Comparison of immunoglobulin chains made in ascites extract and reticulocyte lysate programmed with mRNA from four mouse myelomas

303

Biochimica et Biophysica Acta, 476 (1977) 303--320 © Elsevier/North-HollandBiomedicalPress

BBA 98937

COMPARISON OF IMMUNOGLOBULIN CHAINS MADE IN ASCITES EXTRACT AND RETICULOCYTE LYSATE PROGRAMMED WITH mRNA FROM FOUR MOUSE MYELOMAS *

B.J. S C H M E C K P E P E R

**, S U Z A N N E C O R Y and J E R R Y M. A D A M S

* F r o m the Biochemistry and Biophysics Unit, Walter and Eliza Hall Institute,P.O. Royal Melbourne Hospital, Melbourne, Victoria, 3050 (Australia)

(Received December 27th, 1976)

Summary To learn more about the cell-free synthesis of immunoglobulin (Ig) chains, the translation of mRNA from microsomes of four murine plasma cell tumors (myelomas) has been compared in a supplemented Krebs II ascites cell extract and in a rabbit reticulocyte lysate. (i) In the ascites system, translation of Ig light chain mRNAs was enhanced by addition of 18-S rRNA, ascites tRNA and KC1 concentrations higher than the optimum for total amino acid incorporation. Reticulocyte initiation factors strongly stimulated translation of exogenous and endogenous mRNAs. The background of endogenous incorporation was not eliminated by preincubation of the extract. (ii) Messenger RNA from each of the myelomas directed synthesis predominantly of light chains and their presumptive precursors, P chains, in the ascites system. The P chains ranged in size from 1300 to 2200 daltons larger than the corresponding L chains. In response to mRNA from a particular tumor, P chains synthesized in the ascites extract and in the reticulocyte lysate (and in a wheat germ system) were indistinguishable in size. (iii) Small amounts of large polypeptides serologically related to a and 7 heavy chains were made in the ascites system and large amounts of an a chain related product were made in the reticulocyte system, probably in a precursor form.

* Publication No. 2249. ** Present address: T r a y l o r R e s e a r c h B u i l d i n g R o o m 9 3 3 , J o h n s H o p k i n s U n i v e r s i t y S c h o o l o f Medicine, 7 2 0 R u t l a n d A v e n u e , B a l t i m o r e , M d . 2 1 2 0 5 , U . S . A .

304 Introduction

Studies on the cell-free translation of mRNAs from mouse myelomas have revealed that the m R N A s for immunoglobulin light chain often yield polypeptides larger than the secreted light chain. These cell-free products, designated here as P chains, are serologically and chemically related to the Ig light chains [1--9] b u t have extra residues at the amino terminus [1,9] and possibly at the carboxy terminus [9]. It has been suggested that P chains correspond to precursors to the light chain [1,2] b u t a precursor-product relationship has n o t been firmly established and it could be argued that P chains are artifacts of cell-free translation, resulting from faulty initiation on the light chain m R N A . Considerably less is known a b o u t the translation of Ig heavy chain m R N A , although there is good evidence that a ~/ chain m R N A can be translated in the reticulocyte system and yields a product which is structurally similar though n o t identical to that secreted by the t u m o r [10]. Similarly, an chain m R N A can be translated in an Ehrlich ascites system, yielding a product antigenically and structurally related to the secreted ~ chain [ 11 ]. As most previous studies have involved translation of the m R N A from a single m y e l o m a in a single cell-free system, it is difficult to compare the results obtained (but see also ref. 11). For example, P chains have been variously estimated to be from 800 to 4700 daltons larger than the corresponding light chains, b u t it is unclear whether this range reflects the different cell-free systems used, errors of measurement, or true variation in size. We have studied the translation of microsomal m R N A from four mouse myelomas in three commonly u s e d cell-free systems. We report here our studies with a supplemented Krebs II ascites extract similar to that described by Metafora et al. [12] and a rabbit reticulocyte lysate and compare the results with those we obtained using a wheat germ extract [6]. Our results strengthen the case that P chains are a general feature of Ig light chain m R N A translation. We also show that both the ascites and reticulocyte systems are capable of some synthesis of ~ and ~ heavy chain-related products. Methods Plasma cell tumors and radiolabelled marker Ig

The plasmacytomas (myelomas) used and their secreted immunoglobulins were MOPC 41A, K light chain [13]; P3K, IgGl (K) [14]; MOPC 315, IgA (~2) [15] ; and HPC-108, IgA (K) [6]. All the myelomas were maintained as subcutaneous tumors in Balb/c mice; the P3K, MOPC 315 and HPC-108 lines were also maintained in continuous cell culture [16] and MOPC 41A in short-term culture [17]. The source of radiolabelled secreted ~mmunoglobulin was the culture fluid prepared by incubating 2.5 • 106--3 • 106 cells for 20 h in 6 ml Dulbecco's Modified Eagle's medium (supplemented with 10% heat-inactivated fetal calf serum) in which [12C]leucine or [32S]methionine had been replaced with 10 pM [~4C]leucine (Amersham, 100 mCi/mmol) or 10 ~M [3SS]methionine (Amersham, 5 Ci/mmol). Krebs H ascites extract

Krebs II cells were stored in ascitic fluid plus 6% glycerol under liquid N2.

305 When cells were needed for extract, 3 • 106 viable cells/mouse were injected intraperitoneally into 2--3 outbred mice (Hall Institute strain). After 1 week, the ascitic fluid was harvested and injected intraperitoneally into 10--15 mice (3 • 104 viable cells/mouse). 7 days later all white ascitic fluids were collected sterilely and added to ice-cold wash buffer [18]. Extract was prepared by a procedure similar to that of Mathews [18] except that after homogenization tonicity was restored by addition of 0.2 cell volume of 175 mM Tris • HC1 (pH 7.5), 725 mM KC1, 25 mM Mg(CH3COO)2, 66 mM 2-mercaptoethanol. Twenty extracts were used in these studies.

Rabbit reticulocyte lysate and initiation factors Reticulocytosis was induced in rabbits with phenylhydrazine [19]. Blood was collected and the cells (generally 80% reticulocytes) were washed as described [20], then lysed by adding 1.5 volumes distilled water. The preparation was stirred for 1 rain, then centrifuged for 15 rain at 27 000 × gma~. The supernatant (lysate) was stored in small portions under liquid N2. Initiation factors were prepared from the 0.5 M KC1 wash of reticulocyte ribosomes as described for IF fraction A by Schreier and Staehelin [20], except that 0.25 M rather than 0.3 M KCI was used in the step elution from DEAEcellulose. Recovery was 1 A2so unit of factors per 500--700 A260 units polysomes. This method yielded more active factors than another procedure [12].

