THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 hv The American Society for Biochemistry and Molecular Biology, Inc

Vol. 264, No. 15, Issue of May 25, pp. 8779-8785, 1989 Prrnted in U.S.A.

Interaction of Human Rheumatoid Synovial Collagenase (Matrix Metalloproteinase 1) and Stromelysin (Matrix Metalloproteinase 3) with Human a2-Macroglobulin and Chicken Ovostatin BINDING KINETICS AND IDENTIFICATION OF MATRIX METALLOPROTEINASE CLEAVAGE SITES*

(Received for publication, November 21, 1988)

Jan J. Enghild, Guy Salvesen, Keith Brew$, and Hideaki Nagasetll From the Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, the $Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, and the §Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, Kansas 66103

The homologous proteinase inhibitors, human a2- macroglobulin (a2M) and chicken ovostatin, have been compared with respect to their “bait” region sequences and interactions with two human matrix metallopro- teinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region se- quence was determined and the matrix metallopro- teinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the a2M bait region at the Glye79-Le~680 bond, and stromelysin at Glye7s-Le~s80 and Phe684-Tyres5 bonds. Sequence similarity in the bait region of members of the Q-

macroglobulin family is strikingly low. The kinetic studies indicate that a2M is a 150-fold better substrate for collagenase than type I collagen. Structural predic- tions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequi- site for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to a2M is slower than that of collagenase. Stromelysin reacts with ovo- statin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human a2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that a2M may play an important role in regulating the ac- tivities of matrix metalloproteinases in the extracel- lular space.

Connective tissue cells produce a group of related metallo- proteinases that digest various extracellular matrix macro- molecules (1-3). One of them, collagenase (matrix metallo- proteinase l),’ specifically digests collagen types I, 11, and I11 at a Gly-(Ile or Leu) bond located in the triple helical region approximately three-fourths of the distance from the NH, terminus to the COOH terminus (4-6). However, when these

* This work was supported by National Institutes of Health Grants AR 39189, CA29589, and GM 21363. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence should he addressed Dept. of Biochem- istry, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS 66103.

connective tissue cells follow the proposal by Okada et al. (3). The numerical names for matrix metalloproteinases secreted from

substrates are heat-denatured to form gelatin, collagenase has little or no action on them, indicating that the helical struc- ture of collagen molecules is important for enzyme recogni- tion. On the other hand, stromelysin (matrix metallopro- teinase 3), another metalloproteinase secreted from the con- nective tissue cells, has a wide spectrum of action on extra- cellular matrix macromolecules. It digests proteoglycans, fi- bronectin, laminin, type IV collagen, and gelatins and removes the NH,-terminal propeptides of type I procollagen (3, 7, 8). Stromelysin also digests casein and reduced carboxymethyl- ated transferrin (3, 7). Collagenase and stromelysin are about 55% identical in sequence (9-12), but stromelysin does not digest interstitial collagen types I and I1 although it has limited activity on type I11 collagen.’ The enzymic activities of these matrix metalloproteinases are thought to be regulated by inhibitors present in the tissue or body fluid, in particular by a tissue inhibitor of metalloproteinases (TIMP)3 which is also secreted from most of the connective tissue cells (13-15). However, the level of TIMP in body fluids such as plasma and rheumatoid synovial fluid is very low, and the predomi- nant inhibitory activity for collagenase resides in a,M (16, 17). Thus, a,M appears to be one of the major regulatory factors for the activity of matrix metalloproteinases in the extracellular space.

a,M is a major plasma proteinase inhibitor with M , = 725,000 (18, 19). The inhibitory mechanism of a,M is unique. Unlike many other proteinase inhibitors, it inhibits almost all endopeptidases from all four catalytic classes of protein- ases regardless of their specificities (20). The reaction of a proteinase and a,M requires proteolytic attack of the enzyme on a particular locus located near the middle of the quarter subunit, the so-called “bait” region. The cleavage of a peptide bond in this region triggers a conformational change in the a,M molecule that, in turn, entraps the enzyme without blocking the active site (21). Thus, the enzyme within the complex is able to catalyze the hydrolysis of low molecular weight substrates but is restricted from reaction with large protein substrates. Although the specificity of collagenase is limited to native interstitial collagens, the ability of a,M to inhibit collagenase indicates that a,M serves as a collagenase substrate. Earlier work of Werb et al. (22) indicates that this reaction takes place very slowly. On the other hand, our

Biochem. J. 258, 115-119. * Gunja-Smith, Z., Nagase, H., and Woessner, J. F., Jr. (1989)

The abbreviations used are: TIMP, tissue inhibitor of metallopro- teinases; a2M, a2-macroglohulin; APMA, 4-aminophenylmercuric acetate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis.

