167

Biochimica et Biophysica Acta, 5 8 3 ( 1 9 7 9 ) 1 6 7 - - 1 7 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 28834

PROTEOLYTIC ENZYMES IN NORMAL AND TRANSFORMED CELLS

V I J A K M A H D A V I * a n d R I C H A R D O. H Y N E S **

Center for Cancer Research, Department of Biology, E17-227, Massachusetts Institute of Technology, Cambridge, MA 02139 (U.S.A.) (Received July 17th, 1978)

Key words: Proteases; Transformation

Summary Virally transformed cells show an increased production of proteolytic enzymes. These might be involved in transformation-dependent alterations of cell surface glycoproteins. The possibility arises that some of these proteases might be membrane-bound. To investigate this possibility, we have undertaken a comparative study of the reactivity of intact normal and transformed cells with the tritium labelled protease inhibitor diisopropylfluorophosphate, in parallel with fibrinolytic assays. Using these two approaches in concert, it was possible to identify and localize in the transformed cells several proteases which were present in the particulate cell fraction and were probably membrane bound. In particular, a diisopropylfluorophosphate-reactive polypeptide of 62 000 was increased 5--8-fold on tranformation. It comigrated with a fibrinolytic activity. Other particle-bound activities were also detected. While diisopropylfluorophosphate-labelling can be useful for detecting proteases inside cells, it does not appear to be specific for surface proteases.

Introduction Virus-transformed fibroblasts exhibit a number of characteristics which are different from those of their normal counterparts. These include several properties which could reflect altered cell surfaces; abnormal growth behavior, altered morphology and adhesion, increased rates of nutrient transport and altered interactions with lectins. Several lines of evidence indicate alterations in cell

* P r e s e n t address: T h e R o c k e f e l l e r University, 1230 York Avenue, New York, NY 10021, U.S.A. ** To whom correspondence is t o b e s e n t .

168 surface glycoproteins associated with transformation (reviewed in Ref. 1). Some of these transformation-related properties can be induced in normal cells by addition of exogenous proteases [2--6]. Transformed cells also show increased production and release of proteolytic enzymes [4,7--18], although some normal cells also produce proteases [19--28]. It has been suggested that some of the alterations in surface proteins may be a reflection of increased proteolytic activity in t u m o r cells [4,5]. Furthermore, it has been shown that some of the altered properties of transformed cells correlate with proteolytic activities produced by these cells [15,16,29,30]. In particular, an enzyme, called plasminogen activator, which activates the serum zymogen, plasminogen, to plasmin [9--11,31,32] has been implicated in some altered properties of transformed cells [16,29,30,33] and in tumorigenicity in vivo [12,13,16]. Recent studies have demonstrated the localization of plasminogen activator activity in the plasma membrane [34]. Proteolytic enzymes secreted by cells have been studied by assaying their activities [7--18] and have also been identified [9,32,35] using diisopropylfluorophosphate (DFP), a reagent which reacts with the active site of serine esterases, including proteases [36]. Since DFP can be used to introduce radioactivity into serine proteases, it has potential for localizing protease activities in intact cells and the possibility arises of using it as a surface label, since it is highly polar and might be non-permeating. In this paper, we have investigated the use of [3H]DFP to identify serine proteases in cells. We have also assayed proteolytic activities in parallel with the DFP labelling. Materials and Methods

