Purification and characterization of a cellular fibrinolytic factor associated with oncogenic transformation: The plasminogen activator from SV-40-transformed hamster cells

339

Biochimica et Biophysica A cta, 340 (1974) 339--347 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 97958

PURIFICATION AND CHARACTERIZATION OF A CELLULAR FIBRINOLYTIC FACTOR ASSOCIATED WITH ONCOGENIC TRANSFORMATION: THE PLASMINOGEN ACTIVATOR FROM SV-40-TRANSFORMED HAMSTER CELLS

JUDITH K. CHRISTMAN and GEORGE ACS

Institute for Muscle Disease, Inc., 515 East 71st Street, N e w York, N.Y., 10021 (U.S.A.) {Received October 5th, 1973)

Summary Neoplastic cells, whether transformed by oncogenic viruses or chemical agents, release a fibrinolytic factor not released by normal cells. We have purified this factor 14000-fold from the supernatant culture fluid of SV40-transformed hamster cells. It is a plasminogen activator with a molecular weight of approx. 50 000 consisting of subunits linked by disulphide bridges. It is irreversibly inhibited by diisopropylfluorophosphate, indicating that it is a serine protease. The subunit contianing the diisopropylfluorophosphate binding site has a molecular weight of approx. 25 000.

Introduction

The importance of elucidating tumor-specific functions has long been recognized. The biochemical characterization of such differences between normal and neoplastic cells would be expected to give insight into the mechanism for initiating and maintaining the transformed state. However, neoplastic cell types form a continuum ranging from those that have most of the characteristics of the parental cell type to those whose origin is no longer recognizable [1]. Thus most of the differences between normal and tumor cells have proven to be quantitative rather than qualitative in nature. One parameter of the neoplastic state is a change in hydrolytic enzymes [2--4]. Recently, Reich and his co-workers [5,6] began an intensive study of a fibrinolytic activity associated with the transformation of fibroblasts by oncogenic viruses. They have now established that these fibroblasts produce and release into serum-free medium a proteolytic enzyme which activates plasminogen [7]. A comparable fibrinolytic activity, resulting from the activation of plasminogen in the growth medium, has been shown to be present in a large variety of cell types, whether transformed spontaneously, by viral or by chemi-

340 cal agents. The activity is not detectable in the parental primary cultures or in fetal tissues [5,6]. It is known, however, that several plasminogen activators are produced by normal eukaryotic cells (serum plasminogen activator, tissue plasminogen activators and urokinase [8--10] ). Therefore, it is of great importance to fully purify and characterize the plasminogen activator from transformed cells, n o t only to clarify its role in the transformation process, but to determine h o w or if it differs from these normal activators. This paper described the 14 000-fold purification of the plasminogen activator or "cell factor" produced by SV-40-transformed hamster fibroblasts. The results reported here indicate that it is a serine protease with a molecular weight of approx. 50 000, consisting of subunits linked by disulphide bridges. Materials and Methods

Cell culture and factor harvest SV-40-transformed hamster cells from a clone isolated in the laboratories of Dr E. Reich [5] were maintained at 37°C in Eagle's medium as modified by Dulbecco [11] supplemented with 10% fetal bovine serum. The harvesting of serum-free medium containing cell factor has been previously described [5]. Cells grown to confluence on plastic petri dishes (150 mm diameter) were washed carefully with 0.13 M NaC1, 0.01 M KCI, 0.074 M Tris--HC1 (pH 7.4) and then incubated in Eagle's minimal essential medium [12] for 16--18 h. After collecting the culture fluid, the cells were allowed to recover for 5 h in growth medium. This cycle was rountinely repeated four times before the cultures were discarded. Fibrinolytic assay The assay for plasminogen activating activity was based on release of 125 I-labeled fibrinopeptides from ~ 2 s I-labeled fibrin-coated petri dishes [ 5]. Each 35-mm dish was coated with approx. 0.1 mg fibrinogen containing 6 0 0 0 0 cpm. The fibrinogen was converted to fibrin by incubating plates containing 2 ml of modified Eagle's minimal essential medium with 10% fetal bovine serum for 2 h at 37 ° C. The plates were then washed thoroughly with 0.1 M Tris--HCl (pH 8.1). The standard assay consisted of 100 pmoles Tris-HC1 (pH 8.1), 25 pl dog serum (as plasminogen source) and the solution or polyacrylamide gel slice to be assayed in a total volume of 1 ml. The m a x i m u m release of radioactivity per plate was approx. 30 000 cpm as measured in a Nuclear Chicago scintillation counter with Aquasol as fluor. Using aliquots such that the release of radioactivity is proportional to the a m o u n t of cell factor added, the activity of any factor preparation is reported as cpm 12 s I-labeled fibrinopeptides released in 2 h into a 1-ml reaction mixture multiplied by total sample volume per volume assayed. Diiso[ 3H] propylfluorophosphate labeling of cell factor 1 ml of reaction mixture containing enough of the cell factor activity to release 3 . 1 0 7 cpm of 12 s I-labeled fibrin in 2 h, 100 pmoles of Tris--HC1 (pH 8.1) and 50 pl of [3 H] DFP {0.9 Ci/mM, 0.2 mg/ml) was incubated at 22°C for 3 h at which time no detectable proteolytic activity remained. The reaction

