IMMUNOGLOBULlN NASCENT CHAINS
Mestecky, J., ('Zikan,J., and Butler, W. T. (1971), Science 171, 1163. Mihaesco, E., Seligman, M., and Frangione, B. (1971), Nature (London),New Biol.232,220. Miller, F., and Metzger, H. (1966), J. Bioi. Chem. 241,1732. Shuster, J. (1971), Immunochemistry 8,405. Smyth, D. S., and Utsumi, S. (1967), Nature (London) 216,332. Tomasi, T. (1972), Progr. Allergy 16,81. Virella, G., and Parkhouse, R . M. E. (1971), Immunochemistry
8,243, Warner, N. L., and Marchalonis, J. J. (1972), J. Immunol. 109,657. Weber, K., and Osborn, M. (1969), J. Bioi. Chem. 244,4406. Williams, D. E., and Reisfeld, R . A. (1964), Ann. N . Y. Acad. Sci. 121,373. Wofsy, L., andBurr, B. (1969), J . Immunol. 103,380. Wolfenstein-Todel, C., Frangione, B., and Franklin, E. C. (1972), Biochemistry I I , 3971.
Purification and Characterization of Nascent Chains from Immunoglobulin Producing Cellst D. Cioli$ and E. S. Lennox*
A method is described for preparing immunoglobulin nascent chains from mouse myeloma cells. Nascent chains are isolated by ion-exchange chromatography based on the strongly acidic charge conferred to peptidyl-tRNA by its nucleic acid moiety. The method is rapid, simple, gives essentially quantitative recoveries, and permits the subsequent serological isolation of a single protein species. Peptidyl-tRNAs isolated in this way have chromatographic ABSTRACT:
T
he study of early events in immunoglobulin biosynthesis by mouse myeloma cells requires a reliable method of preparing nascent polypeptide chains in good yields, uncontaminated by other cell proteins, and identifying them by specific antisera. Currently used techniques range from one based on assuming that after short-term labeling all protein radioactivity in the polysome region of a sucrose gradient is nascent chains, to more elaborate procedures of degradation and analysis of the polysomal complexes. However, ribosome preparations are usually contaminated by nonribosomal components, and we show that such contamination confuses kinetics of protein synthesis even for short labeling periods. Methods that recover nascent chains after ribonuclease attack or, alternatively, salt precipitation of the nucleic acids and subsequent recovery of the protein components give poor yields of a mixture of nascent polypeptides with ribosomal proteins and possible other ribosome contaminants. A more selective approach is the release of nascent proteins from isolated polysomes by addition of puromycin in uitro. This has the disadvantage of inconsistent and generally low recoveries. To avoid these difficulties, we adapted to myeloma cells
t From The Salk Institute, San Diego, California 92112, and the Laboratory of Cell Biology, C.N.R., Rome, Italy. ReceicedJune 2, 1972. The work reported in this paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer. Research funds were provided through a grant from the National Institutes ofHealth (AI-06544) to Dr. E. S. Lennox. $ Present address: Laboratory ofCell Biology, 00196 Rome, Italy. * Address correspondence to this author at The Salk Institute.
properties which are sensitive to alkali and ribonuclease. They form a rapidly turning over pool on ribosomes, are released upon puromycin treatment, turnover is blocked by actidione, and they have a disperse size profile on sodium dodecyl sulfate-acrylamide gel electrophoresis. An immunoglobulin fraction devoid of tRNA is also present on ribosomes and some of its unusual chemical, kinetic, and physical characteristics are also described.
a method devised for isolating nascent protein chains from bacterial lysates (Ganoza and Nakamoto, 1966) based on ion-exchange chromatography at pH 4.7 of peptidyl-tRNAs cia the negative charge of the RNA moiety at this pH. The method is easily adapted to the analysis of myeloma cells, is rapid, and permits a quantitative recovery of nascent chains. Subsequent serological methods allow isolation of a single protein species (a light-chain immunoglobulin in this study). Chemical, kinetic, biological, and physical characteristics of the nascent chain fractions thus isolated are in accord with its identification as peptidyl-tRNA. Other proteins, associated with ribosomes, but not identifiable as nascent chains, were also investigated. In the accompanying paper (Cioli and Lennox, 1973), these methods of isolating and identifying nascent chains are used to determine whether light chain is synthesized on membrane-bound or free ribosomes. Materials and Methods A flow sheet of the procedures is given in Figure 1. Cells and Cell Suspensions. MOPC-46 tumors producing a K-type light chain (Melchers et ai., 1966) were maintained by subcutaneous transfer in female Balb/c mice and used 2-3 weeks after transfer. Transplant generations 42-51 were used for this study. Cell suspensions from freshly excised tumors were prepared as described in Choi et ai. (1971a) in Eagle's medium (Dulbecco modified) without leucine and supplemented with 2.5 % undialyzed horse serum and freshly dissolved glutamine. Cell densities between 1 and 5 x lo7 cells/ml were used for the short incubations (1-5 min) and between 2 and 8 x lo6cells/ml for longer incubations. BIOCHEMISTRY,
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ClOLl AND LENNOX MOPC-46 CELL SUSPENSION + 3H-LEU SPIN 1
0 x~ 5 ~ MIN i
PI CELLS + I % NP-40
SI MEDIUM
SPIN 104q x 10MIN
p2
SPIN 16 HRS AT 140,OOOg
RIBOSOME SUPERNATANT (“CY TOPLASM”)
p3
-
J
i
WASH I x IN BUFFER WITHOUT SUCROSE 1 p4 $4
r
”
RIBOSOMAL PELLET I’
4 ADD: 0 . 0 5 % SDS 6 M UREA 0.1% BRIJ-35 0.1 M FORMATE pH 4.7 0.1 M NaCl SPIN 2
1 0 ~ 9x
s MINUTES
INCREASE NaCl FROM O.I--I.OM WASH SAME BUFFER
3 STICKING CPM 4](11~~$${
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SPIN ?O CLEAR SEROLOGY
FLGURE 1 : Scheme
of cell fractionation for the analysis of total ribosomes. For details, see Materials and Methods.