Culture and labelling o f cells NIL8 hamster cells and their transformed derivatives NIL8-HSV [37,38] were grown to confluence in Dulbecco's modified Eagle's medium + 5% fetal calf serum. For labelling they were harvested by treatment in 2 mM EGTA/ phosphate buffered saline ('buffer'}, Ca 2÷, Mg2÷-free at 37°C until detached from the substrate. They were washed five times and resuspended at approx. 4 • 107 cells/ml in buffer. Labelling was with 40 mCi/1 of tritiated diiosopropylfluorophosphate ([3H]DFP, 3.9 Ci/mmol; 21 Ci/g), at 37°C, for 30 min unless otherwise specified. The labelling reaction was stopped by washing the samples five times in buffer, until the radioactivity in the washes became constant, or by adding 1/3 vol. of 45% glycerol/6% SDS/0.3 M dithiothreitol. Membrane fractionation Isopycnic gradient centrifugation was performed following the procedure described by Graham et al. [39]. The labelled cells were resuspended at 2 107 cells/ml in 0.25 M sucrose/0.2 mM Mg2÷/5 mM Tris-HC1 (pH 7.4) and homogenized by nitrogen cavitation (750 lb/inch 2 for 15 min at 4°C). Nuclei were pelleted by centrifugation at 1000 × g for 5 min. The post nuclear supernatant was made 1 mM EDTA, and 5-ml samples were loaded onto 30-ml discontinuous sucrose gradients. The sucrose solutions were in 1 mM EDTA/5 mM Tris-HC1 (pH 7.4). Density, expressed in w / w sucrose, and volumes are indi-

169 cated in the figure legends. Centrifugation was at 4°C, 24 000 rev./min for 16 h in a SW27 rotor (large buckets). For SDS polyacrylamide gel electrophoresis analysis, fractions were pooled, diluted to 10% w / w sucrose, pelleted at 4°C for 60 min in a SW27 rotor at 26 000 rev./min, and resuspended in electrophoresis sample buffer.

SDS polyacrylarnide gel electrophoresis Samples were reduced unless indicated and boiled for 2 min. The slab gels were made according to Laemmli [40]. 5% and 9% acrylamide were used in the stacking and the running portion of the gels, respectively. For detection of the 3H-radioactivity, the gels were dehydrated in dimethyl sulfoxide, (Me2SO), soaked in 2,5-diphenyloxazole in Me2SO (22.2%), dried and exposed on Kodak RP Royal " X - O m a t " film at --70°C [41]. Autoradiograms were scanned on a Zeineh densitometer. Alternatively, the lanes were sliced, dissolved in 0.5 ml H202 overnight at 45°C and counted in 4 ml of Triton/PPO scintillation mix. Molecular weight markers were run in parallel: fl-galactosidase, 130 000; bovine serum albumin, 68 000; ovalbumin, 45 000; chymotrypsin, 23 000.

Detection of fibrinolytic activity 12SI-labelled fibrin-coated multiwell dishes were prepared as previously described [31,32]. Each well contained approximately 140 000 cpm 12sI in fibrin. Cell samples were extracted in 0.5% NP40/0.1 M Tris-HC1, pH 6.8/15% glycerol. The 20 000 × g supernatants were electrophoresed on SDS polyacrylamide slab gels. The gels were washed for 1 h in 100 ml of 0.025 M Tris base/ 0.192 M glycine/0.1% NP40. The lanes were cut transversely in 1-mm slices. For comparison between fibrinolytic activity and [3H]DFP radioactivity, slices were cut longitudinally in two equal parts. Half of each slice was assayed on '2SI-labelled fibrin, in 0.5 ml 0.1 M Tris-HC1, pH 8.1, containing 2.5% acidtreated fetal calf serum, and incubated at 37°C. The other half was dissolved in H202 and assayed for 3H-radioactivity [32].

Materials [1,3-3H]Diisopropylfluorophosphate (3.9 Ci/mmol) was obtained from the Radiochemical Center, Amersham/Searle, IL, U.S.A. Tissue culture media and serum were from Flow Laboratories, Inc. Acrylamide, bisacrylamide and sodium dodecyl sulfate were from Bio-Rad Laboratories. Results

[3H]DFP labelling Intact NIL and NIL-HSV hamster cells were labelled with [3H]DFP. The lysed cells were analyzed on SDS polyacrylamide slab gels. The autoradiograms demonstrated major bands with apparent molecular weights of 80 000-35 000, 62 000 and 25 000--28 000, respectively (Fig. 1). The 62 000-dalton band was prominent in the NIL-HSV cells b u t nearly absent from the NIL cells. Its mobility remained unchanged whether analysed in reducing or non-reducing conditions (not shown). Similar results were obtained when the cells were

