Two Latent Metalloproteases of Human Articular Cartilage That

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Two Latent Metalloproteases of Human Articular Cartilage That Powered By Docstoc
					THEJOURNAL BIOLOGICAL
         OF        CHEMlSTRY                                                                        Vol. 259, No. 6, Issue of March 25, pp. 3633-3638,19&1
0 1984 by The American Society of Biological Chemist%Inc.                                                                               Printed in U.S.A.



Two Latent Metalloproteasesof Human Articular CartilageThat
Digest Proteoglycan"
                                                                                                (Received forpublication, September 6 , 1983)


                J. Frederick Woessner,Jr. and MarieG . Selzer
                From the Departments Biochemistry and Medicine, University of Miami School of Medicine, Miami,Florida 33101
                                   of



   Human articular cartilage contains very low levels                    treatment. Due to thepossible presence of tissue inhibitors of
of metalloprotease activity; the activity in 1 g of car-                 proteases in cartilage, it would be necessary to extract the
tilage is approximately equivalent to the activity of 1                  enzymes and to separate them from such inhhkors to be
pg of trypsin. Development of a  sensitive assay, based                  certain that theirfull activity was expressed.
on the digestion radioactive proteoglycan, has
                 of                                made                     In this report we describe the extraction and characteriza-
it possible to study protease activity in 1-2-g speci-                   tion of metalloprotease activities from single specimens of
mens of cartilage. Cartilage was extracted with Tris                     human cartilage which have wet weights of 1-2 g. The sensi-
buffer in the coId and with Tris buffer containing 10                    tivity of existing assay methods has been increased about 30-
m CaCh at 60 "C. The extracts were passed through
  M                                                                      fold, and methods have been developed for homogenizing and
Sepharose 6B; two major and two minor metallopro-                        extracting these small amounts of tissue. It was discovered
tease activities were detected. A neutral metallopro-




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                                                                         that both neutral and acid metalloproteases are present in
tease activity, pH optimum 7.4, was found as a latent
form of M, = 56,000. It could be activated with ami-                     cartilage in latent forms. These forms can be activated with
nophenylmercuric acetate or trypsin with a resultant                     mercurials or trypsin, with a concomitant decrease in molec-
decrease of M, to 40,000. An acid metalloprotease,   pH                  ular weight. The metal requirements of these proteases have
optimum 5.3, also occurred as a latent form of M, =                      been established. Although more than 20 metalloproteases of
50,000. Activationconverted this to M, = 35,000.                         mammalian origin have been reported in the literature, the
Removal of calcium ions by dialysis reduced theactiv-                    cartilage metalloprotease is the only one that has an acid pH
ity of the neutral enzyme by 8 0 4 5 % and of the acid                   optimum.
enzyme by 100%.Both activities were restored by 10
m Ca". Both enzymes were completely inhibited by
  M                                                                                         MATERIALSANDMETHODS
    M
1 m o-phenanthroline in the presence of excess cal-                         Enzyme Assay-We have previously described an assay for pro-
cium. This inhibition was overcome by l m Zn2+and,
                                            M                            teases based on the digestion of proteoglycan monomer prepared from
to a lesser extent, by Co2+. These proteases maybe                       bovine nasal cartilage (3). The monomer is entrapped in polyacryl-
important in the metabolism of the cartilage matrix                      amide gel beads, the pore size is adjusted to retain monomers, but to
and in its destruction in osteoarthritis.                                permit the escape of digestion products smaller than approximately
                                                                         200,000 daltons. This assay is used in order to identify proteases in
                                                                         cartilage that can attack the protein core of proteoglycans in the
  ~~