[ 3H]Leucine Incorporation. Cell suspensions were preincubated in plastic petri dishes for 10-15 min in the leucine free medium at 37 in a humidified atmosphere of 95 % air5 % CO,. [3H]Leucine (Schwarz BioResearch, 40 Ci/mmol) was added in a small volume of medium to a final concentration of 25-250 pCi:“l. Incorporation was stopped by quickly pipetting the cell suspension into 5 vol of ice-cold medium. Cells were pelleted (PI) by centrifugation gt lOO0g for 5 min at 4”.The [ 3H]leucine-containing supernatant (SI) was sometimes reused for new incubations. Cell Fractionafion. Lysis of cells and all subsequent operations were performed at 0-4” in a buffer consisting of 0.075 M NaC1-0.01 M MgCl-0.025 M phosphate (pH 6.7) (pH 6.7 buffer) (Becker and Rich, 1966). This buffer gave the best recoveries of total ribosomal nascent chains. At lower p H , the amount of radioactivity insoluble after detergent lysis increased. At higher pH, radioactivity was released from the ribosomes. The pellet of labeled cells was resuspended in 2 ml of 1 Nonidet P-40 in p H 6.7 buffer and after 10 min at 0 ” centrifuged at 10,OOOg for 10 min. The pellet (P,) was usually counted but not processed any further. To prepare “total cytoplasmic ribosomes” according to Blobel and Potter (1967a,b), the supernatant (S?) was applied to a discontinuous sucrose gradient consisting of 2 ml of 2.0 M sucrose and 2 ml of 1.38 M sucrose in p H 6.7 buffer, and was spun overnight (-16 hr) in the SB-239 rotor of the International B-60 centrifuge at 38,000 rpm (140,00Oga,). The pellet (P3) was resuspended in p H 6.7 buffer and pelleted again (PJ in 1 hr at 180,OOOg,, in the A-321 rotor of the International centrifuge. The sucrose layers and the original sample layer (S,) were pooled into a “postribosomal supernatant” or “cytoplasm.” Analysis of the Ribosornal Pellef. Peptidyl-tRNA was isolated by a modification of the technique of Ganoza and Nakamoto (1966). Without modification, their technique
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released about 50 of [ 3H]leucine radioactivity from plasma cell ribosomes. However, addition of sodium dodecyl sulfate to their solubilizing mixture increased release to near 100 %. To achieve this release, the following solutions were added to the washed ribosomal pellet (P4)in the order given: 0.05 ml of a 10% solution of sodium dodecyl sulfate in HnO; 0.1 ml of 1 M NaCl-1 M formate buffer (pH 4.7); 0.10 ml of 10% Brij-35; 0.25 ml of H 2 0 ; 0.6 ml of a solution of 10 M urea freshly deionized by passage through a mixed-bed Amberlite column (Bio-Rad MB3). The ribosomal pellet was almost completely solubilized in this mixture by using a Bellco homogenizer pestle fitting a screw-capped centrifuge tube. The insoluble residue (P,,, comprising 1-5% of the original radioactivity in the pellet, was separated from the supernatant (Sj) by a 5-min centrifugation at 2000g in a clinical centrifuge. The stepwise addition of reagents to the original ribosomal pellet was adopted to inhibit with sodium dodecyl sulfate the activity of ribosomal nucleases liberated by urea (Spahr and Hollingsworth, 1961). and t o inhibit Nith low -pH formate buffer the stripping of nascent peptides from tRNA (Bresler ef ctl., 1966). The S5 supernatant was then diluted to 10 mi with 0.1 xi formate (pH 4.7). 0.1 M NaCI-6 M urea-0.1 >? Brij-35 (starting buffer), lowering the sodium dodecyl sulfate concentration to 0.05 %, and then passed through a 0.5-ml column of Cellex-E (Bio-Rad, Ecteola, 42 eyuiv,:mg) equilibrated with starting buffer and packed in a standard Pasteur pipet plugged with a small glass-wool ball. This quantity of resin is just sufficient to retain the nucleic acid from the ribosomes collected from 1 to 2 X 10s myeloma cells; more than this overloads the column. An initial 10-ml fraction is collected (fraction 1 ) as the sample is run on. The column is then washed with 3 ml of starting bulkr and the effiuent is collected separately (fraction 2). The material sticking to the resin is eluted by increasing the molarity of NaCl from 0.1 to 1.0 M, leaving unchanged all the remaining ingredients of the buffer (the eluting buffer Brijis 0.1 M formate (pH 4.7)-1 bf NaCl-6 M urea-0.1 35). Most of the adhering radioactivity is eluted with the first 3 nil of buffer (fraction 3) as shown by the low level of radioactivity in the subsequent 3 ml of washing with the same buffer (fraction 4). Serohgicul Anci1j.se.s. Fractions to be analyzed serologically for their L-chain content were dialyzed overnight against PBS and centrifuged to eliminate any insoluble of the C1,CCOOH-precipitable material. About 20- 25 radioactivity M B S l a c during dialysis and an additional small amount (1 --5%) in the subsequent centrifugation. Serology was done