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mixture was then diluted 10-fold with 0.05 M ammonium acetate (pH 5.0), 1 M urea and concentrated to approx. 0.5 ml on an Amicon PM-10 membrane. This dilution and reconcentration was repeated at least five times to remove the unreacted [3 H] DFP.

Materials Bovine fibrinogen, Fraction I was obtained from Miles Laboratories, Inc. Ultra-pure urea, ammonium sulfate, sucrose and carrier-free Na 125I from Schwarz/Mann Biochemicals; specially pure dodecylsulphate from B.D.H. Chemicals, Ltd; [3 H] DFP, Aquasol and Liquifluor from New England Nuclear Corp.; Soluene from Packard Instrument Co.; acrylamide, ammonium persulfate, N,N'-methylene bisacrylamide, N,N,N',N'-tetramethylene diamine and Coomassie brilliant blue from Bio-Rad Laboratories; SP-C25 Sephadex from Pharmacia Fine Chemicals; sera from Flow Laboratories, and powdered culture media from Gibco. Ampholites were from LKB Industries. Results

Purification of cell factor protein The results of a typical purification are summarized in Table I. Immediately after collection, the serum-free harvest fluid was centrifuged at 5000 X g for 10 min to remove cell debris, acidified to pH 3.0 by careful addition of I M HC1 and then made to 50% of saturation with (NH4): SO4 (31.3 g/100 ml). After a minimum of 30 min at 4 ° C, the precipitate was collected by centrifugation (7000 × g for 15 min) and resuspended in one-eighteith of the initial volume in 0.05 M glycine--HC1 (pH 3.0), and stored at 4°C. At this point all of the activity and a b o u t 80% of the protein in the harvest fluid was recovered. This slurry (Stage 2) could be stored for several months at 4°C with no detectable loss of activity.

TABLE

I

TYPICAL PURIFICATION*

Stage

1 2 3 4 5 6

HF 50% ( N H 4 ) 2 S O 4 s u s p e n s i o n Supernatant O 0 . 4 M ( N H 4)2 SO4 e l u a t e f r o m SP-C25 Amicon concentrate Gel e l u a t e

Total protein** (mg)

Total activity* ** (X 10 - 8 )

Specific activity (cpm/mg protein)

Recovery (%)

Purification (fold)

950 720 130

4.0 4.0 4.1

4.2 . i 0 S 5.5-105 3.2"106

-I00 102

--

2.5 2.1 1.8

2.8.107 1.8.108 6.0"109

62 52 45

8.8 1.2 O.030t

1.3 7.5 70 400 14000

* Based o n H F f r o m o n e w e e k o r 8 0 0 0 cc. ** L o w r y et al. [ 1 3 ] d e t e r m i n a t i o n . *** c p m / m l p e r 2 h, t a k e n f r o m l i n e a r c o n c e n t r a t i o n r a n g e , m u l t i p l i e d b y t o t a l v o l u m e p e r v o l u m e assayed, t Fluorescamine determination [ 1 4 J ( K i n d l y p e r f o r m e d b y Dr S. Stein).

