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Characterization of a high-molecular weight glycoprotein in bovine rod photoreceptor outer segment by… Wong, Simon Yuk Chun 1984

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CHARACTERIZATION OF A HIGH-MOLECOLAR WEIGHT GLYCOPROTEIN IN BOVINE ROD PHOTORECEPTOR OOTER SEGMENT BY A MONOCLONAL ANTIBODY By SIMON YUK CHUN WONG B . S c . , The U n i v e r s i t y o f B r i t i s h Columbia,1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY We accept t h i s the s i s as conforming to the reguired standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1984 fO Copyright by Simon Yuk Chun Wong, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Biochemistry  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 2 7 , 1984-A B S T R A C T A m o n o c l o n a l a n t i b o d y d e s i g n a t e d as 4B2 was u s e d t o i n v e s t i g a t e the molecular p r o p e r t i e s and s t r u c t u r a l organiza t ion of a 220,000 M g l y c o p r o t e i n , r e f e r r e d to as ROS 1.2, i n bovine r e t i n a l rod p h o t o r e c e p t o r o u t e r segment d i s k membranes. The approaches taken were f i r s t to p r o d u c e , c h a r a c t e r i z e , and p u r i f y the 4B2 a n t i b o d y , then to use the p u r i f i e d 4B2 a n t i b o d y as a l i g a n d f o r t h e p u r i f i c a t i o n o f ROS 1 .2 b y a f f i n i t y c h r o m a t o g r a p h y , and f i n a l l y t o d e t e r m i n e t h e amino a c i d composit ion of ROS 1.2 by amino a c i d a n a l y s i s of p u r i f i e d ROS 1.2. The 4B2 ant ibody was produced by growing the 4B2 hybridoma c e l l l i n e as a s c i t e s tumors i n mice o r as mass c u l t u r e . The former method proved to be e a s i e r and more economical . A l s o , the antibody concentra t ion i n a s c i t e s f l u i d was shown by sol id-phase radioimmune a s say to be 30-100 f o l d g r e a t e r than i n c u l t u r e supernatant. The 4B2 ant ibody was shown by radioimmune assay and sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s to be an IgM molecule w i t h p. heavy c h a i n s o f apparent 1X1^=55,000 and k l i g h t c h a i n s o f a p p a r e n t M r = 2 2 , 0 0 0 . The 4B2 a n t i b o d y was p u r i f i e d f rom a s c i t e s f l u i d b y D E A E - S e p h a c e l i o n exchange chromatography. The y i e l d o f t h i s 4B2 a n t i b o d y was low (0.5 mg/ml a s c i t e s f l u i d ) compared to the y i e l d of an ant i - rhodops in antibody (7 mg/ml a sc i t e s f l u i d ) ob ta ined from 1D4 hybridoma c e l l l i n e . ROS 1.2 c o u l d o n l y be p a r t i a l l y p u r i f i e d by a f f i n i t y chromatography on a 4B2 a n t i b o d y - a g a r o s e c o l u m n . As shown by sodium d o d e c y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , a r e s i d u a l amount o f r h o d o p s i n , the major rod outer segment (ROS) p r o t e i n , c o p u r i f i e d w i t h ROS 1 .2 . T h e r e f o r e , the amino a c i d composit ion of ROS 1.2 could not be determined. The 4B2 a n t i b o d y was u s e d t o p r o b e t h e s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f ROS 1.2 by d e t e r m i n i n g w h e t h e r i m m u n o l o g i c a l c r o s s - r e a c t i v i t y e x i s t s b e t w e e n ROS 1.2 and p r o t e i n s o f s i m i l a r i n o t h e r c e l l t ype s , n a m e l y , myosin i n muscle, s p e c t r i n i n red b l o o d c e l l s , and b r a i n s p e c t r i n ( f o d r i n , or c a l s p e c t i n ) i n b r a i n c e l l s . The 4B2 monoclonal a n t i b o d y was shown b y r a d i o immune c o m p e t i t i o n a s s a y s t o c r o s s - r e a c t w i t h r a b b i t s k e l e t a l muscle m y o s i n , b o v i n e b r a i n homogenate , and human red b l o o d c e l l membranes. Radioimmune l a b e l i n g s tudies indica ted t h a t the c r o s s - r e a c t i v i t y was due to a 1 6 0 , 0 0 0 M f p r o t e i n i n b o v i n e b r a i n h o m o g e n a t e . S i m i l a r radioimmune l a b e l i n g s t u d i e s were unab le to demonstra te which p r o t e i n i n red b l o o d c e l l membranes c r o s s - r e a c t w i t h the 4B2 m o n o c l o n a l a n t i b o d y . T h e s e s t u d i e s a l s o i n d i c a t e d t h a t the degraded fragments o f ROS 1.2 and myosin w i t h apparent o f 140,000 r e t a i n e d 4B2 a n t i