©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Matrix Metalloproteinase-2 Is an Interstitial Collagenase
INHIBITOR-FREE ENZYME CATALYZES THE CLEAVAGE OF COLLAGEN FIBRILS AND SOLUBLE NATIVE TYPE I COLLAGEN GENERATING THE SPECIFIC ¾- AND ¼-LENGTH FRAGMENTS (*)

(Received for publication, November 18, 1994)

Ronald T. Aimes (1)(§) James P. Quigley (2)(¶)

From the  (1)Department of Biochemistry and Cell Biology and (2)Department of Pathology, State University of New York, Stony Brook, New York 11794

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The 72-kDa gelatinase/type IV collagenase (MMP-2) is a member of the matrix metalloproteinase (MMP) family of enzymes. This enzyme is known to cleave type IV collagen as well as degrade denatured collagens. However, native interstitial collagens are reportedly resistant to MMP-2 and are thought to be susceptible only to the interstitial collagenases MMP-1 and MMP-8. In this study we report that both human and chicken MMP-2, free of tissue inhibitors of metalloproteinases (TIMPs) are capable of cleaving soluble, triple helical type I collagen generating the ¾- and ¼-length collagen fragments characteristic of vertebrate interstitial collagenases. MMP-2 cleaves at the same Gly-Ile/Leu bond in the collagen alpha chains as interstitial collagenases with k and K values similar to that of MMP-1. MMP-2 also is capable of degrading reconstituted type I collagen fibrils. The closely related 92-kDa gelatinase/type IV collagenase (MMP-9) is unable to cleave soluble or fibrillar collagen under identical conditions indicating that the specific collagenolytic activity of MMP-2 is not a general property of gelatinases. That MMP-2, a potent gelatinase, also can cleave fibrillar collagen provides an alternative to the proposal that two enzymes, an interstitial collagenase and a gelatinase, are required for the complete dissolution of stromal collagen during cellular invasion.


INTRODUCTION

Extracellular matrix (ECM) (^1)degradation is mediated by a battery of secreted enzymes produced by various cell types(1) . The matrix metalloproteinases (MMPs) are a family of at least 10 zinc-dependent endoproteinases that function at neutral pH and cooperatively hydrolyze most of the proteins in the ECM (2) . Interstitial collagens (collagens I, II, and III), the most abundant proteins of vertebrate connective tissue, are particularly resistant to proteases including trypsin, plasmin, and other members of the serine and sulfhydryl proteinase families(1, 3) . The interstitial collagenases, MMP-1 (4) and MMP-8(5) , belong to an MMP subfamily that specifically cleaves native triple helical collagens, yielding ¾- and ¼-length collagen fragments as a result of the hydrolysis of a single Gly-Ile/Leu bond in each alpha chain of the collagen molecule(2, 6, 7) .

The collagen fragments produced by the interstitial collagenases are susceptible to the gelatinases (MMP-2 and MMP-9), a second MMP subfamily, that rapidly degrade denatured collagens and collagen fragments(8, 9) . MMP-2 has been identified in a wide range of normal and malignant cells and in several species(2, 9, 10) . Cooperation between the interstitial collagenases and the gelatinases is thought to be essential for clearing interstitial collagens in connective tissue during inflammatory and invasive processes. While capable of cleaving Gly-Leu and Gly-Ile bonds (along with other peptide bonds) in denatured collagens(11, 12) , MMP-2 is thought to be unable to cleave the same bonds in native interstitial collagens(9, 13, 14) . However, MMP-2 is capable of cleaving native type IV and V collagens(2) . The expression of the gelatinases/type IV collagenases have been correlated with the metastatic state(15) , and these enzymes are believed to influence the ability of a cell to invade and metastasize due to their ability to degrade basement membrane type IV collagen(16) .

The study of the activation and substrate specificity of the gelatinases has been hampered by the co-isolation of the tissue inhibitors of metalloproteinases (TIMPs). TIMP-1 and TIMP-2 usually co-purify in tight, noncovalent complexes with both the zymogen and active forms of the 92- and 72-kDa gelatinases, respectively(8, 17) . The ability to purify TIMP-free native enzyme (18, 19) and the production of recombinant progelatinases (20, 21) has allowed for more detailed and accurate assessments of the properties of these enzymes.

