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Activation of leucocyte collagenase proenzyme by rheumatoid synovial fluid
436
BIOCHIMICAET BIOPHYSICAACTA
BBA 36290 A C T I V A T I O N OF LEUCOCYTE COLLAGENASE P R O E N Z Y M E BY R H E U M A T O I D SYNOVIAL F L U I D
DARIUSZ KRUZE* AND ELZBIETA WOJTECKA Department of Biochemistry, Institz*te of Rheumatology, Warsaw (Poland)
(Received June i3th, 1972)
SUMMARY
A collagenase proenzyme was found in crude human leucocyte extract. An activating agent was present in all the ten studied rheumatoid synovial fluids taken from knee joints. The activator was found to be heat labile and nondialyzable. Activation occurred after mixing c:ude extract with synovial fluid (protein ratio I :I) and heating the mixture for several hours at 37 °C. Collagenase was produced from proenzyme both when the activator acted on the crude leucocyte extlact and when it acted on the same extract previously devoid of free collagenase; however, activation did not occur in crude extracts partially purified with dioxan. The amount of proenzyme in the extract was about 3 times greater than that of free collagenase. Activated proenzyme was capable of releasing soluble hydroxyproline peptides from reconstituted collagen fibrils at 37 °C, and of decreasing the viscosity of collagen in solution at 26 °C. Both the activated proenzyme and the free collagenase from leucocytes produced the same degradation products from soluble native collagen. An alternative possibility, that the activator was present in crude extract and the proenzyme in synovial fluid, was experimentally ruled out. The role of collagenase proenzyme from leucocytes in the inflamatory state of tissues is discussed.
INTRODUCTION At neutral p H and physiological temperature native collagen is degraded only by collagenase. However, in these conditions it has not been possible to demonstrate collagenolytic activity in direct extracts of any tissue. I0 years ago Gross and Lapiere I demonstrated a collagenase in tadpole skin culture fluids. Since that time m a n y authors have obtained collagenolytic enzymes from m a n y m a m m a lian tissues b y using culture techniques 2.
land.
Present address: Dept of Pharmacology, Biocenter of the University of Basel, Switzer-
Biochim. Biophys. Acta, 285 (1972) 436-446
ACTIVATION OF COLLAGENASEPROENZYME
437
The lack of collagenase in direct extracts could be explained either by the synthesis of the enzyme during tissue culture, or b y the occurrence of the enzyme in tissues as an inactive precursor 3. The latter supposition has been recently supported b y Harper et al. 4, who have isolated a precursor of tadpole collagenase from extracts of tadpole tail-fin tissue. The sole exception is provided b y human granulocyte collagenase, which is directly extractable from granulocytes and does not require tissue culture to produce detectable enzyme 5. The occurrence of collagenase in the supernatant fraction of human white cell homogenates does not exclude the possibility of active enzyme occurring together with inactive proenzyme. In this report we describe the detection and mode of activation of a proenzyme found in direct extracts of human leucocytes. METHODS
Preparation of crude enzyme The white cells were obtained from fresh whole blood taken from normal adult donors. The blood was mixed with a solution containing 0.9% NaC1 and 6% dextran. After allowing the red cells to settle at room temperature, the white cell-rich supernatant was decanted and centrifuged. All subsequent steps were carried out at 4 °C. The cell pellet was resuspended in saline and the remaining red cells removed by brief hypotonic hemolysis. The white cells were then washed with saline, collected by centrifugation, and homogenized in o.oi M Tris-HC1 containing 0.005 M CaC12 and 0.2 M NaC1. The homogenate was then frozen, thawed and centrifuged. The supernatant was dialyzed against the same buffer, after which it was clarified by centrifugation.
Partial purification of crude enzyme To the crude extract of leucocyte in an ice bath was added dioxan in the ratio I :I (v/v) and the suspension was allowed to stand for I h before removing the precipitate b y centrifugation at IO ooo × g for 15 rain. The supernatant solution, containing collagenase, was dialyzed against o.oi M Tris-HC1, p H 7.5, containing 0.005 M CaC12 and 0.2 M NaC1, and then concentrated.
Preparation of substrate Acid-extracted calf skin collagen was purified by the method of Kang et al. 6. Collagen solution for use in the assay of collagenolytic activity was prepared by dissolving lyophilized collagen in cold 0.05% acetic acid at a concentration of 0.20/0 . The solution was dialyzed against 0.05 M Tris-HC1, p H 7.5, containing 0.005 M CaC12 and 0.2 M NaC1, which was followed by centrifugation to remove any undissolved collagen.
Measurement of collagenolytic activity Collagenase activity was determined by the containing collagen degradation products from a enzyme and reconstituted native trypsin-resistant fibrils were incubated with trypsin (at a protein
release of soluble hydroxyprolinereaction mixture which contained collagen fibrils. When the collagen ratio of 2 :I) only 6% of the sub-
Biochim. Biophys. Acta, 285 (1972) 436-446
438
D. KRUZE, E. WOJTECKA
strate was solubilized. A typical reaction mixture contained I ml of 0.2% collagen fibrils which had been allowed to gel for 6 h at 37 °C, i ml of o.o5 M Tris-HC1, p H 7.5, containing 0.003 M CaC12 and I ml enzyme solution containing approx, o.2-1.o mg of protein. The mixture was incubated for 18 h at 37 °C, and then filtered to remove insoluble collagen fibrils. Aliquots of the supernatant were hydrolysed in 6 M HC1 and their hydroxyproline contents were determined. Control tubes contained thermally denatured enzyme solution and were treated in a similar way.
Removal of active collagenase and activation of proenzyme Removal of active collagenase was performed by the method of Harper et al. 4, as follows. A suspension of reconstituted collagen fibrils in o.oi M Tris-HC1, p H 7-5, containing o.oo5 M CaC12 and o.2 M NaC1 was added to the supernatant of a white cell homogenate in an ice bath, in order to remove active collagenase by adsorption onto its solid substrate. The ratio of suspended collagen to protein in solution was I : 5 on a weight basis. The mixture was gently stirred for 2 h, followed by centrifugation and separation of the clear supernatant. Synovial fluids were mixed with the supernatant at a protein concentration ratio of I :I, and the mixture was incubated with reconstituted collagen fibrils for 1 8 h a t 37°C.
Molecular sieve filtration of crude leucocyte extract Gel filtration of crude leucocyte extract was performed at 4 °C on a 2 cm × 8o cm column of Sephadex G-2oo equilibrated with o.oi M Tris-HC1, p H 7.5, containing o.oo5 M CaCI~ and o.2 M NaC1. Fractions which had collagenase activity were pooled, concentrated using Aquacide, Calbiochem., Los Angeles, Calif. and further fractionated by gel filtration on a column, o. 9 cm × 6o cm, of Sephadex G-/oo which had been equilibrated with the same buffer.
Disc gel electrophoresis Thermally denatured products from incubation of collagen with activated proenzyme were subjected to electrophoresis in polyacrylamide gel according to the method of Nagai et al. 7. The enzyme was inactivated by the addition of sufficient o.I M HC1 to the reaction mixtule to reduce the p H to approximately 2.0 before denaturation at 4 ° °C.
Synovial fluid Synovial fluids were aspirated from rheumatoid arthritis patients with knee effusions.