170 80000 62000

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£ Migration Fig. 1. [ 3 H ] D F P l a b e l l i n g p r o f i l e s o f N I L a n d N I L - H S V cells a n a l y z e d b y S D S 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 . N I L a n d N I L - H S V cells w e r e l a b e l l e d in s u s p e n s i o n w i t h 4 0 m C i f l o f [ 3 H ] D F P . Cell h o m o g e h a t e s w e r e p r e p a r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . E q u a l p r o t e i n s a m p l e s w e r e r e d u c e d , b o i l e d a n d a p p l i e d o n 9% S D S p o l y a c r y l a m i d e gel. T h e a u t o r a d i o g r a m s o f t h e gels w e r e s c a n n e d n o r m a l i z i n g f o r t h e 2 8 0 0 0 d a l t o n b a n d . U p p e r line: N I L - H S V ceils; l o w e r line: N I L cells. A r r o w s i n d i c a t e t o p a n d b o t t o m o f gels.

labelled either as monolayers, cell suspensions or cell homogenates. More than 95% of [3H]DFP proteins were recovered in the supernatant when labelled intact cells were extracted with 0.5% NP40. Conversely, cell proteins solubilized in 0.5% NP40 and labelled showed a [3H]DFP incorporation profile very similar to that of the whole cells (see Fig. 6). Subcellular location We next attempted to localize the DFP-labelled proteins in the cell using isopycnic sucrose density gradients. NIL and NIL-HSV cells were prepared for this analysis as described in Materials and Methods. Fig. 2 shows that most of the protein-bound radioactivity (trichloroacetic

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Pooled fractions Fig. 2. E q u i l i b r i u m s u c r o s e d e n s i t y g r a d i e n t a n a l y s i s o f [ 3 H ] D F P l a b e l l e d N I L a n d N I L - H S V cells. P o s t n u c l e a r s u p e r n a t a n t s o f l a b e l l e d cells p r e p a r e d as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s w e r e s e p a r a t e d o n a discontinuous gradient of 6 ml each of 60, 50, 40, 30, 30% w/w sucrose solutions. The effluent from the g r a d i e n t w a s m o n i t o r e d a t 2 8 0 m m a n d c o l l e c t e d in 2 m l f r a c t i o n s . 100-#1 a l i q u o t s o f e a c h f r a c t i o n w e r e a s s a y e d f o r t r i c h l o r o a c e t i c a c i d - p r e c i p i t a b l e 3 H . T h e f r a c t i o n s w e r e s u c c e s s i v e l y p o o l e d as i n d i c a t e d , f o r S D S p o l y a c r y l a m i d e e l e c t r o p h o r e s i s a n a l y s i s (Fig. 3). a, N I L - H S V cells; b , N I L cells.

171

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acid-precipitable) is concentrated in the slowly sedimenting material (10--25% sucrose) in both NIL and NIL-HSV cells. The NIL-HSV cells showed a peak of label at higher density (35% sucrose) which was less obvious in the NIL cells. Fractions were pooled as indicated in Fig. 2, pelleted and analysed by SDSpolyacrylamide gel electrophoresis (Fig. 3). The 62 000 protein was concentrated in fractions 4, 5 and 6 (sucrose density of 27--38%, p = 1.118--1.173). Very little was present in any fraction from the NIL cells (Fig. 3b). The higher molecular weight proteins (80 0 0 0 - - 8 5 000) were not detectable in the pelleted material, which indicate, that they are soluble proteins. Very little of the 5oo~

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Fig. 4. E q u i l i b r i u m d e n s i t y analysis o f [ 3 H ] D F P - l a b e l l e d N I L - H S V cells. S a m p l e s w e r e p r e p a r e d as d e s c r i b e d in Fig. 2. T h e g r a d i e n t s c o n t a i n e d 5 m l of 60%, 7 m l o f 35% a n d 2 0 m l of 19% w / w sucrose, a, T r i c h l o r o a c e t i c a c i d - p r e e i p i t a b l e r a d i o a c t i v i t y d i s t r i b u t i o n across g r a d i e n t o f N I L - H S V Post n u c l e a r supern a t a n t . 1 0 0 #1 a l i q u o t s f r o m 2-ml f r a c t i o n s , b - - d . D i s t r i b u t i o n o f t h e light p o r t i o n o f t h e g r a d i e n t , ( p o o l e d f r a c t i o n s 1 6 - - 2 0 f r o m a) r e r u n a f t e r r e h o m o g e n i z a t i o n , b, With N I L - H S V cells; c, w i t h N I L cells; d, alone; e, d i s t r i b u t i o n of t h e d e n s e p o r t i o n o f the g r a d i e n t ( p o o l e d f r a c t i o n s 6 - - 7 f r o m a) r e r u n a f t e r

rehomogenization.

