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The induction of collagenase and a neutral proteinase by their high molecular weight substrates in Achromobacter iophagus
J. Mol. Biol. (1976) 107, 293-305
The Induction of Collagenase and a Neutral Proteinase by their High Molecular Weight Substrates in Achromobacter iophagus V. KEIL-DLOUHA, R. M.ISRAI~[t AND B. Kv.m
Service de Chimie des Protdines, Institut Pasteur 28, rue du Docteur Roux, 75024 Paris Cedex 15, France (Received 12 April 1976) The synthesis of collagenase in Aehromobacter iophagus has been shown to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the macromolecular inducer of peptide bonds which could be digested by the collagenase is indispensable for the effect of induction. On the other hand, an addition of a low molecular weight substrate or inhibitor of collagenase does not stimulate the enzyme synthesis. Lack of collagenase induction was also observed in the case of ~-casein which is a macromolecular substrate with four peptide bonds digestible by the collagenase. Nevertheless in the presence of fi-casein the induction of a neutral caseinolytic proteinase was found. The probable role of conformation structure of a macromolecular substrate in the mechanism of induction is discussed. The dependence of induction on the growth phase of the culture was studied. The collagenase activity appears only after the last phase of the exponential growth. It was proved that no zymogen or cell-accLunulated enzyme is present in the first stage of exponential growth and that the collagenase synthesis is in direct correlation with a particular state of the bacterial growth cycle.
1. Introduction The collagenase from a non-pathogenic aerobic Achromobacter iophagus strain is an extracellular metallo-enzyme (EC 3.4.24) which was first described b y Woods et al. (1972) and which could be isolated from the culture medium as described previously (Lecroisey et al., 1975; Keil-Dlouha, 1976). The molecular weight of this eollagenase is 104,000. Its amino acid composition is quite different from that of Clostridium collagenase. The enzyme cleaves the X-Gly bond in the sequence Pro-X-Gly-Y where Y is generally proline or alanine and X is a neutral amino acid (Keil et al., 1975). The Achromobacter collagenase splits this kind of bond in the helical regions of native collagen as well as in a number of synthetic peptide substrates. The recent study in our laboratory proved that pure Achromobacter collagenase can also split analogous bonds in fl-casein (Gilles & Keil, 1976). According to current ideas the extracellular proteinases are in general considered as constitutive enzymes. Nevertheless clear evidence for the constitutive or inducible nature of extracellular proteinases is rather difficult to obtain since most growth media used contain complex nitrogenous substances such as peptone, meat extracts, etc. which could supply inducers for proteinase synthesis. Institut Pasteur Production. 293
294
V. K E I L - D L O U H A ,
R. M I S R A H I A N D B. K E I L
T h e first i n d i c a t i o n t h a t a n e x t r a c e l l u l a r p r o t e i n a s e m a y b e i n d u c e d can be f o u n d in t h e s t u d y on t h e b i o s y n t h e s i s o f a p r o t e i n a s e in Serratia marcescens ( C a s t a n e d a Agullo, 1955). T h e e x p e r i m e n t s p r e s e n t e d in this p a p e r were designed to p r o v i d e evidence t h a t coUagenase o f A . iophagus is a n i n d u c i b l e e x t r a c e l l u l a r e n z y m e . T h e results show t h a t t h e i n d u c t i o n of collagenase t a k e s place o n l y a t t h e e n d o f t h e e x p o n e n t i a l p h a s e o f b a c t e r i a l g r o w t h a n d t h a t this d e l a y could be e x p l a i n e d n e i t h e r b y e v e n t u a l z y m o g e n a c t i v a t i o n nor b y t h e l i b e r a t i o n o f t h e i n t r a c e l l u l a r s t o c k o f t h e e n z y m e . T h e s y n t h e s i s of collagenase is i n d u c e d e i t h e r b y collagen or b y its h i g h m o l e c u l a r weight f r a g m e n t s . Collagen also induces t h e f o r m a t i o n of a n e u t r a l p r o t e i n a s e . Different results were o b t a i n e d w i t h fl-casein, which u n d e r t h e s a m e c o n d i t i o n s induces t h e s y n t h e s i s of a n e u t r a l p r o t e i n a s e only.
2. Materials and Methods (a) Materials ~-Chymotrypsin (lot CD1), trypsin (TRL) and pepsin (PM) were obtained from Worthington Biochemical Corp. Crude collagenase from A . iophagus (spec. act. 170 nkat/mg) was a gift from I n s t i t u t P a s t e u r Production. Calf skin collagen (acid-soluble, C-1633) was purchased from Sigma. Casein (LAB) was a product of Merck. fl-casein was a gift from Dr B. Ribadeau-Dumas. The synthetic collagenase substrate 4-phenylazobenzyloxy-carbonyl-L-prolyl-L-leucyl-glyeyl-L-prolyl-D-arginined i h y d r a t e was purchased from Fluka. The synthetic collagenase inhibitor prolyl-leucyl-sarcosyl-proline was a gift from Dr O. Siffert. (b) Bacterial culture, growth control and induction method The collagenase-producing strain of A.. iophagu~ was the same as isolated a n d previously described b y Woods et al. (1972). I t was routinely m a i n t a i n e d on a complex agar m e d i u m (Welton & Woods, 1973). The culture medium consisted of a 2.5% solution of Casamino acids (a vitamin-free acid-hydrolyzed casein, Difco Laboratories) in 0.1 M-Tris-HC1 buffer (pH 7"6) which contained 0.4 M-NaC1 and 2 mM-CaC12. The bacterial suspension (100 ml with O.D. 4-0 to 5"0 at 600 nm) was inoculated to 1000 ml of culture medium a n d incubated at 29~ in the 2-1 Biolafitte fermenter under standardized conditions. The samples were withdi'awn a t 1-h intervals. The mass of bacterial growth was estimated b y the t u r b i d i t y of suitable dilutions of the culture samples at 600 inn using a Zeiss spectrophotometer. The products whose inducing properties were being tested were dissolved in 100 ml of sterile 0-1 M-Tris-HC1 buffer (pH 7"6) and then a d d e d to the growing culture. (e) Assay of proteolytic activity Collagenase activity, in the culture samples, was measured colorimetrically using 4-phenylazo -benzyloxycarbonyl -L-prolyl-L-leucyl -glycyl - t. -prolyl - D-arginine d i h y d r a t e (Pz-Pro-Leu-Gly-Pro-D-Arg) according to Wfinsch & Heidrich (1963). Numerical d a t a have been recalculated on the basis of 1 p k a t = 0-09 units according to Wfinsch & Heidrich. General protease a c t i v i t y was measured on casein as substrate according to Laskowski (1955). I t was impossible to determine proteinase activity directly in the culture samples because of the presence of acid-hydrolyzed casein. The assays were performed after chromatographic purification of the dialysed a m m o n i u m sulphate precipitate of culture supernatant. (d) Attempts at zymogen activation Culture samples (10 ml) were centrifuged at 4~ for 20 rain at 10,000 revs/min in a SorvaU centrifuge RC2B. The cells were washed rapidly once with 10 ml of ice-cold 0-1
INDUCTION
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I N A. iophagus
295
M-Tris. HC1 buffer (pH 7"6) and then resuspended in 10 ml of the same buffer. The cells in the suspension were sonicated in a Sonifier B12 a t 130 W for 2 min. The eollagenase assays were performed either directly after t r e a t m e n t or after incubation of the suspension of the broken cells with 1 ml of the sample from the middle of the enzyme production phase. I n the second series of experiments the supernatants (10 ml) of samples from the early growth phase were mixed with sample (1 ml) from the middle of the enzyme production phase. The incubation of samples was done either at 4~ for 16 h or at 30~ for 1 h or 2 h.
