238

Biochimica et Biophysica Acta, 539 (1978) 238--247 © Elsevier/North-Holland Biomedical Press

BBA 28448 BINDING OF LATENT R H E U M A T O I D SYNOVIAL COLLAGENASE TO COLLAGEN FIBRILS

CAROL A. VATER, CARLO L. MAINARDI and EDWARD D. HARRIS, Jr. Connective Tissue Disease Section, Department of Medicine, Dartmouth-Hitchcock Medical Center, Hanover, N.H. 03755 (U.S.A.)

(Received May 17th, 1977) (Revised manuscript received October 26th, 1977 )

Summary 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 b o u n d to fibrils equally well at 24°C and 37°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 n o t inhibited by the presence of ~2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase b u t 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.

Introduction In the past five years it has been recognized that many mammalian collagenases are released in a latent precursor form by cells which synthesize them. Collagenases from bone [1--3] and polymorphonuclear leukocytes [4--6] were isolated in latent form after Gross and co-workers [7,8] demonstrated that collagenase from tadpole tail tissue in culture contained a 'procollagenase' and an 'activator' proteinase. Similar latent collagenases activated in vitro by proteinases have been found in medium from cultures of bovine gingival explants [9] and guinea pig [10] and rabbit [11] bones, uterus and skin, and monolayers of fibroblasts from bovine gingiva [ 12 ], human skin [ 13 ], and rabbit synovium [11,14]. Dayer et al. [15] have found a latent collagenase in very high concentrations in culture medium of isolated and adherent rheumatoid synovial cells. In serum-free medium, the latent collagenase

239 produced by rheumatoid synovial cells could be activated by non-enzymatic means as well as by limited proteolysis [16,17]. The latent collagenase produced by gingival fibroblasts [12] and rhumatoid synovial cells [16,17] did not form a complex with a2 macroglobulin, the plasma protein responsible for more than 90% [18,19] of circulating anti-collagenase activity. Subsequently, we have demonstrated that isolated, adherent rheumatoid synovial cells produced plasminogen activator as well as latent collagenase [17]. To activate latent collagenase in cultures of rheumatoid synovial cells it was sufficient to add only plasminogen, which was activated by a plasminogen activator to plasmin which, in turn, activated latent collagenase. Since plasminogen is present in increased concentration in synovial fluids from inflamed joints [ 20], we have suggested that plasmin is one enzyme which might well be involved in vivo in activation of latent collagenase at the synovial (pannus) junction with cartilage, bone or tendon in the destructive phase of this disease. Central to this hypothesis has been the unproved assumption that collagenase in latent form would be capable of binding to fibrillar substrate while remaining incapable of catalysis of cleavage of the triple helix (i.e., its normal function in activated form). In two other systems, accumulated data has been contradictory to this assumption. Neither the zymogen of collagenase from tadpole tissue characterized by Harper et al. [7,8] nor the zymogen of polymorphonuclear leukocyte collagenase [4] was capable of binding to substrate. In contrast, Gillet et al. [21] have used collagen bound to Sepharose to purify latent collagenase from mouse bone culture medium. The collagen prepared in this fashion was not in fibril form, however, and the reactions were carried out at 4°C. In the experiments presented here we observed that latent rheumatoid synovial collagenase bound to collagen fibrils over a broad temperature range and that binding as well as activation by proteinases occurred in the presence of a2 macroglobulin.

Materials and Methods Chemicals used in the experiments were obtained from the following sources: bacterial collagenase (crude and CLSPA) and TosPheCH2Cl trypsin from Worthington Biochemical Corporation; Dulbecco's modified Eagle's medium, lactalbumin hydrolysate and dog serum from Grand Island Biological Corporation; purified a m m o n i u m sulfate from Schwartz-Mann; Ultrogel A c A 34 from L K B Instruments; trypsin inhibitor (soybean) from Sigma Chemical Corporation; rabbit anti-human a2 macroglobulin (a2M) and antihuman IgM from Cappell Laboratories; Sepharose 4B from Pharmacia; 14Clabelled glycine and proline from N e w England Nuclear Corporation; [all]acetic anhydride from ICN Radiochemicals. All other chemicals were the highest reagent grade available.

