Coordinate regulation of collagen and proteoglycan synthesis in costal cartilage of scorbutic and acutely fasted, vitamin C-supplemented guinea pigs

The effects of ascorbic acid deficiency and acute fasting (with ascorbate supplementation) on the synthesis of collagen and proteoglycan in costal cartilages from young guinea pigs was determined by in vitro labeling of these components with radioactive proline and sulfate, respectively. Both parameters were coordinately decreased by the second week on a vitamin C-free diet, with a continued decline to 20-30% of control values by the fourth week. These effects were quite specific, since incorporation of proline into noncollagenous protein was reduced by only 30% after 4 weeks on the deficient diet. The time course of the decrease in collagen and proteoglycan synthesis paralleled the loss of body weight induced by ascorbate deficiency. Hydroxylation of proline in collagen synthesized by scorbutic costal cartilage was reduced to about 60% of normal relatively early, and remained at that level thereafter. Neither collagen nor proteoglycan synthesis was returned to normal by the addition of ascorbate (0.2 mM) to cartilage in vitro. Administration of a single dose of ascorbate to scorbutic guinea pigs increased liver ascorbate and restored proline hydroxylation to normal levels by 24 h, but failed to increase the synthesis of collagen or proteoglycan. Synthesis of both extracellular matrix components was restored to control levels after four daily doses of ascorbate. A 96-h total fast, with ascorbate supplementation, produced rates of weight loss and decreases in the synthesis of these two components similar to those produced by acute scurvy. There was a linear correlation between changes in collagen and proteoglycan synthesis and changes in body weight during acute fasting, scurvy, and its reversal. These results suggest that it is the fasting state induced by ascorbate deficiency, rather than a direct action of the vitamin in either of these two biosynthetic pathways, which is the primary regulatory factor.     

Decreased extracellular matrix production in scurvy involves a humoral factor other than ascorbate

Our recent studies suggested that decreased collagen synthesis in bone and cartilage of scorbutic guinea pigs was not related to ascorbate-dependent proline hydroxylation. The decrease paralleled scurvy-induced weight loss and reduced proteoglycan synthesis. Those results led us to propose that the effects of ascorbate deficiency on extracellular matrix synthesis were caused by changes in humoral factors similar to those that occur in fasting. Here we present evidence for this proposal. Exposure of chick embryo chondrocytes to scorbutic guinea pig serum, in the presence of ascorbate, led to effects on extracellular matrix synthesis similar to those seen in scorbutic animals. The rates of collagen and proteoglycan synthesis were reduced to approximately 30-50% of the levels in cells cultured in normal guinea pig serum plus ascorbate, but proline hydroxylation and procollagen secretion were unaffected. Similar results were obtained with serum from fasted guinea pigs supplemented in vivo with ascorbate. The growth rate of the chondrocytes was not significantly affected by scorbutic guinea pig serum.     

Rates of collagen synthesis in lung, skin and muscle obtained in vivo by a simplified method using [3H]proline.

Methods for measurement of rates of collagen synthesis in vivo have thus far been technically difficult and often subject to quite large errors. In this paper a simplified method is described for obtaining synthesis rates of collagen and non-collagen proteins, for tissues of rabbits. This involves an intravenous injection of [3H]proline, administered with a large dose of unlabelled proline, and measurement of the specific radioactivity of proline and hydroxyproline in body tissues up to 3 h later. The specific radioactivity of [3H]proline in plasma and the tissue free pools rises rapidly to a plateau value which is maintained for at least 2 h, when the specific radioactivity of the type I collagen precursors, isolated from the skin, was similar to that of the plasma and tissue-free pool. Furthermore, over this period, the increase in the specific radioactivity of proline in collagen and non-collagen protein was linear with respect to time. These results suggest that the large dose of proline floods the precursor pools for protein synthesis, and that this effect can be maintained for quite long periods of time. Such kinetics greatly simplified the method for obtaining collagen synthesis rates in vivo, which were calculated for lung, heart, skin and skeletal muscle, and shown to be quite rapid, ranging between about 3 and 10%/day. The lung was a particularly metabolically active tissue, with synthesis rates of about 10%/day for collagen and 35%/day for total non-collagen proteins, indicating rapid turnover of both intracellular and extracellular proteins of this tissue.     