Preparation of mRNAs, rRNA and tRNA Microsomal mRNA from myelomas was prepared as described elsewhere [ 21]. Briefly, microsomes isolated from tumor h omogenates were digested with proteinase K and extracted from phenol/chloroform/isoamyl alcohol. Polyadenylated mRNA was selected from total microsomal RNA by adsorption to oligo(dT)-cellulose. Rabbit globin mRNA Was prepared similarly from saltwashed reticulocyte ribosomes [20]. 18-S RNA was prepared by glycerol gradient centrifugation of myeloma microsomal RNA which did not bind to oligo(dT)-cellulose [22]. Transfer RNA was prepared from Krebs II ascites cells [23].

Cell-free protein synthesis The standard assay with the ascites extract contained (in 50 gl): 1 mM ATP, 0.1 mM GTP, 10 mM creatine phosphate (K ÷ salt), 60 pg/ml creatine phosphokinase, 6 mM 2-mercaptoethanol, 30 mM Tris • HC1 (pH 7.4), 80 mM KC1, 3-3.5 mM Mg(CH3COO)2 (optimized for each extract), 30--200 pM EDTA, 7 uCi/ml of 14 14C-labelled L-amino acids (Amersham), 20--25 pM of each of the unlabelled L-amino acids, 1% glycerol, 100--165 pg/ml ascites tRNA, 100 ug/ml myeloma 18-S rRNA, 4--12 pg/ml mRNA, 5 #1 ascites extract (0.25 A2~0 units), and 5 ~1 (approx. 0.03 A2s0 units) reticulocyte initiation factors. Assay variations are noted in legends. Samples were incubated at 37°C for 1 h and the reaction stopped by chilling. The standard reticulocyte lysate assay contained (in 50 ~l): 20 ~1 lysate, 1 mM ATP, 0.1 mM GTP, 15 ml~ creatine phosphate (K ÷ salt), 60 pg/ml creatine phosphokinase, 6 mM 2-mercaptoethanol, 10 mM Tris • HCL (pH 7.4), 30 pM each of 19 unlabelled L-amino acids, 10 uM [3SS]methionine (Amersham,

306 approx. 30 Ci/mmol), 75 mM KC1, 1.25 mM Mg(CH3COO)2, 40 pM EDTA, 65 pg/ml mRNA and 20 pM heme. Samples were incubated at 30°C for 1 h.

Analysis of cell-free reaction products Hot trichloroacetic acid-precipitable radioactivity was measured as described [24]. Cell-free products (from 20---40 ul of reaction mixtures) were analyzed by disc electrophoresis on 15% polyacrylamide gels containing dodecyl sulfate [25]. Gels were stained, sliced, dried and autoradiographed (Kodak RP/S Xomat film) for 1--52 days [26]. 12SI-Labelled standards [6], run in a parallel gel with as much extract or lysate as experimental gels, had the following molecular weights [27] : bovine serum albumin (68 000), catalase (60 000), glutamate dehydrogenase (53 000), ovalbumin (43 000), glyceraldehyde-3'-phosphate dehydrogenase (36 000), chymotrypsinogen A (27 500), trypsin (23 300) arid rabbit globin (15 500). Ig-related polypeptides among the cell-free products were detected by immunoprecipitation and subsequent electrophoresis. Up to 50 pl of reaction mixture was adjusted to 100 pl with ice-cold buffered saline (0.12 M NaC1, 16 mM Na2HPO4, 4 mM NaH2PO4) containing carrier antigen (either 1 pg of HPC-108 K chain or 1.5 #g of MOPC 315 IgA). To this was added 400 pl of cold buffered saline containing 10--50 ~l of titered antiserum and 3.8% Triton X-100. After 15 h at 2°C, immunoprecipitates were collected and washed 3 times by centrifugation (8000 ×g, 20 min) with cold buffered saline containing 3% Triton X-100 and 1% casein hydrolysate, then dissolved at 37°C in electrophoresis sample buffer [25 ], and analyzed by electrophoresis.

Antisera Non-immune serum was prepared from adult rabbits, which were then injected intramuscularly with antigen in complete Freund's adjuvant as follows: 1 mg (day 1), 1 mg (day 26), 0.1 mg (day 63). Blood was collected on days 49, 66, 73, and 80, and antisera prepared [28]. The antigens used were purified Ig light chains from urine of mice bearing HPC-108 myelomas, a gift from Dr. N.L. Warner, and purified IgA from serum of mice with MOPC 315 myelomas, a gift from Dr. H. Eisen; the resulting antisera were respectively called anti108 K serum and anti-315 IgA (~2) serum. Both antigens were electrophoretically homogenous on dodecyl sulfate-polyacrylamide gels. Each antiserum was titered with its respective antigen, labelled ,with 12sI [29]. At the optimal ratio of antigen to antiserum, the addition of diluted Cell-free components (previous section) did not affect the efficiency of precipitation of either 12SI-labelled antigen. The antisera were then tested for cross-reactivity with 125I-labelled proteins. The anti-108 K serum had some cross-reactivity with MOPC 315 IgA: on a molar basis, it was 3.4 times as efficient in precipitating HPC-108 light chains as MOPC 315 IgA. Similarly, the anti-315 IgA (~2) serum on a molar basis was 12.5 times as efficient in precipitating MOPC 315 IgA as 108 light chain. These cross-reactions are most likely due to a slight contamination of the HPC-108 light chain preparation by fragments of HPC-108 ~ chain. Sera from unimmunized rabbits did not precipitate detectable amounts of 12SI-labelled MOPC 315 IgA, HPC-108 K chain, or IgG myeloma proteins, even when mixed with components of the cell-free systems. An adsorbed antiserum specific for murine

307 7~ heavy chains (anti~l serum), a gift from Dr. N.L. Warner, did not precipitate 12SI-labelled MOPC 315 IgA or an IgG2 myeloma protein. Results

Enhanced translation in an ascites system

Our initial studies on the translation of myeloma microsomal mRNA using an unsupplemented ascites system [18] gave very poor synthesis of Ig chains, so it was necessary to improve the system. Addition of rRNA has been reported to enhance translation, apparently by slowing degradation of mRNA by ascites ribonucleases [30]. In the system used here, 18-S rRNA at 100/~g/ml stimulated total incorporation in the presence of globin mRNA or myeloma (P3K) mRNA by only 10--20%, while endogenous incorporation was stimulated by 100% (Fig. 1A). Thus 18-S rRNA actually reduced "net incorporation", i.e., incorporation with exogenous mRNA minus endogenous incorporation. Nevertheless, electrophoretic analysis of the reaction products revealed that adding 18-S rRNA had actually about doubled synthesis of the myeloma m RNA-specific polypeptides P and L (gels I and 2, Fig. 2A). The rRNA also increased synthesis of completed globin chains and endogenous polypeptides. There are conflicting reports on the tRNA dependence of ascites systems [12,18,23]. In our hands, ascites tRNA at 100 ~g/ml stimulated total incorporation in the presence of globin or P3K mRNA by 25--35% and net incorporation was increased by up to 2.7-fold (Fig. 1B). Moreover, the tRNA markedly improved synthesis of completed P and L chains (cf. gels 2 and 3, Fig. 2A) and of globin chains (not shown).