172 80000

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Migration Fig. 5. L a b e l l i n g p a t t e r n o f t h e s o l u b l e a n d p a r t i c u l a t e [ 3 H ] D F P m a t e r i a l . 1 0 0 - # l a l i q u o t s t a k e n f r o m t h e peak fractions containing the soluble and particulate material (fractions 19 and 6 of the sucrose gradient in Fig. 4 a ) w e r e e l e c t r o p h o r e s e d o n S D S p o l y a c r y l a m i d e gel. R a d i o a c t i v i t y w a s d e t e c t e d b y f l u o r o g r a p h y . F o r s c a n n i n g , t h e s a m p l e s w e r e n o r m a l i z e d f o r t h e 6 2 0 0 0 d a l t o n p r o t e i n . U p p e r line: f r a c t i o n 1 9 ; l o w e r line: f r a c t i o n 6.

labelled material from the light portion of the gradient was recovered on pelleting. Since most of the radioactive material which sedimented lighter than the particulate fraction was soluble, we analysed this material directly from the gradient w i t h o u t pelleting. The post-nuclear supernatant of [3H]DFP labelled NIL-HSV cells was applied on a shallow sucrose gradient which allowed better separation between fast and slow sedimenting material (Fig. 4a). The distribution of the radioactivity in the peak fractions (fraction 19 and fraction 6) is shown in Fig. 5. Three main bands of labelled protein (80 000-85 000, 62 000 and 28 000) were present in the soluble fraction [39] whereas the largest labelled protein (80 000--85 000) was almost absent in the particulate fraction (fraction 6). The 62 000 protein comprised 44% of the total radioactivity in the heavy fraction whereas it represented only 18% of the radioactive material in the light fraction. When corrected for the total radioactivity in the two fractions, the proportions of the 62 000 protein present in soluble and particle-associated form were 57 and 43%, respectively. The radioactive material associated with the particle-bound fraction might sediment due to artefactual sticking to membranes during the nitrogen cavitation of the cells. To test this possibility, the light portion of the gradient (tubes 16--20, Fig. 4a) was pooled and rehomogenized either alone or with NIL-HSV or NIL cells. This material was then rerun on sucrose gradients. As seen in Figs. 4b,c,d; sedimentation of the radioactivity was not affected and it banded exclusively above 25% w/w sucrose. The heavy material (tubes 6--7, Fig. 4a) treated in a similar way was also recovered in its original position (Fig. 4e). Thus, the distribution of the 62 000 protein in the DFPolabelled material is in both soluble and particulate cell fractions. Relative to other DFP-labelled bands, the 62 000 band is enriched in particulate material (Fig. 5).

Test for proteolytic activity To characterize further the proteolytic activities, NIL-HSV cell proteins sepa-

173

rated by SDS gel electrophoresis were assayed o n '2sI fibrin for proteolytic activity. This type of assay selects for enzymes which are still active in presence of 1% SDS and does not allow characterization of proteases which could be active under other conditions. Two major peaks of activity were detected by this method, one comigrating with the [3H]DFP labelled protein at 62 000 dalton, the other, showing higher activity, at approx. 39 000 daltons, was not associated with a [3H]DFP labelled band (Fig. 6). The 39 000 dalton activity could be due either to a non-serine hydrolase or to a protease inaccessible to labelling by low concentrations of [3H]DFP. Minor proteolytic activities were observed migrating at 50 000 and 25 000 daltons. The latter comigrated with one of the lower molecular weight DFP-labelled bands. Control experiments showed all fibrinolysis was plasminogen-dependent. To test further the possible identity of the 62 000 dalton protease and the 62 000 dalton DFP-labelled band, a detergent extract of NIL-HSV cells was preincubated with unlabelled DFP, dialyzed and assayed for fibrinolytic activity. The DFP treatment blocked subsequent labelling with [3H]DFP (greater than 95%) and inhibited fibrinolysis (greater than 98%, Table I). This indicates that both proteolytic activities (39 000 and 62 000 daltons) were inhibited by DFP treatment at the higher levels used in the blocking reaction. This sensitivity of the proteolytic activity to DFP inhibition supports the identity of the two activities (fibrinolytic, DFP-reactive) at 62 000 daltons. Inhibition of the I00

