Purification and partial characterization of an α-chymotrypsin-like protease of rat peritoneal mast cells

BIOCHIMIE, 1979, 61, 653-662.

Purification and partial characterization of an -chymotrypsin-like protease of rat peritoneal

mast

cells c).

Michael T. EVERITT and Hans NEURATH O.

D e p a r t m e n t o[ B i o c h e m i s t r y , University of Washington, Seattle, W a s h i n g t o n 98195.

R6sum6.

Summary.

Une enzyme de type a-chymotryptique a 6t6 isol6e des mastocytes de la cavit6 p6riton6ale de Rat par extraction & l'aide de phosphate de potassium 0,8 M, 2 p. cent sulfate de protamine, suivie d'une chromatoqraphie d'affinit6 sur aqarose - ovoinhibiteur de poule et d'une adsorption sur sulfate de barium. Ce protocole fournit plus de 9 mq de prot6ase & partir des liquides de l a v a q e p6riton6al de 100 rats, correspondant & 44 p. cent de l'activit6 initiale. La prot6ine purifi6e est homoq~ne en 61ectrophor~se sur qel de polyacrylamide-sodium dodecyl sulfate, en focalisation iso61ectrique analytique et p a r a n a l y s e de la s6quence N-termihale. La prot6ase ne contient pas de qlucides li6s de fa~on covalente e t a un poids mol~culaire d'environ 26000. L'enzyme est form6e d'une seule chalne polypeptidique, avec une s6quence N-terminale homoloque de celle de la chalne B de l'~-chymotrypsine bovine. Les param~tres cin6tiques. Km et keat, de l'hydro1yse du N-benzoyl-L-tyrosine ethyl ester sont & pH 8.0 et & 25°C, 1,1 X 10-s M e t 84 sec -1, respectivement. La valeur de la constante de vitesse d'ordre 2 pour l'inactivation de la prot6ase de mastocytes par le diisopropyl phosphofluoridate est 300 fois inf6rieure & ceUe trouv6e pour l'~-chymotrypsine bovine.

An a-chymotrypsin-like enzyme was isolated from mast cells of the rat peritoneal cavity b y extraction with 0.8 M potassium phosphate, 2 per cent protamine sulfate followed b y affinity chromatography on hen ovoinhibitor-agarose and adsorption on barium sulfate. This procedure yielded over 9 mq of protease from the peritoneal l a v a g e fluid of 100 rats, equivalent to 44 per cent of the initial activity. The purified protein w a s homoqeneous as judqed b y sodium dodecyl sulfate-polyacrylamide qel electrophoresis, analytical isoelectric focusinq, and amino-terminal sequence analysis. The protease contains no covalently bound carbohydrate a n d has a molecular weiqht of approximately 26,000. The enzyme molecule is a sinqle polypeptide chain with an amino-terminal sequence homoloqous to that of the B chain of bovine a-chymotrypsin. The kinetic parameters, Km and keat, for the hydrolysis of N-benzoyl-L. tyrosine ethyl ester were determined at pH 8.0 and 25°C as 1.1 X 10-~ M and 84 sec -1, respectively. The value of the second-order rate constant for inactivation of mast cell protease b y diisopropylphosphofluoridate was 300 times lower than for bovine a-chymotrypsin.

(*) This article is dedicated to the memory of JeanFrancois Pech~re, a dedicated scientist and a valued friend, who was associated with H. N. in the Department of Biochemistry of the University of Washington as a Postdoctoral Fellow from October, 1955 until August, 1958.

0 To whom all correspondence should be addressed. The following abbreviations are used : BzTyrOEt, a-N-benzoyl-L-tyrosine ethyl ester ; DEAE, dielhylaminoethyl ; DFP, diisopropylphosphofluoridate ; Tris-HCl, tris(hydroxllmelhyl)aminomethane hydrochloride.

Key words : Mast cells, serine protease, chymotrypsin-like specificity affinity chromatography.