I

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.......... /./,.......... x "............

200

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ol~jlo 60 ?0 80 910 I(~O KCIiroN)

Fig. 1. E f f e c t o f 1S-S r R N A , t R N A , a n d KCI o n a m i n o ,+Ao m c o r p o r a t i o n in the ascites s y s t e m . R e s u l t s s h o w n are a v e r a g e s o f d u p l i c a t e assays+ e x p r e s s e d as i n c o r p o r a t i o n per 50 ~1 assay. A . E f f e c t o f m y e l o m a 18-S r R N A in a b s e n c e o f ascttas t R N A a n d w i t h o u t a d d e d m R N A (X . . . . . . X) o r w i t h 8 / ~ g / m l P 3 K m i c r o s o m a l m R N A (o . a ) o r 8 1 4 / m l g l o b i n m R N A (o . . . . . -o). B. E f f e c t o f ascites t R N A u n d e r s t a n d a r d c o n d i t i o n s w i t h o u t a d d e d m R N A (X . . . . . . X) o r w i t h 8 ~lg/ml o f P 3 K m R N A (o l) or of g l o b i n m R N A (o . . . . . -o). C, E f f e c t o f KCI c o n c e n t r a t i o n u n d e r s t a n d a r d c o n d i t i o n s w i t h o u t a d d e d m R N A (X . . . . . . X) o r w i t h 10 Mg/ml o f MOPC 3 1 5 m R N A (Jr Jr) o r o f g l o b i n m R N A (o . . . . . +o). [ 14 C ] aa, 14 C-labeled a m i n o acid.

308 B

A

P3K

M315

Endog.

i

P L

I

2

3

L

4

KC! (mM) Fig. 2. E f f e c t o f l S - S r R N A , t R N A , a n d KC1 o n s y n t h e s i s o f Ig c h a i n s in t h e ascites s y s t e m . R e a c t i o n m i x t u r e s w e r e a n a l y z e d b y e l e c t r o p h o r e s i s o n d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gels. A. A u t o r a d i o g r a p h y o f p r o d u c t s m a d e in t h e p r e s e n c e o f 8 /~g/ml PSK m R N A w i t h o u t 18-S r R N A or t R N A (gel 1), w i t h 8 p g / m l P 3 K m R N A a n d 1 0 0 D g / m l 18-S r R N A (gel 2), or w i t h 8 /zg]ml P 3 K m R N A , 1 0 0 # g ] m l 18-S r R N A a n d 1 0 0 # g / m l t R N A (gel 3). Gel 4 s h o w s t h e e n d o g e n o u s p r o d u c t s m a d e in t h e p r e s e n c e o f 1 0 0 # g / m l e a c h o f 18-S r R N A a n d t R N A . B. A u t o r a d i o g r a p h of p r o d u c t s m a d e in s t a n d a r d assays w i t h 10 p g / m l MOPC 3 1 5 m R N A at t h e i n d i c a t e d KC1 c o n c e n t r a t i o n .

As we found with the wheat germ system [6], polypeptide chain completion appears to be improved at KC1 concentrations higher than the optimum for total amino acid incorporation. Fig. 1C displays the effect of KC1 concentration on total incorporation in reactions stimulated by globin and myeloma (MOPC 315) mRNA and Fig. 2B shows an electrophoretic analysis of the products directed by MOPC 315 ml%NA at different KC1 concentrations. Although total incorporation peaked at 70 mM KC1, optimal synthesis of completed P chain occurred at 80 mM KC1 and nearly as much P chain was made at 100 mM KC1 as at 80 mM, despite the reduced total incorporation. However, with globin mRNA there was no marked difference between the KC1 concentration for optimal total incorporation and that for optimal synthesis of complete globin chains.

309

In accord with previous studies [12,31], reticulocyte initiation factors strongly stimulated incorporation in our ascites system (Fig. 3). They stimulated incorporation up to 20-fold in samples with added MOPC 41A or globin mRNA; however, endogenous incorporation was stimulated to about the same extent (mRNA curve, Fig. 3). The factors were not contaminated by globin mRNA, since electrophoretic analysis revealed no radioactive globin chains in endogenous or myeloma mRNA~timulated samples (see below). In the presence of the factors, incorporation increases steadily, albeit not linearly, for up to 120 min. The effect of mRNA concentration on net incorporation is shown in Fig. 4. Without added initiation factors, the ascites system responded only slightly to either globin or MOPC 41A mRNA, even when each was added at a concentration 25-fold higher than that needed to detect synthesis in the presence of fac. tors. With added factors, the response to globin mRNA peaked at about 10 #g/ ml and then declined somewhat. The response to MOPC 41A mRNA reached a plateau at 10 #g/ml which was only one-quarter that given by globin mRNA. The difference in activities of globin and myeloma mRNAs was actually even greater than depicted in Fig. 4 (and Figs. 1 and 3) since globin mRNA was

+ F .=.,

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Fig. 3. E f f e c t o f r a b b i t r e t i c u l o c y t e i n i t i a t i o n f a c t o r s o n a m i n o acid i n c o r p o r a t i o n in t h e ascites s y s t e m . R e s u l t s are e x p r e s s e d as a v e r a g e i n c o r p o r a t i o n in d u p l i c a t e 50 ~I assays. S t a n d a r d assays c o n t a i n e d n o a d d e d m R N A (X . . . . . . X), 3.8 ?tg/ml o f MOPC 4 1 A 14-S m R N A ( e -') o r o f globin 9-S m R N A (o . . . . . . o). [ 14 C ] aa, 14 C-labeled a m i n o acid. Fig. 4. E f f e c t o f m R N A c o n c e n t r a t i o n o n n e t a m i n o acid i n c o r p o r a t i o n in t h e ascites s y s t e m . R e s u l t s are e x p r e s s e d as a v e r a g e n e t i n c o r p o r a t i o n in d u p l i c a t e 50 /~1 assays. T h e assays, w h i c h c o n t a i n e d 9 0 m M KC1, w e r e p e r f o r m e d w i t h o u t ( o p e n s y m b e l s ) o r w i t h (closed s y m b o l s ) 5 #1 r e t i c u l o c y t e f a c t o r s a n d w i t h g l o b i n m R N A (. . . . . . ) o r MOPC 4 1 A m i c r o s o m a l m R N A ( ) as i n d i c a t e d . T h e e n d o g e n o u s i n c o r p o r a t i o n has b e e n s u b t r a c t e d ( 4 5 8 0 c p m w i t h o u t i n i t i a t i o n f a c t o r s ; 71 9 8 0 c p m w i t h i n i t i a t i o n f a c t o r s ) .

