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The specificity of collagenase
298
Vm~LIM]NARV No'r~;s
eeL. 3,~ (z9.~9)
The specificity of collagenase Collagenase is known as a proteinase with a v e r y high degree of s n b s t r a t e specificity; in order to elucidate t h e n a t u r e of this specificity, earle,as s y n t h e t i c peptides have been i n v e s t i g a t e d as s u b s t r a t e s . M.~,',,'DJ. el a l . 1 test6d L e u . Pro" Gly, P r o . L c u , Gly. N H 2, L c u . P h e . Pro, L e u . Phc P r o ' OCi-I 3. polyproline a n d several proline- or h y d r o x y p r o l i n e - c o n t a i n i n g dipeptides; (]ALLOP dt a 1 3 t e s t e d polyproline a n d some origopeptides; M O N I E R el al. a a n d BID~VELL AND VAN HF.YNINGFN 4 tried a large n u m b e r of s y n t h e t i c s u b s t r a t e s with a c r u d e collngenasc. None of tLese c o m p o u n d s were h y d r o l y s e d . I t has also been e s t a b l i s h e d t h a t a m o n g proteins o n l y collagen a n d gelatin are a t t a c k e d ; c~sein, hemoglobin, serum albumin, tibrln, k e r a t i n a n d elastin do n o t react. We have also i n v e s t i g a t e d several s y n t h e t i c p e p t i d e s of t h e t y p e G-P-Rs, 6 as po.~sible s u b s t r a t e s for purified eollagenase 7, n a m e l y Pro" Leu-Gly*, G l y . P r o - L o t # , poly (Pro. Lt,u- Gly) wizh aver:t go molecular weight s of 3ooo, a n d ( ;ly- Pro. !,eu. GlyPro. N H 0'~. It was f o u n d t h a t t h e purified collagen~se was able to h y d r o l y z e t h e l a t t e r two s y n t h e t i c peptidcs. Tim m e t h o d of synthesis of these peptides, which are m e d e | s for coI}ag~u, has been lmblishedS. Their characteristics will be publi.nhed elsewhere 9. After hydrolysis of poly (Pro. L e u - G l y ) with col]~lgenasc, t h r e e peptides were found on paper c h r o m a t o g r a p h y , with the a mine acid sequence Pro. L e u . G l y - P r o . Leu, G,ly. P r o - L e u a n d C,l y - P r o - L e u - G l y respectively. The molar ratio for these peptides was o . 8 : : t 9 : i . No free amino acid could be f o u n d a m o n g t h e p r o d u c t s of digestion. I t is e v i d e n t t h a t the fir.~t p e p t i d e was d e r i v e d from the N - t e r m i n a l p o r t i o n of poly( P r o - L e u - G l y ) , a n d th<: third from the C-terminal portion, a n d t h a t the p o l y m e r was almost completely split into tripeptides. The m o l a r ratio of Gly- Pro- L e u to the t e t r a pcptide d e r i v e d fronl the C-terminal portion was larger t h a n the c a l c u l a t e d r a t i o for the melee'liar weight of 3000 of the p o l y m e r used. This d i s c r e p a n c y m a y be due to poor recovery of peptides from t h e ends, or due to an error in t h e d e t e r m i n a t i o n of the average molecular weight, or both. There was no, evidence of the a p p e a r a n c e of Pro. Leu. Since no glycine a n d Pro. Leu were f o u n d a m o n g t h e p r e d u c t s of digestion, a n d the p c n t a p e p t i d e from the N - t e r m i n a l p o r t i o n a n d the t e t r a p e p t i d e from t h e Ct e r m i n a l portion were f o u n d in c o m p a r a b l e a m o u n t s , it a p p e a r s t h a t collagenase does not a t t a c k P r o - L e u . G l y . P r o . Leu, in which the imino g r o u p of N - t e r m i n a l proline is u n m a s k e d . It can be f u r t h e r c o n c l u d e d t h a t t h e presence of an imino acid o n l y in a positiot~ p e n u l t i m a t e to glycine does n o t s a t i s f y the specificity r e q u i r e m e n t s of collagenase, a n d t h a t two proline residues in the sequence P r o . R . R ' , Pro are required. G l y - P r o . Leu" G l y . P r o . N H , , which from t h e above c o n s i d e r a t i o n s h o u l d be t h e simplest s u b s t r a t e for collagena.se, was i n c u b a t e d with purified collagenase. B y p a p e r electrophoresis a n d paper c h r o m a t o g r a p h y , it was found t h a t t~is p e p t i d e was split into two pcptides with a m i n o acid sequences G l y - P r o . L e u a n d G l y . P r o . N F l v respectively. No free a m i n o acid was found. The above, results are consistent w i t h the conclusion t h a t t h e sequence Pro- R . R r. Pro is necessary in ~, substrc.te for collagenase. O t h e r possibilities are, however, n o t yet c o m p l e t e l y excluded, a n d the s t u d y of t h e n a t u r e of collagenase specificity is now in progress. Results on t h e eollagenase digestion of purified r a t - t a i l - t e n d o n collagen", are in a g r e e m e n t with the present results.
