Radioimmunoassay for ribosomes

lX.4LTTIC.41,

BIOCHEMISTRY

48, 339-3‘k?

(1972)

Radioimmunoassay JOAN

for

Ribosomes’

WIKMAN-COFFELT’

Departments of Biochemistry and Pediatks, Oklahoma University iVfe&al School, Oldahoma Gil y, Oklahoma Rcwivcd

Septcmher

23, 1971

In view of the importance of quantification of ribosomes in assessing various physiological disorders (l-3)) a radioimmunoassay was developed to quantitate ribosomea in tissue homogenates. For this assay an antiserum against the liver GOS ribosomal subunit was developed in goats (4). With this immunoassay it is possible to quantitnte nanogram quantities of monosomes in small l)iol>sy samplch. MATERIALS

ASD

METHODS

Develop,!~ent of Antb.se/,rrj)l. To obtain au opt.imnl level of antibody, the 60s ribosomal subunit of rat liver n-as injected along with Freund’s adjuvant into axillae of goats with initial doses of 5 mg each, 10 days apart, followed by similar monthly doses (4). Preparation of Ribosortces md Ribosomal Srsb~cGts (5-7). For preparation of liver ribosomes, the livers were removed and minced in 0.05 M Tris HCl, pH 7.4, buffer containing 0.05 M KCl, 0.005 M MgCl,, and 0.25 1M sucrose. After centrifugation for 10 min (6,OOOg), the pelleted liver cells were homogenized in this buffer in a Teflon-glass homogenizer. After centrifugation for 10 min (S,OOOg), the pellet was separated from the red blood cells and homogenized in a Teflon-glass homogenizer with a tight pestle in the same buffer but which lacked sucrose, and again centrifuged 10 min (12,OOOg) ; the supernatant was made 0.5% deoxycholate, homogenized, and centrifuged 10 min (30,OOOg). The supernat,ant was underlayed with ?(3 volume of 1.0 &I sucrose containing the above buffer and centrifuged IF hr at 78,OOOg. For preparation of monosomes the same procedure was used except that the underlayered sucrose was made 2 M and the intermediate layer was used instead of t,he pellet (8). ‘Supported by a grant from Development 05365-01. ’ Present address: Departments Universi@r of California Davis,

the

iSntionn1

of Intwnal Davis, California 339

@ 1972 by

Academic

Press,

Inr.

Institute Medic& 95616.

of Child and

Health Biological

and

Human

Chemktry,

340

JOAN

WIIMAN-COFFELT

For preparation of ribosomal subunit’s, the pelleted polysomes (liver) were suspended in 0.05 M Tris HCl, pH 7.4, with gentle homogenization to give a concentration with an absorbance at 260 rnp of 30 OD/ml. The suspension was made 0.008 M EDTA and layered on a 5-4570 sucrose gradient with 0.01 LW Tris HCl, pH 7.4, and centrifuged 16 hr in a Beckman BIV rotor at 70,OOOy. The 40s and 60s fract,ions were collected and pellet’ed in a Beckman 60-Ti rotor for 8 hr at 240,OOOg (4). Foi preparation of myocardial monosomes the tissue was minced in 0.05 JI Tris HCl, pH 7.4 buffer, containing 0.001 M EDTA, 0.025 M KCl, 0.01 211 MgCl,, and 0.25M sucrose. After centrifugation for 10 min (S,OOOg), the pelleted cells were suspended in 2 vol of the above buffer, homogenized, and again centrifuged for 10 min (12,000g). The supernatant was underlayered with 1/s volume of 1.0 M sucrose containing the above buffer and centrifuged 2 hr (120,OOOg). Preparation of Homogenate for Ribosome Determination. The tissue was minced in 0.05 M Tris HCl, pH 7.4, containing 0.20 M NH,Cl, 0.15 ,%I KCl, 0.001 M EDTA, and 0.1% sodium deoxycholate. The mixture was sheared with a Virtis homogenizer for 1 min at medium speed, homogenized with a Teflon-glass homogenizer, and then stirred for 30 min (37°C). The homogenate was centrifuged at 6,000g (37”). For ribosomal quantification the supernat.ant was diluted in the same buffer. Dilutions were made according to standard determinations. Ozcchterlony Double-Diffusion. For immunodiffusion plates, 1.0% agarose was made up in 0.05 M Tris HCl, pH 7.4, 0.025 M KCl, 0.001 M EDTA, 0.1% deoxycholate, 0.01% mercaptoethanol, and 0.01% Merthiolate. The mixt.ure was heated to boiling and cooled to about 45°C and then poured (25 ml/Petri dish). A center well and a number of circumferential wells were cut equal distance apart so that the distance from the edge of the center well to that. of each surrounding well was 1 mm. The antisera were diluted 1:5 and 100 ~1 was added to the center well. About 0.5 mg of solubilized ribosomes or ribosomal subunits to be tested was added to the side wells. The ribosomes were solubilized in 0.05 M Tris HCl, pH 7.4, 0.20M NH,Cl, 0.15&f KCl, 0.001 M EDTA, and 0.1% deoxycholate and brought to 37” for 10 min. They were stored in 0.05 J!l Tris HCl, pH 7.4, 0.20 M NH,Cl, 0.15 M KCl, and 0.001 M EDTA at 4”; this solution was made to 0.1% deoxycholate and warmed to 37” for 10 min before using. Deoxycholate, in the concentration used here, did not cause breakdown of ribosomes. Incubations were made at room temperature. After the samples were absorbed from the wells, a wet paper towel was cut and fitted to the lid; the plates were inverted for development. The plates could also be developed more slowly at

