Non-pancreatic proteases of the chymotrypsin family. II. Two proteases from a mouse mast cell tumor

BIOCHIMICA ET BIOPHYSICA ACTA

395

BBA 66431 N O N - P A N C R E A T I C P R O T E A S E S OF T H E C H Y M O T R Y P S I N FAMILY I I TWO P R O T E A S E S FROM A MOUSE MAST CELL TUMOR

W I L L I A M H VENSEL*, J A N U S Z K O M E N D E R * * AND ERIC A BARNARD*"*

Det)artmenls of Bzochemzstry and Bwehemzeal Pharmacology, State University of New } ork, Buffalo, N Y z42z4 ( U S A ) (Recel~ed Ma'v 25th, I97 I)

SUMMARY

A chymotrypsin-hke protease has been prepared from the mouse P815Y mastcell tumor It is similar in most respects to the first such enzyme purified from the normal peritoneal mast cells of the rat It, too, has a molecular weight (estimated by gel filtration) of about 25 ooo The apparent molecular weight is lower, unless tngh salt concentrations are used to overcome affinity of this enzyme for the dextran or polyamide gel columns The binding of lndole, and the binding of potato chymotrypsln inhibitor I, are characteristically strong Specific alkylations and phosphorylation suggest a serme histldine active center system The protein is basic, u l t h strong affinity for the heparm of the mast cell granule High K + concentrations ( > I M), rather than Na +, are needed to displace fully the enzyme from heparin in solution A trypsin-like enzyme is also prominent in the mouse (but not the rat) cells The trypsin-like enzyme has a specificity and active center reactivity characteristic of pancreatic trypsin, and a molecular weight, estimated by gel filtration, of about 35 ooo

INTRODUCTION

As part of a study of the molecular evolution of chymotryptic enzymes, a chymotrypsIn-hke protease has been purified from the peritoneal mast cells of the rat 1 Another source is the rat thyroid, where a protease has been attributed to the mast cells 2 However, mast cells comprise only about 4% of the m a m m a h a n peritoneal population 3 and less than 1% of the thyroid cells * Mast cells may, on the other hand, Abbre~latlons BTEE, benzoyl-L-tyrosme ethyl ester, DFP, dusopiopslphosphorofluorldate, TAME, tosyl-L-arglnme m e t h y l ester, TLysCK, a-N-tosyl-L-lysme chloromethyl ketone, FLeuCK, N-tosyl-L-leucme chloromethyl ketone, TPCK, N-tosyl-L-ptlenyalanme chloromethv1 ketone * Present address D e p a r t m e n t of Biochemistry, University of Chxcago, Ill, U S A ** On lea~e of absence from t h e A c a d e m y of Medmme, Warsaw, Poland (present address), while engaged in this work *** E n q m r m s a n d reprint requests should be addressed to this a u t h o r

Bzoch,m Btophys Acta, 250 (1971) 395-407

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be obtained in high concentrations from propagated mast cell tumors3, 4 The P815Y mastocytoma4 possesses most of the characteristic features of mast cells, including the production of hepann, hlstamme and 5-hydroxytryptamlne 5 We report herein on the preparation and characteristics of a chymotrypsm-hke protease obtained from these tumor cells, and on a trypsln-hke protease also present MATERIALS AND METHODS

Unless otherwise specified, materials and methods were as described previously i a-N-Tosyl-L-lysme chloromethyl ketone (TLysCK), N-tosyl-L-leuclne chloromethyl ketone (TLeuCK) and tryptophan substrates were obtained from Cyclo Chemical Corp TEAE-cellulose was Cellex-T (Blo-Rad Labs ) and was prepared and used as recommended by the manufacturer Chymotrypslnogen A (6 × crystalhzed) and human O / ) were from Mann, and bovine trypsmogen serum albumin (crystallized, iOO/o (crystallized) from Sigma Collagenase (from Clostrzd~cum h,stolyt,cum) was fromWorthlngton (Purified), rat-tall collagen was a pure preparation kindly donated by Dr J Bello (Roswell Park Mere Institute, Buffalo) and elastm-orcem was from Sigma

Propagat,on of the tumor The P815Y tumor was obtained from Inoculated mice kindly supphed by I)r Glenn Fischer (Roger Wllhams General Hospital, Providence, Rhode Island) and maintained in our laboratory in the peritoneal cavities of female mice of the DBA/2 Ha strain The tumor was transferred by mtrapentoneal mlectlon of a suspension of the mastocytoma in sterile Hanks' medium 5-7 days later, the animals were killed and their peritoneal cavities washed either with sterile Hanks' solution (for moculatlons) or with modified Tyrode's solution 6 (for homogenization and subsequent enzyme isolation)