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174 TABLE I INHIBITION OF FIBRINOLYTIC

ACTIVITY BY DFP PRETREATMENT

C o n f l u e n t N I L - H S V cells w e r e l y s e d in 0.5% T r i t o n X - 1 0 0 / 0 . 1 M T r i s - H C l , p H 8.1 a n d t h e l y s a t e w a s s p u n at 20 0 0 0 X g f o r 10 rain. T h e s u p e r n a t a n t w a s m a d e 30 m M in d i i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) b y t h r e e s u c c e s s i v e a d d i t i o n s o f D F P in i s o p r o p a n o l at 1-h i n t e r v a l s d u r i n g i n c u b a t i o n at r o o m t e m p e r a t u r e . C o n t r o l s a m p l e s r e c e i v e d n o t h i n g or i s o p r o p a n o l . A f t e r e x t e n s i v e d i a l y s i s , s a m p l e s w e r e a n a l y z e d f o r fibrin o l y s i s as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . A l i q u o t s w e r e a s s a y e d f o r i n c o r p o r a t i o n o f [ 3 H ] D F P i n t o trichloroacetic acid-precipitate material. Sample

12 S I r e l e a s e d ( c p m )

No addition 0.5% Triton

50 pl 1 0 0 #1 0.25% Trypsin NIL-HSV control lysate NIL-HSV + isopropanol NIL-HSV, DFP-treated NIL-HSV control + DFP-treated

15 17 16 15

460 592 666 419 084 638 699 238

18 16 14 14

[3 H ] D F P i n c o r p o r a t e d ( c p m ) 265 441 880 107 875 844 617 673

4921 229

39 000 dalton activity by DFP is considered in the Discussion. We next compared the activity of the 39 000 and 62 000 dalton proteins in NIL-HSV and NIL cells (Fig. 7). The activity corresponding to the 39 000 dalton protein was two-fold higher in the transformed cells. The 62 000 dalton protein was also much more (5.-10 times) active in the transformed cells. This is in good agreement with the [3H]DFP labelling pattern of the protein of the same molecular weight in the two cell types. We have shown that the particulate cell fraction was enriched in the [3H]-

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Fig. 7. F i b r i n o l y t i c a c t i v i t y o f 6 2 0 0 0 a n d 39 0 0 0 p r o t e i n s , a s s a y e d as in Fig. 6. a a n d b, A n a l y s i s o f Part i c u l a t e a n d s o l u b l e f r a c t i o n s o f N I L - H S V cells. N I L - H S V cells w e r e h o m o g e n i z e d a n d c e n t r i f u g e d f o r 6 0 r a i n at 39 0 0 0 r e v . / m i n at 4 ° C in a S W 5 0 - 1 r o t o r . E q u a l a m o u n t s o f s o l u b l e a n d p a r t i c u l a t e f r a c t i o n s w e r e a n a l y z e d o n gels as in Fig. 6. a, 6 2 0 0 0 b a n d ; b, 39 0 0 0 b a n d ; • •, particulate; • • soluble. e a n d d. C o m p a r i s o n o f N P 4 0 e x t r a c t s o f N I L a n d N I L - H S V as in Fig. 6. c 62 0 0 0 b a n d ; d, 39 0 0 0 b a n d ; N I L - H S V , /~ •; N I L , o o.

175

DFP labelled 62 000 dalton protein (Figs. 4 and 5). The proteolytic activities of both 62 000 and 39 000 dalton proteins were concentrated 7--10-fold in the microsomal pellet (Fig. 7). This observation gives further support for the identity of the [3H]DFP labelled and the fibrinolytically active protein migrating at 62 000 daltons.

