IgG was unable to inhibit the binding of the PN-1.protease complexes. ..... Imnopreeipitarion of RI-I m d RI-1:thrombin Compleree: I~unopreeipIta~ion of RI-I:m ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.
Val. 261, No. 2, Issue of January 15, pp. W-689, 1986 Printed in U.S.A.
Human Protease Nexin-I FURTHER CHARACTERIZATION USING A HIGHLY SPECIFIC POLYCLONAL ANTIBODY* (Received for publication, July 1, 1985)
Eric W. Howard and Daniel J. Knauer From the Department of Developmental and Cell Biology, School of Biological Sciences, Universityof California, Iruine, California 9271 7
We have used purified protease nexin-I (PN-I)from human fibroblasts to develop a polyclonal antibody that specifically blocks the PN-I-mediated cellular binding ofthrombinand urokinase. Anti-PN-I IgG didnot inhibit the binding of l2’1-epidermal growth factorbinding protein to fibroblasts, which is mediated by protease nexin-11, another cell-secreted, serine protease inhibitor that is distinct from PN-I. Thisfurthers the belief that the protease nexins are distinct from one another. In addition, while anti-PN-I IgG immunoprecipitated PN-Iathrombincomplexes, it did not do so with antithrombin-III*thrombin.Metabolically labeled PN-I was alsoimmunoprecipitatedby IgG, indicating that the protein can be labeled in vivo. The antibody also recognized primarily one band on Western transfers of conditioned medium from fibroblast cultures. These results suggest that anti-PN-I will be useful in probing the physiological role of PN-I aswell as its biosynthesis.
Human fibroblasts in culture secrete three known protease inhibitors referred to as protease nexins (1). PN-I’ binds thrombin and urokinase (2), whereas PN-I1 binds EGF-BP (3) and PN-I11 binds y-nerve growth factor (4). Protease nexins are similar to plasma protease inhibitors, such asAT111, al-antitrypsin, and C1 inactivator. Inhibition of the protease occurs when it binds to the inhibitor, cleaves a portion of the inhibitor, and remains covalently attached through an acyl linkage (2). The complexes formed between plasma protease inhibitors and active proteases are cleared from the circulation, presumably by binding to receptors found in liver (5). Protease nexins are found in nonvascular areas and are produced and taken up by various cells, including those derived from lung, kidney, embryo, and connective tissue (2,6). Like plasma protease inhibitors, they are cleared from their
environment by the same cells that produce them, but only after forming complexes with specific serine proteases. Possible physiological roles of PN-I include the mediation of plasminogen activation duringtissue damage (7) andthe modulation of thrombin-stimulated mitogenesis (8). PN-I is similar to AT-I11 in that their activities are accelerated by heparin (2). AT-111, like PN-I, binds a variety of proteases, including thrombin and urokinase (9). While their specificities aresimilar, PN-I andAT-I11appear to be unique proteins. Polyclonal antibodies raised against PN-I do not recognize AT-111, and vice versa (Ref. 2 and this report). It is not known if the protease nexins are derived from a common precursor or if PN-I is a product of PN-I1 processing. We have raised a polyclonal antibody that recognizes PN-I exclusively, and we have shown that, while it blocks the binding of 1251-thrombinto cells, it does not block the binding of lZ51-EGF-BP.Since thrombin binds to human fibroblasts almost entirely through its interaction with PN-I (10) and EGF-BP bindssimilarly via PN-I1 (3), one can claim, at least tentatively, that the two nexins aredistinct entities. We report here the purification of PN-I aswell as theproduction of a polyclonal antibody which blocks the interaction of PNI . protease complexes with cells and which can immunoprecipitate both PN-I .protease complexes and metabolically labeled PN-I. MATERIALS AND METHODS AND RESULT$ DISCUSSION
