Edward Day HARRIS

Monolayer cultures of rabbit synovial fibroblasts can be stimulated experimentally to produce large quantities of latent collagenase by treatment with phorbol myristate acetate (PMA) or by formation of multinucleated giant cells with poly(ethylene glycol). We studied the ability of all-trans-retonoic acid and/or corticosteroids to modulate collagenase production induced experimentally. Concomitant addition of PMA along with retinoic acid (10−6 M) inhibited production of collagenase by ...

Two active forms (Mr 45,000 and 28,000) of a metalloendopeptidase that digest proteoglycans and other extracellular matrix components of connective tissues have previously been purified from rheumatoid synovial cells and characterized [Okada, Nagase & Harris (1986) J. Biol. Chem. 261, 14245-14255]. To study the mechanisms of activation the precursor of this metalloendopeptidase has now been purified. The final products are homogeneous on SDS/polyacrylamide-gel electrophoresis and ...

To elucidate the mechanism of synovial damage in rheumatoid arthritis, we studied the activation of latent collagenases released from adherent rheumatoid synovial cells in culture. Latent enzyme was not complexed with alpha2 macroglobulin, the prinicpal proteinase inhibitor in serum, and could be activated by trypsin in the presence of alpha2 macroglobulin if sufficient proteinase was added to saturate inhibitor. Latent collagenase bound half as effectively to collagen fibrils as active ...

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ...

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular ...

Human rheumatoid synovial cells in culture stimulated with the conditioned culture medium of rabbit macrophages secrete three distinct latent metalloproteinases. One of them, a proteinase that digests proteoglycan and other connective tissue matrix components, was purified as two active forms after activation with 4-aminophenylmercuric acetate. The two forms were homogeneous on sodium dodecyl sulfate-gel electrophoresis with Mr = 45,000 and Mr = 28,000, whereas the latent precursor was ...

An enzyme which degrades native collagen at neutral pH has been isolated from cultures of rheumatoid synovium in vitro, but little or no collagenolytic activity has been found in homogenates of fresh rheumatoid synovium. Similar to most other mammalian collagenases this synovial enzyme is readily inhibited by serum proteins. Proteins of synovial fluid are derived largely from serum and synovial fluid from noninflamed joints was found to inhibit synovial collagenase; the inhibitor was ...

Monolayer cultures of rabbit synovial fibroblasts stimulated with phorbol myristate acetate to produce large amounts of collagenase (EC 3.4.24.7) were used to study the biosynthesis and secretion of this enzyme. [3H]Leucine was added to cell cultures for pulse-chase and continuous-labelling experiments. The labelled procollagenase synthesized was identified by immunoprecipitation followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The amounts of ...

We studied mechanisms governing production of the neutral proteinase collagenase by synovial cells. We used a model system of monolayer cultures of rabbit synovial fibroblasts stimulated to produce collagenase by treatment with phorbol myristate acetate or crystals of monosodium urate monohydrate. mRNAs from these and untreated cells were translated in a wheat germ cell-free system. Collagenase was not present in the culture medium or in the in vitro translation products of mRNA from ...

Rheumatoid synovial cells dissociated from matrix and adherent to culture dishes released a latent form of collagenase into culture medium. Previous studies have shown that the latent enzyme does not complex with alpha2-macroglobulin and binds to fibrillar substrate. We now show that serum-free culture medium of the synovial cells contains an inhibitor of collagenase as well as latent enzyme; the two were separated on a column of acrylamide/agarose. Latent collagenase (estimated mol wt ...

1.1.|An endopeptidase, capable of degrading gelatin to small fragments, has been found in the medium from cultures of rheumatoid synovium. This peptidase, which does not degrade undenatured collagen, has numerous characteristics in common with the rheumatoid synovial collagenase: It is inhibited by EDTA, by serum proteins and by sulfhydryl compounds such as cysteine; it has a broad pH optimum in the neutral range; its synthesis and release does not seem to be stimulated by addition of ...

A series of intracellular events occurring after treatment of rabbit synovial fibroblasts with 0.01 micrograms/ml phorbol myristate acetate (PMA) were measured. Ten minutes after addition of PMA, there was a temporary increase in intracellular cyclic AMP levels, followed by a transient decrease in incorporation of 3H-thymidine into DNA. Approximately 500 ng/mg cell protein of PGE2 were found in culture medium from the 12- to 24-hour incubation period, but significant collagenase was not ...

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS ...

Crude synovial collagenase, an enzyme that may affect joint cartilage in rheumatoid arthritis, can destroy whole articular cartilage. To study this phenomenon under more specific conditions, we purified collagenase synthesized by primary cultures of rheumatoid synovium and used it to degrade collagen fibrils purified from human articular cartilage. These highly cross-linked fibrils were resistant to solubilization in acetic acid-pepsin extracts and, in comparison with skin collagen ...

Rheumatoid synovium, cultured in vitro in media not enriched with serum, synthesized and released a specific collagenase. This enzyme appeared in measurable quantities only after three or more days of culture. Enzyme activity correlated well with hydroxyproline content in the culture media, indicating that extracellular matrix collagen in synovial explants was being degraded. Collagenase activity was found to be 2 to 10 times greater when colchicine (0.1 μg/ml) was added to the culture ...