In the present study we show that both human and chicken TIMP-free MMP-2 cleave collagen fibrils and soluble native type I collagen specifically generating the ¾- and ¼-length fragments characteristically produced by the interstitial collagenases. Values for the Michaelis-Menten constants K and k were determined using soluble collagen I as a substrate, and MMP-2 was found to be comparable with MMP-1 in catalyzing this reaction. Implications of these findings to the roles of MMP-2 in cell invasion are discussed.


EXPERIMENTAL PROCEDURES

Materials

Bovine type I collagen, p-aminophenylmercuric acetate (APMA), Clostridium histolyticum collagenase, TPCK-treated trypsin, and Brij-35 were purchased from Sigma. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) reagents, protein molecular weight standards, and Coomassie Brilliant Blue R-250 were purchased from Bio-Rad. Immobilon polyvinylidene difluoride membrane was purchased from Millipore (Bedford, MA). [^3H]Acetic anhydride was purchased from Amersham Corp. Recombinant human pro-MMP-9 pro-MMP-2 and TIMP-2 were gifts of Dr. R. Fridman (Wayne State University, Detroit, MI). Recombinant human MMP-1 and MMP-3 was a gift of Dr. G. McGeehan (Glaxo, Research Triangle Park, NC). Human pro-MMP-1 was a kind gift of Dr. H. Nagase (University of Kansas, Kansas City, KS).

Enzyme Purification

Chicken TIMP-free 72-kDa progelatinase was purified from serum-free conditioned medium harvested from Rous sarcoma virus-transformed chicken embryo fibroblasts as described previously(19, 22) . Protein concentrations were determined by silver-stained SDS-PAGE or spectrophotometrically. pro-MMP-2 was activated with 2 mM APMA for 3 h at 25 °C; pro-MMP-9 and pro-MMP-1 were activated for 6 h at 37 °C using 2 mM APMA.

Collagen Purification and Radiolabeling

Native rat tail type I collagen was isolated from adult rat tail tendons and used either before labeling or after acetylation with [^3H]acetic anhydride as described previously(23) .

SDS-Polyacrylamide Gel Electrophoresis

SDS-PAGE was performed according to Laemmli(24) . Silver staining was performed as described previously(25) . Gelatin zymography was performed on 10% polyacrylamide gels as described previously(22) . Analysis of collagenase assays was performed on 5-15% polyacrylamide gradient gels using reducing conditions. Gels were stained with 0.2% Coomassie Brilliant Blue in 50% methanol, 10% acetic acid (v/v) and destained with 20% methanol, 5% acetic acid (v/v).

Enzyme Assays

[^3H]Gelatin degradation assays were performed as described previously(22) . Soluble, triple helical collagen cleavage was assayed in 50 mM Tris (pH 7.5), 200 mM NaCl, 10 mM CaCl(2), 0.05% Brij-35 (CAB/Brij) containing the indicated concentrations of collagen and enzyme at 25 °C unless otherwise indicated. ^3H-Labeled rat type I collagen fibril degradation was performed using a modification of the method of Johnson-Wint(26, 27) . Aliquots (30 µl) of collagen (1.6 mg/ml) were allowed to polymerize at 37 °C for 2 h in individual wells of a 96-well microtiter plate. Active proteinases were added to the fibrillar collagen in 100 µl of CAB/Brij and allowed to incubate at 37 °C for 18 h, and the supernatants were analyzed for soluble ^3H-labeled material by liquid scintillation spectroscopy. Trypsin (100 µg/ml) was used as a control for nonspecific protease-sensitive material. C. histolyticum collagenase (1000 units/ml) was used to determine total counts/min available.

Determination of K(m) and k

Values for K(m) and k were determined using acid-soluble rat tail type I collagen as a substrate(28) . APMA-activated MMPs were incubated at 25 °C with 0.1-2.2 µM collagen in 100 µl of CAB/Brij and analyzed as above. Reaction rates were determined from the stained gel using scanning imaging densitometry and the equation: % collagen cleaved = (TC^A) ((TC^A) (alpha1))(28) . The amount of active enzyme in each experiment was determined by titration with TIMP-2 in a [^3H]gelatin degradation assay for MMP-2 or in a triple helical collagen cleavage assay for MMP-1.