In the present report, we demonstrate the binding properties of a polyclonal antibody to the cell-secreted protease inhibitor PN-I. Purified IgG specifically blocked the binding of both PN-I-urokinase and PN-I-thrombin tohuman foreskin fibroblast cells by up to 90%. The inhibition was shown to be due to thespecific affinity of anti-PN-I, since anti-EGF IgG was unable to inhibit the binding of the PN-1.protease complexes. The purified IgGwas also shown to behighly * This work was supported by Research Grant CA 22400 from the specific for PN-I, since it recognized primarily one protein in National Institutes of Health. The costs of publication of this article Western blot analysis of concentrated conditioned media from were defrayed in part by the payment of page charges. This article human foreskin fibroblast cell cultures. In addition, the major must therefore be hereby marked “aduertisement” in accordance with metabolically labeled species that IgG was able to immuno18 U.S.C. Section 1734 solelyto indicate this fact. ‘The abbreviations used are: PN-I, protease nexin-I; EGF-BP, precipitate from conditioned medium was PN-I. Anti-PN-I .PNepidermal growth factor-binding protein; AT-Ill, antithrombin-111; IgG also immunoprecipitated complexes of 1251-thrombin PN-I:Th, protease nexin-I complexed with thrombin; PN-I:UK, protease nexin-I complexed with urokinase; HF cells, human foreskin fibroblasts; PBS, phosphate-buffered saline; D-PBS,Dulbecco’sphosphate-buffered saline; DTT, dithiothreitol; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium; CHAPS, 3- [(3-chloramidopropyl)dimethylaminol-1-propanesulfonate;PMSF, phenylmethanesulfonyl fluoride; BSA, bovine serum albumin; CGU, chloroglycouril; NP-40, Nonidet P-40; HEPES, 4-(2-hydroxyetbyl)-l-piperazineethanesulfonic acid.
Portions of this paper (including “Materials and Methods,” “Results,” Figs. 1-6, and Table 1) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85111-2171, cite the authors, and include a checkormoney order for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.
684
Immunologic Characterizationof PN-1 I, but not '251-thrombin.AT-III or lZ5I-thrombin alone. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell-bound label showed that anti-PN-I decreases the presence of PN-I. thrombin in a concentration-dependent manner. The inhibition of binding was independent of the protease providing the protease binds PN-I. Urokinase may be substituted for thrombin in cellular binding studies; both are inhibited by equal concentrations of anti-PN-I IgG. It is likely that these proteasesbecome linked to PN-I aatcommon active site. There are three protease nexins known to be secreted by cultured HF cells (1).We conclude that two of the nexins, PN-I and PN-11, are unrelatedproteins, at least in their secreted forms. PN-I1 binds EGF-BP with high affinity and is different than PN-I in molecular weight, ability to bind heparin, and method of purification (3). In addition, we have shown that anti-PN-I is unable to block the binding of lZ5Ilabeled binding protein to cells, indicating that thetwo nexins are separate entities. It is unlikely that PN-I is derived from PN-I1 based on their lack of immunologic cross-reactivity. In addition, previous work suggests that the protease nexins do not share a common receptor, since they do not appear to compete with one another in cellular binding studies (6). The phenomenon of heparin competition of PN-I .protease binding to cells has been reported previously (6), yet the mechanism, as well as thephysiological basis of this effect, is not understood. Dermatan sulfate, a related molecule, does not exhibitthe blocking properties of heparin (18).Inhibition of PN-1.protease binding by anti-PN-I approached that