THE JOURNAL OF BIOLOQXCAL CHEMISTRY Vol. 250, No. 6,Issua of March 25, pp. 2135-2142, 1975

Printed in U.S.A.

Identification and Purification of Collagen-synthesizing Polysomes with Anti-Collagen Antibodies*

(Received for publication, May 10, 1974)

PHILIP J. PAWLOWSKI, MICHAEL T. GILLETTE, JAMES MARTINELL, AND LEWIS N. LUKENS

From the Department of Biology, Wesleyan University, Middletown, Connecticut 06467

HEINZ FURTHMAYR

From the Department of Pathology, Yale University, New Haven, Connecticut 066.20

SUMMARY

Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase- free. lz51-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of g-day chick embryo wings and legs on sucrose gradients by measuring the poly- some associated radioactivity. The 1251-anti-collagen anti- bodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particu- lar tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system.

The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of short- ening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.

Antibodies against collagen would appear to have considerable promise as tools for aiding in the isolation of collagen mRNA, as reagents for measuring the collagen produced in response to the addition of collagen mRNA to cell-free protein-synthesizing systems, and as probes that might be able to distinguish between

* This work was supported by Research Grants AM16162-01 and AM1616-02 from the National Institute of Arthritis and Met- abolic Diseases and a grant-in-aid from the American Heart As- sociation with funds contributed in part by the Middlesex Heart Association.

the different types and amounts of collagen within a single orga- nism. In particular, our attempts to date to isolate the mRNAs for collagen have been hampered by the relatively low propor- tion of the total protein synthesis (around 797,) that is devoted to collagen in the chick embryo tissues used. One way of over- coming this problem would be to develop a method for specifi- cally precipitating collagen-synthesizing polysomes by means of collagen-specific antibodies. Such an approach has proved suc- cessful in isolating polysomes synthesizing ovalbumin (1, 2), and immunoglobulin L chain (3,4). This paper reports that such an approach is also feasible in the case of collagen-synthesizing polysomes. In addition, the location of collagen-synthesizing polysomes on sucrose gradients has been made more precise, both by means of antibody binding studies and by a modification of the previously reported cell-free system for elongating poly- some-bound nascent chains (5).

EXPERIMENTAL PROCEDURE

Methods

Polysome Preparation-The homogenization of the wings and legs of 8-day (0.5 g) chick embryos and the low speed centrifuga- tion of the homogenate were as described previously (5) except that the centrifugation was reduced to 4 min at 2000 X g and, in the experiments involving collagen antibodies, sodium heparin at 40 fig/ml was added to the buffer as an RNase inhibitor. The buffer used for homogenizing and for all sucrose solutions con- sisted of 0.25 M KCl, 0.01 MgC12, and 0.01 M Tris-HCl, pH 7.4 (6), and is referred to below as Buffer A. The gradients consisted of 11.7 ml of 15 to 40% (w/v) sucrose in Buffer A. To get reproduci- ble preparations of polysomes it was found necessary’ to use sterile conditions and to chill the tissues as rapidly as possible from 37 to 0”; all subsequent steps involving polysomes were done at 4’. For the experiments reported in the present paper, the polysomes were subjected to a preliminary purification before use. The 2000 X g-supernatant was layered on top of 4 ml of 1 M sucrose which were layered over 2 ml of 2.5 M sucrose made up in Buffer A, in a Beckman SW 41 cellulose nitrate tube. The tube was then centrifuged for 90 min at 40,000 rpm in the SW 41 rotor of a Beck- man centrifuge and the polysomes, relatively free from mono- somes, were collected in a volume of about 0.7 to 0.8 ml from the 2.5 M to 1 M sucrose interface by puncturing the side of the tube with a sterile syringe. To layer directly on a 15 to 40% sucrose gradient, as in Fig. 6, the polysome sample was simply diluted with 3.3 volumes of buffer. In the antibody binding studies, however, the polysome sample was first dialyzed at 4’ against Buffer A to

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remove the sucrose which has been reported to inhibit the binding of antibodies (3). Polysomes prepared in this way had a ratio of absorbance at 260 to 280 nm of 1.65 to 1.9.

Antibody Preparation-White, male, 7-pound New Zealand rabbits were injkcted intradermally with 5.mg of purified chick bone collagen dissolved in 2.0 ml of 0.01 N acetic acid and mixed with 2.0 my of complete Freund’s adjuvant. Two weeks later the rabbits were given 10 mg of bone collagen in 4.0 ml of 0.01 N acetic acid intraperitoneally. Ten days later the rabbits were bled and tested for anti-collagen antibodies with a passive hemagglutina- tion technique (7). The two-step injection series was then re- peated u&l no increase of titers from the previous injection series was found. Maximum production of antibodies was usually reached after five or six such series of injections after which time the rabbits were bled and checked periodically for maintenance of high levels of antibodies.

