Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A collagenous trimeric cross-linked peptide has been isolated from the insoluble matrix of calf aorta, using trypsin solubilisation, and purified by gel filtration, cation-exchange chromatography and reversed-phase HPLC. Molecular mass and amino acid composition indicated that the C-terminal, non-helical region of type I collagen in its dimer form, designated as [ColC(I)]2, is cross-linked to a tryptic peptide TN(I) from the N-terminal helical cross-link region of an adjacent type I molecule, forming the cross-linked peptide [ColC(I)]2 X TN(I). Amino acid sequence analysis of the peptide yielded a series of sequences corresponding to the cross-linking domains ColC(I) and TN(I) and furnished the first direct chemical evidence for the 4D staggered arrangement of type I molecules within native fibers. The trifunctional cross-linking amino acid pyridinoline was shown to occur in the peptide, confirming the peptides three-chain structure. Pyridinoline was isolated from the cross-linked peptide by preparative amino acid analysis and reversed-phase HPLC and identified by its ultraviolet absorption spectra, its fluorescence excitation and emission spectra and, for the first time, its time-of-flight secondary ion-mass spectrum. The high sensitivity of the latter method, exceeding that of fast-atom-bombardment mass spectroscopy by three orders of magnitude, allowed detection of pyridinoline in the picomole range. The occurrence of pyridinoline in non-stoichiometric amounts, the presence of hydroxylysine in hydrolysates of all cross-linked peptides and the finding that hydrolysates also contained an unidentified component indicated that there is at least one cross-link form that is different from pyridinoline and is hydrolysable.
...
PMID:Characterisation of a type-I collagen trimeric cross-linked peptide from calf aorta and its cross-linked structure. Detection of pyridinoline by time-of-flight secondary ion-mass spectroscopy and evidence for a new cross-link. 359 96

In the present study we show that adhesion of normal rat liver epithelial cells (RL34) to substratum coated with type I collagen (collagen substratum) is promoted by a factor involved in 80% ammonium sulfate precipitated proteins from serum-free conditioned medium (PCM) of rat embryo fibroblasts. Adhesion of RL34 cells to collagen substratum was promoted dose dependently by whole PCM and the maximum effects on adhesion could be achieved by 200 micrograms/ml whole PCM. Kinetics studies with 100 micrograms/ml whole PCM showed that adhesion proceeded very slowly, taking 16 h to reach a plateau. Adhesion-promoting activity in whole PCM was sensitive to treatments with trypsin, acid, and heat but stable to dithiothreitol treatment. Further purification of whole PCM was performed using a combination of chromatography on blue Sepharose column, gel filtration column and heparin Sepharose column. The partially purified proteins, referred to as heparin PCM, are not bound or only weakly bound to heparin under physiological ion strength and pH, and the apparent molecular weight (Mr) range is estimated to be 40,000 to 60,000 from gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. When whole PCM or heparin PCM was used for coating on plastic or collagen substratum, they no longer exerted the promoting activity.
...
PMID:Adhesion-promoting factor from embryonic fibroblasts for normal liver epithelial cells. 359 33

A metalloproteinase with activity against type IV collagen, type I collagen and gelatin has been purified from the cytosol of a highly metastatic mouse melanoma by anion-exchange, zinc-chelated and lectin-affinity column chromatography. The purified enzyme has a molecular mass of approx. 59 kDa and on isoelectric focusing in two-dimensional gels produced three spots with apparent isoelectric points (pI) between 5.7 and 6.1. Enzymic activity with collagen, but not gelatin, substrates was latent, requiring activation by trypsin or organomercurials. Trypsin activation of this metalloproteinase was accompanied by a change in molecular mass, whereas autoactivation after 1 month's storage, was not. The degradation of types I and IV collagen by the melanoma enzyme yielded products of lower molecular masses than those yielded by mammalian collagenases, this characteristic thus differentiating this metalloproteinase from classical collagenases.
...
PMID:Purification and characterization of a connective-tissue-degrading metalloproteinase from the cytosol of metastatic melanoma cells. 366 69

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.
...
PMID:Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament. 376 22

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).
...
PMID:Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form. 390 16

Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of pro alpha 1(I) chains. Trimers of alpha 1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the alpha 1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the alpha 1(I) trimers compared to control type I collagen. We next examined the thermal stability of the alpha 1(I) trimers using digestion with a combination of trypsin and alpha-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36 degrees to 40 degrees C, the alpha 1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each alpha 1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the alpha 1(I) trimers was probably explained by the absence of alpha 2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the alpha 1(I) chains contributed to the altered helical structure.
...
PMID:Altered helical structure of a homotrimer of alpha 1(I)chains synthesized by fibroblasts from a variant of osteogenesis imperfecta. 405 61

