Assignments, secondary structure and dynamics of the inhibitor-free catalytic fragment of human fibroblast collagenase

Fibroblast collagenase (MMP-1), a 169-residue protein with amolecular mass of 18.7 kDa, is a matrix metalloproteinase which has beenassociated with pathologies such as arthritis and cancer. The assignments ofthe ¹H, ¹⁵N, ¹³CO and¹³C resonances, determination of the secondary structure andanalysis of ¹⁵N relaxation data of the inhibitor-freecatalytic fragment of recombinant human fibroblast collagenase (MMP-1) arepresented. It is shown that MMP-1 is composed of a -sheet consistingof five -strands in a mixed parallel and antiparallel arrangement(residues 13–19, 48–53, 59–65, 82–85 and94–99) and three -helices (residues 27–43, 112–124and 150–160). This is nearly identical to the secondary structuredetermined from the refined X-ray crystal structures of inhibited MMP-1. Themajor difference observed between the NMR solution structure ofinhibitor-free MMP-1 and the X-ray structures of inhibited MMP-1 is thedynamics of the active site. The 2D ¹⁵N-¹H HSQCspectra, the lack of information in the ¹⁵N-edited NOESYspectra, and the generalized order parameters (S²) determinedfrom ¹⁵N T₁, T₂ and NOE datasuggest a slow conformational exchange for residues comprising the activesite (helix B, zinc ligated histidines and the nearby loop region) and ahigh mobility for residues Pro¹³⁸-Gly¹⁴⁴ in thevicinity of the active site for inhibitor-free collagenase. In contrast tothe X-ray structures, only the slow conformational exchange is lost in thepresence of an inhibitor.     

NMR solution structure of the catalytic fragment of human fibroblast collagenase complexed with a sulfonamide derivative of a hydroxamic acid compound.

The solution structure of the catalytic fragment of human fibroblast collagenase (MMP-1) complexed with a sulfonamide derivative of a hydroxamic acid compound (CGS-27023A) has been determined using two-dimensional and three-dimensional heteronuclear NMR spectroscopy. The solution structure of the complex was calculated by means of hybrid distance geometry-simulated annealing using a combination of experimental NMR restraints obtained from the previous refinement of the inhibitor-free MMP-1 (1) and recent restraints for the MMP-1:CGS-27023A complex. The hydroxamic acid moiety of CGS-27023A was found to chelate to the "right" of the catalytic zinc where the p-methoxyphenyl sits in the S1' active-site pocket, the isopropyl group is in contact with H83 and N80, and the pyridine ring is solvent exposed. The sulfonyl oxygens are in hydrogen-bonding distance to the backbone NHs of L81 and A82. This is similar to the conformation determined by NMR of the inhibitor bound to stromelysin (2, 3). A total of 48 distance restraints were observed between MMP-1 and CGS-27023A from 3D 13C-edited/12C-filtered NOESY and 3D 15N-edited NOESY experiments. An additional 18 intramolecular restraints were observed for CGS-27023A from a 2D 12C-filtered NOESY experiment. A minimal set of NMR experiments in combination with the free MMP-1 assignments were used to assign the MMP-1 (1)H, 13C, and 15N resonances in the MMP-1:CGS-27023A complex. The assignments of CGS-27023A in the complex were obtained from 2D 12C-filtered NOESY and 2D 12C-filtered TOCSY experiments.     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy

