Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A genetic approach to define the role of collagenase in physiological and pathological bone remodeling is to identify spontaneous mutations in the collagenase gene which alter enzymatic activity. Alternatively it is possible, though site-directed mutagenesis, to alter genes encoding critical amino acid sequences in the collagen substrate, in a manner analogous to the successful development of animal models for osteogenesis imperfecta. We have thus utilized this approach to alter the Col1a1 gene to encode amino acid substitutions in sequences around the known collagenase cleavage site (glycine-isoleucine at positions 775-776) in type I collagen, and transfect these genes into homozygous Mov-13 fibroblasts, in which the endogenous Col1a1 gene is inactive. Nonconservative substitutions of proline for isoleucine at the P1' site and double substitutions of proline for glutamine (P2) and alanine (P2') resulted in type I collagen resistant to hydrolysis by collagenase. Furthermore, in normal fibroblasts transfected with a mutant Col1a1 gene encoding collagenase resistance in which an additional methionine substitution at position 776 provided a marker for the mutant protein, mutant and wild type triple helical molecules were synthesized and secreted as heterotrimers. A single mutant alpha 1(I) chain did not prevent cleavage of the wild type alpha 1(I) chain but it is likely that the uncleaved alpha 1(I) chain would prevent dissociation of the triple helical fragments containing the other cleaved chains. Introduction of these genes into transgenic mice should result in abnormal phenotypes characterized by altered connective tissue remodeling.
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PMID:Site-directed mutagenesis of type I collagen: effect on susceptibility to collagenase. 148 89

1. The effect of vasoactive intestinal polypeptide (VIP) upon adenylate cyclase activity was determined in purified cortical basolateral membranes and in glomeruli and tubular elements obtained from rabbit kidney. 2. In purified basolateral membranes prepared from cortex, 1 microM-VIP consistently stimulated adenylate cyclase activity above basal levels (1.55 +/- 0.09-fold (mean +/- S.E. of mean), n = 10 animals). Half-maximal stimulation was observed at 17 +/- 11 nM-VIP (S.D., n = 9). 3. Related peptides, e.g. secretin, glucagon, gastric inhibitory peptide, human pancreatic growth hormone releasing factor, and peptide having N-terminal histidine and C-terminal isoleucine amide (PHI), were without effect or gave lower stimulations of adenylate cyclase activity when tested at 1 microM. 4. Significant VIP degradation was observed under the assay conditions used but this did not substantially alter the response or selectivity to VIP. 5. In separate preparations of isolated glomeruli and proximal tubules addition of 1 microM-VIP resulted in a 3.3 +/- 1.1-fold (S.D., n = 3) and 2.2 +/- 1.0-fold (S.D., n = 3) stimulation (respectively) of adenylate cyclase activity. 6. In isolated medullary tubule suspensions, isolated by collagenase-hyaluronidase digestion of outer (red) medulla, and in thick ascending-limb-enriched preparations prepared by Percoll density gradient fractionation, 1 microM-VIP significantly increased adenylate cyclase activity by 2.4 +/- 0.6-fold (S.D., n = 3) and 2.1 +/- 0.7-fold (S.D., n = 3) respectively. 7. A possible role for VIP in the regulation of renal function in the rabbit is discussed in relation to the occurrence of VIP stimulation of adenylate cyclase activity in several renal cellular elements.
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PMID:Vasoactive intestinal polypeptide regulation of rabbit renal adenylate cyclase activity in vitro. 365 72

Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [ 3H ]proline-labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly-Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin-A--Sepharose affinity chromatography and further purified by QAE-Sephadex ion-exchange chromatography and gel filtration on Sephacryl S-200. The purified enzyme had a molecular weight of approximately 105000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non-helical form, but not when in native triple-helical conformation. Dansyl-monitored end-group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly-Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly-Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non-helical as a result of collagenase cleavage.
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PMID:Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence. 634 59

