RESUMO
Here we summarized what is known at the present about function, structure and effect of mutations in the human prolidase. Among the peptidases, prolidase is the only metalloenzyme that cleaves the iminodipeptides containing a proline or hydroxyproline residue at the C-terminal end. It is relevant in the latest stage of protein catabolism, particularly of those molecules rich in imino acids such as collagens, thus being involved in matrix remodelling. Beside its intracellular functions, prolidase has an antitoxic effect against some organophosphorus molecules, can be used in dietary industry as bitterness reducing agent and recently has been used as target enzyme for specific melanoma prodrug activation. Recombinant human prolidase was produced in prokaryotic and eukaryotic hosts with biochemical properties similar to the endogenous enzyme and represents a valid tool both to better understand the structure and biological function of the enzyme and to develop an enzyme replacement therapy for the prolidase deficiency (PD). Prolidase deficiency is a rare recessive disorder caused by mutations in the prolidase gene and characterized by severe skin lesions. Single amino acid substitutions, exon splicing, deletions and a duplication were described as causative for the disease and are mainly located at highly conserved amino acids in the sequence of prolidase from different species. The pathophysiology of PD is still poorly understood; we offer here a review of the molecular mechanisms so far hypothesized.
Assuntos
Dipeptidases/deficiência , Dipeptidases/genética , Dipeptidases/fisiologia , Mutação , Prolina/química , Sequência de Aminoácidos , Dipeptidases/química , Ativação Enzimática , Terapia Enzimática , Genótipo , Humanos , Metais/química , Dados de Sequência Molecular , Fenótipo , Pró-Fármacos , Estrutura Terciária de Proteína , Pyrococcus/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Prolidase deficiency (PD) is a rare autosomal recessive connective tissue disorder caused by mutations in the prolidase gene. The PD patients show a wide range of clinical outcomes characterised mainly by intractable skin ulcers, mental retardation and recurrent respiratory infections. Here we describe five different PEPD mutations in six European patients. We identified two new PEPD mutant alleles: a 13 bp duplication in exon 8, which is the first reported duplication in the prolidase gene and a point mutation resulting in a change in amino acid E412, a highly conserved residue among different species. The E412K substitution is responsible for the first reported phenotypic variability within a family with severe and asymptomatic outcomes.
Assuntos
Dipeptidases/deficiência , Dipeptidases/genética , Duplicação Gênica , Mutação/genética , Adulto , Sequência de Aminoácidos , Criança , Pré-Escolar , Doenças do Tecido Conjuntivo/enzimologia , Doenças do Tecido Conjuntivo/genética , Doenças do Tecido Conjuntivo/patologia , Análise Mutacional de DNA , Dinamarca , Saúde da Família , Feminino , Genótipo , Humanos , Deficiência Intelectual/patologia , Itália , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Linhagem , Fenótipo , Homologia de Sequência de Aminoácidos , Úlcera Cutânea/patologia , TurquiaRESUMO
As indices of triple helix stability of type I collagen CNBr peptide homotrimers, deltaG degrees for monomer-trimer transitions and melting temperatures were obtained from circular dichroism measurements at increasing temperatures. The data were compared with the stability of the parent native molecule. Peptides were found to have a lower stability than the whole collagen molecule. The general implication is that the coordinated water molecules play a key role in determining collagen triple helical stability and high cooperativity at melting. Other factors (monomer stability, ionic and hydrophobic factors, variations of composition, specific sequences) could also contribute towards peptide stability; these factors could explain the data obtained in the case of peptide alpha1(I) CB3.
