a kinetic study of collagen biosynthesis

THE JOURNAL OF BIOLOGICAL CHEMI~TIW Vol. 247, No. 3, Issue of February 10, pp. 85tXG2, 1972

Printed in U.S.A.

A Kinetic Study of Collagen Biosynthesis

(Received for publication, August 24, 1971)

JENS VEST* AS-D ILUXL A. PIEZ

From the Laboratory of Biochemistry, National Institute of Dental Research, National Institutes of Health,

Bethesda, Marylard %‘0014

SUMMARY

The kinetics of incorporation of [X!]glycine into the cyanogen bromide peptides of the al and ot2 chains of pulse-labeled collagen was analyzed in a system of cranial bone (calvaria) from newborn rats. From the time dependence of the difference in specific activity between peptides in the same chain, it was estimated that the rate of translation of the mRNA for a collagen chain is about 209 residues per min and is approximately equal for both otl and (r2. The data indicate that hydroxylation and helix formation occur either during translation or very rapidly thereafter.

It was determined that the samples consisted in part of collagen obtained by conversion of precursor (procollagen) during neutral salt extraction. The pro oil chain from acid- extracted samples was found to have a molecular weight of 115,000 compared to 95,000 for an (Y 1 chain. The calculated assembly time for a pro 0~ chain, and for a completed procollagen molecule, is then 6.0 min, assuming linear polypeptide chains of 1,250 residues and a constant translation rate. The kinetic analysis allows the prediction that the extra peptide material that is lost during conversion of procollagen is at the amino-terminal ends of the chains.

The time previously calculated (VUUST, J., AND PIEZ, K. A. (1970) J. BioZ. Chem., 245, 6201) from pulse-labeling studies for the biosynthesis of a complete fully hydroxylated collagen molecule, 4.8 min, can now be seen to be an apparent value obtained as a result of the then unknown conversion of procollagen to collagen during extraction. Re-evaluation of those data leads to a biosynthesis time of 5.8 min for a procollagen molecule, in good agreement with the present study. The rate of conversion to collagen is not known.

The intracellular biosynthesis of collagen includes amino acid assembly of the cz chains on polyribosomes, hydroxylation of certain prolyl and lysyl residues in peptide linkage, and aggrega- tion of two otl chains and one a2 chain into a triple helix (l-3). An earlier investigation by us (4) indicated that the time required for one a! chain to complete these biosynthetic steps is about 4.8 min at 37”. This time was calculated from the slopes

* Fellow of the Helen Hay Whitney Foundation. Present ad- dress, Institute of Biochemistry, University of Aarhus, DK 8000 Aarhus C, Denmark.

of the radioactivity gradients along collagen o( chains obtained from pulse-labeled rat calvaria. However, the time required for the individual steps could not be determined from these data. Other studies (5, 6) have now demonstrated that hydroxylation of proline occurs primarily on nascent chains, but it can also occur on completed chains (1). V I o data arc available on molecular assembly in viva but it has been suggested (7, 8) that it may begin before the a! chains are completed.

It has recently been reported (9-11) that collagen is initially synthesized in a precursor form, designated “procollagen,” that may function in transport and in triple chain helix formation. Procollagen apparently contains polypeptide chains that are larger than (Y chains (10) and is converted in vitro by pepsin to a collagen-like product (9, 10) in a process that may mimic the natural step. These results must be taken into account in an) consideration of collagen biosynthesis.

We have now measured the translation step independently and determined its rate by analyzing the kinetics of radioactivity incorporation into the cyanogen bromide peptidcs of native colla- ven synthesized by rat calvaria in culture. The kinetic ap- Froach was originally introduced by Krropf and Lamfrom (12) and has been successfully used by other investigators (13). We have also studied the properties of procollagen in our system. These results allow a more complete description of collagen biosynthesis.