Scorbutic and fasted guinea pig sera contain an insulin-like growth factor I-reversible inhibitor of proteoglycan and collagen synthesis in chick embryo chondrocytes and adult human skin fibroblasts

Chick embryo chondrocytes cultured in sera from scorbutic and fasted guinea pigs exhibited decreases in collagen and proteoglycan production to about 30-50% of control values (I. Oyamada et al., 1988, Biochem. Biophys. Res. Commun. 152, 1490-1496). Here we show by pulse-chase labeling experiments that in the chondrocyte system, as in the cartilage of scorbutic and fasted guinea pigs, decreased incorporation of precursor into collagen was due to decreased synthesis rather than to increased degradation. There was a concomitant decrease in type II procollagen mRNA to about 32% of the control level. As in scorbutic cartilage, proteoglycan synthesis by chondrocytes in scorbutic serum was blocked at the stage of glycosaminoglycan chain initiation. Scorbutic and fasted guinea pig sera also caused a 50-60% decrease in the rates of collagen and proteoglycan synthesis in adult human skin fibroblasts, which synthesize mainly type I collagen. Decreased matrix synthesis in both cell types resulted from the presence of an inhibitor in scorbutic and fasted sera. Elevated cortisol levels in these sera were not responsible for inhibition, as determined by the addition of dexamethasone to chondrocytes cultured in normal serum. Insulin-like growth factor I (IGF-I, 300-350 ng/ml) reversed the inhibition of extracellular matrix synthesis by scorbutic and fasted guinea pig sera in both cell types and prevented the decrease in type II procollagen mRNA in chondrocytes. Therefore, in addition to its established role in proteoglycan metabolism, IGF-I also regulates the synthesis of several collagen types. An increase in the circulating inhibitor of IGF-I action thus could lead to the negative regulation of collagen and cartilage proteoglycan synthesis that occurs in ascorbate-deficient and fasted guinea pigs.     

Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth muscle cells in culture.

Rates of collagen and non-collagen protein synthesis in rabbit arterial smooth muscle cells (SMC) were determined by using the specific (radio)activity of [3H]proline in the extracellular, intracellular, and prolyl-tRNA pools. The intracellular free proline specific activity was only 25% of the extracellular value in cultures incubated for 12 h in 0.25 mM-proline. The specific activity of prolyl-tRNA was less than 10% of the extracellular specific activity. Increasing the extracellular proline concentration 10-fold (to 2.5 mM), while keeping the extracellular specific activity of proline constant, resulted in equilibration of the specific activities of intracellular and extracellular free proline, but the specific activity of prolyl-tRNA remained at less than 10% of the extracellular specific activity. Therefore, calculated rates of collagen and non-collagen protein synthesis were greatly underestimated using the intracellular or extracellular specific activity of proline. SMC were also incubated with 0.1 mM-[14C]ornithine in 0.25 nM or 2.5 mM non-labelled proline to examine synthesis de novo of proline and prolyl-tRNA from ornithine. In SMC cultures containing 0.25 mM unlabelled proline, the specific activity of intracellular ornithine was approx. 45% of the extracellular specific activity, due to the production of unlabelled ornithine. The specific activity of ornithine-derived intracellular free proline in SMC incubated with 2.5 mM-proline was significantly lower than in SMC incubated in 0.25 mM-proline, due to the influx of unlabelled proline. However, a corresponding difference in the specific activity of [14C]prolyl-tRNA between SMC in 0.25 mM- or 2.5 mM-proline was not observed. Ornithine-derived [14C]proline was incorporated into proteins in a manner different from that of exogenously added radiolabelled proline. A much higher proportion of the proline synthesized de novo was channelled into collagen synthesis relative to total protein synthesis. Together, these results show that intracellular proline pools are highly compartmentalized in arterial SMC. They also suggest that proline synthesized from ornithine may enter a prolyl-tRNA pool separate from that of proline entering from the extracellular medium.     

Complement component C1q is sensitive to acute vitamin C defficiency in guinea pigs

In acutely scorbutic guinea pigs, where interstitial collagen synthesis is markedly impaired, there was no significant reduction in total complement component C1 activity measured by a functional assay, and no significant reduction in the ratio of protein-bound hydroxyproline to protein-bound proline or to total serum protein, in comparison with pair-fed controls. There was a moderate increase in non-protein-bound hydroxyproline in the serum of the deficient animals.These result suggest that component C1q is largely resistant to the effects of severe acute scurvy, adn that some hydroxyproline-containing proteins may respond differently others, during vitamin C deficiency.     