(e) Estimation of synthetic substrate in the broken cells I n order to see if the synthetic substrate could penetrate the bacterial cell, the cells grown in the presence of the synthetic substrate were broken as described above. One half of the sample was acidified to p H 3.0 and e x t r a c t e d directly b y 5 ml of ethyl acetate. The other half was t r e a t e d for 2 h at 30~ with 5 mg of crude collagenase, acidified to p H 3-0 and then e x t r a c t e d b y 5 ml of ethyl acetate as in the routine enzyme assay. This method permits one to measure even traces of Pz-Pro-Leu-Gly-Pro-i)-Arg. (f) Isolation of collagenase and neutral proteinase.from the culture medium All steps of the purification were performed at 4~ The cells from 1-1 of bacterial culture were removed b y centrifugation. The supernatant was made 60% in a m m o n i u m sulphate. The precipitate after centrifugation was dialysed and the resulting solution was applied to a DE32 cellulose column (10 cm • 1 cm). The chromatography was performed according to Lecroisey et al. (1975). The samples from the fractions were tested for the presence of collagenolytic or caseinolytic activities, as described previously. (g) Peptic hydrolysate of collagen and its further degradation by proteolytic enzymes F i v e grams of denatured collagen were hydrolysed with pepsin at 37~ p H 2 at an e n z y m e / s u b s t r a t e ratio of 1 : 100. After 2 h of digestion the solution was lyophilized, then dissolved in 20 in[ of 0.01 ~-NH4HCO3 buffer (pH 8.5) and applied to a column of Sephadex G25. The macromolecular fraction which was obtained from 3 successive separations was lyophilized, thon dissolved in 200 ml 0"05 M-Tris.ttC1 buffer (pH 8"5). 100 ml of this solution were digested b y crude Aehromobaeter collagenase. The other 100 ml portion was digested with trypsin and then chymotrypsin. I n all eases, the digestion was performed at 30~ for 16 h with an enzyme/substrate ratio of 1 : 100. Two-thirds of each digest were used for the induction experiments, the rest was lyophilized, redissolved in 10 ml 0"01 M-NH4HCO3 buffer and separated on Sephadex G25 as described for the peptic hydrolysate of collagen. 3. R e s u l t s (a) Induction of extraceUular collagenase anal its relation to the age of the culture T h e f o r m a t i o n o f collagenase b y A. iophagus was s t u d i e d in t w o cultures g r o w i n g in t h e buffered m e d i u m c o n t a i n i n g 2 . 5 % C a s a m i n o acids (Fig. l(a)) a n d c o n t a i n i n g 2 . 5 % p e p t i c h y d r o l y s a t e o f t h e d e n a t u r e d collagen (Fig. l(b)). T h e final level o f g r o w t h was higher in t h e m e d i u m c o n t a i n i n g t h e h y d r o l y s e d collagen. W h e n collagenase p r o d u c t i o n was e x a m i n e d t h e p r i n c i p a l difference b e t w e e n t h e t w o cultures was e v i d e n t . N o t r a c e o f collagenase a c t i v i t y was d e t e c t e d in t h e case o f c u l t u r e (a) (Fig l(a)). On t h e o t h e r h a n d , w h e n t h e c u l t u r e m e d i u m c o n t a i n s d i g e s t e d collagen (Fig. l(b)) f o r m a t i o n o f collagenase can be observed. I t s t a r t s a t t h e e n d o f t h e e x p o n e n t i a l phuse of g r o w t h . T h e e n z y m e p r o d u c t i o n t h u s c o n t i n u e s to a c c e l e r a t e w h e n t h e b a c t e r i a l m a s s has become c o n s t a n t . M a x i m u m collagenase a c t i v i t y was a c h i e v e d on a v e r a g e a f t e r 11 hours in t h e c u l t u r e s c o n t a i n i n g 2 . 5 % h y d r o l y s e d collagen. I t is e v i d e n t f r o m t h e a b o v e results (Fig. l(b)) t h a t e v e n w h e n t h e c u l t u r e m e d i u m c o n t a i n s t h e i n d u c e r (digested collagen) f r o m t h e beginning, t h e f o r m a t i o n o f col20
296
V. K E I L - D L O U H A ,
R. MISRAHI
(a)
A N D B. K E I L
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FIo. 1. Dependence of eollagenase formation on culture medium. A. iophagus was grown in buffered medium as described in Materials and Methods. (a) I n the presence of 2-5% Casamino acids. (b) I n the presence of 2.5% peptic hych.olysate of denatured collagen. --l--l--, Bacterial growth as turbidity measured at 600 nm. - - O - - O - - , Collagenase activity against synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg.
lagenase starts only at the end of the exponential phase of bacterial growth. In order to see the effect of inducer at various stages of culture, buffered medium containing 2-5% Casamino acids was inoculated with bacteria twice washed with the same medium in three simultaneous running cultures under the same conditions as in Figure l(a). Then peptic hydi'olysate of collagen was added (to a final eoncn of 0-25%) to each culture, at the different stage of growth: simultaneously with inoculum (Fig. 2(a)), after five hours, at the end of the exponential phase (Fig. 2(b)) and after 7 hours (Fig. 2(c)). I t is evident that if hydrolysed collagen is added to the culture at the end of the exponential phase, then the appearance of the collagenase activity could be detected after one hour (Fig. 2(b)). When the inducer is added to the culture medium simultaneously with inoeulum the activity again appears only at the end of the exponential phase of growth (Fig. 2(a)). On the other hand, when the inducer is added at a later stage (Fig. 2(c)), the collagenase formation is rather slow. The maximum amount of enzymic activity is proportional to the final concentration of the inducer in the culture medium (Fig. 3). Relatively low concentrations of inducer were chosen for this study in order not to stimulate the bacterial growth. As was shown above, the peptic hydrolysate of denatured collagen is a good source of nutrition for A. iophagus. I t can be seen that addition of higher quantities of inducer, at the end of the exponential phase, causes additional bacterial growth (Fig. 3(c)). Consequently a retardation of collagenase formation by two hours was observed. This retardation corresponds to the end of the second exponential phase in the growth curve. These results indicate that the induction of collagenase synthesis may be associated with some particular phase of the division cycle. The cells are not able to synthesize this enzyme during exponential growth,
INDUCTION 10
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5
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0
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Fro. 2. Influence of age of the bacterial culture on the induction effect. 5 g peptic hydrolysate of d e n a t u r a t e d collagen ( $ ) were added to 1 1 of Achromebacter culture which was grown on 2.5% Casamino acids. (a) A t zero time. (b) After 5 h of bacterial growth. (c) After 7 h o f b a c t e r l a l growth. -- 9 9 Bacterial growth; - - O - - O - - , collagenase activity.
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FIG. 3. Dependence of the collagenase synthesis on the q u a n t i t y of the added inducer. Different quantities of peptic hydrolysate of d e n a t u r e d collagen were added ( $ ) to 3 cultures growing u n d e r the same conditions on Casamino acids. Final concentration of the peptic hydrolysate was 0.25% (a), 0.5% (b), a n d 2-5% (c). Bacterial growth (a) - - 0 - - 0 - - ; (h) - - B - - m - - ; (e) - - A - - A - - . Collagenase activity (a) - - O - - O - - ; (b)--~--~--; (o) --A--A--.
