Programmed synthesis of collagen during postembryonic development of the nematode Panagrellus silusiae.

The relative rate of collagen synthesis in the free-living nematode Panagrellus silusiae during postembryonic development was found to be discontinuous by measuring either the incorporation of tritium into material extracted as collagen or the amount of collagen-bound tritiated proline and hydroxyproline after 2-hr incubations of whole worms with [³H]proline. A peak of collagen production preceded each of the three molts that were examined. Moreover, protocollagen prolyl hydroxylase activity during each intermolt period paralleled the pattern of collagen synthesis. On the other hand, a triphasic pattern was not observed when noncollagenous proteins were labeled with either [³H]tryptophan or [³H]leucine. In addition, the level of soluble radioactive proline that accumulates in whole organisms after 2-hr incubation periods did not fluctuate appreciably during postembryonic development. The mean ratio of hydroxy-proline to proline in a number of collagen samples extracted at various times during the maturation phase was 0.113 ± 0.040. Pulse and chase experiments with [³H]proline indicated that most of the collagen synthesized during a peak period is lost after the second ecdysis following the labeling interval. In contrast, a considerable proportion of the collagen synthesized during nonpeak periods is retained throughout the postembryonic period. It is postulated that the modulated pattern of collagen biosynthesis in Panagrellus reflects, for the most part, a quantitative regulation of the production of cuticular collagen during postembryonic development.     

Rapid intracellular degradation of newly synthesized collagen by bone cells. Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesized collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [3H]proline into collagenase-digestible proteins or by the conversion of [3H]proline into [3H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

心室成纤维细胞条件培养液促进成纤维细胞胶原合成和增殖

采用心室成纤维细胞条件培养液培养心室成纤维细胞,通过测定[^3H]－脯氨酸（[^3H]－proline）的掺入率来了解心室成纤维细胞总胶原合成速率,通过测定[^3H]－胸腺嘧啶核苷（[^3H]－TdR）的掺入率以及c-fos基因的表达丰度来了解心室成纤维细胞的增殖速率。结果显示：心室成纤维细胞条件培养液（FCGM）能增加细胞自身的[^3H]－proline的掺入率和[^3H]－TdR的掺入率,并具有剂量依赖性;FCGM也能促进细胞自身c-fos基因的表达,刺激后1h达高峰。ETA受体拮抗剂BQ123能部分阻断FCGM增加成纤维细胞胶原合成的增殖作用,而AT1受体拮抗剂CV11974和α肾上腺素受体拮抗剂regitin无此效果。结果提示：心室成纤维细胞具有自分泌功能,能分泌内皮素等生物活性物质,促进成纤维细胞胶原的合成和增殖。     

Rapid intracellular degradation of newly synthesized collagen by bone cells Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesizes collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [³H]proline into collagenase-digestible proteins or by the conversion of [³H]proline into [³H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Growth effect of lithium on mouse mammary epithelial cells in serum-free collagen gel culture

The effect of lithium on the growth of mammary epithelial cells from adult virgin and midpregnant BALB/c or BALB/cfC3H mice was tested in a serum-free collagen gel culture system. The serum-free medium consisted of a 1:1 mixture of Ham's F12 and Dulbecco's Modified Eagle's medium supplemented with insulin, transferrin, cholera toxin, epidermal growth factor (EGF), and bovine serum albumin fraction V (BSA V). A multifold increase in cell number occurred during 10–12 days of culture in this medium. In dose-response studies in which the concentration of each component of this serum-free medium was varied in turn, the addition of LiCL (10 mM) enhanced growth at most concentrations of each factor. However, LiCL could not enhance growth in the absence of insulin or BSA V, but could replace EGF. The optimal concentration of LiCl was 5–10 mM; higher concentrations (20–80 mM) were toxic. KCl (1–10 mM) when added to the serum-free medium slightly stimulated growth; the addition of NaCl to the medium had little effect on growth. LiCl did not enhance the growth of cells from spontaneous mammary tumors of BALB/cfC3H mice.     

The formation and thermal stability of in vitro assembled fibrils from acid-soluble and pepsin-treated collagens.

The role of the non-helical regions of the collagen molecule in fibrillogenesis has been investigated by comparing the kinetics of fibril formation of pepsin-treated acid-soluble collagen, acid-soluble collagen and mixtures of the two and by comparison of the thermal stabilities of the fibrils formed. The acid-soluble collagen was found to aggregate more rapidly than the pepsin-treated collagen under physiological conditions of pH and ionic strength. Variations in ionic strength, at physiological pH, were found to have differing effects on the aggregation of these two forms of soluble collagen. Fibrils formed from the pepsinized-collagen had a lower thermal stability tha n those formed from the intact collagen. The behavior observed with mixtures of acid-soluble and pepsin-treated collagens was found to be quantitatively consistent with the pepsinized collagen being able to utilize the nuclei formed by the acid-soluble collagen for subsequent growth. However, the use of the acid-soluble nuclei by the pepsinized collagen for growth did not enhance its rate of precipitation during the growth phase, nor did it enhance the thermal stability of the fibrils formed from the pepsinized collagen.     