Enzyme extraction and labehng Homogenization of the washed mastocytoma cells was carried out in o 25 M sucrose-o oI M E D T A - o o1% Tween 80 solution, adjusted to pH 6 o with HC1 (5 ml of buffer per ml of packed cells), using a glass homogenizer with a driven teflon pestle For more than 5 ml of cells, a \ lrtls Model 45 homogenizer with a I25-ml chamber was used instead, at a setting of 55 Following a Io-mm homogenization, the cell debris was collected at 900 × g (IO ram), and re-homogenized, twice The combined supernates ~ ere centrifuged at 24 ooo × g for 30 ram, to yield the granule fraction, which could be stored frozen at --IO ° The enzyme was extracted from the granules In I 4 M KCI-o 05 M succmateo oI M EDTA solution (pH 6 o), either by stlrnng at room temperature for 30 ram, or by using the glass/teflon homogenizer for 3 ram, followed by centnfugatlon of the homogenate at 24 ooo × g for 30 mm After three homogemzatlons the supernates were combined For labeling, E~H]dnsopropylphosphorofluorldate ([3HIDFP) was added to a final concentration of lO -4 M in the sucrose-EDTA medium at pH 6 o at 25 °, either in the granule stage or after extraction of the enzyme The activity on B T E E was measured at intervals (and was seen to become extinguished) After 3 h, the D F P molarlty was raised to lO _3 M by the addition of unlabeled D F P and incubation was Bzochtm Btophys Acta,

25 ° ( 1 9 7 1 ) 3 9 5 - 4 0 7

TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

397

continued for another 3 h at 25 °, for saturation and exchange The granules were then washed with IO 2 M unlabeled D F P in the same medium, and collected by centnfugatlon

Gel filtration In the purification steps, the extracts were filtered in 5-ml batches on a column (usually 88 c m x 2 o cm) of Sephadex G-Ioo, at 4 ° Elution was by o 05 M succmateo oi M E D T A - I 4 M KC1 solution (pH 6 o) at the rate of 8 ml/cm 2 per h Desalting of each enzyme peak was effected on a column of Blo-Gel P-2 or Sephadex G-25, firstly in 50 mM HC1 (ref I) and subsequently in water

Other methods Electrophoresls was conducted at room temperature in a Beckman Mlcrozone apparatus with cellulose acetate membranes Staining of the strips for protein was with Buffalo Black N B R (Allied Chemical Corporation) in IO~o trlchloroacetlc acid solution Collagenase activity was determined ~ b} quantitative nlnhydrin reaction of the products released from rat-taft collagen, with bacterial collagenase used as a reference Elastase was assayed by the elastm-orcem colorimetric method 8 RESULTS

Isolat, on of proteases Upon removal of the mastocytoina cells from the peritoneal cavity of the mouse, over 9 o ~ of the suspended cells were shown, upon tolmdme blue staming 8, to be granulated mast cells Normal (non-mahgnant) mast cells, readily recognized by their cytology3, comprised much less than I /ooJ of the population Upon homogenization of these ceils in I 2 M NaC1 (or KCI) medium to prepare the granule fraction, they released into solution all of their protease and benzoyl-L-tyrosme ethyl ester (BTEE) esterase activity On the other hand, homogenization in water or o 05 or o 2 M NaC1o oI M EDTA solution released about two-thirds of the protease activity, in contrast to the case of the rat normal mast cells 1 where the activity then remained in the granules The extracted protease was associated with soluble heparin Cell homogenization was, therefore, routmely performed in o 25 M sucrose (which released only 4°~o of the BTEE-esterase from the granules), and tile isolated granule fraction subsequently extracted in I 4 M KC1 medium Alternatively, the enzyme was isolated similarly after inactivation by [3H]D F P This reaction could be conducted either on the granules or on the extracted enzyme in solution, both methods giving the same final result The extracts were fractlonated by gel filtration on Sephadex G-Ioo While it was necessary, for separation to use an ionic strength > I, the profile obtained was dependent upon the precise ionic medium used (Figs I and 2) Using the IaH]DFP-mactlvated material, extraction, of the granules in I I M NaC1 and filtration in that medium (Fig I) resulted in a large peak (I) of labeled protein soon after the ~ oid volume, with a satellite (IA), and a smaller, more retarded peak (II) However, when the procedure was performed, instead, in I 4 M NaC1 medium, Peak II was increased at the expense of Peak I, and a further such redistribution occurred when the initial extract was gel-filtered in I 4 M KC1 medium, Peak I I then being about 8 times larger (in 3H content) than Peak B~och~m Bzophys Acta, 25o (1971) 395-4o7

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Fig I Gel filtratlon on a 5ephadex G-leo column (i c m > 75 cm) of [~H~DlTP-mhlbzted mastoc ~ t o m a proteases, in I I ~I iWaC] (buffered ~Ith o 05 s o d m m succinate-o el M F D T A (pH 6 o)) Fractions were sampled for prote]n (A2s 0 nm, A), and for 3 H content (counts/ram per m], C~) ~lhe peaks centered near 6o and 7o m] contained no protein the former is attributcd to [3H]DI~I' h% dro]~sls product~ - AZSO

