Quantitative and metabolic changes of hepatic collagens in rats after carbon tetrachloride poisoning.
ABSTRACT: 1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.
Project description:1. Collagenolytic activity towards acid-soluble collagen labelled with [(14)C]-proline was assayed in rat liver with and without carbon tetrachloride poisoning. The products of enzymic digestion were found to be free amino acids and peptides. 2. The hepatic collagenolytic activity increased under conditions of single-dose and subacute carbon tetrachloride poisoning, and correlated with hydroxyproline content. The highest activity was found during recovery from subacute poisoning. 3. Under the same experimental conditions, hepatic acid-proteinase activity changed independently of the collagenolytic activity and also of hepatic hydroxyproline content. 4. The increased collagenolytic activity during carbon tetrachloride poisoning was found mainly in the supernatant fraction. 5. The ratio of the collagenolytic activity to hepatic hydroxyproline content increased during recovery from single-dose and subacute poisoning, and decreased during subacute poisoning.
Project description:Thermal stabilities of mature insoluble collagen, salt-precipitated fibrils of acid-soluble collagen and acid-soluble collagen in solution were compared as a function of acid pH. Both insoluble and precipitated collagens showed large parallel destabilization with decrease in pH, whereas the intrinsic stability of individual collagen molecules in dilute solution was comparatively unaffected.
Project description:Rats of synthesis and degradation in vivo of collagens in 0.5 M-acetic acid-soluble and -insoluble extracts from skins of three growing rats were determined by using a labelling procedure involving exposure of the animals to an atmosphere of 18O2 for 36 h. For comparison, rats also received injections of [2H]proline. Serial skin biopsies were taken at frequent intervals over 392 days. Enrichment of 18O and 2H in the hydroxyproline of the collagen fractions was determined by gas chromatography-mass spectrometry. Changes in size of the soluble and insoluble collagen pools were considered in the evaluation of isotope kinetic data. The insoluble collagen fraction showed no degradation. The efflux (mean +/- S.D., expressed as mumol of hydroxyproline) from the soluble collagen pool was estimated to be 59.9 +/- 1.9 per day from the 18O data, and 25.5 +/- 7.5 per day from the 2H results. The finding indicates significant reutilization of 2H-radiolabelled proline for hydroxyproline synthesis. From these isotope data and estimates of size of the collagen pools it was determined that 55% of the collagen disappearing from the soluble pool was due to maturation into insoluble collagens and 45% of the disappearance was a result of actual degradation of soluble collagen. These results confirm the utility of 18O2 as a non-reutilizable label for studies of collagen turnover in vivo.
Project description:1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.
Project description:1. Collagen- and total-protein-synthesis rates were determined in rabbit muscle by continuous infusion of radioactive proline. 2. The precursor pool of free proline used for collagen synthesis was defined by measuring the specific radioactivity of hydroxy-proline in isolated type I procollagen. The specific radioactivities of type I procollagen were about 40% of those for free proline in the homogenate. 3. The mean ratio (+/- S.E.M.) between the fractional synthesis rates of muscle collagen and total protein was 0.99 +/- 0.10, where the total protein values were based on specific radioactivities of the homogenate free proline pools. 4. Types I, III and V collagen were solubilized by pepsin and isolated by fractional precipitation with NaCl. The fractional synthesis rates of types I and III collagens were very similar. Type V collagen samples had higher specific radioactivities than the other collagens, but this was not necessarily indicative of a higher rate of synthesis because of uncertainty about the cellular origin of this collagen and, hence, the specific radioactivity of its precursor proline pool.
Project description:The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice.Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction.Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated.TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.
Project description:Collagens are the most abundant proteins in marine animals and their degradation is important for the recycling of marine nitrogen. However, it is rather unclear how marine collagens are degraded because few marine collagenolytic proteases are studied in detail. Deseasins are a new type of multidomain subtilases. Here, the collagenolytic activity of deseasin MCP-01, the type example of deseasins, was studied. MCP-01 had broad substrate specificity to various type collagens from terrestrial and marine animals. It completely decomposed insoluble collagen into soluble peptides and amino acids, and was more prone to degrade marine collagen than terrestrial collagen. Thirty-seven cleavage sites of MCP-01 on bovine collagen chains were elucidated, showing the cleavage is various but specific. As the main extracellular cold-adapted protease from deep-sea bacterium Pseudoalteromonas sp. SM9913, MCP-01 displayed high activity at low temperature and alkaline range. Our data also showed that the C-terminal polycystic kidney disease (PKD) domain of MCP-01 was able to bind insoluble collagen and facilitate the insoluble collagen digestion by MCP-01. Site-directed mutagenesis demonstrated that Trp-36 of the PKD domain played a key role in its binding to insoluble collagen. It is the first time that the structure and function of a marine collagenolytic protease, deseasin MCP-01, has been studied in detail. Moreover, the PKD domain was experimentally proven to bind to insoluble protein for the first time. These results imply that MCP-01 would play an important role in the degradation of deep-sea sedimentary particulate organic nitrogen.
Project description:The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.
Project description:1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymal regions of bovine lungs were determined. The collagen content was approx. 70% (g/100g of salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleura and 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura and parenchyma of bovine lungs by various methods was attempted. The collagen fractions isolated after incubation of the pulmonary tissues with the proteolytic enzymes collagenase ("collagenase-soluble" fraction) or pancreatic elastase ("elastase-insoluble" fraction) each contained approx. 87% of the total collagen initially present. 3. Both collagen fractions were chemically analysed for their amino acid and carbohydrate contents and were found to be similar to those of the intact interstitial collagens isolated from skin, bone and tendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovine pulmonary collagen fractions, and were found to decrease with increasing age of the animals, and were similar to the values found in intact collagens from bone and tendon.
Project description:Synthesis of collagens in vitro was studied on minced mouse skins incubated with [3H]-proline in organ-culture conditions. A comparative study was carried out on genetically diabetic mice (KK strain) and control mice (Swiss strain). After incubation, neutral-salt-soluble and acid-soluble collagens were extracted. The insoluble dermis was digested by pepsin and type I and type III collagens separated by differential precipitation in neutral salt solutions. Type I and Type III collagens were characterized by ion-exchange and molecular-sieve chromatography, amino acid analysis and by the characterization of CNBr peptides. In diabetic-mouse skin, the relative proportion of type III collagen was significantly higher than in control-mouse skin. The incorporation of radioactively labelled proline into hydroxyproline of type III collagen was significantly faster in diabetic-mouse skin than in control-mouse skin. No significant modifications in the total collagen content of the skin or of their rates of synthesis were observed between the two strains. Alteration in the ratio of type III to type I collagen in the diabetic-mouse skin can be interpreted as a sign of alteration of the regulation of collagen biosynthesis and may be related to the structural alterations observed in the diabetic intercellular matrix.