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Fibroblast procollagen production rates in vitro based on [3H]hydroxyproline production and procollagen hydroxyproline specific activity
ANALYTICAL
BKKHEMISTRY
14.478-485
(1984)
Fibroblast Procollagen Production Rates in Vitro Based on [3H]Hydroxyproline Production and Procollagen Hydroxyproline Specific Activity’ JOANG.CLARKANDJAMESN.HILDEBRAN Pulmonary Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405 Received December 16, 1983 In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [‘Hlhydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [‘Hlproline, and [‘Hlhydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [“C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. B&hem. 96, 45-551. Procollagen production rates were readily determined by this method using 5 to 20 PCi [‘Hlproline and approximately lob cells. It was found that )H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/pg DNA - h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro. KEY WORDS: procollagen; collagen; hydroxyproline; fibroblast.
procollagen production is required. Procollagen production2 in vitro is frequently quantitated by incubating cells in the presence of tracer amounts of radiolabeled proline, and then measuring radiolabeled hydroxyproline in newly synthesized protein ( 10). Since posttranslational hydroxylation of prolyl residues occurs primarily in procollagen, the amount of radiolabeled hydroxyproline is proportional to the amount of newly synthesized procol-
Collagen, a major component in extracellular matrix, is synthesized as a precursor macromolecule, procollagen, by fibroblasts and other cells in a variety of tissues (1). Cultured procollagen-producing cells have been a useful model for the study of the kinetics of procollagen synthesis (2-4) and for investigation of procollagen production after exposure to a variety of agents (5-7) and in pathological disorders (8,9). In many of these experiments, precise determination of ’ This work was supported in part by United States Public Health Service Grants HL29594 and HLl4212 (SCOR) from the National Institutes of Health. 0003-2697184 $3.00 Copyright Q 1984 by Academic Pmm, Inc. All rights of npmduction in my form reserved.
478
* Procollagen production refers to the total (radioactive plus nonradioactive) soluble, nondialyzable procollagen in culture medium and reflects the net accumulation that results from both synthesis and degradation.
FIBROBLAST
PROCOLLAGEN
lagen. If the degree of procollagen prolyl hydroxylation and the number of prolyl and hydroxyprolyl residues in procollagen are known, the amount of radiolabeled procollagen can be deduced. However, to calculate actual procollagen production from the isotope incorporation data, the specific activity3 of the newly synthesized procollagen must also be known (10-13). As an alternative to direct measurement of procollagen specific activity, proline precursor pool specific activities have been measured ( 14). Intracellular free proline specific activity is the most frequently measured pool (15- 17). This approach is based on the assumption that the specific activity of newly synthesized procollagen is equal to the specific activity of this proline pool. However, several proline pools exist within mammalian cells, and they are not homogeneous (18). Hildebran et al. (19) demonstrated that the specific activity of intracellular free proline in cultured fibrobiasts is higher than that of prolyl-tRNA, the immediate precursor for protein synthesis. Their results indicate that valid procollagen production rates may be calculated if the specific activity of the latter pool is known. However, measurement of prolyl-tRNA specific activity is technically difficult and requires large amounts of isotope. Moreover, this approach is valid only when prolyl-tRNA specific activity is constant throughout the incubation period. Because of the technical and theoretical difficulties encountered in the analysis of proline precursor pools, we used a simple microanalytical method to directly measure fibroblast procollagen specific activity (13). The method is based on the analysis of the specific activity of hydroxyproline in newly synthesized procollagen. Hydroxyproline specific activity was measured because essentially all the hydroxyproline in medium from fibroblast cultures isotopically labeled with proline in fresh medium is in newly synthesized procollagen. 3 Specific activity refers to radioactivity of amino acid or protein (i.e., dpm/pmol).
per unit weight
PRODUCTION
479
RATES
Therefore, extensive purification of procollagen is unnecessary. The hydroxyproline in the newly synthesized procollagen was reacted with [‘4C]dansyl chloride of known specific activity to determine the hydroxyproline specific activity (20). We used this approach to determine procollagen production rates by cultured lung fibroblasts as well as free proline pool specific activities. The specific activities of extracellular and intracellular free proline pools were not constant during the labeling period, nor were they equal to procollagen specific activity, indicating that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach that we used obviates the need to identify or characterize the true precursor pool from which procollagen is synthesized, and may be readily applied to determine actual fibroblast procollagen production in vitro. EXPERIMENTAL
PROCEDURES
Cell Culture Human fetal lung fibroblasts (IMR-90, Institute for Medical Research, Camden, N. J.; passage lo- 12) were cultured in 6-well tissue culture plates (9.62 cm2, Flow Laboratories, McLean, Va.) using 1.5 ml Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 200 U/ml penicillin, 200 pg/ml strep tomycin, and 2 mM glutamine (basal medium) in a 95% air-5% CO2 atmosphere at 37°C. When the fibroblasts were confluent, basal medium was supplemented with ascorbic acid (50 /*g/ml). After 24 h, basal medium was removed and replaced with fresh basal medium containing 50 &ml ascorbic acid and 5 to 20 &i [3H]proline (100 Ci/mmol; Amersham, Arlington Heights, ill.). In some experiments, proline (0.2 mM) was also added to the labeling medium. Labeling medium was removed after an incubation period of 2 to 24 h, and protease inhibitors (1 pM phenylmethanesulfonyl fluoride, 10 mM n-ethyl maleimide, and 20 mM EDTA, in final concentration) were added.