654

M. T. E v e r i t t a n d H. N e u r a t h .

Introduction. A l t h o u g h t h e a - c h y m o t r y p s i n - l i k e e n z y m e of m a s t cells h a s b e e n u n d e r s t u d y for n e a r l y t h r e e d e c a d e s , t h e p h y s i o l o g i c a l r o l e s of t h e e n z y m e a n d its h i s t o l o g i c a l s o u r c e , c o n n e c t i v e t i s s u e m a s t cells, a r e r e l a t i v e l y o b s c u r e [1-3]. E a r l y s t u d i e s , utilizing histochemical substrates, demonstrated t h a t m a s t cells c o n t a i n a powerf, ul e s t e r a s e activ i t y [4]. S u b s e q u e n t i n v e s t i g a t i o n i n d i c a t e d t h a t t h e e s t e r a s e r e s e m b l e d b o v i n e a - e h y m o t r y p s i n in specificity toward ester substrates and susceptib i l i t y to D F P [5-7]. L a t e r s t u d i e s [8, 9] d e m o n s t r a t e d t h a t t h e e n z y m e w~as an a l k a l i n e p r o t e a s e . which was intimately associated with the major c o m p o n e n t s of t h e m a s t cell s e c r e t o r y g r a n u l e , i.e. heparin, histamine, and serotonin. P u r i f i c a t i o n of t h e m a s t cell e n z y m e h a s b e e n reported by several laboratories. Pastan and A l m q v i s t [10, 11] i s o l a t e d t h e e n z y m e f r o m r a t thyroid tissue employing extraction with 1 M K C1 f o l l o w e d b y gel f i l t r a t i o n a n d D E A E - c e l l u l o s e c h r o m a t o g r a p h y . A l t h o u g h t h e .overall y i e l d of act i v e e n z y m e w a s love, t h e i r p r e p a r a t i o n h y d r o l y z e d a n u m b e r of p r o t e i n s a n d s y n t h e t i c s u b s t r a t b s a n d it w a s n o t i n h i b i t e d b y c h e l a t i n g o r s u l f h y dryl modifying reagents. A similar protease has b e e n i s o l a t e d f r o m a v a r i e t y of tissues i n c l u d i n g r a t m a s t cell g r a n u l e s [12], m o u s e m a s t o c y t o m a t i s s u e E13], r a t s k e l e t a l muscl,e [14-16], r a t s k i n E17], a n d r a t l i v e r [18]. R e c e n t l y , Yurt a n d A u s t e n E19] i s o l a t e d t h e e n z y m e f r o m p u r i f i e d rat m a s t cells u s i n g i o n e x c h a n g e c h r o m a t o g r a p h y on D o w e x - 1 , gel f i l t r a t i o n , a n d a f f i n i t y c h r o m a t o g r a p h y on D 4 r y p t o p h a n m e t h y l est,er. A l t h o u g h t h e y i e l d of a c t i v e e n z y m e w a s l o w , t h e y w e r e able to p r e p a r e a s u f f i c i e n t q u a n t i t y of h o m o g e n e o u s e n z y m e for a p r e l i m i n a r y i n v e s t i g a t i o n of its int e r a c t i o n w i t h o t h e r m a s t cell g r a n u l e c o m p o nents. I n t h e c o u r s e of s t u d i e s of the i n i t i a t i o n of p r o t e i n d e g r a d a t i o n , it yeas n o t e d t h a t a p r o t e a s e in rat skel~etal m u s c l e r e a d i l y i n a c t i v a t e d t h e a p o e n z y m e f o r m s of s e v e r a l p y a ' i d o x a l p h o s p h a t e r e q u i r i n g e n z y m e s [20, 21]. S u b s e q u e n t l y , it w a s dem o n s t r a t e d t h a t ,this s k e l e t a l m u s c l e e n z y m e w a s i d e n t i c a l to t h e m a s t c e l l d e r i v e d p r o t e a s e E22]. T h e p r e s e n t w o r k d e s c r i b e s t h e i s o l a t i o n of t h e a - e h y m o t r y p s i n - l i k e m a s t cell p r o t e a s e u s i n g a h i g h y i e l d a f f i n i t y t e c h n i q u , e, a n d r e p o r t s a p a r t i a l c h a r a c t e r i z a t i o n of t h e ,enzyme.

Materials and Method~ Materials. Sodium lauryl sulfate was obtained from British Drug House. et-N-Benzoyt-r-tyrosine ethyl ester and BIOCHIMIE, 1979, 61, n ° 5-6.

u-N-tosyl-r-phenylalanine chloromethyl ketone were f r o m Cyelo Chemical Corporation. Barium sulfate (X-ray grade) was obtained from Matheson, Coleman, and Bell and bovine albumin (Pathoeyte 4) from Miles Laboratories. Cyanogen bromide, DFP, and dimethylsulfoxide (Sequenal grade) were purchased from Pierce. N-Aeetyl neuraminic acid, bovine serum albumin (Fraction V), Coomassie Brilliant Blue R-250. o-nitrobenzaldehyde, pyridoxal-5'-phosphate, ornithine-HC], 2-oxoglutaric acid, protamine sulfate (Grades I and II), toluidine blue and Tris-HC1 were obtained from Sigma. Bovine basic pancreatic trypsin inhibitor, bovine a-chymotrypsin, bovine chymotrypsinogen A, lima bean trypsin inhibitor, and soybean trypsin inhibitor were purchased from Worthington Biochemical Corporation. Sepharose CL-6B was obtained from Pharmacia Fine Chemicals. Turkey ovomucoid was a gift f r o m Dr. M. Laskowski, Jr. Benzyloxycarbonyl-L-phenylalanine bromomethyl ketone was a gift from Dr. J. LonsdaleEccles. Hen ovoinhibitor was a gift from Dr. H. Ako. o-Aminobenzaldehyde was prepared from o-nitrobenzaldehyde according to the procedure of Smith and Opie [23]. Sprague-Dawley rats (250-400 g) were purchased from Tyler Laboratories (Bellevue, Washington). Ornithine aminotransferase was isolated from rat liver as described by Chee and Swiek [2~]. The apoenzyme was prepared according to the procedure of Sanada et al. [25]. Methods. 1. Determination of enzymatic activities. Esterase activity was determined by a procedure similar to that of Hummel [26] using BzTyrOEt as the substrate. The ester substrate (1.07 raM) dissolved in 50 per cent methanol (w/w) was combined with assay buffer (0.2 M Tris-HC1, 0.2 M NaC1, pH 8.0) and preincubated for 5 min at 25°C. The reaction was initiated by adding 100 ~tl of appropriately diluted enzyme and the rate of hydrolysis determined from the increase in absorbanee at 256 nm. Proteolytie activity was determined by monitoring the reaetivation of the apoenzyme form of ornithine aminotransferase upon addition of the coenzyme [27]. Protcase (2.5-10 units) was added to 0.25 mg of ornithine aminotransferase apoenzyme in a final volume of 750 Ix1. Aliquots (150 Ixl) were removed at 0, 10, 20, and 30 min after protease addition and diluted with 2.0 ml of 0.2 M Tris-HC1, containing 0.35 M NaC1, 0.1 mM pyridoxal phosphate (pH 8.0), to convert the remaining apoenzyme to the more stable holoenzyme form. Residual ornithine aminotransferase activity was assayed as described by Jenkins and Tsai [28]. The activity remaining (per cent) was plotted against time to determine the time o f half-inactivation of the apoenzyme. One unit of protease activity was defined as that amount which inactivated 50 per cent of the apoenzyme activity in 30 rain. Duplicate samples containing approximately a 100 fold excess of lima bean trypsin inhibitor were used as controls. 2. Amino acid analyses. Samples were prepared and analyzed by the methods of Moore and Stein [29] and Spaekman et al. [30] employing a Durrum Model D-500 amino acid analyzer and utilizing a single column procedure. Duplicate samples were hydrolyzed in 6 M HC1 at 110°C for 24, 48, 72 and 96 h in evacuated tubes. The values of threonine and serine were extrapolated to zero hydrolysis