Other methods Hydroxyproline was determinated by the method of Stegemann and Stalder s, and protein by the method of Lowry et al. 9. RESULTS
Activation of crude leucocyte preparation by synovial fluid The effects of synovial fluids on the collagenotytic activity of crude white cell Biochim. Biophys. Acta, 285 (t972) 436-446
ACTIVATION
OF COLLAGENASE
439
PROENZYME
TABLE I I N F L U E N C E OF S Y N O V I A L F L U I D ON T H E C O L L A G E N O L Y T I C A C T I V I T Y OF C R U D E E X T R A C T
The reaction mixture consisted of I ml of crude extract containing i mg of protein, 0.o2-0.o25 ml of synovial fluid containing I m g of protein, i ml of o.2 % collagen (gel) and i ml of o.o 5 M Tris-HC1, pH 7.5, containing 0.o03 M CaC12. These components were mixed and incubated at 37 °C for 18 hl
Crude extract
Crude extract Crude extract + synovial fluids without collagenolytic activity Crude extract + synovial fluids with collagenolytic activity
l~g of released hydroxyproline
i 2 3 4 5 i 2 3 4
Expt I
Expt I I
2o. 4 81.o 8o.9 83.1 82.o 85.1 84.2 8o.0 81.6 --
25.o 112. 5 120. i lO8.8 11o.9 118.o I i 1.9 114.o I 16.9
e x t r a c t s are shown in T a b l e I. Two series of e x p e r i m e n t s were carried o u t on the crude e x t r a c t o b t a i n e d from two donors. S y n o v i a l fluids from knees joints of nine p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s were used for the e x p e r i m e n t s . F i v e of the fluids used were f o u n d to be d e v o i d of collagenolytic a c t i v i t y , a n d four were positive. The s y n o v i a l fluids were a d d e d to crude leucocyte p r e p a r a t i o n a t a r a t i o o f I : i p r o t e i n c o n c e n t r a t i o n (details see T a b l e I). The a m o u n t of soluble h y d r o x y p r o l i n e p e p t i d e s released from the collagen gels was used as a m e a s u r e of collagenolysis. I n the c o n t r o l samples, account was t a k e n of t h e collagenolytic a c t i v i t y of s y n o v i a l fluid, o f the h y d r o x y p r o l i n e c o n t a i n e d in t h e fluid, a n d also o f the v e r y s m a l l a m o u n t of soluble collagen which d i d n o t u n d e r go t h e change into r e c o n s t i t u t e d fibrils. I n these r e a c t i o n conditions, two different crude leucocyte p r e p a r a t i o n s released a m o u n t s of h y d r o x y p r o l i n e peptides, 20.4 a n d 25.0/~g h y d r o x y p r o l i n e which c o n s t i t u t e d a b o u t 12% of the s u b s t r a t e used. A considerable increase of collagenolytic a c t i v i t y occurred after the a d d i t i o n of s y n o v i a l fluids a n d an a c t i v a t e d crude leucocyte e x t r a c t released from 8 0 # g to I20/~g of h y d r o x y p r o l i n e peptides, which a m o u n t e d to 40-60~o of the r e c o n s t i t u t e d fibrils. No differences in t h e c a p a c i t y for a c t i v a t i o n were found b e t w e e n the s y n o v i a l fluids which showed collagenolytic a c t i v i t y a n d those which d i d not.
Effect of synovial fluids on partially purified collagenase E n z y m e which h a d been p a r t i a l l y purified b y the use of d i o x a n was a c t i v a t e d b y t h e s y n o v i a l fluids. The e x p e r i m e n t s were carried out on two samples of purified e n z y m e a n d using four s y n o v i a l fluids. E v e n t h o u g h t h e e x p e r i m e n t s were carried out u n d e r tile same conditions as for the crude leucocyte e x t r a c t a n d the s a m e s y n o v i a l fluids were used, there was no increase of collagenolytic a c t i v i t y , a n d in fact some small decrease was f o u n d (Table II). The results show t h a t the increase of collagenolytic a c t i v i t y a f t e r the a d d i t i o n of s y n o v i a l fluid is n o t caused b y increased a c t i v i t y of free leucocyte collagenase, nor is it caused b y a s i m u l t a n e o u s effect of collagenase a n d the e n z y m e s occurring in s y n o v i a l fluid.
Biochim. Biophys. Acta, 285 (1972) 436-446
44 °
I). KRUZE, E. WOJTECKA
T A B L E 1[ INFLUENCE DIOXAN
OF SYNOVIAL
FLUIDS
ON THE
ACTIVITY
OF PARTIALLY
PURIFIED
COLLAGENASE
(AFTER
PURIFICATION)
The reaction m i x t u r e consisted of i ml of partially purified collagenase, o.2 mg of protein, I ml of o.2% collagen (gel) and I ml of o.o 5 M Tris-HC1, p H 7.5, containing o.oo 3 M CaCI v These c o m p o n e n t s were mixed and i n c u b a t e d at 37 °C for 18 h. F o r the s t u d y of activation, the incub a t i o n s were carried o u t as above, with the addition of o.o4-o.o 5 ml of synovial fluids, o.2 m g of protein, diluted I :io with Tris-HC1 buffer.
I*g of released hydroxyproline
Partially purified collagenase Partially purified i collagenase + 2 synovial fluids 3 4
Expt I
Expt [I
5o.o 45.o 45.8 47 .o 48.3
38.o 35.o 34.8 37 .2
Effect of synovial fluid on crude leucocyte extract freed from collagenase To confirm the above, and to prove that the increase of activity is connected with the activation of some other component of the crude extract, the following experiments were carried out. In order to bind the free collagenase with substrate and thus remove it from the solution, crude leucocyte preparation was shaken with insoluble collagen. Following such treatment, the crude extract was found to be devoid of collagenolytic activity; no hydroxyproline was released from collagen fibrils, nor was there any decrease in the viscosity of the collagen solution. After the addition of synovial fluid, the enzymatically inactive solution released a considerable amount of hydroxyproline from the collagen gel (Table III). Experiments were carried out on three crude leucocyte extracts, and in each case collagenolytic activity was noted at a level several times greater than the initial activity of the crude leucocyte collagenase. TABLE In ACTIVATION BY SYNOVIAL NOLYTIC ACTIVITY
FLUIDS
OF THt~ CRUDE
EXTRACT
FROM LEUCOCYTES
DEVOID
OF COLLAGE-
The reaction m i x t u r e contained I ml of crude e x t r a c t or crude e x t r a c t devoid of collagenase, i mg of protein, i ml o f o . 2 % collagen (gel) a n d i ml o f o . o 5 M Tris-HC1, p H 7-5, containing 0.003 M CaC1 v Cotlagenase was r e m o v e d f r o m the crude e x t r a c t as described in Methods. F o r the activ a t i o n studies, 0.025 ml of synovial fluid, i mg of protein, was added to the reaction mixture.
#g of released hydroxyproline
Crude e x t r a c t Crude e x t r a c t p r e t r e a t e d with collagen at o °C Crude e x t r a c t p r e t r e a t e d with collagen at o °C + synovial fluid
Expt I
Expt I I
21. 7
28.0
16.o
o
o
o
75.0
77.0
12o.o
Biochim. Biophys. Acta, 285 (1972) 436-446
Expt I I I
441
ACTIVATION OF COLLAGENASE PROENZYME 0.7 0.6
)<
120
o
11o t_6 loo 9o~= 8o~ 7o _~
E 0.5 o 40~
E O.Z o
00 u c
~o.3
O.4
~0.3
c
20 ~z l
~o.2 <~o.~
o
5O 40~
< 0.2
3o~ 20> lO ~
0.1
o
lb
20 30 40 50 Fraction number
60
E c W
~0-~/10
20
30
40
Fraction number
c W
Fig. i. C h r o m a t o g r a p h y on S e p h a d e x G-2oo of t h e c r u d e l e u c o c y t e e x t r a c t . A s a m p l e of i 7 o m g p r o t e i n in a v o l u m e o f 4 m l w a s applied to t h e c o l u m n , a n d 4-ml effluent f r a c t i o n s were collected a t a r a t e of io m l / h ; O - - - O , a b s o r b a n c e a t 280 n m (solution d i l u t e d i : i o ) ; x - - - x , e n z y m e a c t i v i t y ; 0 - - - 0 , e n z y m e a c t i v i t y a f t e r a d d i n g s y n o v i a l fluid. F r a c t i o n s 30-42 were pooled a n d concentrated. Fig. 2. R e c h r o m a t o g r a p h y on S e p h a d e x G-I oo o f a p e a k f r o m a c r u d e l e u c o c y t e e x t r a c t p r e v i o u s l y s e p a r a t e d on S e p h a d e x G-2oo ( F r a c t i o n s 30-42). A s a m p l e o f 13 m g p r o t e i n in a v o l u m e of 2 ml w a s applied to t h e c o l u m n , a n d 1.5-ml effluent f r a c t i o n s were collected a t a r a t e of 5 m l / h ; © - - - O , a b s o r b a n c e a t 280 n m ; × - - - × , e n z y m e a c t i v i t y ; O---O, e n z y m e a c t i v i t y a f t e r a d d i n g s y n o v i a l fluid; V0, void v o l u m e .