                                                                         extracellular matrix. The present assay method follows the published
                                                                         description quite closely, except that the incubation time is prolonged
  In 1976 (1) we discovered that human articular cartilage               and thesubstrate is labeled with tritium. Labeling was by the method
contains metalloprotease activity capable of cleaving the core           of Montelaro and Rueckert (4). Lyophilized proteoglycan monomer
                                                                         was allowedto swell overnight in 0.3 M sodium phosphate buffer, pH
protein of cartilage proteoglycan at physiological pH. This              7.2, at 4 "C. This suspension was then diluted 2-fold to a final
activity was blocked by chelating agentsand could be restored            concentration of 10 mg of proteoglycan/ml. To 750 mg of   proteoglycan
by Znz+or Co2+.Studies of the enzyme were impeded by the                 in 75 ml buffer were added 10 mCi of 13H]acetic anhydride (New
very low levels of activity found. However, it was possible to           England Nuclear; 50 mCi/mmol). The mixture was stirred for 30 min
achieve partial purification and characterization ( 2 ) by start-        at 24 "C. The product was dialyzed exhaustively against water a t
ing with large quantities of tissue (1200 g from approximately           4"C, then frozen and lyophilized. Polyacrylamide beads were pre-
                                                                         pared using this radioactive substrate (3). For enzyme assays, 4 mg
500 pairs of patellae). Although the proteolytic activities are          of dry beads (approximately 1 mg of proteoglycan, 20,000 cpm) were
low, theyappear to be important in regulating the slow                   weighed and placed in a tube. Enzyme and buffer were added to a
metabolic turnover of the matrix proteoglycans. Protease                 total volume of0.2 ml, and incubation was for 18 h at 37 'C. The
activity may also play a central role in degenerative changes            assay buffer contained 0.05 M Tris/HC\, pH 7.5,0.01 M CaCl,, 0.05%
of the cartilage, particularly in aging and osteoarthritis. In           Brij-35, 0.02% sodium azide, and 0.2 M NaCl. After incubation, 0.5
this connection, it would be very useful to be able to measure           ml of water was added, the beads were removed (3), and 0.2 ml of
                                                                         supernatant was transferred to a 20-ml glass scintillation vial. Ten
enzyme activities in small samples of cartilage (1-2 g). This            ml of Aquasol(New England Nuclear) were added and shaken.
would permit the study of tissue from a single patient to                Counting was carried out in a Tricarb Model 3003 (Packard) liquid
detemine the effect of factors such as age, disease, and drug            scintillation counter at 18 "C. Results are expressed as counts/min
                                                                         released per mg of dry bead. No correction was made for the fact that
   * This work was supported by Grant AM-16940 from the National         the aliquot was only 2/7 of the total digestion volume. The assay
Institutes of Health. A preliminary report of part of this work was      method was shown to give a linear response with time for 18 h and
presented at the Symposium on Connective Tissue in Health and            with enzyme concentration, using cartilage enzymes and trypsin (0.3-
Disease, Melbourne, Australia, p.  59 (abst.), 1982. The costs of        3.0 ng). At this level of sensitivity there is appreciable noise; repro-
publication of this article were defrayed in part by the payment of      ducibility is approximately f a%.
page charges. This article must therefore be hereby marked "aduer-          Extraction of Enzymes from Tissue-Human patellae were removed
tiernent" in accordance with 18 U.S.C. Section 1734 solely to indicate   at autopsy and frozen in smail plastic bags at -20 "C. Several speci-
this fact.                                                               mens were also obtained at surgery under sterile conditions, placed

                                                                    3633
3634                                Two Latent Metalloproteasesof Human Articular Cartilage
 in a sterile dish, and carried directly to the laboratory for freezing.                               RESULTS
 Tissues have been kept longerthan 1year without significant loss      of
enzyme activity. In all further steps, buffer solutions and glassware           Crude Extracts.-Due to its elastic nature, cartilage is a
were autoclavedand instruments were soaked in 70%         ethanol. Sterile   verydifficult tissue to homogenize. Originally, the VirTis
technique was used insofar as practicable to minimize introduction                                                 did
                                                                             homogenizer was used (l), but this not thoroughly disperse
and growth of microorganisms. A frozen patella was mounted on a              all of the fragments of cartilage. Sectioning of the cartilage
dissecting board with pins through soft surrounding tissues. The
                                      the                                    into 20-pm slices with a freezingmicrotome followed by
cartilage surface was scraped with  a scalpel to remove ice,fingerprints,    homogenization in Potter-Elvehjem glass homogenizer was
and other possible contaminants. The cartilage was sliced off and            satisfactory but  time-consuming. A Polytron homogenizer has
collected in sterile weigh boats on ice. Weighed slices were  transferred
to a Petri dish on ice and diced finely (1-2-mm cubes) with scalpels.        proven to give the best dispersion of the tissue in a short
The mince was transferred to a 40-ml polycarbonate centrifuge tube.          time. Brij-35 is added after  homogenization to minimize losses
For each gram of tissue (approximately 2 g/batch), 3.3 ml of 0.05 M          of the cartilageproteases.
Tris/HCl buffer, pH 7.5, were added. Buffer and tissuewere kept in              Bacterial contamination was found to be a serious problem
an ice-water bath. The tissue was homogenized in the tube with a             in the early stages     of this work. Microorganismsmay be
Polytron instrument (Brinkmann Instruments) using the PT-1OST                introduced at autopsy or during enzyme preparation. Pseu-
                                                     a
sawtooth generator.This step was carried out in laminar flow-hood            domonas and Corynebacterium species were particularly prev-
to prevent the dispersion of possible infectious agents intothe air. A       alent. It was possible to detect microbialmetalloprotease
burst of 30 s was followed by a period of cooling and the addition of
                                                                             production during the course of an 8-h working day, even
another 3.3 ml of buffer/g of tissue. A third repetition of these steps
resulted in a final volume of homogenate of approximately 11 ml for          though the preparationswere kept cold and contained azide.
each g oftissue.At this stage, several loops ofhomogenatewere                These problems were overcome by the introduction of the
streaked on a Lennox agar plate to monitor the possible presence of          sterile techniques outlined under “Materials and Methods.”
bacteria. Sodium azide was then added to a final concentration of            Occasional contaminated preparations were discarded. Sev-
0.02%. The homogenate was centrifuged for 20 min at 17,600 X g               eral sterilespecimens of cartilage were obtained from surgical