VOL. 34 (x959)
Vm:~Lt.~Xl:;alIV X~)n.:S
299
The present a u t h o r s ,Are g r e a t l y indebted to Professor AKAI:ORt a n d Dr. GA~,:AKTIIA!¢A Of O s a k a UniversitV for their helpfuI discttssion~ a n d their kindness in providing tts with ttte necessary peptidc.~. Y U-EAKA NAGAI Deparl~r~em o/ 13iophssivs ,.emt Hio, l~emistry, FavMl~, o/S~:ieuvv, tLx/IgaiKo Nt)l).X
Lrnivemity o/ Tokyo (Japan.) * 1. MANDL, 1[, Zlppt'.ll AND L. T. F t ~ G U S O N , ,olrch[ ldiochc,,t, l~i,,plO's., 74 (x~35'4) 4¢~5• 2 S. I~,'[ICI-IAIgLB, 1 ~. i'll. (~rALLOt'. S. S~IlYTI£R AND E .~|I':ILMAN, llit'chitJt. I¢iophys...;clef, 29 (1958) .t5,~. a R . MONII~R, G . LIT'~VAI2.N, ~'I. SOZILO ANn l']. LABOUI'.;SSE, BiocbiOt. t ¢ i o p h y s . ,,tct~t. 18 [1955) 7 t . 4 E . B1D'WlNLL AND ~¢V. E . "I~'AN }[I~VNINGI';N, B i o c h e m . j . , q_, (194,~ } t 4 o . ~l ~.V, A. SCllttol.rI~l~.li, L. M . KAY, J. i./~Gt~:'r'rl~, I.. l l o N x ~ N AND |:. C. GREEN, J . ,-IIH. (.~-kV~l¢..~;r~t;., 76 (I954) 3556. M . L . H u G G I r c s , laroteitt Ghet;zisteF, V e t . 5, 1,2y~3ritsu . % h u p p a n , Tt~k.~.t~. ~957, p. 6 t . ~e" ~NAGAI AND H . L~ODA, ill preparation. S I-T. I~.[TAOI,I:A, S. SAKAKIHAHA ANI) 1I, "l',',xl. lltt?l. G'h*m..~ot'. jtep~t~t, 3t {1958) st~2. .~. GAIKAKIFtARA et al., ttlr|publiMlt."el'. "
Received March 23rd, 1959
Some chemical changes associated with prothrombin act~'~ration In the a c t i v a t i o n of purified p r o t h r o m M n prepar~rti,m~ .~everaI stages in tile developmerit of t h r o m b i n a c t i v i t y h a v e been recognized t. First, p r o t h r o m b i n loses the cap a c i t y to become thronlbii~ in the Frcsence of lung t h r o m b o p l a s t i n . Ac-globulin a n d Ca ++ (z-stage a n a l y t i c a l reagent.~). T h e n tile protein regains its s e n s i t i v i t y to th~:.~e s u b s t a n c e s , a n d lastly t h r o m b i n a c t i v i t y arises. The sequence which a c c o u n t s for e x p e r i m e n t s c o m p l e t e d thu.~ far m a y bc described as follows: P r o t h r o m b i n (sensitive to Ca ++ + Ac-globu!in -i- brain thromhopta~tin) --> P m t h r o m b i n - d e r i v a t i v e I (not sensitive to Ca ~ - .!- Ac-gtobulin + brain t hlxmabopl:,~ii,u -~ P r o t h r o m b i n - d e r i v a t ire I I (sensitive to Ca++ -+- Ac-glohulin : I,rain t h r o m b o p l a , l i n ) --- t h r o m b i n a n d other reaction products. This u n i q u e c a p a c i t y of the z y m o g e n to undergo modifications implies an intermediate(s) in tt~e a c t i v a t i o n process. For ii;s~,,ncc intermediati~s |lave been ft~un,[ in the a u t o c a t a l y t i c a c t i v a t i o n of prothroml~in in 25 '7,, stadium citrate". We have now f o u n d t h a t the first step in p r o t h r o m b i n a c t i v a t i o n is ;tssoeiatcd with the a p p e a r a n c e of N - t e r m i n a l proli~te, a n d this is true with the use of three di[fcrent materials: n a m e l y , t h r o m b i n , puritied platelet factor 3. a n d scdimctttablc tung t h r o m b o p l a s t i n . In the a c t i v a t i o n with s e d i m e n t a b l c lung e x t r a c t t h r o m b o p l a s t i n (x mg/ml) a n d Ca ++ (o.o23 M) a b o u