Gel Slicing

Collagenase activity in polyacrylamide gels was assayed as described previously (29) with minor modifications. Enzyme samples were electrophoresed on nonreducing SDS-12% polyacrylamide gels and then washed twice for 30 min in 2.5% Triton X-100 followed by water for 1 h. Each lane was cut into 2-mm slices (30 slices/lane), and individual slices were added to 150 µl of CAB/Brij containing 10 µg of rat tail type I collagen and 2 mM APMA. Reactions were incubated for 72 h at 25 °C. An aliquot (10 µl) was removed from each sample for gelatin zymography, and the remaining material was analyzed by SDS-PAGE for collagen cleavage products as indicated above.


RESULTS

TIMP-free Preparations of MMP-2 Are Capable of Cleaving Native, Triple Helical Type I Collagen

The ability of MMP-2 to degrade native, triple helical type I collagen was assessed using TIMP-free preparations of native chicken pro-MMP-2 (19) and human recombinant pro-MMP-2 (20) (Fig. 1). An assay temperature of 25 °C was used to ensure that the collagen remained native and triple helical(27) . Human and chicken MMP-2 cleave rat type I collagen in an enzyme concentration-dependent manner generating ¾- and ¼-length fragments (Fig. 1A, lanes3-6) with similar electrophoretic mobility to the collagen fragments generated by human recombinant MMP-1 (lanes7 and 8). The collagenolytic activity of MMP-2 is dependent on activation as latent chicken pro-MMP-2 fails to generate the and collagen fragments (lane2). Neither human MMP-9 (lanes9 and 10) nor recombinant MMP-3 (data not shown) is capable of cleaving native type I collagen, demonstrating that the cleavage is specific to MMP-2 and MMP-1. Chicken and human MMP-2bulletTIMP-2 complexes treated with APMA displayed markedly reduced collagenolytic activity (lanes11 and 12) compared with TIMP-free MMP-2 assayed using identical conditions. That the cleavage of type I collagen is not particular only to rat collagen is demonstrated in Fig. 1B. Human (lane3) and chicken (lane2) MMP-2 as well as MMP-1 (lane4) are able to cleave the alpha chains of native bovine type I collagen yielding proteolytic products with similar electrophoretic mobility; human MMP-9 (lane5), MMP-3, and trypsin (data not shown) are unable to cleave bovine collagen. Specific cleavage of native chicken type I collagen yielded similar results (data not shown). In all reactions with different type I collagens two to four times more MMP-2 than MMP-1 was required to generate equivalent amounts of specific TC^A and TC^B cleavage fragments. Time-dependent collagenolysis of rat type I collagen (17.5 µg) was linear during a 4-h period for both MMP-1 (25 ng) and MMP-2 (100 ng), and the TC^A fragments were apparent within 15 min of incubation (Fig. 1C).


Figure 1: Specific cleavage of rat tendon and bovine skin type I collagen by chicken and human MMP-2. Native type I collagen was incubated in the presence of latent or active MMPs in 0.1 ml of CAB/Brij for 4 h at 25 °C, and the reactions were reduced, electrophoresed on 5-15% polyacrylamide gels, and stained with Coomassie Blue. A, rat collagen I (17.5 µg) was incubated alone (NoEnzyme) or with chicken pro-MMP-2 (cproMMP-2), chicken MMP-2 (cMMP-2), recombinant human MMP-2 (hMMP-2), recombinant human MMP-1 (hMMP-1), recombinant human MMP-9 (hMMP-9), chicken or human MMP-2/TIMP-2 complexes (cMMP-2/TIMP-2 and hMMP-2/TIMP-2, respectively). The amount of enzyme used is indicated in µg at the top of each lane. B, bovine collagen I (10 µg) was incubated alone (No Enzyme) or with the indicated amounts of chicken MMP-2, recombinant human MMP-2, recombinant human MMP-1, and recombinant human MMP-9. C, rat collagen (17.5 µg, 1.35 µM) was incubated with 0.1 µg of human MMP-2 or 0.025 µg of human MMP-1 for 0-4 h at 25 °C and analyzed by SDS-PAGE, and scanning densitometry and the results were plotted as µg of collagen cleaved versus time. Inset shows the SDS-PAGE profile at the indicated times used in the densitometric analysis. In A and B the positions of molecular mass standards are indicated on the left (times 10 Da). alpha1 and alpha2 indicate the alpha1(I) and alpha2(I) peptide chains of type I collagen, respectively. beta indicates two cross-linked alpha1 chains and beta an alpha1 chain cross-linked to an alpha2 chain. TC^A and TC^B indicate the ¾ NH(2)-terminal and COOH-terminal collagen cleavage products, respectively.