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(15,000 rpm) at 4°C. If a pellicle formed at the top of the tube, it was     operations to verify that the activities reported here not
                                                                                                                                     were
retained and combined with the pellet. The supernatant was drawn             microbia1 in origin.
off and stored in a sterile conical graduated screw-capped tube.     The        Enzyme assays were performed for both neutral and acid
pelletwasrehomogenizedin          5 ml of buffer/g of originaltissue.
                                                                             metalloprotease activity at the   crude extractstage. About 95%
Another streak plate was prepared and the homogenate centrifuged
as before. The second supernatant wasadded to the first (=Tris               of the activity is latent, so the assay included 1 mM amino-
extract).                                                                    phenylmercuric acetate to reveal the fullactivity. Blanks
   The remaining pellet was resuspended in 50 m Tris/HCl buffer
                                                      M                      contained 0-phenanthroline to assure that only metallopro-
containing 10 mM CaCIZand 0.02% sodium azide (10 ml of buffer/               tease activity was measured. Cathepsin D was present in the
original g of tissue). This was homogenized for s and again streak
                                                   30            a           extracts, but was blocked by pepstatin. The mercurial acti-
was made, The tube was covered and heated with agitation for 4 min           vator blocks any cysteine proteases that might be present.
in a 60 “C water bath.It was then cooled in ice water     and centrifuged    There are two complications in assaying the crude extracts.
as before. The supernatant was retained ( 5 60 “C extract).                  The extracts contain     proteoglycan, which may compete with
   Chromatography on Sepharose GB-The volume of the two extracts
was determined,then 0.5%Brij-35 was added a final concentration
                                                 to                          the proteoglycan substrate in the beads, and cartilage inhibi-
of 0.05%and solid NaCl to a molarity of 1.0. One extract was held in         tors of metalloproteases are present. Attempts to     remove the
the cold for later processing, and the other was concentrated on a           proteoglycan by precipitation or ultrafiltration were not SUC-
YM-5 membrane (Amicon) to a final volumeof 7 ml. Six ml were                 cessful. Amicon XM-100 or XM-50 membranes retained the
applied to the column, and the remainder wasusedfor bacterial                proteoglycan, buttheproteaseactivities         could not be re-
streak plates and enzyme assay. For the assay, 0.1 ml of extract was         covered oneither side of the membrane.Cetylpyridinium
mixedwith0.1 ml of assay buffer containing 1 m APMA’ and M                   chloride precipitated the proteoglycan, but the precipitates
incubated for 18 h.                                                          entrapped the protease activities. It was possible to remove
   A column (1.6 X 90 cm) of Sepharose 6B (Pharmacia Fine Chem-              the proteoglycan by chromatography on Sepharose 6B. For-
icals, Piscataway,NJ) was packedand washed ina buffer correspond-
ing to that of the concentrated Tris extract: 0.05 M Tris/HCl, 0.01 M        tuitously, this also separated  a metalloprotease inhibitor from
CaCl2,1 M NaCI, 0.05% Brij, 0.02% azide, pH 7.5 Sample and buffer            the enzymes. The full recovery of neutral activity from this
were pumped through the column at a rate of 12 ml/h and fractions            column indicated that there is minimal interference in assays
of4.6 m1 were collected. The individual fractions were dialyzed in           of crude extracts either proteoglycan or inhibitor. However,
                                                                                                by
Visking tubing (Union Carbide) against buffer withM0.15 prior  NaCl          the acid activity was recovered in increased amount from the
to assay, since 1M NaCl interferes in the enzyme assay. Protein was          column, indicating less reliability of the assay in crude ex-
determined by absorbance at 280 nm and proteoglycan by the di-               tracts.
methylmethylenebluedyemethod            (5). A 0.1-mlportion of each            A study of extraction methods was conducted using the
fraction was assayed for enzyme activity in the presence of 1 m        M     assay of neutral metalloprotease activity as a guide. Homog-
APMA. o-Phenanthroline (1mM) was added to a corresponding tube               enization in Tris   buffer yieldedboth acid and neutral   activity.
to provide a blank. For assayat acid pH, 0.1 ml of enzyme was mixed
with 0.1 ml of 0.2 M Tris maleate buffer, pH 5.0, containing 10 m      M     Various second extraction steps were tried overnight in 1 M
CaC12. This brought the finalpH to 5.3. APMAwasadded at a                    NaCl, 2 M MgC12, incubation a t 37 “C, etc. The most useful
concentration of 1 m to activate latent enzyme, and pepstatin was
                       M                                                     method was a heat extraction at60 “C in Trisbuffer with 10
added at a concentration of 5 pg/ml to block cathepsin D. Any                mM CaC12. This method was previously developed to extract
cathepsin B activity would be blocked by APMA.                               collagenase from uterine tissue (6); however, it worked well
   Properties of the Enzyme Activities-Since most of         the enzyme      for cartilage after the  calcium ion concentration was reduced
activities were found to be latent, it was first necessaryto activate by     from 100 to 10 mM. This second extract recovers an amount
incubation with 1 mM APMA for 4-5 h at 37 “C. APMA was then                  of enzyme at least as great as that obtained in first extract
                                                                                                                                   the
                                                       It
removed by overnight dialysis against cold buffer. is important to
activate first so that studies of pH, metal requirements, etc. do not        (ratio in15 experiments was 4555, Tris extract:60” extract).
reflectcombined effects on the activation step and the substrate                Demonstration of Multiple EnzymeActivities-The two se-
digestion. Further details will be found in figure and table
                                            the                  legends.    quential extracts (Tris and 60 “C) are made to 1 M in NaCl
~-                                                                           andconcentratedon         Amicon YM-5 membranes.Variable
  I   The abbreviation used is: APMA, aminophenylmercuricacetate.            losses are experienced in this  step, probably duet o adsorption
                                 Two Latent Metalloproteases of Human Articular Cartilage                                            3635
on the membranes or other surfaces. The concentrates are               Again, the acid activity was almost entirely latent and could
then chromatographed on Sepharose 6B (Fig. 1). The Tris                be activated with aminophenylmercuric acetate. Both acid
extract (Fig. 1A)    gives a high peak of protein (not shown) and      and neutral activity could also be activated by treatment with
proteoglycan near the void volume. It is important that the            trypsin, 10 pg/ml for 3 min at 37 “C.
sieve column produce a large separation between the enzyme                Chromatography of the 60 “C extracts gives a somewhat
and the proteoglycan. These compounds tend to interact,                different pattern (Fig. 16). The proteoglycan emerges as a
spreading the enzyme peaks toward the higher molecular                 sharp peak at V,, indicating that only intact monomers are
weights. In this event, a second chromatography of the inter-          present, whereas the Tris extracts contain additional degra-
mediate region is required to produce a clean separation of            dation products or small forms of proteoglycan. The major
enzyme and proteoglycan. The major peak of neutral metal-              peak I of neutral metalloprotease falls in exactly the position
loprotease activity (I) emerges slightly beyond the position of        found with the Tris extract. A small shoulder to the right is
the bovine serum albuminmarker. Some spreading to theleft              due to theactive form of the enzyme, whereas the main peak
is due to interaction with proteoglycan. The activity is almost        is latent enzyme. Peaks I1 and I11 are virtually absent from
entirely latent (95%)but is activatable by aminophenylmer-             the profile. Again, the acid activity coincides almost exactly
curic acetate. A smaller peak (11)is found at an apparentM,            with the neutral peak I and is not illustrated.
= 9,000. This metalloprotease is fully active. However, it is             Properties of the Metalloproteases-Since peak I fell in the
not derived directly from peak 1. If the enzyme in peak I is           same position for both Tris and 60 “C extracts, it appeared
activated and rechromatographed, it emerges at fraction 31             likely that the same enzyme was being extracted by both
 ( A I r = 40,000). A further peak (111) containing latent metal-      procedures. However, the presence of both acid and neutral
loprotease activity emerges in fraction 47,well beyond the             activities in the same peak suggested that a single enzyme
column volume. Peaks I1 and 111 were generally present in              might account for both. A study of pH curves helped to resolve
quite small amount (most favorable cases shown) and could              this question. First, it was necessary to activate the latent
                                                                       enzymes so that the measured pH effects would depend only