of heparin but did not exceed it, indicating that specific, PN-Imediated binding can be analyzed using either antibody or heparin.Heparinisa convenient indicator of nonspecific binding of labeled thrombin because of cost and availability. It is also used routinely to analyze specific low density lipoprotein binding (11).Both thrombin and PN-I have heparinbinding sites, and their interactionis accelerated by heparin, as is the reaction between AT-I11and thrombin. It is conceivable that heparin serves as a factor that limits proteolytic activity at wound sites by catalyzing the rapid inactivation of at least two key proteases, thrombin and plasminogen acti-
685
vator, both of which control the cascade of proteolytic reactions leading to thrombus formation and breakdown. By closely mediating the proteolysis that occurs during tissue damage, protease inhibitors, both plasma-borne and ceil-secreted, could serve to limit the clotting response to a defined area. Acknowledgments-We wish to thank Genesio Murano for supplying highly purified human urokinase in generous amounts and John Fenton I1 for providing large quantities of human thrombin. We also express our gratitudeto Suzel Deas for her expert technical assistance. REFERENCES 1. Knauer, D. J., and Cunningham, D. D. (1984) Trends Biochem. Sci. 9,231-233 2. Baker, J. B., Low, D. A., Simmer, R. L., and Cunningham, D. D. (1980) Cell 2 1 , 37-45 3. Knauer, D. J., and Cunningham, D. D. (1982) Proc. Natl. Acad. Sci. U. 5'. A . 7 9 , 2310-2314 4. Knauer, D. J., Scaparro, K. M., and Cunningham, D. D. (1982) J. Biol. Chem. 257,15098-15104 5. Fuchs, H. E., M. A. Shifman, G. Michalopoulos, and S. V. Pizzo (1984) J. Cell. Biochem. 2 4 , 197-206 6 . Knauer, D. J., J. A. Thompson, and D. D. Cunningham (1983) J. Cell. Physiol. 1 1 7 385-396 , 7. Scott, R. W . , D. L. Eaton, N. Duran, and J. B. Baker (1983) J. Eiol. Chem. 258,4397-4403 8. Low, D. A., R. W . Scott, J. B. Baker, and D. D. Cunningham (1982) Nature 298,476-478 9. Rosenberg, R. D., and P. S. Damus (1973) J. Biol. Chem. 2 4 8 , 6490-6505 10. Moss, M., H. S. Wiley, Fenton, J. W . , 11, and D. D. Cunningham (1983) J. Biol. Chem. 2 5 8 , 3996-4002 11. Goldstein, J. L., Basu, S. K., Brunschede, G. Y., and Brown, M. S. (1976) Cell 7 , 85-95 12. Eaton, D. L., and J. B. Baker (1983) J. Cell Eiol. 97,323-328 13. Hjelm, H., K. Hjelm, and J. Sjoquist (1972) FEES Lett. 2 8 , 7376 14. Scott, R. W . ,and J. B. Baker (1983) J. Biol. Chem. 258, 1043910444 15.Fraker, P. J., and Speck, J. C. (1978) Biochem. Eiophys.Res. Commun. 80,849-857 16. Laemmli, U. K. (1970) Nature 227,680-685 17. Burnette, W . N. (1981) Anal. Biochem. 112,195-203 18.Low, D. A. (1982) Ph.D dissertation, University of California, Irvine
Continued on next page.
Immunologic Characterization of PN-I
686 Supplementary Macer181 t o "Human Roteaee Nexin-I: S p e c i f i cR l y e l o n a l
F u r t h eC r haracterization
Antibody" by E r i e U. HwardandDaniel
U ~ i n g A Highly
J. Xnauer.
PN-I k r i f l c a t i a n Serum-free.conditioned medium f r o ms p i n n e rf l a s k sc o n r a i n i n g HF fells on microearrier beads viu c o l l e c t e d a ncdo n c e n t r a t e d by h o l l o w - f i b e fr i l t r a t i o n (Amicon, Danvers. n a s s . ) , f o l l o w e d by re~ircularion on B 53 al h e p a r i n - S e p h a r o s e column. as d e s c r i b epdr e v i o u s l y (14). B r i e f l yt.h e colvvl VaB washed several times w i t h PBS. and PN-I was e l u t e dw i t h a l i n e agr r a d i e n t 0.2 of I to 1.5 U NaCl. The maferial from the column deemed PN-I by Chrombin-binding assay wae c o n c e n t r a t aesndtdo r e d i n aliquot8 containing LOO nlT a t -7O'C. -re-purified For t h e material was f r a c t i o n a c e d u s i n g BioCel P-60. This r e s u l t e d in II s i n g l e band on a n a l y s i s by SDS-FACE.
RI-I.