METHODS

Translation Rate for Collagen OL Chains-In our experiments, the collagen-synthesizing system was calvaria from newborn Sprague-Hawley rats cultured in modified Eagle’s ;\Iiminum Essential Medium containing B-aminoyropionitrile to inhibit collagen cross-linking (4), A preliminary experiment was done to measure the time required for added radioactiv-e glycine to reach equilibrium in the free amino acid pool of the system. Five calvaria were placed in each of ten 25.ml Erlenmeyer flasks, rinsed with 10 ml of the incubation medium, and then incubated in another 5 ml of medium for 30 min in a water bath at 37” under constant slow shaking. The medium was poured off and replaced with 5 ml of fresh medium containing [UJ4C]glycine (> 100 mCi per mmole), 5 &i per ml, pre- heated to 37”. Incubation was allowed to continue, and every 2 min a flask was removed from the v,-ater bath. Incorporation was stopped by rapidly pouring off the medium, placing the flask in ice water, and adding 10 ml of ice-cold distilled water. Free radioactive amino acid was removed by extensive dialysis against distilled water at 5”. The calvaria were homogenized

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and lyophilized, and a weighed amount from each of the 10 samples was hydrolyzed under nitrogen in sealed tubes with 6 K HCl for 24 hours at 108”. After removal of the HCl in a rotatory evaporator, the hydrolysates were each dissolved in 5.0 ml of water and l&ml aliquots were assayed for 14C activity.

The experiment designed to measure translation rate was carried out as follows. Thirty calvaria in each of five 50.ml Erlenmeyer flasks were treated as described above with 15 ml of medium per flask and an isotope concentration of 10 $Zi of [YJglycine per ml. Incorporation was continued for 8, 10, 12, 14, and 16 min. After removal of the medium and cooling of the sample, 20 ml of ice-cold 1 M NaCl (0.05 M Tris, pH 7.5) was added to each flask, and collagen was extracted for 3 days at) 5” with constant stirring. The insoluble residue was removed by centrifugation at 8000 X g for 30 min. Collagen was precipitated with 20*& NaCI, collected by centrifugation at 8000 X g for 30 min, and dissolved in the buffer used in the subsequent chromatographic separation on CM-cellulose1 (sodium acetate, 0.06 ionic strength, pH 4.8) followed by exhaustive dialysis against this same buffer. Collagen uniformly labeled with trit- iated glycine was obtained by incubating 10 calvaria in 10 ml of medium for 24 hours wit’h 5 PCi per ml of [2-3H]glycine (5 Ci per mmolc) ; collagen extraction and purification were achieved as described above. Portions of this 3H-labeled collagen solution were then added as internal standards (4) to the collagen samples pulse-labeled with [Wlglycine. To make the data from the different points in the time course directly comparable, the [3H]collagen and the [‘%]collagen had to be mixed in identical molar proportions for each 14C-labeled sample (12, 13). There- fore, aliquots from each of the [W]collagen samples wrre dena- tured at 45” for 15 min, filtered, and taken to dryness in a rotatory evaporator. After hydrolysis for 24 hours in 6 N HCl and removal of the HCl by evaporation, the hydrolysates were assayed for hydroxyproline by Procedure B of Bergman and Loxley (14). Volumes of the YXabeled samples containing identical amounts of hydrosgproline (and therefore of collagen) were then mixed with a constant volume of the [3H]collagen solution. Lathyritic rat, skin collagen, isolated by salt extraction (15), 100 mg l)er sample, was added as a carrier. The collagen was purified and separation of the cul and ot2 chains was accomplished by ion exchange chromatography under denaturing conditions on a CWcellulosc column (Whatman (X-32) as described (4, 16). Flnthcr purification of the CY chains was achieved by molecular siere chromatography on an Agarose column (Bio-Gel ,4 1.5m, 200-400 mesh) (17). Cleavage of the N chains with cya~~ogcn bromide and chromatographic separation of the resulting pepbides were done as previously described (4). 3H and 14C activities of the isolated pept,ides were then determined in a liquid scintillation counter (4).

Wydroxylation oj Pulse-labeled Collagen-Fifteen calvaria in each of two flasks were incubated for 7.5 min with 10 ml of medium containing 10 &i per ml of L-[U-14C]proline ( >180 mCi per mmole). Incubation was stopped in the usual manner with 20 ml of ice-cold 1 M NaCl. To one of the flasks, 1.5 mM (Y, cr’-dipyridyl was added in the NaCI solution to inhibit hydroxylation that might occur during the extraction period. Collagen extraction and isolation of crl were carried out as described above. Portions of [W]proline labeled cul thus obtained were

1 The abbreviation used is: CM-cellulose, carboxymethylcellu- lose.

hydrolyzed in 6 N HCl and applied to the column of an automa- tic amino acid analyzer (18) ; the effluent was collected directly from the column in l.O-ml fractions which were then counted in a liquid scintillation counter to determine the ratio of [14C]hydroxyproline to [14C]proline. In a separate experiment, IO calvaria were labeled with [14C]proline for 24 hours; in this case both whole al and the al cyanogen bromide peptides were isolated and analyzed for radioactive hydroxyproline to proline ratio.