Anti-collagen antibodies were purified from whole serum by affinity chromatography on colur&ns of p-aminobenzyl cellulose- collagen according to Timpl et al. (8) except that 1.0 N acetic acid was used to elute the bound antibodies. The peak (A280 absorb- ing) fractions eluted from the column were neutralized with 4.0 M Tris and precipitated by adding an equal volume of saturated am- monium sulfate. The precipitate was dissolved in phosphate- buffered saline (2.73 g oi KH&‘Oa, 6.63 g of Na2HPOI, and 35 g of NaCl adjusted to DH 7.2 wit,h NaOH and made un to 5 liters) and was dialyzed against more phosphate-buffered saline to remove traces of ammonium sulfate. After dialysis the antibody solution was passed through a column of CM-cellulose stacked on top of DEAE-cellulose (1) equilibrated with sterile phosphate-buffered saline. The same saline solution was used to wash the column after application of the sample. The peak (AtaO absorbing) frac- tions of the flow through and wash from this column were pooled, precipitated with sterile ammonium sulfate, and were dissolved and dialyzed against phosphate-buffered saline as above. These dialyzed samples were stored frozen at -2O”, until needed. Nor- mal rabbit immunoglobulin (IgG)2 was prepared from the serum of uninjected rabbits as above using ammonium sulfate precipita- tion and DEAE-CM-cellulose chromatography. Chromatogra- phy on p-aminobenzyl cellulose-collagen columns was omitted. Goat anti-rabbit IgG was prepared by <he same procedure used for normal rabbit IgG. from serum obtained from Miles Laboratories.

Labeling of &&bodies with 1261-In vitro labeling of antibody preparations with lzaI was done according to Marchalonis (9) ex- cept that glucose and glucose oxidase were used to control the amount of Hz02 present in the reaction (10). The specific activity of the antibodies resulting from this procedure was usually be- tween 3 and 5 X 106 cpm/mg of protein.

After labeling, the antibodies were precipitated with 50% am- monium sulfate, redissolved in phosphate-buffered saline, and passed through a column (0.7 X 20 cm) of Sephadex G-100 equili- brated in phosphate-buffered saline. The peak (A280 absorbing) fractions were again passed through a column of CM and DEAE- cellulose as described above.

Binding of Antibodies to Polysomes-In our first experiments, antibodies were added to polysomes after dialysis, but it was found subsequently to be more convenient to add the antibodies before dialysis to allow a longer exposure time for the antibody-antigen complex to reach equilibrium. This procedure requires that the antibody preparation be completely free of RNase activity as shown by the absence of polysome degradation in its presence.

To measure the I*61 counts in fractions from sucrose gradients, trichloroacetic acid was added to the samples to 8% and after 34 hour at 4”. the Drecinitates were collected on Millinore filters and

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subsequently counted in 5 ml of scintillation fluids in a Beckman scintillation counter.

In Vitro Assay of Collagen-synthesizing Polysomes-These poly- somes could be assayed by their ability to incorporate radioactive proline into collagen chains, when incubated in a cell-free system for protein synthesis. The system was adapted from that of Aviv et al. (11); although an S-30 from Krebs II Ascites cells, as described by these authors, was active in elongating the nascent collagen chains,’ a similarly prepared S-30 from decapitated 8- or g-day chick embryos was used for the experiments reported here. The tRNA preparation was also made from these embryos by the described procedure (ll), except that the isopropyl alcohol pre-

z The abbreviation used is: IgG, immunoglobulin G.

cipitation step was omitted. The other components of the system were as described (11) except that the polysomes to be assayed re- placed the added mRNA and the labeled amino acid was 13,4-3H]- proline. The extent of collagen synthesis was measured, after removal of the free radioactive nroline bv dialvsis. bv incubation of the nondialyzable material with collagen p”ioline hydroxylase and collection and counting of the 3HzO produced, as previously described (5).

Materials

Collagen was prepared from the leg bones of 16- to 17-dav chick embryos previously injected on the chorioallantoic membrane at Day 14 with 24 mg of a-aminonronionitrile in 0.1 ml of sterile H&; the method ofKang et al. (12)was followed but only acid ex- tractions were used. Cartilage-type collagen was identically prepared from sternum except that only salt extractions were used. Rat skin collagen and mouse skin collagen were also pre- Dared bv this method.

The &rity of the collagens used in this study was routinely checked by neutral sodium dodecvl sulfate eel electronhoresis (13) and ai1 preparations used were seen to by uncontaminated with other protein.

