01 M-sodium phosphate, pH 7.1, overnight at 37°C. In some cases the dialysis step was omitted when it was later realized that the subsequent electrophoresis was unaffected. Samples were then electrophoresed on 7.5% acrylamide gels (in 0.1% sodium lauryl sulfate, 0.5 M-urea, 0.01 M-sodium phosphate, pH 7.1) with an internal control of [14C]leucine-labeled secreted immunoglobulin which was carried through the same procedure. Electrophoresis and counting of the gels was carried out as previously described (Schubert $ Cohn, 1968). Fractions are numbered from the negative to the positive electrodes. Since this gel system contains lauryl sulfato, separation of non-glycoproteins is primarily on the basis of molecular weight (Shapiro, Vinuela & Maizel, 1967), but the mobility of glycoproteins is retarded with respect to proteins devoid of carbohydrate (Schubert, 1970).
3. Results (a) Polysomal
sites of immunoglobulin
synthesis
A tissue culture of S194 cells was labeled with [3H]leucine for ten minutes and the distribution of polysomes assayed on a sucrose gradient as O.D.zso (Fig. 1). Also illustrated is the profile of total and serologically precipitable radioactivity. The presence of heavy and light chains were assayed in the pooled fractions (Fig. 1, fractions 14 to
IMMUNOGLOBULIN
BIOSYNTHESIS
Fraction
307
no.
FIG. 1. Polysome profile of the tissue culture line of mouse myeloma S194. Polysomes were generated as described in Materials and Methods after a lo-min pulse with [3H]leucine. Portions of each fraction were serologically precipitated as described in Materials and Methods. Normal rabbit serum was used as a control for non-specific precipitation and all data are expressed as the difference between the specific and non-specific precipitates. ) O.D. as,,; -O--O-, trichloroaoetic acid-precipitable radioactivity (total); ( - i: - x -, serologically precipitable radioactivity.
19 and 22 to 27) corresponding to the peaks of immunoglobulin-related nascent chains. The serological precipitate from each pool was reduced with 2-mercaptoethanol, alkylated, and electrophoresed on acrylamide gels containing sodium lauryl sulfate (Fig. 2(a) and (b)). Figure 2(c) shows the electrophoretic pattern of the serologically precipitable protein recovered from the top of the gradient. The heavier (Fig. 2(a)) and lighter (Fig. 2(b)) polysome fractions containing immune precipitable material are thus responsible for heavy and light chain synthesis, respectively; light chains are also found complemented with heavy chain on polysomes (Fig. 2(a) and Schubert, 1968). The two proteins migrating more slowly than light chain are both heavy chain fractions (Fig. 2(c)). The heavy chain migrating identically with the secreted heavy chain marker contains carbohydrate, while the faster moving heavy chain fraction is devoid of carbohydrate (Schubert, 1970). Acrylamide electrophoresis also revealed a protein on light chain polysomes which is precipitable by the antiserum and migrates faster than light chain (Fig. 2(b)). Smaller amounts of this material were also found in the polysome supernatant (Fig. 2(c)). From the mobility of this protein relative to a series of proteins of known molecular weights in this acrylamide gel system (Shapiro et al., 1967; Schubert, 1970), it was calculated that the fastest migrating protein has a molecular weight of 12,000 f 1000. This molecular weight was confirmed by sizing on Sephadex GlOO equilibrated with 8 M-urea, 0.01 Macetic acid following complete reduction and alkylation of an immune precipitate from the pulse-labeled polysome supernatant. A similar fast migrating protein has been
303
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SCHUBERT
(b) 6
AND
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COHN
H i
t
Fraction
no.
FIG. 2. Acrylamide electrophoresis of proteins serologically precipit8ted from S194 polysomes asd polysome supernat8nt. The two regions of the polysome profile shown in Fig. 1 containing the maximum amount of serologically precipitable protein (fractions 22 to 27 and 14 to 19), and the polysome supernatant, were separately pooled 8nd precipitated with anti-S194 immunoglobulin plus anti-b-chein sera. The precipitetes were weshed, reduced and elkylated, and eleotrophoresed on acrylamide gels 8s described in Materials and Methods. H and L mark the positions of [1’%]leuoine-labeled secreted heavy and light chain, respectively, which were electrophoresed 8s markers in the same gels. (8) Heavy chain region of polysome gradient (frections 14 to 19); (b) lighter region of polysomu gradient (fractions 22 to 27); (c) polysome supernatant.
repeatedly observed on the ribosomes and in the polysome supernatants of several other mouse myelomss (Schubert & Cohn, 1968; Schubert, 1970). This protein, henoeforth celled immunoglobulin fragment, is the subject of this investigation. Two other features of the polysome profile, namely the size of the polysomes making heavy and light chains, and the specific activity across the gradient, should be noted. As shown in Figure 1, there is a trimodal distribution of total radoactivity associated with nascent chains. Such isotope proties are characteristic of myeloma lines which make up to 30 %of their total protein 8s immunoglobulin (Schubert, Munro & Ohno, 1968; Schubert, 1968; Kimmel, 1969), but are not seen in immunoglobulin negative myeloma cells (Schubert t Cohn, 1968). If the relationship between the number of ribosomes in a polysome and migration distance in the gradient is calculated according to Kuff & Roberts (1967), and the number of ribosomes per polysome is plotted as a function of specific activity, deviations from the expected linear function are observed with the immunoglobulin producing cell lines (Kimmel, 1969; Schubert, 1968)
IMMUNOGLOBULIN
309
BIOSYNTHESIS
but not with immunoglobulin negative mutants (Fig. 3). The two peaks of high specific activity are on polysomes of an average size of 4 and 11 ribosomes which are those making light and heavy chains, respectively (Figs 1 and 2). The polysome size designations are exact, for the linear extrapolations of the experimental specific activity curves (Fig. 3) fit the theoretical curves calculated according to Kuff & Roberts (1967). The excess radioactivity on heavy chain polysomes is predictable
I
I 4
1 8
!
I
12
16
Rlbosomes per polysome
3. Relationship between specific activity and the number of ribosomes per polysome for immunoglobuhn producing and mutant non-producing mouse myelomas. Polysome profiles were prepared as described in Fig. 1, and the specific aotivity across the gradient plotted as a function of the number of ribosomes per polysome as described in text. (a) IgA producing 5194; (b) immunoglobulin negative mutant XSf33. FIG.
since there is complementation of labeled light chain with heavy chain while the latter is associated with the ribosome on which it is synthesized (Shapiro, Soharff, Maize1 & Uhr, 1966; Schubert, 1968; Schubert & Cohn, 1968). By this argument, however, one would not have expected a disproportionate amount of radioactivity associated with light chain polysomes. The third peak of total radioactivity (Fig. 1) is antigenically unrelated to immunoglobulin and does not show anomalously high specific activity. For this reason it will not be considered further. The peak of specific activity on the tetramer polysomes has been observed by other investigators, and attributed to polysome breakdown due to endogenous ribonuclease activity (Kuff &I Roberts, 1967). This observation cannot, however, be explained by ribonuclease breakdown for the following reasons. (i) Myeloma cell lines not synthesizing immunoglobulin show no deviation from linearity in specific activity plots (Fig. 3(b) and Kuff $ Roberts, 1967). (ii) There is minimal, if any, polysome breakdown, for heavy chains are not found in the region of light chain polysomes (Fig. 2 and Schubert, 1968). (iii) When a cell extract is treated with amall amounts of
310
1). SCHUBERT
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Bl. COHN
ribonuclease, there is a general increase in specific activit’y on dimcr through octamer polysomes, not a higher specific activity uniquely associated kth t,rimers and tetramers as observed in the untreated preparations (Kuff $ Roberts, 1967). The relatively higher increase in specific activity shown in Figure 3(a) over that observed by Kuff & Roberts probably reflects the fact that those investigators were looking only at non-membrane bound polysomes, while membrane bound ribosomes may be most extensively involved in the synthesis of secretory immunoglobulin. The synthesis of light chain on polysome tetramers has been demonstrated previously (La Via, Vatter, Hammond & Northup, 1967; Schubert, 1968). Since hemoglobin, a protein of 16,000 molecular weight per chain is synthesized on pentamers (Warner, Knopf & Rich, 1963), it follows that ribosome tetramers are not likely to be involved in the synthesis of a complete light chain of 23,000 molecular weight. It may be argued that the packing of ribosomes on messenger RNA4 is different in reticulocytes and myelomas. In that case, one might compare two polysome fractions within a cell, one engaged in light and the other in heavy chain synthesis. If a tetramer were involved in the synthesis of a complete light chain, then an octamer should make the heavy chain. In fact, the heavy chain polysome has eleven, not eight, ribosomes (Fig. 1). It has been argued that light and heavy chains are synthesized on polysomes of the size predicted for the synthesis of complete chains (Shapiro et al., 1966; Williamson & Askonas, 1967; Becker & Rich, 1966). However, it should be not’ed that none of these investigations combined the necessary criteria to support this argument, e.g. identification of nascent immunoglobulin chains on ribosomes, and evidence that no breakdown of polysomes occurred during the preparation of the profiles. These criteria are met by the data presented in Figures 1, 2 and 3. The above observations are consistent with the assumption that light chain is synthesized as two separate polypeptide chains of 12,000 molecular weight each. Actually this idea would require that a tetrameric polysome be involved in the synthesis of three proteins, each of molecular weight 12,000. These are the variable regions of both light and heavy chain and the constant region of t,he light chain. The heavy chain constant region would be three times this size, i.e. around 36,000 molecular weight. It would therefore be expected to be synthesized on polysomes containing an average of 12 ribosomes, close to t,hat found (Figs 1 and 2). (b) Distribution
of irnnaunoglobulin fragment in myelomas
The existence of proteins precipitable by anti-immunoglobulin sera which migrate more rapidly than light chain in sodium lauryl sulfate-acrylamide gels has also been demonstrated in two mouse myelomas, MOPC46 and RPCZO, which only synthesize K-type and X-type light chains, respectively. Figure 4 shows that the immunoglobulin fragment is precipitable from the polysome supernatant after a one minute pulse with [3H]leucine ; it is also found on ribosomes. To try and reveal light chain breakdown during the lysis and precipitation procedures, l*C-labeled MOPC46 and RPCBO secreted light chain were added to the cell suspension before labeling with [3H]leucine and carried through the same procedure. Similarly, partially reduced and alkylated l*C-labeled S194 IgA and fully reduced and alkylated MOPC46 light chain were added to the initial cell suspensions and precipitated with the polysome supernatant. No breakdown of the input marker protein was noted in any preparation (Fig. 4). The protein migrating slightly faster than marker MOPC46 light chain (Fig. 4(c) and (a)) is probably a light chain fraction devoid of carbohydrate, thus having enhanced
IMMUNOGLOBULIN
30
BIOSYNTHESIS
33
40 Fraction
311
40
no.