Preparation of latent collagenase from rheumatoid synovial cells Rheumatoid synovial tissue was obtained at time of arthroplasy and/or synovectomy performed on patients at Mary Hitchcock Memorial Hospital.

240 Lining layers of synovium were dissected and cells were dissociated using bacterial collagenase and trypsin [15]. Isolated rheumatoid synovial cells adherent to plastic dishes were cultured in Dulbecco's modified Eagle's medium in 10% fetal calf serum until confluent. Serum was then removed from the medium and cells were washed. Medium was then supplemented with 0.2% lactalbumin hydrolysate. Medium from unpassaged cell cultures containing latent collagenase but no detectable active enzyme was concentrated by pressure filtration using Amicon UM10 membranes, or by precipitation in ammonium sulfate (0--90% saturation at 4°C) and re-solubilization and dialysis against 0.1 M Tris'HC1/pH 7.6, 0.2 M NaC1/0.005 M CaC12/0.02% NaN3 (Buffer I). The enzyme was activated by limited proteolysis (see below) and using standard methods [22] was demonstrated to meet criteria established for a partially purified mammalian collagenase, i.e., capacity to cleave collagen in solution into two fragments (TC A and TC B) at 27°C with minimal capacity to degrade gelatin fragments further at 37°C and neutral pH, and susceptibility to inhibition by chelating agents (e.g., EDTA, 1-10 phenanthroline).

Purification of ~2M Whole human serum was dialyzed extensively against H20 at 4°C. The precipitating euglobulin fraction was discarded. The supernatant was concentrated by pressure dialysis using an Amicon XM-100A membrane and passed through a column (2.5 X 90 cm) of Ultrogel AcA 34 in Buffer I. Fractions eluted from the column were screened by immunodiffusion in agar plates using rabbit anti-human ~2M. Fractions containing high concentrations of ~2M and no IgM (assayed by immunodiffusion against anti-human IgM) were pooled.

Preparation of plasmin Plasmin was activated from plasminogen with urokinase (Calbiochem., one Plough unit/2.4 pg plasminogen, 2--3 h, 25°C). Plasminogen was partially purified from dog serum by affinity chromatography from a column of lysylSepharose using the method of Chibber et al. [23].

Collagen purification and preparation 14C-labelled collagen was extracted by 0.5 M acetic acid from minced skins of guinea pigs and purified as described previously [24]. 3H-labelled collagen was prepared by the method of Gisslow and McBride [25] from purified acidsoluble guinea pig skin collagen using [3H]acetic anhydride. Collagen was dissolved in 0.1 M acetic acid (2--3 mg/ml) and dialyzed against either 0.05 M Tris" HC1, pH 7.6/0.2 M NaC1/0.02% NaN3 or 0.06 M sodium cacodylate, pH 7.5/0.2 M NaCl/0.02% NaN3 (use of the latter buffer solution was a helpful suggestion of Yves Eeckhout, Louvain, Belgium). Native reconstituted collagen fibrils were formed by incubation of 100 pl aliquot portions at 37°C overnight in glass tubes (6 X 50 mm, KIMAX). Collagen fibrils were rinsed twice with Buffer I or 0.06 M sodium cacodylate, pH 7.5/0.2 M NaC1/0.005 CaC12/0.02% NaN3 (cacodylate buffer) before use.

Assay for binding of latent collagenase to collagen fibrils Latent collagenase was concentrated as described above and incubated with