8779

8780 Binding of Matrix Metalloproteinases to Macroglobulins

kinetic studies of the binding of rabbit synovial collagenase to chicken ovostatin, an a,M-like protein from egg white (23), demonstrated that ovostatin is an excellent substrate for collagenase (24) in spite of the fact that the specificity of mammalian collagenase is believed to be limited to helical regions in collagen types I, 11, and 111. This observation suggested that the triple helices of collagen might not be an absolute requirement for the binding of collagenase to a substrate, since it is very unlikely that ovostatin contains a collagen-type helix in its “bait” region. Thus, information regarding the amino acid sequence of the collagenase-suscep- tible bait region of ovostatin and a,M may be important in developing an understanding of the interaction of collagenase with its substrates.

We report here the amino acid sequence of a &residue bait region of chicken ovostatin and the locations of collagen- ase cleavage sites in both chicken ovostatin and human aZM determined using human rheumatoid synovial collagenase. The sites of cleavage in the two inhibitors by stromelysin, a metalloproteinase homologous to collagenase, have also been determined. Detailed kinetic studies of the binding of human synovial collagenase with a2M and ovostatin indicate that azM is the best substrate for collagenase that has been char- acterized to date.

EXPERIMENTAL PROCEDURES4

RESULTS

Cleavage Sites of a2M by Collagenase and Stromelysin-The “bait” region of human a,M has been characterized with various proteinases. However, the sites of azM cleavage by connective tissue metalloproteinases have not been deter- mined, although a collagenase cleavage site has been predicted at the Gly679-Leu660 bond (40) since the sequence around this bond, Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly, is somewhat simi- lar to the collagenase cleavage site in interstitial collagens. Cleavage of aZM by either collagenase or stromelysin gener- ated fragments of M, = 98,000 and M, = 80,000 (Fig. 1). Two separate sequence analyses of the upper fragments of M, = 98,000 reveal a single sequence of Leu-Arg-Val-Gly-Phe-Tyr- Glu-Ser-Asp-Val, indicating that collagenase cleaved the Gly679-Le~680 bond as predicted. On the other hand, the NH2- terminal sequence of the fragment generated by stromelysin showed two sequences in an equal amount, Leu-Arg-Val-Gly- Phe-Tyr-Glu-Ser-Asp-Val and Tyr-Glu-Ser-Asp-Val-Met- Gly-Arg-Gly, indicating cleavage sites at Gly679-Le~680 and Phe684-Tyr685 (Fig. 2). Both the collagenase and stromelysin cleavage sites in azM are located in the region cleaved by other proteinases (40).

Sequence Analyses of the Ovostatin "Bait I'Region-Reaction of chicken ovostatin with human collagenase resulted in the cleavage of the M, = 165,000 subunit into two fragments of M, = 88,000 and M , = 78,000 (Fig. 1). Electrophoretic elution of both bands and subsequent sequence analyses of the frag- ments indicated that the fragment of M, = 88,000 was the COOH-terminal half of the ovostatin subunit and the frag- ment of M, = 78,000 the NHZ-terminal half. Two separate sequence analyses of the M, = 88,000 fragment generated by collagenase gave a single sequence as shown in Fig. 3, indi- cating that collagenase cleaves ovostatin subunits at a single site. Similar sequence analyses of the M , = 88,000 fragment

Portions of this paper (including “Experimental Procedures,” Figs. 1, 3, and 5-7, and Equations 1-4) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

generated by stromelysin were conducted. The major cleavage site of stromelysin in ovostatin was found at the Gly-Phe bond 4 residues on the COOH-terminal side of the collagenase cleavage site (Fig. 3). In the second experiment a minor sequence starting at the same site as collagenase was observed, but the major sequence started with the phenylalanine resi- due.