These results seem to indicate that a precursor of collagenase, which does not bind with collagen, undergoes activation by synovial fluid.
Properties of collagenase obtained after the activation of the proenzyme A crude leucocyte preparation was separated on a Sephadex G-2oo column (Fig. I). Collagenolytic activity was estimated by measuring the release of hydroxyproline-containing peptides from collagen. Activity was estimated in each tube twice : before and after the addition of synovial fluid. Overlapping peaks of activity were obtained. The smaller peak represented the activity of free collagenase, and the larger represented the sum of the activity of free collagenase plus the activity obtained after activation by synovial fluid. The appropriate effluents (Fig. I) were pooled and concentrated, and the solution was passed through Sephadex G-Ioo. After the determination of collagenolytic activity it was found that, just as in the previous experiment, the free collagenase was eluted together with its proenzyme, moreover, at a level close to the exclusion limit of the gel (Fig. 2). The appropriate effluents were then pooled and free collagenase was bound by the addition of insoluble collagen. The inactive form of collagenase remaining in the supernatant was incubated with synovial fluid (at a protein ratio of I :I) for 6 h at 37 °C. Activated proenzyme was added to the collagen solution and after incubation for 6 h at 26 °C a decrease in viscosity of about 40% was found. In the control sample, in which inactivated supernatant was used, the viscosity was reduced by only 4% (Fig. 3). The collagen reaction products obtained under these conditions were subjected Biochim. Biophys. Acta, 285 (1972) 4 3 6 - 4 4 6
442
D. KRUZE, E. WOJTECKA
100
90 80
70
o
60
50
To Hours
Fig. 3. Viscometric assay of collagenolytic activity generated b y t r e a t m e n t of inactive p r o e n z y m e w i t h synovial fluid. I ml of inactive proenzyme, 1.2 m g of protein, and o.o3 ml of synovial fluid, (1.2 mg of protein, were mixed a n d i n c u b a t e d for 6 h at 37 °C. This solution was t h e n transferred to a 26 °C w a t e r b a t h and mixed w i t h I ml of o.2% collagen and I ml o.o 5 M Tris-HC1, p H 8. 5, containing o.o15 M CaCI~ a n d o. 5 M NaC1. × - - - × , p r o e n z y m e ; Q---O, p r o e n z y m e p r e i n c u b a t e d w i t h synovial fluid.
10o
1.0
Z
80 ~
E Q8 0 co
6o _~
('4
~o.6 c
o4
tS~
e~
2o
~0.2 10 20 30 40 Fraction number
£
50
._> e0
E E kd
Fig. 4. Acrylamide gel electrophoreses of activated p r o e n z y m e - c o l l a g e n reaction m i x t u r e after t h e r m a l d e n a t u r a t i o n . On the left is s h o w n a zero-time reaction m i x t u r e ; on the right is a reaction m i x t u r e after a 4o% reduction in specific viscosity, a, single polypeptide chains; /3, a covalently linked dimer of a-chains, ; y, t h r e e - s t r a n d e d polypeptide chain ; /5A and a A, TCA ; a B, TC B. Fig. 5. C h r o m a t o g r a p h y on S e p h a d e x G - i o o of the activated crude e x t r a c t peak. A sample of 12 mg p r o t e i n (Fractions 3o-42 f r o m S e p h a d e x G-2oo) in a v o l u m e of 2 ml was mixed w i t h synovial fluid at a protein ratio of I :I, and i n c u b a t e d for 6 h at 37 °C. The m i x t u r e was t h e n applied to the c o l u m n and effluent i .5-ml fractions of were collected at a rate of 5 ml/h. © - - - Q , absorbance at 280 n m ; × - - - × , e n z y m e activity.
to disc gel electrophoresis. The activated proenzyme cleaved the collagen molecule to produce a pattern consistent with TCA and TC B (Fig. 4). This pattern is essentially identical with that previously described for other animal collagenasesS, n.
Comparison of molecular weights or free eollagenase and of that released from proenzyme The fraction obtained after chromatography on Sephadex G-2oo, containing Biochim. Biophys. Acta, 285 (1972) 436-446
443
ACTIVATION OF COLLAGENASE PROENZYME
free collagenase and its proenzyme (Fig. I), was concentrated to about 2 ml, preincubated with synovial fluid (protein ratio of I :I) for 6 h at 37 °C and then rechromatographed on Sephadex G-Ioo. Only a single and symmetrical peak of collagenase activity was obtained (Fig. 5). This indicates that the free leucocyte collagenase and the collagenase originating from proenzyme have very similar, or even the same, molecular weights
Effect of synovial fluid on previously activated crude leucocyte extract The foregoing experiments were based on the assumption that the activating factor was present in synovial fluid. However, it could not be ~uled out that the activator was present in the crude leucocyte preparation, and the proenzyme in the synovial fluid. The following experiment was carried out to clarify this question. After chromatography on Sephadex G-ioo, the effluents with collagenolytic activity were pooled (Fig. 5)- This fraction contained free collagenase as well as that obtained after activation of the ploenzyme. I f it is assumed that the proenzyme is present in synovial fluid, this fraction should also contain an activator from the crude leucocyte extract (see results in Fig. 2). The collagenolytic activity was determined in this fraction, and then one of the previously used synovial fluids was added (protein ratio I :I and 1:2). The collagenolytic activity was again determined. Instead of an increase, a decrease of about lO-15% in the collagenolytic activity was found (Table IV). This indicates the absence of an activator in crude leucocyte preparation, thus refuting the considered suggestion. Moreover, the decrease in activity indicates the presence of some inhibitor in synovial fluid. TABLE
IV
I N F L U E N C E OF SYNOVIAL FLUID O N A PREVIOUSLY ACTIVATED FRACTION OF C R U D E E X T R A C T T h e activated fraction was obtained as follows. A peak of crude extract from Sephadex G-zoo, Fractions 3o-4 z, was activated b y synovial fluid (at a protein ratio of I :I) and further fractioned on Sephadex G-loo. Fractions having collagenase activity were pooled and concentrated. T h e reaction mixture, containing i m l of activated fraction, i m l of o . 2 % collagen (gel) and i ml o.o 5 M Tris-HCl, p H 7.5, was incubated at 37 °C for 18 h. For further studies, synovial fluid was added.
