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A J Docherty

Publications and source records attributed to A J Docherty.

At least 19 recordsLinked to original sources
PubMed1992

Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.

Amino Acid Sequence↗
PubMed1992

Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases.

The latent precursor of matrilysin (EC 3.4.24.23; punctuated metalloproteinase (PUMP) was purified from transfected mouse myeloma cell conditioned medium and was found to contain one zinc atom per molecule which was essential for catalytic activity. Promatrilysin could be activated to the same specific activity by (4-aminophenyl)mercuric acetate, trypsin, and incubation at elevated temperatures (heat activation). Active matrilysin hydrolyzed the fluorescent substrate 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond with a maximum value for kcat/Km of 1.3 x 10(4) M-1 s-1 at the pH optimum of 6.5 and pKa values of 4.60 and 8.65. Activity is inhibited by the tissue inhibitor of metalloproteinases-1 in a 1:1 stoichiometric interaction. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with N-terminal sequencing revealed that, as with all other matrix metalloproteinases similarly studied, promatrilysin activation was accompanied by the stepwise proteolytic removal of an M(r) 9000 propeptide from the N-terminus. The intermediates generated were dependent on the mode of activation used but, in all cases studied, activation terminated with an autocatalytic cleavage at E77-Y78 to yield the final M(r) 19,000 active matrilysin. From an analysis of the stability of the various intermediates, we propose that the sequence L13-K33 is particularly important in protecting the E77-Y78 site from autocatalytic cleavage, thereby maintaining the latency of the proenzyme.

Amino Acid Sequence↗
PubMed1992

The role of the C-terminal domain in collagenase and stromelysin specificity.

Recombinant human interstitial collagenase, an N-terminal truncated form, delta 243-450 collagenase, recombinant human stromelysin-1, and an N-terminal truncated form, delta 248-460 stromelysin, have been stably expressed in myeloma cells and purified. The truncated enzymes were similar in properties to their wild-type counterparts with respect to activation requirements and the ability to degrade casein, gelatin, and a peptide substrate, but truncated collagenase failed to cleave native collagen. Removal of the C-terminal domain from collagenase also modified its interaction with tissue inhibitor of metalloproteinases-1. Hybrid enzymes consisting of N-terminal (1-242) collagenase.C-terminal (248-460) stromelysin and N-terminal (1-233) stromelysin.C-terminal (229-450) collagenase, representing an exchange of the complete catalytic and C-terminal domains of the two enzymes, were expressed in a transient system using Chinese hamster ovary cells and purified. Both proteins showed similar activity to their N-terminal parent and neither was able to degrade collagen. Analysis of the ability of the different forms of recombinant enzyme to bind to collagen by ELISA showed that both pro and active stromelysin and N-terminal collagenase.C-terminal stromelysin bound to collagen equally well. In contrast, only the active forms of collagenase and N-terminal stromelysin.C-terminal collagenase bound well to collagen, as compared with their pro forms.

Amino Acid Sequence↗
PubMed1992

The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases.

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

Base Sequence↗
PubMed1992

The matrix metalloproteinases and their natural inhibitors: prospects for treating degenerative tissue diseases.

Uncontrolled matrix metalloproteinase activity is thought to be a cause of the tissue damage observed in many disease processes. None of the drugs currently in use can prevent tissue destruction, and strategies for the development of synthetic inhibitors have been hampered by a poor understanding of the biochemistry of matrix metalloproteinases. Recent cDNA cloning efforts and characterization of recombinant human matrix metalloproteinases have permitted structure-function analysis of the enzymes and their inhibitors. Progress in this area should help indicate a route to rational strategies for designing lead therapeutic compounds.

Amino Acid Sequence↗
PubMed1992

The matrix metalloproteinases and their inhibitors.

A number of metalloproteinases that degrade the extracellular matrix of connective tissues and two specific tissue inhibitors of metalloproteinases (TIMPs) have now been isolated, characterized, and cloned. Comparison of the enzyme sequences has allowed the delineation of domain structures, and initial studies have been carried out to assess the contribution of these domains to their biochemical and biologic properties, including activation, inhibition by TIMPs, and matrix binding. Such events represent the major levels of extracellular regulation of metalloproteinase activity, which is thought to be an important aspect of their control. Activation is probably a cell surface phenomenon, involving the plasminogen activator cascade or other membrane-associated mechanisms. The inhibitory action of TIMPs is postulated to be as important in activation as in the subsequent regulation of enzyme degradation of the matrix.

