Incorporation of proline analogs into procollagen. Assay for replacement of imino acids by cis-4-hydroxy-L-proline and cis-4-fluoro-L-proline

Previously, several proline analogs have shown to be incorporated into protein and, in particular, into procollagen polypeptides. Here a new technique was used to determine the extent to which two proline analogs, cis-4-hydroxy-l-proline and cis-4-fluoro-l-proline, replaced proline and hydroxyproline in newly synthesized pro-α and pro-γ chains of procollagen. Matrix-free chick embryo tendon cells, when incubated with 1.53 mm, cis-4-hydroxy-l-proline, synthesized collagenous polypeptides in which from 13 to 19% of the total imino acid residues were replaced with the analog. Incubation of cells with 1.50 mm, cis-4-fluoro-l-proline resulted in the synthesis of polypeptides in which 27% of the imino acid residues were replaced by the analog. With lower concentrations, proportionally less of the analog was incorporated into protein. The observations here extend previous indications that proline analogs in relatively low concentrations may have a specific effect on the synthesis of collagen.     

Decreased thermal stability of collagens containing analogs of proline or lysine

Fibroblasts were incubated with analogs of proline or lysine and the thermal stability of procollagen molecules containing the analogs was investigated using pepsin digestion at different temperatures as an enzymatic probe of conformation. The procollagens containing either 4-cis-hydroxy-l-proline, 3,4-dehydroproline, or 4,5-trans-dehydrolysine were less stable than normal procollagen and these abnormal collagens were largely in a non-triplehelical conformation within the cells at 37 °C. These results support the idea that procollagen molecules which are not in a triple-helical conformation are not secreted at a normal rate. Procollagens containing both 4,5-trans-dehydrolysine and a proline analog were much less stable than molecules containing a single type of analog. This result suggests that simultaneous administration of both types of analogs may have a greater effect on collagen accumulation in whole-animal experiments than administration of a single analog.     

Molecular insights into prolyl and lysyl hydroxylation of fibrillar collagens in health and disease

Collagen is a macromolecule that has versatile roles in physiology, ranging from structural support to mediating cell signaling. Formation of mature collagen fibrils out of procollagen α-chains requires a variety of enzymes and chaperones in a complex process spanning both intracellular and extracellular post-translational modifications. These processes include modifications of amino acids, folding of procollagen α-chains into a triple-helical configuration and subsequent stabilization, facilitation of transportation out of the cell, cleavage of propeptides, aggregation, cross-link formation, and finally the formation of mature fibrils. Disruption of any of the proteins involved in these biosynthesis steps potentially result in a variety of connective tissue diseases because of a destabilized extracellular matrix. In this review, we give a revised overview of the enzymes and chaperones currently known to be relevant to the conversion of lysine and proline into hydroxyproline and hydroxylysine, respectively, and the O-glycosylation of hydroxylysine and give insights into the consequences when these steps are disrupted.     

Inhibition of tropoelastin secretion by incorporation of DL-3,4-dehydroproline

Cells isolated from embryonic chick aorta were incubated in suspension culture with labeled amino acids and proline analogs. Incorporation of 4-cis-hydroxy-l-proline inhibited the secretion of labeled procollagen but not tropoelastin, while incorporation of dl-3,4-dehydroproline inhibited the secretion of both proteins and caused them to accumulate intracellularly. Protein synthesis did not appear to be significantly diminished during the 2-h incubation period. Incorporation of dl-3,4-dehydroproline may alter the conformation of tropoelastin leading to abnormal intracellular processing and a decreased rate of secretion.     

The disulphide-bonded nature of procollagen and the role of the extension peptides in the assembly of the molecule.