It is well-known that the hypothalamus predominantly exerts an inhibitory control on prolactin secretion and that dopamine (DA) is the main prolactin inhibiting factor (PIF). In addition, the hypothalamus contains prolactin-releasing factors (PRF). Thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide (VIP) and peptide-histidine-isoleucine (PHI) are the components of PRF. However, the detailed mechanism by which the peptides release prolactin (PRL) at the pituitary level is still unknown. Therefore, in this paper, an in vitro perifusion system using the cell column of cultured rat pituitary cells attached on Cytodex beads was employed to investigate the mechanism of PRL release. The rat anterior pituitary cells were isolated using collagenase, and the dispersed pituitary cells were cultured with swollen Cytodex beads in Dulbecco's modified Eagle medium (DMEM) containing fetal calf serum at 37 degrees C in 5% CO2 and 95% air for 2--3 days. The cultured anterior pituitary cells attached on Cytodex beads were packed in a column and perifused with DMEM at a constant flow rate of 0.4 ml/min using a peristaltic pump. The following results were obtained. A five minute perifusion with 100 pg/ml to 100 ng/ml TRH caused a significant increase of PRL in a dose-related manner. A continuous perifusion with 2 ng/ml or 10 ng/ml DA inhibited PRL release in a dose-related manner. When TRH at a dose of 1 ng/ml, 10 ng/ml or 100 ng/ml was perifused for 120 min at a rate of 0.4 ml/min, a large amount of PRL was released during the early period of the TRH infusion, and then the PRL release gradually decreased to the basal levels in spite of the continuous TRH infusion. An additional TRH, of which the concentration was ten-fold higher than the TRH level in the continuous infusion, when added at the end of the continuous TRH infusion, had no effect on PRL release. On the other hand, a 5 minute TRH infusion given at 30 min after the end of the continuous TRH infusion caused a significant increase in PRL release. A continuous perifusion with 1 mM 8-bromo-cyclic AMP caused a small but continuous PRL release. An additional continuous 8-bromo-cyclic AMP infusion during the late period of a continuous TRH infusion caused a continuous PRL release similar to that induced by the continuous infusion of cyclic AMP only. A short period perifusion with 1 X 10(-9)M to 1 X 10(-7)M of vasoactive intestinal polypeptide (VIP) enhanced a significant increase of PRL release in a dose-related manner, but the amounts of PRL release induced by VIP were smaller than those induced by TRH.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:[A study on the prolactin releasing mechanism using an in vitro perfusion system with a cell column of cultured rat anterior pituitary cells]. 644 Aug 13

Hepatocytes prepared by collagenase perfusion from Antarctic nototheniid fish of genus Trematomus are active in uptake of [14C]leucine at 0, 5, and 10 degrees C. The system is saturable with apparent Km about 1.0 mM. Isoleucine and phenylalanine were major competitors, valine was about one-half as effective, while alanine, glycine and histidine had no effect. Temperature dependency of rates in the 0-10 degrees C range yielded Ea = 65 kJ/mol (Q10 = 2.7). The average first-order rate constant at 0 degrees C was 0.1 min-1, one-third the value of 0.3 min-1 estimated for clearance of [14C]leucine by liver of these species in vivo. Affinity and specificity agreed well with in vivo data on liver clearance of leucine, both in Antarctic fish at 0 degrees C and in temperate fish acclimated to 10 degrees C and 20 degrees C. The results indicate similar modifications of leucine transport associated with evolutionary cold adaptation and seasonal acclimation in fish.
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PMID:Characteristics of leucine transport by isolated hepatocytes of Antarctic fish at low temperatures. 717 5

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.
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PMID:Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). 782 72

Entactin is the basement membrane protein which bridges laminin and type IV collagen. Entactin is known to be degraded by serine proteinases, but its susceptibility to matrix metalloproteinases has not been determined. We have studied the capacity of three matrix metalloproteinases (interstitial collagenase, 92-kDa gelatinase, and matrilysin) to degrade entactin. While all three metalloenzymes cleaved entactin, matrilysin was approximately 100-fold as effective as collagenase and 600-fold as effective as 92-kDa gelatinase. The Km of matrilysin for entactin was 8.9 x 10(-7) M. A Vmax of 21 molecules of entactin degraded/molecule of matrilysin/min at 37 degrees C was observed. An Arrhenius plot relating matrilysin's catalytic activity to temperature was linear from 15 to 37 degrees C and indicated an activation energy of 10,060 calories/mol. Matrilysin produced multiple, but distinct, cleavages in entactin resulting in peptide fragments ranging from 115 to 29 kDa. The precise sites of cleavage of six fragments were determined by Edman degradation. Cleavage sites consistently occurred amino-terminal to leucine or isoleucine. These data indicate that entactin is a substrate for matrix metalloproteinases. The effectiveness of matrilysin is noteworthy, however, particularly in relation to the minimal ability of other much more well described matrix metalloproteinases to attack this substrate. Our results suggest a potentially important role for matrilysin in disruption of basement membranes by tumor or inflammatory cells.
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PMID:Degradation of entactin by matrix metalloproteinases. Susceptibility to matrilysin and identification of cleavage sites. 838 May 88