Activated fraction Activated fraction + synovial fluid
I 2
Enzyme protein (l~g)
Synovial fluid protein
Released hydroxyproline (l~g)
Inhibition (%)
ioo ioo IOO
-ioo 20o
72 65 61
-io 15
(~,g)
DISCUSSION
The collagenase found in direct extracts of human white cells b y Lazarus et a/.5, TM has been partially purified, and some of its properties have-been described. Up to now, no report has appeared to confirm and complete the characterization of leucocyte collagenase. In general, our enzymatic preparation has similar properties, but we were not able to confirm some of the previous observations. After one or even Biochim. Biophys. Acta,
285 (1972) 4 3 6 - 4 4 6
444
D. KRUZE, E. WOJTECKA
three steps of partial purification, the capacity of the enzyme to degrade fibrils was not reduced 13, and our leucocyte extract had a m a n y times greater specific activity. Further studies m a y be able to dispel doubts as to whether pure leucocyte collagenase is itself able to degrade the native collagen fibrils which exist in the human body. The enzyme obtained after activation by synovial fluid, both from crude extract and from its fractions after sieve chromatography, complies with the requirements foi true collagenase. It degrades reconstituted collagen fibrils, below its denaturation temperature at a neutral pH, to soluble hydroxyproline peptides, and it decreases the intrinsic viscosity of collagen molecules in solution at 26 °C at physiological pH, under which conditions the collagen is cleaved into two fragments. On the basis of its action on collagen in solution or in fibrils, of the degradation products separated on acrylamide gel electrophoresis, and also of its behaviour on gel filtration on Sephadex G-ioo, one can assume that the collagenase obtained in the extract and the collagenase obtained activation by synovial fluid, are one and the same enzyme. When two biological fluids are mixed and the resulting activity is increased, the question arises as to which fluid contains the activator and which contains the activated compound. On the basis of the results obtained, we think that the activator was present in synovial fluid. I f the activator was present in crude leucocyte extract, then it would have to elute from Sephadex G-2oo and Sephadex G-ioo together with flee collagenase (Figs I and 2), and, assuming that proenzyme was present in the synovial fluid, the collagenase formed would have to possess the same molecular weight as the free leucocyte collagenase (Fig. 5). Moreover, adding synovial fluid to the fraction of crude leucocyte extract previously activated by the same fluid, did not result in an increase of collagenolytic activity (Table IV). The increase of collagenolytic activity after adding synovial fluid to the crude extract of white cells, or to fractions obtained after gel filtration on Sephadex, was not caused by the immediate influence of the activator on the enzyme itself, because the synovial fluid did not increase the activity of the partially purified enzyme in the supernatant obtained after fractionation with dioxan. These results exclude the possibility that the increase in activity is due to the combined action of enzyme with some synovial fluid agent. A convincing explanation of this phenomenon is that the collagenase either appears in proenzyme form, or forms a complex with some inhibitor. In the first case, the activation would be due to the cleavage of covalent bonds, and in the second, as a result of the destruction of inhibitor (or at least its partial damage), followed by dissociation of the inhibitor's fragments. The leucocyte collagenase had a molecular weight of approximately 7 ° ooo (ref. i2), and if one of the two known collagenase inhibitors, ax-antitrypsin (mol. wt 45 ooo) or az-macroglobulin (tool. wt ~ 8 o o ooo), had formed a complex with the enzyme, the complexed molecule would have been much bigger than the molecule which we obtained in our experiments (Fig. 2). Moreover, it should be assumed that the amount of inhibitors is always insufficient to bind all enzyme, because we have found free collagenase in all our preparations. On this basis we consider that the collagenase exists as a proenzyme and not as an inactive complex. The amount of proenzyme was always greater than that of free collagenase, and, in various samples of crude leucocyte extract it was about 3-fold greater than the active enzyme. Only the upper limit of molecular weight of collagenase has been Biochim. Biophys. Acta, 285 (1972) 436-446
ACTIVATION OF COLLAGENASE PROENZYME
445
stated in this paper. Collagenase was eluted from the Sephadex G-Ioo column soon after the void volume. Thus, based on the properties of Sephadex G-Ioo (ref. 14), the molecular weight of collagenase was smaller than 80 ooo. The proenzyme appeared in the same fraction as the enzyme or somewhat earlier (Figs I and 2). The nature of the activation process is admittedly unknown, but if the activator is a proteolytic enzyme, as shown b y Harper et al. 4 for tadpole tail-fin procollagenase and by Vaes 15 for mouse bone latent collagenase, the rupture of a peptide bond or the cleaving of a small peptide fragment from proenzyme would take place during the process of activation. However, other activation mechanisms can also occur. The activating factor appeared in all ten synovial fluids studied, but comparisons of its levels of activity in the various fluids was not made. This factor was nondialyzable and thermolabile, but no other properties have been studied. In comparison to the " a c t i v a t o r " found in cultured tadpole medium 4, its activity was considerable ; incubation of crude leucocyte preparation with only an equal amount of synovial fluid protein was sufficient to release a considerable amount of collagenase. Unlike the activator of procollagenase described in tadpole skin culture fluids (ref. 4), and the inactive form of activator in mouse bone explant culture medium 15, the activator found in this study originated from knee joint synovial fluid of rheumatoid arthritis patients. Thus, it did not require tissue culture techniques and it was obtained in an active form. The existence of collagenase in the form of proenzyme together with an activator, would be another regulating mechanism of enzyme activity in addition to the known mechanism involving inhibitor action. The occurrence of a considerable amount of collagenase proenzyme in leucocytes m a y play an important role in collagen degradation in inflammatory states of various tissues, concomitant with leucocyte infiltration. Attention should be drawn to the fact that both leucocytes and the activator discovered in this report occur in joints involved in the rheumatic process. Admittedly Harris et al. n did not note any correlation between the leucocyte count and collagenase activity in synovial fluid, but they did demonstrate the dependence on inhibitor(s). Moreover, inhibitors should simultaneously be included in the determination of the actual amount of collagenase. Furthermore, both collagenolytic activity and amount of inhibitor in synovial fluid probably v a r y with the different stages of disease. Also, the possibility cannot be excluded that an activator occurs not only in synovial fluid but also in other tissues. In this case, in relation to the process of collagen resorption in the human body, account should be taken not only of the considerable amount of free collagenase in leucocytes, but also of the greater amount of procollagenase which is potentially capable of degrading collagen. The evidence of the inactive form of collagenase in human leucocytes, makes it desirable to test for the presence of procollagenase in homogenates or extracts of other human tissues. REFERENCES I Gross, J. and Lapiere, C. M. (1962) Proc. Natl. Acad. Sci. U.S. 48, lO14 2 Evanson, J. M. (1971) in Tissue proteinases (Barret, A. J. and Dingle, J. T., eds) p. 327, North-Holland Publishing Company, Amsterdam Biochim. Biophys. Acta, 285 (1972) 436-446
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D. KRUZE, E. WOJTECKA
3 Lapiere, C. M. and Gross, J. (1963) in Mechanism of Hard Tissue Destruction (Sognnaes, R. E., ed.) American Association for the Advancement of Science, New York Publication No. 75 4 Harper, E., Bloch, K. J. and Gross, J. (I97t) Biochemistry io, 3035 5 Lazarus, G. S., Daniels, J. R. and Brown, R. S. (1968) J. Clin. Invest. 47, 2622 6 Kang, A. H., Nagai, Y., Piez, K. A. and Gross, J. (1966) Biochemistry 5, 509 7 Nagai, Y., Gross, J. and Piez, K. A. (1964) Ann. N . Y . Acad. Sci. 121, 494 8 Stegemann, H. and Stalder, K. (1967) Clin. Chim. Acta 18, 267 9 Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265 Io Sopata, I., Wize, J., Gietka, J., Jakubowski, S. and Kruze, D. (1972) Paper presented at I I I Czechoslovahian Congress of Rheumatology, Piestany II Harris, Jr, E. D., Di Bona, D. R. and Krane, S. M. (1969) J. Clin. Invest. 48, O-lO4 12 Daniels, J. R., Lian, J. and Lazarus, G. (1969) Clin. Res. 17, 154 13 Wojtecka, E. and Kruze, D., in the press I4 In Separation News "Selectivity curves and the choice of sephadex" January, 1972, Pharmacia 15 Vaes, G. (1972) Biochem. J. 126, 275 Biochim. Biophys. Acta, 285 (1972) 436-446
BIOCHIMICAET BIOPHYSICAACTA
BBA 36290 A C T I V A T I O N OF LEUCOCYTE COLLAGENASE P R O E N Z Y M E BY R H E U M A T O I D SYNOVIAL F L U I D
DARIUSZ KRUZE* AND ELZBIETA WOJTECKA Department of Biochemistry, Institz*te of Rheumatology, Warsaw (Poland)
(Received June i3th, 1972)
SUMMARY
A collagenase proenzyme was found in crude human leucocyte extract. An activating agent was present in all the ten studied rheumatoid synovial fluids taken from knee joints. The activator was found to be heat labile and nondialyzable. Activation occurred after mixing c:ude extract with synovial fluid (protein ratio I :I) and heating the mixture for several hours at 37 °C. Collagenase was produced from proenzyme both when the activator acted on the crude leucocyte extlact and when it acted on the same extract previously devoid of free collagenase; however, activation did not occur in crude extracts partially purified with dioxan. The amount of proenzyme in the extract was about 3 times greater than that of free collagenase. Activated proenzyme was capable of releasing soluble hydroxyproline peptides from reconstituted collagen fibrils at 37 °C, and of decreasing the viscosity of collagen in solution at 26 °C. Both the activated proenzyme and the free collagenase from leucocytes produced the same degradation products from soluble native collagen. An alternative possibility, that the activator was present in crude extract and the proenzyme in synovial fluid, was experimentally ruled out. The role of collagenase proenzyme from leucocytes in the inflamatory state of tissues is discussed.