FIG. 4. Synthesis of immunoglobulin fragment in light chain secreting mouse myelomas. Mouse myelomas MOPC46 and RPCSO were labeled for 1 min with [sH]leucine. Ribosomes and ribosome-free supernatants were prepared as described in Materials and Methods, and precipitated with anti-light chain sera. The immune precipitates were washed, reduced and alkylated, and electrophoresed on acrylamide gels as described in Materials and Methods. [14C]Leucine-labeled secreted light chain was added to t,he cell suspensions before the labeling experiment as a control for light chain breakdown. L marks the position of these marker proteins. (a) RPCZO ribosome supernatant; (b) RPC20 ribosome-bound nascent chains; (c) MOPC46 ribosome supernatant ; (d) MOPC46 ribosome-bound nascent chains. - X-X -, [3H]Leucine-labeled protein; -O-O-, [‘W]leucine-labeled secreted lightchain marker proteins.
mobility relative to the carbohydrate-containing secreted protein (Schubert, 1970). The immunoglobulin fragment is not covalently assooiated with light chain, for if the immune precipitates are dialyzed against O*1o/osodium lauryl sulfate and electrophoresed on acrylamide without reduction and alkylation, the light chain to immunoglobulin fragment ratio is identical to the reduced and elkylated preparation. To demonstrate that the immunoglobulin fragment is indeed related to light chain, a study of the competition between unlabeled light chain and immunoglobulin fragment for specific antisera was carried out. Polysome supernatant fractions were prepared, and divided into two equal portions. To one portion, 200 pg of purified light chain were added and both were then precipitated with a titrated minimal amount of anti-light chain serum. Figure 5 shows that the highly purified light chain competed for the labeled immunoglobulin fragment and light chain equally well. In the case of 5194 (Fig. 5(c)), anti-light chain serum (prepared against a K-type chain from Adj PC5 IgG2a immunoglobulin) precipitated heavy chain from the ribosome-free supernatant in addition to light chain and immunoglobulin fragment. This reflects the fact that light chain exists in two forms in immunoglobulin producing cells. One form is that covalently associated with heavy chain in immunoglobulin, and the other consists
312
D.
SCHUBERT
AND
M.
COHN
‘Cl
(b)
25
35
Fraction no.
FIG. 5. Inhibition of precipitation by purified light chain. RPC20, MOPC46 and 5194 myeloma cells were labeled for 1 min with [3H]leucine and the ribosome-free supernatants prepared as described in Materials and Methods. Two equal portions from each supernatant fraction were taken, and to one portion 200 pg of the purified urinary light chain (RPC20 and MOPC46) or light chain prepared from serum immunoglobulin (S194), were added. The supernatant fractions were then precipitated with anti-light chain serum. The precipitates were washed, reduced and alkylated, and electrophoresed on acrylamide gels as described in Materials and Methods. The resultant radioactivity profiles were then superimposed. (a) RPC20; (b) MOPC48; (c) 5194. precipitation in presence Precipitation in absence of added protein; --O--o-, -x -x-, of 200 pg of light chain.
of monomeric free light chain (Schubert, 1968). Anti-light chain sera usually react with both forms, but the competing light chain is in the free form and thus competes better for the unbound light chain. This conclusion is supported by the observation that there is an equimolar amount of heavy and light chain remaining after competition (Fig. 5(c)). An immunoglobulin fragment antigenic related to A-type light chain has also been found in mouse myeloma XMOPC104E, a mutant derived from IgM producing MOPClO4E in which the complement&ion between heavy and light chain is defective. In this c&se, anti-h-type light chain aerum precipitates light chain and immunoglobulin fragment, but not the heavy chain which it makes and destroys; anti-immunoglobulin serum precipitates all three fractions in the mutant as well as the wild-type MOPC104 (Schubert & Cohn, 1968). When mutant cell lines derived from immunoglobulin producing mouse myelomas completely lose their ability to synthesize immunoglobulin (Schubert & Horibata, 1968) the ability to make immunoglobulin fragment is lost concomitantly (Fig. 6). In both experiments (Figs 5 and 6) involving 5194 and 563 heavy chain was being synthesized by the cell. Presumably the variable region intermediate of the heavy, as well as that of the light chain, should be present. The heavy chain variable intermediate was not detected, probably because the anti-immunoglobulin sera used did not react with this fragment. The light chain variable intermediate would have been missed also if anti-light chain sera had not been employed in addition to the rtnti-immunoglobulin sera (see Materials and Methods). In fact, this fragment has not been found in
313
Fraction
no.
FIG. 6. The presenceof the fragment in an immunoglobulin producing cell line (S63) and its absence in the immunoglobulin-negative derivative (XS63). IgA producing S63 and its derivative XSf33 were labeled for 1 min with [3H]leucine. The immunoglobulin-related proteins were precipitated from the polysome-supernatants and electrophoresed simultaneously on acrylamide gels as described in Materials and Methods. The two radioactivity profiles were superimposed. H and L mark the positions of 14C-labeled heavy- and light-chain markers from an IgG mouse myeloma protein. -x-x-, 563; --O-O--, XS63.
MOPC21 (IgG,) and 5563 (IgG,,) myelomas presumably because the available antisera used to detect it were inadequate. (c) Kinetics of immunoglobulin
synthesis
(i) Continuous labeling 5194 cells were labeled with [3H]leucine in the presence of 14C-labeled secreted S194 immunoglobulin. In this manner the recovery of immunoglobulin could be corrected for sampling error. Aliquots were withdrawn, the cells lysed with detergent, and the ribosome-free cell fraction prepared. Immunoglobulin related proteins were precipitated with antisera, reduced, alkylated, and electrophoresed on acrylamide gels. The radioactivity in the immunoglobulin fragment, heavy, and light chains, were determined, and plotted as a function of labeling time (Fig. 7(a)). There was a continuous linear uptake of the isotope into intracellular heavy and light chain for up to 15 minutes, but the steady state level in the pool of immunoglobulin fragment reached a plateau after about one minute. Figure 7(b) shows the radioactivity (weight) ratio of light chain (L) to immunoglobulin fragment (I.F.) and heavy (H) to light chain as a function of the time of labeling. If the assumptions are made that (1) the immunoglobulin fragment is a precursor to the light chain and (2) light chain is made and released from ribosomes at the same rate as the immunoglobulin fragment, at pulse times up to the synthetic time of half of a light chain an equal amount of isotope should be incorporated into released light chain and immunoglobulin fragment. The fact that a light chain to immunoglobulin fragment ratio of less than one is observed at the shortest pulse times (a ratio of 0.71 at 16 set) indicates that an excess of the immunoglobulin fragment may be synthesized. At times longer than the synthetic time of a variable or
314
I).