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Next, the glycine slurry was centrifuged for 15 min at 10000--12000 × g. The supernatant contained cell factor activity equal to or slightly greater than that in the harvest fluid but only 14% of the protein (Stage 3). Stage 3 supernatant was diluted 4-fold to a final (NH4)2 SO4 concentration of less than 0.1 M with 0.05 M a m m o n i u m acetate, (pH 5.0) and the activity bound to SP-C25 Sephadex equilibrated with the same buffer. After washing the column to remove the bulk of the protein (Fig. 1), the activity was eluted with 0.4 M (NH4)2 SO4 in acetate buffer (Stage 4). The fractions containing activity were combined, made to 1 M urea, desalted and concentrated on an Amicon PM-10 membrane (usually 25--50-fold). The purification at this step resulted both from loss of low molecular weight components and from the removal by centrifugation of an inactive precipitate formed during concentration. The final stage of purification was achieved by preparative polyacrylamide gel electrophoresis. Since the factor activity is stable for more than 72 h in the presence of 0.1% dodecylsulphate, it was possible to employ a standard analytical polyacrylamide gel system [15] for preparative purposes. (It should be noted, however, that more than 50% of the factor activity is lost after 30 min exposure to 1% dodecylsulphate and that concentrations of 0.005% or greater in the assay mixture inhibit formation of fibrinopeptides.) For large-scale preparations, a gel (16 mm X 170 mm) was layered with approx. 1 • 108 cpm of Stage 5 factor activity in a total of 2 ml. The gel system is described in the caption to Fig. 3. After electrophoresis for 60 h at 8°C with a current fo 20 mA/gel, the gel was cut into 2-mm slices and a small plug from each slice placed directly into a standard assay mixture. All activity was found in a region 4--5 slices in width. Although factor activity could be assayed from these gel cores in 2 h or less, no detectable activity could be soaked out of the gel slices, even after 24 h

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Fig, 1. T y p i c a l s t e p w i s e e l u t i o n of cell f a c t o r a c t i v i t y f r o m an S P - 6 2 5 S e p h a d e x c o l u m n , e - ~ , A 2 8 0 n m ; ~. _,), 12 S I-labeled f i b r i n o p e p t i d e s r e l e a s e d f r o m a s t a n d a r d assay p l a t e b y 50-#1 ( F r a c t i o n s 1 8 - - 4 2 ) or 5-#1 ( F r a c t i o n s 44- 50) a l i q u o t s o f c o l u m n e l u a t e Stage 3 p r o t e i n c o n t a i n i n g 75 • 105 c p m o f f a c t o r a c t i v i t y was a p p l i e d to the c o l u m n ; 70 ' 106 c p m of a c t i v i t y w e r e r e c o v e r e d w i t h a 10-fold p u r i f i c a t i o n . T h e b u l k of t h e p r o t e i n was e l u t e d b y w a s h i n g t h e resin first w i t h 0 . 0 5 M a m m o n i u m a c e t a t e ( p H 5,0) ( n o t s h o w n , no a c t i v i t y is e l u t e d ) a n d t h e n w i t h 0 . 1 5 M ( N H 4 ) 2 S O 4 in t h e s a m e b u f f e r . W h e n n o m o r e p r o t e i n c o u l d be r e m o v e d (as d e t e r m i n e d b y a b s e n c e of m a t e r i a l w i t h a b s o r b a n c e at 2 8 0 n m ) , t h e cell f a c t o r a c t i v i t y was e l u t e d w i t h 0.4 M ( N H 4 ) 2 S O 4 in a c e t a t e b u f f e r .

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Fig. 2. I n a c t i v a t i o n o f cell f a c t o r b y DFP. T h e r e a c t i o n m i x t u r e was i d e n t i c a l to t h a t u s e d for [3I-I] D F P labeling of cell f a c t o r (Materials a n d M e t h o d s ) . S a m p l e s (5 #l) w e r e r e m o v e d at t h e i n d i c a t e d intervals a n d p l a c e d in 1 m l of 0.1 M Tris--HCl ( p H 8.1) c o n t a i n i n g 1.5 #g dog p l a s m i n o g e n ( p u r i f i e d b y lysine a f f i n i t y c h r o m a t o g r a p h y [ 1 8 ] , gift o f Dr D. R i f k i n ) a n d k e p t at 0 ° C . A t t h e e n d o f 2 h, all o f t h e s a m p l e s w e r e a s s a y e d at 3 7 ° C with a l i q u o t s t a k e n at 1 a n d 2 h. T h e a c t i v i t y o f t h e c o n t r o l r e a c t i o n m i x t u r e ( - - D F P ) was u n c h a n g e d a f t e r 2 h at 2 2 ° C . T h e final c o n c e n t r a t i o n of 0 . 2 7 #M D F P in the assay did n o t inhibit fibrinolysis (0 t i m e + D F P = c o n t r o l = 100%). A l i q u o t s w e r e c o u n t e d d i r e c t l y in a g a m m a c o u n t e r a n d are c o r r e c t e d f o r b a c k g r o u n d . All p o i n t s are an a v e r a g e o f six d e t e r m i n a t i o n s . T h e d e v i a t i o n f r o m a v e r a g e is indicated.