INTRODUCTION At neutral p H and physiological temperature native collagen is degraded only by collagenase. However, in these conditions it has not been possible to demonstrate collagenolytic activity in direct extracts of any tissue. I0 years ago Gross and Lapiere I demonstrated a collagenase in tadpole skin culture fluids. Since that time m a n y authors have obtained collagenolytic enzymes from m a n y m a m m a lian tissues b y using culture techniques 2.
land.
Present address: Dept of Pharmacology, Biocenter of the University of Basel, Switzer-
Biochim. Biophys. Acta, 285 (1972) 436-446
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437
The lack of collagenase in direct extracts could be explained either by the synthesis of the enzyme during tissue culture, or b y the occurrence of the enzyme in tissues as an inactive precursor 3. The latter supposition has been recently supported b y Harper et al. 4, who have isolated a precursor of tadpole collagenase from extracts of tadpole tail-fin tissue. The sole exception is provided b y human granulocyte collagenase, which is directly extractable from granulocytes and does not require tissue culture to produce detectable enzyme 5. The occurrence of collagenase in the supernatant fraction of human white cell homogenates does not exclude the possibility of active enzyme occurring together with inactive proenzyme. In this report we describe the detection and mode of activation of a proenzyme found in direct extracts of human leucocytes. METHODS
Preparation of crude enzyme The white cells were obtained from fresh whole blood taken from normal adult donors. The blood was mixed with a solution containing 0.9% NaC1 and 6% dextran. After allowing the red cells to settle at room temperature, the white cell-rich supernatant was decanted and centrifuged. All subsequent steps were carried out at 4 °C. The cell pellet was resuspended in saline and the remaining red cells removed by brief hypotonic hemolysis. The white cells were then washed with saline, collected by centrifugation, and homogenized in o.oi M Tris-HC1 containing 0.005 M CaC12 and 0.2 M NaC1. The homogenate was then frozen, thawed and centrifuged. The supernatant was dialyzed against the same buffer, after which it was clarified by centrifugation.
Partial purification of crude enzyme To the crude extract of leucocyte in an ice bath was added dioxan in the ratio I :I (v/v) and the suspension was allowed to stand for I h before removing the precipitate b y centrifugation at IO ooo × g for 15 rain. The supernatant solution, containing collagenase, was dialyzed against o.oi M Tris-HC1, p H 7.5, containing 0.005 M CaC12 and 0.2 M NaC1, and then concentrated.
Preparation of substrate Acid-extracted calf skin collagen was purified by the method of Kang et al. 6. Collagen solution for use in the assay of collagenolytic activity was prepared by dissolving lyophilized collagen in cold 0.05% acetic acid at a concentration of 0.20/0 . The solution was dialyzed against 0.05 M Tris-HC1, p H 7.5, containing 0.005 M CaC12 and 0.2 M NaC1, which was followed by centrifugation to remove any undissolved collagen.
Measurement of collagenolytic activity Collagenase activity was determined by the containing collagen degradation products from a enzyme and reconstituted native trypsin-resistant fibrils were incubated with trypsin (at a protein
release of soluble hydroxyprolinereaction mixture which contained collagen fibrils. When the collagen ratio of 2 :I) only 6% of the sub-
Biochim. Biophys. Acta, 285 (1972) 436-446
438
D. KRUZE, E. WOJTECKA
strate was solubilized. A typical reaction mixture contained I ml of 0.2% collagen fibrils which had been allowed to gel for 6 h at 37 °C, i ml of o.o5 M Tris-HC1, p H 7.5, containing 0.003 M CaC12 and I ml enzyme solution containing approx, o.2-1.o mg of protein. The mixture was incubated for 18 h at 37 °C, and then filtered to remove insoluble collagen fibrils. Aliquots of the supernatant were hydrolysed in 6 M HC1 and their hydroxyproline contents were determined. Control tubes contained thermally denatured enzyme solution and were treated in a similar way.
Removal of active collagenase and activation of proenzyme Removal of active collagenase was performed by the method of Harper et al. 4, as follows. A suspension of reconstituted collagen fibrils in o.oi M Tris-HC1, p H 7-5, containing o.oo5 M CaC12 and o.2 M NaC1 was added to the supernatant of a white cell homogenate in an ice bath, in order to remove active collagenase by adsorption onto its solid substrate. The ratio of suspended collagen to protein in solution was I : 5 on a weight basis. The mixture was gently stirred for 2 h, followed by centrifugation and separation of the clear supernatant. Synovial fluids were mixed with the supernatant at a protein concentration ratio of I :I, and the mixture was incubated with reconstituted collagen fibrils for 1 8 h a t 37°C.
Molecular sieve filtration of crude leucocyte extract Gel filtration of crude leucocyte extract was performed at 4 °C on a 2 cm × 8o cm column of Sephadex G-2oo equilibrated with o.oi M Tris-HC1, p H 7.5, containing o.oo5 M CaCI~ and o.2 M NaC1. Fractions which had collagenase activity were pooled, concentrated using Aquacide, Calbiochem., Los Angeles, Calif. and further fractionated by gel filtration on a column, o. 9 cm × 6o cm, of Sephadex G-/oo which had been equilibrated with the same buffer.
Disc gel electrophoresis Thermally denatured products from incubation of collagen with activated proenzyme were subjected to electrophoresis in polyacrylamide gel according to the method of Nagai et al. 7. The enzyme was inactivated by the addition of sufficient o.I M HC1 to the reaction mixtule to reduce the p H to approximately 2.0 before denaturation at 4 ° °C.
Synovial fluid Synovial fluids were aspirated from rheumatoid arthritis patients with knee effusions.