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Bzoch~,n Btophys Acta, 250 (1971) 395-4o7

TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

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l?lg 3 Gel filtration of material from the labeled Peak II obtained from separations as m Fig 1 Practlons ~ e r e sampled for protein (A2s0 nm, A), counts/mm per ml (@) and salt concentratmn measurement by conductivity (dotted hne) (A) On a Sephadex G-25 column, in i o mM HCI (B) On the same column as in A, but in 5 ° mM HC1 When a similar BmGel P-2 column was used, in ,~ater, three labeled protein peaks were obtained at the positions of the arro~s (in A), contaming radmactlvzty m the ratio I 4 6 I I n e a c h c a s e r e f i l t r a t l o n o f t h e m a t e r i a l i n t h e c e n t r a l f r a c t i o n o f P e a k I r e s u l t e d in two peaks in the positions of I and II Gel f i l t r a t i o n o f t h e a c t i v e e x t r a c t s u n d e r i d e n t i c a l c o n d i t i o n s r e s u l t e d i n profiles e q u i v a l e n t t o t h o s e o b t a i n e d w i t h t h e l a b e l e d p r o t e i n , in e a c h c a s e ( F i g 2) I n a l l i n s t a n c e s P e a k I c o n t a i n e d h e p a r l n (Fig 2) w h i l e P e a k I I d i d n o t H e n c e , in summary, Peak I contains a heparln-bound form of the DFP-reactlve enz3me of Peak II This residual bound enzyme can be released from the heparln by a second t r e a t m e n t i n h i g h s a l t c o n c e n t r a t i o n ( e s p e c i a l l y w h e n K + is u s e d ) A t t e m p t s t o d e s a l t P e a k I I (from t h e l a b e l e d e n z y m e p r e p a r a t i o n ) b y gel f i l t r a t i o n o n a B l o - G e l P - 2 c o l u m n i n d i s t i l l e d w a t e r o r o n a S e p h a d e x G-25 c o l u m n i n I o m M HC1 ( F i g 3A) r e s u l t e d in a m i n o r P e a k I w h i c h e m e r g e d a t t h e v o i d v o l u m e and a much larger all-containing peak which overlapped the salt peak On the P-2 c o l u m n , t h e r e w a s also a s m a l l e r l a b e l e d p e a k r e p r e s e n t i n g a n I n t e r m e d i a t e r e t a r d a Btoch~m B~ophys Acta, 250 (1971) 395-407

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F i g 4 C h r o m a t o g r a p h v on a T E A E - c e l l u l o s e c o l u m n (o 8 c m "< i o cm) a t 4 ~ The enz-vme cxtrac* was first f r a c t m n a t e d on G - i o o as m F i g 2 t h e m a j o r l a b e l e d p r o t e i n p e a k in t h e p o s i t i o n oI P e a k I I t h e r e was used, a±ter d i a l y s i s Q)--C), aH ( c o u n t s / m m p e r mI), d o t t e d hne , s a l t g r a d i e n t , b y c o n d u c t t v ~ t y r e a d m g s , &, protexn (by 4z~0 nm) 15o ml ~ e r e first c ol l e c t e d d u r i n g a d s o r p t i o n a n d w a s h i n g ( ~ l t h one c o l u m n ~ o l u m e of t h e e q u a h b r a t l n g 20 mM T n s - H C 1 (pH 7 o) buffer) oI t h e s a m p l e on t h e c o l u m n t h e c h r o m a t o g r a p h y s h o ~ n (with a gra da e nt of o - I o M KCI In t h e s a m e buffer) u a s t h e n c o m m e n c e d

tlon Equivalent anomalous retardation was also found when the non-inhibited enzyme was refiltered similarly in distilled water on a P-6 gel column Refiltratlon of the radioactive Peak II on G-25 in 5 ° mM HC1, on the other hand, resulted in the radloactlwty emerging from the column at the breakthrough position (Fig 3B), aa expected for a protein on this gel