E$ect of Preliminary Purification of Polysomes-The A2c0 pro-

files obtained on 15 to 40% sucrose gradients before and after

purification of polysomes by collection at the 1 .O to 2.5 M sucrose

interface are compared in Fig. 1. This purification step may be seen to separate the heavier polysomes from most of the mono-

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FIG. 1. Polysome profiles of wing and leg supernatant (-) and of polvsomes obtained after nurification bv collection onto a 2.5 M s&&e “cushion” describedunder “Meth>ds” (- - -). One milliliter of a 2,000 X g supernatant from a wing and leg homoge- nate, or 1 ml of a polysome sample obtained after “cushioning” and dialysis overnight against buffer were layered onto 15 to 40% sucrose gradients in the same buffer and centrifuged for 60 min at 40,000 rpm in the SW 41 rotor of a Beckman L3-50 centrifuge. The optical density was measured by pumping the gpadients through a Gilford recording spectrophotometer.

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somes and from some of the lighter polysomes. Such polysomes were also found to be free from contaminating soluble proteins and from free tRNA.

Purijication of Antibodies to Chick Bone Collagen-Chick bone collagen was found to be a poor immunogen with specific anti- collagen antibody titers ranging in the order of 3 to 5% of the total immunoglobulin fraction as determined by radio-immuno- precipitation. Since our intended studies required a high degree of specificity, the anti-collagen antibody fraction was purified from whole sera by affinity chromatography on columns of de- natured bone collagen covalently linked to p-aminobenzyl cellu- lose (8). Elution of the bound antibodies by competition with tryptic peptides from bone collagen as in Timpl et al. (8) proved unsatisfactory for our preparations in that the recovered anti- bodies had very low activity, probably due to difficulties in completely removing the peptides from the antibodies. Higher activities of eluted antibodies were achieved by eluting with 1.0 N acetic acid. Final activities per /Ig of protein achieved by this method were found to be 80- to go-fold higher than whole anti- collagen sera and 200 to 300 pg of this purified antibody fraction could be recovered from each milliliter of whole sera.

On reduction and subsequent gel electrophoresis of the purified ‘251-labeled antibodies, two major peaks were observed (Fig. 2) corresponding to the heavy and light chains of the antibody molecule; the radioactivity profile also shows that the purified antibodies are relatively free from contamination by other serum proteins. The absence of RNase activity after passage of the purified antibodies through CM-cellulose and DEAE-cellulose columns (1) was confirmed routinely by observing no degrada- tion of polysomes that had been exposed to antibody prepara- tions for periods as long as 18 hours.

Anti-Collagen Antibody Binding to Ribosomes-It has been found (3) that in some cases antibodies will bind nonspecifically

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FIG. 2. Sodium dodecyl sulfate acrylamide gel electrophoresis of 12KI-anti-collagen antibodies. A sample of purified and iodi- nated anti-collagen antibodies was reduced by heating for 15 min at 100” in the presence of 1% sodium dodecyl sulfate and 1% mer- captoethanol and was subjected to electrophoresis according to Maize1 (13) on 7.5y0 sodium dodecyl sulfate acrylamide gels (0.6 X 8 cm) at 9 ma per gel until the bromphenol blue tracking dye had reached the bottom of the tube. Gels were fractionated using

to polysomes, giving a broad pattern corresponding to the ribo- somal concentration rather than to the nascent antigen. Other cases have been reported, however (l), where this lack of specific- ity was not found. To determine the character of the anti-col- lagen antibody fraction we had prepared, polysomes were reacted with 1251-labeled antibodies and analyzed on 15 to 409ib sucrose gradients. The results of such an experiment (Fig. 3) showed that the binding of antibodies per unit of ribosome (histogram) is not constant throughout the gradient, but is higher in the region of larger polysomes than in the region of smaller poly- somes. This result rules out nonspecific binding of the anti- collagen antibodies to ribosomes which would result in an even distribution of radioactivity per unit of ribosome throughout the gradient.

Specificity of Anti-Collagen Antibodies-Passive hemagglutina- tion tests showed that the anti-collagen antibody fraction was unable to react with skin collagen of rat or mouse, and reacted poorly with chick cartilage-type collagen. On the other hand the antibodies showed equal capability of reacting with either native or denatured chick bone collagen.

To test whether the anti-collagen antibodies were binding to polysomes at a fixed number of specific sites (presumably nascent collagen chains), radioactive antibodies were allowed to bind to polysomes in the presence of either unlabeled anti-collagen anti- bodies or normal rabbit IgG.