by a “sandwich” technique consisting of the addition of a n excess of rabbit anti-L-chain serum to the sample with subsequent precipitation of the antigen-. antibody complexes by sheep (or goat) anti-rabbit serum added in slight excess. As a control, we showed that L chain secreted by MOPC-46 retained its precipitability (98 %) by antiserum after the same treatment used for release and chromatography of nascent polypeptide chains. Characterization of antisera and details of the serological assays have been described by Choi rt al. (1971a). Acrj.lamide Gel Elrcfrophoresis. A serological precipitate of the fraction to be analyzed was washed two times with PBS by resuspending in 3 ml of PBS and centrifuging 5 niin at 10,000g. The pellet was then completely reduced and alkylated and run on a sodium dodecyl sulfate containing gel 1 Abbreviation used is: PBS, phosphate-buffered saline (0.5 0.01 M potassium phosphate buffer (pH 8)).
!d
NaCL-
IMMUNOQLOBULIN NASCENT CHAINS
TABLE 1 :Characterization
of Ecteola Fractions.= C
B
A Fraction (Concn (M))
z
Expt 1 Input 122,100 l(O.1) 6,000 6 . 9 2 (0.1) 400 3 (1 .O) 82,300 93.1 4(1.0) 4,600 Recovery 93,300 76.4 Expt 2 Input 70,000 l(O.1) 3,550 8 , 4 2 (0.1) 470 3 (1 .O) 40>160 91.6 4 3,490 Recovery 47,670 68.1 _
_
~ ~
___
Fract 3 Rerun cpm
Fract 3 RNase cpm
8000 100 90 5500 820 6510
8000 4880 500 250 120 5750
_____
Ribosomal Pellet cpm
D
z
9000 70 50 4790 510 5420
2’9
97.1 81.4
2’2 97.8 60.2
z
F
E
+
+
~
Fract 3 pH 10 cpm
z
8000
+
Fract 1 Rerun “Cytoplasm” on A New Column Cold Ribosomes cpm CPm
z
6000
93.5
5470 440
96.1
4800 390
96.5
6’5 71.8
180 60 6150
3.9
150
4.4
78.6
5430
90.5
9000 5970 520 200
95.7
6780
75.3
z
9000
4’3
420
250 80 6890
65,000 57,320 4,670
95.2 4.8
gg,7 0.3
‘lo 60 62,160
76.6
~
~
~
95.6 ~
_
_
Results were from two independent experiments. MOPC-46 cells were labeled for 1 min with [3H]leucine and a “ribosomal pellet” prepared according to Figure 1. Column A: the ribosomal pellet was fractionated on Ecteola as a described in Figure 1. Three equal aliquots from fraction 3 were diluted with HzO, 10 M urea, formate buffer, and Brij-35 to lower the NaCl concentration to the original 0.1 M of the Ecteola “starling buffer” and leave the other components unchanged. One aliquot was kept at 37” for 30 min and then reapplied directly to a new column (B); one aliquot was treated with 10 pg/ml of pancreatic ribonuclease for 30 min at 37” before reloading on a new column (C); one aliquot was brought to pH 10 with NaOH, left at 37” for 30 min, adjusted back to pH 4.7 with formic acid. and applied to a new column (D). Fraction 1 from experiment 1 was reapplied directly to a new Ecteola column (E). In column F, 0.05 ml of the [3H]leucine ribosome supernatant (“cytoplasm”) from experiment 2 was added to an unlabeled ribosomal pellet and the mixture processed as described in Figure 1 and applied to Ecteola in the usual 10 ml of “starting buffer.” All the values are total C13CCOOH-precipitableradioactivity. a
(7.5 % acrylamide) using the procedure of Maize1 (1966). A small amount of reduced and alkylated 14C-labeled yglobulin was usually added to the sample as internal markers for heavy- and light-chain positions. The gels were usually about 7 cm long, but only the 5-cm portion farthest from the origin was cut into 50 slices, while the remaining portion was counted as a single piece together with slice 1. Radioactivity Measurements. Aliquots to be counted were precipitated with 10% C1,CCOOH in the presence of carrier bovine serum albumin, twice redissolved in 0.2 N KOH and precipitated with C1,CCOOH and finally dissolved in 0.5 ml of 0.2 N KOH and counted in a Nuclear-Chicago counter after addition of 10 ml of a scintillation cocktail containing naphthalene (1800 g), butyl-PBD (96 g), Cab-0-si1 (420 g), andp-dioxane to bring thetotal volume to 12 1. Whenhydrolysis of aminoacyl-tRNA was required, the samples were left at 37” for 30 min in 0.2 N KOH. Dissolving and counting of acrylamide gel slices were performed according to Choules and Zimm (1965). All the values given are corrected for background and IH/14Ccross-contamination. Chemicals. Protease-free pancreatic ribonuclease A and puromycin dihydrochloride were purchased from Sigma. Actidione was purchased from Calbiochem. Results Ecteola Isolation of Peptidyl-tRNAs. Table I presents the results of two experiments showing that the [ 3H]leucine-labeled proteins from the ribosomal fractions that absorb to Ecteola do so via their covalently bound tRNA