0
I
SCHUBERT
I
,
30
60
AND
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I
90 I20 TlmeCsec)
I
I
150
I80
O
FIG. 7. Continuous labeling of 5194. S194 cells were resuspended in media containing [14C]leucine-labeled secreted S194 immunoglobulin. [aH]leucine was added and samples of the suspension withdrawn at the indicated times. Cells were lysed, and the polysome (ribosome-free) supernatant fractions were prepared. Following serological precipitation, the proteins were reduced and alkylated, and electrophoresed on acrylamide gels. The amount of labeled heavy and light chain and immunoglobulin fragment was determined. The recovery of the pulse-labeled 3H proteins was normalized at the 30-sac pulse to the 14C markers and plotted as a function of time. (a) Total radioactivity in heavy (--O-O-), light (- x - x -) and immunoglobulin fragment (--n--n--). (b) Heavy to light chain ratio (H/L, -O-O-) and light to immunoglobulin fragment (L/I.F., -x-x-).
constant region, increasingly more isotope should appear in released light chain than immunoglobulin fragment, reflecting a saturation of the precursor pool. This was observed (Figs 7 and 9(a)). These results are not compatible with any model of light chain catabolism which could be invoked to explain the origin of the immunoglobulin fragment. The kinetics of heavy and light chain synthesis reflect the fact that two moles of light chain are synthesized for each mole of heavy chain, and have been discussed in detail previously (Schubert, 1968). (ii) Pube-chase 5194 cells were labeled for one minute with [3H]leucine, followed by the addition of a 200-fold molar excess of unlabeled leucine. Ribosome-free supernatants were prepared as described in Figure 7, precipitated with antisera, and the immune precipitate electrophoresed on acrylamide after reduction and alkylation. The recovery of the immunoglobulin fractions was normalized to [14C]leucine 5194 standards as described above. After addition of unlabeled leucine, there was a rapid loss without lag of the label from the soluble pool of the immunoglobulin fragment (Fig. 8). No radioactivity in the immunoglobulin fragment was detectable after two minutes. The acrylamide
IMMUNOGLOBULIN
315
BIOSYNTHESIS
Chase tune (mm)
FIQ. 8. Pulse-chase experiment. 5194 cells were labeled for 1 min with [3H]leucine, followed by the addition of a 200-fold molar excess of leucine. Ribosome-free supernatants were prepared and the results plotted as described in Fig. 7. Total radioactivity incorporated into heavy (-O-@-), light (- x- X-), and immunoglobulin fragment (-AA-) protein.
gels of S194 after a one-minute pulse and an eight-minute chase (Fig. 9) clearly illustrate the passage of the immunoglobulin fragment out of the serologically precipitable pool. The radioactivity shift in heavy chain fractions was due to the acquisition of carbohydrate by the faster migrating heavy chain which is devoid of carbohydrate (Schubert, 1970). To test whether the immunoglobulin fragment was released from ribosomes and enzymically catabolized, X194 cells were labeled for one minute with [3H]leucine; then
Fraction
FIG. 9. Acrylamide electrophoresis of Acrylamide electrophoresis profiles positions of [‘Wlleucine-labeled heavy as marker proteins. (a) l-min pulse with [sH]leucine; (b)
no
serologically precipitable material before and after chase. of experiment described in Fig. 8. H and L indicate the and light chain from secreted 5194 immunoglobulin used S-min chase with unlabeled
leucine.
316
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AN11
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Time (mid
FIG. 10. Inhibition of protein synthesis with cycloheximide. S194 cells were labeled with [3H]leucine for 1 min. Cycloheximide was then added to a final concentration of 50 pg/ml. The zero time sample was immediately withdrawn, and diluted into iced buffer containing detergent, as described in Materials and Methods. The polysome supernatants were prepared and the data plotted as described in Fig. 7. (a) Total radioactivity in heavy (-O-O-), light (- X-X-), and immunoglobulin fragment (-A-A--) proteins; (b) triohloroacetic acid-(5%) precipitable radioactivity in polysome supernatant.
protein synthesis was inhibited with cycloheximide. globulin fragment from the polysome supernatant in (Fig. 10). Therefore, the removal of immunoglobulin dent on protein synthesis; protein degradation is not
There was no loss of immunothe absence of protein synthesis fragment from its pool is depen(Feldman & Yagil, 1969).
4. Discussion Immunoglobulin producing cells synthesize a protein of molecular weight 12,000, one-half the size of a light chain. There is no doubt that this protein corresponds to a part of the light chain, for it competes with purified light chain for anti-light chain serum (Fig. 5). Furthermore, this fragment is not found in mutants which have lost the ability to synthesize heavy and light chain (Fig. 6). However, for technical reasons it has not been formally established whether the immunoglobulin fragment is the variable or constant half. Nevertheless, whatever data bear on this question favor that it is the variable portion. The most direct argument is that many antisera specific for the constant region do not react with this fragment which contains determinants (idiotypic or sub-group specific) restricted to a small number of light chains unlike those in the constant region which are shared by all. With a human myeloma, due to the existence of an anti-idiotype serum for the light-chain variable region, it was established that a similar immunoglobulin fragment is the variable region (Matsuoka, Yagi, Moore & Pressman, 1969). In the present study the variable region of the heavy chain has not been found as a fragment. Possibly our antisera do not recognize this region. Attempts to extend this finding to heavy chain are in progress. The limits on any hypothesis concerning the biosynthesis of light and heavy chain
IMMUNOGLOBULIN
BIOSYNTHESIS
317
have been set by the pulse-labeling experiments on the mouse light chain, MOPC46, Figure 6, (Lennox, Knopf, Munro & Parkhouse, 1967) and of mouse and rabbit heavy chains (Lennox et al., 1967; Fleischman, 1967). These studies (1) demonstrated that immunoglobulins, like other proteins, are synthesized from the amino-terminal residue and (2) ruled out chain assembly through a pool of completed constant region proteins. However, the above 6ndings are compatible with the hypothesis that a small rapidly turning over pool of a variable region intermediate is linked to the nascent constant region peptide. Since nearly completed light chains are found on ribosomes, Figure 2 (Shapiro et al., 1966; Schubert, 1968,1970), and since synthesis begins from the amino terminal residue, the only way in which any proposed mechanism for joining the variable and constant peptides could operate is through a pool of variable regions. Since it is generally believed that the variable and constant regions of the light chain are joined at the DNA level, it could be expected that this fragment would result from light chain catabolism. In fact it has been shown that some of the urinary light chain fragments are catabolic products of whole light chain (Cioli & Baglioni, 1967 ; Solomon 8: McLaughlin, 1969). However, in this system, no light chain degradation could be detected by the following experiments : (i) Purified, secreted light chain carried through the usual procedure of cell lysis, serological precipitation, reduction, alkylation, and acrylamide electrophoresis, shows no breakdown to immunoglobulin fragment (Fig. 4) ; this is also true if secreted proteins completely reduced and alkylated in 9.8 M-urea are processed. Such a procedure should expose any labile peptide bond which may be protect,ed from proteolysis by a conformational change following the release of nascent chain. (ii) The kinetics of the appearance of the immunoglobulin fragment in the ribosomefree immunoglobulin pool are incompatible with light chain catabolism, for at short pulse times mre isotopically labeled fragment is released from ribosomes than light chain. If light chain were synthesized in one piece and enzymically cleaved, the radioactivity incorporated into light chain at short pulse times would necessarily be greater. (iii) The disappearance of t.he fra#gment from the pool is dependent upon protein synthesis (Fig. 10). (iv) The fragment pool reaches maximum specific activit’y in one minute whereas t.he light chain pool requires more than ten minutes (Fig. 7). It could be argued that the immunoglobulin fragment results from the release of nasaent chains into theribosome-free supernatant during the preparation of polysomes. This appears unlikely since no transfer RNA is associated with protein found in the ribosome-free supernatant (Schubert & Cioli, unpublished observation), although ribosome-bound nascent light chain and nascent immunoglobulin fragment in MOPC46 are associated with transfer RNA (Cioli, personal communication). The anti-K serum used in this work has a specificity for the completed variable and constant regions of the light chains studied here. This fact permits two important arguments to be made. If light chain were synthesized as a single unit from N- to C-terminal, then at the completion of the variable half, the antiserum would recognize it as a nascent chain independent of the state of completion of the constant region. Consequently, a spectrum of sizes of nascent light chain between half and completed should appear. This is not seen, for the non-overlapping peaks in acrylamide gels of serologically isolated light chain and immunoglobulin fragment (Figs 2, 4 and 5) indicate two distinct size classes of proteins, not a continuous spectrum of them
318
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M.