1. The molecular weights of chick tendon and cartilage procollagens, and their constituent polypeptides, were determined by gel filtration and gel electrophoresis. The values obtained are in good agreement and indicate that the mol.wts. of the secreted procollagens (types I and II) and their individual pro-alpha-chains are of the order of 405 000-445 000 and 137 000-145 000 respectively.2. Digestion of tendon procollagen with human rheumatoid synovial collagenase gave products consistent with the presence of large non-helical peptide extensions at both N-and C-termini. Electrophoretic analysis gave apparent mol.wts. of 17 500 and 36 000 for the respective N- and C-terminal extensions of pro-alpha1(I)-and pro-alpha2-chains, and inter-chain disulphide bonds were restricted to the C-terminal location. 3. During the biosynthesis of procollagen by tendon and cartilage cells a close correlation was observed between the extent of inter-chain disulphide bonding and the proportion of procollagen polypeptides having a triple-helical conformation. These processes appeared to commence in the rough endoplasmic reticulum and be completed in the smooth endoplasmic reticulum, but the rate at which they occur in cartilage cells is markedly slower than that found in tendon cells. 4. When the intracellular [14C]procollagen polypeptides present in the rough-endoplasmic-reticulum fractions of tendon and cartilage cells were analysed under non-reducing conditions on agarose/polyacrylamide composite gels, no significant pools of dimeric intermediates were detected. 5. In both cell types, inter-chain disulphide-bond formation occurred even when hydroxylation, and hence triple-helix formation, was inhibited. The presence of pro-alpha1- and pro-alpha2-components in a ratio of 2:1 in the disulphide-linked unhydroxylated procollagen isolated from tendon cells demonstrated that correct chain association occurs in the absence of hydroxylation. This observation is consistent with a model for the assembly of pro-gamma112-chains in which the recognition and selection of pro-alpha1-and pro-alpha2-chains in a 2:1 ratio are directed by the non-helical C-terminal extension peptides of tendon procollagen.     

Lysosomal function in the degradation of defective collagen in cultured lung fibroblasts

Human fibroblasts when induced to make nonhelical , defective collagen have mechanisms for degrading up to 30% of their newly synthesized collagen intracellularly prior to secretion. To determine if at least a portion of the degradation of defective collagen occurs by lysosomes, extracts of cultured HFL-1 fibroblasts were examined for proteinases capable of degrading denatured type I [3H]procollagen. The majority of the proteolytic activity against denatured [3H]-procollagen had a pH optimum of 3.5-4; it was stimulated by dithiothreitol and inhibited 95% by leupeptin, 10% by pepstatin, and 98% by leupeptin and pepstatin together. Extracts of purified lysosomes from the fibroblasts were active in degrading denatured [3H]procollagen and were completely inhibited by leupeptin and pepstatin. To demonstrate directly that human lung fibroblasts can translocate a portion of their defective collagen to lysosomes, cultured cells were incubated with cis-4-hydroxyproline and labeled with [14C]proline to cause the cells to make nonhelical [14C]procollagen. About 3% of the total intracellular hydroxy[14C]proline was found in lysosomes. If, however, the cells were also treated with NH4Cl, an inhibitor of lysosomal function, 18% of the intracellular hydroxy[14C]proline was found in lysosomes. These results demonstrate that cultured human lung fibroblasts induced to make defective collagen are capable of shunting a portion of such collagen to their lysosomes for intracellular degradation.     

The route of secretion of procollagen. The influence of alphaalpha''-bipyridyl, colchicine and antimycin A on the secretory process in embryonic-chick tendon and cartilage cells.

I. Embryonic-chick tendon cells were pulse-labelled for 4 min with [14C]proline and the 14C-labelled polypeptides were chased with unlabelled proline for up to 30 min. Isolation of subcellular fractions during the chase period and their subsequent analysis for bacterial collagenase-susceptible 14C-labelled peptides demonstrated the transfer of procollagen polypeptides from rough to smooth microsomal fractions and thence to the extracellular medium. Parallel analyses of Golgi-enriched fractions indicated the involvement of this organelle in the secretory pathway of procollagen. Sodium dodecylsulphate/polyacrylamide-gel electrophoresis of the 14C-labelled polypeptides present in the Golgi-enriched fractions demonstrated that the procollagen polypeptides were all present as disulphide-linked pro-gamma components. 2. When similar kinetic studies of the intracellular transport of procollagen were conducted with embryonic-chick cartilage cells almost identical results were obtained, but the rate of translocation of cartilage procollagen was significantly slower than that observed for tendon procollagen. 3. When hydroxylation of procollagen polypeptides was inhibited by alphaalpha'-bipyridyl, the nascent polypeptides accumulated in the rough microsomal fraction. 4. When cells were pulse-labelled for 4min with [14C)proline and the label was chased in the presence of colchicine, secretion of procollagen was inhibited and an intracellular accumulation of procollagen 14C-labelled polypeptides was observed in the Golgi-enriched fractions. 5. The energy-dependence of the intracellular transport of procollagen was demonstrated in experiments in which antimycin A was found to inhibit the transfer of procollagen polypeptides from rough to smooth endoplasmic reticulum. 6. It is concluded that procollagen follows the classical route of secretion taken by other extracellular proteins.     