480
CLARK
[3H]Hydroxyproline
AND HILDEBRAN
Production Rate
One-half of the medium was dialyzed against running tap water for 24 h, hydrolyzed in 6 N HCl at 110°C for 18 h, and dried by evaporation. The [3H]hydroxyproline in the sample was isolated chromatographically and measured in a liquid scintillation system as previously described (5). In some experiments, [3H]hydroxyproline in the cell layer was also measured (17). Hydroxyproline
Specific Activity
Procollagen was isolated from the remaining half of the medium by precipitation with ammonium sulfate (114 mg/ml) at 4°C for 24 h and centrifugation at 12,000g for 1 h. The precipitate was resuspended in 0.15 M NaCl, 0.05 M Tris/HCl (pH 7.5) containing protease inhibitors as above and dialyzed against running tap water for 24 h and distilled water for 8 h. The sample was hydrolyzed in 6 N HCl for 18 h at 1 lO”C, evaporated to dryness, resuspended in 100 ~1 distilled water, transferred to siliconized glass tubes (10 X 75 mm), and again evaporated to dryness. The sample was then dissolved in 10 ~1 of 0.2 M bicarbonate-carbonate buffer (pH 9.6). The pH of the sample was monitored with pH paper and adjusted to pH 9 by the addition of 2-~1 aliquots of 1 M bicarbonate-carbonate buffer (pH 9.6). [‘4C]DNS-C14 (110 pCi/pmol in acetone; New England Nuclear, Boston, Mass.) was diluted with a stock solution of DNS-Cl (1 mg/ml in acetone) to obtain a solution of [‘4C]DNS-Cl containing 40 or 50 dpm/pmol. Ten microliters of this [ 14C]DNSCl was added to the sample and incubated at 37°C for 45 min. The dansylated amino acid derivatives were dried and then extracted 3 times into 100 jd water-saturated ethyl acetate, dried by evaporation, and resuspended in 10 ~1 acetone:water (1: 1). Two microliters of nonradioactive DNS-hydroxyproline (1 ms/rnl) 4 Abbreviations used: DNS-Cl, dansyl chloride (l-dimethylaminonaphthalene-Ssulfonyl chloride); !%A., specific activity.
was added to facilitate identification of the DNS-hydroxyproline spot on the chromatogram. The entire sample was then spotted onto polyamide thin layer chromatography plates (7.5 X 7.5 cm; Schleicher & Schuell, Keene, N. H.). The dansyl derivatives were separated by two-dimensional ascending chromatography (first-dimension solvent = formic acid:water, 2: 100; second-dimension solvent = benzene:acetic acid, 90: 10). After identifying the DNS-hydroxyproline spot under ultraviolet light, the spot was cut from the plate, and ‘H and 14C measured in a liquid scintillation counter (Beckman Model LS 8000 equipped with automatic quench correction). Hydroxyproline specific activity was calculated by the formula (units are in parentheses): (1) Hydroxyproline S.A. (dpm/pmol) (dpm)/14C (dpm)] X DNS-Cl S.A. pmol).
= [3H (dpm/
Other Procedures
Extracellular and intracellular proline specific activity were measured as previously described (19,20). Procollagen prolyl hydroxylation was measured by incubating medium from parallel cultures with bacterial collagenease (Sigma Type VI, St. Louis, MO.) purified as described by Peterkofsky and Diegelmann (2 1). Dialyzable r3H]proline and [3H]hydroxyproline were separated chromatographically and the radioactivity was determined. Details of this procedure have previously been described (17). DNA in cell layers was measured by the method of Burton (22). Calculations
(2) [3H]Hydroxyproline production rate (dpm/pg DNA. h) = [nondialyzable [3H]hydroxyproline (dpm)/DNA (pg)] + incubation time (h). (3) Hydroxyproline production rate (pmol/ pg DNA - h) = [3H]hydroxyproline production rate (dpm/pg DNA - h) i hydroxyproline S.A. (dpmhmol).
FIBROBLAST
PROCOLLAGEN
(4) Procollagen prolyl hydroxylation = [3H]hydroxyproline released by collagenase (dpm) f total radioactivity released by collagenase (dpm). (5) Procollagen production rate (pmol/pg DNA * h) = hydroxyproline production rate (pmol/pg DNA - h) f procollagen prolyl hydroxylation + prolyl and hydroxyprolyl residues per procollagen molecule. Precautions (1) Since measurement of amino acids by reaction with [‘4C]DNS-Cl is sensitive in the picogram range, care must be taken to avoid contamination of the samples with hydroxyproline. This does not require elaborate precautions since hydroxyproline is not a ubiquitous amino acid. However, because our laboratories are involved in other collagen studies, glassware used for the procedure is not in general laboratory circulation, disposables are used when possible, and reagent blanks are analyzed periodically. (2) The presence of excess amino acids derived from fetal calf serum and excess salts in samples may compromise dansylation and separation of the dansyl derivatives by chromatography. This can be minimized by partial purification of procollagen by ammonium sulfate precipitation, dialysis against distilled water, complete evaporation of acid from hydrolysates to minimize the amount of buffer required in the dansylation reaction, and extraction of dansylated derivatives into watersaturated ethyl acetate. (3) DNS-glycine chromatographs near DNS-hydroxyproline in this system. Clear separation is required since the presence of [‘4C]DNS-glycine in the DNS-hydroxyproline spot would alter the ratio of ‘H to 14C used in the calculation of hydroxyproline specific activity. Separation may be improved by viewing the plates after chromatography in the first dimension and rechromatographing if necessary. Likewise, chromatography may be repeated in the second dimension if sep aration is inadequate. Simultaneous chro-
PRODUCTION
RATES
481
matography of a mixture of DNS-hydroxyproline and DNS-glycine on a control chromatography plate may facilitate the assessment of adequate separation. (4) Since this method yields an actual value of hydroxyproline in medium the sample to be analyzed should not contain hydroxyproline produced prior to the labeling period. To this end, old medium is removed before labeling, and the labeled sample is dialyzed to remove free hydroxyproline which may be present as a contaminant in [3H]proline or in serum. Cell layers which contain previously synthesized, extracellular collagen would be an inappropriate sample in which to measure hydroxyproline specific activity. (5) Since two-channel scintillation counting is required to obtain the ratio of 3H to 14C radioactivity in DNS-hydroxyproline, it is essential that quenching and subsequent changes in spillover of 14C into the 3H channel as well as background radioactivity are accurately assessed with appropriate quench curves and standards. It is also necessary to adjust the specific activity of [‘4C]DNS-CI such that 14C counted in the 3H channel is small compared to 3H counts. RESULTS
Confluent lung fibroblasts were cultured in 9.6cm2 tissue culture wells (approximately lo6 cells) under conditions that enhance procollagen synthesis (23). In our initial experiments we varied the proline concentration and the amount of [3H]proline included in the labeling medium. The labeling time was 24 h. [3H]Hydroxyproline production and hydroxyproline specific activity increased in direct proportion to the amount of [3H]proline included in the medium (Fig. 1). Supplemental proline (0.2 mM) in the culture medium decreased the hydroxyproline specific activity, but actual hydroxyproline production rate was the same in all cultures. The results indicate that these determinations can be made with as little as 5 &i of [3H]proline even in cultures containing supplemental proline.