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not be studied in detail. Serine protease        activity was also
found in small amount in the region of peaks I and 1 1 The 1.                                            not the
                                                                       on digestion of the substrate and on activation process.
peak of acid metalloprotease activity coincides almost exactly         The activated enzymes were incubated with substrate and
with the neutral activity of peak I and is not illustrated.            withpepstatin to block aspartic proteases. The activator
                                                                       aminophenylmercuric acetate blocked cysteine protease. The
        650       I   1     I      I     I      I      I       I       results are shown in Fig. 2. A bimodal curve was obtained
                                                                       with greatly different ratiosof acid and neutral activity in the

        6oo   t                                TRlS EXTRACT
                                                           A
                                                                       two types of extracts. Furthermore,the activated preparations
                                                                       lost acid activity upon standing for several days. The neutral
                                                                       enzyme then gave a pH curve that fell monotonically from a
                                                                       maximum at pH 7.4 to zero at pH 5.5-6.0. Careful study of
                                                                       seven Sepharose chromatographies showed that, on the av-
                                                                       erage, the acid activity emerged about 1/2 fraction ahead of
                                                                       the neutral activity. Further confirmation that there were
                                                                       indeed two separate enzymes was obtained by additional study
                                                                       of molecular weights.




                            FRACTION NUMBER                               FIG. 2. pH activity curve of activated metalloprotease       from
   FIG. 1. Chromatography of cartilage extracts on Sepharose           peak I of the Sepharose 6B chromatography. Fractions were
6B. Details of the chromatography are given under “Materials and       pooled from peak I and activated with APMA as described under
Methods.” Six ml of concentrated extractfrom 1.8 g of cartilage were   “Materials and Methods.” Aliquots of 0.1 ml wereadjusted to various
applied to thecolumn, and fractions of 4.6 ml werecollected. Proteo-   pH-values by adding 0.1 ml of 0.2 M buffers (acetic acid/NaOH, Tris
glycan content and proteoglycan-digesting activity at pH 7.5 are       maleate, or Tris/HCl) to provide 0.2 ml at thedesired final pH. After
recorded. The blanks for the two measurements are 0.350 and 350        incubation for 18 h at 37 “C,  the final pH was determined with a
cpm, respectively. APMA was added to theenzyme assays. Molecular       microelectrode (Radiometer). Pepstatin (5 pg/ml) was added to all
weight markers, indicated along the top of B, were bovine serum                                                            M
                                                                       tubes below pH 7. Tubes containing enzyme and 1 m o-phenanthro-
albumin (67,000), ovalbumin (45,000), and cytochrome c (13,000). 0,    line were added at each pH to be certain that only metalloprotease
proteoglycan; 0,protease.                                              was being measured. 0,Tris extract; 0, 60 “C extract.
3636                              Two Latent Metalloproteases of Human Articular
                                                                           Cartilage
    The molecular weight of the neutralenzyme was examined
                                                                                                     ~          ~     ~~~       ~