moiety. Comparison of radioactivity among the various Ecteola fractions (Table I, column A) indicates that after short [3H]leucinepulses (1 min) more than 90% of the labeled material in the ribosomal pellet adheres to the column and can be eluted by increasing the NaCl molarity. This material (fraction 3), diluted to restore the low salt molarity of the starting buffer, and reapplied to a new column was-as expected-almost completely retained by the resin (Table I, column B). But when an aliquot of the same material was treated with ribonuclease before being rerun, essentially all radioactivity was now recovered in fraction 1 (Table I, column C), an indication that absorption to Ecteola of the radioactive material is via ribonucleic acid. The results in column D of Table I yield a similar conclusion. Treatment of the sample at pH 10 for 30 min at 37” destroys its ability to adhere to Ecteola at low salt concentration. This indication that the linkage between the protein and the RNA responsible for its chromatographic behavior is alkali labile is in accord with the known property of the ester bond between tRNA and nascent polypeptides (Zachau et al., 1958). An additional control further characterized the chromatographic behavior of fraction 1 (experiment 1, Table I (column E)). In a second passage on a new column essentially all the radioactivity again passes through and thus is different from fraction 3. Chromatographic behavior of cytoplasmic proteins was examined in another experiment (experiment 2 in Table I (column F)). An aliquot of the supernatant after removing ribosomes (SI) was applied to the Ecteola column under the same conditions as ribosomal material. No radioactivity was retained by the column. BIOCHEMISTRY,
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FIGURE 2: Kinetics of L-chain labeling in Ecteola fractions. A batch of MOPC-46 cells was incubated with [3H]leucine at 37". At the times indicated an aliquot of the cell suspension was quickly pipeted into 5 vol of ice-cold medium and kept in ice until the end of the incubation. Cells from each time point were processed separately in parallel tubes, as indicated in Figure 1. Ecteola fractions were prepared and their L-chain content determined by serology. Values given represent net (specific minus nonspecific) radioactivity precipitable as L chain.
From these results we conclude that Ecteola columns as used selectively absorb polypeptides covalently linked t o tRNA molecules and thus allow isolation of nascent polypeptide chains from solubilized ribosomes. Kinetics of Lubeling of Ecteola Fractions. In Table I it is shown that after a 1-min pulse less than 10% of the radioactivity in the ribosomal pellet is in fraction 1. Experiments were done to measure the amount and kinetics of labeling of the light chain. In Figure 2 are presented the data from the serological analysis of the Ecteola fractions. By contrast, Table I lists the total C1,CCOOH-precipitable radioactivity in the fractions. In this and subsequent experiments total counts per minute of fraction 2 are pooled with those of fraction 1, and similarly fractions 4 and 3 are pooled. No striking differences were found in the relative L-chain content of each fraction after different labeling times; net L-chain content (specific minus nonspecific values) of fraction 1 was found to be roughly 10% of the total C1,CCOOH-precipitable proteins, whereas in fraction 3 it fluctuated around the average of 20 % of total protein. The amount of L chain in fraction 3 very quickly (1-2 min) approaches a constant value. Such kinetics are expected for nascent polypeptides, with the total amount of label determined by the number of active ribosomes and the total number of leucine residues in the chains. Moreover, the time required to reach the constant value is very close to the average synthetic time previously measured by the rate of labeling of different tryptic peptides of this L chain (Lennox et ul., 1967). In fraction 1, on the other hand, not only does the total amount of C1,CCOOH-precipitable radioactivity increase with time, perhaps due to progressive labeling of structural ribosomal proteins, but so does the amount of serologically precipitable L-chain radioactivity continue to increase. In
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3: Effect of antibiotics and "chase" after a short pulse ( 5 min). Cells were labeled with [3H]leucinefor 5 min at 37"; the cell suspension was then split into four aliquots and incubated for 5 min after the following additions: (0-0) no addition (control) ; (0-0)addition of a 1000-fold excess of nonradioactive leucine ("chase"); (A---A) addition of 5 X M puromycin; (E---E) addition of 5 X M actidione. Cells were lysed with Nonidet P40 and an aliquot of the lysate was used to determine total C1,CCOOH-precipitable radioactivity incorporated (A). Ecteola fractions were prepared from the rest of the lysate and their L-chain content determined by serology (values in B and C represent net radioactivity in L chain).