Influence of ascorbic acid on ribosomal patterns and collagen biosynthesis in healing wounds of scorbutic guinea pigs

Scorbutic guinea pigs were wounded and the influence of administering ascorbic acid 6 days later was studied with respect to cellular morphology, ribosomal distribution and protein synthesis. Electron-microscopic studies revealed that the dilated endoplasmic reticulum observed in the fibroblasts of scorbutic wound tissue had reverted to a normal configuration 24h after intraperitoneal injection of 100mg of ascorbate. Quantitative determination of the distribution of free and membrane-bound ribosomes indicated a significant increase in membrane-bound ribosomes in wound tissue from ascorbate-supplemented (recovery) animals. Sucrose-density-gradient centrifugation indicated a significant increase in the proportion of large membrane-bound polyribosomes in the range 300-350S and a concomitant decrease in 80S monoribosomes in the ribosome sedimentation profile of recovery tissue. Determination of the synthesis of non-diffusible [(3)H]hydroxyproline in scorbutic and recovery wounds showed a 3-4-fold stimulation in peptidyl-proline hydroxylation in recovery tissues. Studies carried out in which scorbutic and recovery tissues were incubated with [(14)C]leucine indicated that general protein synthesis, as measured by (14)C incorporated into non-diffusible material/mug of DNA, was unaltered by ascorbate supplementation. Similar studies of [(3)H]proline incorporation suggested that in recovery tissues there was a small but significant increase in [(3)H]proline incorporated/mug of DNA, which probably represents an increase in protocollagen synthesis. This observation correlates well with the increase seen in recovery tissues of large polyribosomes on which collagen precursor polypeptides are known to be synthesized. Preliminary characterization of the repair collagen synthesized by recovery animals showed it to be a typical Type I collagen having the chain composition (alpha(1))(2)alpha(2). The extent of glycosylation of the hydroxylysine of the newly synthesized collagen was greater than that reported for either normal guinea-pig dermal collagen or dermal scar collagen.     

Longitudinal gradients of collagen and elastin gene expression in the porcine aorta

The physical and chemical properties of the mammalian aorta are known to vary as a function of distance from the heart. These properties are highly dependent collagen and elastic fibers. In order to evaluate the mechanisms which regulate the accumulation of these two connective tissue proteins, gene expression was evaluated at both the biosynthetic and messenger RNA levels. Short-term (3 h) explant cultures of the medial portion of four segments of the descending aorta in newborn pigs were incubated in the presence of [3H] proline. Collagen production was quantified by collagenase digestion and elastin production was determined by immunoprecipitation. Between the conus arteriosus and the bifurcation of the iliac arteries, relative collagen synthesis increased 2-fold (from 5.8 to 12.0% of total protein synthesis), while relative elastin synthesis declined 10-fold (from 16.4 to 1.6% of total protein synthesis). Similarly, collagen production increased more than 7-fold (from 6.7 to 49.8 X 10(3) molecules/cell/h) while elastin production was reduced more than 3-fold (from 71.8 to 21.0 X 10(3) molecules/cell/h) along this developmental gradient. Elastin synthesis appeared to be controlled to a significant extent by the availability of elastin mRNA, since both cell-free translation and molecular hybridization to a cloned elastin gene probe showed gradients of elastin gene expression. Similarly, collagen synthesis was apparently regulated, at least in part, by an inverse gradient of collagen mRNA, as measured with a cloned cDNA for the pro-alpha 1(I) collagen gene. Marked changes in the amount of non-elastin protein synthesis accompanied differentiation and accounted for larger changes in relative synthesis. These results suggest that the phenotype of the cells of the porcine artery wall is distinct in different regions of this organ at this developmental stage.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

cAMP Level Modulates Scleral Collagen Remodeling,a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.     