k r i f i c a c i o n of Rotease Nexin-I: PN-I ha8 been p u r i f i e d t o a p p a r e n t homogeneity p r e v i o u s l y ( 1 4 ) . I n an a t t e m p t f o maximize b o tyhi e l d and ~ ~ t l v l t yan , a c c e l e r a t e d p r o c e d u r e involving h e p a r i n - S e p h a r o s e chromatogrephy was "sed to p u r is fuyf f i c i eqnuta n t i tr ioeefl sa t i v e l y puce RI-I. R e p a r a t i o n s of PN-I l o s e a c t i u l ~ y r a p i d l y . even when mtored PC -7O.C; a d d i t i o no f LOO YU D T I b e f o r e concentration m a r k e d liyn c r e a s etdheec t i v i toyf RI-I. as w e l l an i c a l o n g e v i t y . F i g u r e I shave t h ep r o d u c to b t a i n e df r o mh e p a r i n - S e p h s r o s ep u r i f i c a t i a no f Pt-I, f o l l o w e d by g e l f i l r r a f i o n on BioGel P-60. The g e l f i l r r a t i o n s t e p can be eliminated when p r e p a r i n g PN-1 a c r l v i t y and e l a r g e r p r o t e i ny i e l d are f o r most usee, e s p e c i a l l y when a h i q h e ri n h i b i t o r y around 43,000 Da i n SDS-PACE. The a d d i t i o n of thrombin desired. PN-I a p p e a r s 88 a doubler t o p u r i f i e d PN-I caused thedisappearanceof one o tfh eb a n d (s d a t a not shown); i f is not k n w n why t h e nom-reactive material i n t h eo t h e r band Ill p r e s c n tt.h o v g h it may be a nonreactive f o r mn o r m a l l ys e c r e t e d . since bothhands are seen when PN-I is m e t a b o l i c a l l yl a b e l e d ( f h l er e p o r r ) . I n h i b i t i o n of RI-I-Mediated
AnribodyRoducrion
Pcofeaee Binding by Anti-PN-I
IgC:
and IgC k r i f l c a t i a n a
Female. New Zealand Uhife r a b b i t s were s u b c u t a n e o u s l yi n j e c r e d in a v l r i p l el o c a t i o n s on rhebackwith a I : l d i l u t i o n of LOO yg PN-I and PBS i n completeFreund's adjuvant. f o l l o w e d by a booet t h r e e weeks later w i it nh c o m p l eFt er e m n d ' s adjuvant. Blood was c o l l e c t e d . t o isalate serum. IgC was o b t a i n e d by p a s s i n g serum, d i l u c e d I : I w i t h d e f i b r i n a t e d , andspun PBS. over p r o t e i n A-Sepharose, followed by e l u t i o nw i t h 0.2U glycine-HCI, pH 2.7(13). The d m e d i s r ewl yi t h PI T r i e t l C l . p8H.8. IgG was e l u t e d IgG f r a c t i o n vas n e u t r a l i z iem Its concentration d e t e r m i n e d c o n c e n t r a t e d using u l t r I I - f i l t r P t i o n (Amicon Rl-30 membrane),and byab@arblmee a t 280 nm. Storage was a t -7O'C.
HF c e l l s r e r e p l a t e d in 2 4 - r e lpl l a t e s , g r w n t o confluence, washed With D-PBS, and 0.5 ml serum-free medium. k r l f i e d IgC at V B I ~ O Y B eoncenrra~ionswiu added i n c u b e r e dw i t h f a r2h o u r 8t o cells h a v i n gc o n d i t i o n e d or f r e s hm e d i a .f o l l o w e d by t h ea d d i t i o no fi o d i n a t e d l i g a n df o r2 hours. P l a t e s were thenwashed 3X w i t h PBS at 4*C. t h e nl y s e dw i t h hot In NaOH and counted i n II g a m a counter. Non-specific binding RI-I:proterse of complexes was d e t e r m i n e d by h e p a r icno m p e t i t i o(n0 . 2 w i d ) . iu d e s c r i b e(d6 A ) . n a l y s iocsfe l l - b o u n d 11 NP-40. b o i l i n g , and l i g a n d was aceompliahed by l y s i n gc e l l s In 20 dl HEPES. pH 7.5.with l o a d i n g onto SDS-PAGE g e l s . I l e a s u r e m e ~Of ~ U n k a g e Between Il2SII-rhrombinand
RI-I by SDS-PACE.