Thermolysin (23) and Serratia metalloproteinase (45) have been shown to be stoichiometrically inhibited by chicken ovostatin. The results of sequence analyses of the similar M, = 88,000 fragments generated by these enzymes are also summarized (Fig. 3). Although chymotrypsin is not inhibited by chicken ovostatin (241, the initial reaction of chymotrypsin with ovostatin generates the M, = 88,000 and M , = 78,000 fragments (24). Sequence analyses of the M , = 88,000 frag- ment are also shown (Fig. 3). From alignment of the NH,- terminal sequences of the proteolytic fragments generated by five different enzymes, a stretch of 34 amino acid residues in the proteinase-susceptible “bait” region of chicken ovostatin was assembled (Fig. 3). Although there is some sequence similarity in the bait region of azM and ovostatin, the level is quite low (Fig. 4). Out of 34 residues in the bait region of ovostatin, only 8 residues (24%) are found to be identical with human a2M, 11 residues (32%) with rat aZM, and 6 residues (18%) with the major isoform of a,-inhibitor 3.5

Binding of Collagenase to azM and Ovostatin-The rate of binding of human rheumatoid synovial collagenase to azM or ovostatin was determined using ‘251-labeled collagenase. The inhibitor was incubated with ‘251-collagenase (8 rg/ml) at 25 “C, and the reaction was terminated by the addition of 1,lO-phenanthroline and EDTA to give final concentrations of 17 and 8.3 mM, respectively. ‘251-Collagenase bound to a2M or ovostatin was quantified by measuring the radioactivity incorporated into the inhibitor band after electrophoresis of the sample on 5% polyacrylamide gels. A typical analysis is shown in Fig. 5. The addition of ‘z51-collagenase to apM in the presence of the chelating agents at the above concentra- tions completely prevented binding of the enzyme, indicating that the enzyme reaction was instantaneously terminated (Fig. 5).

The binding of collagenase to a2M was very rapid, and the data were analyzed to give an apparent pseudo first-order rate constant, kaPp, using Equation 3 (Miniprint Supplement). A plot of the data is shown in Fig. 5. The values of k,, obtained with various concentrations of a2M in this manner were plotted according to Equation 4 (see plot in Fig. 6A). The line was drawn according to the medium values of K, and kz obtained by the method of Eisenthal and Cornish-Bowden (38). The dissociation constant, K,, and the first-order rate constant for the irreversible formation of the a,M-collagenase complex, k,, were 171 nM and 0.48 s-’, respectively. The kz/ K, value of 2.8 X lo6 M-’ s-’ indicates that azM is approxi- mately 150-fold better a substrate for collagenase compared with human type I collagen (kcat/Km = 1.8 X lo4 M” s” (see Table I)).

The rate of binding of human collagenase with chicken ovostatin was slower compared with that to a Z M ; the K, and the k, values were 323 nM and 5.8 X s-’, respectively (Fig. 6B). However, the k2/K; = 1.8 X lo3 ”’ s-’ is comparable to the k J K , values reported for various types of interstitial collagens (see Table I).

Binding of Strornelysin to a2M and Ovostatin-The kinetic constants for the formation of a,M-stromelysin were deter- mined by the same method as for collagenase. Although stromelysin has proteolytic activity against various protein

J. J. Enghild, unpublished data.

Binding of Matrix Metalloproteinases to Macroglobulins

%" T . P L , T H . T L . S = l

8781

T . SGT . PA

TL CS C S

collagenase Stromelysin stromelysin

FIG. 2. Collagenase and stromelysin cleavage sites in azM bait region. Human aaM (1 nmol) was incubated with 0.8 nmol of collagenase for 2 min at 37 "C, and the reaction was terminated by 1 mM 1,lO- phenanthroline. Proteolytic fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described under "Experimental Procedures" and subjected to NH,-terminal sequence analysis. To identify the stromelysin cleavage sites, a2M (300 pmol) was reacted with 206 pmol of stromelysin for 10 min at 37 'C, and the reaction was terminated by 1 mM 1,lO-phenanthroline. After sodium dodecyl sulfate-polyacrylamide gel electro- phoresis proteins were transferred to polyvinylidene difluoride-Millipore Immobilon transfer membrane (35) and subjected to NHa-terminal analysis. The cleavage sites by other proteinases are shown: papain (PA), Streptomyces griseus trypsin (SGT), calf chymosin (CS), bovine chymotrypsin (CT), human plasmin (PL), bovine thrombin (TH), thermolysin (TL), and subtilisin ( S ) are after Mortensen et al. (40); bovine trypsin (T) and Staphylococcus aureus V8 proteinase ( S P ) after Mortensen et al. (40) and Hall et al. (42); porcine pancreatic elastase ( E ) after Sottrup-Jensen et al. (43); and human neutrophil cathepsin G (CG) and elastase (HLE) after Virca et al. (44).