Imino acid biosynthesis in Delonix regia

The biosynthesis of l-azetidine-2-carboxylic acid and trans-3-hydroxy-l-proline has been studied in Delonix regia seedlings by labelled precursor feeding techniques. α,γ-Diaminobutyric acid was incorporated into azetidine-2-carboxylic acid more efficiently than homoserine, methionine or aspartic acid. More radioactivity from proline was found in trans-3-hydroxyproline after 2 day's than after 4-day's metabolism, indicating a continuous turnover of the hydroxyimino acid in seedlings.     

Kinetics for the secretion of nonhelical procollagen by freshly isolated tendon cells

Fibroblasts isolated by enzymic digestion of chick embryo tendons have previously been used to examine the kinetics for the secretion of procollagen (Kao, W. W.-Y., Berg, R. A., and Prockop, D. J. (1977) J. Biol. Chem. 252, 8391-8397). The results indicated that the kinetics approximated the sum of two first order processes with half-times of 14 and 115 min. Here, the same fibroblasts were incubated in the presence of 1.53 mM cis-4-hydroxyproline, an analogue of proline, or in the presence of 0.3 mM alpha,alpha'-dipyridyl, an inhibitor of prolyl hydroxylase, so that the cells synthesized procollagen which could not assume a triple helical conformation characteristic of procollagen. Measurements of the secretion of nonhelical procollagen indicated that the kinetics for secretion differed from the kinetics for the secretion of procollagen and approximated a single first order process with a half-time of approximately 130 min. The nonhelical procollagen synthesized and secreted in the presence of either cis-4-hydroxyproline or alpha,alpha'-dipyridyl consisted of disulfide-bonded pro gamma chains of type I procollagen. The results suggested that the intracellular nonhelical procollagen was present in a single metabolic pool and secretion from this pool occurred with a different rate-limiting step than for helical procollagen. Further results indicated that nonhelical procollagen had a high affinity for prolyl hydroxylase and the affinity for the enzyme was greatly reduced if the procollagen was allowed to assume the triple helical conformation characteristic of normal procollagen. The results are consistent with the hypothesis that the secretion of procollagen is influenced by its conformation-dependent interaction with prolyl hydroxylase or other post-translational enzymes.     

Subunit structure of wheat germ agglutinin

Cells isolated by enzymic digestion of embryonic tendon were incubated under N₂ so that they synthesized and accumulated the unhydroxylated form of procollagen which is known as protocollagen and which is largely comprised of pro-α chains linked by interchain disulfide bonds. The cells were then exposed to O₂ so that the intracellular protocollagen was hydroxylated and secreted as procollagen. When the hydroxylation was allowed to proceed at 31° or 34°, the procollagen secreted into the medium was triple-helical but its hydroxyproline content was less than two-thirds and its hydroxylysine content was less than half the control. Even when the hydroxylation was allowed to occur at 37°, the procollagen secreted by the cells was under-hydroxylated by about 15% in terms of its hydroxyproline content and about 45% in terms of its hydroxylysine content. The results may have consequences for collagen synthesis by tendons and similar tissues $\text{in vivo}$, since temporary anoxia in such tissues may well lead to the synthesis of a less stable procollagen or to fibers of decreased tensile strength.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Synthesis of procollagen by matrix-free cells from embryonic-chick arteries

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [(14)C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[(14)C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-alpha-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.     