241 fibrils in either Buffer I or cacodylate buffer. The final concentration of calcium was adjusted to 0.01 M. The temperature of latent enzyme and duration of its exposure to fibrils were varied and are detailed in the Results section. At the conclusion of each period of incubation fibrils were centrifuged at 5000 rev./min at 20°C for 10 rain in a temperature-controlled centrifuge (Sorvall RC 2B). Supernatant solutions were removed with disposable glass pipettes and fibrils were repeatedly washed by resuspension in 600/al Buffer I or cacodylate buffer and subsequent aspiration of the wash solution w i t h o u t prior centrifugation. After washing, fibrils were resuspended either in Dulbecco's modified Eagle's medium and 0.2% lactalbumin hydrolysate or buffer solution to a final volume equal to that of the original reaction mixtures. Tos-Phe-CH2C1 trypsin or plasmin was added to activate latent collagenase and the calcium concentration adjusted to 0.01 M. Collagenase assays were stopped before any one set of fibrils was lysed to more than 75% of the original volume of gel. After incubation at 37°C to measure collagenolysis, the reaction mixtures were centrifuged at 14 000 rev./min for 10--15 min at 22°C and aliquot portions of the supernatant were added to 10 ml of modified Bray's solution [26] and counted in a scintillation spectrometer (Packard Instruments) as a measure of collagenase activity. The total radioactivity of collagen fibrils was determined by total digestion of washed control fibrils at 37°C by bacterial collagenase and subsequent counting of an aliquot portion of the reaction products. EDTA (Sigma) in sufficient concentration to exceed that of ionized calcium present was added to certain tubes during each assay of collagenase after activation to give assurance that bound, activated b u t inhibited collagenase had no capacity to solubilize 3H- or 14C-labelled fibrils. In summary, four steps were repeated for test and control samples in all experiments. First, latent collagenase was exposed to fibrils for binding to take place; second, the fibrils were washed to rid the assay of enzyme n o t b o u n d or weakly attached to fibrils; third, latent collagenase b o u n d to fibrils was activated; and finally each tube was assayed for collagenase by incubation of the fibrils at 37°C. One unit of collagenase represented 1/~g collagen in fibril form degraded per h at 37°C.

Results

Effects of multiple washing of collagen fibrils with bound collagenase Preliminary experiments showed that incubation of latent collagenase with fibrils at temperatures from 17--35°C resulted in significant association of the latent enzyme with fibrils. In the following discussion, the word 'binding' is used to signify this association and does n o t imply specific binding such as that between enzyme and substrate at a catalytic site. The following experiments were designed to test the effect of multiple washing of fibrils u p o n residual b o u n d enzyme. Latent coUagenase was b o u n d to [3H]collagen at 17°C for 2.5 h and after removal of s u p e m a t a n t solutions the fibrils were washed one, three or five times before activation and assay for collagenase activity. As shown in Fig. 1, the amount of enzyme associated with fibrils was stable after three washes under the conditions used. As another measure of the effectiveness o f

242

5o

57 °C

,.~4c

. ~

/ "

o

~ 2C

w I0

o'.5

,:5 ~

1.5

ASSAY TIME (HOURS) F i g . 1. E f f e c t o f w a s h i n g u p o n a p p a r e n t b i n d i n g o f l a t e n t c o l l a g e n a s e t o c o l l a g e n fibrils. L a t e n t e n z y m e w a s i n c u b a t e d w i t h 3 H - l a b e l l e d c o l l a g e n f i b r i l s at 17°C f o r 2 . 5 h . F i b r i l s w e r e w a s h e d e i t h e r o n e (a []), t h r e e (A . . . . . . A), o r five t i m e s (o o) in 6 0 0 #1 o f 0 . 0 5 M T r i s . HC1, p H 7 . 6 / 0 . 0 0 5 M CAC12/0.02% N a N 3 a t 1 7 ° C f o r 10 r a i n e a c h t i m e . A f t e r a c t i v a t i o n w i t h 10 #1 t r y p s i n ( 6 0 0 # g / m l , 2 2 ° C , 3 0 r a i n ) f o l l o w e d b y a d d i t i o n o f 1 0 #l s o y b e a n t r y p s i n i n h i b i t o r ( 2 4 0 0 # g / m l ) t o a c t i v e b o u n d l a t e n t c o l l a g e n a s e , t h e fibrils w e r e a s s a y e d f o r b o u n d a c t i v i t y b y i n c u b a t i n g t w o f i b r i l s f r o m e a c h g r o u p f o r 1, 1 . 5 , a n d 2.5 h. T h e d a t a are s h o w n as a m e a n o f t w o d e t e r m i n a t i o n s a t e a c h p o i n t .

washing the fibrils, A560nm for each wash was recorded. The original culture medium had an absorption of 1.640, reflecting the phenol red content of the medium. In four consecutive washes the As60 readings were 0.055, 0.015, 0.007 and 0.000, respectively, with reference to cacodylate buffer. This was supportive evidence that fibril washing was complete. In subsequent experiments reported here, all fibrils were washed at least three times after binding.