Chicken Ovostatin ** * ** * * * ** * * **** LNAGFTASI--HTVALSAEVAREERGKRHILETIRL

R a t a p KPKVCERLRD-----

Human a p KPKMCPQLQQYEMHGPEGLRVGFYESDVM-GRGHARLVHVEEPHTETVRKY

R a t a, I n h i b i t o r 3 LPWAVKSP----LPQEP-PRKDPPPKDPVIETIRN

I l l I I I I I I I I I I I I NKGIPAAY--HLVSQSHMDAFLESSESP-TETRRSY

FIG. 4. Alignment of ovostatin bait region with human and rat azM and rat al-inhibitor 3. The bait region sequences of human aaM, rat a2M, and rat a,-inhibitor 3 (clone pRLA113/2J) are after Sottrup-Jensen et al. (19), Gehring et al. (46), and Braciak et al. (47), respectively. The gaps (-) are introduced to optimize the alignment; * indicates residues where ovostatin is identical with one or more of the other sequences; 1 indicates identical residues in the two a,Ms.

TABLE I Comparison of kinetic parameters for hydrolysis of macroglobulins, collagens, and octapapeptides

by human tissue collagenase and stromelysin All the values were obtained at 25 "C exceut for octaueutides (49) at 30 "C.

Enzyme Substrate K, or K , kZ or kcak k,/K, or LtlKrn

Ref.

(A) Tissue collagenase (human) a,M (human) Ovostatin (chicken)

Tissue collagenase (rabbit) Ovostatin (chicken) Stromelysin (human) a,M (human)

(B) Tissue collagenase (human) Type I collagen Human Calf

Rat Guinea pig

Type I1 collagen Human Calf Rat

Human Guinea pig

Type 111 collagen

Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln ( l e ) Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln (5") Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln ( 1 0 " ) Gly-Pro-Gln-Gly-Ile-Ala-Gly-Thr ( 1 1") Gly-Pro-Asp-Gly-Ile-Ala-Gly-Gln (8')

The numbers for octapeptides are after Fields et al. (49).

@ 0.17 0.32 0.57 0.10

0.8 0.8 0.9 0.9

2.1 1.6 1.1

1.4 0.7

3300

3600

2800

2400

s" x la M' s - ~ x 10"