FIGURE
the experiment of Figure 2 , L-chain radioactivity of fraction 1 increases almost linearly for the first 30 min and after about 20 min of labeling it roughly equals the amount in fraction 3. In other experiments, the time at which the L-chain content of the two fractions became equal was rather variable (from 10 to 30 min), and the slope of the labeling curve of fraction 1 was constant for only about 30 min, after which it decreased. We did other experiments to determine the relationship of the L chain in fraction 1 to that in fraction 3 and to extraribosomal material. Effect of Pulse Chase and cf Antibiotics. Cells were labeled with [3H]leucine for 5 min at 37" and divided into four aliquots: one, the control, was unchanged; to a second, for a chase, was added a large excess of nonradioactive leucine; to a third, puromycin was added; and to a fourth, actidione was added. All were then incubated for another 5 min a t 37". In Figure 3A it is shown in the form of a graph that total CI3CCOOH-precipitableradioactivity in the control continues to increase, doubling from 5 to 10 min, whereas the chase and both antibiotics treatments inhibit almost completely further uptake. The kinetic behavior of the Ecteola fractions from these cells is also shown in Figure 3. The kinetics of fraction 3 (Figure 3C) are those expected from nascent chains. Between 5 and 10 min, the amount of radioactivity in L chain remains essentially constant in the control, in accord with the experiment of Figure 2 . It also remains unchanged after the addition of actidione, as expected from its known inhibition of protein synthesis without disruption of polysomal struc-
IMMUNOGLOBULIN NASCENT CHAINS
I
1
FIGURE 4: Effect of “chases” after short- and long-term labeling. A cell suspension was labeled with [3H]leucine; aliquots were withdrawn after 5 min (-) and 30 min (.---.) of labeling and “chased” with a 1000-fold excess of nonradioactive leucine for 5 and 30 min more. Ecteola fractions were prepared and their L-chain content determined by serology. Values given represent net (specific minus nonspecific) radioactivity in L chain.
tures or release of nascent chains (Wettstein et al., 1964; Trakatellis et af., 1965; McKeehan and Hardesty, 1969). In cells treated with puromycin, on the contrary, essentially no radioactivity (and no L chain) remains in fraction 3, as expected from the known effect of this antibiotic in releasing peptidyl-tRNA from ribosomes as a consequence of its structural competition with tRNA (Nathans, 1964). A rapid loss of radioactivity in fraction 3 is also obtained after chase with nonradioactive leucine. This is also expected of nascent chains still present on ribosomes since between 5- and 10min labeled L chains have been replaced by new chains containing essentially nonradioactive leucine, In the control, fraction 1 (Figure 3B) shows a n approximate doubling of counts per minute in L chain (as in Figure 2) during the period from 5 to 10 min but, unexpectedly, puromycin, chase, and, to a slightly lesser extent, actidione treatment also decrease the amount of L-chain radioactivity a t 10 min t o less than half the amount a t 5 min. These kinetics imply that not all of the L chain in fraction 1 is a cytoplasmic contaminant of ribosomes, but that at least half might be a rapidly turning over ribosomal intermediate lacking tRNA. The results of the puromycin and chase experiments may indicate that part of fraction 1 is nascent chains stripped of their tRNA during isolation, thus really belonging to fraction 3. The results with actidione, however, seem to rule out this possibility for L-chain radioactivity in fraction 3 remains constant, but drops in fraction 1 . An experiment designed to find out whether fraction 1 is a mixture did show two kinetically different components : a relatively small, chaseable one comprising a constant amount of radioactivity irrespective of the length of the labeling period, and a nonchaseable one steadily increasing in size with increasing labeling time. This is shown in Figure 4. A cell suspension was labeled with [3H]leucine for 5 min gnd then a n aliquot chased with nonradioactive leucine; another aliquot was labeled for 30 min before chasing in the
FRACTION X I
FRACTlONX3
‘\y,0x PURO CHASE
! t
30
60
MIN
’H L E U
FIGURE 5 : Effect of antibiotics and of “chase” upon long-term labeled cells. Cells were labeled with [3H]leucinefor 30 min and then subjected to 30 more min of incubation after the following additions: (0-0) no addition (control); (0-0) addition of a 1000-fold excess of nonradioactive leucine (“chase”); (A---A) addition of 5 x actidione. Ecteola lo-‘ M puromycin; (o-0) addition of 5 X fractions were prepared and their L-chain content determined by serology. Values given represent net (specific minus nonspecific) radioactivity in Lchain.