M a s t cell p r o t e a s e . t i m e . T h e v a l u e s of i s o l e u e i n e a n d v a l i n e w e r e c a l c u l a t e d f r o m tl~e 96 h o u r h y d r o l y s i s . T r y p t o p h a n w a s d e t e r m i n e d a f t e r a l k a l i n e h y d r o l y s i s a c c o r d i n g to H u g l i a n d Moore [31]. H a l f c y s t i n e w a s d e t e r m i n e d as c y s t e i e a c i d [32]. 3. C a r b o h y d r a t e a n a l y s e s . Neutral sugar was determined by both the phenols u l f u r i c a c i d m e t h o d o f D u b o i s et al. [33] as d e s c r i b e d b y A s h w e l l [34] a n d t h e a n t h r o n e m e t h o d of Spiro [35] u s i n g a 1:1 m i x t u r e of m a n n o s e a n d g a l a c t o s e as a s t a n d a r d . F o r t h e d e t e r m i n a t i o n of h e x o s a m i n e s , d u p l i c a t e s a m p l e s w e r e h y d r o l y z e d i n 2 N HC1 f o r 24 h a t 110°C a n d a n a l y z e d d i r e c t l y f o r g a l a e t o s a m i n e a n d g l u e o s a m i n e w i t h t h e D u r r u m (D-500) a m i n o a c i d a n a l y zer. Sialic a c i d w a s d e t e r m i n e d a s f r e e N - a c e t y l n e u r a m i n i c a c i d b y t h e t h i o b a r b i t u r i c a c i d m e t h o d of W a r r e n [36] a f t e r h y d r o l y s i s of d u p l i c a t e s a m p l e s f o r 1 h a t 80°C in 0.1 M s u l f u r i c acid. 4. S e q u e n a t o r a n a l y s e s . Automated Edman degradations were performed with a B e c k m a n s e q u e n a t o r Model 890B, u s i n g a m o d i f i e d f o r m [37] o f t h e t e c h n i q u e o f E d m a n a n d Begg [38]. Pth-amino acids were identified by gas- and high press u r e l i q u i d c h r o m a t o g r a p h y (HPLC) as d e s c r i b e d b y W o o d b u r y et al. [39]. S a m p l e s w e r e r e d u c e d a n d p y r i d y l e t h y l a t e d a c c o r d i n g to t h e m e t h o d of H e r m o d s o n et al. [40]. 5. P r o t e i n c o n c e n t r a t i o n . T h e m i e r o b i u r e t m e t h o d o f Z a m e n h o f f [41] w a s u s e d with bovine serum albumin as the reference protein. When extinction coefficients were known, protein concentration was determined spectrophotometrically from t h e a b s o r b a n c e a t 280 n m , u s i n g a 1 c m q u a r t z c u v e t t e . 6. E l e e t r o p h o r e s i s i n s o d i u m d o d e e y l s u l f a t e , 8 M urea. The discontinuous, Tris-HC1 b u f f e r s y s t e m of L a e m m l i [42] a s m o d i f i e d b y S t u d i e r [43] f o r a s l a b gel a p p a r a t u s w a s u s e d . D e - i o n i z e d u r e a w a s i n c l u d e d in t h e s a m p l e b u f f e r a n d b o t h t h e s p a c e r (5 p e r c e n t a c r y l a m i d e ) a n d r e s o l v i n g (10 p e r c e n t a c r y l a m i d e ) gels. M a s t cell p r o t e a s e a n d r e f e r e n c e s t a n d a r d s (10 lxg25 l~g) w e r e d i s s o l v e d in 25 Ixl of b o i l i n g s a m p l e b u f f e r a n d i n c u b a t e d f o r 20 m i n in a b o i l i n g w a t e r b a t h . W h e n c o n d i t i o n s f o r r e d u c i n g d i s u l f i d e b o n d s were r e q u i r e d , 2 - m e r c a p t o e t h a n o l (1 p e r cent, v / v ) w a s i n c l u d e d in t h e i n c u b a t i o n b u f f e r . E l e c t r o p h o r e s i s w a s i n i t i a t e d a t a c u r r e n t of 20 m A , r e a d j u s t e d to 35 m A a f t e r t h e s a m p l e s h a d e n t e r e d t h e r e s o l v i n g gel, a n d c o n t i n u e d f o r 4 h. T h e s l a b w a s s t a i n e d f o r p r o t e i n w i t h 0.1 p e r c e n t C o o m a s s i e B r i l l i a n t B l u e R-250 f o r I h a t 60°C, d e s t a i n e d f o r 1 h in m e t h a n o l , acetic acid, w a t e r (5:1:5), a n d c l e a r e d o v e r n i g h t in m e t h a n o l , acetic acid, w a t e r (1:1:9). C a l i b r a t i o n c u r v e s w e r e c o n s t r u c t e d using proteins of known molecular weights as outlined b y W e b e r a n d O s b o r n [44]. 7. P o l y a c r y l a m i d e gel i s o e l e c t r i c f o c u s i n g . Analytical isoelectrie focusing was performed with a n LKB M u l t i p h o r a p p a r a t u s a n d p r e c a s t p l a t e s of p o l y a c r y l a m i d e gel. P r i o r to s a m p l e a p p l i c a t i o n , t h e p l a t e w a s i m m e r s e d f o r 2 h in 100 m l of d e i o n i z e d 7 M u r e a w h i c h c o n t a i n e d 6.0 m l of A m p h o l i n e 3.5-10. A f t e r p l a c e m e n t of t h e s p e c i a l l y t r e a t e d p l a t e o n t o t h e c o o l i n g b l o c k o f t h e M u l t i p h o r c h a m b e r , 25 ~g of s a m p l e , d i s s o l v e d in 7 M u r e a , w e r e a p p l i e d to t h e s u r f a c e of t h e gel a n d e l e e t r o f o e u s i n g w a s i n i t i a t e d . U p o n

BIOCH1MIE, 1979, 61, n ° 5-6.