Other methods Hydroxyproline was determinated by the method of Stegemann and Stalder s, and protein by the method of Lowry et al. 9. RESULTS
Activation of crude leucocyte preparation by synovial fluid The effects of synovial fluids on the collagenotytic activity of crude white cell Biochim. Biophys. Acta, 285 (t972) 436-446
ACTIVATION
OF COLLAGENASE
439
PROENZYME
TABLE I I N F L U E N C E OF S Y N O V I A L F L U I D ON T H E C O L L A G E N O L Y T I C A C T I V I T Y OF C R U D E E X T R A C T
The reaction mixture consisted of I ml of crude extract containing i mg of protein, 0.o2-0.o25 ml of synovial fluid containing I m g of protein, i ml of o.2 % collagen (gel) and i ml of o.o 5 M Tris-HC1, pH 7.5, containing 0.o03 M CaC12. These components were mixed and incubated at 37 °C for 18 hl
Crude extract
Crude extract Crude extract + synovial fluids without collagenolytic activity Crude extract + synovial fluids with collagenolytic activity
l~g of released hydroxyproline
i 2 3 4 5 i 2 3 4
Expt I
Expt I I
2o. 4 81.o 8o.9 83.1 82.o 85.1 84.2 8o.0 81.6 --
25.o 112. 5 120. i lO8.8 11o.9 118.o I i 1.9 114.o I 16.9
e x t r a c t s are shown in T a b l e I. Two series of e x p e r i m e n t s were carried o u t on the crude e x t r a c t o b t a i n e d from two donors. S y n o v i a l fluids from knees joints of nine p a t i e n t s w i t h r h e u m a t o i d a r t h r i t i s were used for the e x p e r i m e n t s . F i v e of the fluids used were f o u n d to be d e v o i d of collagenolytic a c t i v i t y , a n d four were positive. The s y n o v i a l fluids were a d d e d to crude leucocyte p r e p a r a t i o n a t a r a t i o o f I : i p r o t e i n c o n c e n t r a t i o n (details see T a b l e I). The a m o u n t of soluble h y d r o x y p r o l i n e p e p t i d e s released from the collagen gels was used as a m e a s u r e of collagenolysis. I n the c o n t r o l samples, account was t a k e n of t h e collagenolytic a c t i v i t y of s y n o v i a l fluid, o f the h y d r o x y p r o l i n e c o n t a i n e d in t h e fluid, a n d also o f the v e r y s m a l l a m o u n t of soluble collagen which d i d n o t u n d e r go t h e change into r e c o n s t i t u t e d fibrils. I n these r e a c t i o n conditions, two different crude leucocyte p r e p a r a t i o n s released a m o u n t s of h y d r o x y p r o l i n e peptides, 20.4 a n d 25.0/~g h y d r o x y p r o l i n e which c o n s t i t u t e d a b o u t 12% of the s u b s t r a t e used. A considerable increase of collagenolytic a c t i v i t y occurred after the a d d i t i o n of s y n o v i a l fluids a n d an a c t i v a t e d crude leucocyte e x t r a c t released from 8 0 # g to I20/~g of h y d r o x y p r o l i n e peptides, which a m o u n t e d to 40-60~o of the r e c o n s t i t u t e d fibrils. No differences in t h e c a p a c i t y for a c t i v a t i o n were found b e t w e e n the s y n o v i a l fluids which showed collagenolytic a c t i v i t y a n d those which d i d not.
Effect of synovial fluids on partially purified collagenase E n z y m e which h a d been p a r t i a l l y purified b y the use of d i o x a n was a c t i v a t e d b y t h e s y n o v i a l fluids. The e x p e r i m e n t s were carried out on two samples of purified e n z y m e a n d using four s y n o v i a l fluids. E v e n t h o u g h t h e e x p e r i m e n t s were carried out u n d e r tile same conditions as for the crude leucocyte e x t r a c t a n d the s a m e s y n o v i a l fluids were used, there was no increase of collagenolytic a c t i v i t y , a n d in fact some small decrease was f o u n d (Table II). The results show t h a t the increase of collagenolytic a c t i v i t y a f t e r the a d d i t i o n of s y n o v i a l fluid is n o t caused b y increased a c t i v i t y of free leucocyte collagenase, nor is it caused b y a s i m u l t a n e o u s effect of collagenase a n d the e n z y m e s occurring in s y n o v i a l fluid.
Biochim. Biophys. Acta, 285 (1972) 436-446
44 °
I). KRUZE, E. WOJTECKA
T A B L E 1[ INFLUENCE DIOXAN
OF SYNOVIAL
FLUIDS
ON THE
ACTIVITY
OF PARTIALLY
PURIFIED
COLLAGENASE
(AFTER
PURIFICATION)
The reaction m i x t u r e consisted of i ml of partially purified collagenase, o.2 mg of protein, I ml of o.2% collagen (gel) and I ml of o.o 5 M Tris-HC1, p H 7.5, containing o.oo 3 M CaCI v These c o m p o n e n t s were mixed and i n c u b a t e d at 37 °C for 18 h. F o r the s t u d y of activation, the incub a t i o n s were carried o u t as above, with the addition of o.o4-o.o 5 ml of synovial fluids, o.2 m g of protein, diluted I :io with Tris-HC1 buffer.
I*g of released hydroxyproline
Partially purified collagenase Partially purified i collagenase + 2 synovial fluids 3 4
Expt I
Expt [I
5o.o 45.o 45.8 47 .o 48.3
38.o 35.o 34.8 37 .2
Effect of synovial fluid on crude leucocyte extract freed from collagenase To confirm the above, and to prove that the increase of activity is connected with the activation of some other component of the crude extract, the following experiments were carried out. In order to bind the free collagenase with substrate and thus remove it from the solution, crude leucocyte preparation was shaken with insoluble collagen. Following such treatment, the crude extract was found to be devoid of collagenolytic activity; no hydroxyproline was released from collagen fibrils, nor was there any decrease in the viscosity of the collagen solution. After the addition of synovial fluid, the enzymatically inactive solution released a considerable amount of hydroxyproline from the collagen gel (Table III). Experiments were carried out on three crude leucocyte extracts, and in each case collagenolytic activity was noted at a level several times greater than the initial activity of the crude leucocyte collagenase. TABLE In ACTIVATION BY SYNOVIAL NOLYTIC ACTIVITY
FLUIDS
OF THt~ CRUDE
EXTRACT
FROM LEUCOCYTES
DEVOID
OF COLLAGE-
The reaction m i x t u r e contained I ml of crude e x t r a c t or crude e x t r a c t devoid of collagenase, i mg of protein, i ml o f o . 2 % collagen (gel) a n d i ml o f o . o 5 M Tris-HC1, p H 7-5, containing 0.003 M CaC1 v Cotlagenase was r e m o v e d f r o m the crude e x t r a c t as described in Methods. F o r the activ a t i o n studies, 0.025 ml of synovial fluid, i mg of protein, was added to the reaction mixture.
#g of released hydroxyproline
Crude e x t r a c t Crude e x t r a c t p r e t r e a t e d with collagen at o °C Crude e x t r a c t p r e t r e a t e d with collagen at o °C + synovial fluid
Expt I
Expt I I
21. 7
28.0
16.o
o
o
o
75.0
77.0
12o.o
Biochim. Biophys. Acta, 285 (1972) 436-446
Expt I I I
441
ACTIVATION OF COLLAGENASE PROENZYME 0.7 0.6
)<
120
o
11o t_6 loo 9o~= 8o~ 7o _~
E 0.5 o 40~
E O.Z o
00 u c
~o.3
O.4
~0.3
c
20 ~z l
~o.2 <~o.~
o
5O 40~
< 0.2
3o~ 20> lO ~
0.1
o
lb
20 30 40 50 Fraction number
60
E c W
~0-~/10
20
30
40
Fraction number
c W
Fig. i. C h r o m a t o g r a p h y on S e p h a d e x G-2oo of t h e c r u d e l e u c o c y t e e x t r a c t . A s a m p l e of i 7 o m g p r o t e i n in a v o l u m e o f 4 m l w a s applied to t h e c o l u m n , a n d 4-ml effluent f r a c t i o n s were collected a t a r a t e of io m l / h ; O - - - O , a b s o r b a n c e a t 280 n m (solution d i l u t e d i : i o ) ; x - - - x , e n z y m e a c t i v i t y ; 0 - - - 0 , e n z y m e a c t i v i t y a f t e r a d d i n g s y n o v i a l fluid. F r a c t i o n s 30-42 were pooled a n d concentrated. Fig. 2. R e c h r o m a t o g r a p h y on S e p h a d e x G-I oo o f a p e a k f r o m a c r u d e l e u c o c y t e e x t r a c t p r e v i o u s l y s e p a r a t e d on S e p h a d e x G-2oo ( F r a c t i o n s 30-42). A s a m p l e o f 13 m g p r o t e i n in a v o l u m e of 2 ml w a s applied to t h e c o l u m n , a n d 1.5-ml effluent f r a c t i o n s were collected a t a r a t e of 5 m l / h ; © - - - O , a b s o r b a n c e a t 280 n m ; × - - - × , e n z y m e a c t i v i t y ; O---O, e n z y m e a c t i v i t y a f t e r a d d i n g s y n o v i a l fluid; V0, void v o l u m e .