The rate and extent of binding of latent collagenase to fibrils compared with that o f activated enzyme Collagenase activity bound to fibrils was examined as a function of time of binding at 17°C for both activated and latent forms. This temperature was

r-~ 2oo z .::) o rn ch lz I00

z cD

g, DURATION of BINDING (rains) Fig. 2. Binding o f a c t i v a t e d a n d l a t e n t c o l l a g e n a s e as a f u n c t i o n o f time at 17°C. E n z y m e s w e r e p r e p a r e d as d e s c r i b e d in t h e t e x t a n d a d d e d to fibrils. A f t e r i n t e r v a l s n o t e d i n t h e f i g u r e , t h r e e t u b e s c o n t a i n i n g activated enzyme/collagen and three containing latent collagenase/collagen were removed from the bath a n d w a s h e d t h r e e t i m e s . T h e n t h e t u b e s c o n t a i n i n g l a t e n t c o l l a g e n a s e / c o l l a g e n w e r e a c t i v a t e d a n d all w e r e a s s a y e d f o r c o l l a g e n a s e a c t i v i t y at 37°C. R e s u l t s w e r e e x p r e s s e d as t h e m e a n _+S . D . E L , l a t e n t c o l l a g e n a s e (o . . . . . . o); E', a c t i v e c o l l a g e n a s e (e e).

243 below that at which active mammalian collagenases cleave significant amounts of collagen fibrils. A sample of latent collagenase was divided into two equal portions. One part was activated with trypsin (10 ~g/ml, 22°C for 30 min, followed by soybean trypsin inhibitor, 4 0 p g / m l ) . Latent and activated collagenases were incubated with pre-formed fibrils for times shown in Fig. 2. Active enzyme was shown to bind faster and more completely to fibrils than did the latent form. After 22.5 h, binding of latent collagenase had reached 67% of that achieved by the active form.

Temperature dependence of binding In additional experiments, binding of latent collagenase at 24 and 37°C was compared. Approximately 65% of the collagenase added b o u n d to fibrils at both temperatures. In tubes to which EDTA had been added prior to activation negligible radioactivity was found in supernatant solutions after incubation at 37°C, ruling out the possibilities that non-collagenolytic enzyme activity (e.g., trypsin) or non-enzymatic mechanisms were responsible for fibril degradation after activation of the latent collagenase.

Extent of binding of latent collagenase to fibrils The percentage of total available latent enzyme which bound to collagen fribils varied with conditions of binding and concentration of enzyme and substrate used, b u t generally fell within a range of 40--70%. The total enzyme added in each sample was determined as follows. Control fibrils were incubated with culture medium never exposed to cells {unconditioned medium) throughout the binding period and were washed in parallel with fibrils incubated with latent enzyme. After binding and washing, an identical quantity of latent collagenase as that added to the test fibrils was then added to the fibrils not previously exposed to enzyme, and unconditioned medium was added to fibrils with latent collagenase b o u n d to them. Then all samples were activated with trypsin or plasmin and final incubation at 37°C was carried out. Comparable results were obtained when either trypsin or plasmin was used to activate latent collagenase. In general, better binding was achieved using the cacodylate than in Tris buffer, and cacodylate was used in later studies of binding. In one such experiment, 100 pl of concentrated latent collagenase was incubated with 100 gl [14C]collagen fibrils in cacodylate buffer, pH 7.5, at 35°C for 17.5 h. The fibrils were washed four times (10 min at 35°C each} with cacodylate buffer, resuspended in medium with or without collagenase as described above and activated with approx. 0.38 mg activated plasminogen during the final 37°C incubation. 45.1 ± 1.7 units out of a total of 68.4 +_ 5.5 units of collagenase (1 unit = 1 ug collagen degraded/h at 37°C) were b o u n d to the fibrils, a bound: total ratio of 66%. It was not known whether latent collagenase which did not bind to fibrils in a given experiment was incapable of binding, or whether all available binding sites had become saturated. This was examined in the following manner. Latent collagenase was incubated with one set of 14C-labelled collagen fibrils (B) for 6 h at room temperature. The supernatant was removed and added to a second tube of fresh fibrils (C) for 17.5 h. To each of a matched set of fibrils (A) was added the total amount of latent collagenase for the entire 23.5 h. All fibrils were then washed and