Passage in monolayer influences the response of chondrocytes to dynamic compression

This study tests the hypothesis that expansion by passage in monolayer influences the response of isolated articular chondrocytes to dynamic compression. Chondrocytes, isolated from bovine articular cartilage, were seeded in monolayer and passaged 4 times (P1-4). For assessment of chondrocytic and fibroblastic phenotype, freshly isolated and passaged cells were seeded on glass coverslips or in 2% alginate beads and cultured for 7 days in DMEM + 10% FCS. Samples were assayed for DNA and GAG content and stained for collagen types I and II. In separate experiments, freshly isolated or passaged chondrocytes were seeded at 10 x 10(6) cells.ml(-1) in 4% cylindrical agarose constructs and subjected to 15% dynamic compressive strain at 1 Hz for 24 hours. [(3)H]-thymidine incorporation, SO(4) incorporation and nitrite release were analysed. Immediately following isolation (P0), chondrocytes seeded in alginate expressed high levels of type II collagen, but did not stain for type I collagen. Following repeat passage the cells expressed enhanced levels of type I collagen, with an associated reduction in type II collagen staining. These data indicate a modulation to a fibroblastic phenotype during monolayer expansion which was not rapidly reversed by culture in a 3D hydrogel. Dynamic compression down-regulated SO(4) incorporation at P0, but did not affect [(3)H]-thymidine incorporation. By contrast the incorporation of both SO(4) and [(3)H]-thymidine was enhanced by dynamic compression at both P1 and to a lesser extent P2. SO(4) and [(3)H]-thymidine incorporation were inhibited at P3 and P4. Nitrite release was down-regulated by dynamic compression at all passages. These data demonstrate a clear modulation in the response of bovine articular chondrocytes to dynamic compression following passage in monolayer.     

Characterization of recombinant human prolyl 3-hydroxylase isoenzyme 2, an enzyme modifying the basement membrane collagen IV

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.     

Human skeletal growth factor stimulates collagen synthesis and inhibits proliferation in a clonal osteoblast cell line (MC3T3-E1)

Human skeletal growth factor (hSGF), an 11-kD polypeptide purified from human bone, has been proposed to be a local regulator of bone formation. To investigate the underlying cellular mechanisms in an in vitro model system, we examined the effects of hSGF on proliferation and collagen synthesis in cells of the clonal osteoblast cell line MC3T3-E1. This line was isolated from newborn mouse calvarial cells and retains many characteristics of mature osteoblasts (Sudo, H., et al., (1984) J. Cell Biol. 96:191). A 14-hr treatment with hSGF increased noncollagenous protein synthesis to 215% of unstimulated controls and increased collagen synthesis to 630% of controls as determined by [3H]proline incorporation and high-pressure liquid chromatographic separation of [3H]proline and [3H]hydroxyproline in acid hydrolysates of trichloroacetic acid-insoluble protein. HSGF did not increase cell number over a 48-hr period and caused a reversible inhibition of DNA synthesis. Half-maximal hSGF concentration for stimulation of [3H]proline incorporation and inhibition of [3H]thymidine incorporation was 100 ng/ml. HSGF also inhibited DNA synthesis in cells stimulated by serum. In contrast, hSGF stimulated both collagen synthesis and DNA synthesis in primary cultures of chick embryo bone cells, which may be developmentally less mature than MC3T3-E1 cells. The results suggest that hSGF directly stimulated mature osteoblast matrix synthetic activity and that hSGF has differential effects on proliferation of osteoblast progenitor cells and mature osteoblasts.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Inhibition of normal rat kidney cell growth by transforming growth factor-beta is mediated by collagen

We have investigated the mechanism of inhibition of the serum-free monolayer growth of normal rat kidney (NRK) cells by transforming growth factor-beta (TGF-beta). NRK cells grown on fibronectin-coated dishes exhibited a biphasic response to TGF-beta. Monolayer growth was slightly stimulated by subpicomolar concentrations, while picomolar concentrations of TGF-beta inhibited NRK cell growth in the presence or absence of epidermal growth factor. NRK cells exhibited a similar biphasic growth response to exogenous type I collagen. TGF-beta induced a 3-5-fold increase in the deposition of type I collagen-like proteins into the extracellular matrix of NRK cells during serum-free growth. Type I collagen-like proteins were identified by their sensitivity to degradation by purified bacterial collagenase and by Western blot analysis. The TGF-beta dose-response curves for induction of extracellular matrix-localized collagen and inhibition of NRK cell growth were similar. Finally, the inclusion of a purified bacterial collagenase, which did not degrade TGF-beta or TGF-beta receptors, or alter control NRK growth, prevented exogenous collagen or TGF-beta from inhibiting the serum-free growth of NRK cells. Our results demonstrate that an increase in collagen secretion plays an important role in the inhibition of the growth of NRK cells by TGF-beta.     