RADIOIMMU~OASSAY

FOR

RIBOSOMES

341

4”. They could be kept at 4” for several weeks without diffusion of precipitin lines. Labeling of Ribosomes. To 2 mCi of Na1311 (New England Nuclear) at high specific activity in 0.1 M NaOH was added 0.25 ml of 0.5 M phosphate buffer, pH 7.5. Then 5 mg of 60s ribosomal subunits was mixed with 0.25 ml of 0.05 M phosphate buffer, pH 7.5, and added to the vial containing the isotope. After mixing, the sample was incubated 15 min at room temperature followed by the addition of 0.05 ml of fresh chloramine-T (4 mg/ml in 0.05 M phosphate buffer, pH 7.5). After mixing, 0.2 ml of sodium metabisulfite (2.4 mg/ml in 0.05 M phosphate, pH 7.5) was added. This was mixed and 0.30 ml of KI (10 mg/ml) in 0.05 M phosphate buffer, pH 7.5, was added. The iodinated ribosomal subunits were purified from free isotope by passing the material through a Sephadex column, collecting and pooling the labeled material obtained from the first peak of eluent, and dialyzing this against a phosphate buffer overnight. Before passing the sample through a column (a small column of Sephadex G-25 was made in a Pasteur pipet), 1 ml of 5% albumin (in 0.0175 M phosphate, pH 7.5) was passed through the column. After the sample was added, the column was washed with 1 ml of KI (10 mg/ml) and eluted with phosphate buffer (0.0175 M phosphate, pH 7.5, in 0.15 M KCl) . The dialyzed material containing iodinated ribosomal subunit’s was diluted with 20 ml of eluent buffer. The samples were frozen in 100 ~1 aliquots (about 1.5 X lo5 cpm/tube). The following analyses were made to demonstrate that only the proteins were iodinated: Ribosomal proteins were extracted from the 1311-60S subunit by the urea-LiCl method (9). No isotope was associated with the precipitated RNA. The proteins were electrophoresed and the stained bands cut out of the gels and examined for labeled iodine incorporation. 90% of the isotope was recovered in the protein bands. Radioimmunoassay for Ribosomes. Unlabeled ribosomes were solubilized in fresh buffer containing 0.05 M Tris HCl, pH 7.4, 0.20 M NH&I, 0.15 M KCI, 0.001 .M EDTA, and 0.1% deoxycholate (37°C). Ribosomes were incubated (37”) for 10 min in this buffer. The stock solution was made 100 pg/ml and dilutions of this were made with the same buffer for the following assays. A tube of labeled ribosomes was t,hawed and brought to 37”. The above buffer (3 ml) was added to a tube of labeled 1311-ribosonles and this was made 0.5% with albumin; 100 ~1 of the labeled ribosomes was used for each experimental assay, SO that each tube had approximately 5 X lo4 cpm. (The specific activity of the labeled ribosomal subunits was approximately 5 X 109 cpm/OD at. 260 mp.) The antiserum was diluted 1:lOO in 0.05 M Tris HCl, pH