t 5 o % of the prothrornbin (2oo0 units/rot) was c o n v e r t e d to t h r o m h i n in 5 h, a n d tile r e m a i n i n g prothromt~in became a derivat ire (not c o n v e r t a b l e to t h r o m b i n in 2-stage a n a l y t i c a l reagents). We t h e n r e m o v e d the t h r o m b o p l a s t i n b y high-speed c e n t r i f u g a t i o n a n d precipitated the protein b y a d d i n g acetone to a eoncentra.tion of 50 % at 0% TI~e precipitated protein wa_~ dried fr'.~m the frozen s t a t e a n d was f o u n d to c o n t a i n N-terminM proline a n d g l u t a m i c acid. F r o m o t h e r experim e n t s it is k n o w n t h a t the N - t e r m i n a l a m i n o acid of bovine p r o t h r o m b i n is alanine a a n d for t h r o m b i n it is g l u t a m i c acid L A n o n - t h r o m b i n derivz~tive of p r o t h r o m b i n t h u s has proline as the N - t e r m i n M a m i n o acid.
Vm~LIM]NARV No'r~;s
eeL. 3,~ (z9.~9)
The specificity of collagenase Collagenase is known as a proteinase with a v e r y high degree of s n b s t r a t e specificity; in order to elucidate t h e n a t u r e of this specificity, earle,as s y n t h e t i c peptides have been i n v e s t i g a t e d as s u b s t r a t e s . M.~,',,'DJ. el a l . 1 test6d L e u . Pro" Gly, P r o . L c u , Gly. N H 2, L c u . P h e . Pro, L e u . Phc P r o ' OCi-I 3. polyproline a n d several proline- or h y d r o x y p r o l i n e - c o n t a i n i n g dipeptides; (]ALLOP dt a 1 3 t e s t e d polyproline a n d some origopeptides; M O N I E R el al. a a n d BID~VELL AND VAN HF.YNINGFN 4 tried a large n u m b e r of s y n t h e t i c s u b s t r a t e s with a c r u d e collngenasc. None of tLese c o m p o u n d s were h y d r o l y s e d . I t has also been e s t a b l i s h e d t h a t a m o n g proteins o n l y collagen a n d gelatin are a t t a c k e d ; c~sein, hemoglobin, serum albumin, tibrln, k e r a t i n a n d elastin do n o t react. We have also i n v e s t i g a t e d several s y n t h e t i c p e p t i d e s of t h e t y p e G-P-Rs, 6 as po.~sible s u b s t r a t e s for purified eollagenase 7, n a m e l y Pro" Leu-Gly*, G l y . P r o - L o t # , poly (Pro. Lt,u- Gly) wizh aver:t go molecular weight s of 3ooo, a n d ( ;ly- Pro. !,eu. GlyPro. N H 0'~. It was f o u n d t h a t t h e purified collagen~se was able to h y d r o l y z e t h e l a t t e r two s y n t h e t i c peptidcs. Tim m e t h o d of synthesis of these peptides, which are m e d e | s for coI}ag~u, has been lmblishedS. Their characteristics will be publi.nhed elsewhere 9. After hydrolysis of poly (Pro. L e u - G l y ) with col]~lgenasc, t h r e e peptides were found on paper c h r o m a t o g r a p h y , with the a mine acid sequence Pro. L e u . G l y - P r o . Leu, G,ly. P r o - L e u a n d C,l y - P r o - L e u - G l y respectively. The molar ratio for these peptides was o . 8 : : t 9 : i . No free amino acid could be f o u n d a m o n g t h e p r o d u c t s of digestion. I t is e v i d e n t t h a t the fir.