Evidence for pretranslational regulation of collagen synthesis by procollagen propeptides

We present, here, evidence for a pretranslational role of procollagen propeptides in the regulation of collagen synthesis. Amino- and carboxyl-terminal type I procollagen propeptides were isolated and purified from chick calvaria and tendon cultures. Human lung fibroblasts (IMR-90) were incubated in medium containing varying concentrations of propeptides. Amino-propeptides at 10 nM caused an 80% decrease in collagen synthesis compared to control. Higher concentrations of amino-propeptides did not decrease collagen synthesis further and no significant effect on non-collagen synthesis was found throughout the entire concentration range. Carboxyl-propeptides also inhibited collagen synthesis. At 10 nM, collagen synthesis was decreased by 30% and a concentration of 40 nM caused an 80% reduction. However, at the latter concentration non-collagen synthesis was also affected, decreasing by 20% relative to control. To assess possible pretranslational effects of propeptides, IMR-90 fibroblasts were treated with varying concentrations of each propeptide and levels of type I procollagen mRNA was determined by dot hybridization with a 32P-alpha 2(I) cDNA probe. Both propeptides caused significant concentration-dependent decreases in procollagen type I mRNA levels. At 10 nM, the amino-propeptide resulted in a 55% decrease in collagen mRNA levels while at 40 nM these levels decreased by 72% compared to control. Carboxyl-propeptides were also inhibitory, decreasing mRNA levels by 33% at 10 nM and 73% at 40 nM. Messenger RNA levels of a representative noncollagenous protein, beta-actin, were unaffected by either propeptide throughout the concentration range.     

Type X collagen gene expression is transiently up-regulated by retinoic acid treatment in chick chondrocyte cultures

During endochondral ossification, resting and proliferating chondrocytes mature into hypertrophic chondrocytes that initiate synthesis of type X collagen. The mechanisms regulating the differential expression of type X collagen gene were examined in confluent Day 12 secondary cultures of chick vertebral chondrocytes in monolayer treated with the vitamin A analog retinoic acid (RA). Preliminary results showed that major effects of RA on chondrocyte gene expression occurred between 24 and 48 h of treatment. Thus in subsequent experiments cultures were treated for 24, 30, 36, 42, 48, 72, 96, and 120 h. Total RNAs were isolated and analyzed by hybridization with ³²P-labeled plasmid probes coding for five matrix macromolecules including type X collagen. We found that the steady-state levels of mRNAs for the large keratan sulfate/chondroitin sulfate proteoglycan (KS:CS-PG) core protein and type II collagen decreased several fold between 24 and 48 h of treatment compared to untreated cells, and remained low with further treatment. In sharp contrast, the level of type X collagen mRNA increased threefold by 42 h of treatment; thereafter it began to decrease and reached minimal levels by 72–120 h of treatment. The changes in steady-state mRNA levels during RA regimen paralleled similar changes in relative rates of protein synthesis. The transient up-regulation of type X collagen gene expression at 42 h of treatment was preceded by a five-fold increase in fibronectin gene expression, was followed by a several fold increase in type I collagen gene expression, and was accompanied by cell flattening and loss of the pericellular proteoglycan matrix. Thus, RA treatment leads to a unique biphasic modulation of type X collagen gene expression in maturing chondrocyte cultures. The underlying, RA-sensitive mechanisms effecting this modulation may reflect those normally regulating the differential expression of this collagen gene during endochondral ossification.     

Collagen synthesis by monkey arterial smooth muscle cells during proliferation and quiescence in culture

Monkey arterial smooth muscle cells (SMC) which are stimulated to proliferate in the presence of 5% monkey blood serum (MBS) and which remain quiescent in 5% monkey platelet-poor plasma serum (MPPPS) were examined for their ability to synthesize collagen in each of these conditions in culture. Collagen synthesis was measured by determining amounts of newly formed labeled hydroxyproline, following labelling in the presence of [³H]proline and ascorbic acid. Ascorbate requirements of SMC were examined to assure maximal hydroxylation. SMC synthesize the same amount of collagen/cell in 5% whole blood serum (MBS) during the early phase of rapid proliferation as during slow growth in later phases in culture. SMC grown in the presence of serum-lacking platelet factors synthesize 60–90% less collagen and 60–90% less non-collagen protein (per cell or per mg protein) than cells grown in MBS. Non-collagen protein synthesis was measured as incorporation of both [³H]proline and of [³H]leucine, determined as trichloroacetic acid (TCA)-precipitable material. Previous studies indicate that a factor derived from platelets is the principal mitogen present in whole blood serum for diploid cells such as SMC and fibroblasts in culture. Similarly derived factors are potent stimulators of both collagen and non-collagen protein synthesis by SMC. SMC, quiescent in medium lacking platelet derived material (MPPPS), is being used to investigate factors important in SMC proliferation since this is a significant event in atherogenesis in vivo. An increased deposition of collagen also occurs during atherogenesis. Consequently it will be useful to employ similar cultures of quiescent SMC to examine agents which affect production of this connective tissue matrix protein.     