Equilibrium denaturation of streptomycin adenylyltransferase (SMATase) has been studied by CD spectroscopy, fluorescence emission spectroscopy, and binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid (ANS). Far-UV CD spectra show retention of 90% native-like secondary structure at 0.5 M guanidine hydrochloride (GdnHCl). The mean residue ellipticities at 222 nm and enzyme activity plotted against GdnHCl concentration showed loss of about 50 and 75% of secondary structure and 35 and 60% of activity at 0.75 and 1.5 M GdnHCl, respectively. At 6 M GdnHCl, there was loss of secondary structure and activity leading to the formation of GdnHCl-induced unfolded state as evidenced by CD and fluorescence spectroscopy as well as by measuring enzymatic activity. The denaturant-mediated decrease in fluorescence intensity and 5 nm red shift of λ_max point to gradual unfolding of SMATase when GdnHCl is added up from 0.5 M to a maximum of 6 M. Decreasing of ANS binding and red shift (∼5 nm) were observed in this state compared to the native folded state, indicating the partial destruction of surface hydrophobic patches of the protein molecule on denaturation. Disruption of disulfide bonds in the protein resulted in sharp decrease in surface hydrophobicity of the protein, indicating that the surface hydrophobic patches are held by disulfide bonds even in the GdnHCl denatured state. Acrylamide and potassium iodide quenching of the intrinsic tryptophan fluorescence of SMATase showed that the native protein is in folded conformation with majority of the tryptophan residues exposed to the solvent, and about 20% of them are in negatively charged environment. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 11, pp. 1514–1523.     

1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.     

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady‐state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high‐throughput drug screening campaigns.     

Structural heterogeneity of the various forms of apomyoglobin: implications for protein folding.

Temperature-induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surrounding do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native-like (tightly packed) and molten globule-like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two "nucleation" events whereby native-like contacts are formed. One of these events, which involves AGH "core" formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermodynamical properties as well as formation kinetics.     

Alternate succession of steps can lead to the folding of a multidomain oligomeric protein

The beta 2 subunit of Escherichia coli tryptophan synthase can be either unfolded in 6 M guanidine, or extensively denatured at acidic pH. These two denatured forms of beta 2 have different circular dichroism spectra and thus correspond to distinct physical states. Here we compare the folding pathways of these two different denatured forms of beta chains. We describe the kinetics of regain of a variety of physical, functional, and immunochemical signals characteristic of six successive steps previously identified on the folding pathway of guanidine unfolded beta 2. It is shown that whereas identical molecular events occur with the same kinetics, the two folding pathways are different, and involve different structural intermediates.     

Global and local dynamics of the human U1A protein determined by tryptophan fluorescence.

Tryptophan residues have been introduced into two domains of the human U1A protein to probe solution dynamics. The full length protein contains 282 residues, separated into three distinct domains: the N-terminal RBD1 (RNA Binding Domain I), consisting of amino acids 1-101; the C-terminal RBD2, residues 202-282; and the intervening linker region. Tryptophan residues have been substituted for specific phenylalanine residues on the surface of the beta-sheet of either RBD1 or RBD2, thus introducing a single solvent exposed tryptophan as a fluorescence reporter. Both steady-state and time-resolved fluorescence measurements of the isolated RBD domains show that each tryptophan experiences a unique environment on the beta-sheet surface. The spectral properties of each tryptophan in RBD1 and RBD2 are preserved in the context of the U1A protein, indicating these domains do not interact with each other or with the linker region. The rotational correlation times of the isolated RBDs and the whole U1A, determined by dynamic polarization measurements, show that the linker region is highly flexible such that each RBD exhibits uncorrelated motion.     

Metal ion specificity at the catalytic site of yeast enolase.