482
CLARK
AND HILDEBRAN
was constant after 4 h. The procollagen hydroxyprolyl specific activity initially increased linearly, suggesting that it was derived from a proline precursor pool of relatively constant specific activity (Figs 2 and 3A). The specific activities of the free proline precursor pools measured here were neither constant nor equal to the procollagen hydroxyproline specific activity. Examination of procollagen production over time in labeling medium revealed that [3H]hydroxyproline in the culture medium increased linearly from 1.6 to 9 h (Fig. 3B; r = 0.996). Actual hydroxyproline production
I Oo
I 5 C3H1 PROLINE
1 IO
I
20 ()rCi/culture)
FIG. I. In vitro procollagen production rate based on [3H]hydroxyproline production and hydroxyproline specific activity. Fibroblasts (IMR-90) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 200 U/ml penicillin, 200 j&ml streptomycin, and 50 &ml ascorbic acid. At confluence, medium was removed and replaced with 1.5 ml of fresh medium containing [‘Hlproline in varying amounts for 24 h. Cultures contained either no additional proline (0) or 0.2 mM proline (0). [“HlHydroxyproline production (A), hydroxyproline specific activity (B), and hydroxyproline production rate (C) were determined as described under Experimental Procedures using [‘4C]DNS-Cl (50 dpm/ pmol). Values are the mean + SD (n = 4 cultures). The hatched area in (C) indicates the mean f SD of all values.
A second experiment was designed to assess the relationship between the specific activity of free proline pools and the specific activity of procollagen hydroxyproline over time. Extracellular proline specific activity decreased significantly over the incubation period of 24 h. After an initial increase in intracellular proline specific activity, the specific activity of this pool also decreased (Fig. 2). The ratio of intracellular to extracellular specific activity
...*..,~ / O--CL--L-L0246 TIME
.. .... b . .. .... .. . ... .. . ... ... . ... .. . .. . ... . .. ..A 24
9 IN LABELING
MEDIUM
(hours)
FIG. 2. Specific activity of extracellular free proline, intracellular free proline, and hydroxyproline in procollagen during isotopic labeling. Confluent fibroblasts were cultured as described in Fig. I. At intervals after addition of the labeling medium containing 20 &i [‘Hlproline, extracellular free proline (medium) specific activity (O), intracellular free proline (trichloroacetic acid nonprecip itated) specificactivity (0), and procollagen hydroxyproline (medium hydroxyproline) specific activity (A) were measured as described under Experimental Procedures using [“C]DNS-CI(40 dpm/pmol). Values are mean + SD (n = 3 or 4 cultures). Values of procollagen hydroxyproline specific activity are also shown in Fig. 3A.
FIBROBLAST
PROCOLLAGEN
160 1”
T
PRODUCTION
RATES
483
parameters were considered: (a) procollagen prolyl hydroxylation = 0.40 -+ 0.01 (mean + SD; n = 4); (b) prolyl and hydroxyprolyl residues per procollagen molecule = 24 1 ( 15); and (c) medium [3H]hydroxyproline in these cultures represented 63% of the total [3H]hydroxyproline. We assumed that the specific activity of newly synthesized hydroxyproline in the cell layer was equal to that in the medium, although this is only true when procollagen specific activity has reached a plateau value. The value corresponds to 1.3 X 1O6 molecules of procollagen produced per cell per hour using the calculation method of Breul et al. (15). DISCUSSION
%
01
0246
’
”
I
’
24
9
TIME IN LABELING
MEDIUM
(hours)
FIG. 3. Kinetics of fibroblast procollagen production in vitro. Fibroblasts were cultured as described in Fig. 1 using 20 &i [3H]proline in the labeling medium. After incubation for 2 to 24 h, procollagen hydroxyproline specific activity (A), [‘Hlhydroxyproline production (B), hydroxyprohne production (C), and hydroxyproline production rate (D) were measured as described under Experimental Procedures using [%]DNS-Cl (40 dpm/pmol). The hatched area is the mean f SD of all values obtained in the first 9 h (n = 14). Individual values are the mean f SD (n = 3 or 4 cultures).
into medium based on the specific activity of hydroxyproline was constant from the beginning of the incubation through 9 h (Fig. 3C; I = 0.986). Thereafter, the production decreased. This was also evident when hydroxyproline production rate was calculated from data obtained in the first 9 h (13.9 + 3.4; II = 15) and from data obtained at 24 h (5.4 +- 1.6; n = 4; p < 0.001). Procollagen production rate calculated from data obtained after 6 h incubation was 0.23 pmol procollagen/pg DNA h. To make this calculation (Eq. [5]) the following additional l
These studies demonstrate the application of a sensitive method of measuring hydroxyproline specific activity to obtain actual procollagen production rate of fibroblasts in vitro. The procedure, which does not require quantitative recovery of the hydroxyproline in the sample, can be done relatively rapidly and inexpensively. Hydroxyproline specific activity was readily measured in confluent lung fibroblast cultures containing approximately 1O6 cells and 5 to 20 &i of [3H]proline. Inclusion of proline at physiologic concentration (0.2 mM) decreased the hydroxyproline specific activity, but did not preclude the measurement. The culture conditions employed in these studies enhance procollagen synthesis (23), but measurement of hydroxyproline specific activity is also probably feasible under suboptimal conditions (e.g., serum-free medium). Since application of the method requires sufficient amounts of [3H]hydroxyproline in the sample, larger cultures and/or more [3H]proline in the labeling medium may be required in some instances. In these studies we also examined extracellular and intracellular free proline pool specific activities and their relation to specific activity of hydroxyproline in procollagen synthesized over 24 h. We observed that (1) extracellular free proline specific activity (i.e., proline specific activity in labeling medium)
484
CLARK
AND HILDEBRAN