Assuntos
Colágeno/química , Brometo de Cianogênio/química , Peptídeos/química , TermodinâmicaRESUMO
Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been associated with a family of chondrodysplasias that includes diastrophic dysplasia (DTD), atelosteogenesis type 2 (AO2) and the lethal condition achondrogenesis type 1B (ACG1B). There is a correlation between the nature of the mutations and the clinical phenotype, but our understanding of the pathophysiology of the disorder, which involves defective sulfation of cartilage proteoglycans, is far from complete. To evaluate the degree of proteoglycan undersulfation in vivo, we have extracted chondroitin sulfate proteoglycans from cartilage of twelve patients with sulfate transporter chondrodysplasias and analyzed their disaccharide composition by HPLC after digestion with chondroitinase ABC. The amount of non-sulfated disaccharide was elevated in patients' samples (controls, 5.5%+/-2.8 (n=10); patients, 11% to 77%), the highest amount being present in ACG1B patients, indicating that undersulfation of chondroitin sulfate proteoglycans occurs in cartilage in vivo and is correlated with the clinical severity. To investigate further the biochemical mechanisms responsible for the translation of genotype to phenotype, we have studied fibroblast cultures of patients with DTD, AO2 and ACG1B, and controls, by double-labelling with [35S]sulfate and [3H]glucosamine. The incorporation of extracellular sulfate, estimated by the 35S/3H ratio in proteoglycans, was reduced in all patients' cells, with ACG1B cells showing the lowest values. However, disaccharide analysis of chondroitin sulfate proteoglycans showed that these were normally sul fated or only moderately undersulfated; marked undersulfation was observed only after addition of the artificial glycosaminoglycan-chain initiator, beta-D-xyloside, to the culture medium. These results suggest that, while utilization of extracellular sulfate is impaired, fibroblasts can replenish their intracellular sulfate pool by oxidizing sulfur-containing compounds (such as cysteine) and thus partially rescue PG sulfation under basal conditions. This rescue pathway becomes insufficient when GAG synthesis rate is stimulated by beta-D-xyloside. These findings may explain why phenotypic consequences of DTDST mutations are restricted to cartilage, a tissue with high GAG synthesis rate and poor vascular supply, and imply that pharmacological therapy aimed at restoring the intracellular sulfate pool might improve PG sulfation in DTD and related disorders.
Assuntos
Proteínas de Transporte/metabolismo , Cartilagem/metabolismo , Exostose Múltipla Hereditária/metabolismo , Sulfatos/metabolismo , Proteínas de Transporte de Ânions , Transporte Biológico , Proteínas de Transporte/genética , Células Cultivadas , Criança , Exostose Múltipla Hereditária/genética , Feto/patologia , Humanos , Lactente , Recém-Nascido , Proteínas de Membrana Transportadoras , Mutação , Fenótipo , Transportadores de SulfatoRESUMO
The underlying defect in patients affected by a form of osteogenesis imperfecta (OI) clarified at the molecular level regards the amount or the structure of type I collagen synthesized. This leads to a decreased and/or abnormal mineral deposition in bone and affects bone mass and/or strength. Abnormal interactions between collagen molecules in the presence of mutant trimers could give rise to abnormal fibrils, which, in turn, can determine incorrect interactions with noncollagenous matrix macromolecules. The interactions can be disturbed or modulated by an abnormal distribution on the collagen fibril surface of electrically charged or hydrophobic groups, or by an increased presence of sugar moieties linked to hydroxylysyl residues due to chain post-translational overmodifications (lysyl overhydroxylation and hydroxylysyl overglycosylation) of the portion of the triple helical domain of abnormal type I collagen molecules N-terminal with respect to the defect localization.
Assuntos
Colágeno/metabolismo , Osteogênese Imperfeita/etiologia , Sequência de Aminoácidos , Colágeno/química , Glicosilação , Humanos , Dados de Sequência Molecular , Osteogênese Imperfeita/metabolismo , Conformação ProteicaRESUMO
We describe a girl with Marfan syndrome in whom the clinical expression of the disease was much more evident on the left side of the body.
Assuntos
Síndrome de Marfan/patologia , Pré-Escolar , Colágeno/metabolismo , Ectopia do Cristalino/genética , Feminino , Humanos , Deformidades Congênitas dos Membros , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Miopia/genética , Pele/metabolismoRESUMO
Serum levels of procollagen type III aminopropeptide (P-III-NP), a peptide released during conversion of type III procollagen to collagen, were abnormally low in six of 10 patients with Ehlers-Danlos syndrome (EDS) type IV (arterial-ecchymotic type) and low-normal in 4. The serum P-III-NP levels correlated with the amount of type III procollagen secreted by the patients' cultured fibroblasts. Serum P-III-NP determination is a simple test that identifies a major subgroup of patients with this life-threatening, dominantly inherited connective tissue disorder and may be especially helpful in making the diagnosis in children.