Ion-e~change cellulose chromatography An improvement over the chromatographic results obtained 1 with the rat enzyme was found here when a purification step by gel filtration preceded chromatography on TEAE-cellulose (Fig 4) About 80°0 of the protein present emerged m the break-through fraction with less than I % of the total radioactivity A gradient of KC1 ~ as then apphed (Fig 4), when an unlabeled protein peak was followed by tx~ o labeled, overlapping peaks When the material an the combined labeled peaks x~as again gel-filtered on Sephadex G-Ioo in I 4 M KC1 medium, all of its radioactivity was pre~ent in two peaks corresponding to Peaks IA and U of Fig 2, in the respecUve proportions of the two labeled peaks of Fig 4 The results indicate that httle, if an5, of the label used an following the enzyme m the above-described experiments 1~ attached to proteins other than the chymotrypsm-hke and trypsm-hke enzymes However, the yield of these enzymes in the TEAE-cellulose chromatography ~xa~ only about 30% (by 3H recovery) A reasonable degree of purification for present purposes was, in practice, obtained by a series of gel filtrations (starting with Peak II f, om the crude extract) alternately in i 4 M KC1 solution and in low salt medium, as described for the rat mast cell chymotrypsln 1 B~ochzm B~ophys Acta 25 ° (I97 I) ~95-4o7

TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

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Ve/Vo Fig 5 Molecular welght determination on Sephadex gel columns The calibration plot was constructed m each case using the data obtained on separate runs of each marker protein (O) s h o w n (CTg = b o v i n e c h y m o t r y p s m o g e n A, Tg = b o v i n e t r y p s l n o g e n , P T I = p a n c r e a t i c t r y p sin I n h i b i t o r ~°) Ve = e l u t l o n v o l u m e of t h e c o m p o n e n t s t u d i e d , V 0 = v o i d v o l u m e of t h e c o l u m n ( t a k e n as t h e e l u t l o n v o l u m e of Blue D e x t r a n ) Sohd line e x p e r l m e n t s u s m g G - i o o gel (7 ° c m I cm) in t 4 M KC1 m e d i u m (buffered b y o 05 M s u c c m a t e - o oI M E D T A , p H 6 o) B r o k e n line e x p e r i m e n t s u s m g G-75 gel (5 ° cm × I o cm) in o i M KC1 m e d i u m (buffered b y o o2 M T r l s H C I - o o i M E D T A , p H 7 5) Ve for t h e m a s t o c y t o m a e n z y m e ([7) ~ a s r e a d m e a c h case, b y SH c o u n t i n g u s m g t h e [ S H j D F P - m h l b l t e d e n z y m e , as well as b y A~s0 nm m e a s u r e m e n t in p a r a l l e l e x p e r i m e n t s u s i n g t h e a c t i v e e n z y m e , ~ h e n Ve v a l u e s i d e n t i c a l t o t h o s e sho,~n for t h e l a b e l e d e n z y m e were o b t a i n e d

Molecular we,ght The purified p r e p a r a t i o n o b t a m e d was used for Mw d e t e r m i n a t i o n 1,9 on a gel column (Fig 5) I n I 4 M KC1 m e d i u m , a value of 26 ooo was obtained, b o t h for the active a n d the [ a H ] D F P - l n a c t l v a t e d e n z y m e On a gel column in o I M KC1 m e d i u m , on the o t h e r hand, a m u c h smaller a p p a r e n t Mw value was o b t a i n e d (Fig 5), as f o u n d also z for the r a t m a s t cell c h y m o t r y p s l n in lower salt concentrations, due to b i n d i n g to t h e gel column I n t e r m e d i a t e a p p a r e n t Mw values ~ ere found, as before 1, at Interm e d i a t e salt c o n c e n t r a t i o n s (using a column of Agarose A - o 5 m gel in KC1 solutions from o 2 M c o n c e n t r a t i o n upwards), the c o n s t a n t value of Mw = 26 ooo ( ± iooo) being reached at a n d a b o v e I I M c o n c e n t r a t i o n of KC1 In the m e d i u m

Trvpsan-hke protease On a G - i o o column (Fig 2), a protease p e a k (IA) was e l u t e d on the t r a l h n g side of P e a k I P e a k I A was c h a r a c t e r i z e d b y its considerable a c t i v i t y on t h e t r y p s i n s u b s t r a t e , tosyl-L-arglnme m e t h y l ester (TAME), a n d its t o t a l I n a c t i v i t y on the c h y m o t r y p s l n s u b s t r a t e , B T E E I t possessed protease a c t i v i t y on casein (Table I) at a b o u t t h e same r a t i o to its esterase a c t i v i t y as is found in t h e case of bovine p a n c r e a t i c t r y p s i n I n t h e gel filtration of t h e labeled enzyme, a r a d i o a c t i v e p e a k (enzymlcally inactive) was found (Fig I) in the c o r r e s p o n d i n g position The l a t t e r a l w a y s r e m a i n e d B*ochzm B~ophys Acta, 25o (t97 I) 395-4o7

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in the position of Peak IA, even after repeated filtration of that fraction in I 4 M KC1 medium, when it was completely separated from the heparin The position of this peak m the G-Ioo column effluent (in I 4 M KC1) corresponded, by comparison with the cahbratmn of Fig 5, to Mw about 35 ooo