If the binding was specific for a fixed number of sites, then competition for these available sites should take place only with homologous anti-collagen antibodies and not with antibodies from normal rabbit sera. This was in fact the observed result, as shown in Fig. 4, where the binding of 1251-labeled anti-collagen antibodies was the same in the absence (solid histogram) or pres- ence (dashed hislogram) of unlabeled IgG from uninjected rab- bits. The addition of unlabeled anti-collagen antibodies (dotted histogram) does, however, effectively lower the binding of the labeled antibodies by competing for the same binding sites on the polysomes. These results indicate that the binding of anti- collagen antibodies is specific for a fixed number of sites on the

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FIG. 3. Binding of ‘261-labeled anti-collagen antibodies to wing and leg polysomes. Thirty micrograms of 1261-labeled anti-col- lagen antibodies were added per 10 AZCO units of “cushioned” polysomes and this mixture was dialyzed overnight at 4” as de- scribed under “Methods.” One milliliter of the solution was then centrifuged on a 15 to 40y0 sucrose gradient; the gradient was passed through a Gilford flow cell as in Fig. 1, and 0.57-ml fractions were collected. The radioactivity in each fraction was measured a Gilson gel fractionator and counted in a Beckman scintillation

counter after adding 5 ml of Aquasol (New England Nuclear). and the radioactivity per Ate0 unit was calculated (histogram).

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FIG. 4. Binding of Y-anti-collagen antibodies to wing and leg polysomes in the presence of either unlabeled anti-collagen anti- bodies or unlabeled normal rabbit IgG. “Cushioned” wing and leg polysomes were divided into three fractions and reacted with: 1261-anti-collagen antibodies alone using 50 pg/lO Also units of polysomes (-); 12sI-anti-collagen antibodies plus 250 rg of un- labeled anti-collagen antibodies (. . . . ); 1261-anti-collagen anti- bodies plus 250 pg of unlabeled normal rabbit IgG (- - -). After dialysis equal amounts of polysomes were layered over sucrose gradients, centrifuged and analyzed as in Fig. 1. Histograms represent the specific activity (cpm X 10-3/A~~~ units of poly- somes).

polysomes and that serum from rabbits which have not been im- munized with collagen does not contain antibodies that bind to these sites.

Further evidence of an indirect kind both for the specificity of these antibodies and for their ability to measure the quantity of collagen-synthesizing polysomes in a gradient, was obtained by reacting labeled anti-collagen antibodies with polysomes isolated from embryonic chick brain and liver. Since both these tissues would be expected to be synthesizing much less collagen than those of wings and legs, it would be expected that the degree of antibody binding per unit of ribosome would be much less. The results of this experiment (Fig. 5), confirm this prediction, in that both brain and liver polysomes (dashed and dotted histograms, respectively) bind much less antibody than polysomes from wings and legs (solid histogram).

It should be noted that the major difference in the amount of antibody bound occurs in the heavy region of the polysome pro- files while the brain and liver polysomes show almost as much radioactivity in the lighter regions. This latter radioactivity, as shown below, seems to represent antibody aggregation as it is RNase-insensitive.

Localization of Collagen-synthesizing Polysomes by in Vitro Activity-The reliability of the binding of anti-collagen anti- bodies for identifying polysomes carrying nascent collagen chains could be most convincingly assessed by examining the degree of correlation between the localization of collagen-synthesizing polysomes by antibody binding and by another, independent, method. Therefore, the previously reported (5) localization of collagen polysomes by their ability to elongate nascent collagen chains in a cell-free incubation, was extended. It became feasi- ble to analyze narrower regions of sucrose gradients than before, by using a more active cell-free system for elongating polysome- bound nascent chains (11). When [3,4-aH]proline is incorpo- rated into nascent chains as a result of incubating polysomes in this cell-free system, the [3H]proline residues in collagenous pep- tides can be subsequently hydroxylated by incubation with col- lagen proline hydroxylase. Since this hydroxylation releases

FIG. 5. Binding of ‘261-anti-collagen antibodies to polysomes from chick embryo brain, liver, wing, and leg. Polysomes were prepared from the wings and legs, brain, and liver of &day chick embryos and reacted with 1261-anti-collagen antibodies (60 pg/lO AZOO units of polysomes) as described under “Methods.” Equal amounts of polysomes were then analyzed on sucrose gradients and the radioactivity bound per A260 units of polysomes was de- termined for each fraction. The Atao profile shown is that ob- tained for the wing and leg polysome preparations; those of both brain and liver were similar except for a lower proportion of mono- somes. Wing and leg (-); brain (- - -); liver (....). Histo- grams represent specific activity (cpm X 10-a A2e0 units of poly- somes).

the 3H atom on Cq to the aqueous medium, equal amounts of tritiated hydroxyproline and tritiated Hz0 are formed. By measuring the radioactivity released to HzO, a measure of the collagenous peptides synthesized by the polysomes in the cell- free system is obtained.

