Characterization of type I collagen from the skin of blue grenadier (Macruronus novaezelandiae)

The skin collagen of a fish, blue grenadier (Macruronus novaezelandiae), has been purified and characterized. The fish skin was readily soluble in dilute acetic acid, with no pepsin treatment needed. The collagen was purified by salt precipitation. Skin samples from fish of various ages showed that even in the oldest sample, more than 8 years of age, the collagen was still readily acid soluble. The purified collagen had a melting temperature of 22 degrees C; the shrinkage temperature for the skin was 48 degrees C. Its tissue distribution, examined by immunohistology, and its chemical properties indicated a close homology to mammalian type I collagen. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that three distinct alpha-chains were present. These were purified by ion-exchange chromatography on CM-cellulose and by gel permeation chromatography on Superose 6. The three purified alpha-chain fractions were examined by amino acid analysis and by SDS-PAGE of their cyanogen bromide fragments. These data indicated that the additional chain was genetically distinct, and most closely related to the alpha 1-chain, from which it was poorly resolved on SDS-PAGE.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.     

Characterization of the skin collagen of a surface water fish Scomberoides commersonianus.

The molecular organization of the skin collagen in the fish S. commersonianus has been investigated. The contents of imino acids proline and hydroxyproline are less in the skin collagen. The major collagenous component of fish skin is homologous to type I collagen. The number of CNBr peptides in fish skin collagen alpha 1 chains is two times that of rat skin alpha 1 CNBr peptides. The proline-hydroxyproline ratio in the six peptides studied was 1.66-3.5 as compared to that of rat skin collagen (0.67-1.94). This indicates that proline and hydroxyproline are not uniformly distributed in the collagen molecule in fish skin collagen.     

Collagen from diamondback squid (Thysanoteuthis rhombus) outer skin

Collagens (acid-solubilized and pepsin-solubilized collagens) were prepared from diamondback squid outer skin and partially characterized. The yields of acid-solubilized and pepsin-solubilized collagens were about 1.3 and 35.6%, respectively, on a dry weight basis. Pepsin-solubilized collagen was heterotrimer with a chain composition of ala2a3. The patterns of peptide fragments were different from that of porcine skin collagen. Denaturation temperature was 27.5 degrees C, about 10 degrees C lower than that of porcine collagen. The amino acid composition of pepsin-solubilized collagen from diamondback squid outer skin was similar to that from cuttlefish outer skin. This squid is big among squid species, and its skin is thick. It is clear that diamondback squid outer skin has a potential as an alternative source of collagen to bovine skin and bone. At present, collagen using aquatic materials such as skin (cod and a deep-sea fish) and scale (sea bream and anchovy) is the development stage in the related industries. Unless the problem of BSE infection in land animals is resolved aquatic materials as an alternative source of collagen will attract much attention in the cosmetic and medical fields.     

Complete primary structure of rainbow trout type I collagen consisting of alpha1(I)alpha2(I)alpha3(I) heterotrimers.

The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be alpha1(I)alpha2(I)alpha3(I) and [alpha1(I)](2)alpha2(I), respectively. The occurrence of alpha3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP-13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 degrees C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N-terminal and/or a part of triple-helical domains of proalpha(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple-helical domain of each proalpha(I) chain had 338 uninterrupted Gly-X-Y triplets consisting of 1014 amino acids and was unique in its high content of Gly-Gly doublets. In particular, the bonyfish-specific alpha(I) chain, proalpha3(I) was characterized by the small number of Gly-Pro-Pro triplets, 19, and the large number of Gly-Gly doublets, 38, in the triple-helical domain, compared to 23 and 22, respectively, for proalpha1(I). The small number of Gly-Pro-Pro and the large number of Gly-Gly in proalpha3(I) was assumed to partially loosen the triple-helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proalpha3(I) had diverged from proalpha1(I). This study is the first report of the complete primary structure of fish type I procollagen.     

Glucosylation of galactosylhydroxylysyl residues in collagen in vitro by collagen glucosyltransferase. Inhibition by triple-helical conformation of the substrate.