same way. From each sample, a ribosome protein fraction was prepared, fractionated on Ecteola, and analyzed serologically for L chain. The lower part of Figure 4 shows that in fraction 3 the total amount of nascent L chains remains essentially constant between 5 and 30 min, as expected. Irrespective of the length of the incubation, essentially all the radioactivity disappears permanently from fraction 3 (and obviously from its L chain) as soon as a large excess of cold leucine is added to the medium. Fraction 1 (upper portion of Figure 4) shows, in the solid curve, essentially a repetition of the experiment of Figure 3, i.e., more than half of the radioactivity incorporated in 5 min of labeling is chased in the subsequent incubation with cold leucine. When the labeling period is extended to 30 min (dotted curve) approximately the same absolute amount of L chain is lost in the first 5 min of chase, but it is now a much smaller proportion of the original radioactivity. Moreover, when the chase period is prolonged to 30 min, fraction 1 seem to regain its original amount of radioactive L chain, a phenomenon which was not noticeable after 5 min of labeling. These data suggest that fraction 1 contains a t least two components: one, chaseable, is perhaps an intermediate just detached from its tRNA prior to passing to the extraribosoma1 stage, and the other, nonchaseable, increasing in specific activity with time is perhaps a contamination of ribosomes by extraribosomal L chains. An experiment was designed to determine whether the latter component requires the presence of nascent light chain on ribosomes and is thus a result of dimerization or polymerization between nascent and released L chains. To decide this, the behavior of fraction 1 was analyzed under two differBIOCHEMISTRY,
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V
% I
I
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I
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X
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I
2
2
X
E
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30
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r
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SLICE #
SLICE #
Sodium dodecyl sulfate-acrylamide gel electrophoresis of serological precipitates of various MOPC-46 fractions. Cells were labeled with [3H]leucinefor the following times: A, 6 hr; B, 1 hr; C and D. 5 min. A serological precipitate was obtained from the extracellular fluid (secreted), from the postribosomal supernatant (cytoplasm), and from Ecteola fractions 3 and 1 , respectively. The precipitate was reduced, blocked with iodoacetamide, and run on sodium dodecyl sulfate containing gels (7.5 %' acrylamide) together with a small amount of reduced and blocked MOPC-21 y G (secreted) which had been labeled with [ Wlleucine. Fractions are numbered from the negative (origin) to the positive electrode. FIGURE 6:
ent conditions: after a chase with cold leucine, which leaves nascent unlabeled chains on ribosomes, and after puromycin treatment, which releases nascent chains from ribosomes. If nascent chains were required to bind the nonchaseable component of fraction 1, puromycin treatment would eliminate this component. Cells were labeled for 30 min and then treated for another 30 min as indicated in Figure 5. In the lower part is shown the usual behavior of the nascent L chains of fraction 3 after chase. In fraction 1 (Figure 5, upper part) the radioactivity remains almost constant after 30 min of chase (same result as in Figure 4) as well as after 30 min of puromycin, thus indicating that nascent chains are not needed to bind fraction 1. The slight loss of radioactivity after actidione treatment is not easy to interpet. Size of Various L-Chain Fractions. Secreted MOPC-46 L chain is a glycoprotein containing about 12% by weight of carbohydrate (Melchers et al., 1966). The intracellular form does not contain the full carbohydrate complement and the various component sugars are added in a characteristic sequence during the intracellular transport of the L chain (Choi et al., 1971b). The presence of large amounts of carbohydrate on the relatively small L chain confers to this glycoprotein a characteristic mobility in sodium dodecyl sulfate-acrylamide gel electrophoresis, a sensitive technique known to separate different molecules according to their size (Shapiro and Maizel, 1969). Experiments were done to compare the sizes of MOPC46 light-chain fractions in various stages of completion with
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other completed light chains. A small amount of reduced and alkylated yG with [ 'T]leucine was added to the tested fraction as internal marker for the H- and L-chain positions. The H and L chains of MOPC-21 have the same mobility as the corresponding subunits of Adj-PC-5 yG where the H chain has carbohydrate and the L chain does not (Parkhouse, 1969). Disregarding possible minor differences in the total number of amino acid residues in the various L chains (Gray et al., 1967) the mobility of MOPC-21 L chain is taken as that of a light chain without carbohydrate. Figure 6A shows that the electrophoretic mobility of secreted MOPC-46 L chain containing a full complement of carbohydrate is intermediate between the mobility of the marker H and L chains (it runs only 5 0 z of the distance between H and L chains). Cytoplasmic (postribosomal) L