The results of measuring the collagen-synthesizing ability of the polysomes in 0.5-m] fractions from a 15 to 40% sucrose gradi- ent are shown in Fig. 6 (dashed histogram). The collagen-syn- thesizing polysomes may be seen to constitute a discrete, roughly bell-shaped peak in the same region of the gradients as was pre- viously reported (5). It should be noted that the centrifugation time used in this experiment was 50 min compared to 73 min used in the earlier experiments (5), but control experiments showed that the polysomes in the heavy region of the gradient move very little down the tube as a result of the longer centrif- ugation. To get a measure of the amount of collagen synthe- sized, relative to the total protein synthesized by these polysomes, the ratio (dpm in [3H20] x 2 x IOO)/(dpm in total protein) is plotted in Fig. 6 (solid histogram). The numerator is multiplied by 2 to facilitate comparison with experiments using [%]proline, since the radioactivity that appears in 3H20 and in hydroxy- proline is equal, but represents only half of the radioactivity present in the precursor proline molecules (14). This ratio is atypically low (5% maximum) in the experiment of Fig. 6, since ratios of 9 to 13 y. have been observed in a number of experiments with polysomes which were not “cushioned” prior to layering on the gradient. An identical localization of collagen-synthesiz- ing polysomes is obtained if the preliminary purification of the polysomes is omitted, although in this case the pellet has more activity, in some cases equal to that found in the polysome peak.

Localization of Collagen-synthesizing Polysomes by Binding of Anti-CoZZagen Antibodies-The experiments already reported (Figs. 3, 4, and 5) show that the polysomes which bind the most anti-collagen antibodies per unit of polysome (Az,o) are in Frac- tions 2 and 3 from the bottom of the gradient. Since the total amount of polysomes increases in the fractions further from the

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FIG. 6. Location of collagen-synthesizing polysomes on sucrose gradients by in vi&o assay. The polysomes from the wings and legs of 27 g-day chick embryos were prepared and collected on a 2.5 M sucrose cushion as described under “Methods” and then layered in O.&ml aliquots on six 15 to 4001, sucrose gradients. The gradients were centrifuged for 50 min at 40,000 rpm in a Beckman SW 41 rotor. Three gradients were used to monitor Atao; since the profiles agreed closely, one is shown. The polysomes from the other three gradients were collected in 11 0.5-ml fractions by puncturing the bottoms of the tubes; an additional fraction of 3.75 ml was collected from one of these gradients. The corre- sponding 0.5-ml fractions were pooled and the polysomes were pelleted by centrifuging for 150 min at 43,000 rpm in the SW 50.1 rotor or for 180 min at 40,000 rpm in the SW 41 rotor. The pellets were stored at -70” until incubation in the cell-free system for completing nascent chains in a final volume of 50 ~1. After incu- bation at 37” for 60 min, samples were put on ice and 0.7 ml of 30 mM Tris-HCl, pH 7.4, and 2 ~1 of 100 mM n-proline were added. After extensive dialysis against 30 mM Tris at 4”, samples of 25 ~1 were taken from each dialysate to determine total nondialyzable [aH]proline present. The amount of [3H]Hg0 produced after in- cubation of 0.6 ml of each dialysate with proline hydroxylase was then determined (dashed histogram). The ratio of 3H~0 produced, x2, to total nondialyzable [3H]proline is also shown (solid histo- gram) as a measure of the fraction of total polysomes in each frac- tion that is devoted to collagen synthesis.

bottom, the greatest total amount of antibody binding is found in Fractions 3 and 4.

It has been noted by other workers (2) and observed by our- selves, that when polysomes are mixed with radioactive anti- bodies prior to centrifugation through sucrose gradients, some of the radioactivity that is found in the polysomal regions of the gradients is resistant to RNase, probably because of antibody aggregation. Fig. 7 compares the AzcO profiles and radioactive distribution of bound i251-anti-collagen antibody in control poly- somes and polysomes treated with RNase after reaction with antibody. It is seen that even though polysomes are removed from the heavy regions of the gradient, after RNase treatment, a substantial portion of radioactivity remains in the polysome re- gion. This result would indicate that some of the radioactivity found in the polysome region of gradients not treated with RNase is due to non-polysomal associated antibody. Better localiza- tion of collagen-synthesizing polysomes might be possible if the spurious non-polysomal associated radioactivity were subtracted from the total radioactivity of the polysome region, so experi- ments were done in which antibody-treated polysome prepara- tions were split in halves and one half was treated with RNase. Both halves were then analyzed on sucrose gradients and the radioactivity in the polysome region of the RNase-treated sam- ple was subtracted from the untreated sample. This type of analysis, although valid for the heavy regions of the gradients, results in negative values for the control gradients in the lighter

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FIG. 7. Optical density profiles and radioactive distribution of polysomes reacted with ‘261-anti-collagen antibody with and with- out subsequent RNase treatment. “Cushioned” wing and leg polysomes were reacted with 1*61-anti-collagen antibodies (50 fig/l0 A260 units of polysomes). After dialysis the sample was split and one-half was treated for 15 min at 4” with 10 fig/ml of pancreatic ribonuclease. The RNase-treated and untreated sam- ples were then layered over 15 to 40% sucrose gradients and cen- trifuged for 60 min as in Fig. 3. The gradients were then frac- tionated and each fraction assayed for radioactivity. Polysomes treated with anti-collagen antibody; --, A260; O- - -0, radio- activity. Polysomes treated with anti-collagen antibody fol- lowed by RNase : . . . . , AzG~; X- - -X, radioactivity.