Effect of L-3,4-dehydroproline on collagen synthesis and prolyl hydroxylase activity in mammalian cell cultures.

The incorporation of DL-3,4-dehydro[14C]proline into collagen and total protein of 3T3 cells occurred at approximately one-fifth the rate observed for L-[14C]proline. Addition of L-3,4-dehydroproline to the culture medium inhibited markedly the incorporation of [14C]glycine and L-[3H]lysine into the collagen of 3T3 cells, but there was only slight inhibition of the incorporation of the radiolabeled amino acids into total cellular proteins, indicating that the action of L-3,4-dehydroproline is specific for collagen. When 1 mM L-3,4-dehydroproline was added to the culture medium the [14C]hydroxyproline content was reduced 40% in the cell layer and 70% in the medium. The D isomer of 3,4-dehydroproline did not inhibit [14C]hydroxyproline formation. These findings indicate that L-3,4-dehydroline reduced the hydroxylation of the susceptible prolyl residues in the collagen molecule and the secretion of collagen from the cell. The reduction in the hydroxyproline content is probably related in part to a reduction in the activity of prolyl hydroxylase; when various mammalian cell cultures were exposed to 0.2 mM L-3,4-dehydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroproline, the specific activity of prolyl hydroxylase was reduced markedly, while that of lysyl hydroxylase was not affected. Under these conditions, cell growth and lactic dehydrogenase required protein synthesis. Removal of L-3,4-dehydroproline from the growth medium resulted in a time-dependent increase in the specific activity of prolyl hydroxylase.     

Influence of bleomycin on the metabolism of dermal collagen in albino rats

The metabolism of collagen in male rats by treatment with bleomycin was studied following the injection of [3H]proline and the determination of specific and total activity of [3H]hydroxyproline in skin collagen fractions and urine. In the case of the bleomycin-treated animals, there was found to be an increase in the neutral salt soluble collagen content with no change in insoluble collagen content as compared to the control group. The specific and total radioactivity of [3H]hydroxyproline in soluble and insoluble collagen fractions was also increased. Examination of [3H]hydroxyproline activity in soluble and insoluble collagen showed that the conversion of soluble to insoluble collagen was improved by the bleomycin-treated group. It was found that this was accompanied by a decrease in urinary excretion of total hydroxyproline and [3H]hydroxyproline during the first 12 hr after the administration of [3H]proline. Therefore, the results of the present investigation clearly indicate that the maturation of soluble to insoluble collagen is promoted and accompanied by a decrease in the catabolism of soluble collagen in the bleomycin-treated animals. In addition, administration of bleomycin increased the synthesis of collagen.     

Transforming growth factor-beta isoforms differently stimulate proalpha2 (I) collagen gene expression during wound healing process in transgenic mice

The role of many growth factors and cytokines in the process of wound healing has been intensively investigated in recent two decades. Among them, transforming growth factor-betas (TGF-betas) are well known to have a potent stimulatory effect on collagen synthesis as shown in various in vivo experimental systems. In the present study, we examined the effects of various growth factors on the promoter activity of the proalpha2 (I) collagen gene (COL1A2) during the wound healing process. For this purpose, we utilized transgenic mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase or a bacterial beta-galactosidase gene. These mice exhibited normal phenotypic expression and the wound healing process was not impaired. Full thickness wounds were made by punch biopsy. We examined the effects of TGF-beta1, -beta2, -beta3, basic fibroblast growth factor, platelet-derived growth factor, and connective tissue growth factor by applying them locally to the open wound every 2 days. Among the growth factors examined, all of the three isoforms of TGF- exhibited a more potent stimulatory effect on COL1A2 promoter activity than did other factors. In addition, while TGF-beta1 and -beta2 significantly increased the number of fibroblasts which were positive for X-Gal staining, TGF-beta3 treatment did not change the number of beta-galactosidase expressing cells. Accumulation of collagen fibers was observed to the same extent in the mice treated with TGF-beta1 and those with TGF-beta3. These findings suggest that TGF-beta1 and -beta3 have similar but not identical regulatory mechanisms of COL1A2 expression, and that their pathophysiological roles in wound healing might be different from each other.     

Removal of glycosaminoglycans from cultures of human skin fibroblasts.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.