Amino Acid Sequence↗
PubMed1991

Tissue inhibitor of metalloproteinases-2 inhibits the activation of 72 kDa progelatinase by fibroblast membranes.

We report that monolayers of human fibroblasts stimulated with concanavalin A were able to activate 72 kDa progelatinase but not 95 kDa progelatinase. The activating capacity of fibroblasts appeared approx. 6 h after concanavalin A stimulation and was blocked by cycloheximide. The activation of 72 kDa progelatinase was readily inhibited by TIMP-2 but only poorly by TIMP-1. Plasma membranes isolated from the fibroblasts were capable of activating 72 kDa progelatinase. The cleavage products of the plasma membrane-mediated activation of 72 kDa progelatinase corresponded to those of organomercurial-induced self-cleavage. Only inhibitors of metalloproteinase self-cleavage inhibited the activating capacity of plasma membrane preparations, although the activating capacity was destroyed by trypsin and heat. As with the fibroblast monolayers, TIMP-2 was a potent inhibitor of the membrane-mediated activation whereas TIMP-1 was less so.

Binding Sites↗
PubMed1991

The N-terminal domain of tissue inhibitor of metalloproteinases retains metalloproteinase inhibitory activity.

Recombinant tissue inhibitor of metalloproteinases (TIMP-1) and a truncated version containing only the three N-terminal loops, delta 127-184TIMP, have been expressed in myeloma cells and purified by affinity chromatography and gel filtration. delta 127-184TIMP was found to exist as two main glycosylation variants of molecular mass 24 kD and 19.5 kDa and an unglycosylated form of 13 kDa. All forms of the truncated inhibitor were able to inhibit and form complexes with active forms of the matrix metalloproteinases, indicating that the major structural features for specific interaction with these enzymes resides in these three loops. Stable binding of delta 127-184TIMP to pro 95-kDa gelatinase was not demonstrable under the conditions for binding of full-length TIMP-1.

Amino Acid Sequence↗
PubMed1991

Matrix metalloproteinase degradation of elastin, type IV collagen and proteoglycan. A quantitative comparison of the activities of 95 kDa and 72 kDa gelatinases, stromelysins-1 and -2 and punctuated metalloproteinase (PUMP).

The abilities of the matrix metalloproteinases 95 kDa and 72 kDa gelatinases (type IV collagenases), stromelysins-1 and -2 and punctuated metalloproteinase (PUMP) to degrade insoluble elastin, type IV collagen films and proteoglycan have been compared. The gelatinases and PUMP were markedly more active in the degradation of elastin than were the stromelysins. PUMP and the stromelysins were more potent proteoglycan-degrading enzymes. All of the enzymes studied degraded soluble native type IV collagen, but the gelatinases were more effective at higher temperatures. These quantitative data allow an analysis of the potential relative roles of these metalloproteinases in the breakdown of the key components of connective tissue matrices.

Cell Line↗
PubMed1991

Purification and characterization of human 72-kDa gelatinase (type IV collagenase). Use of immunolocalisation to demonstrate the non-coordinate regulation of the 72-kDa and 95-kDa gelatinases by human fibroblasts.

Human gingival fibroblast gelatinase (type IV collagenase) has been purified to homogeneity using a combination of ion exchange chromatography, gel filtration and affinity chromatography. The purified proenzyme electrophoresed under reducing conditions as a single band of 72 kDa which could be activated to a species of 65 kDa. Gelatinase was activated by organomercurials by a process apparently initiated by a conformational change and involving self-cleavage. It was not activated by trypsin or plasmin unlike the other family members, collagenase and stromelysin. Gelatinase otherwise exhibited properties typical of the metalloproteinases: it was inhibited by metal chelating agents and by the specific inhibitor TIMP (tissue inhibitor of metalloproteinases). Its major substrate was shown to be denatured collagen although it was also able to degrade native type IV and V collagens. A polyclonal antibody was raised in a sheep using the purified enzyme as antigen. The antiserum recognised and specifically inhibited the 72-kDa gelatinase but not a 95-kDa gelatinase from pig leukocytes. It was used in immunolocalisation studies on human fibroblasts to investigate the regulation of the production of the two Mr forms of gelatinase. These studies clearly demonstrate that human fibroblasts constitutively synthesize and secrete 72-kDa gelatinase but that 95-kDa gelatinase was inducible by agents such as cytokines. The significance of these results in relation to the likely in vivo rôle of gelatinases is discussed.

Amino Acid Sequence↗
PubMed1990

Immunoassays for the detection of human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and enzyme-inhibitor complexes.