483 280 0.58 0.18 6.0 1.1 5.8 5.6

0.15 1.8 9.5 1.2 6.0 0.70 5.5 0.60

0.28 0.013 0.75 0.047 1.3 0.11

~~~

160 11 5.0 0.72

200 0.0060

330 0.0093

270 0.0097

270 0.011

0.0018

This study This study

24 This study

48 48 48 48

48 48 48

48 48 49

49

49

49

49

substrates, the rate of binding with a2M was considerably with chicken ovostatin was even slower. The cleavage of the slower compared with that of collagenase; the K; and k2 values ovostatin subunit into the M, = 88,000 and M , = 78,000 obtained were 104 nM and 5.8 X s-' (k2/Ki = 5.6 X lo4 fragments was not completed even after a 2-h incubation at M-' s-I), respectively (Fig. 6C). The reaction of stromelysin 37 "C (Fig. 7). The complex formation was not clearly detected

Binding of Matrix Metalloproteinases to Macroglobulins 8782

by the gel electrophoresis method indicating that chicken ovostatin is not an effective inhibitor of stromelysin.

DISCUSSION

The kinetic data presented here demonstrate that the bind- ing of mammalian tissue collagenase to aZM is very rapid. Since proteolytic cleavage of the a2M quarter subunit is prerequisite for a proteinase to bind to a2M, the kz/K, values obtained in our study can be compared with kJKm values reported for human skin collagenase with various types of collagen (48) and synthetic substrates (49). As summarized in Table I, aZM serves as a far better substrate than any other substrate so far examined with tissue collagenase. The se- quence analysis of the COOH-terminal fragment of a2M gen- erated by collagenase indicated that this enzyme cleaves the predicted Gly679-Le~G80 bond furthest upstream in the bait region sequence. The sequence around the collagenase cleav- age site, Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly, is similar to Gly- Pro-Gln-Gly-(Ile or Leu)-(Ala or Leu)-Gly-X in collagen, but the azM bait region possesses an acidic and a basic amino acid residue at the P, and P'z sites, respectively (subsites are according to Schechter and Berger (50)). Recent studies by Fields et al. (49) on the sequence specificity of human skin fibroblast collagenase using a series of synthetic octapeptides with sequences based on potential collagenase sites indicate that amino acid substitutions influenced the rate of hydrolysis of peptides. Interestingly, the substitution at the Pp site with an acidic residue, aspartic acid (peptide 8 in Table I), de- creased the susceptibility about 3-fold (49). Nevertheless, the sequence specificity alone could not account for the hydrolysis of native interstitial collagens at a single site. Fields et al. (49) re-emphasized the view that the local conformational features of the collagen molecule at the cleavage site govern the specificity of collagenase. Indeed, the k,,,/K,,, values for all of the synthetic peptides were considerably lower than those for collagen types I, 11, and 111 although their kcat values are higher than those for collagens (Table I). azM is a better substrate than type I collagen for tissue collagenase by a factor of about 150. The high susceptibility of a,M to colla- genase is also likely to be controlled by the local conformation of the apM bait region. Gettins and Cunningham (51) have concluded from 'H NMR studies of native and methylamine- treated human apM that the bait region is a flexible loop with high mobility. This was extended by Gettins et al. (52), with secondary structure predictions from the amino acid sequence, to provide a model for the bait region as a loop composed of two antiparallel strands of p structure (residues 680-685 and 695-700) separated by a turn, with the majority of proteinase cleavage sites being located within the two strands of p sheet. We have carried out similar secondary structure predictions for the sequences around the bait region in both rat and human a,Ms, as well as for the ovostatin bait region sequence reported here using both the Chou and Fasman procedure (53) and the DELPHI program (both were utilized on-line through the Protein Identification Resource of the National Biomedical Research Foundation: programs PRPLOT and DELPHI). Neither prediction program supported the pro- posed two-stranded p structure of Gettins et al. (52) for any of the three sequences, although a turn centered around residue 678 was predicted for both human and rat a2Ms. A weak p structure prediction was obtained for residues 680- 685 by the Chou and Fasman procedure, but this was not confirmed by the DELPHI program, while the region corre- sponding to the second strand of Gettins et al. (52), residues 695-700 in human azM, showed a higher probability for helix formation than for p structure by the method of Chou and

Fasman but was predicted to form a coil structure by the DELPHI program. This was also the case for the correspond- ing sequence in the rat protein. Based on these results, we propose that the bait region, although perhaps having a loop structure and being flexible and accessible to solvent and proteinases, does not have any dominant secondary structure of a conventional type. In fact, the high level of susceptibility of a large number of the peptide bonds in this region (Figs. 2 and 4) would be inconsistent with their involvement in exten- sive H-bonding arrangements of the type proposed by Gettins et al. (52). The high susceptibility of the Glys79-Le~680 bond in human a2M to collagenase cannot be readily explained at present. Similarly, the lower susceptibility of chicken ovo- statin to collagenase, although the kz/Ki values are compa- rable to kcat/Km values for type I collagen, must reflect the combination of the sequence in this region and the local conformation of the bait region. It may be speculated that the flexible nature of this region allows it to exist in a multiplicity of conformational states which could fit specificities of various proteinases.