310

such an effective competitor with ascites mRNA that it virtually abolished synthesis of endogenous products (cf. gels 1 and 6, Fig. 5), while the myeloma mRNAs competed much less well (Fig. 5 and below).

Effect of preincubation on activity of ascites extracts Ascites extracts are commonly preincubated to reduce endogenous protein synthesis, although this also reduces the response to exogenous mRNA [18]. We tested ascites extracts preincubated for 0, 10 or 45 min before chromatography on Sephadex G-25 (see Methods). Endogenous activity tested without added initiation factors declined during the 45 min preincubation to one-tenth

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Fig. 5. Cell-free products synthesized in the ascites system stimulated with m y e l o m a microsomal m R N A s or globin m R N A . Standard assays (90 m M KCl) were incubated with the indicated m R N A s and 40/~l of each reaction mixture was analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiographs: Gel 1, n o added m R N A ; Gel 2, 9.6 /~g/ml M O P C 4 1 A m R N A ; Gel 3, 10.1 ~tg/ml P 3 K m R N A ; Gel 4, 9.8 /~g/ml H P C - 1 0 8 m R N A ; Gel 5, 9.6 #g/ml M O P C 3 1 5 m R N A ; Gel 6, 11.9 pg/ml globin m R N A . T h e line at the left indicates the migration of the 8 standards. P, L and G indicate positions of cell-free synthesized P chains, Ig light chains, and globin chains and arrows indicate products thought to be related to Ig heavy chains. Since M O P C 41A mRNA gave m u c h higher synthesis of P and L chains than the other m R N A s , the exposure of Gel 2 was reduced by one half.

311 its original activity, but high levels of endogenous activity were demonstrable in the presence of the factors (Table I). This suggests that the major effect of preincubation was not, as often thought, degradation of ascites mRNA. This conclusion was reinforced by electrophoretic analysis which showed that the endogenous polypeptides synthesized (with added factors) by either preincubated extract were the same size as those made by the unpreincubated extract (not shown). Even the unpreincubated extract was stimulated over 3-fold by the factors (Table I). Surprisingly, endogenous activity tested with factors was actually higher in the preincubated extract. In the presence of factors, myeloma mRNAs stimulated incorporation by 25--62% in all three extracts, although the extract preincubated for 10 min gave the largest response (Table I). In the absence of factors there was little or no stimulation by myeloma mRNAs in any of the extracts. Immunoglobulin chains made in the ascites system Microsomal mRNA from all four myelomas stimulated total incorporation in the ascites system, although none was as active as globin mRNA (Table II). The appropriate antisera precipitated up to 15% of the total incorporated radioactivity and up to 24% of the net incorporation (Table II), but this may not be a precise measure of the amount of Ig chains synthesized since, for example, short nascent chains presumably would not be immunoprecipitable. Although a number of endogenous polypeptides were synthesized in the ascites system (Fig. 5, gel 1), the background immunoprecipitation of these products was low (Table II) and no endogenous polypeptides were specifically precipitated by either antiserum used (e.g., Fig. 6, gel 13). Electrophoretic analysis (Fig. 5) revealed that mRNA from each of the myelomas synthesizing K light chains (MOPC 41A, P3K, and HPC-108) directed synthesis of two prominent polypeptides (P and L, gels 2--4). The L products TABLE I E F F E C T OF P R E I N C U B A T I O N ON A C T I V I T Y OF T H E ASCITES E X T R A C T A s s a y s ( 5 0 /~1) w e r e as d e s c r i b e d in M e t h o d s e x c e p t t h a t i n c u b a t i o n was f o r 2 h a n d r e t i c u l o c y t e i n i t i a t i o n f a c t o r s w e r e a d d e d as i n d i c a t e d . T h e c o n c e n t r a t i o n s o f H P C - 1 0 8 a n d MOPC 3 1 5 m i c r o s o m a l m R N A s w e r e r e s p e c t i v e l y 42 a n d 4 1 / ~ g / m l ( w i t h o u t i n i t i a t i o n f a c t o r s ) o r 9.4 a n d 8 . 9 / ~ g / m l ( w i t h i n i t i a t i o n f a c t o r s ) . Minutes of preincubation

Myeloma mierosomal mRNA added

Without initiation factors

With i n i t i a t i o n f a c t o r s

cpm 14 C-labeled a m i n o acids incorporated

cpm 14 C-labeled a m i n o acids incorporated

0 10 45

None None None

20 700 8 740 2 lS0

----

68 220 114 790 100 410

----

0 10 45

HPC-108 HPC-108 HPC-108

21 2 2 0 7 860 2 000

520 0 0

100 800 148 880 125 010

32 5 8 0 34 090 24 6 0 0

0 10 45

MOPC 3 1 5 MOPC 3 1 5 MOPC 3 1 5

20 5 4 0 7 620 1 770

0 0 0

102 070 185 380 153 760

33 8 5 0 70 5 9 0 53 3 5 0

Net cpm incorporated

Net cpm 'incorp orated

312 T A B L E II S T I M U L A T I O N O F A M I N O A C I D I N C O R P O R A T I O N BY m R N A s I N T H E A S C I T E S E X T R A C T All results are expressed per 50 /tl assay. T h e assays in E x p . 1 c o n t a i n e d 9 0 m M KCI a n d 4% ( v / v ) glyce r o l ; t h e r a R N A s w e r e h e a t e d at 6 5 ° C f o r 5 rain in 10 raM KC1/1 raM E D T A i m m e d i a t e l y b e f o r e t h e react i o n . The assays in E x p . 2 also c o n t a i n e d 9 0 raM KCI. Since n o iraraunoprecipitate was f o r m e d w i t h n o n i m m u n e s e r u m (see M e t h o d s ) , non-specific trapping of radioactivity in the i m r a u n o p r e c i p i t a t e was m o n i t o r e d b y adding a n t i s e r u m a n d c a r r i e r a n t i g e n t o e n d o g e n o u s assays, n . d . , n o t d e t e r m i n e d . Experiment