244 TABLE I EFFECT OF ADDITIONAL LAGEN FIBRILS (22°C)

SUBSTRATE

UPON BINDING OF LATENT

COLLAGENASE

TO COL-

Source of E L *

[14C]Collagen fibril substrate (pg)

Exposure of latent c o l l a g e n a s e to substrate ( b i n d i n g t i m e , h)

Collagenase bound (units)

A 3 8 0 p l C M ** B 380 plCM C S u p e r n a t a n t f r o m B at 6 h * * *

200pg 200 pg 200 pg

0 ~--~->6

213 ±8.1 183.5 + 9.8\ J 264 8 0 . 7 + 7.0

>23.5 6

> 23.5

* EL, latent collagenase. * * CM, 3 0 0 #I i n c o n c e n t r a t e d culture m e d i u m f r o m r h e u m a t o i d s y n o v i a l cells plus 8 0 DI Buffer I. * * * V o l u m e o f supernatant adjusted t o 3 8 0 / z l .

assayed for b o u n d collagenase activity. The availability of additional substrate provided in the B--C series resulted in a 24% increase in b o u n d latent enzyme activity over that apparent in the limited substrate A set (Table I). These data indicated that the geometry of the assay system we used was a factor which limited binding potential of latent enzyme and made it unlikely that enzyme which did n o t bind was altered in some way to be incapable of binding.

Binding in the presence of ~2M Latent enzyme was b o u n d to 3H-collagen fibrils in cacodylate buffer, pH 7.5, at 35°C for 21 h in the presence of sufficient partially purified ~2M to inhibit completely an identical a m o u n t of enzyme activated prior to addition of ~2M. This quantity of a2M (approx. 135 pg) was worked o u t empirically before beginning the assay. After washing and activation, assay revealed that there

TABLE II E F F E C T O F c¢2M O N B I N D I N G O F L A T E N T C O L L A G E N A S E

Reconstituted

Binding

TO COLLAGEN FIBRILS

Washes

A c t i v a t i o n ** *

Percent lysis of c o l l a g e n fibrils at 3 7 ° C

3 3 3

T T T

3 4 ± 2.6 4 5 _+ 4 . 4 29 ± 1 3 . 0 **

3 3 3 3 3

EL+T EL + T (E L + T ) + S B T I (E L + T ) + a 2 M T

61 54 42 10 5

c o l l a g e n fibrils *

Fibrils + E L Fibrils + E L Fibrils + E L Fibrils Fibrils Fibrils Fibrils Fibrils

+ + + + +

------

+ ~2 M + BSA

+ ~2M

(15 #g) (15 #g)

± -+ -+ + _+

5.0 3.2 1.5 1.5 1.2

* 3H-labeBed collagen fibrils (0.3%) in cacodylate b u f f e r , ** T h e large S . D . f o r t h e s e s a m p l e s w a s d u ~ t o o n e v a l u e in t r i p l i c a t e s a m p l e s b e i n g o f f ~ 1 0 0 % f r o m

the o t h e r t w o . * * * A c t i v a t i o n w a s a t 3 0 m i n a t 2 2 ° C u s i n g 1 0 / ~ g / m l t r y p s i n ( T ) in each set o f t u b e s . A b b r e v i a t i o n s : E L , l a t e n t c o l l a g e n a s e ; B S A , b o v i n e s e r u m a l b u m i n ; T, t r y p s i n ( 1 0 /~g/ml); S B T I , soybean trypsin inhibitor.