298
V. K E I L - D L O U H A ,
R. MISRAHI
AND B. KEIL
(b} Demonstration of absence of zymogen or of intracellular
coUagenase in the early stages of growth As previously shown (Fig. l(b)), collagenase activity appears in the culture at the end of the exponential phase of bacterial growth. A possible explanation of this phenomenon could be a progressive activation of an eventual zymogen. I f an activation system for a zymogen had existed, it would have been present in the sample with the active enzyme. Therefore supernatants of samples from the early growth phase of the culture were mixed with samples from the end of the enzyme production phase (10 : 1 (v/v)). One series of these mixtures was incubated 16 hours at 4~ another one was incubated at 30~ for one or two hours and then assayed in the usual way. I t was found that no activation of the samples from early growth phase by the later one occurred. From these results it can be concluded that no measurable amount of extracellular zymogen is present in the culture medium. A delay in the formation of collagenase would also be consistent with an accumulation of the enzyme, eventually of the zymogen, inside the cells. To test this possibility samples were taken at different stages of growth, and centrifuged. The cells were resuspended in 0.1 M-Tris.HC1 buffer, broken by sonication and incubated under the same conditions as described above. Neither free collagenase fior a zymogen which could be activated was found. (c) Induction of collagenase by denatured collagen and by its high
molecular weight fragments Thermally denatured collagen represents a mixture of polypeptide chains of average molecular weight 100,000. When a solution of this gelatinized protein is added to the culture at the end of its exponential phase of growth collagenase activity appears in less than an hour (Fig. 4). I t was shown in Figures 1 to 3 that the peptic hydrolysate of denatured collagen causes the induction of collagenase. In order to characterize better the nature of this
IO20
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FIG-. 4. The induction of collagenase b y thermally denatured collagen. Collagen solution preincubated for 1 h a t 80~ was added ( $ ) in final concentration 0.5% to a culture grown a s d e s c r i b e d in Fig. 3. --t--O--, Bacterial growth; - - O - - O - - , collagenase activity.
INDUCTION
OF COLLAGENASE
I N A. iophagua
299
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FIo. 5. Preliminary characterization of the inducer contained in the peptic hydrolysate of collagen. (a) Separation of peptic hydrolysate (5 g), on Sephadex G25. The column (180 cm • 4 cm) was equilibrated with 0.01 M-NH4HCO3 buffer (pl:[ 8.5). 7-ml fractions were collected in 10-min intervals. 4 The position of molecular weight markers on the elution diagram : 25,700, Chymotrypsinogen; 11,700, cytochromc c; 6500~ pancreatic trypsin inhibitor. ( - ) Optical density a t 280 nm. The fractions 100 to 180 (I), 181 to 230 (II), 231 to 300 (III) were pooled a n d lyophflized. (b) Effect of isolated fraction I on the eollagenase induction. Fraction I (2.5 g) was dissolved in 50 ml 0.1 ~-Tris-HC1 buffer and added to the culture grown on Casamino acids. - - $ - - O - - , Bacterial growth; - - O - - O - - , eollagenase activity. (e) The effect of fractions I I and I I I under the same conditions. Fraction I I (2-5 g) and fraction I I I (2.5 g), which were obtained from 2 preparation runs, were added to the appropriate cultures. Bacterial growth in cultures: - - t - - $ - - , :II; and - - V - - Y - - , I I I . Collagenase activity in cultures after addition of fraction I I ( - - O - - O - - ) and I I I ( - - V - - V - - ) .
inducer we separated the peptic hydrolysate into three main fractions by Sephadex G25 gel filtration (Fig. 5(a)). The induction capacity was tested by separate simultaneous additions of each fraction to three cultures of A. iophagus which were grown up in the buffered medium containing 2.5% Casamino acids. Figure 5(b) shows that after the addition of maeromolecular fraction 1, the induction of collagenase takes place. On the other hand, no trace of the collagenase activity was found after addition of low molecular weight fractions II and I I I (Fig. 5(c)). In the next experiment the macromolecular fraction I (from Fig. 5(a)) was digested
300
V. K E I L - D L O U H A ,
R. MISRAHI
AND B. KEIL
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either by collagenase or by trypsin and chymotrypsin. A comparison of the elution diagrams from a gel filtration on a Sephadex G25 column shows that the action of collagenase as well as that of trypsin and chymotrypsin resulted in the liberation of a number of small molecular weight products (Fig. 6(a) and (b)). When the macromolecular fraction I which was digested by collagenase was added to the bacterial culture under the same conditions as in the previous experiment, no induction of enzyme was observed as shown in Figure 7(a). The same results were obtained with non-fractionated, denatured collagen which was also digested by collagenase. On the other hand, fraction I which was hydrolysed by trypsin and chymotrypsin provoked the same effect of induction (Fig. 7(b)) as in the case of non-digested fraction I. These results show that the presence of peptide bonds susceptible to the enzymic action of collagenase is indispensable for the induction of the enzyme. (d) An attempt to induce collagenase formation in the presence of its low molecular weight substrate or inhibitor In order to see whether low molecular weight substrate or inhibitor of collagenase could provoke the induction of this enzyme two types of experiment were done. First of all we chose the synthetic peptide Pz-Pro-Leu-Gly-Pro-D-Arg which is the best low molecular weight substrate. The concentration of added substrate was 2 • 10-4 ~I (0-016% }. Nevertheless collagenase was not induced. After l0 hours of
INDUCTION
I N A . iophagus
OF COLLAGENASE
(0)
301
(b)
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7. E f f e c t o f the enzymic t r e a t m e n t on the inducing properties o f the collagen or its m a c r o -
fraction I. (a) Loss of inducing properties of collagen or of its macromolecular fraction I after their digestion b y Achromobac~er collagenase. Two cultures were grown on the buffered m e d i u m containing 2.5% Casamino acids. After 5 h growth, 2.5 g of collagen or fraction I digested b y collagenases were added ( ~ ) . The conditions of the digestion are described in Materials a n d Methods. - - O - - Q - - , Bacterial growth, a n d - - O - - O - - , eollagenase activity, in the culture to which t;he collagen hydrolysed b y collagenase was added. - - 9 1 4 9 Bacterial growth a n d - - ~ 7 - - V - - , collagenase activity in the culture to which the fraction I hydrolysed b y collagenase was added. (b) Collagenase ind,action b y the fraction I digested b y trypsin a n d ehymotrypsin. The culture conditions the same as in (a). After 5 h growth 2.5 g fraction I digested b y trypsin a n d chymotrypsin were added ( ~ ). - - O - - O - - , Bacterial growth; - - O - - O - - , eollagenase activity. molecular
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Fzo. 8. A t t e m p t to induce collagenase b y its inhibitor Pro-Leu-Sar-Pro. (a) The culture (1 1) was grown on Casamino acids, as described previously. After 5 h of bacterial growth, 150 m g of Pro-Leu-Sar-Pro were added ( Je ). - - O - - O - - , Bacterial growth; - - O - - O - - , collagenase activity. After 10 h the culture s u p e r n a t a n t was separated b y centrifugation, treated as described in Materials a n d Methods a n d t h e n applied to the DE32 cellulose column (10 em X 1 era). (b) Elution profile from the DE32 cellulose column. - - 0 - - 0 - - , Optical density a t 280 n m ; 0 . - - O - - , colla~enasc activit~r; - - 9 - - 9 - - , caseinol~rtic activit~r.
302
V. KEIL-DLOUttA, R. MISRAHI AND B. KEIL
culture growth, the cells were separated from the culture medium by centrifugatlon, washed twice and broken by sonication as described in Materials and Methods. No trace of substrate was detected in the broken cells. The quantity of intact substrate in the culture medium was tested. A correction which was determined for spontaneous degradation during a six hour period at 30~ represented 8% of the total substrate added to the culture. No additional enzymic degradation of the substrate was observed. This experiment proves that low molecular weight substrate did not induce any detectable traces of collagenase. Absence of induction was also shown in the case of the peptide Pro-Leu-Sar-Pro, which is a collagenase inhibitor (Svensson et al. 1975) (Fig. 8(a)). Nevertheless in this case it was necessary to prove that the absence of detectable enzymic activity in the supernatant is not due to inhibition by excess of this tetrapeptide. To this end, the supernatant of this baterial culture was precipitated with ammonium sulphate, the precipitate was dialysed and submitted to chromatography on DE32 cellulose as described in Material and Methods. Under these conditions a possible complex of the enzyme with inhibitor could no longer exist. Collagenase would be displaced from this ion-exchange column with Tris.HC1 buffer containing 1 ~-NaC1. Figure 8(b) shows that no collagenase activity was detected. Those experiments indicate that the low molecular weight substrate and inhibitor of collagenase do not induce synthesis of the enzyme.