decreased dramatically in these cultures that were not supplemented with proline. This probably occurred primarily as the result of net proline production by the cells (19,24), and illustrates the non-steady state of the cultures with respect to proline concentration. (2) The intracellular free proline specific activity increased over the first 4 h and then decreased, presumably reflecting in part trans port of [3H]proline from the changing extracellular pool as well as endogenous proline production. (3) Procollagen hydroxyproline specific activity increased over the culture period. We assume the initial change is due to the secretion of intracellular procollagen formed all or in part prior to the addition of isotope and production of procollagen prior to equilibration of the prolyl-tRNA pool. (4) Extracellular proline specific activity, intracellular proline specific activity, and procollagen hydroxyproline specific activity were not equal at any time over 24 h, and the relationship between these pools and procollagen specific activity was not constant. It is evident that neither of these free proline pools can be used to determine procollagen production. Although we did not examine prolyl-tRNA specific activity in these studies, the specific activity of this pool has previously been shown to be relatively constant for 4 h and equal to cellular protein specific activity by about 8 h in fibroblast cultures containing 0.2 mM proline (19). Thus, prolyl-tRNA specific activity may be a valid estimate of protein specific activity under these conditions. However, based on the data from this study, one can predict that in prolinedeficient cultures and/ or after prolonged labeling periods (e.g., 24 h), the specific activity of prolyl-tRNA (measured at the end of a labeling period) would be different from the mean specific activity of prolyl-tRNA used for procollagen synthesis during the labeling period. For the final prolyltRNA specific activity to be a valid measure of the specific activity of newly synthesized protein, its specific activity must be constant throughout the labeling period. Examination of the kinetics of fibroblast procollagen production based on hydroxy-
proline specific activity revealed that [‘HIhydroxyproline in medium increases linearly from 1.6 to 9 h in labeling medium. The initial lag period of 1.6 h may be attributed to isotope transport into the cells, equilibration with the true precursor pool, and production and secretion of procollagen derived from nonradioactive proline. When actual hydroxyproline production rate was calculated on the basis of hydroxyproline specific activity, no lag period was observed, indicating that procollagen production was constant from time zero through 9 h. This predictable result, similar to that of Schmid and Conrad ( 13), emphasizes that the method described here (a modified measurement of hydroxyproline by isotope dilution) quantitates total procollagen secreted into culture medium regardless of the precursor specific activity. This is highly desirable given the inhomogeneity and inconstancy of proline precursor pools. It should also be noted that if the experimental conditions are changed at the time labeling medium is added, the calculated value of procollagen production rate will reflect in part the control (basal) procollagen production rate (i.e., the contribution of intracellular procollagen and nascent procollagen chains formed prior to addition of isotope) as well as the production rate of newly synthesized procollagen. This can be minimized by changing the experimental conditions prior to labeling and/or prolonging the labeling time. The procollagen production rate by IMR90 fibroblasts determined in this study is similar to the value based on prolyl-tRNA specific activity ( 19), and approximately twice that of another human fetal lung fibroblast strain (HFL- 1) cultured under different conditions (15). In the latter study, intracellular free proline specific activity was used to calculate the rate of procollagen production, and probably contributes to the discrepancy. Had we based our calculation on the specific activity of this pool, our value would have been reduced. In these studies, we made the unexpected observation that hydroxyproline production rate decreased after about 9 h in culture. This decrease was not so apparent when
FIBROBLAST
PRGCOLLAGEN
[3H]hydroxyproline production was examined over 24 h (r = 0.984). Actual hydroxyproline production over 24 h, however, was obviously not constant. Although we did not examine the mechanism of this change in hydroxyproline production rate, such factors as density inhibition of procollagen synthesis, decreased efficiency of prolyl hydroxylation, nutrient depletion, or incorporation of collagen into insoluble matrix could contribute. ACKNOWLEDGMENT We thank Dr. R. B. Low for helpful discussions in the preparation of this manuscript.
REFERENCES 1. Bomstein, P., and Sage, H. (1980) Annu. Rev. B&hem. 49,95?-1003. 2. Prockop, D. J., Berg, R. A., Kivirikko, K. I., and Uitto, J. (1976) in Biochemistry of Collagen (Ramachandran, G. N., and Reddi, A. H., eds.), pp. 163-273, Plenum, New York. 3. Prockop, D. J., Kivirikko, K. I., Tuderman, L., and Guzman, N. A. (1979) N. Engl. J. Med. 301, 1323, 75-85. 4. Kao, W. W.-Y., Prockop, D. J., and Betg, R. A. (1979) J. Biol. Chem. 254, 2234-2243. 5. Clark, J. G., Starcher, B. C., and Uitto, J. (1980) Biochim. Biophys. Acta 631, 359-370. 6. Berg, R. A., Moss, J., Baum, B. J., and Crystal, R. G. (1981) J. C/in. Invest. 67, 1457-1462. 7. Tan, E. M. L., RyhHnen, L., and Uitto, J. (1983) J. Invest. Dermatol. 80, 26 1-267.
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8. Uitto, J., Batter, E. A., and Eisen, A. 2. (1979) J. Clin. Invest. 64, 92 l-930. 9. Byers, P. H., Holbrook, K. A., Barsh, B. S., Smith, L. T., and Borstein, P. (198 I) Lab. Invest. 44,336341. 10. Berg, R. A. (1982) in Methods in Enzymology (Cunningham, L. W., and Frederiksen, D. W., eds.), Part A, Vol. 82, pp. 372-398, Academic Press, New York. I I. Robins, S. P. (1979) Biochem. J. 181, 75-82. 12. Kao, W. W.-Y., Prockop, D. J., and Berg, R. A. (1977) J. Biol. Chem. 252, 8391-8397. 13. Schmid, T. M., and Conrad, H. E. (1982) J. Biol. Chem. 257, 12,451-12,457. 14. Rannels, D. E., Low, R. B., Youdale, T., Volkin, E., and Longmore, W. J. (1982) Fed. Proc. 41,28332839. 15. Breul, S. D., Bradley, K. H., Hance, A. J., Schaffer, M. P., Berg, R. A., and Crystal, R. G. (1980) J. Biol. Chem. 255, 5250-5260. 16. Pietihi, K., and Nikkari, T. (1982) Artery 10, 172179. 17. Clark, J. G., Kostal, K. M., and Ma&o, B. A. (1982) J. Biol. Chem. 257, 8098-8105. 18. Kipnis, D. M., Reiss, E., and Helmreich, E. (1961) Biochim. Biophys. Acta 51, 5 19-524. 19. Hildebran, J. N., Airhart, J., Stirewalt, W. S., and Low, R. B. (1981) Biochem. J. 198, 249-258. 20. Airhart, J., Kelley, J., Brayden, J. E., Low, R. B., and Stirewalt, W. S. (1979) Anal. Biochem. 96,45-55. 21. Peterkofsky, B., and Diegelmann, R. (1971) Biochemistry 10, 988-994. 22. Burton, K. (1956) B&hem. J. 62, 315-323. 23. Booth, B. A., Polak, K. K., and Uitto, J. (1980) Biochim. Biophys. Acfa 607, 145-160. 24. Kruse, P. F., and Miedema, E. (I 965) J. Cell Biol. 27,273-279.