245

was no decrease in activity in samples in which binding had been performed in the presence of crzM (Table II). The slight increase in activity noted in the samples might reflect non-specific stabilization of enzyme in the presence of CQM. Since CQM has the capacity to inhibit activated enzyme, it was of interest to determine whether crzM bound as an active proteinase inhibitor to collagen fibrils. Active enzyme was added to fibrils previously incubated with LY?Mand then washed three times, as were samples to which latent enzyme had been bound. No difference was noted in the activity of activated enzyme (Table II) indicating that azM was washed without difficulty from the fibrils. Discussion Use of reconstituted fibrils from purified collagen, partially purified collagenase, and defined artificial buffer systems rather than natural tissue substrates and serum-containing medium and buffers enabled us to minimize unknown variables which might seriously affect binding. Conversely, we cannot extrapolate ’ completely these data to conditions in vivo where collagen is immersed within a matrix of proteglycans and the extracellular fluid. However, assurance that latent collagenase does bind to collagen substrate under conditions proscribed here should facilitate experiments designed to ‘trap’ enzyme secreted by cells on collagen gel, to localize by immunofluorescent studies the cells in heterogeneous populations (such as rhumatoid synovial cells) which ‘procedure and release collagenase, to define the true nature (zymogen versus enzyme - inhibitor complex) of latent rheumatoid synovial collagenase, and to study effects of proteoglycans on binding of latent enzyme. The concepts of how mammalian collagenases are secreted and act upon substrate in tissues have been evolving rapidly. In recent years it has become apparent that many collagenases are released from cells synthesizing them as inactive or latent forms [l-17]. Activation has been achieved in vitro by limited proteolysis, although there is no concensus that the latent collagenases are true zymogens or procollagenases activated by specific cleavage of covalent bonds. Indeed, the evidence that several different proteinases as well as nonenzymatic methods can activate collagenase [3,6,11,17] supported the concept that latent collagenase represent an inhibitor - enzyme complex. But what of the fate of latent collagenases once they are released into the extracellular space? There was a wide range of possibilities. One was that collagenases in latent form were, in essence, ‘dead’ enzymes which could be cleared from tissues and catabolized. A second was that the latent enzyme not complexed by cllzM or other proteinase inhibitors could nevertheless remain within the extracellular tissues associated with substrate and essentially await activation. The data reported here support this latter hypothesis. We have not proved that a specific recognition or binding site separate from the catalytic site exists. Our data are consistent with that hypothesis, but the nature of collagen with its general cationic charge and strong non-specific attraction for many substances, including IgG [ 271, may be sufficient to hold latent collagenase in extracellular tissues until it is activated. Previous studies have demonstrated that helical structure is esential for maximal rates of

246

catalysis by a rabbit t u m o r collagenase [28] and the r h e u m a t o i d synovial enzyme [29]. It is certainly possible, b u t n o t tested by these experiments, that the helical and/or fibrillar structure of native collagen facilitates binding of latent collagenase. It is difficult to design experiments to study binding of collagenase (which is available only in partially purified f o r m ) t o non-helical collagen (e.g., gelatin) in solution. Use of collagen or gelatin fixed to solid support matrices would n o t resemble the precise fibrillar tertiary structure of collagen as it is found in vivo. The system developed here should serve as a model study of important and as y e t not studied questions. H o w is latent enzyme bound to substrate catabolized? Does binding result in loss of stability of enzyme so that, in the absence of proteolytic activators, potential activity of b o u n d enzyme decays rapidly? Or is the converse true? Does b o u n d latent enzyme decay more or less rapidly than active enzyme in the absence of proteinase inhibitors? Our knowledge of the nature of latent collagenase is t o o meagre for understanding of w h y these molecules bind more slowly and less completely to fibrils than does the activated form at temperatures where the comparison can be made (i.e., temperatures t o o low for effective collagenolysis). It is of interest that whereas there was no significant difference in the a m o u n t of binding of latent collagenase to fibrils at 24 and 36°C demonstrated in our studies here, previous data have shown an as much as 10-fold increase in the rate of cleavage of fibrils by active rheumatoid synovial collagenase over a temperature change from 30 to 36°C [29]. This difference between effects of temperature upon binding and catalysis axe additional support for the probability that these t w o functions are governed by separate determinants on the enzyme and/or substrate.

Acknowledgements This work was supported by USPHS grant AM14780 from the NIAMDD, by a Clinical Research Center grant from the Arthritis Foundation, and by a generous gift from the New Hampshire Chapter of The Arthritis Foundation. Synovium removed at times of therapeutic surgery was kindly provided by Drs. Stuart Russell, Forst Brown, Leland Hall, Michael Mayor and R o b e r t Porter. Ms. Laura Lefkowitz kindly prepared the manuscript.