(e) The different effects of collagen partial hydrolysate and of fl-casein on the induction of proteinases in the culture of Achromobacter iophagus We tested the possibility of collagenase induction by addition of the peptic hydrolysate of collagen and by addition of another macromolecular substrate of collagenase, fl-casein (GiUes & Keil, 1976), to two cultures g r o ~ simultaneously on the medium containing 2-5% Casamino acids. The addition of fl-casein did not induce eollagenase. We have precipitated the supernatant of these two cultures by ammonium sulphate, dialyzed and ehromatographed them on DE32 cellulose. In this way, we observed the presence of collagenase and a caseinolytic neutral proteinase in the experiment induced by collagen hydrolysate (Fig. 9(a)) whereas in that induced by fl-casein only a single peak of easeinolytic activity appeared (Fig. 9(b)). Its position in the elution curve corresponds to that found after induction of the culture by collagen hydrolysate.
4. D i s c u s s i o n
The synthesis of collagenase in the culture of A. iophagus is induced by collagen and by its high molecular weight fragments. This induction is associated with a particular phase of bacterial growth. Even if the inducer is present from the beginning of culture, the collagenase activity appears only at the end of the exponential phase of growth. This delay could not be explained by the activation of inactive precursor or by the absence of permeability in the bacteria. The results show that neither inactive precursor nor cell accumulated enzyme nor zymogen is present in the exponential phase of growth. The absence of collagenase zymogen is not surprising. Nearly all of the plant and microbial proteinases are produced in their active form. Nevertheless, the results do not exclude the possibility that the synthesis of collagenase is in some way connected with the competence of the bacteria.
INDUCTION
OF
COLLAGENASE
I N A. iophagua
303
200
1.0
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100
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FIG. 9. D e m o n s t r a t i o n of collagenase a n d neutral protease formation in the presence of peptic hydrolysate of collagen or of E-casein. A. iophagus culture was grown in t h e buffered solution of 2-5~o Casamino acids. After 5 h of culture growth, 5 g of peptic digest of collagen or of E-casein were added. The s u p e r n a t a n t of each culture was precipitated b y a m m o n i u m sulphate, dialysed a n d s u b m i t t e d to c h r o m a t o g r a p h y on DE32 cellulose. Separation of s u p e r n a t a n t s from culture induced b y the peptic hydrolysate of collagen (a) a n d b y fl-casein (b). --Q--O--, Optical density a t 280 n m ; - - 0 - - 0 - - , collagenase activity; - - , - - V - - , caseinolytic activity.
304
V. KEIL-DLOUHA, R. MISRAHI AND B. KEIL
The extent of collagenase induction is proportional to the quantity of the added inducer. When the inducer is added to a final concentration of 2.5~ a high level of collagenase activity is observed (Fig. 3). Nevertheless, in this case, the appearance of enzymic activity is delayed by the additional growth. In order to avoid the changes in the growth curve, all our studies on collagenase induction were performed with 0.25 to 0.5% final concentration of the inducer. The results illustrated in Figure 7(a) show that the digestion of specific peptide bonds in the collagen or in its high molecular weight fragments by collagenase leads to a loss of induction properties. Therefore, a collagenase-sensitive bond is important for the induction mechanism. However, neither the low molecular weight substrate of collagenase Pz-Pro-Leu-Gly-Pro-D-Arg nor the inhibitor Pro-Leu-Sar-Pro cause the induction of the enzyme. In the case of Pz-Pro-Leu-Gly-Pro-D-Arg which is the best synthetic substrate of Achromobacter collagenase it was possible to prove that it does not penetrate the bacterial cell. Also, low molecular weight fragments from the peptic hydrolysate of collagen (Fig. 5(a)) do not possess inducing properties (Fig. 5(c)). A comparison of the inducing capacity of macromolecular and low molecular weight collagenase substrates and inhibitor indicates that the collagenase-sensitive bond plays an important role in the induction mechanism, but it is not sufficient in itself to initiate collagenase synthesis. Another possible explanation of collagenase induction was that the collagenasesensitive bond could induce only if it is situated in a long polypeptide sequence. To test this possibility, we chose fl-casein. This protein of known primary structure (Ribadeau-Dumas et al., 1971) has a molecular weight of 24,100. In the recent study of Gilles & Keil (1976) the positions of four peptide bonds which are split by Achromobacter collagenase were identified, fi-Casein appeared to be an example of collagenase macromolecular substrate whose primary structure is quite different from that of collagen. The induction experiments with fl-casein have sho~m that it does not induce the formation of collagenase. On the contrary, the induction of another enzyme, a caseinolytic neutral proteinase, was observed. It is well estabhshed that the amino acid sequence of a protein determines the folded conformation appropriate to its biological function. The polypeptide chains of collagen are mainly composed of X-Gly-Pro sequences coiled as well as twisted about a common axis in a rope-like structure. It is probable that this particular structure of collagen and its macromolecular fragments are responsible for the effect of collagenase induction. This hypothesis is in agreement with our results. The whole collagen as well as its macromolecular fraction I provoke induction of collagenase (Figs 4 and 5(b)). The digestion of collagen or its macromolecular fraction by collagenase results in the scission of Pro-X-Gly-Pro bonds all over the substrate polypeptide chain and in the loss of the characteristic coiled structure. In consequence of this the collagen and its macromolecular substrates lose their inducing properties. This hypothesis could also explain the absence of induction by the low molecular weight substrate Pz-Pro-Leu-Gly-Pro-D-Arg, by the inhibitor Pro-Leu-Sar-Pro and by another high molecular weight substrate of collagenase, fl-casein. None of them have the coiled structure characteristic of collagen and its macromolecular fragments. A study of collagenase induction by collagen-like polymers could elucidate the natur.e of this phenomenon.
INDUCTION
O F C O L L A G E N A S E I N A . iophagus
305
The authors t h a n k Dr O. Siffert for the gift of the synthetic p e p t i d e Pro-Leu-Sar-Pro and D r B. R i b a d c a u - D u m a s for the gift of E-casein. REFERENCES Castaneda-AguUo, M. (1955). J. Gen. Physiol. 39, 369-375. Gilles, A.-M. & Keil, B. (1976). JFEBS Letters, 65, 369-372. Keil, B., Gilles, A.-M., Lccroisey, A., Hurion, N. & Tong, N. T. (1975). JFEBS Letters, 56, 292-296. Keil-Dlouha, V. (1976). Biochim. Biophys. Acta, 429, 239-251. Laskowski, M. (1955). I n Methods Enzymol. (Colowick, S. P. & Kaplan, N. O., eds), vol. 2, pp. 26-35, Academic Press, New York. Lccroisey, A., Keil-Dlouha, V., Woods, D. R., Perrin, D. & Keil, B. (1975). ~FEBS Letters, 59, 167-172. Ribadeau-Dumas, B., Grosclaude, F. & Mercier, J.-C. (1971). Eur. J. Biochem. 18, 252-257. Svensson, B., Siffert, O. & Kei|, B. (1975). Eur. J . Biochem. 60, 423-425. Welton, R. L. & Woods, D. R. (1973). J . Gen. Microbiol. 75, 191-196. Woods, D. R., Welton, R. L. & Thomson, J. A. (1972). J. Appl. Bacteriol. 35, 123-128. Wiinsch, E. & Heidrich, H. G. (1963). Z. Physiol. Chem. 333, 149-151.