E f f e c to f ECP-BP. and p r o t e i n A were i o d l n s r e d u s i n g t h e CGU method. as Thrombin. urokinase, d ith m r o m b l n ( 3 0 us) Y ~ B i n c u b a t ew 1 m C i Na112511, 0.4 og d e e c r i b e d elsewhere (15). b e n r l m i d i n e . and 0.2 n Nan, pH 7.0 (YI Y l total volume) f o2r 0 mi". a t 4'C in a r v b e ug CGU. ?he reaction wlul quenched by removing the reacrion mixfnre f r o mt h e c o n t a i n i n g2 5 CCU t u b e and . d d l n g 0.45 m l R S p l u s I &INaI. f o l l o w e d by t h ea d d i f i o no f 0.5 m l ofthe above plua 2 og/d o r a l b u m i n . The thrombin Yd i e l y r eadg a i n s t P U S . and the s p e c i f i c a c ~ i v i t l e s o f VLL~IOYS preparations ranged betveen 12,530 and 20.000 c p d n gU . rokinase (16 ug) w u i o d i n a t e d i n a similar f a s h i o n , save t h a t a 0.1 d solution of 1 m C i Na[125]I, 0.2 og b e n r a r i d i n e , 0.7 I N d l . and 0.2 U NaPi. pH 7.0. wae used. along w i t h 53 ug CGU. Specific a c t i v i t y was found t o be 3000 cpdng. ECF-BP (IO us) vae i o d i n a t e d t o a S p e c i f i ca e f i v i r y of 142.000 c p d n g by i n c u b a t i n g it w i t h I d i N a l l Z S I I i n (I 0.5 mI volume of 0.2 U Nan, pH 7.0 g CCU f o r 20 mi". LL 4'C. R o f e i n A (IO us) I- I o d i n a t e d uaing 53 ug CGU w i t h2 w i t h2 5 d l NeL2$ and 0.2 U N a n . pH 7.5, i n a volume of 53 "1. After 20 min on lee, t h e mixture wm added co 50 y l of 1 dl NaI i n PBS. f o l l o w e d by 100 u l NaI/WS p l u s2 q/ml ovalbumin. Thin m t e r l a l w.8 8 e p e r a f e d on Sephader G-25 e q ( u i 1 l b r s t ew d ith RS. Specific activities rangedfrom 40,000 t o 70.000 e p d n g . R4-I:Th Y U p r e p a r e d by l n c v b a t i n g1 1 2 5 I I - t h r o m b i ow i t h c o n d i t i o n e dm e d i af o r I h r a t 37-C. f a l l o w e d by p u r i f i c a t i o n an Bia-Cel P-LOO, whichhad been e q u i l i b r a t e d VlLh PBS.
D-PBS a n di n c u b a t e df o r 12 C o n f l u e n t XF e e l 1 CultYres (35 ma p l a t e s ) were washedwith h r sw i t h I d s e r u m - f r e e ,m e t h i o n i n e - f r e e medium p l u s 100 mCi/ml 1356lmerhlonine. Medium vas I8 u g / d p u r i f i e d Igc f o r I h r a t 37%. S u b s e q u e n t l y ,p r o r e i n Ar e a v e d a n di n c u b a t e dw i t h S e p h a r o s e( 2 s "1 b e a d s ) w i u added f o r I hr., f o l l w e d by 3 r a l h e s w i t hi c e - c o l d 20 .L( HEFES, pH 7.5. p l u s IO dl CHAPS. I dl EDTA. 1 dl MSF and a f i n a l wash with RS. NOnreduced SDS sample buffer was t h eand d e df o , llwed by b o i l i n g . Samples yere a n a l y z e d SDS-PACE. by I 1 . 0 l n o p r e e i p i t a t i o n of RI-I:1125II-rhrombin was accomplished by i n c u b a t i n g cells w i t h 1 5 "g 11251-Th f o 2rh r sf. o l l o w e d by 3 r a s h e ew i t hc o l d PBS. C e l l s were t h e nl y e e dw i t h 20 d l HEFES, pH 7.5, plus 1% NP-40. 0.5% SDS, I M MSF, and 0.1 sgld ovalbumin. Reei~itation proceeded a~ above.butwashes were performed using t h el y l l i sb u f f e r jusr d e s c r i b e d . E l e c t r o p h o r e s i sa n d
Western BlOLCing
SDS-PACE was performed IS p r e v i o ~ s l yd e s c r i b e d (16). Gels were d r i e du n d e r V P E Y Y ~ a f t e r 8celnlng i n Coomassie Brilliant Blue,and were e r p o e e d t o Yodak 7 . m - 5 f i l m at -7O'C w i t ht h e acreen. T c a n a f eorefl e c t r o p h o r e s e pd r o t e i n s to a i d of a CIODBX L i g h t n i nPg l uesn h a n c i n g w . 8 performed iu d e s c r i b e d ( 1 7 )B . lots n i c r o c e l l ~ l o l l e ( S e h l e i c h e ra n dS c h u l l ) yere p r o c e e s e d w i t h Y) ug/ml p u r i f i e d anti-PN-I IgC, f o l l o r e d by I I 2 5 1 I - p r o r e l n A (10,000 c p d m l ) .