COHN
(Schubert, 1968,197O). This is true whether the serological precipitates were from nascent chains on polysomes or the polysome supernatant. Since a broad spectrum of nascent chain sizes could not be found on polysomes, no additional arguments can be made as to whether the supernatant light chain and fragment are a consequence of the isolation procedures stripping nascent chains. The failure to find a distribution of sizes on polysomes is paradoxical and needs an explanation, for the above argument would be valid if the variable and constant regions were synthesized as one unit. If, however, the variable regions were coupled to constant regions which were half completed, the techniques used here would not have detected a heavily skewed size distribution. One argument frequently used against a peptide joining model comes from a,n analysis of the sequence of two proteins from heavy chain disease, Zuc (Frangione & Milstein, 1969) and Hi (Terry & Ohms, 1970). Both proteins are derived from deletions of different lengths covering distinct portions of both the v and c regions. For example, Zuc is deleted from positions 19 to around 220 whereas the Hi deletion covers positions 34 to around 140. The argument is that such deletions must have occurred in a vc cistron and therefore the v and c genes must have been joined by translocation at the DNA level prior to the deletion event. This argument is weak, however, for it is likely that the subgroup v genes are tandem and closely linked to the c genes (Cohn, 1968). Therefore, any large deletion (expected for repeated homologous genes) could join any v to c by a mechanism not normally used. Consequently, for the above heavy chains resulting from deletions covering parts of v and c, the peptide chain would be synthesized in one piece. These deletions may show that (1) the v and c genes are linked and (2) each gene is tandem on the chromosome in a 5’ to 3’ (N to C) direction. Furthermore, since the probability of joining a somatically selected upon v-gene is about one out of five (the approximate number of vu subgroups), these deleted heavy chains probably are the translation of a part of a germ-line gene. There are many examples of deletions linking together two genes which originally coded for two polypeptide chains, such that one chain is made consisting of an N-terminal portion of one and a C-terminal portion of the other. At least at the level of sophistication of t(ranslocation models, it is possible to construct a peptide-joining model compatible with the present data. A variable region peptide intermediate is released from the ribosome. If there is a normal chain termination event, the immunoglobulin fragment must be activated. One model for this is the synthesis of peptide antibiotics by a reaction of the C-terminal residue with ATP to form an acyl adenylate (Kleinkauf, Gevers & Lipmann, 1969). The activating enzyme could recognize the variable fragment by any of its invariant residues, e.g. Phe.Gly*X*Gly*Thr (positions 98 through 102), a sequence which is common to mouse and human K and human h chains. If there is a special chain termination signal, the fragment could be released activated, e.g. by cleavage of the transfer RNA. A peptide bond would have to be formed between the activated C-terminal of the variable region and the N-terminal of the constant region before the latter is half completed on the ribosome, a limitation dictated by the pulse label data (Lennox et al., 1967). The joining enzyme would have to recognize the activated C-terminal group on the variable region and some sequence or determinant of the constant region such as Ala. Ala*Pro*X*Val (positions 111 through 115). Joining enzymes of different, recognition specificities would have to operate on heavy and light chains. In the absence of an in vitro system which synthesizes immunoglobulin de novo, the
IMMUNOGLOBULIN
BIOSYNTHESIS
319
peptide joining could be distinguished from the translocation model in myelomas by any one of three experiments. (1) The hybridization of two myelomas synthesizing immunoglobulin marked in their variable and constant regions by unique sequences. If the hybrid produced the parental chains as well as recombinant chains the translocation model would be eliminated. (2) The demonstration that a stable cloned cell line synthesizes simultaneously two classes of heavy or light chain with the same variable region. (3) The isolation of mutant cell lines which can not link the constant and variable regions. Experiments are in progress to test these predictions. In summary, the following findings are not expected if the simple hypothesis is made that the subunits are synthesized as single polypeptide chains. (i) Heavy and light ch ains are synthesized on polysomes containing 11 and 4 ribosomes, respectively (Fig. 1). These are smaller than expected for the synthesis of proteins of molecular weight 48,000 and 23,000. We have analyzed this finding by pointing out that the state of packing of the message with ribosomes is not what is in question, but the size relationship between the heavy and light chain polysomes. If tetramers make light chain, octamers, not the observed undecamers, (Fig. 1) should make heavy chain. (ii) There is an anomalously increased specific activity associated with nascent chains on steady-state labeled tetrameric ribosomes (Fig. 3). This can be explained if isot’opically labeled protein taken from a pool were associated with these polysomes. (iii) A protein of 12,000 molecular weight and sharing antigenic determinants with light chain is specifically precipitable by an anti-light chain serum from ribosomes making light chain (Figs 2 and 4). (iv) This immunoglobulin fragment is released from ribosomes and enters a small, rapidly turned-over pool (Figs ‘7, 8 and 9). The kinetics of the appearance of immunoglobulin fragment in the ribosome supernatant are compatible with a two-growing point model, but not with an origin based on light chain catabolism. Furthermore, its removal from the pool is dependent on protein synthesis (Fig. 10). It is well to close with a note of caution. These studies do not formally establish that the variable and constant regions are linked at the peptide level. Rather they stress that the generally accepted view that it occws by translocations at the DNA level should be considered an open question. This work was supported by a National Institutes Training Grant no. CA0521301 to one of us (M.C.).
of Health grant no. A105875, and a
REFERENCES Becker, M. J. & Rich, A. (1966). Nature, 212, 142. Cioli, D. & Baglioni, C. (1967). In y-~Zo&uZna, Nobel Symposium no. 3, ed. by J. Killander, p. 401. Cohn, M. (1968). In Rutgers Sympotium on Nucleic Acti in Immunology, ed. by 0. J. Plescia & W. Braun, p. 6’71. New York, N.Y. : Springer-Verlag. Cohn, M., Notani, G. & Rice, S. (1969). Immunocbmistry, 6, 111. Feldman, M. & Yagil, G. (1969). Biochem. Biophys. Res. Comm. 37, 198. Fleischman, J. B. (1967). Cold Spr. Harb. Symp. Quant. Biol. 32, 233. Frangione, B. 8: Milstein, C. (1969). Nature, 224, 597. Kimmel, C. B. (1969). Biochim. biophys. Actu, 182, 361. Kleinkauf, H., Gevers, W. & Lipmann, F. (1969). Proc. Nat. Acad. Sci., Wwh. 62, 226. 22
326
D.
SCHUBEBT
AND
Xl.
COHN
Koshland, M. E., Davis, J. J. 85 Fujiba, N. J. (1969). f’roc. Not. ~‘lctrrl. ski., II’rrsl/. 63, 1274. Kuff, E. L. & Roberts, N. E. (1967). J. Mol. Biol. 26, 211. La Via, M. F., Vatter, A. E., Hammond, S. W. & Northup, P. V. (1967). Proc. Xtrt. dctd. Sci., Wash. 57,79. Lennox, E. S., Knopf, P. M., Mum-o, A. J. & Parkhouse, R. M. E. (1967). Cold. 8~‘. H&J. Symp. Quant. Biol. 32, 249. Matsuoka, Y., Yagi, Y., Moore, G. E. & Pressman, D. (1969). J. Immunol. 103, 962. Schubert, D. (1968). Proc. Nat. Acad. Sci., Wash. 60, 683. Schubert, D. (1970). J. Mol. Biol. 51, 287. Schubert, D. & Cohn, M. (1968). J. Mol. Biol. 38, 273. Schubert, D. & Horibata, K. (1968). J. Mol. Biol. 38, 263. Schubert, D., Jobe, A. & Cohn, M. (1968). Nature, 220, 882. Schubert, D., Munro, A. & Ohno, S. (1968). J. Mol. Biol. 38, 253. Shapiro, A. L., Scharff, M. D., Maize], J. V. $ Uhr, J. W. (1966). Proc. Nat. Acad. Sci., Wash. 56, 216. Shapiro, A. L., Vinuela, E. & Maizel, J. V. (1967). Biochem. Biophys. Res. Comm. 28, 815. Solomon, A. & McLaughlin, C. L. (1969). J. Biol. Chem. 244, 3393. Terry, W. D. & Ohms, J. (1970). Proc. Nat. Acad. Sk., Wash. 66, 558. Warner, J. R., Knopf, P. M. & Rich, A. (1963). Proc. Nut. Acad. Sci., Wad. 49, 122. Williamson, A. R. & Askonas, B. A. (1967). J. Mol. Biol. 23, 201.
Immunoglobulin
Biosynthesis
V.t Light Chain Assembly DAVID SCHUBERTANDMELVIN COHN The Salk Institute for Biological Studies San Diego, Calif., U.S.A. (Received 27 November 1969, and in revised forra 28 July 1970) A protein
one-half the size of and antigenically related to light chain was found in immunoglobulin and light chain producing myelomas. The kinetics of its turnover are not consistent with its being a degradation product of light chain. The possibility that this immunoglobulin fragment is a synthetic intermediate in light chain biosynthesis is analyzed and discussed in the context of other data on light chain biosynthesis.