Assuntos
Síndrome de Ehlers-Danlos/sangue , Fragmentos de Peptídeos/sangue , Pró-Colágeno/sangue , Adolescente , Adulto , Células Cultivadas , Criança , Síndrome de Ehlers-Danlos/classificação , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/metabolismoRESUMO
We have characterized a familial form of osteogenesis imperfecta (OI). Following the identification by ultrasound of short limbs and multiple fractures in a fetus at 25 weeks of gestation, the family was referred with a provisional diagnosis of severe OI. We detected subtle clinical and radiological signs of OI in the father and in the paternal grandmother of the proposita, who had never received a diagnosis of OI. Linkage analysis indicated COL1A2 as the disease locus. Heteroduplex analysis of reverse transcription-polymerase chain reaction (RT-PCR) amplification products of pro alpha2(I) mRNA from an affected member and subsequent sequencing of the candidate region demonstrated the presence of normal transcripts and a minority of transcripts lacking exon 26 (54 bp) of COL1A2. Sequencing of PCR-amplified genomic DNA identified an A --> G transition in the moderately conserved +3 position of the IVS 26 donor splice site. The mutant pre-mRNA molecules were alternatively spliced, yielding both full-length and deleted transcripts that represented less than 30% of the total pro alpha2(I) mRNA. The biochemical data on type I collagen synthesized by dermal fibroblasts showed intracellular retention of the mutant protein; failure to detect the shortened alpha2(I) chains either in the medium or in the cell layer may be the consequence of their instability at physiological temperature. These observations justified the mild resulting phenotype.
Assuntos
Colágeno/genética , Mutação , Osteogênese Imperfeita/genética , Adulto , Processamento Alternativo , Sequência de Bases , DNA/genética , Análise Mutacional de DNA , Primers do DNA/genética , Éxons , Feminino , Humanos , Lactente , Masculino , Osteogênese Imperfeita/classificação , Osteogênese Imperfeita/diagnóstico , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal , RNA Mensageiro/genéticaRESUMO
In osteogenesis imperfecta (OI) the effects of mutations in type I collagen genes generally reflect their nature and localization. Unrelated individuals sharing identical mutations present, in general, similar clinical phenotypes. However, in some such cases the clinical phenotype differs. This variable clinical expression could be the result of abnormalities in other connective tissue proteins. Since decorin is a component of connective tissue, binds to type I collagen fibrils and plays a role in matrix assembly, we studied decorin production in skin fibroblasts from OI patients. Cultured fibroblasts from one patient with extremely severe osteogenesis imperfecta (classified as type II/III) who has an alpha 1(I)gly415ser mutation were found to secrete barely detectable amounts of decorin into culture medium. Western blotting using antibodies raised against decorin confirmed the reduction of the decorin core protein and Northern blot analysis showed decorin mRNA levels below the limit of detection. Cells from a patient, with a less severe phenotype, bearing a mutation in the same position of the triple helix (alpha 1(I)gly415) expressed decorin normally. The different clinical phenotypes could be due to the differing genetic backgrounds of the patients so it is tempting to conclude that in our most severely affected patient the absence of decorin aggravates the clinical phenotype.
Assuntos
Osteogênese Imperfeita/genética , Mutação Puntual , Proteoglicanas/genética , Northern Blotting , Western Blotting , Células Cultivadas , Decorina , Proteínas da Matriz Extracelular , Feminino , Fibroblastos/metabolismo , Genes Letais , Triagem de Portadores Genéticos , Glicina , Humanos , Recém-Nascido , Masculino , Osteogênese Imperfeita/classificação , Osteogênese Imperfeita/metabolismo , Fenótipo , Proteoglicanas/análise , Proteoglicanas/biossíntese , RNA Mensageiro/biossíntese , Serina , Pele/metabolismo , Pele/patologiaRESUMO
To reveal the biocompatibility of TiMo12Zr6Fe2 (TMZF), a new titanium alloy used since 1998 for orthopaedic prosthesis, we compared the behavior of primary human fibroblasts and osteoblasts grown on TMZF discs or on plastic tissue culture dishes, a widely used material specifically treated by the manufacturer to enhance cell growth. Proliferation, differentiation. RNA and collagen type I expression level of human cells were carried out. The analysis were performed over a period of 96 h. Fibroblasts behaved at the same way on the two different supports after 48 h, their number increased after 96 h when cells were grown on the alloy. Osteoblasts adhered and proliferated on the alloy discs as well as on plastic. RNA expression level was not affected. Interestingly, cell number at each time point was higher for fibroblasts than for osteoblasts. The RNA expression level was higher for the osteoblasts. Both cell types cultured on the alloy revealed an increase in the amount of type I collagen and a similar electrophoretic pattern was found for collagen produced by fibroblasts and osteoblasts grown on either supports. These results indicate good biocompatibility of the TMZF alloy, which allowed adhesion and proliferation of both the examined cell types and suggest that TMZF is a promising material for orthopaedic implants.