The Induction of Collagenase and a Neutral Proteinase by their High Molecular Weight Substrates in Achromobacter iophagus V. KEIL-DLOUHA, R. M.ISRAI~[t AND B. Kv.m
Service de Chimie des Protdines, Institut Pasteur 28, rue du Docteur Roux, 75024 Paris Cedex 15, France (Received 12 April 1976) The synthesis of collagenase in Aehromobacter iophagus has been shown to be inducible by denatured collagen and by its high molecular weight fragments. The presence in the macromolecular inducer of peptide bonds which could be digested by the collagenase is indispensable for the effect of induction. On the other hand, an addition of a low molecular weight substrate or inhibitor of collagenase does not stimulate the enzyme synthesis. Lack of collagenase induction was also observed in the case of ~-casein which is a macromolecular substrate with four peptide bonds digestible by the collagenase. Nevertheless in the presence of fi-casein the induction of a neutral caseinolytic proteinase was found. The probable role of conformation structure of a macromolecular substrate in the mechanism of induction is discussed. The dependence of induction on the growth phase of the culture was studied. The collagenase activity appears only after the last phase of the exponential growth. It was proved that no zymogen or cell-accLunulated enzyme is present in the first stage of exponential growth and that the collagenase synthesis is in direct correlation with a particular state of the bacterial growth cycle.
1. Introduction The collagenase from a non-pathogenic aerobic Achromobacter iophagus strain is an extracellular metallo-enzyme (EC 3.4.24) which was first described b y Woods et al. (1972) and which could be isolated from the culture medium as described previously (Lecroisey et al., 1975; Keil-Dlouha, 1976). The molecular weight of this eollagenase is 104,000. Its amino acid composition is quite different from that of Clostridium collagenase. The enzyme cleaves the X-Gly bond in the sequence Pro-X-Gly-Y where Y is generally proline or alanine and X is a neutral amino acid (Keil et al., 1975). The Achromobacter collagenase splits this kind of bond in the helical regions of native collagen as well as in a number of synthetic peptide substrates. The recent study in our laboratory proved that pure Achromobacter collagenase can also split analogous bonds in fl-casein (Gilles & Keil, 1976). According to current ideas the extracellular proteinases are in general considered as constitutive enzymes. Nevertheless clear evidence for the constitutive or inducible nature of extracellular proteinases is rather difficult to obtain since most growth media used contain complex nitrogenous substances such as peptone, meat extracts, etc. which could supply inducers for proteinase synthesis. Institut Pasteur Production. 293
294
V. K E I L - D L O U H A ,
R. M I S R A H I A N D B. K E I L
T h e first i n d i c a t i o n t h a t a n e x t r a c e l l u l a r p r o t e i n a s e m a y b e i n d u c e d can be f o u n d in t h e s t u d y on t h e b i o s y n t h e s i s o f a p r o t e i n a s e in Serratia marcescens ( C a s t a n e d a Agullo, 1955). T h e e x p e r i m e n t s p r e s e n t e d in this p a p e r were designed to p r o v i d e evidence t h a t coUagenase o f A . iophagus is a n i n d u c i b l e e x t r a c e l l u l a r e n z y m e . T h e results show t h a t t h e i n d u c t i o n of collagenase t a k e s place o n l y a t t h e e n d o f t h e e x p o n e n t i a l p h a s e o f b a c t e r i a l g r o w t h a n d t h a t this d e l a y could be e x p l a i n e d n e i t h e r b y e v e n t u a l z y m o g e n a c t i v a t i o n nor b y t h e l i b e r a t i o n o f t h e i n t r a c e l l u l a r s t o c k o f t h e e n z y m e . T h e s y n t h e s i s of collagenase is i n d u c e d e i t h e r b y collagen or b y its h i g h m o l e c u l a r weight f r a g m e n t s . Collagen also induces t h e f o r m a t i o n of a n e u t r a l p r o t e i n a s e . Different results were o b t a i n e d w i t h fl-casein, which u n d e r t h e s a m e c o n d i t i o n s induces t h e s y n t h e s i s of a n e u t r a l p r o t e i n a s e only.
2. Materials and Methods (a) Materials ~-Chymotrypsin (lot CD1), trypsin (TRL) and pepsin (PM) were obtained from Worthington Biochemical Corp. Crude collagenase from A . iophagus (spec. act. 170 nkat/mg) was a gift from I n s t i t u t P a s t e u r Production. Calf skin collagen (acid-soluble, C-1633) was purchased from Sigma. Casein (LAB) was a product of Merck. fl-casein was a gift from Dr B. Ribadeau-Dumas. The synthetic collagenase substrate 4-phenylazobenzyloxy-carbonyl-L-prolyl-L-leucyl-glyeyl-L-prolyl-D-arginined i h y d r a t e was purchased from Fluka. The synthetic collagenase inhibitor prolyl-leucyl-sarcosyl-proline was a gift from Dr O. Siffert. (b) Bacterial culture, growth control and induction method The collagenase-producing strain of A.. iophagu~ was the same as isolated a n d previously described b y Woods et al. (1972). I t was routinely m a i n t a i n e d on a complex agar m e d i u m (Welton & Woods, 1973). The culture medium consisted of a 2.5% solution of Casamino acids (a vitamin-free acid-hydrolyzed casein, Difco Laboratories) in 0.1 M-Tris-HC1 buffer (pH 7"6) which contained 0.4 M-NaC1 and 2 mM-CaC12. The bacterial suspension (100 ml with O.D. 4-0 to 5"0 at 600 nm) was inoculated to 1000 ml of culture medium a n d incubated at 29~ in the 2-1 Biolafitte fermenter under standardized conditions. The samples were withdi'awn a t 1-h intervals. The mass of bacterial growth was estimated b y the t u r b i d i t y of suitable dilutions of the culture samples at 600 inn using a Zeiss spectrophotometer. The products whose inducing properties were being tested were dissolved in 100 ml of sterile 0-1 M-Tris-HC1 buffer (pH 7"6) and then a d d e d to the growing culture. (e) Assay of proteolytic activity Collagenase activity, in the culture samples, was measured colorimetrically using 4-phenylazo -benzyloxycarbonyl -L-prolyl-L-leucyl -glycyl - t. -prolyl - D-arginine d i h y d r a t e (Pz-Pro-Leu-Gly-Pro-D-Arg) according to Wfinsch & Heidrich (1963). Numerical d a t a have been recalculated on the basis of 1 p k a t = 0-09 units according to Wfinsch & Heidrich. General protease a c t i v i t y was measured on casein as substrate according to Laskowski (1955). I t was impossible to determine proteinase activity directly in the culture samples because of the presence of acid-hydrolyzed casein. The assays were performed after chromatographic purification of the dialysed a m m o n i u m sulphate precipitate of culture supernatant. (d) Attempts at zymogen activation Culture samples (10 ml) were centrifuged at 4~ for 20 rain at 10,000 revs/min in a SorvaU centrifuge RC2B. The cells were washed rapidly once with 10 ml of ice-cold 0-1
INDUCTION
OF COLLAGENASE
I N A. iophagus
295
M-Tris. HC1 buffer (pH 7"6) and then resuspended in 10 ml of the same buffer. The cells in the suspension were sonicated in a Sonifier B12 a t 130 W for 2 min. The eollagenase assays were performed either directly after t r e a t m e n t or after incubation of the suspension of the broken cells with 1 ml of the sample from the middle of the enzyme production phase. I n the second series of experiments the supernatants (10 ml) of samples from the early growth phase were mixed with sample (1 ml) from the middle of the enzyme production phase. The incubation of samples was done either at 4~ for 16 h or at 30~ for 1 h or 2 h.