both on Sepharose 6B and onUltrogel AcA-54 columns (Fig.                              I         67kDa
                                                                                             45kDa                          25 kDc

 3). Twenty-eightrunsonSepharose6B                gave an average
 elution volume of 127.4 ml, which corresponds toM, = 56,900
in relation to markers of bovine serum albumin, ovalbumin,
and cytochrome c. On AcA-54, five runs each of latent and
active enzymegave M , = 56,000 and 40,000, respectively. Fig.
 3 illustrates a separation of mixed active and latent forms; in
                      two
the other runs, the forms were studied separately.
   The acid  enzyme  proved more difficult          to evaluate. It
emerged just slightly ahead (about0.6 fraction) of the neutral
enzyme on Sepharose 6B, suggesting M , = 65,000. Rechro-
matography of this peak on Sepharose 6B            gave a shift to
M , = 50,000. Other sieve columns were tried, but difficulty
                                        acid
was encountered in that the latent enzyme almost invar-                                             FRACTION NUMBER
iably became self-active upon rechromatography a t neutral               FIG. 4. Chromatography of acid protease activity on Se-
pH. The use of Sephacryl S-200at pH 5.3 gave good recovery            phacryl S-200. Pooled fractions from peakI were chromatographed
of the latentenzyme, but some loss of the activeform. In this         separately as latent or activated forms. Three ml of sample were
                                                                      applied to a column (1.6 X 85 cm) of Sephacryl S-200 (Pharmacia)
system (Fig. 4), M, = 50,000 for latent acid metalloprotease          equilibrated in 0.05 M Tris maleate buffer, p H 5.3, containing 0.2 M
and 35,000 for active enzyme.                                         NaC1, 10 mMCaC12, and 0.02% sodium azide. Fractions of 3 ml were
   Metal Requirements-The active forms of both neutral and            collected. Assays were done at pH 5.3 with 5 pg/ml of pepstatin to
acid enzymes were produced by treating peak I with 1 mM               block cathepsin D and APMA to activate latentenzyme. Markers are
               h
APMA for 4 at 37 "C. These preparations          were then dialyzed   as inFig. 1,with the addition of chymotrypsinogen (25,000). 0, latent