[3H]DFP labelling of other cell types Several other cell types were tested for their reactivity with [3H]DFP. These cells were different clones of hamster fibroblasts, transformed with RNA and DNA t u m o r viruses [37,38], and hamster cells derived from spontaneous tumors [37]. NIL1 and NIL8 cells are two different normal clones of hamster cell; they both showed low levels of [3H]DFP labelling in the 62 000 protein (see Fig. 1). The 80 000--85 000 and 25 000--28 000 bands were labelled. NIL1 grows to a higher saturation density than does NIL8, and shows somewhat higher levels of DFP incorporation into 62 000 protein. Both clones, when transformed by hamster sarcoma virus, showed an approx. 5-fold increase in labelling of the 62 000 protein. Polyoma transformation also produced a 2--3-fold increase in labelling. All the hamster cells, transformed with either hamster sarcoma or p o l y o m a virus showed incorporation of [3H]DFP into the 80 000--85 000, 62 000 and 25 000--28 000 proteins. NIL t u m o r 7 and NIL t u m o r 17 lines were derived from rare tumors arising in hamsters after injection of NIL1 cells [37]. NIL t u m o r 7 cells are transformed for ability to grow in agar, have transformed morphology, and have lost LETS protein from their surfaces, whereas NIL t u m o r 17 cells have 'normal' morphology, and glycolipids [35] and retain LETS protein [1]. Correspondingly, the amount of [3H]DFP present in the 62 000 protein was higher in the NIL t u m o r 7 cells. Thus, several different transformants, R N A virus, DNA virus and spontaneous, show an increase in labelling of the 62 000 protein which correlates with other in vitro parameters of transformation. This 62 000 protein is apparently specific to the cells and does n o t depend on the transforming agent. The data are summarized in Table II. T A B L E II INCORPORATION

OF [3H]DFP INTO VARIOUS CELLS

T h e q u a n t i t a t i o n o f t h e 3 H - l a b e l l i n g in t h e d i f f e r e n t s a m p l e s w a s d e t e r m i n e d b y s c a n n i n g gels. N u m b e r s r e p r e s e n t area u n d e r t h e p e a k o f t h e 6 2 0 0 0 b a n d , a n d are e x p r e s s e d in a n a r b i t r a r y b u t c o n s t a n t u n i t of measure. H a m s t e r cells

3 H-labelling in 62 000 protein

NIL 8 * NIL8-HSV6 NIL8-HSV5 NIL1 NIL1-HSV3 Py-NIL1 Py8-NIL1 NIL1 tumor NIL1 tumor

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7 17

* I n d i c a t e cell c l o n e s u s e d in t h e o t h e r e x p e r i m e n t s .