Conversion of Procollagen to Collagen-Radioactive procollagen has been identified by CM-cellulose chromatography of acetic acid extracts of pulse-labeled rat calvaria (10). -% 1 ;\I NaCl extract of pulse-labeled calvaria, however, yields only material similar to normal collagen (4). This indicates that while newly synthesized collagenous material is extracted in the precurfior form with acetic acid, 1 M NaCl either allows conversion of procollagen to collagen to take place during the extraction or does not extract the procollagen. To distinguish between these pos- sibilities, the following experiment was done. Thirty calvaria were labeled for 10 min with [14C]g1ycine and extracted lvith 0.5 M acetic acid for 3 days. The extract was then dialyzed exten- sively against 0.5 M acetic acid, lyophilized, and dissolved in 20 ml of cold 1 M NaCl (0.05 M Tris, pH 7.5). To a IO-ml aliquot were added 25 fresh calvaria, and the sample was taken t)hrough the usual salt extraction procedure at 5”, including precipitation with 20% NaCl, and chromatographed on CRI-cellulose. hs a control, another lo-ml portion Tvas treated in the same way except that t,he calvaria were omitted. ,The radioactive crl and pro al peaks from the CM-cellulose column were then subjected to molecular sieve chromatography on agarose (17).

RESULTS

Translation Rate for Collagen 01 Chains-The theory behind the met,hod used to measure translation rate is as follows (12, 13). Let t be auy given time after introduction of a radioactive amiuo acid into a protein-synthesizing system, and let tt be the translation t,ime for the polypeptide chain. It follows from the known mechanism of polypeptide chain synthesis, \q-hich also applies to collagen (4), that the amount of radioactivity incorporated into the amino terminus at t + tt will be equal to the radioactivity contained in the carboxy terminus at time t. Con- sequently, tt can be found by determining the time difference between two curves describing radioactivity incorporation with time into the amino- and carboxy termini, respectively. In our experiments, points within the chain as represented by specific peptides were utilized and the translation rate was calculated from the measured times and known distances between the peptides. tt was then calculated from the known length of the chain, assuming a constant translation rate. [‘%]Glycine was used as the radioactive isotope. Except for a short amino- terminal segment which was not included, this amino acid oc- cupies every third position in the collagen chains, and the 1% activity found in each of the cy chain peptides obtained by CNBr cleavage can therefore be assumed to reside in the midpoint of that peptide. For both the oil and the cr2 chains, the order (19-21) and size (22, 23) of the CNBr peptides have been estab- lished so the positions of all these midpoints are known.

It is desirable that this kind of study be performed over a time period during which incorporation of the radioactive amino acid is linear. The results presented in Fig. 1 show that incorporation of the glycine isotope into total protein is linear from


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S5S Collagen Biosynthesis Vol. 247, so. 3

-‘;:~/, , , 4 8 12 I6 20

i

TIME (Mln)

FIG. 1. Total [‘%]glycine incorporation into rat calvaria. The straight line was calculated by least squares analysis of all points beyond the 2-min value.

,': 06. \

k - 05- "

E

/ al CHAIN

/

CB- PEPTIDE: / .=7 /

a=ii+4+5 ,' a//

0=3 . =s

/ / / /

2 4 6 a IO 12 14 I6 TIME IMin)

FIG. 2. Kinetics of [Wlglycine incorporation into CNBr peptides from the al chain of collagen. The peptides are numbered according to Butler et al. (22). The solid lines were calculated by a modified regression analysis (see text), and the theoretical lines for ‘Y-incorporation into the carboxy- and amino-termini were calculated from the translation rate (Table I) and the known size (22) and order (19) of the CNBr peptides.

about 4 min to at least 20 min. The experiment analyzing the kinetics of incorporation into the CNBr peptides of salt-extracted collagen was therefore carried out over the time period 8 to 16 min.