BKKHEMISTRY
14.478-485
(1984)
Fibroblast Procollagen Production Rates in Vitro Based on [3H]Hydroxyproline Production and Procollagen Hydroxyproline Specific Activity’ JOANG.CLARKANDJAMESN.HILDEBRAN Pulmonary Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, and Department of Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405 Received December 16, 1983 In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [‘Hlhydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [‘Hlproline, and [‘Hlhydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [“C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. B&hem. 96, 45-551. Procollagen production rates were readily determined by this method using 5 to 20 PCi [‘Hlproline and approximately lob cells. It was found that )H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/pg DNA - h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro. KEY WORDS: procollagen; collagen; hydroxyproline; fibroblast.
procollagen production is required. Procollagen production2 in vitro is frequently quantitated by incubating cells in the presence of tracer amounts of radiolabeled proline, and then measuring radiolabeled hydroxyproline in newly synthesized protein ( 10). Since posttranslational hydroxylation of prolyl residues occurs primarily in procollagen, the amount of radiolabeled hydroxyproline is proportional to the amount of newly synthesized procol-
Collagen, a major component in extracellular matrix, is synthesized as a precursor macromolecule, procollagen, by fibroblasts and other cells in a variety of tissues (1). Cultured procollagen-producing cells have been a useful model for the study of the kinetics of procollagen synthesis (2-4) and for investigation of procollagen production after exposure to a variety of agents (5-7) and in pathological disorders (8,9). In many of these experiments, precise determination of ’ This work was supported in part by United States Public Health Service Grants HL29594 and HLl4212 (SCOR) from the National Institutes of Health. 0003-2697184 $3.00 Copyright Q 1984 by Academic Pmm, Inc. All rights of npmduction in my form reserved.
478
* Procollagen production refers to the total (radioactive plus nonradioactive) soluble, nondialyzable procollagen in culture medium and reflects the net accumulation that results from both synthesis and degradation.
FIBROBLAST
PROCOLLAGEN
lagen. If the degree of procollagen prolyl hydroxylation and the number of prolyl and hydroxyprolyl residues in procollagen are known, the amount of radiolabeled procollagen can be deduced. However, to calculate actual procollagen production from the isotope incorporation data, the specific activity3 of the newly synthesized procollagen must also be known (10-13). As an alternative to direct measurement of procollagen specific activity, proline precursor pool specific activities have been measured ( 14). Intracellular free proline specific activity is the most frequently measured pool (15- 17). This approach is based on the assumption that the specific activity of newly synthesized procollagen is equal to the specific activity of this proline pool. However, several proline pools exist within mammalian cells, and they are not homogeneous (18). Hildebran et al. (19) demonstrated that the specific activity of intracellular free proline in cultured fibrobiasts is higher than that of prolyl-tRNA, the immediate precursor for protein synthesis. Their results indicate that valid procollagen production rates may be calculated if the specific activity of the latter pool is known. However, measurement of prolyl-tRNA specific activity is technically difficult and requires large amounts of isotope. Moreover, this approach is valid only when prolyl-tRNA specific activity is constant throughout the incubation period. Because of the technical and theoretical difficulties encountered in the analysis of proline precursor pools, we used a simple microanalytical method to directly measure fibroblast procollagen specific activity (13). The method is based on the analysis of the specific activity of hydroxyproline in newly synthesized procollagen. Hydroxyproline specific activity was measured because essentially all the hydroxyproline in medium from fibroblast cultures isotopically labeled with proline in fresh medium is in newly synthesized procollagen. 3 Specific activity refers to radioactivity of amino acid or protein (i.e., dpm/pmol).
per unit weight
PRODUCTION
479
RATES
Therefore, extensive purification of procollagen is unnecessary. The hydroxyproline in the newly synthesized procollagen was reacted with [‘4C]dansyl chloride of known specific activity to determine the hydroxyproline specific activity (20). We used this approach to determine procollagen production rates by cultured lung fibroblasts as well as free proline pool specific activities. The specific activities of extracellular and intracellular free proline pools were not constant during the labeling period, nor were they equal to procollagen specific activity, indicating that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach that we used obviates the need to identify or characterize the true precursor pool from which procollagen is synthesized, and may be readily applied to determine actual fibroblast procollagen production in vitro. EXPERIMENTAL
PROCEDURES
Cell Culture Human fetal lung fibroblasts (IMR-90, Institute for Medical Research, Camden, N. J.; passage lo- 12) were cultured in 6-well tissue culture plates (9.62 cm2, Flow Laboratories, McLean, Va.) using 1.5 ml Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 200 U/ml penicillin, 200 pg/ml strep tomycin, and 2 mM glutamine (basal medium) in a 95% air-5% CO2 atmosphere at 37°C. When the fibroblasts were confluent, basal medium was supplemented with ascorbic acid (50 /*g/ml). After 24 h, basal medium was removed and replaced with fresh basal medium containing 50 &ml ascorbic acid and 5 to 20 &i [3H]proline (100 Ci/mmol; Amersham, Arlington Heights, ill.). In some experiments, proline (0.2 mM) was also added to the labeling medium. Labeling medium was removed after an incubation period of 2 to 24 h, and protease inhibitors (1 pM phenylmethanesulfonyl fluoride, 10 mM n-ethyl maleimide, and 20 mM EDTA, in final concentration) were added.