The biological functions of rat surfactant protein A (SP-A), an oligomer composed of 18 polypeptide subunits derived from a single gene, are dependent on intact disulfide bonds. Reducible and collagenase-reversible covalent linkages of as many as six or more subunits in the molecule indicate the presence of at least two NH2-terminal interchain disulfide bonds. However, the reported primary structure of rat SP-A predicts that only Cys6 in this region is available for interchain disulfide formation. Direct evidence for a second disulfide bridge was obtained by analyses of a set of three mutant SP-As with telescoping deletions from the reported NH2-terminus. Two of the truncated recombinant proteins formed reducible dimers despite deletion of the domain containing Cys6. Edman degradation revealed that each mutant protein was a mixture of two isoforms with and without an isoleucine-lysine-cysteine (IKC) extension at the NH2-terminus, which was derived from the COOH-terminal end of the reported signal peptide. Large variations in the abundance of the IKC isoforms between truncated SP-As suggested that the amino acid sequences located downstream from the signal peptide modulated alternate-site cleavage by signal peptidase. Elution of the newly identified cysteine in the position of DiPTH-Cys indicated participation in disulfide linkage, which was interchain based on the direct correlation between prevalence of the IKC variant and the extent of dimerization for each truncated protein. Sequencing of both native rat SP-A and human SP-A also revealed isoforms with disulfide-forming NH2-terminal extensions. The extended rat SP-A isoforms were enriched in the more fully glycosylated and multimeric SP-A species separated on SDS-PAGE gels. Thus, a novel post translational modification results in naturally occurring cysteinyl isoforms of rat SP-A, which are essential for multimer formation.
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PMID:Alternate amino terminal processing of surfactant protein A results in cysteinyl isoforms required for multimer formation. 918 99

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (PDB ID: 1BKV). A torsional map approach was used to characterize the conformational motion of the molecule that differ between imino-rich and imino-poor regions. At temperatures 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. We found that unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 A, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly.
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PMID:Region-specific role of water in collagen unwinding and assembly. 1838 48

Matrix metalloproteinases (MMPs) are essential for normal collagen turnover, recovery from fibrosis, and vascular permeability. In fibrillar collagens, MMP-1, MMP-8, and MMP-13 cleave a specific glycine-isoleucine or glycine-leucine bond, despite the presence of this sequence in other parts of the protein. This cut site specificity has been hypothesized to arise from a unique, relaxed super-secondary structure in this area due to local hydroxyproline poor character. In this study we examined the mechanism of interaction and cleavage of human type III collagen by fibroblast MMP-1 by using a panel of recombinant human type III collagens (rhCIIIs) containing engineered sequences in the vicinity of the cleavage site. Native and recombinant type III collagens had similar biochemical and structural characteristics, as indicated by transmission electron microscopy, circular dichroism spectropolarimetry, melting temperature and hydroxyproline analysis. A single amino acid change at the I785 cleavage site to proline resulted in partial MMP-1 resistance, but cuts were found in novel sites in the original cleavage region. However, the replacement of five Y-position residues by proline in this region, regardless of I785 variation, conferred complete resistance to MMP-1, MMP-8, MMP-13, trypsin, and elastase. MMP-1 had a decreased specific activity towards and reduced cleavage rate of rhCIII I785P but a K(m) similar to wild-type. Despite the reductions in protease sensitivity, MMP-1 bound to all of the engineered rhCIIIs with comparable affinity, indicating that MMP-1 binding is not sufficient for cleavage. The relaxed tertiary structure in the MMP cleavage region may permit local collagen unwinding by MMP-1 that enables site-specific proteolysis.
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PMID:Matrix metalloproteinase-1 cleavage site recognition and binding in full-length human type III collagen. 1939


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