~t p e p t i d e was d e r i v e d from the N - t e r m i n a l p o r t i o n of poly( P r o - L e u - G l y ) , a n d th<: third from the C-terminal portion, a n d t h a t the p o l y m e r was almost completely split into tripeptides. The m o l a r ratio of Gly- Pro- L e u to the t e t r a pcptide d e r i v e d fronl the C-terminal portion was larger t h a n the c a l c u l a t e d r a t i o for the melee'liar weight of 3000 of the p o l y m e r used. This d i s c r e p a n c y m a y be due to poor recovery of peptides from t h e ends, or due to an error in t h e d e t e r m i n a t i o n of the average molecular weight, or both. There was no, evidence of the a p p e a r a n c e of Pro. Leu. Since no glycine a n d Pro. Leu were f o u n d a m o n g t h e p r e d u c t s of digestion, a n d the p c n t a p e p t i d e from the N - t e r m i n a l p o r t i o n a n d the t e t r a p e p t i d e from t h e Ct e r m i n a l portion were f o u n d in c o m p a r a b l e a m o u n t s , it a p p e a r s t h a t collagenase does not a t t a c k P r o - L e u . G l y . P r o . Leu, in which the imino g r o u p of N - t e r m i n a l proline is u n m a s k e d . It can be f u r t h e r c o n c l u d e d t h a t t h e presence of an imino acid o n l y in a positiot~ p e n u l t i m a t e to glycine does n o t s a t i s f y the specificity r e q u i r e m e n t s of collagenase, a n d t h a t two proline residues in the sequence P r o . R . R ' , Pro are required. G l y - P r o . Leu" G l y . P r o . N H , , which from t h e above c o n s i d e r a t i o n s h o u l d be t h e simplest s u b s t r a t e for collagena.se, was i n c u b a t e d with purified collagenase. B y p a p e r electrophoresis a n d paper c h r o m a t o g r a p h y , it was found t h a t t~is p e p t i d e was split into two pcptides with a m i n o acid sequences G l y - P r o . L e u a n d G l y . P r o . N F l v respectively. No free a m i n o acid was found. The above, results are consistent w i t h the conclusion t h a t t h e sequence Pro- R . R r. Pro is necessary in ~, substrc.te for collagenase. O t h e r possibilities are, however, n o t yet c o m p l e t e l y excluded, a n d the s t u d y of t h e n a t u r e of collagenase specificity is now in progress. Results on t h e eollagenase digestion of purified r a t - t a i l - t e n d o n collagen", are in a g r e e m e n t with the present results.
VOL. 34 (x959)
Vm:~Lt.~Xl:;alIV X~)n.:S
299
The present a u t h o r s ,Are g r e a t l y indebted to Professor AKAI:ORt a n d Dr. GA~,:AKTIIA!¢A Of O s a k a UniversitV for their helpfuI discttssion~ a n d their kindness in providing tts with ttte necessary peptidc.~. Y U-EAKA NAGAI Deparl~r~em o/ 13iophssivs ,.emt Hio, l~emistry, FavMl~, o/S~:ieuvv, tLx/IgaiKo Nt)l).X
Lrnivemity o/ Tokyo (Japan.) * 1. MANDL, 1[, Zlppt'.ll AND L. T. F t ~ G U S O N , ,olrch[ ldiochc,,t, l~i,,plO's., 74 (x~35'4) 4¢~5• 2 S. I~,'[ICI-IAIgLB, 1 ~. i'll. (~rALLOt'. S. S~IlYTI£R AND E .~|I':ILMAN, llit'chitJt. I¢iophys...;clef, 29 (1958) .t5,~. a R . MONII~R, G . LIT'~VAI2.N, ~'I. SOZILO ANn l']. LABOUI'.;SSE, BiocbiOt. t ¢ i o p h y s . ,,tct~t. 18 [1955) 7 t . 4 E . B1D'WlNLL AND ~¢V. E . "I~'AN }[I~VNINGI';N, B i o c h e m . j . , q_, (194,~ } t 4 o . ~l ~.V, A. SCllttol.rI~l~.li, L. M . KAY, J. i./~Gt~:'r'rl~, I.. l l o N x ~ N AND |:. C. GREEN, J . ,-IIH. (.~-kV~l¢..~;r~t;., 76 (I954) 3556. M . L . H u G G I r c s , laroteitt Ghet;zisteF, V e t . 5, 1,2y~3ritsu . % h u p p a n , Tt~k.~.t~. ~957, p. 6 t . ~e" ~NAGAI AND H . L~ODA, ill preparation. S I-T. I~.[TAOI,I:A, S. SAKAKIHAHA ANI) 1I, "l',',xl. lltt?l. G'h*m..~ot'. jtep~t~t, 3t {1958) st~2. .~. GAIKAKIFtARA et al., ttlr|publiMlt."el'. "