(e) Estimation of synthetic substrate in the broken cells I n order to see if the synthetic substrate could penetrate the bacterial cell, the cells grown in the presence of the synthetic substrate were broken as described above. One half of the sample was acidified to p H 3.0 and e x t r a c t e d directly b y 5 ml of ethyl acetate. The other half was t r e a t e d for 2 h at 30~ with 5 mg of crude collagenase, acidified to p H 3-0 and then e x t r a c t e d b y 5 ml of ethyl acetate as in the routine enzyme assay. This method permits one to measure even traces of Pz-Pro-Leu-Gly-Pro-i)-Arg. (f) Isolation of collagenase and neutral proteinase.from the culture medium All steps of the purification were performed at 4~ The cells from 1-1 of bacterial culture were removed b y centrifugation. The supernatant was made 60% in a m m o n i u m sulphate. The precipitate after centrifugation was dialysed and the resulting solution was applied to a DE32 cellulose column (10 cm • 1 cm). The chromatography was performed according to Lecroisey et al. (1975). The samples from the fractions were tested for the presence of collagenolytic or caseinolytic activities, as described previously. (g) Peptic hydrolysate of collagen and its further degradation by proteolytic enzymes F i v e grams of denatured collagen were hydrolysed with pepsin at 37~ p H 2 at an e n z y m e / s u b s t r a t e ratio of 1 : 100. After 2 h of digestion the solution was lyophilized, then dissolved in 20 in[ of 0.01 ~-NH4HCO3 buffer (pH 8.5) and applied to a column of Sephadex G25. The macromolecular fraction which was obtained from 3 successive separations was lyophilized, thon dissolved in 200 ml 0"05 M-Tris.ttC1 buffer (pH 8"5). 100 ml of this solution were digested b y crude Aehromobaeter collagenase. The other 100 ml portion was digested with trypsin and then chymotrypsin. I n all eases, the digestion was performed at 30~ for 16 h with an enzyme/substrate ratio of 1 : 100. Two-thirds of each digest were used for the induction experiments, the rest was lyophilized, redissolved in 10 ml 0"01 M-NH4HCO3 buffer and separated on Sephadex G25 as described for the peptic hydrolysate of collagen. 3. R e s u l t s (a) Induction of extraceUular collagenase anal its relation to the age of the culture T h e f o r m a t i o n o f collagenase b y A. iophagus was s t u d i e d in t w o cultures g r o w i n g in t h e buffered m e d i u m c o n t a i n i n g 2 . 5 % C a s a m i n o acids (Fig. l(a)) a n d c o n t a i n i n g 2 . 5 % p e p t i c h y d r o l y s a t e o f t h e d e n a t u r e d collagen (Fig. l(b)). T h e final level o f g r o w t h was higher in t h e m e d i u m c o n t a i n i n g t h e h y d r o l y s e d collagen. W h e n collagenase p r o d u c t i o n was e x a m i n e d t h e p r i n c i p a l difference b e t w e e n t h e t w o cultures was e v i d e n t . N o t r a c e o f collagenase a c t i v i t y was d e t e c t e d in t h e case o f c u l t u r e (a) (Fig l(a)). On t h e o t h e r h a n d , w h e n t h e c u l t u r e m e d i u m c o n t a i n s d i g e s t e d collagen (Fig. l(b)) f o r m a t i o n o f collagenase can be observed. I t s t a r t s a t t h e e n d o f t h e e x p o n e n t i a l phuse of g r o w t h . T h e e n z y m e p r o d u c t i o n t h u s c o n t i n u e s to a c c e l e r a t e w h e n t h e b a c t e r i a l m a s s has become c o n s t a n t . M a x i m u m collagenase a c t i v i t y was a c h i e v e d on a v e r a g e a f t e r 11 hours in t h e c u l t u r e s c o n t a i n i n g 2 . 5 % h y d r o l y s e d collagen. I t is e v i d e n t f r o m t h e a b o v e results (Fig. l(b)) t h a t e v e n w h e n t h e c u l t u r e m e d i u m c o n t a i n s t h e i n d u c e r (digested collagen) f r o m t h e beginning, t h e f o r m a t i o n o f col20
296
V. K E I L - D L O U H A ,
R. MISRAHI
(a)
A N D B. K E I L
(b)
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~
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FIo. 1. Dependence of eollagenase formation on culture medium. A. iophagus was grown in buffered medium as described in Materials and Methods. (a) I n the presence of 2-5% Casamino acids. (b) I n the presence of 2.5% peptic hych.olysate of denatured collagen. --l--l--, Bacterial growth as turbidity measured at 600 nm. - - O - - O - - , Collagenase activity against synthetic substrate Pz-Pro-Leu-Gly-Pro-D-Arg.
lagenase starts only at the end of the exponential phase of bacterial growth. In order to see the effect of inducer at various stages of culture, buffered medium containing 2-5% Casamino acids was inoculated with bacteria twice washed with the same medium in three simultaneous running cultures under the same conditions as in Figure l(a). Then peptic hydi'olysate of collagen was added (to a final eoncn of 0-25%) to each culture, at the different stage of growth: simultaneously with inoculum (Fig. 2(a)), after five hours, at the end of the exponential phase (Fig. 2(b)) and after 7 hours (Fig. 2(c)). I t is evident that if hydrolysed collagen is added to the culture at the end of the exponential phase, then the appearance of the collagenase activity could be detected after one hour (Fig. 2(b)). When the inducer is added to the culture medium simultaneously with inoeulum the activity again appears only at the end of the exponential phase of growth (Fig. 2(a)). On the other hand, when the inducer is added at a later stage (Fig. 2(c)), the collagenase formation is rather slow. The maximum amount of enzymic activity is proportional to the final concentration of the inducer in the culture medium (Fig. 3). Relatively low concentrations of inducer were chosen for this study in order not to stimulate the bacterial growth. As was shown above, the peptic hydrolysate of denatured collagen is a good source of nutrition for A. iophagus. I t can be seen that addition of higher quantities of inducer, at the end of the exponential phase, causes additional bacterial growth (Fig. 3(c)). Consequently a retardation of collagenase formation by two hours was observed. This retardation corresponds to the end of the second exponential phase in the growth curve. These results indicate that the induction of collagenase synthesis may be associated with some particular phase of the division cycle. The cells are not able to synthesize this enzyme during exponential growth,
INDUCTION 10
o. o
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/
8
OF COLLAGENASE (b)
/
/
.
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9
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5 Time(h)
9
, ~ 1 7o6
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Fro. 2. Influence of age of the bacterial culture on the induction effect. 5 g peptic hydrolysate of d e n a t u r a t e d collagen ( $ ) were added to 1 1 of Achromebacter culture which was grown on 2.5% Casamino acids. (a) A t zero time. (b) After 5 h of bacterial growth. (c) After 7 h o f b a c t e r l a l growth. -- 9 9 Bacterial growth; - - O - - O - - , collagenase activity.
A__..A_--A---cA ~
20
30
~0
E 20~ O
vc
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o
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10 Time (h)
FIG. 3. Dependence of the collagenase synthesis on the q u a n t i t y of the added inducer. Different quantities of peptic hydrolysate of d e n a t u r e d collagen were added ( $ ) to 3 cultures growing u n d e r the same conditions on Casamino acids. Final concentration of the peptic hydrolysate was 0.25% (a), 0.5% (b), a n d 2-5% (c). Bacterial growth (a) - - 0 - - 0 - - ; (h) - - B - - m - - ; (e) - - A - - A - - . Collagenase activity (a) - - O - - O - - ; (b)--~--~--; (o) --A--A--.