Fig. 2 shows the [‘YJglycine incorporation into some of the al peptides, plotted against labeling time. The peptides ~yl- CI32, 4, and 5 were not resolved and appear as a single point. This is valid since they are adjacent in the chain and can be isolated as a group by molecular sieve chromatography (4). They are all small and would give statistically less significant counting data if analyzed separately. For the same reason, the small amino-terminal peptide crl-Cl31 was not counted. The data for the carboxy-terminal peptide al-CR6 were excluded for reasons explained below. The equations for the best four parallel lines combining the fire experimental points for each peptide were calculated by a modified regression analysis in which the common slope and the four individual intercepts were found which gave minimum square deviation.

The time required for translation of the interval represented by the midpoints of two peptides was calculated as the horizontal

2 ' Cl2 CHAIN

,I 0.6 - CB-PEPTIDE.

\ 0'3

N-Termnal Amino Ac,d

TIME (rmn)

FIG. 3. Kinetics of [14C]glycine incorporation into CNBr peptides from the ~2 chain of collagen. The peptides are numbered according to Fietzek and Piez (23). The solid lines were calculated by a modified regression analysis (see text), and the theoretical lines for 14C-incorporation into the carboxy- and amino-termini were calculated from the translation rate (Table I) and the kno\vn size (23) and order (20,21) of the ot2 CNBr peptides.

TABLE I

l’ranslation Tale for oil and a2 chains of collagen

Peptide.

Slopea......

At (min)Q. An (resi-

dues). Rate (resi-

dues/ min)

Average rate......

al-CB ar2-CB - 2

-I -

I- +4+5’ 8 3 7

-i

0.0420 0.0420 0.0420 0.0420 I I

0.848 1.143

101 206

223 180

I

0.876

211

1.85

349

241 189

209

-

4 I 3

0.0526 0.0526

(1 Calculated from a modified regression analysis of the data in Figs. 2 and 3 (see text).

distance between the lines for the two peptides. As shown in Table I, to arrive at the translation rate for each interval, these values were divided int,o the number of amino acid residues, An, between the midpoints. The results for the several intervals do not differ significantly.

The data for the cr2 peptides &Cl33 and 4 are graphically shown in Fig. 3. cr2-CBO, 1, and 2 were not analyzed since these peptides are small. The data for the carboxy-terminal peptide &CB5 were excluded (see below). The data shown at 12 min in parentheses were not included in the calculation of the lines since the)- appear to be aberrant for unexplained rea.sons. The translation rate for the interval between a2-Cl33 and a2- CB4 was not significantly different from the rate found for the crl chain (Table I). I f this value is averaged with the three intervals in the al chain, a rate of 209 residues per min is ob-


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Issue of February 10, 1972 s39

tained. The range of values observed suggests an error in this least the same for all samples. The linearity of the data indi- average value of less than 107;. cates that this was the case. It was further shown in the 12-

The carboxy-terminal peptides, cul-CB6 and cu2-CB5, when min experiment that extraction was essentially complete from isolated and assayed for 14C incorporation gave unexpectedly the following considerations. It was found that 40% of the high and erratic values (not shown). A possible cause may be total [*4C]glycine incorporated was in the salt extract. From sought in the known heterogeneity of these peptides, as described for c&C%6 from rat (22) and calf skin collagen (24), which TABLE II arises at least in part from carboxy-terminal proteolytic attack (24). I f the % and 14C-labeled peptides are heterogeneous to

Degree of hydroxylation of proline

I different degrees, the chromatographic procedures may arti- factually change the 14C :% ratio if the species partially resolve. This problem was encountered in the earlier study with al-CBI, although not with cul-CB6 and &CB5 (4). The test for homo- _ geneity is to show that the 14C:3H ratio is constant across all chromatographic peaks. This was done in the earlier study (4) and for all of the peptides utilized in the present study. After repeated purification in an attempt to improve the analytical data, there was insufficient oil-CB6 and c~2-CB5 to adequately test them. However, the inconsistency of the 14C:3H data and evidence that heterogeneity exists we believe justifies the ex- clusion of the data for these peptides from our calculations.