chain (Figure 6B) is slightly smaller than the secreted one (it runs about 57-64z of the H-L distance) and in other experiments is seen to be somewhat more heterogeneous. In both profiles there is a hint of minor peaks corresponding to the marker H and L chains; these peaks are even more pronounced in the subsequent profiles of Ecteola fractions (Figures 6C and 6D). These minor peaks are probably due to a small proportion of cells synthesizing both H and L chains. Figure 6C shows the profile obtained from a serological precipitate of Ecteola fraction 3. Almost all the radioactivity runs faster than the marker L chain of MOPC-21 and is distributed in a rather broad region of the gel rather than in sharply localized peaks. This behavior correlates with the expected heterogeneity of size of nascent chains. One might
IMMUNOGLOBULIN NASCENT CHAINS
guess that little or no carbohydrate is added even to the semicomplete nascent chains since very little 3H radioactivity runs slower than the marker 14Clight chain. Since the sensitivity of this method to detect a small amount of carbohydrate in the nascent chains is unknown, other kinds of experiments are needed to make this conclusion firmer. The electrophoretic profile of fraction 3 was quite variable from one experiment to the next. Always seen were the broad dispersions of radioactivity with sizes smaller than the marker L chain ; however, the biphasic distribution apparent in Figure 6C was often not seen. There are at least two possible sources of loss of light-chain related material. Fraction 3, originally in urea-detergent buffer, is dialyzed against PBS before serological analysis. Unfinished chains not soluble in PBS or small enough to escape during dialysis would be selectively lost. Second, some of the shorter polypeptide chains might not possess enough antigenic determinants to be precipitated by the anti-L-chain serum in the sandwich technique. Figure 6D shows the electrophoretic profile of a serological precipitate of Ecteola fraction 1 obtained after 5 min of [3H]leucine labeling. An analogous fraction after 60 min of labeling gave essentially the same pattern, except for a slight relative increase of the major peak. Most of the L chain appears to be of the cytoplasmic type, i.e., displaced to the larger side with respect to the marker L chain and rather heterogeneous in size. The amount of radioactivity of the nascent chains size, small in this experiment, was even larger in other experiments. This may be due to contamination of fraction 1 with nascent chains. In any case, the acrylamide results confirm that fraction 1 is probably composite and at least part is of the cytoplasmic type of L chain with a large amount of attached carbohydrate. Polysomes Fractionated on Continuous Sucrose Gradients. We wanted to eliminate the possibility that fraction 1 is an artifact due to contamination of the ribosomal pellet by other cellular fractions during separation on the discontinuous gradients. To do this we fractionated MOPC-46 lysates on continuous sucrose gradients. While this presented some technical difficulties, some of the basic experiments were repeated using ribosomal pellets obtained by pooling, diluting, and centrifuging appropriate fractions from such gradients. After chase or puromycin treatment, there was a certain amount of radioactivity remaining especially in the monoribosomes region. This radioactive material contained L chains, tended to increase with increasing lengths of labeling, and, when analyzed on Ecteola, was found exclusively in fraction 1. It seems unlikely, therefore, that fraction 1 is an artifact due to the use of discontinuous sucrose gradients. Discussion Analysis of early events in the synthesis of a protein requires reliable methods of isolating and identifying nascent chains on ribosomes and of distinguishing them from unrelated proteins or from related ones belonging to later stages of synthesis and secretion. The methods described above and applied here and in the following paper (Cioli and Lennox, 1973) to biosynthesis of immunoglobulin light chain seem to do this. The urea-detergent method of solubilization of ribosomal pellets yields 95-99 % of labeled protein and of this more than 90% is recovered as nascent chains (fraction 3) after short pulses with [3H]leucine (Table I). The losses in preparing nascent chains for serological analyses seem to affect all proteins more or less uniformly, since the amount of nascent L chain isolated by antiserum is about 20% of
total nascent chains, which is only slightly lower than the relative amount of L chain detected in the cytoplasm of the MOPC-46 cells after pulse labeling (Choi et a f . ,1971a). That the fraction identified as nascent chains is in the form of peptidyl-tRNA was shown by the dramatic change in its chromatographic properties (Table I) after alkali or ribonuclease treatment. On the other hand, finished chains released in the cytoplasm do not stick to Ecteola at pH 4.7, as shown by experiment 2 of Table I (last column). That the same is true for ribosomal structural proteins is shown by the fact that ribosomal pellets depleted of nascent chains by the action of puromycin (Figures 3 and 5 ) do not contribute any significant amount of radioactivity to the nascent chain fraction 3. The kinetic properties of the nascent light chain fraction are well in accord with the behavior expected from nascent