regions. This is due to the additional radioactivity contributed to the lighter regions by movement of heavier polysomes and their bound radioactivity to the lighter region of the gradients after RNase treatment. The results of such experiments (Fig. 8) show a much sharper zone of bound radioactivity in the re- gion of collagen-synthesizing polysomes. Fig. 8 also shows the profile obtained under identical conditions when the centrifu- gation time is reduced to 35 min. It can be seen that the major peak of polysome-bound radioactivity is correspondingly moved back in the gradient in much the same relationship to the A260 profile as that seen at the 60.min spin time. It should also be noted that no discernible peak is noticeable sedimenting ahead of the polysome-bound antibodies at 35 min. A similar experi- ment (not shown) in which centrifugation was for 20 min, also failed to show a peak of radioactive antibodies moving ahead of the main collagen-containing polysome peak, which in this case was centered at Fraction 12 from the bottom.

Precipitation of Collagen-synthesizing Polysomes-As shown in other systems (4, 15-18) the binding of specific antibodies to nascent proteins on polysomes can be used to isolate these poly- somes for subsequent extraction of their respective mRNAs. Two techniques employed have involved addition of a slight excess of antigen-specific antibody (direct method) or addition of a second antibody directed against the protein-specific anti- body (indirect method) to cause aggregation and subsequent precipitation. Since we wished to use this antibody precipita- tion procedure to isolate collagen mRNA, we tested the ability of our antibody preparation to precipitate polysomes from the region of polysome gradients responsible for collagen synthesis.

I’olysomes were prepared from isolated wings and legs which had been briefly incubated with [3H]proline to label nascent chains. A third of the cushioned polysome preparation was used

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FIG. 8. Localization of collagen-synthesizing polysomes on gradients bv binding of 12~I-anti-collaaen antibodies. “Cush- Toned” wing”and leg p%ysomes were reactid with 1251-anti-collagen antibodies (50 fig/10 AZ60 units of polysomes). After dialysis the sample was split into four equal fractions and two of the fractions were treated for 15 min at 4” with 10 pg/ml of pancreatic ribonu- clease. The duplicate RNase-treated and untreated samples were then layered over 15 to 40yo sucrose gradients and centri- fuged for 35 or 60 min as in Fig. 3. The gradients were frac- tionated, assayed for radioactivity, and the radioactivity of each fraction of the RNase-treated samples was subtracted from that of the corresponding control fraction. It should be noted as dis- cussed under “Results” that RNase treatment, in removing radio- activity from the heavy regions of the gradients to the light re- gions, results in negative values in the light regions when the ItNase-treated fractions are subtracted from the control frac- tions; these are not plotted.

as an untreated control and received no antibody, while the re- mainder was split into two equal portions, one of which was re- acted overnight during dialysis (see “Methods”) with normal sera IgG and one of which was similarly reacted with anti-col- lagen antibodies. Goat antibody against rabbit IgG was then added to the two polysomal samples that had received rabbit antibodies, in a ratio previously determined to precipitate rabbit antibody. All three polysome samples were allowed to stand 75 minutes at 4’ prior to layering on 15 to 4Ooj, sucrose gradients. The polysome distribution was monitored by both Am and by the presence of [3H]proline-labeled nascent chains with the results shown in Fig. 9. Comparison of the radioactivity profile after treatment with anti-collagen antibodies ( X- - -x) with the profile after treatment with the same amount of normal rabbit IgG (o- - -o), shows that the polysomes from the collagen- synthesizing region of the gradient were preferentially precipi- tated by the anti-collagen antibodies. The A~G~ and radioac- tivity profiles of the polysomes which were dialyzed without exposure to rabbit and goat antibodies (not shown) were identical in shape with the polysomes treated with normal rabbit IgG; uncertainties in relative recoveries prevented conclusions about whether the absolute recoveries of the untreated polysomes and of the polysomes treated with normal rabbit IgG were identical.