Chain or peptide

Hydroxyproline to proline ratio

Radioactive AnalyticaID

al ollb al oil-CB2 + 4 + 5 oil-CBS oil-CB3 oil-CB7 al-CB6

7.5 min 0.77 0.83 7.5 min 0.77 0.83

24 hrs 0.89 0.83 24 hrs 1.05 0.93 24 hrs 1.07 0.87 24 hi-s 1.10 1.00 24 hrs 0.77 0.69 24 hrs 0.69 0.52

For the data in Figs. 2 and 3 to be valid it is necessary that recovery of labeled collagen from the calvaria be complete or at

n Calculated from the data of Butler et al. (22). h cu,&IXpyridyl was present in the extracting solution.

c

c

c

Radioactivi ~A-*--..u \

a2

I 1600

I 800 g

3

; 3

400

EFFLUENT VOLUME, ml

a2

EFFLUENT VOLUME, ml

FIG. 4. CM-cellulose elution patterns of an acetic acid extract of pulse-labeled (10 min) rat calvaria after incubation with un-

were chromatographed under denaturing conditions at 40” (16).

labeled calvaria in 1 M NaCl (top) and, as a control, in 1 M NaCl The distribution of radioactivity in the original acid extracted

alone (bottom). material was not significantly different from that observed after 1

Solid line, absorbance pattern of carrier rat skin M NaCl incubation. collagen; dash line, radioactivity elution pattern. The samples


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860 Collagen Biosynthesis Vol. 247, No. 3

the [14C]hydroxyproline to [14C]proline ratio in a separate 24-hour incorporation experiment, it was determined that 17% of the total protein synthesized was collagen. Correcting for the high glycine content of collagen relative to noncollagenous protein, the 40% [14C]glycine in the extract is equivalent to about 80% of the total collagen synthesized. It can be further calculated from the biosynthesis time of 6 min, as discussed later, that 25% of the counts in collagen after labeling for 12 min will be in nascent chains. The latter fraction would be excluded from the extract. Although approximate, these calculations indicate that recoveries were complete within experimental error.

Since a constant amount of aH-labeled carrier was added to each sample at this point and 14C:aH ratios were measured thereafter, recoveries after the various chromatographic steps do not affect the results. Since purity was of critical importance, low recoveries were acceptable.

Hydroxylation of Pulse-labeled Collagen--As shown in Table II, oil chains isolated from salt extracts of calvaria labeled for 7.5 min with [14C]prolme had a radioactive hydroxyproline to proline ratio of 0.77, whether ar,oc’-dipyridyl, a known inhibitor of hydroxylation (25), was added to the extracting solution or not. This indicates that no hydroxylation took place after the labeling period. The ratio is slightly lower than the value of 0.85 observed before (4), which may be ascribed .to the use of different assay procedures. It is also lower than the value of 0.89 obtained for al labeled for 24 hours (Table II). This was expected since at short times the carboxy-terminal end, where the analytical hydroxyproline to proline ratio is smallest (22),

PRO a.1

06

a

-1600

-400 P d

ol IMP- I I 125 150

IO 175 125 150 175

EFFLUENT VOLUME, ml

FIG. 5. Molecular sieve chromatography on a column (1.8 X 230 cm) of Bio-Gel A 1.5 m of nro 0l1 from an acetic acid extract of pulse-labeled rat calvaria andbf al obtained as a product bv treat- ment of pulse-labeled procollagen with unlabeleh calvaria in 1 M NaCl. Both were isolated bv CM-cellulose chromatoaranhv (Fig. 4). Solid line, absorbance pattern of DL chains and”comp&en& from carrier rat skin collagen; dash line, radioactivity elution pattern of pro CJ (left) and or1 (right). The specific activity gradi- ent appears abuve (A). The samples were chromatographed under denaturing conditions with 1 M CaCll at room temperature (17).

will contain more label and bias the data. This is illustrated in the present system by the finding that, after 24 hours labeling, the radioactive and analytical hydroxyproline to proline ratios in the cyanogen bromide peptides follow a similar pattern (Table II). We, therefore, conclude that within experimental error the pulse-labeled chains analyzed in these experiments were fully hydroxylated.

Conversion of Procollagen to Collagem-To determine whether the procollagen reported by Bellamy and Bornstein (10) was absent from our salt extracts or was present but converted to collagen (see under “Methods”), we repeated the experiment described by them. In agreement with their findings, acetic acid-extracted material after brief labeling (10 min) contained pro crl eluting before al from a CM-cellulose column (Fig. 4). Radioactivity in the region of cr2 also did not coincide with a2, suggesting the presence of a pro cr2. Heterogeneity of the peaks suggests that conversion of procollagen to collagen may not occur as a single step.