1. Introduction Many mouse myelomas contain, but do not secrete, a protein one-half the size of a related to it (Schubert & Cohn, 1968; Schubert, light chain and immunologically 1970). The most obvious hypothesis is that this immunoglobulin fragment represents an intracellular breakdown product of the light chain (Cioli & Baglioni, 1967 ; Solomon & McLaughlin,
1969). However,
as the data from a large number
of experiments
were
analyzed, contradictions to this interpretation arose. The goal of this study is to describe the contradictions which indicate that the question is still open as to whether variable and constant regions of immunoglobulin are joined at the peptide, rather than at the DNA or messenger RNA level. It should be recalled that separate structural genes appear to code for the variable and constant regions of both the heavy and light chain subunits. Therefore, a mechanism must exist for joining these genes or their products to form a single subunit polypeptide chain (reviewed by Cohn, 1968; Koshland, Davis & Fujita, 1969).
2. Materials and Methods (a) Cell lines The tissue culture line of mouse myeloma 5194 produces an IgA (~a) immunoglobulin which reacts specifically with nucleic acid bases (Schubert, Jobe & Cohn, 1968) and grows in suspension culture as described previously (Schubert, 1970). Mouse myeloma RPC20, a gift from Dr K. R. McIntire, produces a X-type light chain only. Mouse myeloma MOPC46, a gift froin Dr M. Potter, produces a K-type light chain only. Neither cell line synthesizes nor secretes detectable heavy chain (Schubert & Cohn, 1968). S63 (~a) and its non-immunoglobulin producing derivative, XS63, have been previously described (Schubert & Horibata, 1968; Cohn, Notani & Rice, 1969). Cells from the solid subcutaneous tumors were dispersed for isotopic labeling as described by Schubert,, Munro & Ohno (1968). t Paper IV in this series is Schubert, 1970. 305
306
D.
SCHUBERT
(b) Pulse labeling
AND
11. COHN
and preparation
of polysomes
Cells were washed twice in modified Eagle’s medium minus loucine and resuspended at about 3 X lo6 cells/ml. in Eagle’s medium minus sodium bicarbonate, pH 6.7, and 1 X 10 -5 M-leuoine. The suspension was gently stirred in a 37°C water bath. After a IO-min preincubation, [3H]leucine (40 c/m-mole) was added to a final concentration of 20 PC/ml. When quantitation of the protein synthesized during a labeling experiment wee required, purified [14C]leucine secreted immunoglobulin or light chain was added before the addition of the [3H]leucine. In this manner, the amount of material recovered during the subsequent purification procedures could be normalized to the input protein. Samples were diluted directly into iced pH 6.7 buffer, 0.025 M-phosphate, 0.075 M-NaCI, 0.01 ivr-Mgcl, (Becker & Rich, 1966), and a final concentration of 1 ye v/v Nonidet P40 detergent (Shell Chemical CO.), and 0.25 M-sucrose. The cell lysate was then layered directly onto 0.3 to 1 M linear sucrose gradients. Centrifugation was carried out for 150 min at 24,000 rev./min in the SW25*1 head of a Beckman model L2; the absorbance at 260 nm was monitored through a Gilford continuous flow spectrophotometer. The determination of labeled protein and the serological precipitation of nascent protein from polysomes and polysome supernatants has previously been described (Schubert & Cohn, 1968). (c) Antisera Rabbit antisera against 5194 and S63 IgA immunoglobulins reacted with monomeric and polymeric IgA, isolated heavy chain, and weakly with light chain dimers. They did not, however, react with light chain monomers. For this reason, all serological precipitations involving 5194 and S63 were performed with a mixture of anti-IgA immunoglobulin and an anti-K-type light chain serum which together precipitated all of the intracellular heavy and light chain. Anti-h type light chain was prepared against RPC20 urinary light chain and anti-K type light chain (a gift from Dr P. Knopf, Salk Institute) was prepared against MOPC46 urinary light chain. The latter serum reacted with IgG and IgA immunoglobulin. (d) Acrylamide
disc electrophoresis
Immune precipitates of intracellular material were washed 3 times in 0.9% N&l and dissolved in 0.3 ml. of a solution containing 9.8 M-urea, 1% sodium lauryl sulfate, 0.5 M-Tris-HCl, pH 8.5 at 37°C. 2-mercaptoethanol was added to 0.25 M, and the mixture incubated at 37°C for 3 hr under nitrogen. Iodoacetamide, dissolved in 2 M-Tris, pH 8.5, was then added to bring the final concentration to 0.30 ivr-iodoacetamide. Incubation was continued for 30 mm, a 10% molar excess of 2-mercaptoethanol was added and the solution was dialyzed against 0.5 M-urea, 0.1% sodium lauryl sulfate, O*2o/o 2-mercaptoethanol, and 0.01 M-sodium phosphate, pH 7.1, overnight at 37°C. In some cases the dialysis step was omitted when it was later realized that the subsequent electrophoresis was unaffected. Samples were then electrophoresed on 7.5% acrylamide gels (in 0.1% sodium lauryl sulfate, 0.5 M-urea, 0.01 M-sodium phosphate, pH 7.1) with an internal control of [14C]leucine-labeled secreted immunoglobulin which was carried through the same procedure. Electrophoresis and counting of the gels was carried out as previously described (Schubert $ Cohn, 1968). Fractions are numbered from the negative to the positive electrodes. Since this gel system contains lauryl sulfato, separation of non-glycoproteins is primarily on the basis of molecular weight (Shapiro, Vinuela & Maizel, 1967), but the mobility of glycoproteins is retarded with respect to proteins devoid of carbohydrate (Schubert, 1970).
3. Results (a) Polysomal
sites of immunoglobulin
synthesis
A tissue culture of S194 cells was labeled with [3H]leucine for ten minutes and the distribution of polysomes assayed on a sucrose gradient as O.D.zso (Fig. 1). Also illustrated is the profile of total and serologically precipitable radioactivity. The presence of heavy and light chains were assayed in the pooled fractions (Fig. 1, fractions 14 to
IMMUNOGLOBULIN
BIOSYNTHESIS
Fraction
307
no.
FIG. 1. Polysome profile of the tissue culture line of mouse myeloma S194. Polysomes were generated as described in Materials and Methods after a lo-min pulse with [3H]leucine. Portions of each fraction were serologically precipitated as described in Materials and Methods. Normal rabbit serum was used as a control for non-specific precipitation and all data are expressed as the difference between the specific and non-specific precipitates. ) O.D. as,,; -O--O-, trichloroaoetic acid-precipitable radioactivity (total); ( - i: - x -, serologically precipitable radioactivity.
19 and 22 to 27) corresponding to the peaks of immunoglobulin-related nascent chains. The serological precipitate from each pool was reduced with 2-mercaptoethanol, alkylated, and electrophoresed on acrylamide gels containing sodium lauryl sulfate (Fig. 2(a) and (b)). Figure 2(c) shows the electrophoretic pattern of the serologically precipitable protein recovered from the top of the gradient. The heavier (Fig. 2(a)) and lighter (Fig. 2(b)) polysome fractions containing immune precipitable material are thus responsible for heavy and light chain synthesis, respectively; light chains are also found complemented with heavy chain on polysomes (Fig. 2(a) and Schubert, 1968). The two proteins migrating more slowly than light chain are both heavy chain fractions (Fig. 2(c)). The heavy chain migrating identically with the secreted heavy chain marker contains carbohydrate, while the faster moving heavy chain fraction is devoid of carbohydrate (Schubert, 1970). Acrylamide electrophoresis also revealed a protein on light chain polysomes which is precipitable by the antiserum and migrates faster than light chain (Fig. 2(b)). Smaller amounts of this material were also found in the polysome supernatant (Fig. 2(c)). From the mobility of this protein relative to a series of proteins of known molecular weights in this acrylamide gel system (Shapiro et al., 1967; Schubert, 1970), it was calculated that the fastest migrating protein has a molecular weight of 12,000 f 1000. This molecular weight was confirmed by sizing on Sephadex GlOO equilibrated with 8 M-urea, 0.01 Macetic acid following complete reduction and alkylation of an immune precipitate from the pulse-labeled polysome supernatant. A similar fast migrating protein has been
303
D.
SCHUBERT
(b) 6
AND
M.
COHN
H i
t
Fraction
no.