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exhaustively against calcium-free buffer. Calcium was then            enzyme, assayed with APMA; @, active enzyme.
restored a t various concentrations (Fig. 5). The activityof the
neutral enzyme could never be fully blocked by calcium re-
moval. There was always 1 5 2 0 % residual activity (five prep-
                                                of
arations). On the other hand, the activitythe acid enzyme
                             the
completely disappeared in absence of calcium. The activity
of both enzymes could be completelyrestored by 10 mM CaC12,
buttheneutral      enzymeresponds to much lower levels of
calcium than does the acid enzyme. It shouldbe kept in mind
that the assays involve 16-18-h incubations at 37 "C. If the
role of calcium is in stabilizing the enzyme, then this long
incubation may magnify calcium effects. The diminished en-
zyme activity at calcium concentrations above 10 mM is due
to an effect of ionic strength on the behavior of the bead
substrate, not to an inhibition of the enzymes. Zinc did not
enhance the    small activity of the calcium-free neutral enzyme;
rather, 1 mMZn" depressed it a further 60%.
   Evidence that both metalloproteases are zinc enzymes is
presented in Table I. In the presence of 10 mM CaCl,, 1 mM
o-phenanthroline totally inhibits both        enzymes. This indi-                            MOLARITY OF CALCIUM ION
catesthat asecond metalionis              required inaddition to
                                                                         FIG.5. Effect of calcium on metalloprotease activity. Frac-
                                                                      tions from peak I were activated with APMA, then dialyzed exhaus-
                                                                                                    of
                                                                      tively against several changes calcium-free buffer at pH Various
                                                                                                                               7.5.
                         67 kDa 45 kDa                                levels of calcium chloride were then added to theusual assay system.
                          t       b         b                         @, assay a t p H7.4; 0, assay at pH5.3.
            z
            0
                 525-
            W                                                          calcium. Zinc restored the activity of both enzymes. Cobalt
            L?
            0                                                                                       completely restore theactivity of
                                                                       was less effective; it did not
             5 450-                                                    either enzyme. In contradistinction to manyzinc metallopro-
             U
             3                                                         teases, the cartilage enzymes are not inhibited by added zinc
            (3
                                                                       at a concentration of 1 mM.
            p   375-
                                                                          Other Properties-That the cartilage enzymesare proteases
             n                                                         and not polysaccharidases was shown in two ways. First, the
                3 0 0 1                                                digestion products produced by the peak I enzymes were
                     14
                                                                       dialyzed in Spectrapor membrane tubingpermeable to mole-
                               FRACTION NUMBER                         cules of mass less than 3500 daltons. Less than 10% of the
   FIG. 3. Chromatography of neutral protease activity on U1- digestion products, based on uronic acid assay             (7), passed
trogel AcA-54. Pooled fractions from peakI were partially activated these membranes. Second, both enzymes of peak I digested
with APMA. The mixture (3 ml) of active and latent enzyme was Azocoll. In this assay, 2 mg of Azocoll were incubated with
then applied to a column (1.6 X 90 cm) of the gel equilibrated in pH enzyme for 18 h at 37 "C in a volume of 1.25 ml. A unit of
7.5 buffer containing 50 mM Tris/HCl, 10 mM CaC12, 0.15 M NaC1,
0.02% sodium azide, and 0.1% Brij-35. Fractions of 3 ml were collected activity is defined as the amountof enzyme that releases 1pg
and assayed. 0, APMA added to reveal latent activity; @, no APMA, of substrate in 1 min. One g of wet cartilage contained 6.8
enzyme that is already active. Markers are as in Fig. 1.               units of proteoglycan- and 6.0 units of Azocoll-digesting ac-
                                    Two Latent Metalloproteases of Human Articular Cartilage                             3637
                                TABLE    I                      why it hasbeen difficult to detectenzyme activity in cartilage
           Effect of metal ions on cartiluge metabproteases     homogenates and extracts.
   Peak I froma Sepharose 6B chromatographywas treated with        The current study has also brought home the need for
APMA to activate neutral and acid protease activities. The sample
                                                                sterile techniques. Bacteria are easily introduced from the air
was dialyzed to remove APMA. The neutral activity was about three
                                                                and can rapidly produce traces of metalloprotease activity,
times that of the acid activity. The activities in the absence of further
                                                                even when all work is done in the cold. This finding has
additions were set equal to 100. Every incubation contained 10 m       M
CaC12.                                                          thrown doubt on our earlier preliminary report of a small
                                           Relative activity
                                                                metalloprotease in bovine nasal cartilage (9). It is likely that
              Additions                                         enzyme activity, which seemed to pass through    dialysis tubing,
                                         pH 7.4        pH 5.3   was formed by traceamounts of bacterial growth in the
       None                               100            100    external dialysis buffer. We have not been able to find signif-
       1 mM o-phenanthroline                0               0   icant amounts of metalloprotease activity in bovine nasal
         + 0.1mM Znz+                      21             25    cartilage using our present methods. Since most of our speci-
         + 0.2 m M Zn2+                    78
                                                                mens of human cartilage were obtained at autopsy, it was
         + 1.0 mM Zn2+                    103              85
                                                                important to   obtain sterilesamples at surgery to make certain
         + 0.2 mM Co2+                      8
         + 1.0 mM Co2+                     77             66    that theenzyme activities reported here were present in fresh
       1 m Zn2+
          M                               106             96    tissue. This was found to be the case. Moreover, we have
       1 m Co2+
          M                                86                   recently completed the study of a large series of surgical
                                                                specimens of control and osteoarthritic cartilages from the
tivity at acid pH. At neutral pH, thevalues were 4 4 and 11.2, human tibial plateau (10). This shows that the neutral pro-
                                                       .
respectively.                                                   tease is present in normal cartilage and is increased in os-
   From these unitsandtheunits           obtained with known teoarthritis. Since the enzyme activity is increased in arthritis,