Structural Aspects of Hydroxyproline-Containing Proteins

Abstract

The occurrence of hydroxyproline (Hyp) in collagen, Clq and acetylcholinesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the β-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific β-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to Clq and acetylcholineesterase both of which share the general structural and functional properties of collagen in their “tail” regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of Clq and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

Effect of proline residues on protein folding

Conformational energy calculations have been used to study the role of the proline residues in the folding of bovine pancreatic trypsin inhibitor. In the calculation, each of the four proline residues of this small protein is forced from the trans to cis peptide isomer while still part of the native folded structure. The cis proline residue can always be accommodated by small changes of the native conformation (< 1 Å root-mean-square deviation). For three of the four proline residues, Pro2, Pro9 and Pro 13, being in the cis form is calculated to destabilize the folded conformation by less than 11 kcal/mol, suggesting that rapid folding to a stable native-like conformation can occur with either isomeric form. For one of these three, Pro13, the destabilization is only 1 kcal/mol, suggesting the existence of an alternative folded native conformation with Pro13 cis. The fourth proline residue, Pro8, is calculated to destabilize the native conformation by so much (33 kcal/mol) that it will block folding in the manner proposed by Brandts et al. (1975).     

The synthesis and secretion of cartilage procollagen.

1. Isolation of free and membrane-bound ribosomes from embryonic chick sternal-cartilage cells labelled for 4min with [14C]proline and their subsequent analysis for hydroxy[14C]proline indicated that cartilage procollagen biosynthesis occurs on bound ribosomes. 2. Nascent procollagen polypeptides on bound ribosomes isolated from cells labelled with [14C]lysine were found to contain hydroxy[14C]lysine indicating that hydroxylation of lysine commences while the growing chains are still attached to the ribosomes. 3. Analysis of bound ribosomes labelled with either [14C]proline or [14C]lysine on sucrose density gradients indicated that cartilage procollagen is synthesized on large polyribosomes in the range 250-400S. 4. Microsomal preparations isolated from cells pulse-labelled for 4 min with [14C]proline were used to determine the direction of release of nascent procollagen polypeptides. Puromycin induced the vectorial release of nascent procollagen polypeptides into the microsomal vesicles suggesting that the first step in the secretion of procollagen polypeptides is their transfer from the ribosomes through the membrane of the endoplasmic reticulum into the cisternal space. 5. The procollagen polypeptides secreted by cartilage cells were shown to be linked by inter-chain disulphide bonds. 6. Examination of the state of aggregation of pro-alpha chains in subcellular fractions isolated from cartilage cells labelled with [14C]proline for various periods of time have provided data on the timing and location of inter-chain disulphide-bond formation. This process commences in the rough endoplasmic reticulum after the release of completed pro-alpha chains from membrane-bound ribosomes. Pro-alpha chains isolated from fractions of smooth endoplasmic reticulum were virtually all present as disulphide-bonded aggregates, suggesting that either disulphide bonding is completed in this cellular compartment, or that procollagen needs to be in a disulphide-bonded form to be transferred to this region of the endoplasmic reticulum. 7. Comparison of these results with previously published data on disulphide bonding in tendon cells suggest that the rate of inter-chain disulphide-bond formation is significantly slower in cartilage cells.     

A post-translational modification, unrelated to hydroxylation, in the collagenous domain of nonhelical pro-alpha 2(I) procollagen chains secreted by chemically transformed hamster fibroblasts.

Transformed Syrian hamster embryo (NQT-SHE) fibroblasts do not synthesize the pro-alpha 1 subunit of type I procollagen, but secrete two modified forms of the pro-alpha 2(I) subunit that migrate more slowly than the normal chain during gel electrophoresis (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). By electrophoretic analysis of cyanogen bromide and V8 protease-derived peptides from the collagenous domains of intra- and extracellular pro-alpha 2(I) chains, we find that the modification occurs almost exclusively in secreted molecules, is located in the region spanned by the cyanogen bromide peptide CB3,5, and persists when hydroxylation is inhibited. Thus, modification is due to a post-translational reaction other than hydroxylation. The modified chains appear to be secreted in the denatured state since: 1) helical structures formed at 4 degrees C under acidic conditions were unstable under neutral conditions at 37 degrees C; 2) conditions that destabilize the type I procollagen helix and thus inhibit its secretion, i.e. inhibition of proline hydroxylation or incorporation of the proline analog cis-hydroxyproline, did not affect secretion of the modified chains. The time courses for secretion of nonhelical modified chains from NQT-SHE and of hydroxylated helical procollagen I from control cells, as a proportion of total collagen synthesized, were similar. Although cis-hydroxyproline did not inhibit the secretion of the modified chains, it induced their rapid intracellular degradation.     