Fig. 3. C o i n c i d e n c e of [ 3 H ] D F P i n a c t i v a t e d cell f a c t o r , cell f a c t o r a c t i v i t y a n d Stage 6 p r o t e i n o n d o d e c y l s u l p h a t e 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 . [3 H ] D F P - l a b e l e d f a c t o r was c o - e l e c t r o p h o r e s e d w i t h active S t a g e 6 p r o t e i n . One gel was c u t i n t o l - r a m slices; each slice was split in half. O n e - h a l f was a s s a y e d d i r e c t l y f o r f a c t o r a c t i v i t y (o - o) a n d t h e o t h e r h a l f was digested in 0.5 m l S o l u e n e for 2 h at 7 0 ° C , in o r d e r to d e t e r m i n e [ 3 H ] D F P c o u n t s ( e - e ) using a scintillation s y s t e m w i t h L i q u i f l u o r . A n identical gel was fixed a n d s t a i n e d a n d split l o n g i t u d i n a l l y . O n e - h a l f was t h e n sliced a n d a s s a y e d f o r [ 3 H ] D F P c o u n t s . T h e a l i g n m e n t of s t a i n e d b a n d s a n d c o u n t s was assured b y t h e use of fine wire m a r k e r pins d u r i n g the c u t t i n g . T h e m o l e c u l a r w e i g h t was a c c u r a t e l y d e t e r m i n e d b y c o - e l e c t r o p h o r e s i n g [ 3 H ] D F P - l a b e l e d f a c t o r with m a r k e r p r o t e i n s a n d c o u n t i n g gel slices a f t e r staining. Gels w e r e 12% a c r y l a m i d e , 0 . 1 5 % bisa c r y l a m i d e , f o r m e d a n d r u n in 0.1 M s o d i u m p h o s p h a t e b u f f e r ( p H 7.2) c o n t a i n i n g 0 . 0 2 M E D T A , 2.5 M u r e a , a n d 0 1% d o d e c y l s u l p h a t e [ 1 5 ] . S a m p l e s w e r e l a y e r e d in 30% sucrose, 0.1% d o d e c y l s u l p h a t e w i t h p H a n d ionic s t r e n g t h a d j u s t e d to a p p r o x i m a t e t h e b u f f e r s y s t e m . R u n n i n g t i m e : 15 h at 4 m A / g e l (16 r a m 2 ) . F o r p r o t e i n staining, gels w e r e fixed f o r 1 h in 12.5% t r i c h l o r o a e e t i c acid, s o a k e d 2--3 h in 0.5% C o o m a s s i e brilliant blue in 50% m e t h a n o l , 12.5% t r i c h l o r o a c e t i c acid, a n d d e s t a i n e d in 12.5% t r i c h l o r o -

344

exposure to a variety of buffers. Apparently mixture can be activated by factor imbedded Electroelution into standard electrophoresis recovery of 80--90% of the factor activity from

the plasminogen in the assay at the surface of the gel slices. buffer, however, allowed the the gel slices in 4--8 h.