The kinetic parameters for the reaction of stromelysin with azM and ovostatin indicate that stromelysin binds to these macromolecules at a much slower rate than collagenase. These results were unexpected, as stromelysin has a broader speci- ficity on various protein substrates (3, 7, 8, 11, 27-29, 54) whereas the action of collagenase is limited to interstitial collagens. Analyses of the cleavage sites in azM and ovostatin revealed that stromelysin hydrolyzes the GlyG79-Le~680 or Phe684-TyrG85 bond in a2M at an equal rate and primarily the Gly-Phe bond in ovostatin. A Gly-Phe bond is also present on the NH,-terminal side of the stromelysin cleavage site in azM, but this bond was not attacked during the formation of the stromelysin-a2M complex. These results indicate that stromelysin favors hydrophobic amino acid residues at both the P, and P', sites and that the substrate specificities of collagenase and stromelysin are clearly distinguishable. The preferential digestion of a bond between the hydrophobic residues indicates that the specificity of stromelysin is similar to that of an acid metalloproteinase purified from human articular cartilage, which has been shown to cleave the B chain of insulin either at the Ala-Leu or Tyr-Leu bond (55).

Ovostatins belong to the family of a-macroglobulins as indicated by their similar NH2-terminal sequences, quater- nary structure, and proteinase inhibition mechanism (24,56- 59). A thiol ester is also present in all ovostatins so far characterized (56, 60, 61) with the notable exception of the one from chicken egg white (24). Complete primary structures have been reported for human a2M (19,62), rat azM (46), and rat al-inhibitor 3 (47), and extensive overall sequence identity has been demonstrated, e.g. human and rat azM share 73% identity (46) and rat a2M and rat a,-inhibitor 3 are 58% identical (47). Partial sequence analysis of human pregnancy zone protein has also shown 68% identity to human a,M (63). However, the sequences around the bait regions of these proteins are highly divergent in comparison to the rest of the sequence, having only 10% identical residues (19, 46, 47, 61), and show no significant sequence correlation in this area when compared using the program ALIGN (64). This diversity of the bait region sequences appears to be a general feature of the proteins in the a-macroglobulin family. The stretch of 34 residues in the chicken ovostatin bait region is also very different from other known bait region sequences (Fig. 4 ) . It is perhaps relevant to this observation that the amino acid residues in the ovomucoid third domain which form close contacts with serine proteinases also show high variability (65). Sequence variations in the bait region may reflect ad-

8783 Binding of Matrix Metalloproteinases to Macroglobulins

aptational changes associated with the emergence of special- ized a-macroglobulins that inhibit specific proteinases. Chicken ovostatin has been shown to bind rapidly and tightly to microbial metalloproteinases such as thermolysin (23) and Serratia proteinase (45). The binding of human tissue matrix metalloproteinases to chicken ovostatin is not as efficient as for these microbial enzymes. Furthermore, chicken ovostatin does not inhibit serine and cysteine proteinases (24). In contrast, although aZM binds and forms stable complexes with almost all endopeptidases, Serratia proteinase-a,M com- plexes are not stable upon prolonged incubation, and about 90% of proteolytic activity can be restored (66). Such an effect was not seen with ovostatin, as it forms a tight complex with the proteinase (46). Thus, ovostatin may function as an anti- bacterial agent in egg white to protect the embryo from infection. On the other hand, the rapid binding of tissue collagenase by a2M suggests that a2M may play an important role in regulating tissue breakdown by acting as an inhibitor of matrix metalloproteinases. Connective tissue cells are ca- pable of producing a tissue metalloproteinase inhibitor of M, = 27,000, TIMP (13-15), and the synthesis of TIMP by the cultured cells can be increased by the agents or factors which induce the production of matrix metalloproteinases (67, 68). However, competition experiments indicate that azM is a much more effective inhibitor of collagenase than TIMP (69), suggesting that a2M may be the major collagenase inhibitor, especially in inflamed lesions. For example, in rheumatoid synovial fluid, the level of a,M can be 0.7-1.0 mg/ml, approx- imately one-third of the normal plasmal level (70,71). Depos- its of a2M in the rheumatoid synovial lining cells (72) and collagenase-a,M complex in rheumatoid synovial fluid (70, 73) further suggest the importance of a2M in regulating col- lagenolysis.

Acknowledgments-We thank Dr. Salvatore V. Pizzo for his en- couragement of this work. We are also grateful to Rikako Suzuki and Ida Thdgersen for their expert technical assistance and L. Denise Byrd for typing the manuscript.

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