298
V. K E I L - D L O U H A ,
R. MISRAHI
AND B. KEIL
(b} Demonstration of absence of zymogen or of intracellular
coUagenase in the early stages of growth As previously shown (Fig. l(b)), collagenase activity appears in the culture at the end of the exponential phase of bacterial growth. A possible explanation of this phenomenon could be a progressive activation of an eventual zymogen. I f an activation system for a zymogen had existed, it would have been present in the sample with the active enzyme. Therefore supernatants of samples from the early growth phase of the culture were mixed with samples from the end of the enzyme production phase (10 : 1 (v/v)). One series of these mixtures was incubated 16 hours at 4~ another one was incubated at 30~ for one or two hours and then assayed in the usual way. I t was found that no activation of the samples from early growth phase by the later one occurred. From these results it can be concluded that no measurable amount of extracellular zymogen is present in the culture medium. A delay in the formation of collagenase would also be consistent with an accumulation of the enzyme, eventually of the zymogen, inside the cells. To test this possibility samples were taken at different stages of growth, and centrifuged. The cells were resuspended in 0.1 M-Tris.HC1 buffer, broken by sonication and incubated under the same conditions as described above. Neither free collagenase fior a zymogen which could be activated was found. (c) Induction of collagenase by denatured collagen and by its high
molecular weight fragments Thermally denatured collagen represents a mixture of polypeptide chains of average molecular weight 100,000. When a solution of this gelatinized protein is added to the culture at the end of its exponential phase of growth collagenase activity appears in less than an hour (Fig. 4). I t was shown in Figures 1 to 3 that the peptic hydrolysate of denatured collagen causes the induction of collagenase. In order to characterize better the nature of this
IO20
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0 0
/
0
_~ E
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5 Time (h)
FIG-. 4. The induction of collagenase b y thermally denatured collagen. Collagen solution preincubated for 1 h a t 80~ was added ( $ ) in final concentration 0.5% to a culture grown a s d e s c r i b e d in Fig. 3. --t--O--, Bacterial growth; - - O - - O - - , collagenase activity.
INDUCTION
OF COLLAGENASE
I N A. iophagua
299
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FIo. 5. Preliminary characterization of the inducer contained in the peptic hydrolysate of collagen. (a) Separation of peptic hydrolysate (5 g), on Sephadex G25. The column (180 cm • 4 cm) was equilibrated with 0.01 M-NH4HCO3 buffer (pl:[ 8.5). 7-ml fractions were collected in 10-min intervals. 4 The position of molecular weight markers on the elution diagram : 25,700, Chymotrypsinogen; 11,700, cytochromc c; 6500~ pancreatic trypsin inhibitor. ( - ) Optical density a t 280 nm. The fractions 100 to 180 (I), 181 to 230 (II), 231 to 300 (III) were pooled a n d lyophflized. (b) Effect of isolated fraction I on the eollagenase induction. Fraction I (2.5 g) was dissolved in 50 ml 0.1 ~-Tris-HC1 buffer and added to the culture grown on Casamino acids. - - $ - - O - - , Bacterial growth; - - O - - O - - , eollagenase activity. (e) The effect of fractions I I and I I I under the same conditions. Fraction I I (2-5 g) and fraction I I I (2.5 g), which were obtained from 2 preparation runs, were added to the appropriate cultures. Bacterial growth in cultures: - - t - - $ - - , :II; and - - V - - Y - - , I I I . Collagenase activity in cultures after addition of fraction I I ( - - O - - O - - ) and I I I ( - - V - - V - - ) .
inducer we separated the peptic hydrolysate into three main fractions by Sephadex G25 gel filtration (Fig. 5(a)). The induction capacity was tested by separate simultaneous additions of each fraction to three cultures of A. iophagus which were grown up in the buffered medium containing 2.5% Casamino acids. Figure 5(b) shows that after the addition of maeromolecular fraction 1, the induction of collagenase takes place. On the other hand, no trace of the collagenase activity was found after addition of low molecular weight fractions II and I I I (Fig. 5(c)). In the next experiment the macromolecular fraction I (from Fig. 5(a)) was digested
300
V. K E I L - D L O U H A ,
R. MISRAHI
AND B. KEIL
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250
Fraclion no. Fie. 6. Separation of fraction I digested b y eollagenase or b y trypsin a n d chymotrypsin on a Sephadex G25 column. The separation was done u n d e r the conditions described in :Fig. 5(a). Position of molecular weight markers on the elution diagram. (a) Separation of fraction I digested b y collagenase. (b) Separation of combined t r y p t i c ehymotryptic hydrolysate of fraction I. ( ) Absorption a t 280 nm.
either by collagenase or by trypsin and chymotrypsin. A comparison of the elution diagrams from a gel filtration on a Sephadex G25 column shows that the action of collagenase as well as that of trypsin and chymotrypsin resulted in the liberation of a number of small molecular weight products (Fig. 6(a) and (b)). When the macromolecular fraction I which was digested by collagenase was added to the bacterial culture under the same conditions as in the previous experiment, no induction of enzyme was observed as shown in Figure 7(a). The same results were obtained with non-fractionated, denatured collagen which was also digested by collagenase. On the other hand, fraction I which was hydrolysed by trypsin and chymotrypsin provoked the same effect of induction (Fig. 7(b)) as in the case of non-digested fraction I. These results show that the presence of peptide bonds susceptible to the enzymic action of collagenase is indispensable for the induction of the enzyme. (d) An attempt to induce collagenase formation in the presence of its low molecular weight substrate or inhibitor In order to see whether low molecular weight substrate or inhibitor of collagenase could provoke the induction of this enzyme two types of experiment were done. First of all we chose the synthetic peptide Pz-Pro-Leu-Gly-Pro-D-Arg which is the best low molecular weight substrate. The concentration of added substrate was 2 • 10-4 ~I (0-016% }. Nevertheless collagenase was not induced. After l0 hours of
INDUCTION
I N A . iophagus
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fraction I. (a) Loss of inducing properties of collagen or of its macromolecular fraction I after their digestion b y Achromobac~er collagenase. Two cultures were grown on the buffered m e d i u m containing 2.5% Casamino acids. After 5 h growth, 2.5 g of collagen or fraction I digested b y collagenases were added ( ~ ) . The conditions of the digestion are described in Materials a n d Methods. - - O - - Q - - , Bacterial growth, a n d - - O - - O - - , eollagenase activity, in the culture to which t;he collagen hydrolysed b y collagenase was added. - - 9 1 4 9 Bacterial growth a n d - - ~ 7 - - V - - , collagenase activity in the culture to which the fraction I hydrolysed b y collagenase was added. (b) Collagenase ind,action b y the fraction I digested b y trypsin a n d ehymotrypsin. The culture conditions the same as in (a). After 5 h growth 2.5 g fraction I digested b y trypsin a n d chymotrypsin were added ( ~ ). - - O - - O - - , Bacterial growth; - - O - - O - - , eollagenase activity. molecular
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Fzo. 8. A t t e m p t to induce collagenase b y its inhibitor Pro-Leu-Sar-Pro. (a) The culture (1 1) was grown on Casamino acids, as described previously. After 5 h of bacterial growth, 150 m g of Pro-Leu-Sar-Pro were added ( Je ). - - O - - O - - , Bacterial growth; - - O - - O - - , collagenase activity. After 10 h the culture s u p e r n a t a n t was separated b y centrifugation, treated as described in Materials a n d Methods a n d t h e n applied to the DE32 cellulose column (10 em X 1 era). (b) Elution profile from the DE32 cellulose column. - - 0 - - 0 - - , Optical density a t 280 n m ; 0 . - - O - - , colla~enasc activit~r; - - 9 - - 9 - - , caseinol~rtic activit~r.
302
V. KEIL-DLOUttA, R. MISRAHI AND B. KEIL
culture growth, the cells were separated from the culture medium by centrifugatlon, washed twice and broken by sonication as described in Materials and Methods. No trace of substrate was detected in the broken cells. The quantity of intact substrate in the culture medium was tested. A correction which was determined for spontaneous degradation during a six hour period at 30~ represented 8% of the total substrate added to the culture. No additional enzymic degradation of the substrate was observed. This experiment proves that low molecular weight substrate did not induce any detectable traces of collagenase. Absence of induction was also shown in the case of the peptide Pro-Leu-Sar-Pro, which is a collagenase inhibitor (Svensson et al. 1975) (Fig. 8(a)). Nevertheless in this case it was necessary to prove that the absence of detectable enzymic activity in the supernatant is not due to inhibition by excess of this tetrapeptide. To this end, the supernatant of this baterial culture was precipitated with ammonium sulphate, the precipitate was dialysed and submitted to chromatography on DE32 cellulose as described in Material and Methods. Under these conditions a possible complex of the enzyme with inhibitor could no longer exist. Collagenase would be displaced from this ion-exchange column with Tris.HC1 buffer containing 1 ~-NaC1. Figure 8(b) shows that no collagenase activity was detected. Those experiments indicate that the low molecular weight substrate and inhibitor of collagenase do not induce synthesis of the enzyme.
