Some properties of latent collagenase from human synovial fluid

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.     

The activation of latent pig synovial collagenase

Latent pig synovial collagenase (EC 3.4.24.7) can be activated by a variety of different treatments to give an active enzyme form of lower molecular weight which rapidly degrades collagen. Trypsin and plasmin effectively activated the latent collagenase whilst elastase and cathepsin G degraded most of the latent enzyme before it was activated. A number of mercurials were compared and maximum activation was achieved using 4-aminophenylmercuric acetate and phenylmercuric chloride. The latent collagenase bound to a mercurial-Sepharose column and was eluted in the active form with NaCl. The latent collagenase also activated spontaneously and the conditions which encouraged and prevented this activation were studied. High NaCl concentration, diisopropylphosphofluoridate, soybean trypsin inhibitor, low Zn2+ concentration and high and low pH all prevented the spontaneous activation of latent pig synovial collagenase.     

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.     

A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase.

1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.     

Activation of latent rheumatoid synovial collagenase by human mast cell tryptase

The functional role of mast cells in rheumatoid synovium was investigated by assessing the ability of mast cell tryptase to activate latent collagenase derived from rheumatoid synoviocytes. Tryptase, a mast cell neutral protease, was demonstrated in situ to reside in rheumatoid synovial mast cells, by an immunoperoxidase technique using a mouse mAb against tryptase, and in vitro to be released by dispersed synovial mast cells after both immunologic and nonimmunologic challenge. Each rheumatoid synovial mast cell contains an average of 6.2 pg of immunoreactive tryptase and the percent release values of this protease correlated with those of histamine (r = 0.58, p less than 0.01). The ability of purified tryptase to promote collagenolysis was demonstrated in a dose-dependent fashion using latent collagenase derived from rheumatoid synovium, synovial fluid, IL-1-stimulated cultured synoviocytes, and partially purified latent collagenase derived from conditioned media, with between 10 and 92% of the collagen substrate degraded. [3H] Collagen, treated with tryptase-activated latent collagenase, was subjected to electrophoresis on SDS polyacrylamide gels and autoradiography showed the collagen degradation pattern (A, B) characteristically produced by collagenase. Mast cell lysates also activated synovial latent collagenase yielding 24% digestion of collagen substrate. This activator in mast cell lysates could be inhibited by diisopropylflurophosphate or by immunoadsorption of tryptase. Thus, mast cells may activate metalloproteinases and play a role in the catabolism of collagen that occurs in rheumatoid synovium.     

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.     

Isolation, purification and properties of a factor from rheumatoid synovial fluid activating the latent forms of collagenolytic enzymes.

1. An activator catalysing specifically conversion of latent forms of human leucocyte collagenase and gelatin-specific protease into the active forms, has been isolated from rheumatoid synovial fluid and purified 55-fold with a yield of 16%. 2. Molecular weight of the activator is about 35 000. 3. The activator is thermolabile, and is irreversibly inactivated at pH below 5.5 or in the presence of low concentrations of trypsin or papain; it is resistant to the action of lysozyme, hyaluronidase, diisopropylfluorophosphate, soybean trypsin inhibitor, p-chloromercuribenzoate, iodoacetamide and dithiothreitol. 4. The activator did not show any activity towards collagen, gelatin, casein, haemoglobin, histones, elastin or p-phenylazobenzyloxycarbonyl-peptide.     

Identification and partial characterization of an inhibitor of collagenase from rabbit bone

Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.     

A latent collagenase from embryonic human skin explants.

Collagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme. 1) Embryonic human skin explants showed a much higher capacity for collagenase production than did adult skin, on the basis of unit weight of tissue. 2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system. 3) The molecular weights of active and latent collagenases were about 40,000 and 50,000, respectively. 4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3 M. The degree of activation produced by this treatment was as high as that by limited proteolysis with trypsin. It was concluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex. 5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.     

Identification of plasma kallikrein as an activator of latent collagenase in rheumatoid synovial fluid

Rheumatoid synovial fluid contains an activator of latent collagenase from culture medium of pig synovium. The activator was purified by gel chromatography on Ultrogel AcA 44 and affinity chromatography on soybean trypsin inhibitor coupled to Sepharose 4B. The purified material was homogeneous on SDS-polyacrylamide gel electrophoresis with Mr 88 000. The activator had limited proteolytic activity against azo-casein, but showed amidase activity on Pro-Phe-Arg-NMec, Z-Phe-Arg-NMec, D-Val-Leu-Arg-NPhNO2 and D-Pro-Phe-Arg-NPhNO2, with an optimum at pH 8.0. Activity was completely inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, leupeptin and Pro-Phe-Arg-CH2Cl, whereas lima bean trypsin inhibitor, Tos-Lys-CH2Cl, a specific inhibitor of factor XIIa from maize, EDTA and iodoacetate were not inhibitory. These properties of the activator suggested that it might be plasma kallikrein (EC 3.4.21.34), and the possibility was further examined. The activator was treated with [3H]diisopropyl fluorophosphate, and run in SDS-polyacrylamide gel electrophoresis with reduction; a radioautograph of the gel showed a pair of [3H]diisopropyl phosphoryl-labelled bands (Mr 36 000 and 34 000) identical to those obtained with authentic plasma kallikrein. Double immunodiffusion with monospecific antiserum against human plasma kallikrein confirmed the identification. This is the first demonstration of collagenase-activating activity of plasma kallikrein, and raises the possibility that activation of prokallikrein in the inflamed joint space may contribute to the disease process not only by the production of bradykinin, but also by activating latent collagenase.     