655

c o m p l e t i o n , t h e gel p l a t e s w e r e fixed a n d s t a i n e d w i t h C o o m a s s i e B r i l l i a n t B l u e R-250 a s s u g g e s t e d b y t h e manufacturer. 8. P r e p a r a t i o n of i m m o b i l i z e d h e n egg w h i t e o v o i n hibitor. S e p h a r o s e CL-6B w a s a c t i v a t e d w i t h c y a n o g e n b r o mide and subsequently coupled with hen ovoinhibitor a c c o r d i n g to a m o d i f i c a t i o n [45] o f t h e m e t h o d of C u a t r e c a s a s [46]. C y a n o g e n b r o m i d e (15 g) w a s d i s s o l v e d in 4 m l of d i m e t h y l s u l f o x i d e ( S e q u e n a l G r a d e ) a n d c o m b i n e d w i t h a s u s p e n s i o n o f 50 m l of w e l l - w a s h e d S e p h a r o s e CL-6B i n 50 m l o f 0.2 M s o d i u m c a r b o n a t e . T h e suspens'_'on w a s c h i l l e d in a n ice b u c k e t a n d m a i n t a i n e d a t p H 11 b y a d d i t i o n o f 10 M s o d i u m h y d r o x i d e . W h e n t h e r e a c t i o n b e g a n to s u b s i d e , t h e a g a r o s e w a s t r a n s f e r r e d to a s i n t e r e d g l a s s f u n n e l a n d w a s h e d w~th 10 v o l u m e s of ice cold d i s t i l l e d w a t e r a n d 10 v o l u m e s of 0.2 M s o d i u m b i c a r b o n a t e . A n a l i q u o t of t h e a c t i v a t e d a g a r o s e (5 m l ) w a s c o m b i n e d w i t h 27 m g o f h e n o v o i n h i b i t o r in 5 m l of 0.2 M s o d i u m b i c a r b o n a t e (pH 9.0) a n d s t i r r e d o v e r n i g h t a t 4°C. P r i o r to use, the ovoinhibitor-agarose was washed extensively with d i s t i l l e d w a t e r a n d 8 M u r e a (pH 2.5) a n d s u b s e q u e n t l y e q u i l i b r a t e d w i t h 0.8 M p o t a s s i u m p h o s p h a t e c o n t a i n i n g 1 m g / m l b o v i n e s e r u m a l b u m i n (pH 8.0). 9. P r e p a r a t i o n of r a t m a s t cells a n d s e c r e t o r y g r a nules. T h e p e r i t o n e a l c a v i t i e s of f r e s h l y sacrificed r a t s w e r e washed with a phosphate buffered saline solution cont a i n i n g 4.1 m M Na~HPOo 2.1 m M KH2PO4, 0.154 M NaC1, 2.7 m M KCI, a n d 0.68 m M CaCl2 (pH 7.2). T h e i n s o l u b l e m a t e r i a l of t h e l a v a g e fluid w a s collected b y c e n t r i f u g a t i o n a t 400 × g f o r 15 m i n a t 4°C, r e s u s p e n d e d in o n e m l of b u f f e r p e r r a t sacrificed, a n d t h e m a s t cells purified by centrifugation through an isotonic solution of b o v i n e p l a s m a a l b u m i n ( P a t h o c y t e 4) a t 400 X g f o r 25 m i n [47]. T h e s u p e r n a t a n t w a s c a r e f u l l y a s p i r a t e d to r e m o v e all cells a n d d e b r i s a t t h e i n t e r f a c e , t h e i n s i d e of t h e t u b e w a s w i p e d , a n d t h e m a s t cell p e l l e t r e s u s p e n d e d in b u f f e r . T o l u i d i n e b l u e s t a i n i n g of a n a l i q u o t d e m o n s t r a t e d t h a t m o r e t h a n 95 p e r c e n t of t h e c e l l s w e r e m a s t cells ( a p p r o x i m a t e l y 106 cells p e r r a t ) . A f t e r w a s h i n g t h e p u r i f i e d cells w i t h 20 m l of b u f f e r a n d 20 m l o f ice cold d i s t i l l e d w a t e r , t h e y w e r e r e s u s p e n d e d i n d i s t i l l e d w a t e r a t a c o n c e n t r a t i o n o f 2 X 106 to 5 X 106 e e l l s / m l a n d a g i t a t e d w i t h a V o r t e x m i x e r until the majority of the granules was released into the medium. Residual material, including intact mast cells a n d n u c l e i , w e r e r e m o v e d b y c e n t r i f u g a t i o n at 800 X g f o r 8 m i n . T h e p u r i f i e d g r a n u l e s w e r e t h e n c o l l e c t e d b y c e n t r i f u g a t i o n a t 1200 X g f o r 25 m i n . 10. K i n e t i c s of i n h i b i t i o n . I n h i b i t i o n e x p e r i m e n t s w e r e d o n e in t h e p r e s e n c e of a n e x c e s s of D F P s u c h t h a t p s e u d o - f i r s t - o r d e r k i n e t i c s w e r e o b s e r v e d , a s o u t l i n e d b y F a h r n e y a n d Gold [48]. U n d e r t h e s e c o n d i t i o n s t h e r a t e of e n z y m e i n a c t f v i i t i o n is p r o p o r t i o n a l to t h e c o n c e n t r a t i o n o f i n h i b i t o r a n d independent of protease concentration, thus simplifying both the experiment and subsequent calculations. F r o m a s e m i l o g a r i t h m i c p l o t of r e s i d u a l e n z y m a t i c a c t i v i t y a g a i n s t t i m e , t~j.~ t h e t i m e r e q u i r e d to r e d u c e t h e a c t i v i t y b y 50 p e r cent, w a s c a l c u l a t e d . T h e p s e u d o f i r s t - o r d e r r a t e c o n s t a n t (min-1) w a s c a l c u l a t e d f r o m the relationship : In 2 kpseud° ~ t1:2