Spec~fic#y The hepann-free enzyme in Peak II (Fig 2), partly purified by further ge, filtration (as noted above), was examined for activity on several substrates (Table II) It acts upon tyroslne and tryptophan esters, as well as on casein Activity on N-acet31tryptophan amlde substrates was found to be, at the most, not greater than the relatively low activity of a-chymotrypsln thereon The apparent Michaehs constant, Kin, for BTEE at pH 7 9, 25 °, ~as determined as previously I for the rat enzyme and was found to be 6 IO-4 M, a value close to that determined for bovine pancreatm a-chymotrypsin under the same conditions TABLE

11

SPECIFICITY

OF THE CHYMOTRYPSIN-LIKE

ENZYME

M e a s u r e m e n t s w e r e m a d e a t 25 o z c o 2 ^, in o 08 M T r l s - o i o M CaC12 ( p H 7 8), e x c e p t t h a t c a s e i n ~ a s in o 05 M p h o s p h a t e ( p H 7 6) S p e c t r o p h o t o m e t r l c r e c o r d i n g w a s m a d e o f t h e a b s o r b a n c e c h a n g e a t 256 n m ( B T E E ) , 247 n m ( T A M E ) , o r 3oo n m ( t r y p t o p h a n s u b s t r a t e ) T h e a c t i v i t y o n t h e s u b s t r a t e r e l a t i v e t o t h a t o n B T E E is e x p r e s s e d ( p e r r a i n o f r e a c t i o n o n e a c h s u b s t r a t e ) i n d e p e n d e n t l y f o r t h e m a s t o c y t o m a p r o t e a s e (MP) a n d f o r box l n e a - c h y m o t r v p s a n (CT) Reaction period, period over which a~erage rate measurement ~as made Relative enzyme c o n c e n t r a t i o n , c o n c e n t r a t i o n o f m a s t o c y t o m a p r o t e a s e r e l a t i v e t o t h e c o n c e n t r a t i o n u s e d in t h e B T E E e x p e r i m e n t T h e r e l a t l ~ e actl~ i t y ~ a l u e s a r e c o r r e c t e d l o r t h e c o n c e n t r a t i o n d i f f e r e n c e s

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B w c h z m B w p h y s Acta, 25o (1971 ) 3 9 5 - 4 o 7

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TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

In view of the possible role of the components of the mast cell granules m degradatlve attack on connective tissues in inflammatory proteases, the enzymes present in the total extract of the granules were examined for collagenase or elastase actlwty Using the elastm-orceln s or the rat-taft collagen solubIlizatmn 7 assay systems, no actawty of either type was detectable in the conditions used, these, involving incubations of 24 h, were such that even a feeble activity would be detectable Inh~b~twns Indole is a strong competitive inhibitor of pancreatic a-chymotrypsln~°, n Since the mdole derivative, 5-hydroxytryptamme, is a physiologically active component of the granules of the P815Y mastocytoma s, the effect of tins compound and of mdole on the chymotrypsln-hke protease was examined 5-Hydroxytryptamine was found to be strongly inhibitory, but exact measurements were not made, since, in the assav system used (employing spectrophotometry In tyrosme or tryptophan ester ~olutions) the absorbance at the useful wavelengths even of concentrations as low as i lO -4 M 5-hydroxytryptamine was so high as to decrease greatly the accuracy of the assay The absorbance at 3oo nm of mdole, however, is low enough to permit its "1A B L E I I I INHIBITIONS

BY INDOLE

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BY CHYMOTRYPSIN

INHIBITOR

I FROM POTATOES

I'he actl~ l t y is e x p r e s s e d as a p e r c e n t a g e of t h a t in a c o n t r o l s a m p l e in t h e a b s e n c e of i n h i b i t o r M P -- m a s t o c y t o m a c h y m o t r y p s m - h k e p r o t e a s e (purified b y T 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 n d gel filtrations) CT = b o v l n e a - c h y m o t r y p s m , used in a s i m i l a r e x p e r i m e n t I n h i b i t i o n b y lndole a s s a y s on a c e t ~ / l - L - t r y p t o p h a n m e t h y l ester (5 1°-4 M, p H 7 8, ~ l t h o 05 M CaC12 p r e s e n t , 25 o i o 2% r e c o r d i n g a t 3oo n m o v e r a 5 - m m p e r i o d a n d p l o t t i n g t h e i n i t i a l ~ e l o c t t y P o t a t o i n h i b i t o r a s s a y s on B T E E in t h e s a m e c o n d i t i o n s E n z y m e mar e i t h e r i n t r o d u c e d ( w i t h o u t prei n c u b a t i o n ) to s t a r t t h e assay, or was p r e m c u b a t e d w i t h i n h i b i t o r for t h e t i m e shown, followed bv substrate addition

Inhzbztor

Indole

Fznal conch (hi)