Received March 23rd, 1959
Some chemical changes associated with prothrombin act~'~ration In the a c t i v a t i o n of purified p r o t h r o m M n prepar~rti,m~ .~everaI stages in tile developmerit of t h r o m b i n a c t i v i t y h a v e been recognized t. First, p r o t h r o m b i n loses the cap a c i t y to become thronlbii~ in the Frcsence of lung t h r o m b o p l a s t i n . Ac-globulin a n d Ca ++ (z-stage a n a l y t i c a l reagent.~). T h e n tile protein regains its s e n s i t i v i t y to th~:.~e s u b s t a n c e s , a n d lastly t h r o m b i n a c t i v i t y arises. The sequence which a c c o u n t s for e x p e r i m e n t s c o m p l e t e d thu.~ far m a y bc described as follows: P r o t h r o m b i n (sensitive to Ca ++ + Ac-globu!in -i- brain thromhopta~tin) --> P m t h r o m b i n - d e r i v a t i v e I (not sensitive to Ca ~ - .!- Ac-gtobulin + brain t hlxmabopl:,~ii,u -~ P r o t h r o m b i n - d e r i v a t ire I I (sensitive to Ca++ -+- Ac-glohulin : I,rain t h r o m b o p l a , l i n ) --- t h r o m b i n a n d other reaction products. This u n i q u e c a p a c i t y of the z y m o g e n to undergo modifications implies an intermediate(s) in tt~e a c t i v a t i o n process. For ii;s~,,ncc intermediati~s |lave been ft~un,[ in the a u t o c a t a l y t i c a c t i v a t i o n of prothroml~in in 25 '7,, stadium citrate". We have now f o u n d t h a t the first step in p r o t h r o m b i n a c t i v a t i o n is ;tssoeiatcd with the a p p e a r a n c e of N - t e r m i n a l proli~te, a n d this is true with the use of three di[fcrent materials: n a m e l y , t h r o m b i n , puritied platelet factor 3. a n d scdimctttablc tung t h r o m b o p l a s t i n . In the a c t i v a t i o n with s e d i m e n t a b l c lung e x t r a c t t h r o m b o p l a s t i n (x mg/ml) a n d Ca ++ (o.o23 M) a b o u t 5 o % of the prothrornbin (2oo0 units/rot) was c o n v e r t e d to t h r o m h i n in 5 h, a n d tile r e m a i n i n g prothromt~in became a derivat ire (not c o n v e r t a b l e to t h r o m b i n in 2-stage a n a l y t i c a l reagents). We t h e n r e m o v e d the t h r o m b o p l a s t i n b y high-speed c e n t r i f u g a t i o n a n d precipitated the protein b y a d d i n g acetone to a eoncentra.tion of 50 % at 0% TI~e precipitated protein wa_~ dried fr'.~m the frozen s t a t e a n d was f o u n d to c o n t a i n N-terminM proline a n d g l u t a m i c acid. F r o m o t h e r experim e n t s it is k n o w n t h a t the N - t e r m i n a l a m i n o acid of bovine p r o t h r o m b i n is alanine a a n d for t h r o m b i n it is g l u t a m i c acid L A n o n - t h r o m b i n derivz~tive of p r o t h r o m b i n t h u s has proline as the N - t e r m i n M a m i n o acid.