342

JOAN

WIKMAK-COFFELT

7.4, 0.2OM NH,Cl, 0.15 M KCl, 0.001 M EDTA, and 0.1% deoxycholatr Immediately after the antiserum was diluted, 100 111was used for each assay. All immunoassays were carried out at 37” for 2 hr. Antigen and antibody were preincubatecl at this temperature before mixing. Labcletl ribosomes (100 ~1) and 100 111of unlabeled ribosome~ mere mixed ancl preincubated 10 min. For controls where unlabeled ribosomes or antibody were omit,ted, the respective buffer was added in himilar volume. After 10 min of preincubation, 100 ,d of antiserum was addccl, mixed, and incubated for 2 hr (37”). The reaction was stopped by addition of Norite A, which binds ribosomes and ribosomal subunits but not the ribosometct-cr:il times and then antibody complex.” Noritc A was prewashetl resuspended in buffer 1: 20 (Sorite: buffer). At, the end of the incubation period 100 ~11of Xorite A in 0.05 114 Tris HCl, pH 7.4 buffer, containing 0.20 M NH&Cl, 0.15 .M Kicl, 0.001 M EDT,4, O.lc/o deoxycholstc, and 0.1% albumin, was added. The solution was vortexed 3 times (5 set each) and centrifuged (300~) for 5 min (37”). The supernatant was decanted and the pellet analyzed for isotope incorporat’ion using an automatic gamma counter (Packard). RESCLTS

AND

DISCUSSIOK

Diffusion of ribosomes in agarose occurred when the agarose was mixed in a buffer containing 0.1% deosycholate and 0.001 M EDTA. Figure 1 is an Ouchtcrlony double-diffusion ljattern of (1) rat liver 60s ribosomal subunits, (2) myocarclial (rat) 60s ribosomnl subunits, (3) homogenate of rat liver after extraction of ribosomes, (4) whole homogenate of rat liver, (5) homogenate of heart, and (6) homogenate of heart after extraction of ribosomes, incubated with (A) antiserum to rat liver 60s ribosomal subunits. Rot,11 homogenate of rat liver and rat heart, as well as purified ribosomal subunits gave a single line of fusion in the Ouchterlony patterns. The fusion of bands indicat’ed like antigens of the two tissues and the single band indicated intact ribosomal subunits as prepared in these assays. Because of the diffusion equilibrium constant of the ribosomal subunits in agarose they acted as a single antigen giving one band. In other studies, not shown here, the antiserum developed precipitin bands when reacted with the purified RXA and also with the purified proteins of the subunits. These results indicated the presence of several determinant’ sites on the nntigcnic ribosomal ‘The optimal proportions point of antigen to antibody determined by the as reportrd here, was the same as that obtained by the preradioimmunoassay, cipitin test (4). Noritc, A for separating free and bound ribosomes was used only for dilute solutions of thr nni.igen-antibody complex. In concentrated solutions (1 mg/ml) the antigen-antihody complex precipitated with the Korite.

RADIOIMMUKOASSAY

FOR

343

RIROSOMES

FIG. 1. Ouchterlony doubly>-tlitlusion of (1) xx1 1irc.r 60s rihxomal subunits, (2) rat myocxdial 60s ribosomal subunits, (3) homopc~uatc of rat liver aft,er extraction of ribosomw, (4) whole Iloulogc:uatc~ of rat, Ii\-r,r. (5) 11oruogen:~k of rat heart, and (6) homogenate of ml, Ilcart nftcr c,stl:idiou ol’ ril~owmw iurwllntcd with (A) antiserum to rat, lircr 60s ril)osomnl subunits.

subunit. Since the proteins which arc native to the ribosomal subunits have not been defined for the mammalian sy~tcm, it cannot be stated at this time whether all determinant sites are an integral part of the ribosome. However, the riboaome?;, either as a purified product, or as found in the homogenate, react as one and the same antigen with the antiserum to the 60s ribosomal subunit,, as shown by the fusion of the precipitin band of the purified ribosome with that of the homogenate (Fig. 2). The ribosomes were completely solubilized at 37°C in 0.05 M Tris

and

FIG. 2. Ouchtcrlony (A) antiserum

pattern of (1) rat liver to rat liver 60s ribosomal

monosomes, subunits.