A new, more gentle enzyme purification for yeast enolase was developed. A series of kinetic experiments was performed with yeast enolase where the concentration of Mg(II) is kept constant and at the Km' level; the addition of Mn(II), Zn(II), or Cu(II) gives a hyperbolic decrease in the enzyme activity. The final velocity of these mixed-metal systems is the same as the velocity obtained only with Mn(II), Zn(II), or Cu(II), respectively. The concentration of the second metal that gives half-maximal effect in the presence of Mg(II) is approximately the same as the apparent Km (Km') value measured for that cation alone. Direct binding of Mn(II) to apoenolase in the absence and presence of Mg(II) shows that Mn(II) and Mg(II) compete for the same metal site on enolase. In the presence of D-2-phosphoglycerate (PGA) and Mg(II), only a single cation site per monomer is occupied by Mn(II). Water proton relaxation rate (PRR) studies of enzyme-ligand complexes containing Mn(II) and Mn(II) in the presence of Mg(II) are consistent with Mn(II) binding at site I under both conditions. PRR titrations of ligands such as the substrate PGA or the inhibitors orthophosphate or fluoride to the enolase-Mn(II)-Mg(II) complex are similar to those obtained for the enolase-Mn(II) complex, also indicating that Mn(II) is at site I in the presence of Mg(II). High-resolution 1H and 31P NMR was used to determine the paramagnetic effect of enolase-bound Mn(II) on the relaxation rates of the nuclei of the competitive inhibitor phosphoglycolate. The distances between the bound Mn(II) and the nuclei were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the 47-kDa talin fragment and the 32-kDa vinculin fragment with acidic phospholipids: a computer analysis.

In recent in vitro experiments, it has been demonstrated that the 47-kDa fragment of the talin molecule and the 32-kDa fragment of the vinculin molecule interact with acidic phospholipids. By using a computer analysis method, we determined the hydrophobic and amphipathic stretches of these fragments and, by applying a purpose-written matrix method, we ascertained the molecular amphipathic structure of alpha-helices. Calculations for the 47-kDa mouse talin fragment (residues 1-433; NH2-terminal region) suggest specific interactions of residues 21-39, 287-342, and 385-406 with acidic phospholipids and a general lipid-binding domain for mouse talin (primary amino acid sequence 385-401) and for Dictyostelium talin (primary amino acid sequence 348-364). Calculations for the 32-kDa chicken embryo vinculin fragment (residues 858-1066; COOH-terminal region) and from nematode vinculin alignment indicate for chicken embryo vinculin residues 935-978 and 1020-1040 interactions with acidic phospholipids. Experimental confirmation has been given for vinculin (residues 916-970), and future detailed experimental analyses are now needed to support the remaining computational data.     

An internal cysteine plays a role in the maintenance of the latency of human fibroblast collagenase.

The cDNA that encodes the proenzyme form of human fibroblast collagenase has been expressed in Escherichia coli. It has been shown by a number of criteria to be functionally identical with the enzyme isolated from human sources. Mutations of each of three cysteine residues found in procollagenase were constructed by site-directed mutagenesis of the cDNA. The relative activities of these mutants were compared to the wild-type enzyme. All of the mutants retained proteolytic activity, but not necessarily on collagen. Mutations that interfere with the formation of the sulfhydryl bridge in the carboxy-terminal domain in some cases increased and in other cases decreased the rate of casein cleavage. On the basis of extensive autolysis within E. coli of a mutant with a replacement of cysteine-73, the procollagenase molecule produced appeared to be either spontaneously active or perhaps more susceptible to autolytic activation, despite the continued presence of the propeptide. Experiments designed to capture the active forms of the mutant by use of the irreversible inhibitor alpha 2-macroglobulin showed that some degree of latency still persisted in the autolytic mutant. These findings suggest that the cysteine at position 73 is important for maintaining the proenzyme in an inactive state but that the maintenance of latency in MMPs may be a complex process, involving a number of interactions between the propeptide domain and the remainder of the collagenase molecule.     

Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.     

Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus

PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase. Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme. On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate. PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V. In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences. The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased. In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C. Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells. Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.     

Solution structure and interaction with basic and acidic fibroblast growth factor of a 3-kDa human platelet factor-4 fragment with antiangiogenic activity.