Characteristics of the cell factor The cell factor can directly activate plasminogen to cause fibrinolysis (i.e. Fig. 2). A rather sharp pH o p t i m u m for this total reaction was observed at approx, pH 8.2. Like the cell factor produced by transformed chick e m b r y o fibroblasts [ 1 6 ] , the cell factor from SV-40-transformed hamster cells is irreversibly inactivated by exposure to diisopropylfluorophosphate. The kinetics of the reaction is first order {Fig. 2) and the DFP cannot be removed by dialysis indicating that the cell factor is a serine protease [17]. The molecular weight of the cell factor is approx. 50 000 as determined by dodecylsulphate polyacrylamide gel electrophoresis. Fig. 3 shows the electropherogram of Stage 6 protein. It can be seen that there is only one protein band visible in this preparation at a mol. wt of 50000. The protein band migrates in exactly the same position as factor activity detected from the gel slices and is completely coincident with the [3 H] DFP-inactivated protease. This suggests that Stage 6 protein consists entirely of cell factor. An additional indication of the purity of the preparation is the determination that for each I • 106 cpm of cell factor activity, 4 pmoles of [3 HI DFP are bound. Assuming that one molecule of DFP reacts per molecule of cell factor, it can be calculated that this protease of mol. wt 5 0 0 0 0 should have a specific activity in the fibrinolytic assay of 5 - 1 0 9 cpm/ml per 2 h per mg when purified to homogeneity. This is in very close agreement with the specific activity actually found for Stage 6 protein (Table I). The final step in the purification procedure, however, by separating according to molecular weight, would select contaminants of the same size class as the cell factor. Thus, it was necessary to assess the homogeneity of Stage 6 protein by a m e t h o d independent of molecular weight. Separation according to isoelectric point was chosen. After removing the dodecylsulphate by D o w e x AG 1-X2 chromatography [19], Stage 6 protein was focused in a polyacrylamide gel containing pH 3--10 ampholites. One major stainable band was found to migrate at the same pH as the factor activity, or approx, pH 9.5 (Fig. 4). A minor stainable band, for which we cannot account at the present time, was visualized within 0.25 pH unit. The staining intensity of the major band, however, was comparable to that of the band on dodecylsulphate acrylamide gel electrophoresis. This observation combined with the predicted specific activity of the protein from DFP-based calculations would indicate that the factor has been purified to a high degree of homogeneity. Any contaminants present would have to have the same binding affinity to SP-C25 Sephadex, the same approximate molecular weight and the same isoelectric point, a highly unlikely coincidence. It should be noted that the gel shown in Fig. 3 is not run under reducing conditions. Exposure of the factor to 1% ~-mercaptoethanol or 0.01 M dithiothreitol irreversibly inhibits the cell factor activity. Since no factor activity can be detected in the gel slices after the factor protein has been exposed to ~-mercaptoethanol, [3 H]DFP-labeled factor was substituted for

345 pH I Irr~,

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i n t o gels w i t h

a f i n a l c o n c e n t r a t i o n o f 5% a c r y l a m i d e , 0 . 1 6 % b i s a c r y l a m i d e , 2 . 5 M u r e a , 5% s u c r o s e a n d 2% o f t h e c h o s e n a m p h o l ] t e r a n g e . A m m o n i u m p e r s u l f a t e w a s u s e d as c a t a l y s t [ 2 0 ] . A t 0 . 2 W p e r gel, t h e a c t i v i t y w a s c o m p l e t e l y f o c u s e d in 6 h a t 1 5 0 V. A f t e r s p l i t t i n g t h e gel l o n g i t u d i n a l l y , o n e - h a l f w a s f i x e d a n d s t a i n e d a n d t h e o t h e r h a l f w a s c u t i n t o l - r a m slices. T w o a d j a c e n t 1 - r a m slices w e r e u s e d f o r e a c h d e t e r m i n a t i o n o f p H a n d a c t i v i t y . T h e s t a i n e d gel is i n d i c a t e d s c h e m a t i c a l l y a b o v e t h e a c t i v i t y g r a p h . (o--o) f a c t o r a c t i v i t y ; (o -o), p H .

active factor. As can be seen in Fig. 5, after reduction only one radioactive band is found at a mol. wt of approx. 2 5 0 0 0 . This suggests that the cell factor consists of subunits linked by disulphide bridges and that these subunits are inactive. The band of stainable protein at mol. wt 50 000 also disappears after exposure to reducing agents, a further proof of the homogeneity of the cell factor. A faint band appears at mol. wt 2 5 0 0 0 , but it is not comparable in intensity to the original band. If this is not due to some change in the staining properties on reduction of the molecule, it may indicate that the other subunits are smaller than 2 5 0 0 0 . (-;I

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346

4' Fig. 6. C o m p a r i s o n o f t h e s e p a r a t i o n o f S t a g e 3 p r o t e i n s b y gel e l e c t r o f o c u s i n g a n d gel e l e c t r o p h o r e s i s . (a) Isofocused Stage 3 protein. The preparation was desalted by Amicon filtration and 2.5 mg of protein was i n c o r p o r a t e d i n t o a 5% p o l y a c r y l a m i d e gel ( M a t e r i a l s a n d M e t h o d s ) . T h e a r r o w i n d i c a t e s t h e p o s i t i o n of f a c t o r a c t i v i t y a s s a y e d f r o m gel slices. (b) D o d e c y l s u l p h a t e 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 o f S t a g e 3 p r o t e i n . T o p , b o v i n e s e r u m a l b u m i n , o v a l b u m i n ; M i d d l e , 1 0 0 /ag S t a g e 3 p r o t e i n d e s a l t e d b y A m i c o n f i l t r a t i o n ; B o t t o m , S t a g e 6 p r o t e i n . All d e t a i l s as in M a t e r i a l s a n d M e t h o d s . A n o d e t o t h e r i g h t . T h e a r r o w i n d i c a t e s t h e p o s i t i o n of f a c t o r activity.