M. T. E v e r i t t a n d H. N e u r a t h .

656

e q u i l i b r a t i o n buffer until a constant base line (Ee80) was o b t a i n e d on the c h r o m a t o g r a m and the protease w a s eluted w i t h 0.025 M f o r m i c acid cont a i n i n g 0.001 M EDTA, 1 m g / m l b o v i n e s e r u m alb u m i n and 20 p e r cent g l y c e r o l (pH 3.0). The flow rate was r e d u c e d to 10 m l / h and one ml f r a c t i o n s w e r e collected in the p r e s e n c e of one ml of 0.2 M a m m o n i u m b i c a r b o n a t e , 1 m g / m l b o v i n e serum albumin (pH 8.6). F r a c t i o n s w e r e m o n i t o r e d for OD2so and esterase a c t i v i t y (BzTyrOEt). The resuits are s h o w n in figure 1. Active fractions w e r e c o m b i n e d , diluted two-fold w i t h distilled w a t e r and b a r i u m sulfate (2.0 g) slowly added with constant mixing. After c e n t r i f u g i n g the suspension at 2,500 × g for 10 rain, the p r e c i p i t a t e w a s collected and w a s h e d t w i c e w i t h 20 ml of 0.01 Tris-HC1 (pH 8.0). The protease was solubilized by w a s h i n g the b a r i u m sulfate pellet w i t h 5 ml p o r t i o n s of 0.01 M Tris-HC1, 1.0 M NaC1 (pH 8.0). A s u m m a r y of the p u r i f i c a t i o n p r o c e d u r e is given in table I. U n d e r the c o n d i t i o n s employed, 44 per cent of the initial a c t i v i t y w a s r e c o v e r e d w i t h a 15 fold

A second-order rate constant, kobsd (liter mol-~ min-1) was calculated by dividing kp~e~do by the concentration of DFP in the inactivation mixture.

Results. 1. Purification of rat peritoneal mast cell protease. A t y p i c a l p u r i f i c a t i o n p r o t o c o l is detailed below. All m a n i p u l a t i o n s w e r e done at 4°C. Peritoneal w a s h e s f r o m 100 rats w e r e c o m b i n e d and c e n t r i f u g e d for 20 rain at 27,000 X g. The supernatant was d i s c a r d e d and the cell pellet resusp e n d e d in 20 ml of buffered saline. The suspension was frozen and t h a w e d t h r o u g h six cycles and the cellular debris and mast cell granules sed i m e n t e d by c e n l r i f u g a t i o n for 30 rain at 27,000 × g. P r o t e a s e w a s solubilized by e x t r a c t i o n of the pellet w i t h t h r e e 20 ml v o l u m e s of 2 per cent p r o t a m i n e sulfate, 0.8 M potassium p h o s p h a t e

IO0 ®

-80

I 4.OI

i i

% 3.0-

|

,L

-60

I

to

1

2.0-

-40 I

-20 \

//, I'0

1'5

50

3'5

4'0

4 '5

c~

-0 50

Fraction number Fw,. 1. - - Elution pattern of rat mast cell protease from ovoinhibitor. Protease was eluted from the column (1 × 4 cm) with 25 mM formic acid, 1 mM EDTA, 20 per cent glycerol, 1 mg/ml bovine serum albumin (pH 3.0) at a flow rate of 10 ml/h. Fractions (1.0 ml) were collected in 1.0 ml of 0.2 M ammonium bicarbonate (pH 8.6). Esterase activity was determined as described under Methods. Fractions that were pooled are indicated by the bar. The insert illustrates the sodium dodecyl sulfatepolyaerylamide gel analysis of the pooled fractions after removal of bovine serum albumin with BaSO~. (--) Absorbancce at 280 n m ; (@. . . . @) Esterase activity. BTEE denotes benzoyl-L-tyrosine ethyl ester.

(pH 8.0). The p r o t e a s e solution w a s a p p l i e d to an o v o i n h i b i t o r - a g a r o s e c o l u m n (1 × 4 era) previously e q u i l i b r a t e d w i t h 0.8 M potassium phosphate, 1 m g / m l b o v i n e serum albumin (pH 8.0). The f l o w r a t e w a s 20 m l / h and 4.5 1111 f r a c t i o n s w e r e collected. The c o l u m n w a s w a s h e d w i t h

BIOCHIMIE, 1979, 61, n ° 5-6.

purification. H o w e v e r , passage of the p u r i f i e d material over a c o l u m n of potato c h y m o t r y p s i n inhibitor I-agarose d e m o n s t r a t e d that 55 per cent of the m a t e r i a l w o u l d n e i t h e r b i n d to the ligand nor h y d r o l y z e BzTyrOEt. The two fractions had identical amino acid c o m p o s i t i o n s and s i m i l a r electro-

Mast cell protease. p h o r e t i c m o b i l i t i e s in s o d i u m d o d e c y l sulfate. C o r r e c t i o n f o r t h e i n a c t i v e m a t e r i a l gave a s p e c i fic a c t i v i t y ,of 58 B z T y r O E t u n i t s / m g of a c t i v e enzyme.