480
CLARK
[3H]Hydroxyproline
AND HILDEBRAN
Production Rate
One-half of the medium was dialyzed against running tap water for 24 h, hydrolyzed in 6 N HCl at 110°C for 18 h, and dried by evaporation. The [3H]hydroxyproline in the sample was isolated chromatographically and measured in a liquid scintillation system as previously described (5). In some experiments, [3H]hydroxyproline in the cell layer was also measured (17). Hydroxyproline
Specific Activity
Procollagen was isolated from the remaining half of the medium by precipitation with ammonium sulfate (114 mg/ml) at 4°C for 24 h and centrifugation at 12,000g for 1 h. The precipitate was resuspended in 0.15 M NaCl, 0.05 M Tris/HCl (pH 7.5) containing protease inhibitors as above and dialyzed against running tap water for 24 h and distilled water for 8 h. The sample was hydrolyzed in 6 N HCl for 18 h at 1 lO”C, evaporated to dryness, resuspended in 100 ~1 distilled water, transferred to siliconized glass tubes (10 X 75 mm), and again evaporated to dryness. The sample was then dissolved in 10 ~1 of 0.2 M bicarbonate-carbonate buffer (pH 9.6). The pH of the sample was monitored with pH paper and adjusted to pH 9 by the addition of 2-~1 aliquots of 1 M bicarbonate-carbonate buffer (pH 9.6). [‘4C]DNS-C14 (110 pCi/pmol in acetone; New England Nuclear, Boston, Mass.) was diluted with a stock solution of DNS-Cl (1 mg/ml in acetone) to obtain a solution of [‘4C]DNS-Cl containing 40 or 50 dpm/pmol. Ten microliters of this [ 14C]DNSCl was added to the sample and incubated at 37°C for 45 min. The dansylated amino acid derivatives were dried and then extracted 3 times into 100 jd water-saturated ethyl acetate, dried by evaporation, and resuspended in 10 ~1 acetone:water (1: 1). Two microliters of nonradioactive DNS-hydroxyproline (1 ms/rnl) 4 Abbreviations used: DNS-Cl, dansyl chloride (l-dimethylaminonaphthalene-Ssulfonyl chloride); !%A., specific activity.
was added to facilitate identification of the DNS-hydroxyproline spot on the chromatogram. The entire sample was then spotted onto polyamide thin layer chromatography plates (7.5 X 7.5 cm; Schleicher & Schuell, Keene, N. H.). The dansyl derivatives were separated by two-dimensional ascending chromatography (first-dimension solvent = formic acid:water, 2: 100; second-dimension solvent = benzene:acetic acid, 90: 10). After identifying the DNS-hydroxyproline spot under ultraviolet light, the spot was cut from the plate, and ‘H and 14C measured in a liquid scintillation counter (Beckman Model LS 8000 equipped with automatic quench correction). Hydroxyproline specific activity was calculated by the formula (units are in parentheses): (1) Hydroxyproline S.A. (dpm/pmol) (dpm)/14C (dpm)] X DNS-Cl S.A. pmol).
= [3H (dpm/
Other Procedures
Extracellular and intracellular proline specific activity were measured as previously described (19,20). Procollagen prolyl hydroxylation was measured by incubating medium from parallel cultures with bacterial collagenease (Sigma Type VI, St. Louis, MO.) purified as described by Peterkofsky and Diegelmann (2 1). Dialyzable r3H]proline and [3H]hydroxyproline were separated chromatographically and the radioactivity was determined. Details of this procedure have previously been described (17). DNA in cell layers was measured by the method of Burton (22). Calculations
(2) [3H]Hydroxyproline production rate (dpm/pg DNA. h) = [nondialyzable [3H]hydroxyproline (dpm)/DNA (pg)] + incubation time (h). (3) Hydroxyproline production rate (pmol/ pg DNA - h) = [3H]hydroxyproline production rate (dpm/pg DNA - h) i hydroxyproline S.A. (dpmhmol).
FIBROBLAST
PROCOLLAGEN
(4) Procollagen prolyl hydroxylation = [3H]hydroxyproline released by collagenase (dpm) f total radioactivity released by collagenase (dpm). (5) Procollagen production rate (pmol/pg DNA * h) = hydroxyproline production rate (pmol/pg DNA - h) f procollagen prolyl hydroxylation + prolyl and hydroxyprolyl residues per procollagen molecule. Precautions (1) Since measurement of amino acids by reaction with [‘4C]DNS-Cl is sensitive in the picogram range, care must be taken to avoid contamination of the samples with hydroxyproline. This does not require elaborate precautions since hydroxyproline is not a ubiquitous amino acid. However, because our laboratories are involved in other collagen studies, glassware used for the procedure is not in general laboratory circulation, disposables are used when possible, and reagent blanks are analyzed periodically. (2) The presence of excess amino acids derived from fetal calf serum and excess salts in samples may compromise dansylation and separation of the dansyl derivatives by chromatography. This can be minimized by partial purification of procollagen by ammonium sulfate precipitation, dialysis against distilled water, complete evaporation of acid from hydrolysates to minimize the amount of buffer required in the dansylation reaction, and extraction of dansylated derivatives into watersaturated ethyl acetate. (3) DNS-glycine chromatographs near DNS-hydroxyproline in this system. Clear separation is required since the presence of [‘4C]DNS-glycine in the DNS-hydroxyproline spot would alter the ratio of ‘H to 14C used in the calculation of hydroxyproline specific activity. Separation may be improved by viewing the plates after chromatography in the first dimension and rechromatographing if necessary. Likewise, chromatography may be repeated in the second dimension if sep aration is inadequate. Simultaneous chro-
PRODUCTION
RATES
481
matography of a mixture of DNS-hydroxyproline and DNS-glycine on a control chromatography plate may facilitate the assessment of adequate separation. (4) Since this method yields an actual value of hydroxyproline in medium the sample to be analyzed should not contain hydroxyproline produced prior to the labeling period. To this end, old medium is removed before labeling, and the labeled sample is dialyzed to remove free hydroxyproline which may be present as a contaminant in [3H]proline or in serum. Cell layers which contain previously synthesized, extracellular collagen would be an inappropriate sample in which to measure hydroxyproline specific activity. (5) Since two-channel scintillation counting is required to obtain the ratio of 3H to 14C radioactivity in DNS-hydroxyproline, it is essential that quenching and subsequent changes in spillover of 14C into the 3H channel as well as background radioactivity are accurately assessed with appropriate quench curves and standards. It is also necessary to adjust the specific activity of [‘4C]DNS-CI such that 14C counted in the 3H channel is small compared to 3H counts. RESULTS
Confluent lung fibroblasts were cultured in 9.6cm2 tissue culture wells (approximately lo6 cells) under conditions that enhance procollagen synthesis (23). In our initial experiments we varied the proline concentration and the amount of [3H]proline included in the labeling medium. The labeling time was 24 h. [3H]Hydroxyproline production and hydroxyproline specific activity increased in direct proportion to the amount of [3H]proline included in the medium (Fig. 1). Supplemental proline (0.2 mM) in the culture medium decreased the hydroxyproline specific activity, but actual hydroxyproline production rate was the same in all cultures. The results indicate that these determinations can be made with as little as 5 &i of [3H]proline even in cultures containing supplemental proline.