1

2

mRNA added Qug/ral)

c p m 14C-labeled amino acids incorporated

None MOPC 4 1 A P3K HPC-108 MOPC 3 1 5

(9.8) (10.2) (9.8) (9.6)

54 120 117 93 86

640 820 720 820 780

None MOPC 4 1 A Globin

(3.6) (4.4)

62 110 81 8 8 0 113 060

Net c p m incorporated

cpra i m m u n o p r e c i p i t a t e d Anti-108 K serum

66 63 39 32

-180 080 180 140

16 10 6 1

480 520 320 900 960

Anti-315 IgA (k2) serum 1 010 n.d. n.d. 3 300 3 270

-19 7 7 0 50 9 5 0

correspond to Ig light chains since each was precipitable by anti-108 K serum and comigrated with light chain secreted by the corresponding t u m o r {Fig. 6, gels 1--9). The P products, which were up to 1800 daltons larger than the corresponding light chains (see below), are related to the light chains, since they were also precipitable by anti-108 K serum (Fig. 6, gels 1--9). No detectable amounts of these x-related products were precipitated by the anti-315 IgA (k2) serum (not shown). As noted by others [1], the P3K P chain sometimes appeared as a doublet. The m R N A from MOPC 315 myelomas, which has a k2-type light chain, also directed synthesis of a P chain (Fig. 5, gel 5), precipitable by anti-315 IgA (k2) serum and 2200 daltons larger than secreted MOPC" 315 L chain (Fig. 6, gels 10--12). No MOPC 315 L chain was evident in the reaction mixture but one can be seen clearly in the immunoprecipitated sample (Fig. 6, gel 10). We think that the MOPC 315 L chain was precipitated more efficiently than the MOPC 315 P chain. Microsomal mRNAs from the myelomas secreting complete Ig molecules (P3K, HPC-108, and MOPC 315) also programmed synthesis of small amounts of large polypeptides (arrows, Fig. 5, gels 3--5) which were not made in the endogenous reaction (Fig. 5, gel 1) or in reactions containing m R N A from a m y e l o m a (MOPC 41A) which secretes light chains (Fig. 5, gel 2). These polypeptides are related to Ig heavy chains since they were precipitated by the following antisera: (1) anti-315 IgA (~2) serum for a MOPC 315 product (Fig. 6, gel 10); (2) anti-71 serum for the P3K products (not shown); and (3) anti-315 IgA (~2) serum or anti-108 K serum (Fig. 6, gel 7) for HPC-108 products. The precipitation by anti-108 K serum presumably was due either to antigenic cross rection between the a and K chains or to a slight contamination of the HPC108 ~ chain immunogen by fragments Of HPC-108 a chain (see Methods). None of these cell-free products comigrated with the corresponding secreted heavy chains, but this was expected since the latter are glycoproteins which

313

M41

P3K

H108

M315

Endog.

43! 0 v

36"

x

7:

253

23~ :

(J 0

15.5

123

456

789

Fig. 6. Size o f i m m u n o p r e c i p i t a t e d Ig c h a i n s s y n t h e s i z e d in t h e ascites s y s t e m c o m p a r e d b y d o d e c y l sulfate-gel e l e c t r o p h o r e s i s t o size o f s e c r e t e d Ig c h a i n s . In e a c h set o f a u t o r a d i o g r a p h s , t h e first gel (Ip) s h o w s i m m u n o p r e c i p i t a t e d cell-free p r o d u c t s , t h e s e c o n d (S) s h o w s i m m u n o g l o b u l i n s e c r e t e d f r o m t h e c o r r e s p o n d i n g m y e l o m a cells, a n d t h e t h i r d (M) s h o w s a m i x t u r e o f i m m u n o p r e c i p i t a t e d a n d s e c r e t e d m a t e r i a l . An anti-108 K serum was used for the products directed by MOPC 41A (M41), P3K and HPC-10S (H108) microsomai mRNAs and an anti-315 IgA (k2) serum for those directed by MOPC 315 (M315) mRNA. T h e i m m u n o p r e c i p i t a t e s l o a d e d c o r r e s p o n d t o 1 7 . 5 - - 5 0 /~I o f r e a c t i o n m i x t u r e . G e l 13 s h o w s a c o n t r o l i m m u n o p r e c i p i t a t e w i t h a n t i - 1 0 8 K s e r u m a n d 5 0 ~I o f e n d o g e n o u s r e a c t i o n p r o d u c t s . P, L a n d H i n d i c a t e P c h a i n s a n d s e c r e t e d Ig l i g h t c h a i n s a n d h e a v y c h a i n s ; a r r o w s i n d i c a t e cell-free p r o d u c t s r e l a t e d t o h e a v y c h a i n s . A d i s c r e p a n c y in t h e l e n g t h s o f gels 5 a n d 6 a c c o u n t s f o r t h e a p p a r e n t d i s p l a c e m e n t o f t h e H b a n d . T h e p o s i t i o n o f t h e s e c r e t e d k2 l i g h t c h a i n w a s c l e a r e r o n t h e o r i g i n a l a u t o r a d i o g r a p h o f gel 1 1 . See M e t h o d s f o r d e s c r i p t i o n o f a n t i s e r a .

migrate more slowly than predicted from their molecular weights [32], while the cell-free products presumably lack carbohydrate.

Immunoglobulin chains made in the reticulocyte lysate system Addition of the mye!oma mRNAs to the reticulocyte lysate decreased total incorporation, but up to 10% of the products were precipitable by appropriate antisera (Table III). Electrophoretic analysis (Fig. 7) showed that all the mRNAs directed synthesis of P chains (gels 3,6,10,14), identified again by their precipitability by anti-108 K serum (gels, 4,7,11) or anti-315 IgA (k2) serum (gel 15) and by their sizes, which were up to 2200 daltons larger than those of the corresponding secreted light chains (gels 5,9,13,16). No L chains were pro-

314 TABLE III S Y N T H E S I S O F I M M U N O G L O B U L I N P O L Y P E P T I D E S IN T H E R E T I C U L O C Y T E L Y S A T E S Y S T E M A s s a y s w e r e as d e s c r i b e d in M e t h o d s e x c e p t t h a t t h e y w e r e i n c u b a t e d f o r 2 h. 1 p m o l m e t h i o n i n e is a p p r o x i m a t e l y 19 5 0 0 c p m , R e s u l t s axe e x p r e s s e d p e r 5 0 pl assay. Since n o i m m u n o p r e c i p i t a t e was f o r m e d w i t h n o n - i m m u n e s e r u m (see Methods)~ n o n - s p e c i f i c t r a p p i n g o f r a d i o a c t i v i t y in t h e i m m u n o p r e c i p i t a t e was m o n i t o r e d b y a d d i n g a n t i s e r u m a n d c a r r i e r a n t i g e n to e n d o g e n o u s assays, n . d . , n o t d e t e r mined. Myeloma microsomaI mRNA added (pg/ml)

cpm [35S]methionine incorporated

None MOPC 4 1 A MOPC 3 1 5 P3K HPC-108

790 760 381 090 318960 283 670 268 960

(36) (65) (66) (67)