FIG. 2. Acrylamide electrophoresis of proteins serologically precipit8ted from S194 polysomes asd polysome supernat8nt. The two regions of the polysome profile shown in Fig. 1 containing the maximum amount of serologically precipitable protein (fractions 22 to 27 and 14 to 19), and the polysome supernatant, were separately pooled 8nd precipitated with anti-S194 immunoglobulin plus anti-b-chein sera. The precipitetes were weshed, reduced and elkylated, and eleotrophoresed on acrylamide gels 8s described in Materials and Methods. H and L mark the positions of [1’%]leuoine-labeled secreted heavy and light chain, respectively, which were electrophoresed 8s markers in the same gels. (8) Heavy chain region of polysome gradient (frections 14 to 19); (b) lighter region of polysomu gradient (fractions 22 to 27); (c) polysome supernatant.
repeatedly observed on the ribosomes and in the polysome supernatants of several other mouse myelomss (Schubert & Cohn, 1968; Schubert, 1970). This protein, henoeforth celled immunoglobulin fragment, is the subject of this investigation. Two other features of the polysome profile, namely the size of the polysomes making heavy and light chains, and the specific activity across the gradient, should be noted. As shown in Figure 1, there is a trimodal distribution of total radoactivity associated with nascent chains. Such isotope proties are characteristic of myeloma lines which make up to 30 %of their total protein 8s immunoglobulin (Schubert, Munro & Ohno, 1968; Schubert, 1968; Kimmel, 1969), but are not seen in immunoglobulin negative myeloma cells (Schubert t Cohn, 1968). If the relationship between the number of ribosomes in a polysome and migration distance in the gradient is calculated according to Kuff & Roberts (1967), and the number of ribosomes per polysome is plotted as a function of specific activity, deviations from the expected linear function are observed with the immunoglobulin producing cell lines (Kimmel, 1969; Schubert, 1968)
IMMUNOGLOBULIN
309
BIOSYNTHESIS
but not with immunoglobulin negative mutants (Fig. 3). The two peaks of high specific activity are on polysomes of an average size of 4 and 11 ribosomes which are those making light and heavy chains, respectively (Figs 1 and 2). The polysome size designations are exact, for the linear extrapolations of the experimental specific activity curves (Fig. 3) fit the theoretical curves calculated according to Kuff & Roberts (1967). The excess radioactivity on heavy chain polysomes is predictable
I
I 4
1 8
!
I
12
16
Rlbosomes per polysome
3. Relationship between specific activity and the number of ribosomes per polysome for immunoglobuhn producing and mutant non-producing mouse myelomas. Polysome profiles were prepared as described in Fig. 1, and the specific aotivity across the gradient plotted as a function of the number of ribosomes per polysome as described in text. (a) IgA producing 5194; (b) immunoglobulin negative mutant XSf33. FIG.
since there is complementation of labeled light chain with heavy chain while the latter is associated with the ribosome on which it is synthesized (Shapiro, Soharff, Maize1 & Uhr, 1966; Schubert, 1968; Schubert & Cohn, 1968). By this argument, however, one would not have expected a disproportionate amount of radioactivity associated with light chain polysomes. The third peak of total radioactivity (Fig. 1) is antigenically unrelated to immunoglobulin and does not show anomalously high specific activity. For this reason it will not be considered further. The peak of specific activity on the tetramer polysomes has been observed by other investigators, and attributed to polysome breakdown due to endogenous ribonuclease activity (Kuff &I Roberts, 1967). This observation cannot, however, be explained by ribonuclease breakdown for the following reasons. (i) Myeloma cell lines not synthesizing immunoglobulin show no deviation from linearity in specific activity plots (Fig. 3(b) and Kuff $ Roberts, 1967). (ii) There is minimal, if any, polysome breakdown, for heavy chains are not found in the region of light chain polysomes (Fig. 2 and Schubert, 1968). (iii) When a cell extract is treated with amall amounts of
310
1). SCHUBERT
4x1)
Bl. COHN
ribonuclease, there is a general increase in specific activit’y on dimcr through octamer polysomes, not a higher specific activity uniquely associated kth t,rimers and tetramers as observed in the untreated preparations (Kuff $ Roberts, 1967). The relatively higher increase in specific activity shown in Figure 3(a) over that observed by Kuff & Roberts probably reflects the fact that those investigators were looking only at non-membrane bound polysomes, while membrane bound ribosomes may be most extensively involved in the synthesis of secretory immunoglobulin. The synthesis of light chain on polysome tetramers has been demonstrated previously (La Via, Vatter, Hammond & Northup, 1967; Schubert, 1968). Since hemoglobin, a protein of 16,000 molecular weight per chain is synthesized on pentamers (Warner, Knopf & Rich, 1963), it follows that ribosome tetramers are not likely to be involved in the synthesis of a complete light chain of 23,000 molecular weight. It may be argued that the packing of ribosomes on messenger RNA4 is different in reticulocytes and myelomas. In that case, one might compare two polysome fractions within a cell, one engaged in light and the other in heavy chain synthesis. If a tetramer were involved in the synthesis of a complete light chain, then an octamer should make the heavy chain. In fact, the heavy chain polysome has eleven, not eight, ribosomes (Fig. 1). It has been argued that light and heavy chains are synthesized on polysomes of the size predicted for the synthesis of complete chains (Shapiro et al., 1966; Williamson & Askonas, 1967; Becker & Rich, 1966). However, it should be not’ed that none of these investigations combined the necessary criteria to support this argument, e.g. identification of nascent immunoglobulin chains on ribosomes, and evidence that no breakdown of polysomes occurred during the preparation of the profiles. These criteria are met by the data presented in Figures 1, 2 and 3. The above observations are consistent with the assumption that light chain is synthesized as two separate polypeptide chains of 12,000 molecular weight each. Actually this idea would require that a tetrameric polysome be involved in the synthesis of three proteins, each of molecular weight 12,000. These are the variable regions of both light and heavy chain and the constant region of t,he light chain. The heavy chain constant region would be three times this size, i.e. around 36,000 molecular weight. It would therefore be expected to be synthesized on polysomes containing an average of 12 ribosomes, close to t,hat found (Figs 1 and 2). (b) Distribution
of irnnaunoglobulin fragment in myelomas
The existence of proteins precipitable by anti-immunoglobulin sera which migrate more rapidly than light chain in sodium lauryl sulfate-acrylamide gels has also been demonstrated in two mouse myelomas, MOPC46 and RPCZO, which only synthesize K-type and X-type light chains, respectively. Figure 4 shows that the immunoglobulin fragment is precipitable from the polysome supernatant after a one minute pulse with [3H]leucine ; it is also found on ribosomes. To try and reveal light chain breakdown during the lysis and precipitation procedures, l*C-labeled MOPC46 and RPCBO secreted light chain were added to the cell suspension before labeling with [3H]leucine and carried through the same procedure. Similarly, partially reduced and alkylated l*C-labeled S194 IgA and fully reduced and alkylated MOPC46 light chain were added to the initial cell suspensions and precipitated with the polysome supernatant. No breakdown of the input marker protein was noted in any preparation (Fig. 4). The protein migrating slightly faster than marker MOPC46 light chain (Fig. 4(c) and (a)) is probably a light chain fraction devoid of carbohydrate, thus having enhanced
IMMUNOGLOBULIN
30
BIOSYNTHESIS
33
40 Fraction
311
40
no.
FIG. 4. Synthesis of immunoglobulin fragment in light chain secreting mouse myelomas. Mouse myelomas MOPC46 and RPCSO were labeled for 1 min with [sH]leucine. Ribosomes and ribosome-free supernatants were prepared as described in Materials and Methods, and precipitated with anti-light chain sera. The immune precipitates were washed, reduced and alkylated, and electrophoresed on acrylamide gels as described in Materials and Methods. [14C]Leucine-labeled secreted light chain was added to t,he cell suspensions before the labeling experiment as a control for light chain breakdown. L marks the position of these marker proteins. (a) RPCZO ribosome supernatant; (b) RPC20 ribosome-bound nascent chains; (c) MOPC46 ribosome supernatant ; (d) MOPC46 ribosome-bound nascent chains. - X-X -, [3H]Leucine-labeled protein; -O-O-, [‘W]leucine-labeled secreted lightchain marker proteins.
mobility relative to the carbohydrate-containing secreted protein (Schubert, 1970). The immunoglobulin fragment is not covalently assooiated with light chain, for if the immune precipitates are dialyzed against O*1o/osodium lauryl sulfate and electrophoresed on acrylamide without reduction and alkylation, the light chain to immunoglobulin fragment ratio is identical to the reduced and elkylated preparation. To demonstrate that the immunoglobulin fragment is indeed related to light chain, a study of the competition between unlabeled light chain and immunoglobulin fragment for specific antisera was carried out. Polysome supernatant fractions were prepared, and divided into two equal portions. To one portion, 200 pg of purified light chain were added and both were then precipitated with a titrated minimal amount of anti-light chain serum. Figure 5 shows that the highly purified light chain competed for the labeled immunoglobulin fragment and light chain equally well. In the case of 5194 (Fig. 5(c)), anti-light chain serum (prepared against a K-type chain from Adj PC5 IgG2a immunoglobulin) precipitated heavy chain from the ribosome-free supernatant in addition to light chain and immunoglobulin fragment. This reflects the fact that light chain exists in two forms in immunoglobulin producing cells. One form is that covalently associated with heavy chain in immunoglobulin, and the other consists
312
D.
SCHUBERT
AND
M.
COHN
‘Cl
(b)
25
35
Fraction no.