(2)

rat

liver

homogenate,

344

JOAN

WIKMAN-COFFELT

HCI, pH 7.4 buffer, containing 0.20M NH&l, 0.15 M KCl, 0.001 M EDTA, 0.1% Na deoxycholate, and 0.1% albumin. Labeled ribosomes did not pellet after solubilization in this solution. If the preparation was contaminated with divalent cations precipitation occurred; in these cases it was necessary to increase the concentration of EDTA to 0.005 M. Higher concentrations of EDTA and deoxycholate were not used so as to avoid dissociation of structural proteins from the ribosomal subunits (10). The ribosomal subunits labeled with 1311 had a high specific activity, approximately 5 X lo9 cpm/OD. The sensitivity of the immunoassay can be increased by increasing the specific activity of the ribosomes or using less 1311-60S ribosomal subunits and consequently less antiserum. However, greater sensitivity is not advantageous in the present assay because of the high concentration of ribosomes in tissue homogenates. 6&70% of the isotope obtained from the pooled material after Sephadex filtration was bound by the antiserum. Norite A was used to precipitate the unbound antigen; the bound antigen remains in solu40 GOAT ANTI-RAT LIVER + RAT LIVER ‘3’l-60S

60s

30 ‘; e *

P

HEART

0

LIVER

l

NORMAL

RIBOSOMES RIBOSOMES SERUM

(NO ANTIBODY)

t 0 20

IO

0 No

RIBOSOMES

ADDED

FIG. 3. Competition of rat myocardial monosomes and rat liver monosomes against ‘J11-60S liver ribosomal subunits for antiserum to rat liver 60s ribosomal subunits. (0) indicates competition of liver ribosomes when goat serum is used instead of goat antiserum.

RADIOIMMUNOASSAY

POR RIROSOMES

345

subunits with no antibody present gives tion. Precipitation of l”‘I-60s total labeled antigen (TA) in any given experiment. After addition of antibody the Korite precipitate gives the total pcrcentagc of unbound antigen (UA) . Thus the bound antigen (BA) is obtained by the equation: (BA = TA - UA). Figure 3 is an isot’ope dilution profile demonst’rating competition of rat myocardial monosomes against 1311-60S ribosomal subunits of rat liver for antiserum to liver 60s ribosomes. The competition of myocardial monosomes was not significantly different from that of liver monosomes, indicating similar competition of the monosomes of the two different types of tissue for t’he antiserum to liver 60s ribosomal subunits. Unknown samples obtained from tissues prepared according to the procedure described in t’he text were analyzed and quantitated according to a standard curve as shown in Fig. 3. SUMMARY

A radioimmunoassay for quantitating ribosomes is presented. The assay allows for precise, efficient, and rapid estimation of ribosomes in small samples of tissue (50-100 mg). ACKNOWLEDGMEXTS

The author wishes to thank Dr. Harris Busch and Bill Spohn (Baylor College of Medicine) for the rat liver ribosomal subunits used in these experiments and Dr. J. Metcoff (Oklahoma Medical School) for the use of laboratory facilities in carrying out this project. REFEREKCES 1. WIKMAN-COFFELT, J., METCOFF, J., MORALES, M., BERNAL, A., AND ROHADO, A., New Eng. J. Med. (submitted). 2. NAIR, G., GROVE, D., ZAK, R., CUTILLETTA, A., AND RARINOWITZ. M., in “Cardiac Hypertrophy” (iV. R. Alpert, ed.), p. 157 Academic Press, iVew York, 1971. 3. ONISHI, T., Biochim. Biophys. Acta 217, 384 (1970). 4. BUSCH, H., BUSCH, R., SPOHN, W., WI~;MAN, J.? .4ND DASKAL, Y., Proc. Exp. Biol. Med. 137, 1470 (1971). 5. WIKMAN, J., HOWARD, E., AND BUSCH, H., Calzcer Res. 30, 773 (1970). 6. WIKMAN, J., HOWARD, E., AND BUSCH, H., J. Biol. Chem. 244, 5471 (1969). 7. WIKMAN, J.. QUAGLIAROTTI, G., HOWARD, E., AND BUSCH, H., Cancer Res. 2749 ( 1970) . 8. WETTSTEIN, F. O., STAEHLIN, T., AND ~SOLL, H., Nature 197, 430 (1963). 9. FALVEY, K., AND STAEIILIN, T.. J. Mol. Biol. 53, 1 (1970). ’ . 10. OLSNES, S., FEBS Let.?. ‘7, 211 (1970).

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