Glucosylation of galactosylhydroxylysyl residues in various collagen polypeptide chains and in small peptides prepared from collagen was studied in vitro using collagen glucosyltransferase purified about 200 to 500-fold from extract prepared from chick embryos. When various denatured polypeptide or peptide chains were compared as substrates for the enzyme, no significant differences were found between citrate-soluble collagens from normal or lathyritic rats and isolated alpha1 and alpha2 chains. In contrast, gelatinized insoluble calf skin collagen, and peptides prepared from collagen and having an average molecular weight of about 500 were clearly less effective substrates as judged from their Km and V values. A marked difference was found between native and heat-denatured citrate-soluble collagen in that no synthesis of glucosylgalactosylhydroxylysine was observed with the native collagen when the reaction was studied at 30 degrees C with different times, enzyme concentrations, and substrate concentrations. When the reaction was studied as a function of temperature, little glucosylation of native collagen was observed below 37 degrees C, but there was a sharp transition in the rate of glucosylation of native collagen at temperatures above 37 degrees C, similar to that observable in the melting curve of collagen. The data suggest that triple-helical conformation of collagen prevents that glucosylation of galactosylhydroxylysyl residues.     

The composition and characteristics of skin and muscle collagens from a freshwater catfish grown in biologically treated tannery effuent water

The skin of catfish Gariepinus spp. reared in tannery effuent water (ETP) had an increased collagen content, decreased acid solubility and high carbohydrate association as compared with skin of normal fish. The skin collagen in either fish was composed of three distinct α chains with mobilities different from that of vertebrate type I α chains and two of these three chains were invariably present in the muscle collagen. The amino acid composition of ETP fish skin and muscle collagens were similar but were characterized by higher degrees of proline and lysine hydroxylation, indicating higher stability. Both the skin and muscle collagens had significantly high denaturation temperatures. Electron microscopy of in vitro reconstituted fibrils of skin and muscle collagens of ETP fish displayed defined periodicities of around 64 nm. This demonstrates that the polymorphism in skin collagen chains is not confined to marine fish. There was a close similarity in the composition of skin and muscle collagens and the ETP environment influences the solubility and stability of the skin collagen. It is hypothesized that the third chain may play a role in the anchorage of skin to muscle, as such chain composition is now evident in fish such as hake, cod and catfish where the musculature is strongly attached to the skin.     

Purification, characterization and inhibition of human skin collagenase

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32mug of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5-8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25 degrees C, producing the two characteristic products TC(A)((3/4)) and TC(B)((1/4)). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25 degrees C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37 degrees C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the alpha-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37 degrees C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins alpha(2)-macroglobulin and beta(1)-anti-collagenase both inhibited the enzyme, but alpha(1)-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.     

Biochemical properties of alligator (Alligator mississippiensis) bone collagen

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) isolated and purified from alligator (Alligator mississippiensis) bone were studied for molecular size, amino acid profile, secondary structure, and denaturation temperature by SDS-PAGE, HPLC, circular dichroism, and viscometry. Two collagen subunits, alpha1 and alpha2 were identified by SDS-PAGE. The molecular masses for alpha1 and alpha2 chains of ASC were 124 kDa and 111 kDa, respectively. The molecular masses were 123 kDa for alpha1 and 110 kDa for alpha2 chains of the PSC preparation. The molecular masses for ([alpha1](2) alpha2) of ASC and PSC were 359 kDa and 356 kDa, respectively. The major composition of alligator bone ASC and PSC was found to be typical type I collagen. The amino acid profiles of alligator ASC and PSC were similar to amino acid profile of subtropical fish black drum (Pogonias cromis, Sciaenidae) bone. Comparison of amino acid profiles with shark cartilage PSC, showed differences in alanine, hydroxylysine, lysine, and histidine contents. The denaturation temperatures (T(d)) of alligator ASC and PSC collagen measured by viscometry were 38.1 and 38.2 degrees C, respectively. Thermal denaturation temperatures, measured by melting point using circular dichroism, were 37.6 and 37.9 degrees C, respectively. Taken together, these results suggest that alligator bone collagen may find a wide range of applications in biological research, functional foods and nutraceuticals, and biomedical and pharmaceutical research.     

Heterogeneity of collagen molecules types I and II according to their resistance to proteolysis

Study of the effects of pepsin treatment on soluble collagens type I of the skin and collagens type II of the costal cartilage of healthy subjects revealed the presence of two classes of molecules differing in the stability of their three-helical structure. In collagen molecules possessing a low stability (their number may amount to 20-30%) within the temperature range of 4-30 degrees C pepsin causes a split-off of N-terminal sites with the formation of short chains, i.e., alpha 1(I), alpha 2(II), and alpha 1(II), whereas at higher temperatures (33 degrees C for collagens type I and 37 degrees C for collagens type II) a complete degradation of these molecules takes place. It was found that collagens types I and II molecules contain a high number of three-helical sites with a high susceptibility to pepsin. The putative functional role of structural heterogeneity of collagen molecules is discussed.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