In order to rule out that the collagenolytic activity exhibited by the TIMP-free preparation of chicken MMP-2 was due to a contaminating interstitial collagenase, a gel-slicing experiment was performed (Fig. 2). Chicken pro-MMP-2 was subjected to SDS-PAGE (Fig. 2A), and after electrophoresis the gel was washed and sliced into 2-mm slices, and each slice was incubated with APMA (to activate the zymogen) and rat type I collagen. Each reaction was analyzed by Coomassie Blue-stained SDS-PAGE (Fig. 2B) and gelatin zymography (Fig. 2C). The specific collagenolytic activity is present in the single fraction corresponding to 68-75 kDa (Fig. 2A and Fig. 2B, lane9) and coincides with the presence of the gelatinolytic activity of MMP-2 (Fig. 2C, lane9). No collagenolytic activity is found in the 30-50 kDa region of the gel where interstitial collagenases would migrate. When recombinant human MMP-1 is subjected to the same analysis, the collagenolytic activity is present only in the 40-45-kDa region of the gel (data not shown). These data support that the collagenolytic activity observed with the TIMP-free MMP-2 preparations is due to MMP-2 and not to the presence of a contaminating interstitial collagenase.


Figure 2: Collagenolytic activity of chicken TIMP-free MMP-2 preparation fractionated by SDS-PAGE. A preparation of chicken pro-MMP-2 (0.2 µg), isolated by gelatin-Sepharose and Mono Q chromatography, was electrophoresed on a 5.5 times 10-cm SDS-12% polyacrylamide gel and washed with 2.5% Triton X-100 and water. The gel was then sliced into 2-mm fragments. Each slice was incubated with 10 µg of rat collagen I and 2 mM APMA in 150 µl of CAB/Brij at 25 °C for 72 h. Following incubation the samples were analyzed for the presence of both the collagen cleavage products and gelatinase activity as described under ``Experimental Procedures.'' A, silver stain of chicken TIMP-free pro-MMP-2 run in a parallel lane of the gel that was sliced. Marks on the right of the gel indicate where the slices were made and numbered. B, Coomassie Blue-stained 5-15% gradient polyacrylamide gel of the collagen cleavage products generated after 72 h. C, a 10-µl aliquot from each reaction was analyzed by gelatin zymography to indicate the slice that contained MMP-2 gelatinolytic activity. Positions of molecular mass standards are indicated on the left (times 10 Da), and slice numbers are indicated at the top. Slice number 9, which contained the 62-kDa active gelatinase, also generated the specific TC^A cleavage fragment.



MMP-2 Cleaves Both Chains of Type I Collagen at the Same Site as MMP-1

Vertebrate interstitial collagenases cleave native type I collagen between Gly and Ile/Leu resulting in two proteolytic products: a ¾-length amino terminus and a ¼-length carboxyl terminus(30) . MMP-2-mediated cleavage of collagen I yields fragments of similar electrophoretic mobility to those produced by MMP-1 (Fig. 1, A and B). The exact site in collagen I cleaved by MMP-2 was determined by partial amino acid sequencing of the TC^B fragments. Bovine type I collagen was incubated with chicken MMP-2 at 25 °C, and the TC^B fragments generated were separated by SDS-PAGE, blotted to Immobilon polyvinylidene difluoride, and microsequenced(31) . The amino-terminal sequences of both the alpha1 and alpha2 carboxyl-terminal fragments are identical to those generated by interstitial collagenases (Table 1), further indicating that MMP-2 behaves as an interstitial collagenase.



Kinetic Properties of the MMP-2-catalyzed Hydrolysis of Type I Collagen

The K(m) and k values for human MMP-2 were determined. These constants also were determined for human MMP-1 and compared with published values. Due to the lack of synthetic active site titrants, recombinant TIMP-2 was used to determine the molar amount of active MMP-2 and MMP-1 in each assay. As can be seen from the data in Table 2the k of human MMP-2 is 16.2 h, nearly identical to the 16.6 hk value obtained for MMP-1 and similar to the 19.5 h value reported for human MMP-1(28) . The K(m) for human MMP-2 (8.5 µM) is higher than that of MMP-1 (1 µM) and may account for the reduced reaction rate of MMP-2 seen in Fig. 1.