The purity of the collagen polysomes after precipitation by this indirect method was examined by measuring the ratio of radioactivity in hydroxyproline to total radioactivity in nascent, antibody-precipitable chains, after labeling polysomes by a brief incubation of S-day wings and legs with [3,4-3H]proline. To ensure complete precipitation of all collagen-synthesizing polysomes the amount of anticollagen antibody used was raised to 150 pg/lO A260 of polysomes. Also to minimize possible pre- mature precipitation during dialysis the antibody was added after dialysis, followed by goat anti-rabbit antibodies. The

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FRACTION NUMBER

FIG. 9. Precipitation of polysomes treated with anti-collagen antibodies or with normal rabbit IgG, by goat anti-rabbit anti- bodies. Wings and legs from 24 8-day embryos were incubated for 15 min at 37” with [3,4-3Hlproline (30 &i/ml) in 8 ml of modi- fied Krebs-ltinger phosphate (19) and polysomes were prepared as described under “Methods.” Polysomes were then treated with unlabeled anti-collagen antibodies (100 pg/lO A260 units of poly- somes) or unlabeled normal rabbit IgG at the same concentration. After overnight dialysis, goat anti-rabbit antibodies were added to each sample at 10 times the concentration of the rabbit anti- bodies. After 75 min at 4” the samples were then analyzed on 15 to 40% sucrose gradients as in Fig. 3. X- - -X, polysomes treated with anti-collagen antibodies; O- - --0, polysomes treated with normal rabbit IgG.

results of such an analysis (Table I) show that the addition of anti-collagen antibodies, followed by goat anti-rabbit antibodies, results in about a 3.6-fold increase in the hydroxyproline content of the polysomes which pellet at the bottom of the 15 to 40% sucrose gradient, as compared with the pellets obtained with normal rabbit IgG. The value of 8.0 for the per cent of total radioactivity present in hydroxyproline in the nascent chains precipitated with anti-collagen antibody indicates that these polysomes are not yet pure collagen-synthesizing polysomes. We do not know at present, however, the theoretical value for the per cent of total radioactivity in hydroxyproline in pure col- lagen nascent chains labeled with [3,4-3H]proline, not only be- cause of the presence of the “pro-a region” (21) but also because the minimum size of nascent chain necessary for hydroxylation is unknown. The theoretical ratio for completed, mature (Y- chains is 27.5%, when the source of the label is [3,4-3H]proline. As the last line on Table I indicates, however, if the radioactivity found pelleted under conditions where no antibody is present (control) is subtracted from the radioactivity precipitated by the anti-collagen and goat anti-rabbit antibody treatment, a value of 14.7% is obtained.

DISCUSSION

This report demonstrates that antibodies to collagen can be used to localize, quantitate, and isolate polysomes synthesizing collagen. Although collagen is a poor antigenic stimulant and antibodies against it are present only in small amounts in the sera of immunized rabbits, the present results show that it is feasible to purify these antibodies so that they are specific for collagen, and free from RNase, two essential requirements if they are to be used to locate and purify polysomes. The exact purity of the final purified antibodies is difficult to determine as elution of the collagen-bound antibodies with 1.0 N acetic acid can result in a variable degree of antibody inactivation. Acrylamide gel analy-

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TABLE I Hydrozyproline content of immunoprecipitated polysomes

Polysomes carrying [3H]proline-labeled nascent chains were were then layered on 15 to 40y0 sucrose gradients and centrifuged prepared as in Fig. 9. After dialysis the “cushioned” polysomes for 35 min at 40,000 rpm in the SW 41 rotor of a Beckman ultra- were divided into three equal samples; two samples were treated centrifuge. The material which had pelleted to the bottom of with anti-collagen antibody or normal rabbit IgG, respectively, the tubes was analyzed after acid hydrolysis for total radioac- for 45 min, followed in each case with goat antibodies for 45 min. tivity, and for radioactivity in hydroxyproline (20). No antibodies were added to the remaining third. The samples

Sample

Polysomes layered on 15 to 40% gradient

Pellet after spinning 15 to 40% gradient

Pelleted radioactivity

Antibody added Total dpm in hydroxyproline Protein

dpm Hydroxypro-

line dpm

/ 488,000 ( 14,000 / r

None 107,000 1,670 21.7 11.9 1.6 Normal rabbit 90,ooo 1,980 18.4 14.1 2.2 Anti-collagen 207,000 16,500 42.5 117 8.0 Anti-collagen - control 100,000 14,800 20.6 105 14.7

Total pellet dpm in hydroxyprolioe

% 2.9

sis of the product, however, indicates that such a preparation is free of any serum proteins other than those migrating as heavy and light immunoglobulin chains. Although antibody prepara- tions were routinely chromatographed on DEAE- and CM-cel- lulose to remove RNase activity we have found that chromatog- raphy on the column of p-aminobenzyl cellulose-collagen alone can produce an antibody fraction free of RNase activity as judged by maintenance of polysomes after prolonged exposure to the antibody.

It should be noted that we have found it unnecessary to iso- late Fab fragments of our antibody in order to avoid the non- specific binding found by Delovitch et al. (3) when using whole antibody to react with polysomes. In this respect our results agree with Palacios et al. (1) who also found it unnecessary to fractionate their anti-ovalbumin antibody. It would seem that the specificity of the reaction of antibodies with nascent chains on polysomes varies with the type of antibodies used and should be determined individually for each antibody.