Assuntos
Ligas/metabolismo , Materiais Biocompatíveis/metabolismo , Fibroblastos/fisiologia , Osteoblastos/fisiologia , Próteses e Implantes , Adesão Celular , Divisão Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Ferro/metabolismo , Teste de Materiais , Molibdênio/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fenótipo , RNA/metabolismo , Titânio/metabolismo , Zircônio/metabolismoRESUMO
Prolidase deficiency occurred in two sisters suffering from recurrent leg ulcers that appeared in early childhood. The patients presented the typical clinical symptoms of the disease, including characteristic facies, dermatologic manifestations of the lower extremities, splenomegaly, and hematologic anomalies. Large amounts of iminodipeptides were excreted into the urine, and prolidase activity in their erythrocytes was virtually absent. Changes associated with a connective-tissue disorder were demonstrated by light and electron microscopic studies of the patients' apparently normal skin. Collagen fibers were smaller than in controls and were irregularly packed; the fibrils had normal aspect but were significantly smaller than in one age-matched control. Elastin fibers appeared altered both in size and structure.
Assuntos
Dipeptidases/deficiência , Úlcera da Perna/genética , Adulto , Doença Crônica , Colágeno , Elastina , Eritrócitos/metabolismo , Feminino , Humanos , Hidroxiprolina/urina , Úlcera da Perna/metabolismo , Úlcera da Perna/patologia , Pele/patologia , Pele/ultraestruturaRESUMO
The use of capillary zone electrophoresis as an efficient method for the identification of urinary imidodipeptides of prolidase-deficient patients has already been reported. However, owing to the complexity of the components excreted, the resolution of electrophoretic patterns obtained was poor. Here we examine the use of micellar electrokinetic chromatography to enhance peak resolution in order to obtain better insight into the electropherograms of patients' urine. The usefulness of sodium dodecyl sulphate as surfactant is reported: refined electropherograms were achieved using 35 mM sodium borate, pH 8.3 containing 65 mM sodium dodecyl sulphate. Almost all peaks were baseline separated, collected and sequenced. This allowed us to define the exact imidodipeptide composition of patients' urine. The possibility of identifying and thus quantifying each single peak means that comparison of urinary imidodipeptide excretion patterns from different patients can be made and the hypothesis that peptide patterns can be correlated with differing clinical severity can be investigated.
Assuntos
Cromatografia/métodos , Dipeptidases/deficiência , Dipeptídeos/urina , Adolescente , Adulto , Eletroforese Capilar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dodecilsulfato de Sódio/farmacologiaRESUMO
Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.