Endog.

cpm [35S]methionine immunoprecipitated

Per c e n t o f t o t a l i n c o r p o r a t i o n w h i c h was immunoprectpitable

Antiol08 serum

Anti-315 IgA (k2) s e r u m

Anti-108 K serum

Anti-315 IgA (k2) s e r u m

7 750 41 4 5 0 n.d. 21 4 5 0 19 1 5 0

4 100 n.d. 13300 n.d. 3 050

1.0 10.9 -7.6 7,1

0.5 -4.7 -1.1

M41

P3K

H108

M315

68 60 53 43 !

(;:,

36

O~

25.7 233

L.) 0

:E 15.5

1

2

3

4

5

Fig. 7. E l e c t r o p h o r e t i c analysis on d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gels o f Ig c h a i n s s y n t h e s i z e d in t h e r e t i c u l o c y t e l y s a t e s y s t e m . T h e a u t o r a d i o g x a p h s s h o w t o t a l cell-free r e a c t i o n p r o d u c t s ( C F ) , i m m u n o p r e c i p i t a t e d p r o d u c t s ( I p ) , o r m i x t u r e s (M) o f t o t a l r e a c t i o n p r o d u c t s a n d Ig c h a i n s s e c r e t e d b y m y e l o m a cells. A n a n t i - 1 0 8 ~ s e r u m was u s e d w i t h t h e p r o d u c t s in gels, 2,4,7 a n d 11, a n anti-T1 s e r u m in gel 8 a n d a n a n t i - 3 1 5 I g A ( k 2 ) s e r u m in gels 12 a n d 15. T h e i m m u n o p r e c i p i t a t e s l o a d e d r e p r e s e n t e d u p t o 20 ~1 o f r e a c t i o n m i x t u r e s . P, L a n d H i n d i c a t e P c h a i n s m a d e in t h e cell-free syst~.m, s e c r e t e d light c h a i n s a n d s e c r e t e d h e a v y c h a i n s ; a r r o w s i n d i c a t e cell-free p r o d u c t s r e l a t e d t o Ig h e a v y chains. Gels 1 a n d 2 s h o w e n d o g e n o u s p r o d u c t s . N o t e t h a t in gel 16 s e c r e t e d h e a v y c h a i n c o - m i g r a t e d w i t h an e n d o g e n o u s product.

315 duced in reactions directed by MOPC 41A, P3K or MOPC 315 mRNAs; unexpectedly, HPC-108 mRNA did program synthesis of an immunoprecipitable polypeptide of L chain size (gels 10,11,13). It is unclear whether this polypeptide, which was observed in three independent experiments using different HPC-108 mRNA preparations and two lysate preparations, represents a true "processed" L chain or a prematurely terminated P chain. Synthesis of some heavy chain-related polypeptides was much better in the reticulocyte than the ascites system (Fig. 7). MOPC 315 mRNA gave excellent synthesis of a 58 000 dalton polypeptide (arrow, gel 14) precipitable by anti315 IgA (k2) serum (gel 15). This product migrated faster than secreted MOPC 315 ~ chain (H, gel 16) presumably because the lysate lacks enzymes which add carbohydrate to Ig chains [10]. HPC-108 mRNA gave some synthesis of a 52 000 and a 56 000 dalton polypeptide (arrows, gel 10), both of which were precipitable by anti-315 IgA (k2) serum (gel 12); these products migrated faster than secreted HPC-108 ~ chain (gel 13). Curiously, the secreted HPC-108 a chain sometimes also migrated as two partially resolved components (Fig. 6, gel 8). P3K mRNA programmed synthesis of very small amounts of a 52 000 dalton polypeptide (arrow, Fig. 7, gel 6) precipitable by anti-7~ serum (gel 8) but not by anti-108 ~ serum or anti-315 IgA (k2) serum. This product, when mixed with authentic ~/~ chain, comigrated on a 15% gel with secreted P3K 7~ chain (gel 9) but on a 10% gel (not shown) the cell-free product migrated somewhat faster than the secreted 7~ chain, which has an apparent molecular weight of 53 000. Size o f immunoglobulin chains made in three cell-free systems We have shown that a wheat germ extract synthesizes P chain in response to the four myeloma mRNAs used here [6]. To establish whether the MOPC 41A P chains made in different systems were the same size, we combined wheat germ reaction mixtures containing this product with the equivalent ascites or reticulocyte reaction mixtures and electrophoresed the mixtures. Fig. 8 shows that the MOPC 41A P chains made in the three systems were indistinguishable in size. Similar mixing experiments showed that MOPC 315 P chains made in alll three systems were the same size, as were the P3K and HPC-108 P chains made in the asictes and reticulocyte systems (wheat germ not tested). Moreover, the MOPC 315 heavy chain-related polypeptide made in the wheat germ system [6] comigrated with that made in the reticulocyte system and the P3K heavy chain-related polypeptide made in the reticulocyte system (Fig. 7) comigrated with the larger product made in the ascites system (Fig. 5). Table IV summarizes the apparent molecular weights of the P and L chains synthesized in the cell-free systems and compares these to the molecular weights calculated for secreted light chains from known amino acid sequences. With the markers used, the gels gave slightly high values for the molecular weights of the light chains, but this should not significantly affect the values for the difference in molecular weight between each P and L chain (4, Table IV). Attempts to convert P chain to light chain If P chain is a precursor to light chain, a processing activity must exist in myeloma cells and it appears that a related activity exists in the ascites extract.

316 +

mRNA !

ASC. W. G. RET.

ASC. + W.G.

RET. + W.G.