As shown under “Results,” our antibodies are capable of re- acting specifically and quantitatively with nascent collagen peptides on polysomes. Such antibodies should therefore be able to provide an accurate measurement of the amount of a specific type of collagen being synthesized by a tissue. To ob- tain precise measurements with such procedures, however, one must be aware of contributing factors such as antibody aggrega- tion (Fig. 7).

The affinity chromatography used would have selected for antibodies to structures on denatured collagen or single poly- peptide chains rather than native collagen. Use of a variety of vertebrate collagens from different species for immunization of rabbits has shown that usually only low amounts of antibodies are produced which require native, triple helical structures for reactivity (22). Extensive immunochemical studies have not been done so far on chick collagen but preliminary results indi- cated that this is true also for rabbit antibodies to chick collagen3 These findings, however, do not exclude the possibility that anti- collagen antibodies, purified on columns of denatured collagen, can cross-react with both native and denatured collagen for rea- sons outlined elsewhere (22). As stated under “Results,” our purified anti-collagen antibodies were in fact found to react with both native and denatured collagen in passive hemaggluti-

3 E. Hahn, R. Timpl, and E. J. Miller, in preparation.

nation tests. Thus our experiments demonstrating binding of such purified antibodies to nascent chains on polysomes do not provide conclusive evidence as to the form or structure of collagen present. These antibodies, however, appear to show extensive tissue specificity as was found by lack of cross-reactivity to chick cartilage collagen (results not shown). This was also indicated in a recent study using a different system (23). The specificity of the antibodies for chick, as compared to mouse or rat collagen, should prove especially useful for assaying the synthesis of chick collagen mRNA, in heterologous protein-synthesizing cell-free systems. Our preparations of the Krebs II ascites system (ll), for instance, have a rather high endogenous level of collagen synthesis,’ which is troublesome in other collagen assays but should not interfere with the assay of chick collagen by the chick- specific antibodies.

The results of the more exact localization of the collagen poly- somes on sucrose gradients, reported here, are chiefly important in showing that these polysomes are distributed in a roughly bell-shaped peak as would be expected for a discrete class of polysomes. As discussed earlier (5) this localization agrees in size with a monocistronic collagen mRNA molecule.

The question of whether the polysomes we observe in the 15 to 40 y0 sucrose gradient are in fact a degradation product of still larger polysomes that would be pelleted during the centrifuga- tion times of 50 or 60 min, is worth considering since in that case the true collagen polysomes might correspond to the size ex- pected for a tricistronic mRNA that coded for all three Q chains destined to form the triple helix of a single tropocollagen mole- cule. In fact, pulse-labeled collagen, as well as material that shows collagen-synthesizing activity in the cell-free protein syn- thesis system, is consistently found pelleted at the bottom of these gradients, Although the amount of collagen-synthesizing activity in the pellet is quite small if the polysomes are first purified by collection on a shelf of 2.5 M sucrose (compare Fig. 6), the activity in the pellet may equal that in the gradient, if this purification is omitted. The failure, however, of 1261-anti- collagen antibody to detect collagen-synthesizing polysomes which move faster than the main collagen-polysome peak when the gradient centrifugation time is reduced to 20 or 35 min (Fig. S), indicates that the collagen-synthesizing polysomes which pellet are sedimenting too rapidly to represent free polysomes even of the size corresponding to a di- or tri-cistronic collagen

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mRNA. Although we do not know the nature of the rapidly sedimenting material, it is plausible that it represents cellular debris to which some collagen-polysomes are bound. In any case, the failure to detect a larger species of collagen-synthesizing polysomes after the short centrifugation times indicates that the collagen-synthesizing polysomes detected in our gradients do not represent degradation products of larger polysomes.

Most importantly, however, as shown by Fig. 9 and Table I, anti-collagen antibodies can prove useful in the isolation of colla- gen-synthesizing polysomes where these polysomes represent only a small fraction of the total polysomes. Although we have not yet succeeded in pelleting collagen polysomes completely free from contamination with other polysomes, we find that vari- ations in the conditions of obtaining this pellet cause large dif- ferences in the purity. Although it has not been rigorously ex- cluded that a lack of specificity of our antibody’ preparation is responsible for the incomplete purity of the pelleted polysomes, the data presented suggest this is unlikely and that refinements in the isolation procedures will allow even more purification. In any case, the purification step described here should aid in attempts to identify and isolate the mRNAs for collagen chains.

Achowledgments-We wish to thank J. Forella and D. Marti- nell for their excellent technical assistance, and Middlesex Memorial Hospital which supplied the blood used in the hemag- glutination tests.

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P J Pawlowski, M T Gillette, J Martinell and L N Lukensanti-collagen antibodies.

Identification and purification of collagen-synthesizing polysomes with

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