Impaired secretion of type III procollagen in Ehlers-Danlos syndrome type IV fibroblasts: correction of the defect by incubation at reduced temperature and demonstration of subtle alterations in the triple-helical region of the molecule

The amount of type III procollagen secreted by fibroblasts from two patients with type IV Ehlers-Danlos syndrome is reduced to 25% and 20%, respectively, of that of control cells after incubation at 37 degrees C, but reverts to 70% and 110% when cells are incubated at 32 degrees C. The type III procollagen molecules secreted only at the lower temperature are of normal size but apparently contain different mutations which disrupt the triple-helical region and lower the thermal stability of the molecule. These data suggest that subtle mutations in the pro alpha 1(III)-chains produce Ehlers-Danlos syndrome type IV by disrupting the triple-helical region of the molecule, lowering its thermal stability, and thus impairing its secretion. At the lower temperature, stabilization of the defective molecules result in more efficient secretion. This approach may be useful for the characterization of other unstable collagens.     

Structural aspects of hydroxyproline-containing proteins

The occurrence of hydroxyproline (Hyp) in collagen, C1q and acetylcholineesterase (AChE) raises important questions concerning the role of this unusual imino acid in the structure and function of these proteins. Available data on collagen indicate that Hyp is necessary for the normal secretion of the protein after its synthesis and for the integrity of the triple-helical conformation. Studies from our laboratory have dealt with the structural aspects of the posttranslational conversion of proline to hydroxyproline in collagen mediated by prolyl hydroxylase. We proposed that the beta-turn conformation at the Pro-Gly segments in the nascent procollagen molecule are the sites of the enzymatic hydroxylation and that this conformation changes over to the collagen-like helix as a result of the hydroxylation process. Recently, we have provided additional experimental support to our proposal by a) synthesizing specific beta-turn oligopeptides containing the Pro-Gly as well as Pro-Ala and Pro-DAla sequences and showing that these act as inhibitors of the enzymatic hydroxylation of a synthetic substrate and b) demonstrating, by circular dichroism spectroscopy, the occurrence of a conformational change leading to the triple-helix as a direct consequence of proline hydroxylation in a non-helical polypeptide substrate. We have also observed that the acquisition of hydroxylation results in a significant enhancement of the rate of folding of the polypeptide chain from the unfolded to the triple-helical conformation. We believe that our observations on proline hydroxylation in collagen should also be applicable to C1q and acetylcholineesterase both of which share the general structural and functional properties of collagen in their "tail" regions. Using the techniques employed in collagen studies, one should be able to assess the role of hydroxyproline in the folding, structural stabilities and functions of C1q and AChE. This would also involve the study of the unhydroxylated and hydroxylated precursors of these proteins which may share common structural features with their collagen counterparts. Finally, a systematic study of hydroxyproline-containing peptides and polypeptides has been initiated by us so as to understand the exact manner in which Hyp participates in the formation and stability of the triple-helical conformation in the proteins in which it occurs.     

An examination of the involvement of proline peptide isomerization in protein folding.

The proline peptide isomerization model of protein folding predicts that the fraction of denatured polypeptide chains which rapidly fold should be quantitatively related to the numbers of cis and trans proline residues in the folded polypeptide conformation. However, we find that neither the comparative nor the absolute kinetic pattern for folding of the homologous proteins, tuna heart and horse heart ferricytochrome c which differ by one proline residue, is compatible with the quantitative predictions of the proline peptide isomerization model.     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. 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 [³H]proline, and [³H]hydroxyproline 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 [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg 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.