Chemical characterization and study of the autodigestion of pure collagenase from Achromobacter iophagus.

Only one collagenase (EC 3.4.24.3) is produced by the non-pathogenic Achromobacter iophagus strain. The chromatography of the crude enzyme on DE-32 cellulose followed by gel filtration on Sephadex G-100 in the presence of 1 M sodium chloride led to the isolation of a homogeneous enzyme. Its specific activity (1.642 mukat/mg) represents the highest value ever obtained for a bacterial collagenase. The amino acid composition of A. iophagus collagenase differs from that of Clostridium histolyticum mainly in the sulfur-containing amino acids. 1 mol of zinc was found for 1 mol of enzyme of molecular weight 104 000. The autodegradation of the A. iophagus collagenase results in the formation of at least three active fractions which can be separated by preparative polyacrylamide gel electrophoresis as well as rechromatography on DE-32 cellulose. They are active towards the synthetic substrate as well as towards the native collagen. The results of ORD have shown that the digestion of the last one occurs in the helical parts of the substrate.     

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.     

Latent collagenase of rheumatoid synovial fluid is not of granulocytic origin

The physicochemical properties of three latent collagenases derived from rheumatoid synovial fluid, polymorphonuclear leucocytes and culture medium of rheumatoid synovium were compared. It has been shown that synovial fluid enzyme is similar to that of synovium collagenase from tissue culture and differs significantly in molecular size and protein charge from granulocyte collagenase. The results indicate that the latent, trypsin-activable collagenase present in rheumatoid synovial fluid is not of granulocytic origin and seems to derive from the synovial membrane.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

Effect of cartilage proteoglycans on human collagenase activities.

No significant inhibition of purified rheumatoid synovial collagenase was found when this enzyme was assayed in the presence of porcine or human cartilage proteoglycans. Reaction mixtures containing up to twice the amount of proteoglycan compared to that of collagen, w/w, had little effect on collagen degradation as judged by the reconstituted [4C]collagen fibril assay and polyacrylamide gel electrophoresis. Proteoglycans were not degraded by the synovial collagenase preparation. Although the human collagenases derived from rheumatoid synoviam, gastric mucosa, skin and granulocytes showed some reduction in activity when exposed to aggregated proteoglycans at high concentrations, disaggregated proteoglycans had no inhibitory effect. It is concluded that cartilage proteoglycans do not directly inhibit human collagenases in vitro, but in vivo they may provide some physical barriers which might limit the accessibility of the enzyme to its collagen substrate.     

Purification and characterization of bovine dental sac collagenase

1. Active type collagenase was purified as much as 140-fold from the explant medium of bovine dental sacs and showed a single band on disc gel electrophoresis. Purified collagenase cleaved native collagen at only one locus under physiological conditions, but hydrolyzed neither gelatin nor alpha-casein. The optimal pH was about 7.8. 2. The molecular weight of active type enzyme was 35,000 by gel filtration and 34,000 by gel electrophoresis. The activation of latent type of collagenase resulted in the reduction of molecular weight from 45,000 to 38,000 by gel filtration. 3. A small but detectable amount of collagenase was directly extracted from frozen and thawed bovine dental sacs. In explant media of frozen and thawed tissue and fresh tissue with actinomycin D, some activity was detected for the first 2 days, but essentially no collagenase activity was detected in the explant medium after day 3. 4. The latent type collagenase was activated by trypsin, 4-aminophenylmercuric acetate (4-APMA), thiocyanate and deoxycholate (DOC). DOC showed irreversible dissociation of latent type enzyme in similar fashion to that exerted by 4-APMA. 5. The purified collagenase was inhibited by bovine serum, EDTA, o-phenanthroline, cysteine and dithiothreitol.     

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes.

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.     

Differences in the physical properties of collagenases isolated from rheumatoid synovium and human skin

During purification of human collagenase from normal skin and rheumatoid synovium differences have been observed with regard to the size and charge properties of the two enzymes. Gel filtration experiments in Sephadex G-100 superfine have allowed molecular weights of approximately 63,000 and 32,000 daltons to be calculated for the skin and rheumatoid synovial enzyme respectively. Ion exchange chromatography using Sephadex QAE, A-50 has shown the rheumatoid synovial enzyme to possess charge properties more basic than that of the skin enzyme. Elution of collagenase activity from $\text{7}\text{1}\text{2}\text{\%}$ polyacrylamide gels has produced no evidence for a ‘fast-moving’ component of either enzyme.     

Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method.

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.