(e) The different effects of collagen partial hydrolysate and of fl-casein on the induction of proteinases in the culture of Achromobacter iophagus We tested the possibility of collagenase induction by addition of the peptic hydrolysate of collagen and by addition of another macromolecular substrate of collagenase, fl-casein (GiUes & Keil, 1976), to two cultures g r o ~ simultaneously on the medium containing 2-5% Casamino acids. The addition of fl-casein did not induce eollagenase. We have precipitated the supernatant of these two cultures by ammonium sulphate, dialyzed and ehromatographed them on DE32 cellulose. In this way, we observed the presence of collagenase and a caseinolytic neutral proteinase in the experiment induced by collagen hydrolysate (Fig. 9(a)) whereas in that induced by fl-casein only a single peak of easeinolytic activity appeared (Fig. 9(b)). Its position in the elution curve corresponds to that found after induction of the culture by collagen hydrolysate.
4. D i s c u s s i o n
The synthesis of collagenase in the culture of A. iophagus is induced by collagen and by its high molecular weight fragments. This induction is associated with a particular phase of bacterial growth. Even if the inducer is present from the beginning of culture, the collagenase activity appears only at the end of the exponential phase of growth. This delay could not be explained by the activation of inactive precursor or by the absence of permeability in the bacteria. The results show that neither inactive precursor nor cell accumulated enzyme nor zymogen is present in the exponential phase of growth. The absence of collagenase zymogen is not surprising. Nearly all of the plant and microbial proteinases are produced in their active form. Nevertheless, the results do not exclude the possibility that the synthesis of collagenase is in some way connected with the competence of the bacteria.
INDUCTION
OF
COLLAGENASE
I N A. iophagua
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FIG. 9. D e m o n s t r a t i o n of collagenase a n d neutral protease formation in the presence of peptic hydrolysate of collagen or of E-casein. A. iophagus culture was grown in t h e buffered solution of 2-5~o Casamino acids. After 5 h of culture growth, 5 g of peptic digest of collagen or of E-casein were added. The s u p e r n a t a n t of each culture was precipitated b y a m m o n i u m sulphate, dialysed a n d s u b m i t t e d to c h r o m a t o g r a p h y on DE32 cellulose. Separation of s u p e r n a t a n t s from culture induced b y the peptic hydrolysate of collagen (a) a n d b y fl-casein (b). --Q--O--, Optical density a t 280 n m ; - - 0 - - 0 - - , collagenase activity; - - , - - V - - , caseinolytic activity.
304
V. KEIL-DLOUHA, R. MISRAHI AND B. KEIL
The extent of collagenase induction is proportional to the quantity of the added inducer. When the inducer is added to a final concentration of 2.5~ a high level of collagenase activity is observed (Fig. 3). Nevertheless, in this case, the appearance of enzymic activity is delayed by the additional growth. In order to avoid the changes in the growth curve, all our studies on collagenase induction were performed with 0.25 to 0.5% final concentration of the inducer. The results illustrated in Figure 7(a) show that the digestion of specific peptide bonds in the collagen or in its high molecular weight fragments by collagenase leads to a loss of induction properties. Therefore, a collagenase-sensitive bond is important for the induction mechanism. However, neither the low molecular weight substrate of collagenase Pz-Pro-Leu-Gly-Pro-D-Arg nor the inhibitor Pro-Leu-Sar-Pro cause the induction of the enzyme. In the case of Pz-Pro-Leu-Gly-Pro-D-Arg which is the best synthetic substrate of Achromobacter collagenase it was possible to prove that it does not penetrate the bacterial cell. Also, low molecular weight fragments from the peptic hydrolysate of collagen (Fig. 5(a)) do not possess inducing properties (Fig. 5(c)). A comparison of the inducing capacity of macromolecular and low molecular weight collagenase substrates and inhibitor indicates that the collagenase-sensitive bond plays an important role in the induction mechanism, but it is not sufficient in itself to initiate collagenase synthesis. Another possible explanation of collagenase induction was that the collagenasesensitive bond could induce only if it is situated in a long polypeptide sequence. To test this possibility, we chose fl-casein. This protein of known primary structure (Ribadeau-Dumas et al., 1971) has a molecular weight of 24,100. In the recent study of Gilles & Keil (1976) the positions of four peptide bonds which are split by Achromobacter collagenase were identified, fi-Casein appeared to be an example of collagenase macromolecular substrate whose primary structure is quite different from that of collagen. The induction experiments with fl-casein have sho~m that it does not induce the formation of collagenase. On the contrary, the induction of another enzyme, a caseinolytic neutral proteinase, was observed. It is well estabhshed that the amino acid sequence of a protein determines the folded conformation appropriate to its biological function. The polypeptide chains of collagen are mainly composed of X-Gly-Pro sequences coiled as well as twisted about a common axis in a rope-like structure. It is probable that this particular structure of collagen and its macromolecular fragments are responsible for the effect of collagenase induction. This hypothesis is in agreement with our results. The whole collagen as well as its macromolecular fraction I provoke induction of collagenase (Figs 4 and 5(b)). The digestion of collagen or its macromolecular fraction by collagenase results in the scission of Pro-X-Gly-Pro bonds all over the substrate polypeptide chain and in the loss of the characteristic coiled structure. In consequence of this the collagen and its macromolecular substrates lose their inducing properties. This hypothesis could also explain the absence of induction by the low molecular weight substrate Pz-Pro-Leu-Gly-Pro-D-Arg, by the inhibitor Pro-Leu-Sar-Pro and by another high molecular weight substrate of collagenase, fl-casein. None of them have the coiled structure characteristic of collagen and its macromolecular fragments. A study of collagenase induction by collagen-like polymers could elucidate the natur.e of this phenomenon.
INDUCTION
O F C O L L A G E N A S E I N A . iophagus
305
The authors t h a n k Dr O. Siffert for the gift of the synthetic p e p t i d e Pro-Leu-Sar-Pro and D r B. R i b a d c a u - D u m a s for the gift of E-casein. REFERENCES Castaneda-AguUo, M. (1955). J. Gen. Physiol. 39, 369-375. Gilles, A.-M. & Keil, B. (1976). JFEBS Letters, 65, 369-372. Keil, B., Gilles, A.-M., Lccroisey, A., Hurion, N. & Tong, N. T. (1975). JFEBS Letters, 56, 292-296. Keil-Dlouha, V. (1976). Biochim. Biophys. Acta, 429, 239-251. Laskowski, M. (1955). I n Methods Enzymol. (Colowick, S. P. & Kaplan, N. O., eds), vol. 2, pp. 26-35, Academic Press, New York. Lccroisey, A., Keil-Dlouha, V., Woods, D. R., Perrin, D. & Keil, B. (1975). ~FEBS Letters, 59, 167-172. Ribadeau-Dumas, B., Grosclaude, F. & Mercier, J.-C. (1971). Eur. J. Biochem. 18, 252-257. Svensson, B., Siffert, O. & Kei|, B. (1975). Eur. J . Biochem. 60, 423-425. Welton, R. L. & Woods, D. R. (1973). J . Gen. Microbiol. 75, 191-196. Woods, D. R., Welton, R. L. & Thomson, J. A. (1972). J. Appl. Bacteriol. 35, 123-128. Wiinsch, E. & Heidrich, H. G. (1963). Z. Physiol. Chem. 333, 149-151.