Assuntos
Catepsinas/metabolismo , Cromatografia Capilar Eletrocinética Micelar/métodos , Elastase de Leucócito/metabolismo , Elastase Pancreática/metabolismo , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Catepsina G , Catepsinas/química , Humanos , Cinética , Elastase de Leucócito/química , Elastase Pancreática/química , Fragmentos de Peptídeos/isolamento & purificação , Serina Endopeptidases , Especificidade por SubstratoRESUMO
BACKGROUND: Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including gingival fibroblasts. Since normal gingival fibroblast functioning is fundamental to the maintenance of the periodontal connective tissue, as well as to wound healing, we examined the effect of acrolein and acetaldehyde, volatile components of cigarette smoke, on proliferation, attachment, and ultrastructure of human gingival fibroblasts (HGFs) in culture. METHODS: Human gingival fibroblast (HGF) strains derived from healthy individuals with non-inflamed gingiva were used in this study. The cells were incubated in the presence of different concentrations of acrolein and acetaldehyde. Cell attachment and proliferation were evaluated after incubation for 3 hours and 5 days, respectively. In addition, the cells were examined with a transmission electron microscope in order to evaluate their morphology. RESULTS: The results show that acrolein and acetaldehyde produced dose-dependent inhibition of HGF attachment and proliferation. The cytotoxic effect was, however, reversible when both substances were removed, after 3 days, from the medium. The main ultrastructural finding for the HGF cytoplasm was the presence of vacuoles and lysosomal structures that became prominent with increasing concentration of acrolein and acetaldehyde. CONCLUSIONS: Our experimental data suggest that acrolein and acetaldehyde, volatile components of tobacco smoke, are detrimental to HGF survival and consequently to the oral connective tissue. According to our morpho-functional evidence, these findings corroborate clinical and epidemiological investigations demonstrating smoke as a risk factor in the development of periodontal disease.
Assuntos
Acetaldeído/efeitos adversos , Acroleína/efeitos adversos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Nicotiana , Plantas Tóxicas , Fumaça/análise , Acetaldeído/administração & dosagem , Acroleína/administração & dosagem , Adulto , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Tecido Conjuntivo/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citotoxinas/efeitos adversos , Relação Dose-Resposta a Droga , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Gengiva/citologia , Gengiva/ultraestrutura , Humanos , Lisossomos/efeitos dos fármacos , Microscopia Eletrônica , Doenças Periodontais/etiologia , Fatores de Risco , Fatores de Tempo , Vacúolos/efeitos dos fármacosRESUMO
Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by inadequate levels of the cytosolic exopeptidase prolidase (E.C. 3.4.13.9), for which there is not, as yet, a resolutive cure. We have investigated whether biodegradable microspheres loaded with prolidase could release active enzyme inside cells, to consider this system as a possible therapeutic approach for prolidase deficiency. Poly(lactide-co-glycolide) microspheres were prepared, modifying the classical double emulsion solvent evaporation method to mitigate the burst effect of the enzyme from the microspheres. Ex-vivo experiments were performed, by incubating microencapsulated prolidase with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to the cells. The microparticulate drug delivery system described carried small amounts of active prolidase inside fibroblasts, ensuring a response to the intracellular accumulation of X-Pro dipeptides, the mechanism that is supposed to be responsible for the development of clinical manifestations of this disorder in man. A positive result of the presence of active enzyme inside cells was an improvement in fibroblast shape.
Assuntos
Dipeptidases/administração & dosagem , Dipeptidases/metabolismo , Fibroblastos/efeitos dos fármacos , Poliglactina 910/química , Biodegradação Ambiental , Células Cultivadas , Dipeptidases/deficiência , Portadores de Fármacos/química , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/enzimologia , Humanos , Microesferas , Pele/citologia , Fatores de TempoRESUMO
Given the importance of hyaluronan (HA) for the homeostasis of connective tissues during embryogenesis and aging and its role in tissue repair, the aim of the present study was to examine the effect of exogenous HA on the synthesis of total protein, collagen and HA by in vitro human dermal fibroblasts. With differences between different cell strains, HA, at concentrations between 0.5 and 1 microM, induced a significant decrease in total protein synthesised and secreted into the medium compared to controls (P < 0.05), and particularly in collagen (-40%; P < 0.05). The ratios between collagen types I and III and between collagen types V and I were normal. Pulse and chase experiments showed that protein degradation was normal. The presence of exogenous HA did not affect HA synthesis. Data strongly indicate that a relatively high concentration of HA in the extracellular space, such as during development and in the first phases of tissue repair, would partially limit the deposition of the extracellular matrix, and of collagen in particular. This would suggest a role for HA in delaying tissue differentiation during embryogenesis and in preventing fibrosis and scar formation in fetus and in the early phases of wound healing.