The effects of different thawing temperatures on morphology and collagen metabolism of -20 degrees C dealt normal human fibroblast

The purpose of present study is to investigate the effects of two different thawing temperatures on normal human fibroblast which dealt with -20 degrees C, hoping to provide a clue for further study in reducing excessive collagen formation after cryotherapy on skin diseases in vitro, as well as in differentiation disorders. In order to elucidate its action mechanism, a programmable freezing device was developed to apply freezing temperatures on cell cultures. The effects of two different thawing temperatures on frozen fibroblast proliferation, viability, collagen synthesis and alpha smooth muscle actin (alpha-SMA) expressing were investigated. We found that compared with 37 degrees C, thawing with 20 degrees C yielded same motility. But there are significant differences in terms of the alpha-SMA expression (P<0.05) of fibroblast and collagen I, III synthesis (P<0.01) between two groups after 72h. The results suggest that comparing with slow thawing; rapid thawing cannot only keep the same cell's damage, but also can modify collagen synthesis and differentiation of fibroblasts. It may be more suitable for the cryosurgical treatment of keloids and benign skin diseases.     

Studies on type I collagen in skin fibroblasts cultured from twins with lethal osteogenesis imperfecta

Studies on type I procollagen produced by skin fibroblasts cultured from twins with lethal type II of osteogenesis imperfecta (OI) showed that biosynthesis of collagen (measured by L-[5-(3)H]proline incorporation into proteins susceptible to the action of bacterial collagenase) was slightly increased as compared to the control healthy infant. SDS/PAGE showed that the fibroblasts synthesized and secreted only normal type I procollagen. Electrophoretic analysis of collagen chains and CNBr peptides showed the same pattern of electrophoretic migration as in the controls. The lack of posttranslational overmodification of the collagen molecule suggested a molecular defect near the amino terminus of the collagen helix. Digestion of OI type I collagen with trypsin at 30 degrees C for 5 min generated a shorter than normal alpha2 chain which melted at 36 degrees C. Direct sequencing of an asymmetric PCR product revealed a heterozygous single nucleotide change C-->G causing a substitution of histidine by aspartic acid in the alpha2 chain at position 92. Pericellular processing of type I procollagen by the twin's fibroblasts yielded a later appearance of the intermediate pC-alpha1(I) form as compared with control cells.     

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.     

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.     

Conformational analysis and stability of collagen peptides by CD and by 1H- and 13C-NMR spectroscopies

Four small type I collagen CNBr peptides containing complete natural sequences were purified from bovine skin and investigated by CD and 1H- and 13C-nmr spectroscopies to obtain information concerning their conformation and thermal stability. CD showed that a triple helix was formed at 10 degrees C in acidic aqueous solution by peptide alpha l(I) CB2 only, and to lesser extent, by alpha 1(I) CB4, whereas peptides alpha 1(I) CB5 and alpha 2(I) CB2 remained unstructured. Analytical gel filtration confirmed that peptides alpha 1(I) CB2 and alpha 1(I) CB4 only were able to form trimeric species at temperature between 14 and 20 degrees C, and indicated that the monomer = trimer equilibrium was influenced by the chaotropic nature of the salt present in the eluent, by its concentration, and by temperature variations. CD measurements at increasing temperatures showed that alpha 1(I) CB2 was less stable than its synthetic counterpart due to incomplete prolyl hydroxylation of the preparation from the natural source. 1H- and 13C-nmr spectra acquired in the temperature range 0-47 and 0-27 degrees C, respectively, indicated that with decreasing temperature the most abundant from of alpha 1(I) CB2 was in slow exchange with an assembled form, characterized by broad lines, as expected for the triple-helical conformation. A large number of trimer cross peaks was observed both in the proton and carbon spectra, and these were most likely due to the nonequivalence of the environments of the three chains in the triple helix. This nonequivalence may have implications for the aggregation of collagen molecules and for collagen binding to other molecules. The thermal transition from trimer to monomer was also monitored by 1H-nmr following the change in area of the signal belonging to one of the two beta protons of the C-terminal homoserine. The unfolding process was found to be fully reversible with a melting temperature of 13.4 degrees C, in agreement with CD results. The qualitative superposition of the melting curves obtained by CD for the peptide bond characteristics and by nmr for a side chain suggests that triple-helical backbone and side chains constitute a single unit.     

Does pulsatile urea excretion serve as a social signal in the gulf toadfish Opsanus beta?