FIG. 5. Inhibition of precipitation by purified light chain. RPC20, MOPC46 and 5194 myeloma cells were labeled for 1 min with [3H]leucine and the ribosome-free supernatants prepared as described in Materials and Methods. Two equal portions from each supernatant fraction were taken, and to one portion 200 pg of the purified urinary light chain (RPC20 and MOPC46) or light chain prepared from serum immunoglobulin (S194), were added. The supernatant fractions were then precipitated with anti-light chain serum. The precipitates were washed, reduced and alkylated, and electrophoresed on acrylamide gels as described in Materials and Methods. The resultant radioactivity profiles were then superimposed. (a) RPC20; (b) MOPC48; (c) 5194. precipitation in presence Precipitation in absence of added protein; --O--o-, -x -x-, of 200 pg of light chain.
of monomeric free light chain (Schubert, 1968). Anti-light chain sera usually react with both forms, but the competing light chain is in the free form and thus competes better for the unbound light chain. This conclusion is supported by the observation that there is an equimolar amount of heavy and light chain remaining after competition (Fig. 5(c)). An immunoglobulin fragment antigenic related to A-type light chain has also been found in mouse myeloma XMOPC104E, a mutant derived from IgM producing MOPClO4E in which the complement&ion between heavy and light chain is defective. In this c&se, anti-h-type light chain aerum precipitates light chain and immunoglobulin fragment, but not the heavy chain which it makes and destroys; anti-immunoglobulin serum precipitates all three fractions in the mutant as well as the wild-type MOPC104 (Schubert & Cohn, 1968). When mutant cell lines derived from immunoglobulin producing mouse myelomas completely lose their ability to synthesize immunoglobulin (Schubert & Horibata, 1968) the ability to make immunoglobulin fragment is lost concomitantly (Fig. 6). In both experiments (Figs 5 and 6) involving 5194 and 563 heavy chain was being synthesized by the cell. Presumably the variable region intermediate of the heavy, as well as that of the light chain, should be present. The heavy chain variable intermediate was not detected, probably because the anti-immunoglobulin sera used did not react with this fragment. The light chain variable intermediate would have been missed also if anti-light chain sera had not been employed in addition to the rtnti-immunoglobulin sera (see Materials and Methods). In fact, this fragment has not been found in
313
Fraction
no.
FIG. 6. The presenceof the fragment in an immunoglobulin producing cell line (S63) and its absence in the immunoglobulin-negative derivative (XS63). IgA producing S63 and its derivative XSf33 were labeled for 1 min with [3H]leucine. The immunoglobulin-related proteins were precipitated from the polysome-supernatants and electrophoresed simultaneously on acrylamide gels as described in Materials and Methods. The two radioactivity profiles were superimposed. H and L mark the positions of 14C-labeled heavy- and light-chain markers from an IgG mouse myeloma protein. -x-x-, 563; --O-O--, XS63.
MOPC21 (IgG,) and 5563 (IgG,,) myelomas presumably because the available antisera used to detect it were inadequate. (c) Kinetics of immunoglobulin
synthesis
(i) Continuous labeling 5194 cells were labeled with [3H]leucine in the presence of 14C-labeled secreted S194 immunoglobulin. In this manner the recovery of immunoglobulin could be corrected for sampling error. Aliquots were withdrawn, the cells lysed with detergent, and the ribosome-free cell fraction prepared. Immunoglobulin related proteins were precipitated with antisera, reduced, alkylated, and electrophoresed on acrylamide gels. The radioactivity in the immunoglobulin fragment, heavy, and light chains, were determined, and plotted as a function of labeling time (Fig. 7(a)). There was a continuous linear uptake of the isotope into intracellular heavy and light chain for up to 15 minutes, but the steady state level in the pool of immunoglobulin fragment reached a plateau after about one minute. Figure 7(b) shows the radioactivity (weight) ratio of light chain (L) to immunoglobulin fragment (I.F.) and heavy (H) to light chain as a function of the time of labeling. If the assumptions are made that (1) the immunoglobulin fragment is a precursor to the light chain and (2) light chain is made and released from ribosomes at the same rate as the immunoglobulin fragment, at pulse times up to the synthetic time of half of a light chain an equal amount of isotope should be incorporated into released light chain and immunoglobulin fragment. The fact that a light chain to immunoglobulin fragment ratio of less than one is observed at the shortest pulse times (a ratio of 0.71 at 16 set) indicates that an excess of the immunoglobulin fragment may be synthesized. At times longer than the synthetic time of a variable or
314
I).
0
I
SCHUBERT
I
,
30
60
AND
I
11. COHN
I
90 I20 TlmeCsec)
I
I
150
I80
O
FIG. 7. Continuous labeling of 5194. S194 cells were resuspended in media containing [14C]leucine-labeled secreted S194 immunoglobulin. [aH]leucine was added and samples of the suspension withdrawn at the indicated times. Cells were lysed, and the polysome (ribosome-free) supernatant fractions were prepared. Following serological precipitation, the proteins were reduced and alkylated, and electrophoresed on acrylamide gels. The amount of labeled heavy and light chain and immunoglobulin fragment was determined. The recovery of the pulse-labeled 3H proteins was normalized at the 30-sac pulse to the 14C markers and plotted as a function of time. (a) Total radioactivity in heavy (--O-O-), light (- x - x -) and immunoglobulin fragment (--n--n--). (b) Heavy to light chain ratio (H/L, -O-O-) and light to immunoglobulin fragment (L/I.F., -x-x-).
constant region, increasingly more isotope should appear in released light chain than immunoglobulin fragment, reflecting a saturation of the precursor pool. This was observed (Figs 7 and 9(a)). These results are not compatible with any model of light chain catabolism which could be invoked to explain the origin of the immunoglobulin fragment. The kinetics of heavy and light chain synthesis reflect the fact that two moles of light chain are synthesized for each mole of heavy chain, and have been discussed in detail previously (Schubert, 1968). (ii) Pube-chase 5194 cells were labeled for one minute with [3H]leucine, followed by the addition of a 200-fold molar excess of unlabeled leucine. Ribosome-free supernatants were prepared as described in Figure 7, precipitated with antisera, and the immune precipitate electrophoresed on acrylamide after reduction and alkylation. The recovery of the immunoglobulin fractions was normalized to [14C]leucine 5194 standards as described above. After addition of unlabeled leucine, there was a rapid loss without lag of the label from the soluble pool of the immunoglobulin fragment (Fig. 8). No radioactivity in the immunoglobulin fragment was detectable after two minutes. The acrylamide
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BIOSYNTHESIS
Chase tune (mm)
FIQ. 8. Pulse-chase experiment. 5194 cells were labeled for 1 min with [3H]leucine, followed by the addition of a 200-fold molar excess of leucine. Ribosome-free supernatants were prepared and the results plotted as described in Fig. 7. Total radioactivity incorporated into heavy (-O-@-), light (- x- X-), and immunoglobulin fragment (-AA-) protein.
gels of S194 after a one-minute pulse and an eight-minute chase (Fig. 9) clearly illustrate the passage of the immunoglobulin fragment out of the serologically precipitable pool. The radioactivity shift in heavy chain fractions was due to the acquisition of carbohydrate by the faster migrating heavy chain which is devoid of carbohydrate (Schubert, 1970). To test whether the immunoglobulin fragment was released from ribosomes and enzymically catabolized, X194 cells were labeled for one minute with [3H]leucine; then
Fraction
FIG. 9. Acrylamide electrophoresis of Acrylamide electrophoresis profiles positions of [‘Wlleucine-labeled heavy as marker proteins. (a) l-min pulse with [sH]leucine; (b)
no
serologically precipitable material before and after chase. of experiment described in Fig. 8. H and L indicate the and light chain from secreted 5194 immunoglobulin used S-min chase with unlabeled
leucine.
316
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Time (mid
FIG. 10. Inhibition of protein synthesis with cycloheximide. S194 cells were labeled with [3H]leucine for 1 min. Cycloheximide was then added to a final concentration of 50 pg/ml. The zero time sample was immediately withdrawn, and diluted into iced buffer containing detergent, as described in Materials and Methods. The polysome supernatants were prepared and the data plotted as described in Fig. 7. (a) Total radioactivity in heavy (-O-O-), light (- X-X-), and immunoglobulin fragment (-A-A--) proteins; (b) triohloroacetic acid-(5%) precipitable radioactivity in polysome supernatant.
protein synthesis was inhibited with cycloheximide. globulin fragment from the polysome supernatant in (Fig. 10). Therefore, the removal of immunoglobulin dent on protein synthesis; protein degradation is not
There was no loss of immunothe absence of protein synthesis fragment from its pool is depen(Feldman & Yagil, 1969).