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o 16 35

inhibition to be measured Such measurements (Table III) showed that lndole is a slightly stronger inhibitor of this chymotrypsm-hke enzyme than of pancreatic chymotrypsln Under similar conditions (at pH 7 6 instead of our 7 8) the pancreatic enzyme has a value of K, for lndolen, 12 of 6 lO -4 M, a slightly smaller value is indicated for the mastocytoma enzyme B,ochzm Bzophys Acta, 250 (1971) 395-407

404

w H VENSEL et al

A highly effective mhlbltor of pancreatic chymotrypsln has been purified from potatoes by BALLS AND RYAN12 It does not inhibit the activity of trypsin on TAME (ref 13) A sample was kindly provided by Dr C A R y a n , and proved to be strongly inhibitory of the mastocvtoma chymotrypsm-hke enzyme (Table nI) The reaction required up to IO mln foi completion, at a concentration of about IO-5 M of the mhlbltor The irreversible mhibitors, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), TLeu('K, and TLysCK, rapidly alkylate, respectively, the active-center histldine residue from mammalian pancreatic chymotrypbin A, chymotrypsln C and trypsinI4 16 As expected, the mastocytoma chymotrypsm ~ a s not Inhibited by TLysCK, but waa Inhibited, at about equal rates, by low concentrations of TPCK or TLeuCK (Fig 6) Although the rates observed are slower than those for bovine pancreatic chymotrypsm A, they are within the range of reactivity observed by MOCKEL A.ND BARNARD17 tor these phenylalanlne- and leucme-based alkylating lnhlbitors acting on other vertebrate chymotrypsins The trypsln-hke enzyme of Peak IA (see above) was fully Inhibited by TL?sCK in an experiment parallel to that of Fig 6 It showed, as expected, no inhibition by T P C K in those condmons I t is evident from the labeling experiments (Fig I) that the ch?motrypsin-like protease is Inhibited by D F P This was further examined the enzyme, when treated with 5 IO-5 M D F P (pH 7 8, 25 °) rapidly lost activity, after 45 rain only 3°0 of the activity on B T E E remained This fast reaction rate is characteristic of an active center serine, although the rate found was approximately 3 times slower than that with bovine pancreatic a-chymotrypsln In the same conditions

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Fig 6 Inactivation of the purified chymotrypsln-hke enzvlne A c t ] ~ a t ~ x~as m e a s u r e d (as a p e r c e n t a g e o f t h e m l t l a l a c t l x l t v ) ~ ] t h ; lO .4 5I T P C K ( C ) - - C ) ) , o r lO .3 M T L e u C K ( & - - - &), i n h i b i t o r ( 0 ) , a l l i n o o 8 5I T r l s - o o2 M CaCl~ (pFI 7 z) i n 3 % ( v / v )

thoch~m B*ophys Acta, z 5 o ( I 9 7 [ ) 3 9 5 - 4 o 7

by specific alkylatlng agents on BTEE, durmg incubation or Io -a M TLysCK (I), or no m e t h a n o l , 25 °

TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

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Fig 7 E l e c t r o p h o r e s l s on cellulose a c e t a t e of t h e [ 3 H ] D F P - m a c t l ~ a t e d c h v m o t r y p s m - h k e enzyme, purified b y t h r e e g e l - f i l t r a t i o n s t e p s R u n in o 04 M T n s - H C l - o o o i M E D T A (pH 7 2), 4 °, 20 ram, 300 V, 3 m A The a r r o w shm~s t h e p o i n t of a p p h c a t l o n A f t e r t h e s t a m m g for p r o t e i n on one h a l f of t h e m e m b r a n e , t h e o t h e r h a l f w a s c u t i n t o s t r i p s as mdacated, for radloactl~ l t v m e a s u r e m e n t (3H c o u n t s / r a m , s h o w n on r e l a t i v e scale)

Stabd~ty I t can be seen from Fag 6 t h a t t h e enzyme, when m c u b a t e d at p H 7 8 at 25 °, is stable for m a n y hours This s t a b f l l t v is g r e a t e r t h a n t h a t shown b y the r a t m a s t cell p r o t e a s e I I t was observed, however, t h a t the t r y p s m - h k e e n z y m e (Peak IA, F i g IC) was u n s t a b l e in the active form, even at 4 ° (pH 6) a b o u t half of the a c t i v i t y on T A M E was lost In one d a y , p r e s u m a b l y due to self-digestion This m i g h t be due to t h e absence of calcium ions m the m e d m m used in t h e purification, since calclum is c o m m o n l y e m p l o y e d to s t a b l h z e p a n c r e a t i c t r y p s l n s