Previous reports have suggested that immune mechanisms are involved in the induction of certain types of pulmonary fibrosis. In this study endotracheal bleomycin was used to induce pulmonary fibrosis in rats, and they were then examined for cellular sensitivity to homologous interstitial collagens isolated from lung. Results indicate that rats treated with bleomycin develop transient cellular sensitivity to homologous collagen. Immunity appears to be selective to type I collagen with minimal response to type III collagen. The kinetics of the development of autoimmunity paralleled the transient increase in collagen synthesis in response to bleomycin treatment. This response was abolished upon prior treatment of the challenging antigen with purified bacterial collagenase, but was resistant to trypsin digestion. This finding confirms the true collagenous nature of the stimulating antigen and eliminates the possibility of a noncollagenous contaminant as the effective agent in the antigen preparation. The data suggest that cellular sensitivity to homologous collagen in response to bleomycin treatment may play a role in the overall fibrotic response.
...
PMID:Cellular sensitivity to collagen in bleomycin-treated rats. 618 Nov 64

The activation energy (EA) and solvent-deuterium kinetic isotope effect (kH/kD) of human skin fibroblast collagenase were studied on the homologous human type I, II, and III collagens in both native and denatured states. Values for EA on human type I and II collagens in solution were 47,000 and 61,000 cal, respectively. The Arrhenius plot for type III collagen, unlike that for the other types, was characterized by a break in EA at approximately 26 degrees C. At temperatures below this point, EA was 42,500 cal; at higher temperatures, EA fell to 29,500 cal. This latter value, intermediate between type I collagen monomers and denatured random gelatin alpha chains, appears to result from a further opening in the already loosened helix of the type III collagen molecule in the region of the 3/4:1/4 collagenase cleavage site. The EA of trypsin on native human type III collagen was also measured and found to be 70,000 cal. This high value calls into question the role of serine proteases in the physiologic degradation of this substrate; a much higher energy expenditure was required for trypsin to cleave type III collagen than for the fibroblast collagenase. Reaction velocity on human collagen types I-III in solution was slowed 15-35% (kH/kD = 1.2-1.5) by the substitution of deuterium for hydrogen in the solvent buffer. This value was far lower than that observed following the aggregation of solution monomers into insoluble fibrils (kH/kD = 9). Denaturation of triple helical monomers into random gelatin alpha chains eliminated any slowing by deuterium, and kH/kD was 1.0 in all cases. Since the same peptide bond hydrolysis accompanies the cleavage of all these forms of the collagen substrate, it would appear that the role of water at the rate-limiting step of collagen degradation may not reside in the hydrolysis of a peptide bond per se, but rather may reflect the difficulty in transporting water molecules to the site of such catalysis, especially following fibril aggregation.
...
PMID:Studies on the activation energy and deuterium isotope effect of human skin collagenase on homologous collagen substrates. 630 32

A procollagenase from monolayer cultures of postpartum rat uterine cells has been purified. The crucial step in the purification is the binding of the procollagenase from crude, fetal bovine serum-containing culture medium to heparin-Sepharose, followed by elution with extremely low concentrations (5-10 nM) of dextran sulfate. Resultant eluates contain 8-10% procollagenase. Purification is completed by ion-exchange chromatography on DEAE-Sepharose, gel filtration on AcA-44, and chromatography on blue-Sepharose. Rat uterine procollagenase appears as a protein doublet of Mr approximately 58,000, as indicated by two polyacrylamide gel electrophoresis systems, by AcA-44 chromatography, and by equilibrium sedimentation ultracentrifugal analysis. The proenzyme forms are converted by trypsin to an active enzyme doublet of Mr approximately 48,000. Small amounts of active enzyme, which are often generated during the purification, are electrophoretically indistinguishable from trypsin-activated collagenase. Active collagenase can be separated from the zymogen forms by DEAE-Sepharose chromatography. The two forms of the proenzyme doublet can be partially separated by gel filtration on AcA-44 and preliminary analysis indicates each has equal collagenolytic activity. The amino acid analysis of rat uterine collagenase reveals it to be markedly different from two other vertebrate collagenases whose composition is known. The uterine proenzyme is unusually rich in glycine and in the hydroxy amino acids and is considerably more acidic than the human skin fibroblast collagenase, consistent with the different ion-exchange behavior of the two molecules. The specific activity of rat uterine collagenase at 37 degrees C is approximately 3000 micrograms collagen/min/mg, using native reconstituted guinea pig skin type I collagen fibrils as substrate. The enzyme cleaves denatured collagen, but fails to attack a variety of noncollagen proteins.
...
PMID:Purification and properties of rat uterine procollagenase. 631 Nov 6

An enzyme that was capable of releasing small fragments containing hydroxynorleucine from the c-terminal extra-helical region of the alpha 1 chain of reduced (tritiated) soluble type I collagen was found, along with collagenase, in medium that had been conditioned by the culture of human gingival fibroblasts (Gin-1). The enzyme was present in a latent form or forms and could be activated by treatment with either trypsin or p-hydroxymercuribenzoate. It was maximally active at neutral pH and inhibited by EDTA. It is suggested that this enzyme, acting within a region of the molecule which is of major importance in stabilizing fibrillar collagen through intermolecular cross-linking, could potentially play an important role in collagen turnover in vivo.
...
PMID:A neutral proteinase from human gingival fibroblasts active against the c-terminal cross-linking region of type I collagen. 631 Nov 93


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---