4. Discussion Immunoglobulin producing cells synthesize a protein of molecular weight 12,000, one-half the size of a light chain. There is no doubt that this protein corresponds to a part of the light chain, for it competes with purified light chain for anti-light chain serum (Fig. 5). Furthermore, this fragment is not found in mutants which have lost the ability to synthesize heavy and light chain (Fig. 6). However, for technical reasons it has not been formally established whether the immunoglobulin fragment is the variable or constant half. Nevertheless, whatever data bear on this question favor that it is the variable portion. The most direct argument is that many antisera specific for the constant region do not react with this fragment which contains determinants (idiotypic or sub-group specific) restricted to a small number of light chains unlike those in the constant region which are shared by all. With a human myeloma, due to the existence of an anti-idiotype serum for the light-chain variable region, it was established that a similar immunoglobulin fragment is the variable region (Matsuoka, Yagi, Moore & Pressman, 1969). In the present study the variable region of the heavy chain has not been found as a fragment. Possibly our antisera do not recognize this region. Attempts to extend this finding to heavy chain are in progress. The limits on any hypothesis concerning the biosynthesis of light and heavy chain
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have been set by the pulse-labeling experiments on the mouse light chain, MOPC46, Figure 6, (Lennox, Knopf, Munro & Parkhouse, 1967) and of mouse and rabbit heavy chains (Lennox et al., 1967; Fleischman, 1967). These studies (1) demonstrated that immunoglobulins, like other proteins, are synthesized from the amino-terminal residue and (2) ruled out chain assembly through a pool of completed constant region proteins. However, the above 6ndings are compatible with the hypothesis that a small rapidly turning over pool of a variable region intermediate is linked to the nascent constant region peptide. Since nearly completed light chains are found on ribosomes, Figure 2 (Shapiro et al., 1966; Schubert, 1968,1970), and since synthesis begins from the amino terminal residue, the only way in which any proposed mechanism for joining the variable and constant peptides could operate is through a pool of variable regions. Since it is generally believed that the variable and constant regions of the light chain are joined at the DNA level, it could be expected that this fragment would result from light chain catabolism. In fact it has been shown that some of the urinary light chain fragments are catabolic products of whole light chain (Cioli & Baglioni, 1967 ; Solomon 8: McLaughlin, 1969). However, in this system, no light chain degradation could be detected by the following experiments : (i) Purified, secreted light chain carried through the usual procedure of cell lysis, serological precipitation, reduction, alkylation, and acrylamide electrophoresis, shows no breakdown to immunoglobulin fragment (Fig. 4) ; this is also true if secreted proteins completely reduced and alkylated in 9.8 M-urea are processed. Such a procedure should expose any labile peptide bond which may be protect,ed from proteolysis by a conformational change following the release of nascent chain. (ii) The kinetics of the appearance of the immunoglobulin fragment in the ribosomefree immunoglobulin pool are incompatible with light chain catabolism, for at short pulse times mre isotopically labeled fragment is released from ribosomes than light chain. If light chain were synthesized in one piece and enzymically cleaved, the radioactivity incorporated into light chain at short pulse times would necessarily be greater. (iii) The disappearance of t.he fra#gment from the pool is dependent upon protein synthesis (Fig. 10). (iv) The fragment pool reaches maximum specific activit’y in one minute whereas t.he light chain pool requires more than ten minutes (Fig. 7). It could be argued that the immunoglobulin fragment results from the release of nasaent chains into theribosome-free supernatant during the preparation of polysomes. This appears unlikely since no transfer RNA is associated with protein found in the ribosome-free supernatant (Schubert & Cioli, unpublished observation), although ribosome-bound nascent light chain and nascent immunoglobulin fragment in MOPC46 are associated with transfer RNA (Cioli, personal communication). The anti-K serum used in this work has a specificity for the completed variable and constant regions of the light chains studied here. This fact permits two important arguments to be made. If light chain were synthesized as a single unit from N- to C-terminal, then at the completion of the variable half, the antiserum would recognize it as a nascent chain independent of the state of completion of the constant region. Consequently, a spectrum of sizes of nascent light chain between half and completed should appear. This is not seen, for the non-overlapping peaks in acrylamide gels of serologically isolated light chain and immunoglobulin fragment (Figs 2, 4 and 5) indicate two distinct size classes of proteins, not a continuous spectrum of them
318
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(Schubert, 1968,197O). This is true whether the serological precipitates were from nascent chains on polysomes or the polysome supernatant. Since a broad spectrum of nascent chain sizes could not be found on polysomes, no additional arguments can be made as to whether the supernatant light chain and fragment are a consequence of the isolation procedures stripping nascent chains. The failure to find a distribution of sizes on polysomes is paradoxical and needs an explanation, for the above argument would be valid if the variable and constant regions were synthesized as one unit. If, however, the variable regions were coupled to constant regions which were half completed, the techniques used here would not have detected a heavily skewed size distribution. One argument frequently used against a peptide joining model comes from a,n analysis of the sequence of two proteins from heavy chain disease, Zuc (Frangione & Milstein, 1969) and Hi (Terry & Ohms, 1970). Both proteins are derived from deletions of different lengths covering distinct portions of both the v and c regions. For example, Zuc is deleted from positions 19 to around 220 whereas the Hi deletion covers positions 34 to around 140. The argument is that such deletions must have occurred in a vc cistron and therefore the v and c genes must have been joined by translocation at the DNA level prior to the deletion event. This argument is weak, however, for it is likely that the subgroup v genes are tandem and closely linked to the c genes (Cohn, 1968). Therefore, any large deletion (expected for repeated homologous genes) could join any v to c by a mechanism not normally used. Consequently, for the above heavy chains resulting from deletions covering parts of v and c, the peptide chain would be synthesized in one piece. These deletions may show that (1) the v and c genes are linked and (2) each gene is tandem on the chromosome in a 5’ to 3’ (N to C) direction. Furthermore, since the probability of joining a somatically selected upon v-gene is about one out of five (the approximate number of vu subgroups), these deleted heavy chains probably are the translation of a part of a germ-line gene. There are many examples of deletions linking together two genes which originally coded for two polypeptide chains, such that one chain is made consisting of an N-terminal portion of one and a C-terminal portion of the other. At least at the level of sophistication of t(ranslocation models, it is possible to construct a peptide-joining model compatible with the present data. A variable region peptide intermediate is released from the ribosome. If there is a normal chain termination event, the immunoglobulin fragment must be activated. One model for this is the synthesis of peptide antibiotics by a reaction of the C-terminal residue with ATP to form an acyl adenylate (Kleinkauf, Gevers & Lipmann, 1969). The activating enzyme could recognize the variable fragment by any of its invariant residues, e.g. Phe.Gly*X*Gly*Thr (positions 98 through 102), a sequence which is common to mouse and human K and human h chains. If there is a special chain termination signal, the fragment could be released activated, e.g. by cleavage of the transfer RNA. A peptide bond would have to be formed between the activated C-terminal of the variable region and the N-terminal of the constant region before the latter is half completed on the ribosome, a limitation dictated by the pulse label data (Lennox et al., 1967). The joining enzyme would have to recognize the activated C-terminal group on the variable region and some sequence or determinant of the constant region such as Ala. Ala*Pro*X*Val (positions 111 through 115). Joining enzymes of different, recognition specificities would have to operate on heavy and light chains. In the absence of an in vitro system which synthesizes immunoglobulin de novo, the
IMMUNOGLOBULIN
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319
peptide joining could be distinguished from the translocation model in myelomas by any one of three experiments. (1) The hybridization of two myelomas synthesizing immunoglobulin marked in their variable and constant regions by unique sequences. If the hybrid produced the parental chains as well as recombinant chains the translocation model would be eliminated. (2) The demonstration that a stable cloned cell line synthesizes simultaneously two classes of heavy or light chain with the same variable region. (3) The isolation of mutant cell lines which can not link the constant and variable regions. Experiments are in progress to test these predictions. In summary, the following findings are not expected if the simple hypothesis is made that the subunits are synthesized as single polypeptide chains. (i) Heavy and light ch ains are synthesized on polysomes containing 11 and 4 ribosomes, respectively (Fig. 1). These are smaller than expected for the synthesis of proteins of molecular weight 48,000 and 23,000. We have analyzed this finding by pointing out that the state of packing of the message with ribosomes is not what is in question, but the size relationship between the heavy and light chain polysomes. If tetramers make light chain, octamers, not the observed undecamers, (Fig. 1) should make heavy chain. (ii) There is an anomalously increased specific activity associated with nascent chains on steady-state labeled tetrameric ribosomes (Fig. 3). This can be explained if isot’opically labeled protein taken from a pool were associated with these polysomes. (iii) A protein of 12,000 molecular weight and sharing antigenic determinants with light chain is specifically precipitable by an anti-light chain serum from ribosomes making light chain (Figs 2 and 4). (iv) This immunoglobulin fragment is released from ribosomes and enters a small, rapidly turned-over pool (Figs ‘7, 8 and 9). The kinetics of the appearance of immunoglobulin fragment in the ribosome supernatant are compatible with a two-growing point model, but not with an origin based on light chain catabolism. Furthermore, its removal from the pool is dependent on protein synthesis (Fig. 10). It is well to close with a note of caution. These studies do not formally establish that the variable and constant regions are linked at the peptide level. Rather they stress that the generally accepted view that it occws by translocations at the DNA level should be considered an open question. This work was supported by a National Institutes Training Grant no. CA0521301 to one of us (M.C.).
of Health grant no. A105875, and a
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