Assuntos
Colágeno/biossíntese , Fibroblastos/metabolismo , Ácido Hialurônico/farmacologia , Biossíntese de Proteínas , Pele/citologia , Adulto , Células Cultivadas , Procedimentos Cirúrgicos Dermatológicos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Pele/lesões , Cicatrização/fisiologiaRESUMO
Glycosaminoglycan-protein complexes were extracted from bovine duodenal mucosa with distilled water, resulting in solubilization of a fraction of the total proteoglycan of the tissue. The extracted material was purified by anion exchange chromatography on DEAE-Sephadex A-25, and then characterized by chemical analysis and by fractionation on Dowex 1. By using these procedures, two major fractions were identified, which were eluted from Dowex with 1.0-1.25 M NaC1 and with 1.5-1.75 M NaC1 respectively. Analyses showed that both fractions were mainly composed of glucosamine-containing, hyaluronidase-resistant polysaccharides, which were identified by their N-sulphate: D-glucosamine and total sulphate: D-glucosamine ratios as heparan-sulphate in the less acidic fraction, and as heparin in the more acidic fraction. Dermatan sulphate molecules were also present in both preparations, with an approximate ratio 1:3 to the glucosamine-containing polysaccharides. Solubility behaviour of the complexes formed by the isolated polyanionic molecules with cetylpyridinium chloride was strongly modified by papain digestion of the duodenal material. This reduction of molecular size of papain treatment suggests that the molecules extracted with water from duodenal mucosa are complex proteoglycans, perhaps in the native state.
Assuntos
Mucosa Intestinal/análise , Proteoglicanas/isolamento & purificação , Aminoácidos/análise , Amino Açúcares/análise , Animais , Bovinos , Duodeno , Glicosaminoglicanos/análise , Peso Molecular , PapaínaRESUMO
In the present paper a relatively rapid and simple method for estimating the ratio of collagen type III to type I in soft tissues is proposed. The ratio Gly/Ala is determined in pure collagen samples obtained from pepsin digests of the tissues. Since this ratio varies linearly depending on the composition of the mixtures of the two collagen types, it is shown that the percentage content of the two collagen types is easily calculated.
Assuntos
Colágeno/análise , Pele/análise , Adulto , Aminoácidos/análise , Eletroforese em Gel de Poliacrilamida , Humanos , Recém-Nascido , Pulmão/análise , MétodosRESUMO
A protective and curative effect of some flavonoids on collagen maturation in lathyrism has been previously demonstrated. This action appeared to involve cross linking of collagen. This paper deals with the effect in vitro of flavonoids and flavonoid-copper complexes on the oxidative deamination of lysine epsilon-amino groups in [4,5-3H]-lysine-labelled elastin. Flavonoids alone do not affect the reaction but it is evident that some flavonoid copper complexes are strongly effective: the aldehyde groups that are quickly formed then enable cross linking to occur. The lysine epsilon-amino groups of elastin are specifically concerned: in fact no effect was observed on free [4,5-3H]-lysine or on [4,5-3H]-lysine-labelled proteins obtained from mouse liver. The lysyl oxidase seems to interfere with the flavonoid-copper complees to regulate the oxidative deamination of lysine epsilon-amino groups.
Assuntos
Cobre , Elastina , Flavonoides , Animais , Aorta , Catequina , Embrião de Galinha , Cinética , Lisina , Ligação Proteica , Conformação ProteicaRESUMO
Hydroxylysine diglycoside was prepared from hydrolyzed sponge by a combination of ion-exchange chromatography and gel filtration. The product obtained was chemically pure on amino acid analyzer and was active as substrate of the enzyme alpha-(1----2)glucosidase. Hyl monoglycoside was prepared by mild acid hydrolysis from diglycoside. A reverse phase HPLC system was devised for Hyl glycosides, hydroxylysine and other basic amino acids. The separation was achieved using an octadecyl bonded silica column on the compounds derivatized with dabsyl chloride to produce di-dabsyl derivatives. Elution was followed in the visible region at 436 nm. In the conditions used no or very low amount of mono-dabsyl derivatives was observed. Hyl di- and monoglycoside resulted a mixture of two diastereoisomers, which form during the alkaline hydrolysis. The separation of the diastereoisomers of each compound depended on pH and ionic strength of the eluent in the HPLC column, whereas they were not separated by our short column on amino acid analyzer. The HPLC system was also used for the analysis of Hyl glycosides on two collagen preparations.