H.teria1* Ns[12511 (13.5 d i l u g ) a n dL - 1 3 B I m e t h i a n i n e( 2 6 7d i / - l ) were obtainedfromAmershm Corp.. A r l i n g t o n HeighLa. IL. ENIWICE was from New England Nvelear. Boston, U A. A f f i 4 e l IO, B i o 4 3 l P-LOO. and a11 e l e c t r a p h o r e . 1 8c h e m i c a l s were from Bio-Rad. R i c h o n d . U. Tiesve c ~ l f u r ' e u p p l i e s were o b t a i n e d a@ follow(l: plaaricware from Fdeon. Oxnard, CA, media and Santa Clara. C A , f e t a lC a l f serum from I r v i n e S C i e n L i f i c , I r v i n e . CA. supplemencefromGibco. RIarmaC1a. PlaeafaWaYLY. NJ. s p i n n felra af kr oB me l l e 0 Glass, I n c . . Cyradex-2 beads from V i n e l a n d , NJ. Thrombin (14W unita/og) was a k i n dg i f t of John Feolon 11, w h i l ue r o k i n a s e VY the generoua g i f t of Cenesio Mureno. ECF-BP Waa p r e p a r e d iu d e s c r i b e (d3 )A. T I I I was p u r i f i e d a8 d e s c r i b e de l s e w h e r e (9).
Anti-RI-I
IgG on t h e Farmation ofComplexes
Between Thrombinand
Pt-I.
Immunologic Characterizationof PN-I I m n o p r e e i p i t a r i o n of RI-I m d RI-1:thrombin
Compleree:
I ~ u n o p r e e i p I t a ~ i oof n RI-I:m e o ~ p l e x e ~ w i t hanti-RI-I IgC wall performed. ae s h m in f i g u r e &a. h r i f i e d RI-1:Th s p e c i f i c a l lpyr e c i p i t a t e dw. h i l ea b e l etdh r o m b i n was not. Rorein A-Sepharoee a l o n de i d mot p r e c i p i t a t e any l a b e l e d material. nor d i d non-specific IgG. It has a l s o been found t h a t anti-RI-I IgC d i d not p r e e i p i t n t e eomplexee of AT-I11 and 112SlI-Chrombin (Figure 4b). "a11 added to c e l l s To dia:over if RI-I could be m e t a b o l i c a l l yl a b e l e d .1 3 5 S I w r h i o n i n e i n the premence of m e t h i o n i n e - f r e e medium. After o v e r n i g h t incubation the mediafrom plates Figure 5 shovechatthe major h n o p r e e i p i t a t e dp r o t e i n was metabolically-labeled RI-I. This is t h ef i r s dt e m o n s t r a t i o nt h a t RI-I can be m e t a b o l i c a l l y l a b e l e d and immunoprecipitated. and t h i s should enable one t o c h a r a c t e r i z e Lhia protease i n h i b i t o r more f u l l y .
was incubatedwithanti-RI-I.
Anti-PN-I IgG Competes f o r RI-I Binding t o Cell.: Thrombin binds LO HF c e l l s i n two ways: M a complex v i t h RI-I. and alone at a r e c e p t o r k n a m to bindDIP-insctivstedthrombin (IO). No knornfunctionhas been a s c r i b e d to t h e 8 0 c a l l e d OIP-Th site; l e s st h a n 20% of addedthrombinbinds t o t h i sr e c e p t o r on HF c e l l s a t Since thrombinbindscellsprimarily as a complex w i t h RI-I. It vas d e e i r a b l et o 37'C (LO). see t haen t i - R I - I a e d i s t edde c r e a s e i n RI-1:Th binding. and in what form the binding Occurred. Cell culturea incubated for three days with serum-free media were t r e a t e dw i t h anti-RI-I IgC f o r two hours, followed by t haed d i t i o n of 112511-thrombin. M d e s c r i b e d earlier. The data shorn in f i g u r e s 6s and 6b d e n o n s t r a t teh de o s e d e p e n d e ndt e c r e a s e in t o anti-RI-I competition. Parallel cultures were c e l l u l a br i n d i n g Of 112511-thrombin due e i t h e er s s a y e df o cr e l l u l a br i n d i n g of [12SII-thrombin or lysed i n SDS sample bufferand analysed by SDS-PAGE and autoradiography. There was a c o r r e l a t i o n between loss ofcell-bound l a b e l due ro i n c r e a s e d anti-PN-I IgC and thedisappearance Of the 68 kD band from g e l s I n t h e p a r a l l e lC u l t u r e s . This i n d i c a r e sd i r e c t l yt h a tt h e action of mti-PN-I i s h i g h l ys p e c i f i c ; namely, if blocks the binding of 68 k0 112511-Th:PN-I complexes t o c e l l s .