482
CLARK
AND HILDEBRAN
was constant after 4 h. The procollagen hydroxyprolyl specific activity initially increased linearly, suggesting that it was derived from a proline precursor pool of relatively constant specific activity (Figs 2 and 3A). The specific activities of the free proline precursor pools measured here were neither constant nor equal to the procollagen hydroxyproline specific activity. Examination of procollagen production over time in labeling medium revealed that [3H]hydroxyproline in the culture medium increased linearly from 1.6 to 9 h (Fig. 3B; r = 0.996). Actual hydroxyproline production
I Oo
I 5 C3H1 PROLINE
1 IO
I
20 ()rCi/culture)
FIG. I. In vitro procollagen production rate based on [3H]hydroxyproline production and hydroxyproline specific activity. Fibroblasts (IMR-90) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 200 U/ml penicillin, 200 j&ml streptomycin, and 50 &ml ascorbic acid. At confluence, medium was removed and replaced with 1.5 ml of fresh medium containing [‘Hlproline in varying amounts for 24 h. Cultures contained either no additional proline (0) or 0.2 mM proline (0). [“HlHydroxyproline production (A), hydroxyproline specific activity (B), and hydroxyproline production rate (C) were determined as described under Experimental Procedures using [‘4C]DNS-Cl (50 dpm/ pmol). Values are the mean + SD (n = 4 cultures). The hatched area in (C) indicates the mean f SD of all values.
A second experiment was designed to assess the relationship between the specific activity of free proline pools and the specific activity of procollagen hydroxyproline over time. Extracellular proline specific activity decreased significantly over the incubation period of 24 h. After an initial increase in intracellular proline specific activity, the specific activity of this pool also decreased (Fig. 2). The ratio of intracellular to extracellular specific activity
...*..,~ / O--CL--L-L0246 TIME
.. .... b . .. .... .. . ... .. . ... ... . ... .. . .. . ... . .. ..A 24
9 IN LABELING
MEDIUM
(hours)
FIG. 2. Specific activity of extracellular free proline, intracellular free proline, and hydroxyproline in procollagen during isotopic labeling. Confluent fibroblasts were cultured as described in Fig. I. At intervals after addition of the labeling medium containing 20 &i [‘Hlproline, extracellular free proline (medium) specific activity (O), intracellular free proline (trichloroacetic acid nonprecip itated) specificactivity (0), and procollagen hydroxyproline (medium hydroxyproline) specific activity (A) were measured as described under Experimental Procedures using [“C]DNS-CI(40 dpm/pmol). Values are mean + SD (n = 3 or 4 cultures). Values of procollagen hydroxyproline specific activity are also shown in Fig. 3A.
FIBROBLAST
PROCOLLAGEN
160 1”
T
PRODUCTION
RATES
483
parameters were considered: (a) procollagen prolyl hydroxylation = 0.40 -+ 0.01 (mean + SD; n = 4); (b) prolyl and hydroxyprolyl residues per procollagen molecule = 24 1 ( 15); and (c) medium [3H]hydroxyproline in these cultures represented 63% of the total [3H]hydroxyproline. We assumed that the specific activity of newly synthesized hydroxyproline in the cell layer was equal to that in the medium, although this is only true when procollagen specific activity has reached a plateau value. The value corresponds to 1.3 X 1O6 molecules of procollagen produced per cell per hour using the calculation method of Breul et al. (15). DISCUSSION
%
01
0246
’
”
I
’
24
9
TIME IN LABELING
MEDIUM
(hours)
FIG. 3. Kinetics of fibroblast procollagen production in vitro. Fibroblasts were cultured as described in Fig. 1 using 20 &i [3H]proline in the labeling medium. After incubation for 2 to 24 h, procollagen hydroxyproline specific activity (A), [‘Hlhydroxyproline production (B), hydroxyprohne production (C), and hydroxyproline production rate (D) were measured as described under Experimental Procedures using [%]DNS-Cl (40 dpm/pmol). The hatched area is the mean f SD of all values obtained in the first 9 h (n = 14). Individual values are the mean f SD (n = 3 or 4 cultures).
into medium based on the specific activity of hydroxyproline was constant from the beginning of the incubation through 9 h (Fig. 3C; I = 0.986). Thereafter, the production decreased. This was also evident when hydroxyproline production rate was calculated from data obtained in the first 9 h (13.9 + 3.4; II = 15) and from data obtained at 24 h (5.4 +- 1.6; n = 4; p < 0.001). Procollagen production rate calculated from data obtained after 6 h incubation was 0.23 pmol procollagen/pg DNA h. To make this calculation (Eq. [5]) the following additional l
These studies demonstrate the application of a sensitive method of measuring hydroxyproline specific activity to obtain actual procollagen production rate of fibroblasts in vitro. The procedure, which does not require quantitative recovery of the hydroxyproline in the sample, can be done relatively rapidly and inexpensively. Hydroxyproline specific activity was readily measured in confluent lung fibroblast cultures containing approximately 1O6 cells and 5 to 20 &i of [3H]proline. Inclusion of proline at physiologic concentration (0.2 mM) decreased the hydroxyproline specific activity, but did not preclude the measurement. The culture conditions employed in these studies enhance procollagen synthesis (23), but measurement of hydroxyproline specific activity is also probably feasible under suboptimal conditions (e.g., serum-free medium). Since application of the method requires sufficient amounts of [3H]hydroxyproline in the sample, larger cultures and/or more [3H]proline in the labeling medium may be required in some instances. In these studies we also examined extracellular and intracellular free proline pool specific activities and their relation to specific activity of hydroxyproline in procollagen synthesized over 24 h. We observed that (1) extracellular free proline specific activity (i.e., proline specific activity in labeling medium)
484
CLARK
AND HILDEBRAN