Collagen Fibril Degradation by MMP-2

Since MMP-2 could cleave soluble monomeric collagen, it was of interest to determine if MMP-2 possessed the most restrictive and unique activity of interstitial collagenases: cleavage of native collagen fibrils. The ability of MMP-2 to degrade fibrillar collagen was examined in a reconstituted fibril assay. Trypsin controls were included to establish the background level of degradation attributed to non-fibrillar collagen. The data in Fig. 3show that at equal molar amounts MMP-2 can degrade fibrillar collagen as efficiently as MMP-1. MMP-2 generated substantially more soluble radiolabeled material than either 100 µg/ml trypsin or equal gelatinolytic amounts of MMP-9. This indicates that the material released from the fibrils by MMP-2 and MMP-1 was not gelatin or non-fibrillar collagen and that MMP-2, like MMP-1, is capable of solubilizing interstitial collagen.


Figure 3: Degradation of fibrillar collagen by MMP-1 and MMP-2 and lack of degradation by MMP-9. Fifty micrograms of ^3H-labeled rat tail tendon type I collagen (1.6 mg/ml) was allowed to gel at 37 °C in microtiter plate wells as described under ``Experimental Procedures.'' After the collagen fibrils were washed and equilibrated with CAB/Brij, proteases were added to the wells in triplicate and allowed to incubate at 37 °C for 18 h. Following incubation, the supernatants were removed and analyzed by liquid scintillation spectroscopy. Buffer alone was used as a control for background and was subtracted from the mean counts/min released by each enzyme. Data are represented as percent of total collagen released above background. Errorbars indicate the standard errors of the mean. Enzymes were used at the indicated concentrations and are labeled as follows: TPCK-treated trypsin (Trypsin), recombinant human MMP-1 (MMP-1), recombinant human MMP-2 (MMP-2), and recombinant human MMP-9 (MMP-9).




DISCUSSION

This study demonstrates that MMP-2 is capable of cleaving native type I collagens under conditions that preclude any helix denaturation (27) . This activity is not common to gelatinases as equal amounts of MMP-9 failed to cleave collagen under identical conditions. That the collagenolytic activity of the MMP-2 preparations was not due to a contaminating protease was shown by a gel-slicing analysis (Fig. 2) in which the single slice containing the 62-kDa MMP-2 gelatinolytic activity also exhibited the specific cleavage of collagen. The use of purified recombinant human MMP-2 further supports that MMP-2 alone is responsible for the observed collagenolysis.

MMP-2 cleaves the same peptide bonds in the collagen alpha chains as the interstitial collagenases. The TC^B fragments produced by MMP-2 (Table 1) have the same amino-terminal sequence as the TC^B produced by other vertebrate collagenases(30) . These data, in conjunction with the apparent identical electrophoretic mobility of the TC^A fragments, suggest that the MMP-2 behaves as an interstitial collagenase cleaving the (P(1))Gly-(P(1)`)Ile/Leu bond in the alpha chains of native triple helical collagen. MMP-2 is known to cleave after glycine with hydrophobic residues, glutamine, asparagine, or serine in the P(1) site(11, 12) . However, it has long been thought that the gelatinases/type IV collagenases were unable to cleave these bonds in native interstitial collagens(2, 8, 9) . Why the ability of MMP-2 to cleave native collagen I has not been detected previously is unclear. When we initially isolated and characterized chicken MMP-2 this collagenolytic activity was not observed (22) as was the case for a number of studies using human MMP-2 isolated from cultured cells(9, 13, 14) . One explanation is the presence of TIMP-2, which co-purifies with MMP-2 from many sources. The presence of TIMP-2 in MMP-2 preparations markedly decreases the rate and extent of activation of pro-MMP-2 and also decreases the specific activity of the enzyme preparations(19, 20, 32) . TIMP-2 also may interfere with the ability of MMP-2 to bind to collagen in a productive fashion. We have shown that an equal molar amount of TIMP-complexed MMP-2, activated in the same manner as TIMP-free MMP-2, generates markedly reduced amounts of specific collagen cleavage products compared with TIMP-free MMP-2 (Fig. 1A). The ability of avian MMP-2 to cleave native type I collagen albeit at reduced levels has been previously reported(33) . However, this activity was thought to be a peculiarity of the avian enzyme, and the effect of the associated TIMP-2 on the reduced activity was not explored. It would appear that any TIMP-2, free or in a complex with the zymogen, in MMP-2 preparations would markedly diminish the specific interstitial collagenolytic activity of MMP-2.