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amounts of trypsin and thermolysin acting on proteoglycan the best starting material for enzyme preparation is cartilage
beads, it is possible to estimate the tissue content of the two that shows some degree of osteoarthritis, such as fibrillation
metalloproteases. One g of wetcartilage contains both neutral of the surface and small erosions. Autopsy material is suitable
and acid activity equivalent to 1 pgof trypsin or 0.1 fig of since the cartilage is usually of advanced age and osteoarthri-
thermolysin. The acid and neutralactivities are found in tic involvement is frequently noted.
varying proportions; this value is only an approximation.          It has not yet been possible to make a complete identifica-
   Minor Protease Activities-Serine protease activity, inhib- tion of the neutral protease described here with the activity
itable by phenyl methanesulfonyl fluoride, was detected under reported earlier (1, 2). In the earlier studies, the need for
peaks I and 111 of the Tris extracts. Since this activity was activation was not recognized, nor the need for calcium ion;
low (5-10% of the total neutral activity), no further charac- indeed, these studieswere done using phosphate buffer. None-
terization was attempted. Peak I1 contained metalloprotease theless, the neutral protease would probably have become
activity that was in a fully active form. It is not derived by active spontaneously after several purification steps, and it
direct activation of the neutral enzyme of peak I, as shown would also have displayed at least 15-20% of its activity in
by its molecular weight properties (discussed above). It has the absence of calcium. The earlier and the present prepara-
an apparent M, = 9000 on Sepharose 6B (average of eight tions are similar in having a pH optimum between 7.2 and
runs) and 4000 as measured on AcA-54 and Bio-Gel P-60 (in 7.5 and having their activities restored by Zn2+and Co2+after
the same buffer, but with NaCl reduced from 1 to 0.15 M). It treatment with chelators. The major differences seems to be
is unlikely that these are true molecular weights; interaction in M, which is 40,000 for the active enzyme in the present
of the enzyme with the gels is probable. This form was study and was reported as 26,000 earlier (2). It is possible that
detected in 8 of 16 Tris extracts and none of seven 60 "C the earlier report dealt with the enzyme found in peak I1 in
extracts. A single preparation was shown to retain complete the present study, an active form of metalloprotease that does
activity after removal of calcium by dialysis and to be inhib- not require calcium and is probably retarded by interaction
ited by o-phenanthroline with 40% restoration by zinc. Peak with the sieve column.
I11 emerged after the column volume of Sepahrose 6B, so its        Acid metalloprotease activity was also noted earlier (1).
molecular weight could not be estimated. It could only be There was some evidence for two distinct activities at acid
detected after activation by APMA. The cartilage also con- pH, both inhibitable by o-phenanthroline. However, in the
tains collagenase, another neutral metalloprotease, but this present study, only a single peak was detected with an opti-
has been reported on elsewhere (8).                             mum at pH 5.3. Again, the latency and calcium requirement
                                                                had not been noted earlier. But restoration of activity after
                           DISCUSSION
                                                                chelator treatment with Zn and Co had been noted.
                                                                   Source of the Metalloproteases-It seems quite probable that
   Advances overPrevious Work-In previous work ( 1 , 2 ) , the the metalloprotease activities of the cartilage are produced by
smallest batch of cartilage that could be studied was on the the resident chondrocytes. Activity is found not only in dis-
order of 100 g, wet weight. With thepresent methodology, we eased cartilage, but also in cartilage from young adults that
can now examine as little as 1 g. This improvement is largely has no evidence of arthritic change. Recently, McGuire et al.
due to theincreased sensitivity of the assay. In thepublished (11)have shown that human chondrocytes in culture produce
method of Nagase and Woessner (3), measurements could be a metalloprotease that can digest proteoglycans. Similar me-
made in the range of 10-50 ng of trypsin. The modified talloproteases that digest proteoglycan at neutral pH and that
method increases the sensitivity to 0.3-3 ng of trypsin. This occur in latent forms activated by mercurials and trypsin have
gain was achieved by the use of radiolabeled proteoglycan and been foundin the culture medium of rabbit (12, 13) and
a 9-fold increase inthe time of incubation. Using these bovine (14,15) chondrocytes. The work of Morales and Kuett-
methods, it was found that human cartilage contains metal- ner (15) is particularly instructive. They showed that bovine
loprotease activities which are equivalent to only 1 pgof       chondrocytes produce metalloprotease under conditions in
trypsin/g of wet tissue. These very low enzyme levels explain which the cell phenotype is maintained, e.g. the cells produced
3638                           Two Latent Metalloproteases of Human Articular Cartilage
only type I1 collagen. However, the bovine enzyme had M, =            We believe that a thorough understanding of the cartilage
33,000 and did not undergo a change in weight upon activa-         metalloproteases will be                             the
                                                                                            important in understanding phys-
tion. The enzyme produced by immature rabbit cartilage in          iology of the extracellular matrix and its degradation in dis-
organ culture (16) is closely similar to the human neutral         ease processes. The method of direct extraction of these
metalloprotease in having M, = 53,000 for the latent form          enzymes, used in conjunction with tissue culture studies of
and 40,500 for the active form. Our laboratory is the only one     enzyme production, would be particularly useful in studying
in which metalloprotease has been successfully extracted di-       the effects of drugs on joint disease.
rectly from cartilage tissues.                                                                REFERENCES
   Relation to Other Metalloproteases-While bacterial metal-
loproteases have been known for a long time, mammalian               1. Sapolsky, A. I., Keiser, H., Howell, D. S., and Woessner, J. F.,
                                                                           Jr. (1976) J. Clin. Invest 5 8 , 1030-1041
proteases capable of endopeptidase action are of relatively          2. Sapolsky, A. I., and Howell, D. S. (1982) Arthritis Rheum. 2 5 ,
recent discovery. Collagenase was first found in 1962 (17) and             981-988
shown to be blocked by EDTA. Evidence that collagenase is            3. Nagase, H., and Woessner, J. F., Jr. (1980) Anal. Biochem. 1 0 7 ,
a zinc enzyme appeared in 1973 (18); calcium is required as                385-392
well. During the 1970s a number of additional metallopro-            4. Montelaro, R. C., and Rueckert, R. R. (1975) J. Biol Chem. 2 5 0 ,
                                                                           1413-1421
teases have been described. These have most commonly been            5. Farndale, R. W., Sayers, C. A., and Barrett,A. J. (1982) Connect.
assayed on gelatin, proteoglycan, Azocoll, and casein. In gen-             Tissue Res. 9,247-248
eral, these enzymes are capable of degrading components of           6. Weeks, J. G., Halme, J., and Woessner, J. F., Jr. (1976) Biochim.
the connective tissue matrix, they are secreted by cultured                Biophys. Acta 446,205-241
                                                                     7. Bitter, T., Muir, H. M. (1962) Anal. Biochem. 4,330-334
                                                                                      and
cells, and they commonly occur in latent forms that can be           8. Pelletier, J.-P., Martel-Pelletier, J., Howell, D. S., Ghandur-
activated by mercurials and trypsin. A second group of me-                 Mnaymneh, L., Enis, J. E., and Woessner, J. F., Jr. (1983)
talloproteases is found intracellularly, commonly in associa-              Arthritis Rheum. 26,68-63