Electrophores,s I n electrophoresls on cellulose a c e t a t e (Fig 7), p a r t of the labeled c h y m o t r y p s l n hke p r o t e m r e m a i n e d a t t h e orlgm, while a l m o s t all of the r e m a i n d e r t r a v e l l e d in a b a n d t h a t was cationic a t p H 7 2 a n d carried essentially all of the rest of the r a d i o a c t i v i t y Since t h e m a t e r i a l r e m a i n i n g a t the orlgm d i d n o t m o v e u n d e r acidic or a l k a h n e p H conditions, it is a t t r i b u t e d to a p a r t i a l d e n a t u r a t i o n due to the d r y i n g of the s a m p l e on the m e m b r a n e (It b e m g o b s e r v e d t h a t f r e e z e - d r y m g i n a c t i v a t e s thl~ enzyme) I f this as so, the u n d e n a t u r e d e n z y m e comprises a single species Bzochzm B*ophvs Acta, 250 (1971) 395-407

406

W H VENSEL et al

DISCUSSION The mouse m a s t o c y t o m a cells are seen to c o n t a m an e n z y m e ~ h l c h has the same characteristics as the e n z y m e s t u d i e d 1 from n o r m a l m a s t cells a n d as p a n c r e a t i c c h y m o t r y p s m , in t e r m s of s u b s t r a t e specificity, apeclfic active center inhibitions, a n d Mw Like the r a t enzyme, it is b o u n d m the granules of the m a s t cells, a n d is r e a & l y linked lOnlcally to h e p a r m in solution and, p r e s u m a b l y , m the granules P o t a s s i u m ions are m u c h more effective t h a n s o d i u m m displacing it from h e p a r m , for b o t h the enzymes Hence, t h e t u m o r m a s t cells of the mouse synthesize an e q u l v a l e n t p r o d u c t The a m o u n t per cell, howe~ er, is r e d u c e d based u p o n m a s t cell counts, the c o n t e n t of e n z y m e (measured b o t h b? a c t l v l t v a n d b y the n u m b e r of molecules of I 3 H I D F P r e a c t e d in t h e c h y m o t r y p s m fraction in each case) was, per mouse m a s t o c y t o m a cell, o n l y a b o u t 5°o of t h a t per r a t m a s t cell The results are c o n s b t e n t with the known c5 t o c h e m l s t r v of this t u m o r m a s t cell It, too, contains m a n y c y t o p l a s m i c granules, b u t these h a v e m u c h less c a p a c i t y t h a n nl the n o r m a l m a s t cell to stain m e t a c h r o m a t lcallv with a p p r o p r i a t e dyes, a n d t h e y a p p e a r to contain c o n s i d e r a b l y fewer sulfated groups of h e p a r m 5 T h e y also contain h i s t a m i n e a n d 5 - h y d r o x y t r y p t a m m e , b u t to a m u c h lesser degree t h a n n o r m a l m a s t cells 5 P r o d u c t i o n of the specific m a s t cell p r o d u c t s is, therefore, being p a r t l y supprebsed in this tmnor, a n d tln~ applies to the c h y m o t r y p s m - h k e protease, to() The position of elutlon of this e n z y m e on the S e p h a d e x G - i o o column used in the p r e p a r a t i o n (at high salt concentration) coincided w i t h t h a t of the r a t enzyme, m a g r e e m e n t with the m e a s u r e m e n t s of M~ The a n o m a l o u s r e t a r d a t i o n of the mouse m a s t o c y t o m a c h y m o t r y p s m on b o t h d e x t r a n a n d p o l y a m l d e gel columns, such t h a t "> i M salt solution or o 0 5 M HC1 solution (Fig 3) i~ required for n o r m a l m o b i l i t y thereon, was found Slmllarl~ i with the r a t m a s t cell c h v m o t r v p s m I n the l a t t e r case, it was a t t r i b u t e d ~ to its baslclty, t o g e t h e r with some other factor such as a concentration of a r o m a t i c groups B a s l c l t v of the mouse m a s t o c v t o m a c h y m o t r y p s m has been d e m o n s t r a t e d here b v electrophoresls The high affinity of the e n z y m e for lndoles is an additional, interesting observation m a d e on the mouse e n z y m e (and not ~et t e s t e d in the rat) Since 5 - h y d r o x ~ t r y p t a m m e is an active agent stored in the m a s t cell a, the presence of b o t h it and this e n z y m e in t h e granule ~ o u l d pro,~lde a m u t u a l l y i n a c t i v a t i n g system W h e n the granules are released into a different mlheu, liberation of b o t h agents ~ ould occur, to p r o m o t e i n f l a m m a t o r y processes This would, indeed, be a reason for the specificity of ttle protease, if it has become a d a p t e d to provide an m d o l e - b m d m g site The m a s t cell t r y p s i n recognlsed here has no m e a s u r a b l e c o u n t e r p a r t in the n o r m a l m a s t cells from the r a t I,I8 H o ~ ever, hlstochemmal evidence, based on the use of b e n z o y l a r g l n l n e naphth5 lamlde or a n a p h t h o h c ester oi e-amino caproate, has led to reports t h a t a tr5 psln-hke enz} me is present in h u m a n , canine a n d bovine m a s t cells a n d in a cat m a s t o c y t o m a ~9-22, as well as a h l s t o c h e m l c a l l y d e t e c t e d chvmot r y p s m - h k e a c t i v i t y The s e p a r a t i o n o b t a i n e d here shows t h a t t ~ o d i s t i n c t proteins are, in fact, m ~ o l v e d N o r m a l m a s t cells from mice , s l t h o u t t u m o r s h a v e not been o b t a i n e d in sufficient q u a n t i t y for us to isolate their proteases, b u t c y t o c h e m i c a l e x p e r i m e n t s have shown 23 t h a t n o r m a l m a s t cells from the mouse p e r i t o n e u m (in the aame i n b r e d strain of mice) a p p e a r to be essentially devoid of the tr? ptlc a c t i v i t y , although t h e y have the c h v m o t r v p t l c a c t i v i t y in a b u n d a n c e I n v e s t i g a t i o n of the 13~o*htm 13zophvs Ac/a, 25° (I971) ~95-4o7