2. Sodium dodecyl eIectrophoresis.

sulfate- polyacrylamide

gel

I n t h e p r e s e n c e of 8 M u r e a t h e e n z y m e p r o t e i n m i g r a t e d as a single, s h a r p b a n d (see i n s e t of

657

fig. 1). T h e a p p a r e n t m o l e c u l a r w e i g h t i n t e r p o l a t e d f r o m s t a n d a r d s of k n o w n m o l e c u l a r w e i g h t , w a s 26,000 b e f o r e a n d a f t e r r e d u c t i o n w i t h 2-merc a p t ~ e t h a n o l , i n d i c a t i n g t h a t the p r o t e i n c o n t a i n s a s i n g l e p o l y p e p t i d e c h a i n . I n s o m e gels a s e c o n d , f a i n t b a n d w a s e v i d e n t , c o r r e s p o n d i n g to a m o l e c u l a r w e i g h t of 52,000, s u g g e s t i n g t h a t e v e n u n d e r d i s s o c i a t i n g c o n d i t i o n s the e n z y m e has a t e n d e n c y to d i m e r i z e .

TABLE I.

Purification of Mast Cell Protease from the Peritoneal Cells of 100 Rats. Purification step

Vol (mL)

Extract Affinity chromatography on ovoinhibitor-agarose

Barium sulfate adsorption

Total protein (mg)

Total Act. Spec. Act. (BzTyrOEt (Units/;mg) Units)

Purilication ([old)

56

330

527

20

--

321

--

61

--

232

25.5

44

15

15

9.1

1.6

Recovery (p. cent)

100

1

TABLE II.

Amino Acid and Carbohydrate Composition of Rat Mast Cell Protease. Components

g/100 g protein

tool amino acid tool protein

Amino acid Alanine

4.3

14

Arginine Aspartic [ NH~

7.0 7.4

12 17

Half-cystine (a)

3.1

8

Glutamic/NH~ Glycine

9.2 5.4

19 21

Histidine Isoleucine (b) Leucine (b)

4.5 5.5 5.1

9 13

Lysine Methiontne Phenylalanine Proline Serine (c) Threonine (c)

Tryptophan (d) Tyrosine Valine (b)

11.2

12 23

2.5 3.8

5 7

5.6 4.2 6.5 1.2 4.2

15 12 16 2 7 22 234

8.6

Total number of residues Carbohydrate Neutral sugars (e) Amino sugars (f) N-Acetylneuraminic acid (g) Total sugar by weight


Molecular weight by composition, Molecular weight by sodium dodecyl sulfate gel electrophoresis,

0 0 0 25 800 26 000

(a) Determined as cysteic acid after performic acid oxidation. (b) Value after 96 h hydrolysis. (c) Value after extrapolation to zero time of hydrolysis. (d) Determined on the amino ae;.d analyzer after base hydrolysis. (e) Hexose determined by anthrone and phenol-sulfuric acid methods. (f) Determined by analysis on the amino acid analyzer. (g) Determined as neuraminie acid.

BIOCHIMIE, 1979, 61, n ° 5-6.

I0

20 lle-lle-Gly-Gly-Val-

Glu-Ala-Val-Pro-GIy-Ser-Trp-Pro-Trp-Gln-VaI-Ser-Leu-GIn-Asp-Lys-Thr-Gly-Phe .....

Bovine chymotrypsinogen A

........ His-Phe-Cys-Gly-GIy-Ser-Leu-Ile-

Bovine chymotrypsinogen A

Fio. 2. - - A m i n o - t e r m i n a l sequences of rat m a s t cell protease and c h g m o t r g p s i n o g e n A. Identical a m i n o acids a r e u n d e r l i n e d . D a s h e s ( - - - - ) r e f e r to spaces i n s e r t e d to b r i n g t h e t w o p r o t e i n s into a l i g n m e n t to o p t i m i z e h o m o l o g y .

Tyr-Lys-Ala-Thr-Cys-Gly-Gly-Phe-Leu-Val-

Rat mast cell protease

4O

G•u-Ser-Arg-Pr•-His-Ser-Arg-Pr•-Tyr-•et-A•a-His-Leu-Glu-I•e-Thr-Thr-Glu-Arg-Gly-

30

Cys-G•y-Val-Pr•-Ala-IIe•Gln-Pr•-Va•-Leu-Ser-G•y-Leu-ser•Arg-••e-Val-Asn-Gly•GIu-

1

Rat mast cell protease

Bovine chymotrypsinogen A

Rat mast cell protease

Mast cell protease. 3. Isoelectric focusing.

659

A single, slightly diffuse, protein band was obs e r v e d w h e n t h e p u r i f i e d m a s t cell e n z y m e w a s s u b j . e c t e d to i s o e t e c t r i c f o c u s i n g i n 7 M u r e a . I n t h e a b s e n c e of u r e a , o n l y a b r o a d s m e a r w a s s e e n . Th, e p r o t e i n h a s a n a p p a r e n t i s o e l e c i r i e p o i n t of p I = 9.5.

less t h a n 1 p e r c e n t n e u t r a l s u g a r a n d n o h e x o s a mine or sialic acid. A minimum molecular vceight of 25,800, b a s e d o n 22 r e s i d u e s of v a l i n e p e r m o l e c u l e of e n z y m e , w a s c a l c u l a t e d . T h i s f i g u r e is e s s e n t i a l l y i d e n t i c a l t o t h e v a l u e of 26,000 e s t i m a t e d f r o m p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s i n sodium dodecyl sulfate and urea.

4. Amino-terminal sequence.

6. Characteristics of rat mast cell protease.

T h e m a s t cell e n z y m e w a s f o u n d to c o n t a i n a single amino-terminal residue, isoleucine, and only a single sequence was observed during subsequen,t a n a l y s i s . T h e r e p e t i t i v e y i e l d f o r e a c h of t h e d e g r a d a t i o n s t e p s w a s a p p r o x i m a t e l y 95 p e r c e n t . T h e a m i n o - t e r m i n a l s e q u e n c e of r e d u c e d and pyridylethylated r a t m a s t cell p r o t e a s e is shown in figure 2 along with the amino-terminal s e q u e n c e of b o v i n e c h y m o t r y p s i n o g e n A [49].