This study evaluated the hypothesis that the pulsatile excretion of urea by toadfish could serve as a social signal. In the first experiment, physiological parameters were measured in pairs of dominant and subordinate toadfish. Subordinate toadfish had elevated concentrations of circulating plasma cortisol, an effect maintained even after cannulation. In the second experiment, one fish of a pair was injected with 14C-urea, and the occurrence of urea pulses during social encounters was documented. Social status did not influence the order of pulsing, that is, whether a dominant or subordinate fish pulsed first during a social encounter. However, in seven out of eight pairs, both toadfish pulsed within 2 h of each other, indicating some form of communication between fish. In the third and final experiment, the response of toadfish to urea (natural or synthetic) was observed. There was a tendency for toadfish to avoid synthetic urea but there was no apparent behavioural response to water containing toadfish urea. Pulsing events do not appear to play an integral role during social encounters as previously hypothesised, but the close timing of pulses in toadfish pairs suggests some transfer of information.     

A novel Gly to Arg substitution at position 388 of the alpha1 chain of type I collagen in lethal form of osteogenesis imperfecta

Cultured skin fibroblasts from a proband with a lethal form of osteogenesis imperfecta produce two forms of type I collagen chains, with normal and delayed electrophoretic migration; collagen of the proband's mother was normal. Peptide mapping experiments localized the structural defect in the proband to alpha1(I) CB8 peptide in which residues 123 to 402 are spaned. Direct sequencing of amplified cDNA covering this region revealed a G to A single base change in one allele of the alpha1(I) chain, that converted glycine 388 to arginine. Restriction enzyme digestion of the RT-PCR product was consistent with a heterozygous COL1A1 mutation. The novel mutation conforms to the linear gradient of clinical severity for the alpha1(I) chain and results in reduced thermal stability by 3 degrees C and intracellular retention of abnormal molecules.     

Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction.

The denatured alpha1(I) chain and the cyanogen bromide peptide, alpha1(I)-CB5, of chick skin collagen cause the relaese of serotonin and leakage of lactic dehydrogenase from human platelets in a manner similar to the release reaction mediated by adenosine diphosphate and native collagen. These peptides also cause a decrease in the level of adenosine 3':5'-monophosphate (cAMP) in platelets. Adenylate cyclase activity of platelets is partially inhibited by these peptides as well as by native collagen, ADP, and epinephrine, but cAMP phosphodiesterase activity is unaltered by these substances. In contrast, the level of platelet guanosine 3':5'-monophosphate (cGMP) is increased by the collagen peptides as well as the other aggregating agents. The increase is associated with increased guanylate cyclase, but normal cGMP phosphodiesterase activities of platelets. Optical rotatory and viscometric measurements of the alpha1 chains and alpha1-CB5 of chick skin in 0.01 M phosphate/0.15 M sodium chloride, pH 7.4, at various temperatures as a function of time indicate that no detectable renaturation occurs at 37 degrees for at least 30 min of observation. Molecular sieve chromatography of alpha1-CB5 in the phosphate buffer at 37 degrees shows that its elution position is identical to that performed under denaturing conditions (at 45 degrees) with no evidence of higher molecular weight aggregates, and the alpha1-CB5 glycopeptide fraction eluting from the column at the position of its monomer retains the platelet aggregating activity. Additionally, electron microscopic examination of the platelet-rich plasma that had been reacted with these peptides fail to show any ordered collagen structures. These data indicate that the denatured alpha1 chain and alpha1-CB5 glycopeptide of chick skin collagen mediate platelet aggregation through the "physiologic" release reaction in a manner similar to that induced by other aggregating agents such as ADP, epinephrine, or native collagen, and support the conclusion that the aggregating activity of the alpha1 chain and alpha1-CB5 is not likely to be due to the formation of polymerized products.     

Proline hydroxylation of collagens synthesized at different temperatures in vivo by two poikilothermic species

Extent of prolyl hydroxylation in newly synthesized viper collagen is decreased at 10 degrees C to approximately 23% of normal on skin and to approximately 57% of normal in bone collagen. At 20 degrees C, prolyl hydroxylation is approximately 50% of normal in skin and normal in bone. At 10 degrees C and 20 degrees C, prolyl hydroxylation is decreased approximately 32% in the skin collagen of carp. In contrast, prolyl hydroxylation is unchanged at 10 and 20 degrees C in bone, scale and lepidotrichia. Prolyl hydroxylation of cartilaginous endoskeleton showed an approximately 25% decrease at 20 degrees C.