decreased dramatically in these cultures that were not supplemented with proline. This probably occurred primarily as the result of net proline production by the cells (19,24), and illustrates the non-steady state of the cultures with respect to proline concentration. (2) The intracellular free proline specific activity increased over the first 4 h and then decreased, presumably reflecting in part trans port of [3H]proline from the changing extracellular pool as well as endogenous proline production. (3) Procollagen hydroxyproline specific activity increased over the culture period. We assume the initial change is due to the secretion of intracellular procollagen formed all or in part prior to the addition of isotope and production of procollagen prior to equilibration of the prolyl-tRNA pool. (4) Extracellular proline specific activity, intracellular proline specific activity, and procollagen hydroxyproline specific activity were not equal at any time over 24 h, and the relationship between these pools and procollagen specific activity was not constant. It is evident that neither of these free proline pools can be used to determine procollagen production. Although we did not examine prolyl-tRNA specific activity in these studies, the specific activity of this pool has previously been shown to be relatively constant for 4 h and equal to cellular protein specific activity by about 8 h in fibroblast cultures containing 0.2 mM proline (19). Thus, prolyl-tRNA specific activity may be a valid estimate of protein specific activity under these conditions. However, based on the data from this study, one can predict that in prolinedeficient cultures and/ or after prolonged labeling periods (e.g., 24 h), the specific activity of prolyl-tRNA (measured at the end of a labeling period) would be different from the mean specific activity of prolyl-tRNA used for procollagen synthesis during the labeling period. For the final prolyltRNA specific activity to be a valid measure of the specific activity of newly synthesized protein, its specific activity must be constant throughout the labeling period. Examination of the kinetics of fibroblast procollagen production based on hydroxy-
proline specific activity revealed that [‘HIhydroxyproline in medium increases linearly from 1.6 to 9 h in labeling medium. The initial lag period of 1.6 h may be attributed to isotope transport into the cells, equilibration with the true precursor pool, and production and secretion of procollagen derived from nonradioactive proline. When actual hydroxyproline production rate was calculated on the basis of hydroxyproline specific activity, no lag period was observed, indicating that procollagen production was constant from time zero through 9 h. This predictable result, similar to that of Schmid and Conrad ( 13), emphasizes that the method described here (a modified measurement of hydroxyproline by isotope dilution) quantitates total procollagen secreted into culture medium regardless of the precursor specific activity. This is highly desirable given the inhomogeneity and inconstancy of proline precursor pools. It should also be noted that if the experimental conditions are changed at the time labeling medium is added, the calculated value of procollagen production rate will reflect in part the control (basal) procollagen production rate (i.e., the contribution of intracellular procollagen and nascent procollagen chains formed prior to addition of isotope) as well as the production rate of newly synthesized procollagen. This can be minimized by changing the experimental conditions prior to labeling and/or prolonging the labeling time. The procollagen production rate by IMR90 fibroblasts determined in this study is similar to the value based on prolyl-tRNA specific activity ( 19), and approximately twice that of another human fetal lung fibroblast strain (HFL- 1) cultured under different conditions (15). In the latter study, intracellular free proline specific activity was used to calculate the rate of procollagen production, and probably contributes to the discrepancy. Had we based our calculation on the specific activity of this pool, our value would have been reduced. In these studies, we made the unexpected observation that hydroxyproline production rate decreased after about 9 h in culture. This decrease was not so apparent when
FIBROBLAST
PRGCOLLAGEN
[3H]hydroxyproline production was examined over 24 h (r = 0.984). Actual hydroxyproline production over 24 h, however, was obviously not constant. Although we did not examine the mechanism of this change in hydroxyproline production rate, such factors as density inhibition of procollagen synthesis, decreased efficiency of prolyl hydroxylation, nutrient depletion, or incorporation of collagen into insoluble matrix could contribute. ACKNOWLEDGMENT We thank Dr. R. B. Low for helpful discussions in the preparation of this manuscript.
REFERENCES 1. Bomstein, P., and Sage, H. (1980) Annu. Rev. B&hem. 49,95?-1003. 2. Prockop, D. J., Berg, R. A., Kivirikko, K. I., and Uitto, J. (1976) in Biochemistry of Collagen (Ramachandran, G. N., and Reddi, A. H., eds.), pp. 163-273, Plenum, New York. 3. Prockop, D. J., Kivirikko, K. I., Tuderman, L., and Guzman, N. A. (1979) N. Engl. J. Med. 301, 1323, 75-85. 4. Kao, W. W.-Y., Prockop, D. J., and Betg, R. A. (1979) J. Biol. Chem. 254, 2234-2243. 5. Clark, J. G., Starcher, B. C., and Uitto, J. (1980) Biochim. Biophys. Acta 631, 359-370. 6. Berg, R. A., Moss, J., Baum, B. J., and Crystal, R. G. (1981) J. C/in. Invest. 67, 1457-1462. 7. Tan, E. M. L., RyhHnen, L., and Uitto, J. (1983) J. Invest. Dermatol. 80, 26 1-267.
PRODUCTION
RATES
485
8. Uitto, J., Batter, E. A., and Eisen, A. 2. (1979) J. Clin. Invest. 64, 92 l-930. 9. Byers, P. H., Holbrook, K. A., Barsh, B. S., Smith, L. T., and Borstein, P. (198 I) Lab. Invest. 44,336341. 10. Berg, R. A. (1982) in Methods in Enzymology (Cunningham, L. W., and Frederiksen, D. W., eds.), Part A, Vol. 82, pp. 372-398, Academic Press, New York. I I. Robins, S. P. (1979) Biochem. J. 181, 75-82. 12. Kao, W. W.-Y., Prockop, D. J., and Berg, R. A. (1977) J. Biol. Chem. 252, 8391-8397. 13. Schmid, T. M., and Conrad, H. E. (1982) J. Biol. Chem. 257, 12,451-12,457. 14. Rannels, D. E., Low, R. B., Youdale, T., Volkin, E., and Longmore, W. J. (1982) Fed. Proc. 41,28332839. 15. Breul, S. D., Bradley, K. H., Hance, A. J., Schaffer, M. P., Berg, R. A., and Crystal, R. G. (1980) J. Biol. Chem. 255, 5250-5260. 16. Pietihi, K., and Nikkari, T. (1982) Artery 10, 172179. 17. Clark, J. G., Kostal, K. M., and Ma&o, B. A. (1982) J. Biol. Chem. 257, 8098-8105. 18. Kipnis, D. M., Reiss, E., and Helmreich, E. (1961) Biochim. Biophys. Acta 51, 5 19-524. 19. Hildebran, J. N., Airhart, J., Stirewalt, W. S., and Low, R. B. (1981) Biochem. J. 198, 249-258. 20. Airhart, J., Kelley, J., Brayden, J. E., Low, R. B., and Stirewalt, W. S. (1979) Anal. Biochem. 96,45-55. 21. Peterkofsky, B., and Diegelmann, R. (1971) Biochemistry 10, 988-994. 22. Burton, K. (1956) B&hem. J. 62, 315-323. 23. Booth, B. A., Polak, K. K., and Uitto, J. (1980) Biochim. Biophys. Acfa 607, 145-160. 24. Kruse, P. F., and Miedema, E. (I 965) J. Cell Biol. 27,273-279.