Cleavage of native type I collagen required two to four times more MMP-2 than MMP-1 to generate similar amounts of the specific cleavage fragments (Fig. 1). This might indicate that MMP-2 is less efficient than MMP-1 in bringing about collagen dissolution. However, determination of the kinetic constants for this reaction revealed that the k values of MMP-2 and MMP-1 were equal (Table 2), although the K(m) value for MMP-2 (8.5 µM) was higher than that of MMP-1 (1 µM). Given these data and the concentration of collagen I used in this study (1.4 µM) the Michaelis-Menten equation indicates that the rate of cleavage of type I collagen by MMP-1 should be 4.1 times greater than that of MMP-2, a value consistent with our observations. Interestingly, the 8.5 µMK(m) value for MMP-2 indicates that assays carried out with collagen concentrations of 100 µg/ml (0.4 µM), 20-fold below the K(m), would yield little specific collagen cleavage products, and this also may explain why this activity has not been observed in other studies. That the k values for MMP-2 and MMP-1 are similar suggests that at suitably high collagen concentrations (i.e. >8 µM) MMP-2 and MMP-1 would cleave native collagen at similar rates. Such high concentrations of collagen might only be encountered in the interstitial stroma.

Most significantly, MMP-2 is capable of extensively degrading reconstituted fibrillar collagen. Since soluble, monomeric collagen exists at negligible levels in the extracellular stroma, fibrillar collagen is a more relevant biological substrate. That MMP-2 degrades fibrillar collagen to an equal or greater extent as MMP-1 (Fig. 3) may indicate that in the fibril assay the K(m) for MMP-2 is exceeded and MMP-2 functions as efficiently as MMP-1. When an equal gelatinolytic amount of MMP-9 was assayed, the level of fibrillar collagen degradation did not exceed that of trypsin (Fig. 3). These data suggest that MMP-2 may have a role as an interstitial collagenase in vivo and be sufficient for the degradation of both the basement membrane collagen IV and the connective tissue collagens.

Degradation of interstitial collagens has long been believed to be dependent on the presence of two enzymes: interstitial collagenases and gelatinases. The data presented here suggest that MMP-2 may be capable of effecting the removal of interstitial collagens in the absence of collagenases. The role of MMP-2 in connective tissue remodeling in normal and pathological processes might, therefore, be re-evaluated. Expression of the gelatinases/type IV collagenases has been linked to the invasive potential of cells(15) . The functional role of the gelatinases/type IV collagenases is thought to be the degradation of type IV collagens, thereby allowing cells to traverse the basement membrane. While this may be true, MMP-2 may also facilitate the invasion of the underlying connective tissue ECM in which type I collagen predominates.

While several malignant cells, including A2058 and MDA-231, express MMP-1 in vitro(34) , others such as HT1080, HT-144, MDA-435, and MCF-7 do not express interstitial collagenase in vitro, yet these cells are invasive and appear to be capable of invading tissues that contain interstitial collagens(34, 35) . MMP-2, which is produced by these cells, could catalyze the degradation of such collagen. Furthermore, there are malignant cells that do not express interstitial collagenase in vivo as determined by in situ hybridization(36, 37) . In the latter case, it is possible that MMP-2 supplies the collagenolytic potential necessary to move across the basement membrane and through the interstitium. Tumor cells have been demonstrated to induce adjacent normal stromal cells to produce MMP-1(38) ; yet, the constitutive expression of MMP-2 may alone suffice for local collagenolysis.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant CA 55852 (to J. P. Q.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Institute for Cell and Developmental Biology predoctoral scholar supported by a fellowship from Merck and Company.

To whom correspondence should be addressed. Tel.: 516-444-3014; Fax: 516-444-3424.

(^1)
The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; APMA, p-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; TC, tropocollagen; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.


ACKNOWLEDGEMENTS

-We thank Ralph Reid at UCSF for his protein sequencing expertise and Dr. Rafael Fridman for providing reagents and for his insightful discussion of the data.


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