References 1 Vaes, G. (1972) Biochem. J. 1 2 6 , 2 7 5 - - 2 8 9 2 Vaes, G. and E e c k h o u t , Y. (1975) in Protides of Biological Fluids (Peters, H., ed.). pp. 391--397, P e r g a m m o n Press, Oxford 3 E e c k h o u t , Y. and Vaes, G. (1974) Proc. Arch. Int. Physiol. Biochim. 82, 786 4 Or on sk y, A.L., Perper, R,J. and Schroder, H.C. (1973) Nature 2 4 6 , 4 1 7 - - 4 1 9 5 Kruze, D. and Wojtecka, E. (1972) Biochim. B i o p h y s . A c t a 285, 436---446 6 Williams, H.R., Lin, T.-Y. and Perper, R.J. (1976) Arthritis R he um. 1 9 , 8 2 9 (Abst.) 7 HarPer, E., Bloch, K.J. and Gross, J. (1971) Biochemistry 10, 3035--3041 8 Harper, E. an d Gross, J. (1972) Biochem. Biophys. Res. C o m m u n . 48, 1 1 4 7 - - 1 1 5 2 9 Birkedal-Hansen, H., Cobb, C.M., Taylor, R.E. and F u n m e r , H.M. (1976) Biochim. Biophys. Acta 429, 229- -2 38 10 Heard, S.B. (1976) J. Oral Pathol. 5, 17--32 11 Sellers, A., Cartwright, E., Murphy, G. and Reynolds, J.J. (1977) Biochem. J. 163, 303--307

247 12 Birkedal-Hansen, H., Cobb, C.M., Taylor, R.E. and Funmer, H.M. (1976) J. Biol. Chem. 251, 3162-3168 13 Bauer, E.A., Stricklin, G.P., Jeffrey, J.J. and Eisen, A.Z. (1975) Biochem. Biophys. Res. Commun. 64, 232--240 14 Harris, Jr., E.D., Reynolds, J.J. and Werb, Z. (1975) Nature 257,243--244 15 Dayer, J.-M., Krane, S.M., Russell, R.G.G. and Robinson, D.R. (1976) I~oc. Natl. Acad. Sci. U.S. 73, 945---949 16 Mainardi, C.L., Vater, C.A., Werb, Z. and Harris, Jr., E.D. (1976) Arthritis Rheum. 19, 809a 17 Werb, Z., Mainardi, C.L., Vater, C.A. and Harris, Jr., E.D. (1977) N. Engl. J. Med. 296, 1017--1023 18 Werb, Z., Burleigh, M.C., Barrett, A.J. and Starkey, P.M. (1974) Biochem. J. 139, 359--368 19 Woolley, D.I~., Robert, D.R. and Evanson, J.M. (1976) Nature (Lond.), 261,325--327 20 Kishner, I. and Somerville, J.A. (1971) Arthritis Rheum. 14, 560--570 21 Gillet, C., Eeckhout, Y. and Vaes, G. (1977) FEBS Lett. 74,126--128 22 McCroskery, P.A., Richards, J.F. and Harris, Jr., E.D. (1975) Biochem. H. 152, 131--142 23 Chibber, B.A.K., Deutsch, D.G. and Mertz, E.T. (1974) Methods Enzymol. 34, 424--432 24 Gllmeher, M.J., Francois, C.J., Richards, L. and Krane, S.M. (1964) Biochim. Biophys. Acta 93, 585-602 25 Gisslow, M.T. and McBride, B.C. (1975) Anal. Biochem. 68, 70--78 26 Evanson, J.M., Jeffrey, J.J. and Krane, S.M. (1968) J. Clin. Invest. 47, 2639--2651 27 McCroskery, P.A. and Harris, Jr., E.D. (1973) Fed. Proc. 3 2 , 6 1 4 28 McCroskery, P.A., Wood, Jr., S. and Harris, Jr., E.D. (1973) Science 182, 70--71 29 Harris, Jr., E.D. and McCroskery, P.A. (1974) N. Engl. J. Med. 290, 1---6