I n t a b l e I I I a r e l i s t e d a n u m b e r of p h y s i c a l - c h e m i c a l c h a r a c t e r i s t i c s of r a t p e r i t o n e a l m a s t cell p r o t e a s e a n d s e v e r a l s i m i l a r e n z y m e s . All r e q u i r e h i g h i o n i c s t r e n g t h f o r e x t r a c t i o n , all h a v e a m o l e c u l a r w e i g h t n e a r 25,000, all h a v e p H o p t i m a n e a r 8.0, a n d all a r e i n h i b i t e d b y D F P . Of p a r t i c u l a r i n t e r e s t is t h e s i m i l a r i t y i n t h e p a t t e r n of inhibition by the protein inhibitors. In all known instances, the proteases are inhibited by soybean trypsin inhibitor, hen ovoinhibitor, and lima bean t r y p s i n i n h i b i t o r , w h i l e n o n e is i n h i b i t e d b y ovom u c o i d . O n l y h m n a n l e u k o c y t e c a t h e p s i n G is inhibited by pancreatic basic trypsin inhibitor. I n a d d i t i o n , m a s t cell p r o t e a s e a n d c a t h e p s i n G

5. Amino acid and carbohydrate composition. T h e r e s u l t s of a m i n o a c i d a n a l y s i s a n d c a r b o hydrate determination for the rat mast cell prot e a s e a r e s h o w n i n t a b l e II. T h e e n z y m e c o n t a i n e d

TABLE III.

Characteristics of Rat Mast Cell Protease and Comparison with Selected Intracellular Proteases. Rat mast cell protease

Rat mast cell protease [t2]

Rat liver mitoehondrial protease [t8]

Rat skin prolease [t7]

Human leukocyte cathepsin G [50]

0 . 8 M potassium phosphate, 2 % protamine-SO 4 26,000 8.0

1.4 M KC1

0.5 M K phosphate

2.0 M KCI

1.0 M NaCl 0.1 ~ Brij 35

25,000 8.0

23,000 8.0

27,500 7.2 - 8.0

28,000 7.5 - 8 . 0

1.1 84

1.1 n.a.

n.a. n.a.

n.a. n.a.

2.6 a 3.1

58.3

- - 60

n.a.

n.a.

1.53

-~+ -~ --[-

-~-~n.a. n.a. n.a.

-~-~n.a. n.a. "4-

-~-4-~-~-4-

+ ----+ -{+

--~--~-

n.a. n.a. n.a. n.a.

n.a. n.a. n.a. n.a.

-n.a. -~-

+ + -~-

Gharaeteristic Solubility Molecular weight p H optimum B z T y r O E t hydrolysis : km (10 -3 M) kcat (see -1) Sp. act. (IU/mg) Inhibition b DFP c TPCK ¢ ZPBK c Cysteine Soybean trypsin inhibitor Pancreatic basic trypsin inhibitor (Kunitz) Hen ovoinhibitor Ovomucoid Lima bean trypsin inhibitor

n . a . = no data available. + ---- S u b s t a n t i a l I n h i b i t i o n , - - = No I n h i b i t i o n , -4- = Slight I n h i b i t i o n or Questionable Conditions. (a) Adjusted to the u n i t s of H u m m e l [51]. (b) Samples were p r e - i n c u b a t e d for 15 m i n u t e s a t 25 o w i t h inhibitor, then assayed for protease a n d esterase activity. (c) Control samples c o n t a i n e d 5 per cent 2-propanol.

BIOCHIMIE, 1979, 61, n ° 5-6.

45

6'6.0

M. T. E v e r i t t a n d H. N e u r a t h .

differ s u b s t a n t i a l l y i n their p a r a m e t e r s of esterase activity t o w a r d BzTyrOEt. 7. Effect of DFP on mast cell protease. W h e n mast cell protease was i n c u b a t e d w i t h 0.1 ram DFP, the pseudo-first-order rate of loss of esterase activity c o r r e s p o n d e d to a half inactivation time of 114 rain. The s e c o n d - o r d e r rate constant for i n a c t i v a t i o n is no.ted in table IV along with the c o r r e s p o n d i n g values for b o v i n e a-chym o t r y p s i n a n d s u b i i l i s i n (Carlsberg). The inactivation rate of the mast cell enzyme is about 10 times lower t h a n that of s u b t i l i s i n and nearly 300 times lower t h a n that of bovine e - c h y m o t r y p sin.

DFP

m~ 100~ S

s

6O DFP 40

5O

¢

I

20

J

I

f

i

i

60 120 Time (m(nutes)

FIG. 3. - - Pseudo-first-order rate of loss of esterase activity upon treatment of rat mast cell protease with DFP. For details of experiment, see table IV. Solid circles represent esterase activity in the presence, and solid triangles in the absence of DFP.

TABLE IV. Second-Order Rate Constants for Reaction w i t h Diisoproptll[luorophosphate. Protein Mast cell protease Bovine a-chymotrypsin Subtilisin (Carlsberg)

K2

(liter mol-' min-~) 61 (a) 17 300 (b) 792 (c)

(a) 0.31 aM mast cell protease, 0.1 M Tris-HC1, 0.2 M NaC1 (pH 8.0) 25°, 0.1 mM DFP 5 per cent 2-propanol. (b) 0.36 t~M bovine a-chymotrypsin, 0.1 M Tris-HC1, 0.2 M NaC1 (pH 8.0) 25°, 0.01 mM DFP, 5 per cent 2-propanol. (c) 0.37 ~M subtilisin (Carlsberg), phosphate buffer (pH 7.6), 0.1 mM DFP, 1 per cent 2-propanol [52]. BIOCHIM1E, 1979, 61, n ° 5-6.