                                                                                                                                               Downloaded from www.jbc.org by guest, on September 6, 2011
tion with microsomal membranes. The rabbit kidney metal-             9. Woessner, J. F., Jr., Sapolsky, A. I., Nagase, H., and Howell, D.
loprotease (19) is typical of this group. These enzymes require            S. (1977) Arthritis Rheum. 2 0 , S116-Sl21
                                                                   10. Martel-Pelletier, J., Pelletier, J.-P., Cloutier, J.-M., Howell, D.
zinc, but not calcium; they are inhibited by 1 m Zn2+.  M                  S., Ghandur-Mnaymneh, L., and Woessner, J. F., Jr. (1984)
Latency and secretion of such enzymes have not been re-                    Arthritis Rheum.27, in press
ported.                                                            11. McGuire, M.K. B., Meats, J. E., Ebsworth, N. M., Gowen, M.,
   The two major cartilage metalloproteases reported here fall             Mumhv.. G.. Revnolds, J. J.. and Russell, R. G. G. (1982) Agents
                                                                                *  - I                                                 -
                                                                           ~ctiow    suppl i l , 131-138'
into the first group, capable of attacking matrix macromole-       12. SaDolskv. A. I.. Malemud. C. J.. Norbv, D. P., Moskowitz. R. W.,
cules. Recent work suggests that the latency phenomenon is                 Matsita, K.,'and Howell, D.'S. (1981) Biochim. Biophys. Acta
due to synthesis of the enzymes in a proenzyme form, rather                658,138-147
than to enzyme-inhibitor   complex formation (20). The neutral     13. Deshmukh-Phadke, K., Lawrence, M., and Nanda, S. (1978)
activity from cartilage has properties similar those reported
                                               to                          Bwchem. Biophys. Res. Commun.85,490-496
                                                                   14. Ridge, S. C., Oronsky, A. L., and Kerwar, S. S. (1980) Arthritis
for the rabbit cartilage enzyme (16) and rabbit bone enzyme                Rheum. 23,448-454
(21). The properties of all three enzymes are not yet known        15. Morales, T.I., and Kuettner, K. E. (1982) Biochim. Biophys. Acta
in sufficient detail to judge just how closely related they are.           705,92-101
   Further characterization of the acid metalloprotease is of      16. Lowther, D. A., Sandy, J. D., Cartwright, E. C., and Brown, H.
                                                                           L. G. (19811 Semin. Arthritis Rheum. 11. SUDD~.     1.65-67
interest because this type of activity has not hitherto been       * "I . Gross, J., and Lapiere, C. (1962) Proc. Nutl. A&. Sci. U.S. A.
                                                                   I
described in mammalian tissues. There isa well known group                 48,1014-1022
of acid metalloproteases found in microorganisms, including        18. Berman, M. B., and Manabe, R. (1973) Ann. Ophthalmol. 5 ,
Penicillium roqueforti and caseicolum and Aspergillus oryzae               1193-1209
and sojae (22). These have molecular weights of 20,000 and         19. Kerr, M. A., and Kenny, A. J. (1974) Biochm. J. 137,489-495
                                                                   20. Nagase, H., Jackson, R. C., Brinckerhoff, C. E., Vater, C. A., and
contain zinc. The cartilage enzyme differs from these enzymes              Harris, E. D., Jr. (1981) J. Biol. Chem. 2 5 6 , 11951-11954
in having a higher molecular weight and occurring in a latent      21. Murphy, G., Cawston, T. E.,Galloway,W.A.,              Barnes, M. J.,
form. Recently, Galloway et al. (23) have described further                Bunning, R. A. D., Mercer, E., Reynolds, J. J., and Burgeson,
details of the rabbit bone metalloprotease. The active form of             R. E. (1981) Biochem. J. 199,807-811
this enzyme has a very broad pH optimum which is almost            22. Gripon, J. C., Auberger, B., and Lenoir, J. (1980) Znt. J. Biochem.
                                                                            12,451-455
flat from pH 5.5 to 9.0. However, the cartilage acid metallo-      23. Galloway, W. A., Murphy, G., Sandy, J. D., Gavrilovic, J. Caws-
protease is distinguished from this enzyme in having a sharp               ton, T. E., and Reynolds, J. J. (1983) Biochem. J. 209, 741-
pH optimum near 5.3.                                                       752

				
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