176 Discussion The results demonstrate the presence of several neutral proteases in transformed hamster fibroblasts by two separate methods. [3H]DFP labelling detected several bands, one of which was increased on transformation. Fibrinolysis assays detected two proteases which appeared to increase on transformation. The two methods are complementary, each having strengths and weaknesses. The DFP labelling approach is, on the one hand, limited to serine proteases and, on the other, not entirely specific for them. The fact that the [3H]DFP labelling profiles of intact cells, homogenates and detergent lysates are very similar suggests that [3H]DFP does not act as a specific marker for externally exposed proteases. This is also suggested by the fact that [3H]DFP labels soluble proteases (e.g., the 80 000--85 000 bands). However, the DFP labelling procedure does provide a ready means of detecting some proteolytic enzymes and following them during subsequent manipulations. The assay of proteolytic activities after gel electrophoresis is limited to those proteases which retain activity after denaturation in sodium dodecyl sulfate. Use of the two procedures in parallel can provide more information than either one alone. Several lines of evidence suggest that the 62 000 protein detected by its reactivity with the inhibitor [3H]DFP is the same as a protein of identical molecular weight which shows proteolytic activity as tested on 12SI-labelled fibrin (Fig. 6). (1) Normal cells, in comparison with transformed cells, had only low levels (10--20%) of the 62 000 proteins, detected either by labelling or by proteolytic assay (Figs. 1, 3 and 7c). (2) A large proportion of the [3H]DFP labelled 62 000 protein was associated with particulate cell material and this band was enriched in this fraction (Figs. 3 and 5). The proteolytic activity of similar molecular weight was also enriched in the microsomat pellet (Fig. 7a). (3) The proteolytic activity was inhibited by preincubation with DFP (Table I). (4) Both 62 000 radioactive and proteolytically active proteins were solubilized after extraction in NP40. Another protein, (approx. 39 000) showed considerable proteolytic activity (Fig. 6); it was associated mainly with the microsomal cell pellet and was more active in transformed cells than in normal cells (Fig. 7). Although this protein did n o t label readily with [3H]DFP (40 or 400 mCi/1, approximately 10--100 pM), its proteolytic activity was inhibited by incubation of a cell extract with 30 mM DFP, suggesting that it is also a serine protease. Consistent with this conclusion, small amounts of DFP incorporation at this molecular weight can be observed in Figs. 1 and 3. The 39 000 activity was the major plasminogen activator activity detected both in the cells and in the culture medium. It may be similar to the activator reported in transformed chicken cells [32]. In contrast, the 62 000 activity was detected only in the cells and not in the medium (not shown). Both enzymes were dependent on plasminogen for activity under the assay conditions used (crosslinked fibrin as a substrate after separation on SDS gels). In addition to these two transformation-related proteases, we detected low amounts of activ-

177 ity at 50 000 and 25 000 daltons. The latter comigrated with a DFP-labelled band. We sometimes, but not always, observed a DFP-labelled band which ran at 50 000 non-reduced and 25 000 after reduction. Since a minor 50 000 dalton proteolytic activity was detected in the culture medium, it is possible that this could correspond with this band. The two major activities (39 000 and 62 000) were both enriched in particulate fractions but were solubilized by non-ionic detergents suggesting association with membranes. Analysis of the 62 000 DFP-labelled protein showed it to be associated with a membrane fraction which bands at a density consistent with its being plasma membrane. This b a n d is too light to be mitochondrial or lysosomal and the inhibition of the activity by DFP also argues against this protein's being lysosomal. Rate zonal sedimentation analysis [39] also showed the 62 000 band to be associated with plasma membrane fractions (unpublised data). However none of these results is conclusive evidence for a plasma membrane location. There have been previous reports of plasminogen activator activity in particulate subcellular fractions [10,21,32] and Quigley [34] has presented extensive evidence for a plasma membrane location of plasminogen activator activity in chicken embryo cells. In conclusion, the use of [3H]DFP is a useful screening approach for the detection and localization of serine enzymes in intact cells. DFP labelling has been used previously for labelling excreted proteases [9,17,27,32]. The method has its limitations, since n o t all proteases are accessible to the inhibitor and not all proteins labelled by this inhibitor are necessarily proteases. However, the introduction of label into the proteins allows one to follow them easily during various subcellular fractionations and manipulations of reaction conditions. This approach, combined with a proteolytic assay, allowed us to detect proteases which are prominent in transformed cells and concentrated in the particulate cell material. The 62 000 protease is of some interest. It shows the largest increase on transformation, is enriched in membrane-bound fractions and does n o t appear to be secreted as such to the culture medium. It could be a precursor form of a secreted protease of different size, or it could function inside the cell. The 39 000 protease may be analogous with the plasminogen activator studied by others. It would be o f particular interest to characterize the substrate specificity of these proteins and compare their activity on other substrates as cell bound or soluble factors. More work will be needed to investigate their role, if any, in transformation. Use of the procedures described should aid in these investigations. Acknowledgements We would like to thank Professor J.P. Quigley for stimulating discussions and for communicating results prior to publication, and Ms. M. Ham for her excellent typing. This research was funded by grants from the National Cancer Institute R01 CA17007 (to R.O.H.) and P01 CA14051 (to the M.I.T. Center for Cancer Research, S.E. Luria). V.M. was supported by a grant of the Fonds National Suisse pour la Recherche Scientifique (3.318.74) to Prof. M. Crippa.

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