Discussion. The p r e s e n t m e t h o d of p u r i f i c a t i o n of an a-chym o t r y p s i n - l i k e protease of rat mast ceils, by affin i t y adsorption, enables the isolation of m i l l i g r a m quantities of the protease from extracts of mast cell granules, p e r i t o n e a l lavage fluid, or rat muscle acetone p o w d e r [22]. Hen o v o i n h i b i t o r is an effective affinity ligand because the protease can be dissociated from the insoluble e n z y m e - i n h i b i tor complex u s i n g relatively moderate c o n d i t i o n s d u r i n g the e l u t i o n cycle of c h r o m a t o g r a p h y , w h i c h s u b s t a n t i a l l y increase t h e yield of active enzyme. Slow flow rates are r e q u i r e d d u r i n g ads o r p t i o n to i n s u r e that the e n z y m e will b i n d to the adsorbent. The final p r o d u c t of p u r i f i c a t i o n has ,a specific activity a p p r o x i m a t e l y one-half of the m a x i m u m value, because both active a n d inactive protease are present. However, this is not necessarily disadvantageous, e.g. for s t r u c t u r a l studies w h i c h do not r e q u i r e active enzymes. The enzyme isolated b y the p r e s e n t p r o c e d u r e from rat p e r i t o n e a l l avage fluid is i d e n t i c a l w i t h the e n z y m e o b t a i n e d b y the m e t h o d of Sanada el al. [16] from rat skeletal muscle [22]. The protease is a single p o l y p e p t i d e c h a i n w i t h a molecular w e i g h t of 26,000, c o n t a i n i n g little, if any, cov a l e n t l y b o u n d c a r b o h y d r a t e . The a m i n o acid composition reveals a high percentage of basic a m i n o acids, especially lysine. The molecule cont a i n s 5 m e t h i o n i n e residues, a n d 8 eysteine residues w h i c h are p r e s u m a b l y paired. The n u m b e r of disulfide b o n d s w o u l d be one less than i n ac h y m o t r y p s i n , a n d one m o r e t h a n i n the protease from ,atypical mast cells [39]. U n l i k e b o v i n e a - c h y m o t r y p s i n , the mast cell protease does not appear to ,exist i n a zymogen form. D a r z y n k i e w i c z a n d B a r n a r d [53] demonstrated that s u b s t a n t i a l a m o u n t s of radioactive D F P could be i n c o r p o r a t e d into mast cell secretory granules. Moreover, e x a m i n a t i o n of the amin o - t e r m i n a l sequence of the p u r i f i e d enzyme indicates the absence of a region c o r r e s p o n d i n g to the A p e p t i d e of a - c h y m o t r y p s i n (fig. 2). This suggents that either the e n z y m e is synthesized w i t h o u t an activation p e p t i d e or t h a t the activation peptide has been lost b y activation, because the mast cell e n z y m e lacks the Cysl-Cys 122 disulfide b o n d w h i c h r e t a i n s this peptide in bovine c h y m o t r y p s i n . Clarification of this p r o b l e m has to await more detailed searches for the presence of a zymogen i n freshly p r e p a r e d tissue extracts. The rate of i n a c t i v a t i o n of rat mast cell protease b y DFP is a p p r o x i m a t e l y 1/300th of that of

Mast cell protease. b o v i n e a - c h y m o t r y p s i n . T h e m a s t cell p r o t e a s e is also less s e n s i t i v e t h a n a - c h y m o t r y p s i n to i n a c t i v a t i o n b y s e v e r a l p r o t e i n p r o t e a s e i n h i b i t o r s . Sev e r a l r e c e n t s p e c i f i c i t y s t u d i e s of e n z y m e s w h i c h are s i m i l a r o r i d e n t i c a l to t h e m a s i cell p r o t e a s e [54, 55] i n d i c a t e d t h a t t h e i r s u b s t r a t e s p e c i f i c i t y is m o r e r e s t r i c t e d t h a n t h a t of b o v i n e a - c h y m o t r y p s i n . A p r e l i m i n a r y r e p o r t f r o m this l a b o r a t o r y [56] d e m o n s t r a t e d t h a t t h e s p e c i f i c i t y of t h e m a s t cell p r o t e a s e l o v e a r d t h e B c h a i n of o x i d i z e d i n s u l i n is s i m i l a r to t h a t of l e u k o c y t e c a t h e p s i n G [57]. I n all cases, p e p t i d e b o n d s on t h e c a r b o x y l s i d e of a r o m a t i c r e s i d u e s (i.e. p h e n y l a l a n i n e , t y r o sine, t r y p t o p h a n ) a n d of l e u c i n e a n d i s o l e u c i n e residues were cleaved. Chymotrypsin-like enzymes have been isolated f r o m a v a r i e t y of .rat t i s s u e s i n c l u d i n g l i v e r , m u s cle, skin, a n d p e r i t o n e a l m a s t cells (table III). U p o n f u r t h e r i n v e s t i g a t i o n , e a c h of t h e s e tissues p e c i f i c p r o t e a s e s yeas f o u n d to be of m a s t c e l l o r i g i n (D. L a g u n o f f , p e r s o n a l c o m m u n i c a t i o n ) . B e c a u s e of t h e u b i q u i t o u s d i s t r i b u t i o n of c o n n e c t i v e ¢issue m a s t cells, t h e r a t s e e m s to be a p o o r m o d e l f o r t h e s t u d y of n o n - m a s t cell r e l a t e d p r o t e o l y t i c a c t i v i t y , as e x e m p l i f i e d b y s t u d i e s of p r o l e i n d e g r a d a t i o n in rat s k e l e t a l m u s c l e [14] a n d of d e n e r v a t i o n a t r o p h y in r a t s [58].

Acknowledgements. We are yrateful to Dr. ]~ichard G. Woodbury and Dr. David Lagunoff for helpful discussions and to Lowell H. Ericsson, Richard R. Granbery, and Helen Wan for their technical assistance. This work has been supported by a grant f r o m the National Institutes of Health (GM 15731).

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