Platelet factor-4 is a protein belonging to the family of ELR-negative CXC chemokines which binds to fibroblast growth factor and inhibits its mitogenic activity. Platelet factor-4 also inhibits tumor growth by mechanisms involving antiangiogenesis. Antiangiogenic activity in vitro has also been shown for the 24-residue C-terminal fragment of the protein, which decreases the affinity between basic fibroblast growth factor and its cell-surface receptor. In this study, the preferential conformation of this fragment in solution has been determined and has been found to be composed of two helical subdomains. In addition, we show that the fragment forms a specific 1:1 complex with acidic and basic fibroblast growth factors and that both subdomains are probably required for inhibition of fibroblast growth factor-driven mitogenesis. Finally, we show that the binding of the fragment alters the structure of the fibroblast growth factors, although some of such alterations do not seem related with the inhibition of mitogenic activity. Since this fragment has recently been shown to inhibit fibroblast growth factor-induced angiogenesis in vivo when injected intraperitoneally, these results are relevant for developing new antiangiogenic treatments.     

Cooperativity in the calcium ion-induced quenching of the intrinsic fluorescence of a series of normal and GLA-deficient bovine prothrombin fragment 1 molecules

Ca²⁺ titrations of the intrinsic fluorescence of a series of -carboxyglutamic acid (GLA)-deficient bovine prothrombin fragments 1 yield response Hill plot parameters useful for characterization of the metal ion-binding process. 11-, 10-, and 9-GLA fragments 1 exhibitT _m (the (Ca²⁺)_total concentration at which ln (B/F)=0 in the response Hill plot) values between 0.2 and 0.3 mM. A 22-fold increase inT _m to 5.4 mM is observed for 8-GLA fragment 1.T _m decreases to 3.8 mM for the 7- and 6-GLA proteins. The value ofh, about 2.8±0.2 for 11-, 10-, and 9-GLA fragments 1, abruptly decreases to 1.2–1.3 for 8-, 7-, and 6-GLA fragments 1. The observed degree of quenching induced by saturating levels of calcium ions is affected by both changes in the intrinsic fluorescence of the metal ion-free proteins and in the maximum possible degree of quenching in the presence of calcium. The kinetic characteristics of the calcium ion-induced quenching of the intrinsic fluorescence of 6-GLA fragment 1 are identical to those observed in 10-GLA fragment 1, suggesting that the fluorescence quenching observed in the 6- and 10-GLA fragments 1, while different in magnitude, involves similar processes. Observation of an abrupt change in the relative electrophoretic mobilities of 11- to 9-GLA fragments 1 compared to 8- to 6-GLA fragments 1, in the absence or presence of Ca²⁺, suggests the existence of a major protein conformation change which occurs concomitantly with the noted changes inT _m andh response Hill plot parameters. Molecular mechanics calculations suggest a structural hypothesis unifying these observations. Central to this model is the presumption of the existence of hydrogen bond-mediated interactions between metal ion-binding sites.     

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Assignments and structure determination of the catalytic domain of human fibroblast collagenase using 3D double and triple resonance NMR spectroscopy

We report here the backbone 1HN, 15N, 13C, 13CO, and 1H NMR assignmentsfor the catalytic domain of human fibroblast collagenase (HFC). Three independentassignment pathways (matching 1H, 13C, and 13CO resonances) were used to establishsequential connections. The connections using 13C resonances were obtained fromHNCOCA and HNCA experiments; 13CO connections were obtained from HNCO andHNCACO experiments. The sequential proton assignment pathway was established from a 3D(1H/15N) NOESY-HSQC experiment. Amino acid typing was accomplished using 13C and15N chemical shifts, specific labeling of 15N-Leu, and spin pattern recognition from DQF-COSY. The secondary structure was determined by analyzing the 3D (1H/15N) NOESY-HSQC. A preliminary NMR structure calculation of HFC was found to be in agreement withrecent X-ray structures of human fibroblast collagenase and human neutrophil collagenase aswell as similar to recent NMR structures of a highly homologous protein, stromelysin. Allthree helices were located; a five-stranded -sheet (four parallel strands, one antiparallelstrand) was also determined. -Sheet regions were identified by cross-stranddN and dNN connections and by strong intraresidue dN correlations, and were corroborated byobserving slow amide proton exchange. Chemical shift changes in a selectively 15N-labeledsample suggest that substantial structural changes occur in the active site cleft on the bindingof an inhibitor.     

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 r?le of gelatinases is discussed.