Collagen degradation in human lung fibroblasts: extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis

Experiments were conducted to determine the extent and variability of collagen degradation in human fetal lung fibroblasts. Cells were incubated with [14C]proline, and degradation was measured by determining the hydroxy[14C]proline in a low molecular weight fraction relative to total hydroxy[14C]proline. Average (basal) degradation in stationary phase HFL-1 cells incubated for 8 h was 16 +/- 3%, and substantial alterations in the composition of the labeling medium, e.g., omitting serum and varying pH between 6.8 and 7.8, had no effect. Organic buffers slightly lowered degradation in a manner that was independent of pH. Collagen degradation in two other lung cell lines, Wl-38 and lMR-90, did not differ from the level in HFL-1. Degradation was significantly higher (23 +/- 5%) in HFL-1 cultures labeled for 24 h rather than 8 h, and pulse-washout experiments showed that the rate of degradation was not uniform: after an 8-h pulse, 11% of the hydroxy [14C]proline in the medium was in the low molecular weight fraction, but 31% was in this fraction after a 16-h washout. The lack of effect of either serum deprivation or elevated pH suggests that lysosomal proteases have no direct role in basal degradation; however, NH4Cl decreased the enhanced degradation observed in ascorbate deficiency to basal level, indicating that abnormal molecules synthesized under those conditions are degraded by lysosomal proteases. The appearance of small hydroxy[14C]proline-containing molecules was inhibited by alpha alpha'dipyridyl and cycloheximide in a dose-dependent and reversible manner, demonstrating that their production depends on enzymatic hydroxylation of proline and protein synthesis.     

Changes of glucose metabolism and skin-collagen neogenesis in vitamin B6 deficiency

The mechanism of pellagrous changes in skin caused by a deficiency of vitamin B6 was studied in respect to neogenesis of proline in skin collagen and glucose metabolism. In vitamin B6 deficiency the insulin/glucagon coefficient in serum decreased significantly from 3.02 to 2.32, indicating a metabolic change towards gluconeogenesis. A deficiency of vitamin B6 caused a decrease in the levels of vitamin B6-dependent enzymes, such as ornithine aminotransferase, alanine aminotransferase, and aspartate aminotransferase, which also contribute to gluconeogenesis. Because the conversion of ornithine to proline via pyrroline-5-carboxylate was suppressed due to the decrease in ornithine aminotransferase activity, the amount of proline in the skin collagen fraction also decreased significantly in vitamin B6-deficient rats compared with the pair-fed control. These results suggest that the pellagrous lesions in vitamin B6-deficiency are caused by an impaired synthesis of proline from ornithine, which results in the suppression of collagen neogenesis in the skin.     

Extrahepatic synthesis of apolipoprotein E

Apolipoprotein E (apoE) synthesis has been examined in rat and guinea pig tissues using in vitro translation and [35S]methionine labeling of tissue slices. A number of tissues not involved in lipoprotein synthesis synthesize a protein very similar to apoE, including the spleen, adrenal, kidney, testis, ovary, heart, and lung. Although the intestine is involved in lipoprotein synthesis, apoE synthesis could not be detected in intestinal mucosa. The protein synthesized by the extrahepatic tissues was identified as apoE by its electrophoretic mobility, its immunologic reactivity with a monospecific antibody and by limited proteolysis mapping with Staphylococcus aureus V8 protease. ApoE represented between 0.02 and 0.7% of the total protein synthesized in the extrahepatic tissues, indicating that apoE mRNA is a fairly abundant mRNA in these tissues. ApoE mRNA was also detected by hybridization with a rat apoE cDNA clone, which hybridized to a single mRNA 1250 nucleotides in length in rat liver and in extrahepatic tissues. Hybridization of the apoE clone to rat genomic DNA demonstrated that the apoE gene was more heavily methylated in intestinal mucosa, which did not synthesize apoE, than in liver, testis, or kidney. 35S labeling of peritoneal macrophages revealed that both rat and guinea pig macrophages synthesized and secreted apoE in vitro. Rhesus aortic smooth muscle cells also synthesized and secreted apoE. The possible functions of apoE synthesized in the peripheral tissues are considered.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.