0' 10
100
10
IgG. rglml
0
0
I12 SI I-Th: PN-I Complexes Formed
m
( X ofConrrol)
No IgC Re-immune IgC (250 u g / o l ) Anti-PN-I I@ (250 ug/nI) Anti-PN-I I& (2% ug/ml)
LOO 88.8 12.2 65.0
Table I . I n h i b i t i o n of Complex Formation Between Rerease Nexin-I and 112SII-rhrombin by Anti-PN-I I@.
M,
1.0
l
x 163
10 IgG a l m l
o
o
100
w
80
946 7-
430 10
100
IgG. elm1
30-
Figure 2. a.
I n h i b i t i o n of the Bindingof
A.
2 0-
RI-1:Rorease
(.
.;
(A.
6,
C0""ter.
b.
128 ng/ml [ 1 2 S I I - u r o k i n a ~ e(3000 cpmlng) or 80 ngld Il251I-EGP-BP (142.000 cpmlng) added t o 0.5 ml/vellconditioned = d i u in place of 11251I-Th. Controlwells and 26.7 pg IIZSII-ECF-BP s p e c i f i c a l l y bovnd 0.65 ng 112SII-urokinase
WM
C.
Figure I.
Complexes LO C e l l s by Anti-RI-I:
Confluentcultures of W c e l l s In 2 4 u e l l c u l t u r e p l a t e s w i t h 0.5 m l f r e s h or Z d s y c o n d i t i o n e d w d i a were incubatedfor 2 hourswith anti-RI-I IgC. followed by 2 hours w i t h M n g / d IIZSII-thrombin. e i t h e r a l o n e (12,Yx) epdng) or complexed u i r h Nonsondirioncd and p u r i f i e d RI-I from conditioned media (60.000 cpm/ng). conditioned wells bound 0 . B 08 and 0.13 ng of112SII-thrombin "oncondicloned; conditioned)respectivelywhilethey bound 1.19 08 and 0.16 n 112SlI-Chrombin in the form added eomp1e;el w i t h RI-I , nonTonditioned: c o n d i t i o n e d ) . a l l i n t h e abaenec of anti-RI-I. P l a t e s were washed with 3X with tcecold PBS. followed by l y s i ( l in boiling IM NaOH. Samples were counted i n II gama
h r i f i e d RI-I:
*(I
C o n d i t i o n e dr d i u m wae collected. concentrated.mdpaesed over heparin-Sepharose. deecribed i n M a t e r i a l s andI(ethods. RI-I was e l u t e dw i t h a g r a d i e n t of 0.2 t o 1.5 M NaC1. Praetiona containing RI-I were collected.concentrated. and p u r i f i e d over BioCel P-60. RI-I ( 1 5 us) vae analyzed by505-PAGE; gel was s t a i n e d w i t h CoomsasieBlue.
(0)
( A )and
Equal d i l u t i o n s of anti-ECP c e l l s c h a t had conditionedfor [IZSI1-Th.
(A).
anti-RI-I (.I were ineubared. 1u1 above, w i t h 2 d a y s , f o l l w e d by 2 hour incubstion with 160 ng/ol
Immunologic Charactcrization o f P.V-1
688
M,
x
H, X 103 '
cy
94-
94-
6 7-
43-
67 -
*
43-
3 0-
30-
A
43-
30-
2 0-
-
B
2014-
A
B
Immunologic Charactprimtion of PN-I
4 3-
302 0-
a b c d e f a h
i
i k
L
T
I ‘n