Illml

P

P

L

1

2

3 "

4

5

Fig. 8. C o m p a r i s o n o f t h e size o f MOPC 4 1 A P c h a i n s y n t n e s i z e d in t h r e e cell-free s y s t e m s . MOPC 4 1 A m R N A was a d d e d to an aseites assay (9.4 # g m R N A / m l ) , a w h e a t g e r m assay ( 7 . 3 / ~ g / m l ) 5, o r a r e t i c u l o c y t e assay (36 /Jg/ml). R e a c t i o n p r o d u c t s w e r e a n a l y z e d b y d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gel e l e c t r o phoresis. Gel 1 , 4 0 /~1 ascites r e a c t i o n m i x t u r e ( a u t o r a d i o g r a p h y for l d ) ; Gel 2, 10 pl w h e a t g e r m r e a c t i o n m i x t u r e ( a u t o r a d i o g r a p h y f o r l l d ) ; Gel 3, 2 /11 r e t i c u l o c y t e r e a c t i o n m i x t u r e ( a u t o r a d i o g r a p h y for 1 3 d ) ; Gel 4, m i x t u r e o f 5 pl aseites r e a c t i o n m i x t u r e a n d 10 /zl w h e a t g e r m r e a c t i o n m i x t u r e ( a u t o r a d i o g r a p h y f o r 1 6 d ) ; Gel 5, m i x t u r e o f 3 /~l r e t i c u l o c y t e r e a c t i o n m i x t u r e a n d 10 #1 w h e a t g e r m r e a c t i o n m i x t u r e ( a u t o r a d i o g r a p h y f o r 1 6 d ) . P a n d L i n d i c a t e MOPC 4 1 A P c h a i n a n d light c h a i n .

We attempted to demonstrate these activities directly. MOPC 41A P chain made in a reticulocyte lysate was separated from unincorporated label by gel filtration. Portions of the macromolecular fraction were added (a) to buffer, (b) to an ascites cell-free system (containing only nonradioactive amino acids) k n o w n to yield both P and L chains, or (c) to subcellular fractions [3] from MOPC 41A tumors: t u m o r homogenate, postmitochondrial supernatant or microsomes, part of which were treated with 1% NP-40 to solubilize membranes. After 2 h of incubation, the mixtures were analyzed electrophoretically to detect any conversion of P to L chain. Only intact P chain was found. This suggests that conversion may be closely coupled to synthesis. Two other experiments with the ascites system support this hypothesis. (1)During a syn-

317 T A B L E IV A P P A R E N T M O L E C U L A R W E I G H T S O F Ig L I G H T C H A I N S A N D P C H A I N S M A D E IN C E L L - F R E E SYSTEMS T h e c a l c u l a t e d m o l e c u l a r w e i g h t s given for t h e acid s e q u e n c e s [ 1 5 , 3 3 , 3 4 ] . T h e e x p e r i m e n t a l l y derived from their elcctroplioretic mobflities c h y m o t r y p s i n o g e n A (25 700) and trypsin (23 and t h e standard d e v i a t i o n o f t h e m e a n is given. Myeloma

Light chain class

Calculated molecular weight

(x MOPC41A P3K HPC-108 MOPC315

K K K A2

10-3)

23.5 23.6 -22.7

s e c r e t e d light c h a i n s w e r e b a s e d u p o n the k n o w n a m i n o d e t e r m i n e d m o l e c u l a r w e i g h t s o f cell-free p r o d u c t s w e r e o n p o l y a c r y l a m i d e gels in r e l a t i o n t o t h e m o b i l i t i e s o f 3 0 0 ) . E a c h v a l u e is b a s e d u p o n at least 14 m e a s u r e m e n t s

Experimentally determined molecular weights of cell-free p r o d u c t s (× 10 - 3 ) Light Chain

P Chain

A,

25.5±0.5 26.021.0 24.1±0.8 23.7±0.6

27.320.7 27.621.0 25.420.9 25.920.7

1.8±0.3 1.6±0.3 1.3±0.3 2.2±0.4

* A m o l e c u l a r w e i g h t = m o l e c u l a r w e i g h t o f P c h a i n m i n u s m o l e c u l a r w e i g h t o f light chain.

thesis directed by HPC-108 mRNA, aliquots were taken at times from 15 to 120 min. Electrophoretic analysis indicated that the ratio of P to L did not change. (2) Synthesis in a reaction containing HPC-108 m R N A was stopped after 1 h with RNAase and incubation continued for another 4 h. The ratio of P to L was n o t altered during this period. Discussion Translation of both globin and myeloma mRNAs in the ascites system was strongly stimulated by reticulocyte initiation factors. The activity of the supplemented system with globin m R N A was comparable to that reported by Metafora et al. [12]: they obtained 4 mol of globin chains per mol of 10-S m R N A , while we obtained 3.0 to 7.5 mol, if we correct for a 40% contamination of our globin m R N A b y rRNA. Globin m R N A was several fold more active than the m y e l o m a m R N A s in this system (Table II), even though both types of m R N A had comparable activity in the wheat germ system [6]. In our hands, the background of endogenous incorporation in the ascites system has consistently been higher than that found b y others [18,35,36], perhaps due to some subtle different in growth or homogenization of the cells. Unlike the situation with bacterial extracts [37], the background could not be abolished b y preincubating the extract, since the major effect of preincubation was loss of an activity replaceable by reticulocyte initation factors rather than degradation of ascites m R N A (Table I). Even an unpreincubated extract was stimulated by reticulocyte factors which suggests that the unpreincubated extract was deficient in some component, such as an initiation factor. Furthermore, preincubated extracts tested with added reticulocyte factors were actually more active than an unpreincubated extract tested with factors (Table I), perhaps indicating activation of some ascites c o m p o n e n t during preincubation or inactivation of an inhibitor. Similarly, we have observed that a preincubated wheat germ extract gives twice the response of an unpreincubated extract to added m R N A s