These results seem to indicate that a precursor of collagenase, which does not bind with collagen, undergoes activation by synovial fluid.
Properties of collagenase obtained after the activation of the proenzyme A crude leucocyte preparation was separated on a Sephadex G-2oo column (Fig. I). Collagenolytic activity was estimated by measuring the release of hydroxyproline-containing peptides from collagen. Activity was estimated in each tube twice : before and after the addition of synovial fluid. Overlapping peaks of activity were obtained. The smaller peak represented the activity of free collagenase, and the larger represented the sum of the activity of free collagenase plus the activity obtained after activation by synovial fluid. The appropriate effluents (Fig. I) were pooled and concentrated, and the solution was passed through Sephadex G-Ioo. After the determination of collagenolytic activity it was found that, just as in the previous experiment, the free collagenase was eluted together with its proenzyme, moreover, at a level close to the exclusion limit of the gel (Fig. 2). The appropriate effluents were then pooled and free collagenase was bound by the addition of insoluble collagen. The inactive form of collagenase remaining in the supernatant was incubated with synovial fluid (at a protein ratio of I :I) for 6 h at 37 °C. Activated proenzyme was added to the collagen solution and after incubation for 6 h at 26 °C a decrease in viscosity of about 40% was found. In the control sample, in which inactivated supernatant was used, the viscosity was reduced by only 4% (Fig. 3). The collagen reaction products obtained under these conditions were subjected Biochim. Biophys. Acta, 285 (1972) 4 3 6 - 4 4 6
442
D. KRUZE, E. WOJTECKA
100
90 80
70
o
60
50
To Hours
Fig. 3. Viscometric assay of collagenolytic activity generated b y t r e a t m e n t of inactive p r o e n z y m e w i t h synovial fluid. I ml of inactive proenzyme, 1.2 m g of protein, and o.o3 ml of synovial fluid, (1.2 mg of protein, were mixed a n d i n c u b a t e d for 6 h at 37 °C. This solution was t h e n transferred to a 26 °C w a t e r b a t h and mixed w i t h I ml of o.2% collagen and I ml o.o 5 M Tris-HC1, p H 8. 5, containing o.o15 M CaCI~ a n d o. 5 M NaC1. × - - - × , p r o e n z y m e ; Q---O, p r o e n z y m e p r e i n c u b a t e d w i t h synovial fluid.
10o
1.0
Z
80 ~
E Q8 0 co
6o _~
('4
~o.6 c
o4
tS~
e~
2o
~0.2 10 20 30 40 Fraction number
£
50
._> e0
E E kd
Fig. 4. Acrylamide gel electrophoreses of activated p r o e n z y m e - c o l l a g e n reaction m i x t u r e after t h e r m a l d e n a t u r a t i o n . On the left is s h o w n a zero-time reaction m i x t u r e ; on the right is a reaction m i x t u r e after a 4o% reduction in specific viscosity, a, single polypeptide chains; /3, a covalently linked dimer of a-chains, ; y, t h r e e - s t r a n d e d polypeptide chain ; /5A and a A, TCA ; a B, TC B. Fig. 5. C h r o m a t o g r a p h y on S e p h a d e x G - i o o of the activated crude e x t r a c t peak. A sample of 12 mg p r o t e i n (Fractions 3o-42 f r o m S e p h a d e x G-2oo) in a v o l u m e of 2 ml was mixed w i t h synovial fluid at a protein ratio of I :I, and i n c u b a t e d for 6 h at 37 °C. The m i x t u r e was t h e n applied to the c o l u m n and effluent i .5-ml fractions of were collected at a rate of 5 ml/h. © - - - Q , absorbance at 280 n m ; × - - - × , e n z y m e activity.
to disc gel electrophoresis. The activated proenzyme cleaved the collagen molecule to produce a pattern consistent with TCA and TC B (Fig. 4). This pattern is essentially identical with that previously described for other animal collagenasesS, n.
Comparison of molecular weights or free eollagenase and of that released from proenzyme The fraction obtained after chromatography on Sephadex G-2oo, containing Biochim. Biophys. Acta, 285 (1972) 436-446
443
ACTIVATION OF COLLAGENASE PROENZYME
free collagenase and its proenzyme (Fig. I), was concentrated to about 2 ml, preincubated with synovial fluid (protein ratio of I :I) for 6 h at 37 °C and then rechromatographed on Sephadex G-Ioo. Only a single and symmetrical peak of collagenase activity was obtained (Fig. 5). This indicates that the free leucocyte collagenase and the collagenase originating from proenzyme have very similar, or even the same, molecular weights
Effect of synovial fluid on previously activated crude leucocyte extract The foregoing experiments were based on the assumption that the activating factor was present in synovial fluid. However, it could not be ~uled out that the activator was present in the crude leucocyte preparation, and the proenzyme in the synovial fluid. The following experiment was carried out to clarify this question. After chromatography on Sephadex G-ioo, the effluents with collagenolytic activity were pooled (Fig. 5)- This fraction contained free collagenase as well as that obtained after activation of the ploenzyme. I f it is assumed that the proenzyme is present in synovial fluid, this fraction should also contain an activator from the crude leucocyte extract (see results in Fig. 2). The collagenolytic activity was determined in this fraction, and then one of the previously used synovial fluids was added (protein ratio I :I and 1:2). The collagenolytic activity was again determined. Instead of an increase, a decrease of about lO-15% in the collagenolytic activity was found (Table IV). This indicates the absence of an activator in crude leucocyte preparation, thus refuting the considered suggestion. Moreover, the decrease in activity indicates the presence of some inhibitor in synovial fluid. TABLE
IV
I N F L U E N C E OF SYNOVIAL FLUID O N A PREVIOUSLY ACTIVATED FRACTION OF C R U D E E X T R A C T T h e activated fraction was obtained as follows. A peak of crude extract from Sephadex G-zoo, Fractions 3o-4 z, was activated b y synovial fluid (at a protein ratio of I :I) and further fractioned on Sephadex G-loo. Fractions having collagenase activity were pooled and concentrated. T h e reaction mixture, containing i m l of activated fraction, i m l of o . 2 % collagen (gel) and i ml o.o 5 M Tris-HCl, p H 7.5, was incubated at 37 °C for 18 h. For further studies, synovial fluid was added.