MOPC-46 light chains (Lennox et al., 1967). Not only does the amount of radioactivity reach a maximal plateau value in 1-2 min of incorporation of [3H]leucine (Figure l), but also chase experiments (Figures 3 and 4, and 5) quickly remove all the radioactivity from the nascent chains fraction. Both actidione and puromycin produce an essentially complete inhibition of protein synthesis in MOPC-46 (Figure 3A). But their different effects upon the nascent chains fraction (Figure 3C) are further confirmation of the proper identification of this fraction. The rapid decrease with puromycin and constancy with actidione are in accord with the known mechanism of action of the two drugs, since puromycin causes release of nascent chains from ribosomes (Yarmolinsky and De La Haba, 1959; Nathans and Lipmann, 1961 ; Nathans, 1964), whereas actidione freezes the whole polysomal structure (Wettstein et al., 1964; Trakatellis et al., 1965; McKeehan andHardesty, 1969). Using acrylamide gels with sodium dodecyl sulfate and appropriate I4C markers, the [ 3H]leucine radioactivity of nascent L chain was shown to be heterogeneous with sizes from that of a complete L chain (without carbohydrate) to that corresponding to smaller peptides. The distribution of radioactivity in some experiments showed a clearly biphasic profile suggestive of models of synthesis in which modulation of translation or subunit assembly of L chain plays a role (Schubert and Cohn, 1970). Alternatively, selective degradation of chains on ribosomes was a possibility. However, the very irregular reproducibility of the biphasic peaks and our failure to find them at all in two other myeloma lightchain producing cell lines (unpublished data) leads us to interpret this as an artifact possibly due to preferential recoveries of nascent chains of certain lengths during the dialysis or serological isolation. We thus feel that the use by Schubert and Cohn (1970) of such data as evidence for a light-chain subunit is suspect. While the behavior of fraction 3 from Ecteola allows its identification with nascent chains, that of fraction 1 indicates the presence of components with different kinetic behavior. Most of the [3H]leucine radioactivity is in fraction 3 after short-term incubations; in fraction 1 there is an approximately linear increase with increasing time of labeling, until more labeled L chain can be recovered in fraction 1 than in fraction 3 (Figure 2). The continuous increase of fraction 1 well after fraction 3 has reached a plateau perhaps reflects that fraction 1 is comprised of finished L chains released from tRNA and contaminating the ribosomes after having passed to another fraction. In this case, the increase in radioactivity of fraction 1 would be due to the increasing specific activity of cytoplasmic L chains. In addition to this component, there BIOCHEMISTRY,
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is a rapidly turning over one. In fraction 1 light chain labeled for 5 min this constitutes more than 50% of the total and is chaseable in a subsequent 5-min incubation with nonradioactive leucine (Figure 3). In light chain labeled for longer periods (Figure 4), this component stays constant in absolute amounts but of course becomes a smaller fraction of the total of fraction 1. From the data of Figures 3 and 4, the amount of this chaseable intermediate is estimated to be between 10 and 20% of the total pool of nascent light chain. An attempt a t showing possible size (;.e., carbohydrate content) differences between this small “intermediate” component and the rest of fraction 1 was unsuccessful. While the present data d o not rule out the chaseable component of fraction 1 as a contaminant from nascent chains of fraction 3, this seems unlikely because after actidione treatment (Figure 3 ) radioactivity drops in fraction 1 but remains constant in fraction 3. Thus, the rapidly turning over portion of fraction 1 suggests that finished polypeptides spend a short time still associated with ribosomes even after the loss of tRNA. In any event, it is clear that with increasing labeling time, more and more radioactivity with the kinetic and physical (Figure 6D) characteristics of the cytoplasmic light chain is present in the ribosomal pellet and is eluted with Ecteola fraction 1. The possibility that finished cytoplasmic L chains returned to the ribosomes by dimerization or polymerization with nascent light chains was ruled out (Figure 5 ) . This leaves open how finished light chains can stick on ribosomes, but it is not surprising that such an adsorption occurs. in view of the finding on ribosomes of various enzymes (Spahr and Hollingsworth, 1961), protein synthesis factors (Gasior and Moldave, 1965), carbohydrates (Molnar et ul., 1965), soluble proteins (Warner and Pene, 1966), and artificially mixed immunoglobulin (Moav and Harris, 1970). In any event, the presence of a significant amount of nonnascent light chains on ribosomes emphasizes once again the need for more refined techniques, like the one described here in the study of bona fide growing polypeptides. Acknowledgments The authors thank Mr. Raleigh Austin for his technical assistance and Dr. Paul Knopf and other members of the Institute for their helpful discussions during the course of the study. References Becker, M J., and Rich, A. (1966), Nu/rrre (London) 212. 142.
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