When the acetic acid-extracted material was incubated in the presence of calvaria in 1 M NaCl, duplicating our extracting procedure, the counts were quantitatively converted to material chromatographing with cul and (r2 (Fig. 4). Therefore, we conclude that the absence of procollagen from 1 M NaCl extracts is caused by conversion to collagen during the extraction procedure. For both samples, 20% NaCl precipitated about 80% of the total counts present in the original acetic acid extracts.

When 14C-labeled pro or1 isolated by CM-cellulose chromatography was applied to an Agarose column with carrier c-rl, it eluted slightly earlier than the presumed 14C-labeled al obtained by the in vitro conversion of procollagen, as demonstrated in Fig. 5. The difference is most evident when the specific act.iv- ities across the peaks are compared. The negative slope results from the earlier elution of a labeled pro arl relative to carrier crl. The slight positive slope of the specific activity after conversion (Fig. 5, right) can be attributed to a small amount of cross-linked fi components in the carrier that are not completely resolved from a chains. The molecular weight of pro oil was calculated from its position relative to carrier B and or1 to be about 115,000. This must be regarded as an approximate value and is within experimental error of the value of 120,000 reported by Bellamy and Bornstein (10). The al formed by in vitro conversion of pro oil has the same molecular weight, 95,000 (26), as carrier otl.

DISCUSSION

The determination of the time required for translation of the a! chains of collagen by the method employed in this study rests upon a measurement of the time difference required to yield a given specific activity at two points along the chain; it does not depend on a measurement of the total labeling time. This is an important point since it means that the time required for the labeled amino acid to reach equilibrium in the intracellular pool, as well as any delay in termination of labeling, will have no effect on the calculated value for the translation rate, provided that the experiment is carried out during a period when the isotope incorporation is linear. The average translation rate, 209 residues per min, can therefore be considered a reliable measure of the true translation rate.

The theoretical line for incorporation into the carboxy-terminal amino acid (Figs. 2 and 3) intersects the abscissa at approximately the same point for both eul and a2 chains. This provides further evidence in support of the earlier suggestion that the


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two chains are synthesized separately (4), since if they were translated sequentially, these two lines would have been sepa- rated by one chain translation time. The possibility that the mRNA might be polycistronic with independent initiation of otl and a!2 has been ruled out by the experiments of Lazarides and Lukens (27).

The data presented in Fig. 1 demonstrate that [14C]glycine comes to equilibrium with the intracellular glycine pool in less than 4 min. The linear part of the curve when extrapolated to zero activity intersects the abscissa at 1.7 min. The correspond- ing points of intersection for the theoretical lines describing [%]glycine incorporation into the carboxy-terminal residues of the cy chains are 1.2 min (cyl) and 0.8 min (ma). The similarity of these times means that we can detect no lag between the incorporation of activity into the carboxy-terminal ends of the Q chains and the appearance of the chains in our samples. Such a lag would be expected if termination and release of the chain from the ribosome or hydroxglation of some or all of the proper prolyl residues (1, 4) required a time period measurable by our methods. This observation is supported by the finding of completely hydroxylated 01 chains in the extracts after pulse-labeling with [14C]prolinc. We, therefore, conclude that the time required for termination and release of the chains is very short compared to the translation time, and, in agreement with other studies (5, 6), that hgdrosylation takes place concurrently with translation.

If we can assume that our samples contained only fully helical native collagen, n-e can also conclude that, helix formation is initiated during translation or occurs very rapidly thereafter. 11-e do not havz direct evidence that random coil chains were not present in our samples, and, if originally present, they might form helix during the extraction period. However, it seems unlikely to us that they would be carried through to the final samples unless helix formation were rapid and efficient, which is our contelltion.

A complete native collagen molecule has carbohydrate attached to certain hydroxylysyl residues (28, 29). It was not possible to analyze our pulse-labeled collagen for carbohydrate and con- sequently we cannot make any conclusions about the rate of glycosylation.