Activated fraction Activated fraction + synovial fluid
I 2
Enzyme protein (l~g)
Synovial fluid protein
Released hydroxyproline (l~g)
Inhibition (%)
ioo ioo IOO
-ioo 20o
72 65 61
-io 15
(~,g)
DISCUSSION
The collagenase found in direct extracts of human white cells b y Lazarus et a/.5, TM has been partially purified, and some of its properties have-been described. Up to now, no report has appeared to confirm and complete the characterization of leucocyte collagenase. In general, our enzymatic preparation has similar properties, but we were not able to confirm some of the previous observations. After one or even Biochim. Biophys. Acta,
285 (1972) 4 3 6 - 4 4 6
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three steps of partial purification, the capacity of the enzyme to degrade fibrils was not reduced 13, and our leucocyte extract had a m a n y times greater specific activity. Further studies m a y be able to dispel doubts as to whether pure leucocyte collagenase is itself able to degrade the native collagen fibrils which exist in the human body. The enzyme obtained after activation by synovial fluid, both from crude extract and from its fractions after sieve chromatography, complies with the requirements foi true collagenase. It degrades reconstituted collagen fibrils, below its denaturation temperature at a neutral pH, to soluble hydroxyproline peptides, and it decreases the intrinsic viscosity of collagen molecules in solution at 26 °C at physiological pH, under which conditions the collagen is cleaved into two fragments. On the basis of its action on collagen in solution or in fibrils, of the degradation products separated on acrylamide gel electrophoresis, and also of its behaviour on gel filtration on Sephadex G-ioo, one can assume that the collagenase obtained in the extract and the collagenase obtained activation by synovial fluid, are one and the same enzyme. When two biological fluids are mixed and the resulting activity is increased, the question arises as to which fluid contains the activator and which contains the activated compound. On the basis of the results obtained, we think that the activator was present in synovial fluid. I f the activator was present in crude leucocyte extract, then it would have to elute from Sephadex G-2oo and Sephadex G-ioo together with flee collagenase (Figs I and 2), and, assuming that proenzyme was present in the synovial fluid, the collagenase formed would have to possess the same molecular weight as the free leucocyte collagenase (Fig. 5). Moreover, adding synovial fluid to the fraction of crude leucocyte extract previously activated by the same fluid, did not result in an increase of collagenolytic activity (Table IV). The increase of collagenolytic activity after adding synovial fluid to the crude extract of white cells, or to fractions obtained after gel filtration on Sephadex, was not caused by the immediate influence of the activator on the enzyme itself, because the synovial fluid did not increase the activity of the partially purified enzyme in the supernatant obtained after fractionation with dioxan. These results exclude the possibility that the increase in activity is due to the combined action of enzyme with some synovial fluid agent. A convincing explanation of this phenomenon is that the collagenase either appears in proenzyme form, or forms a complex with some inhibitor. In the first case, the activation would be due to the cleavage of covalent bonds, and in the second, as a result of the destruction of inhibitor (or at least its partial damage), followed by dissociation of the inhibitor's fragments. The leucocyte collagenase had a molecular weight of approximately 7 ° ooo (ref. i2), and if one of the two known collagenase inhibitors, ax-antitrypsin (mol. wt 45 ooo) or az-macroglobulin (tool. wt ~ 8 o o ooo), had formed a complex with the enzyme, the complexed molecule would have been much bigger than the molecule which we obtained in our experiments (Fig. 2). Moreover, it should be assumed that the amount of inhibitors is always insufficient to bind all enzyme, because we have found free collagenase in all our preparations. On this basis we consider that the collagenase exists as a proenzyme and not as an inactive complex. The amount of proenzyme was always greater than that of free collagenase, and, in various samples of crude leucocyte extract it was about 3-fold greater than the active enzyme. Only the upper limit of molecular weight of collagenase has been Biochim. Biophys. Acta, 285 (1972) 436-446
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stated in this paper. Collagenase was eluted from the Sephadex G-Ioo column soon after the void volume. Thus, based on the properties of Sephadex G-Ioo (ref. 14), the molecular weight of collagenase was smaller than 80 ooo. The proenzyme appeared in the same fraction as the enzyme or somewhat earlier (Figs I and 2). The nature of the activation process is admittedly unknown, but if the activator is a proteolytic enzyme, as shown b y Harper et al. 4 for tadpole tail-fin procollagenase and by Vaes 15 for mouse bone latent collagenase, the rupture of a peptide bond or the cleaving of a small peptide fragment from proenzyme would take place during the process of activation. However, other activation mechanisms can also occur. The activating factor appeared in all ten synovial fluids studied, but comparisons of its levels of activity in the various fluids was not made. This factor was nondialyzable and thermolabile, but no other properties have been studied. In comparison to the " a c t i v a t o r " found in cultured tadpole medium 4, its activity was considerable ; incubation of crude leucocyte preparation with only an equal amount of synovial fluid protein was sufficient to release a considerable amount of collagenase. Unlike the activator of procollagenase described in tadpole skin culture fluids (ref. 4), and the inactive form of activator in mouse bone explant culture medium 15, the activator found in this study originated from knee joint synovial fluid of rheumatoid arthritis patients. Thus, it did not require tissue culture techniques and it was obtained in an active form. The existence of collagenase in the form of proenzyme together with an activator, would be another regulating mechanism of enzyme activity in addition to the known mechanism involving inhibitor action. The occurrence of a considerable amount of collagenase proenzyme in leucocytes m a y play an important role in collagen degradation in inflammatory states of various tissues, concomitant with leucocyte infiltration. Attention should be drawn to the fact that both leucocytes and the activator discovered in this report occur in joints involved in the rheumatic process. Admittedly Harris et al. n did not note any correlation between the leucocyte count and collagenase activity in synovial fluid, but they did demonstrate the dependence on inhibitor(s). Moreover, inhibitors should simultaneously be included in the determination of the actual amount of collagenase. Furthermore, both collagenolytic activity and amount of inhibitor in synovial fluid probably v a r y with the different stages of disease. Also, the possibility cannot be excluded that an activator occurs not only in synovial fluid but also in other tissues. In this case, in relation to the process of collagen resorption in the human body, account should be taken not only of the considerable amount of free collagenase in leucocytes, but also of the greater amount of procollagenase which is potentially capable of degrading collagen. The evidence of the inactive form of collagenase in human leucocytes, makes it desirable to test for the presence of procollagenase in homogenates or extracts of other human tissues. REFERENCES I Gross, J. and Lapiere, C. M. (1962) Proc. Natl. Acad. Sci. U.S. 48, lO14 2 Evanson, J. M. (1971) in Tissue proteinases (Barret, A. J. and Dingle, J. T., eds) p. 327, North-Holland Publishing Company, Amsterdam Biochim. Biophys. Acta, 285 (1972) 436-446
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3 Lapiere, C. M. and Gross, J. (1963) in Mechanism of Hard Tissue Destruction (Sognnaes, R. E., ed.) American Association for the Advancement of Science, New York Publication No. 75 4 Harper, E., Bloch, K. J. and Gross, J. (I97t) Biochemistry io, 3035 5 Lazarus, G. S., Daniels, J. R. and Brown, R. S. (1968) J. Clin. Invest. 47, 2622 6 Kang, A. H., Nagai, Y., Piez, K. A. and Gross, J. (1966) Biochemistry 5, 509 7 Nagai, Y., Gross, J. and Piez, K. A. (1964) Ann. N . Y . Acad. Sci. 121, 494 8 Stegemann, H. and Stalder, K. (1967) Clin. Chim. Acta 18, 267 9 Lowry, O. H., Rosenbrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265 Io Sopata, I., Wize, J., Gietka, J., Jakubowski, S. and Kruze, D. (1972) Paper presented at I I I Czechoslovahian Congress of Rheumatology, Piestany II Harris, Jr, E. D., Di Bona, D. R. and Krane, S. M. (1969) J. Clin. Invest. 48, O-lO4 12 Daniels, J. R., Lian, J. and Lazarus, G. (1969) Clin. Res. 17, 154 13 Wojtecka, E. and Kruze, D., in the press I4 In Separation News "Selectivity curves and the choice of sephadex" January, 1972, Pharmacia 15 Vaes, G. (1972) Biochem. J. 126, 275 Biochim. Biophys. Acta, 285 (1972) 436-446