Immunoassays have been developed for human collagenase, stromelysin, tissue inhibitor of metalloproteinases (TIMP) and TIMP complexed with both of the active enzymes. Selection of antibodies of defined specificity enabled measurement of both the pro and active forms of the metalloproteinase. Free TIMP was quantified by the selection of a monoclonal antibody which did not recognise TIMP when complexed with metalloproteinases. Detection of enzyme-inhibitor complexes was achieved by capturing the TIMP component of the complex and revealing the metalloenzyme using specific antibodies.

Antibodies, Monoclonal↗
PubMed1990

The tissue metalloproteinase family and the inhibitor TIMP: a study using cDNAs and recombinant proteins.

Loss of connective tissue integrity occurs in many disease processes, including rheumatoid arthritis and osteoarthritis. Although there is a high incidence of these diseases in the developed world, there is no treatment that prevents the tissue damage that occurs. Several lines of evidence suggest that uncontrolled connective tissue metalloproteinase activity is responsible for the damage, and as a consequence the inhibition of these enzymes has become the target for therapeutic intervention. Several connective tissue metalloproteinases, including collagenase, stromelysin, and gelatinase, together with tissue inhibitors of metalloproteinases (TIMPs), have been described. Because of difficulties in isolating the metalloproteinases in sufficient quantity as pure separate enzymes, however, very little knowledge has accumulated about their detailed biochemistry. For similar reasons the way in which TIMPs inhibit tissue metalloproteinases is not yet fully understood. In this article it is shown how cloning metalloproteinase and TIMP cDNAs can provide information about the structure of these enzyme and inhibitor families and how the cDNAs can be used to generate recombinant cell lines from which enzymes and inhibitors can be readily purified for further studies.

Amino Acid Sequence↗
PubMed1988

Restriction fragment length polymorphism analysis and assignment of the metalloproteinases stromelysin and collagenase to the long arm of chromosome 11.

Collagenase and stromelysin are two metalloproteinases produced mainly by connective tissue cells and involved in the breakdown of the extracellular matrix. cDNA clones for both of these genes have been isolated and sequencing has shown them to be closely related. The collagenase and stromeylsin cDNA clones have been used to assign these genes to the long arm of chromosome 11 in the regions 11q21-22.1 and 11q22.2-22.3, respectively. This has been achieved using somatic cell hybrids and in situ hybridization. In addition a Taq1 restriction fragment length polymorphism has been demonstrated using the stromelysin cDNA.

Animals↗
PubMed1987

Stromelysin is an activator of procollagenase. A study with natural and recombinant enzymes.

The latent forms of stromelysin and collagenase from human gingival fibroblasts were purified to homogeneity. These latent proenzymes underwent serial small reductions in Mr upon activation by treatment with either 4-aminophenylmercuric acetate or trypsin. Similar shifts in Mr and activation kinetics were observed upon identical treatments of either recombinant prostromelysin or procollagenase. Prostromelysin showed a lag between activation and Mr fall, suggesting an initial activation by conformational change. Collagenase activity was enhanced up to 12-fold by either natural or recombinant stromelysin in the presence of trypsin or 4-aminophenylmercuric acetate. Stromelysin caused a further apparent decrease in the Mr of procollagenase. Since these important connective-tissue-degrading enzymes are usually co-ordinately produced by cells, a cascade mechanism is proposed in which collagenase is activated by stromelysin.

Collagenases↗
PubMed1987

Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor.

Exposure of quiescent MRC-5 human fibroblasts to growth factors such as epidermal growth factor, basic fibroblast growth factor or embryonal carcinoma-derived growth factor resulted in the induction of mRNA transcripts encoding the metalloproteinases collagenase and stromelysin and the specific metalloproteinase inhibitor TIMP, whilst expression of collagen and fibronectin was relatively unaffected. Exposure of quiescent cells to growth factors in the presence of transforming growth factor beta (TGF-beta) resulted in inhibition of collagenase induction and a synergistic increase in TIMP expression. TGF-beta alone did not significantly induce metalloproteinase or TIMP expression. These effects on mRNA transcripts were reflected in increased secretion of TIMP protein and collagenase activity. Nuclear run-off analysis of growth factor-induced transcription revealed that the TGF-beta modulation of TIMP and collagenase expression was due to transcriptional mechanisms. The observations suggest that TGF-beta exerts a selective effect on extracellular matrix deposition by modulating the action of other growth factors on metalloproteinase and TIMP expression.

Cell Line↗