TWO PROTEASES FROM A MOUSE MAST CELL TUMOR

407

differential synthesis of these two enzymes in the malignant growth of these ceils is, therefore, indicated ACKNOWLEDGEMENTS

Th~s work was supported by Grant GM-I6726 from the National Institutes of Health, U S Pubhc Health Serwce, by Grant DRG-867 from the Damon Runyon Memorial Fund, and (for supporting serwces) N A S A Grant NGR33-oI5-OO2 We thank Dr Glenn Fischer for his great assistance in supplying mice bearing the P815Y tumor for our laboratory line, Dr C A Ryan (Washington State University, Pullman) for the gift of a pure specimen of potato chymotrypsln Inhibitor I, and Dr M Laskow~kl, Sr (Roswell Park Memorial Institute, Buffalo) for the gift of pure pancreatic trypsin inhibitor REFERENCES I J KAWIAK, W H VENSEL, J KO~,IENDER AND E A BARNARD, Bzochz*~z Bzophys Acta, 225 (1971) 172 2 I PASTAN AND S ALMQUIST, J B~ol Chem , 241 (1966) 509 ° 3 H SELYE, The Ma~t Cells, Butter~orths, Washington, 1965 4 T B DUNN aND M POTTER, /r Natl Cancer Ins~, 18 (I957) 587 5 D GLICK, D YON REDLICH AND B DIMENT, Bzoche~n Pharmacol, 16 (1967) 553 6 S NORN, Acta Pharmacol, 25 (1967) 281 7 I ~[ANDL, J D MACLENNA~, ~.ND E L HOWES, J Clzn Invest, 32 (1953) 1323 8 L A SACHAR, K K WINTER, N SICHER ~-ND S FR~.NKEL, Proc Soc E v p Bzol M e d , 9 ° (1955) 323 9 P ANDREWS, B~ochem ] , 91 (1965) 222 IO H HUANq AhD (_ NIEMAI~N, J A m Chem Soc , 75 (1953) 1395 I I H I ABRAm1 AND C NIEM~_NN, B~ochem*stry, 4 (1965) 99 12 A K BALLS AND C A RYAN, J Bzol Chem , 238 (1963) 2976 13 C .\ RY~N, Bzochem~strv, 5 (1966) I592 14 E B ()NG, E SHAV~ ~.ND G SCHOELLMAN, J Bzol Chem 240 ( 1 9 6 5 ) 6 9 4 15 "1 TOBITA AND J E FOLK, B~och~m Bzophys .4cta, 147 (1967) 15 16 E SHA'~V, ~ ~ARES-GUIA ~I',D W COHEN, Bzochemzstry, 4 (1965) 2219 1 7 W MOCKEL A.ND E A BARNA.RD, Bloch*m Bzophys .4cta, 141 (1969) 37 ° 18 D LAGUNOFF aND E 1~ BEI~DITT, A n n N ~: Acad Scl , lO 3 (1963) 185 19 G G GLENNER AND L A COHEN, Nature, 185 (196o) 846 20 G G GLENNER, V K H o P s u AND L A COHEN, J Hzstochem Cvtochem, i o ( 1 9 6 2 ) l O 9 21 D LA.GUNOFF, E P BENDITT AND R WATT%, J H,stochem C3,tochem, i o ( 1 9 6 2 ) 6 7 2 22 N ENDE, Y KATA-X~.M~. AND J v AUDITORE, Nature, 2Ol (1964) 1197 23 E A BARNARD, Int Rev Cytol, 29 (197 o) 213

B,ochtm Bzophys Acta, 25 ° (1971) 395-407