318 (our unpublished results), although we do not know what the "activating eff e c t " of preincubation is. The work reported here strengthens the case that P chains are a general feature of L chain m R N A translation. Thus P chains were made in both the ascites and reticulocyte systems, as well as the wheat germ system [6], in response to m R N A from all four myelomas tested, which included myelomas secreting both K- and },-type light chains. Moreover, the P chains made in response to a particular m R N A in each of the three systems were indistinguishable in size {Fig. 8). These observations make it very unlikely that the P chain is produced artifactually in cell-free systems by, for example, initiation of translation at false initiation sites. Evidence favoring the concept of P chain as a biosynthetic precursor is that MOPC 41A cells cultured with a protease inhibitor synthesized an immunoprecipitable polypeptide of the same size as MOPC 41A P chain made in a cell-free system [17]. Two of our observations may mean that the extra portions of different P chains are not identical. First, the ratio of the amounts of P and L chains made in the ascites system was different with different mRNAs (e.g., Fig. 5), perhaps indicating that the ascites "processing mechanism" is more efficient with some P chains than others. Second, the difference between the molecular weight of a P chain and the corresponding light chain (A molecular weight, Table IV) ranged from 1300 to 2200. The differences in A molecular weight were reproducible and were confirmed by co-electrophoresis of the products directed by different mRNAs. Our electrophoretic technique could resolve chains differing in molecular weight by 500. We have obtained limited synthesis of heavy chain-related polypeptides in the ascites system (Fig. 5) and the wheat germ system [6]. The low levels made in these systems may be a consequence of premature chain termination or selective messenger utilization. The possibility that the m R N A preparations were simply deficient in heavy chain m R N A can be discounted at least for the MOPC 315 m R N A since the same MOPC 315 m R N A preparation gave very good synthesis of an ~ chain related polypeptide in the reticulocyte system (Fig. 7). The size of this cell-free product was about 58 000 daltons, while the molecular weight of the protein moiety of ~ chain is 50 100 [38]. Thus it seems likely that the cell-free product is a precursor to the ~ chain, although chemical analysis is needed to substantiate this conclusion. There is chemical evidence that the P3K' (MOPC 21) 7 chain made in the reticulocyte lysate differs from that secreted by the P3K cells [10]. Despite the evidence favoring P chain as a precursor to L chain, we were unable to demonstrate any direct conversion. This could simply mean that the processing enzymes are highly unstable, but we have n o t noted any differences in the ratio of P to L chains made by preincubated and unpreincubated ascites extracts or at early and late times in the ascites reaction. These results may mean that processing is tightly coupled to synthesis of the P chain. Perhaps, as suggested by Milstein et al. [1], extra amino terminal residues are normally excised in vivo even before sy~.~thesis of the chain is completed. The presence of an activity apparently converting P to L in extracts of ascites cells and even in frog oocytes [3,39], neither of which synthesize immunoglobulin, suggests that the cleavage enzymes have some c o m m o n cellular function. There is growing

319 evidence that many proteins, perhaps all those synthesized on membrane-bound polysomes, are made in a precursor form (see e.g., ref. 40). The function of the extra residues has not been established but could be related to formation of membrane-bound polysomes [ 1 ] or to transport of nascent chains through the microsomal membrane. Acknowledgements We thank Miss Jill Jackson for expert technical assistance, Dr. T.E. Martin for the ascites strain, Dr. A.W. Harris and Mr. J. Pye for help with obtaining labelled Ig chains, Dr. H. Eisen for a gift of purified MOPC 315 protein and Dr. N. Warner for an antiserum and for purified HPC-108 light chain. This work was supported by grants from the U.S. National Cancer Institute (RO1 CA 1 2 4 2 1 ) , the American Heart Association and the N.H. and M.R.C. (Canberra). B.J.S. was an American Cancer Society post-doctoral fellow, S.C. a Roche fellow and J.M.A. an Established Investigator of the American Heart Assocation. References 1 Mflstein, C., Browrdee, G.G., Harrison, T.M. and Mathews, M.B. (1972) Nat. New Biol. 239, 117-120 2 Swan, D., Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. U.S. 69, 1967--1971 3 Mach, B., Faust, C. and Vassalli, P. (1973) Proc. Natl. Acad. Sci. U.S. 70, 451--455 4 Tonegawa, S. and Baldi, I. (1973) Biochem. Biophys. Res. C ommun. 51, 81--87 5 Schechter, I. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2 2 5 6 - - 2 2 6 0 6 Schmeckpeper, B.J., Cory, S. and Adams, J.M. (1974) Molec. Biol. R e port s 1 , 3 5 5 - - 3 6 3 7 Kuehl, W.M., Kaplan, B.A., Scharff, M.D., Nau, M., Honjo, T. and Leder, P. (1975) Cell 5, 139--147 8 Green, M., Graves, P.N., Zehavi-Willner, T., Melnnes, J. and Pcstka, S. (1975) Proc. Natl. Acad. Sei. U.S. 72, 224--228 9 Schechter, I., McKean, D.J., Guyer, R. and Terry, W. (1975) Science 188, 160--162 10 Cowan, N.J. and Milstein, C. (1973) Eur. J. Biochem. 36, 1--7 11 Green, M., Zehavi-Willner, T., Graves, P.N., McInnes, J. and Pest]m, S. (1976) Arch. Biochem. Biophys. 172, 74--89 12 Metafora, S., Terada, M., Dow, L.W., Marks, P.A. and Bank, A. (1972) Proc. Natl. Acad. Sei. U.S. 69, 1299--1303 13 Potter, M., Dreycr, W.J., Kuff, E.L. and McIntire, K.R. (1964) J. Mol. Biol. 8, 814--822 14 Horibata, K. and Harris, A.W. (1970) Expt]. Cell Res. 60, 61--77 15 Dugan, E.S., Bradshaw, R.A., Simms, E.S. and Eisen, H.N. (1973) Biochemistry 12, 5400--5416 16 Harris, A.W., Bankhurst, A.D., Mason, S. and Warner, N.L. (1973) J. Immunol . 110,431---438 17 Sehmeckpeper, B.J., Adams, J.M. and Harris, A.W. (1975) FEBS Lett. 5 3 , 95--98 18 Mathews, M.B. (1972) Bioehim. Biophys. Acta 272, 108--118 19 Gilbert, J.M. and Anderson, W.F. (1971) Methods Enzymol. 20, 542--549 20 Schreier, M.H. and Staehelln, T. (1973) J. Mol. Biol. 7 3 , 3 2 9 - - 3 4 9 21 Cory, S., Genin, C. and Adams, J.M. (1976) Biochim. Biophys. Aeta in the press 22 Cory, S. and Adams, J.M. (1975) J. Mol. Biol. 9 9 , 5 1 9 - - 5 4 7 23 Aviv, H., Boime, I. and Leder, P. (1971) Proc. Nat. Acad. Sci. U.S. 68, 2303--2307 24 Bollum, F.J. (1968) Methods Enzymol. 12B, 169--173 25 Laemmli, U.K. (1970) Nature 227, 680---685 26 Fairbanks, Jr., G., Levinthal, C. and Reeder, R.H. (1965) Biochem. Biophys. Res. C ommun. 20, 393--399 27 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4 4 0 6 - - 4 4 1 2 28 Chase, M.W. (1967) in Methods in I m m u n o l o g y and I m m u n o c h e m i s t r y (Williams, C.A. and Chase, M.W., eds.), Vol. 1, pp. 237--254, Academic Press, New York 29 Greenwood, F.C., Hunter, W.M. and Glover, J.S. (1963) Biochem. J. 8 9 , 1 1 4 - - 1 2 3 30 Jacobs-Lorena, M. and Baglloni, C. (1972) Biochemistry 11, 4 9 7 0 - - 4 9 7 4 31 Mathews, M.B., Pragnell, I.B., Osborn, M. and Arnstein, H.R.V. (1972) Biochim. Biophys. Acta 287, 113--123

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