Discussion

We have purified the plasminogen activator from SV-40-transformed hamster cells to apparent homogeneity by t w o criteria: analytical polyacrylamide gel electrophoresis and isoelectric focusing. This protein has a molecular weight of 50 000 and appears to consist of subunits linked by disulphide bridges. The subunit containing the active site, as indicated by the ability to bind diisopropylfluorophosphate, has a molecular weight of 25000. Now that this protein has been purified 14 000-fold, it should be possible to compare it with other known plasminogen activators and to characterize it much more completely. Because of its unusually high isoelectric point, an alternative purification m e t h o d using preparative isoelectric focusing is n o w being investigated. The large initial purification of factor activity from the bulk of Stage 3 protein which can be obtained (Fig. 6), should allow the isolation of relatively pure factor protein in one step and homogeneous protein after the additional step of preparative dodecylsulphate acrylamide gel electrophoresis. With a simple m e t h o d for preparing large amounts of factor protein, antibodies to the factor can be prepared, opening the way to extensive studies on the intracellular location of the factor and its possible precursors. If such antibodies prove to be specific inhibitors of the cellular plasminogen activator, it will also be of great interest to determine the effect of cell factor inactivation on t u m o r growth. Acknowledgements We wish to than Mrs Hanna Klett, Mr Cornelius Whalen and Miss Tina

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Parler for their expert and dedicated assistance and Mr J.C. Unkeless and Dr E. Reich for discussion of their results prior to publication. These studies were supported by U.S.P.H.S. (CA-08751) and the American Cancer Society (NP-3601). References 1 Knox, W.E. (1972) Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues, S. Karger, New Y ork and Basel 2 Kazakova, O.V. and Orckhovich, V.N. (1969) Biokhimiya 34, 73--77 3 Taylor~ J.C., Hill, D.W. and Rogolsky, M. (1972) Exp. Cell Res. 7 3 , 4 2 2 - - 4 2 8 4 Fischer, A. (1925) Arch. Entwicklungsmech, Org. (Wilhelm Roux), 104, 210--219 5 Unkeless, J.C., Tobia, A.~ Ossowski, L., Quigley, J.P., Rifkin, D.B. and Reich, E. (1973) J. Exp. Med. 137~ 85--111 6 0 s s o w s k L L., Unkeless, J.C.~ Tobia, A., Quigley, J.P., Rifkin, D.B. and Reich, E (1973) J. Exp. Med. 137, 112--126 7 Quigley, J. and Unkeless, J. (1973) Fed, Proc. 32, 851 Abstr. 8 Flute, P.T. Proc. 7th Congr. Eur. Soc. Haematol. London, Part II, p. 894 9 Astrup, T. and Pertain, P.M. (1947) Nature 1 5 9 , 6 8 1 - - 6 8 2 10 Williams, J.R.B. (1951) Br. J. Exp. Pathol. 32, 530--537 11 Smith, J.D., Freeman, G., Vogt, M. and Dulbecco, R. (1960) Virology 12, 185--196 12 Eagle, H. (1959) Science 130, 432--437 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L and Randall, R.J. (1951) J. Biol. Chem. 193, 265--275 14 Udenfriend, S., Stein, S., B~hlen, P., Dairman, W., Leimgruber, W. and Weigele, M. (1972) Science 178, 871--872 15 Summers, D.F., Maizel, J.V. and Daxnell, J.E. (1965) Proc. Natl. Acad. Sci. U.S. 54, 505--513 16 Unkeless, J., Dano, K., Kellerman, G.M. and Reich, E. J. Biol. Chem., in the press 17 Cohen, J.A., Oosterhaan, R.A. and Berends, F. (1967) in Methods in E n z y m o l o g y (Hirs, C.H.W., ed.), Vol. XI, pp. 686--702, Academic Press, New York 18 Deutseh, D. and Mertz, E.T. (1970) Science 170, 1095--1096 19 Weber, K. and Kuter, D. (1971) J. Biol. Chem. 246, 4504 --4509 20 Welner, D. (1971) Anal. Chem. 43, 59A--65A