That collagen is synthesized in precursor form must be taken into account in any consideration of the biosynthetic process. Assuming a molecular weight of 115,000 for pro crl and pro ar2, these chains will cont’ain about 1,250 residues, of which about 210 are assigned to the part that is later lost. Assuming that the extra part is colinear with the cx chain and reflects a single messenger that is translated at a constant rate, the biosynthesis time for a pro a! chain will be 6.0 min (1,250/209). The comple- tion of a procollagen molecule will not be significantly longer.

The kinetic analysis strongly suggests that the extra 210 residues are at the amino-terminal ends of the pro QI chains. Their presence at the carboxy-terminal ends would cause an apparent delay of about 1 min in t.he appearance of label at the carboxy- terminal end of the o( chain, which was not observed, whereas loss of amino-terminal material would not appear in the analysis. The amino-terminal location could facilitate triple chain aggre- gation and helix formation while the chains are still being syn- t,hesized on the ribosomes (7, 8), if it is assumed that the extra length is designed to initiate the process. A model of this type has heel) proposed by Speakman (30), which presently seems to

best fit the available data. However, other models have not been eliminated.

If procollagen is a transport form that is converted to collagen at the time of fibril formation (9, 30), its rate of conversion may be slow since it would include transport from the site of synthesis to the growing fibril. Our data do not bear on this sub- ject, but a slow rate would be consistent with the data of Bellamy and Bornstein (10).

Our earlier experiments suggested a value of 4.8 min for the biosynthesis of a complete collagen molecule. This time would include not only translation but any later steps. It is now evident that this is only an apparent value. The demonstration that there is a procollagen with chains longer than o( chains (10) requires a re-evaluation of the earlier data. First,, the finding that procollagen is converted to collagen during extraction means that the conversion step was not included in the measured time. Second, the chains being synthesized were probably not 01 chains with 1040 residues but pro ac chains with about 1250 residues. Inclusion of this data leads to a value of 5.8 min for the biosgn- thesis of a complete fully hydroxylated procollagen molecule. Since we show that t,ranslation requires about the same time, we again conclude that hydroxylation and helix formation do not measurably extend the biosynthesis time for a complete molecule.

Using a system of cranial bone similar to that used in our studies, Lane et al. (31) have recently shown that although [‘“Cl- proline appears in total protein with a lag of about 2 min (in agreement with the 1.7 min for [14C]glycine found here), [‘“Cl- hydroxyproline appears 1.5 min later. Assuming a translation time of 1 to 1.7 min for an cr chain from other data, they then conclude that hydroxylation begins only after the chains are largely complete. However, we show that, translation is several times slower than assumed by them. At a translation rate of 209 residues per min, the 1.5 min lag is equivalent to a separation of about 300 residues along a growing chain between the point of hydroxylation and the point of peptide bond formation. As discussed above, we did not observe such a lag. However, this value is probably within the experimental error of the two studies. Furthermore, a separation would be expected in view of the sizes of the ribosome and the hydroxylating enzyme. Some lag can then be accepted and to a certain extent hydroxylation may continue after translation, but hydroxylation clearly is not normally delayed until translation is complete.

The translation rate found here, 209 residues per min, is about one-third the rate calculated for hemoglobin by the same kinetic technique (13). The reason for the difference is not clear.

Acknowledgments-We wish to thank Dr. Charles J. MacLcan for carrying out the regression analyses and Mrs. Catherine Sullivan for expert technical assistance.

REFERENCES

1. PROCKOP, D. J. (1970) in E. A. BALAZS (Editor), Chemistry and molecular biology of the intercellular matrix, Vol. 1, p. 335, Academic Press, New York.

2. UDENFRIEND, S. (1970) in E. A. BALAZS (Editor), Chemistry and molecular biology of the intercellular matrix, Vol. 1, p. 371, Academic Press, New York.

3. PIEZ, K. A. (1970) J. Cell. Physiol., ‘76, 389. 4. VUUST, J., AND PIEZ, K. A. (1970) J. Biol. Chem., 245, 6210. 5. MILLER, R. L., AND ~JDENFRIEND, S. (1970) Arch. Biochem.

Biophys., 139, 104. 6. LAZARIDES, E. L., LUKENS, L.N., AND INFANTE, A. A. (1971)

J. Mol. Biol., 68, 831.


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Jens Vuust and Karl A. PiezA Kinetic Study of Collagen Biosynthesis

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a kinetic study of collagen biosynthesis

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