g e n i c s i t e s . In a d d i t i o n , a c t i n was shown by radioimmune assay to be present i n rod outer segment prepara t ions . On the b a s i s o f these and o t h e r r e s u l t s , i t i s c o n c l u d e d that ROS 1.2 i s a m y o s i n - l i k e p r o t e i n , and i n c o n j u n c t i o n with other c y t o s k e l e t a l e lements such as a c t i n , s e r v e s to l i n k ROS d i s k s to each other or to ROS plasma membrane for the maintenance o f the h i g h l y - o r d e r e d ROS s t r u c t u r e . I t i s speculated that the l o s s o f t h i s ordered s t ruc ture dur ing the process of d i s k renewal invo lves the p r o t e o l y t i c degradation of ROS 1.2. -jv-ACKNOWLEDGEMENTS I would l i k e to thank D r . Rober t S. Molday for h i s exce l l en t s u p e r v i s i o n , p a t i e n c e , and c o n s t a n t encouragement and support . A l s o , I would l i k e t o t h a n k my c o l l e a g u e s , D a l e L a i r d , Don MacKenzie, Laur ie M o l d a y , and D r s . F i o n a M i l l a r and David Hicks f o r t h e i r superb t e c h n i c a l a s s i s t a n c e s , t h e i r many v a l u a b l e d i s c u s s i o n s , and most o f a l l , t h e i r f r i e n d s h i p s . - v i -LIST OF ABBREVIATIONS ATP adenosine 5'triphosphate BSA bovine serum albumin cGMP c y c l i c guanosine monophosphate CNBr cyanogen bromide Con A concanaval in A CTAB c e t y l t r i m e t h y l ammonium bromide DEAE d ie thylaminoethyl DMSO dimethyl su l fox ide EDTA ethylenediamine t e t r a a c e t i c ac id EGTA e t h y l e n e g l y c o l - b i s (^-aminoethyl e t h y l ) - N , N , N ' , N * - t e t r a a c e t i c ac id FCS f e t a l c a l f serum lg immunoglobulin M molecu la r weight r PBS phosphate-buffered s a l i n e RIA radioimmune assay ROS rod outer segment SDS sodium dodecyl su l f a te T r i s tr i s (hydroxymethyl) aminomethane - v i i -TABLE OF CONTENTS PAGE TITLE PAGE i ABSTRACT i i ACKNOWLEDGEMENTS V LIST OF ABBREVIATIONS v i TABLE OF CONTENTS v i i LIST OF TABLES x i i LIST OF FIGURES x i i i INTRODUCTION 1. Vertebrate r e t ina 1 2. Photoreceptors 1 3. Rods 3 4. Rod outer segment 5 (a) S t ructure (b) Disk renewal (c) Chemical composition 5. V i s u a l e x c i t a t i o n 9 6. Proposed mechanisms for v i s u a l e x c i t a t i o n 10 - v i i i -(a) Ca h y p o t h e s i s (b) cGMP hypothesis 2+ (c) Extended Ca h y p o t h e s i s w i t h a ro le for cGMP 7. Rod outer segment prote ins 17 8. I n i t i a l c h a r a c t e r i z a t i o n of ROS 1.2 18 9. Thes i s i n v e s t i g a t i o n 19 MATERIALS AND METHODS 1. Chemical reagents 20 2. P r o t e i n assay 20 3. Tissue c u l t u r e 20 (a) Maintenance of hybridoma c e l l l i n e s (b) Recloning of e s tab l i shed ant ibody-secret ing 4B2 hybridoma c e l l l i n e 4. Product ion of 4B2 monoclonal antibody 22 (a) Mass c u l t u r e of 4B2 hybridoma c e l l s (b) A s c i t e s tumors of 4B2 hybridoma c e l l s 5. C h a r a c t e r i z a t i o n of 4B2 monoclonal antibody 23 6. P u r i f i c a t i o n of 4B2 monoclonal antibody 24 (a) Chromatography of 4B2 a s c i t e s f l u i d and cu l ture supernatant on a DEAE-Sephacel column (b) Chromatography of 4B2 a s c i t e s f l u i d on a Sephacryl-S-500 column - i x -(c) Chromatography of 4B2 a s c i t e s f l u i d on a DEAE-Af f i -Ge l -B lue column (d) Comparison between rho-lD4 and 4B2 hybridoma c e l l l i n e s i n t h e i r a b i l i t y to produce monoclonal ant ibody 7. Standard so l id-phase radioimmune assay 26 (a) Preparat ion of antigens (b) Preparat ion of rad io labe led goat anti-mouse and goat a n t i - r a b b i t antibody reagents (c) Standard RIA procedure 8. Sol id-phase radioimmune competit ion assay 28 9. SDS-polyacrylamide g e l e l ec t rophores i s 29 and g e l t r ans fe r 125 10. Study on the e l e c t r o t r a n s f e r o f I - l abe led prote ins 30 11. Detec t ion of polypeptides which bind 31 monoclonal ant ibodies 12. P u r i f i c a t i o n of ROS 1.2 32 (a) Chromatography of rod outer segment prote ins on a Sephacryl-S-200 column (b) Chromatography of rod outer segment prote ins on a 4B2 antibody-agarose column RESULTS 1. Product ion of 4B2 monoclonal antibody 2. S p e c i f i c i t y of recloned 4B2 hybridoma c e l l l i n e 3. C h a r a c t e r i z a t i o n of 4B2 monoclonal antibody 34 36 36 -x-4. P u r i f i c a t i o n of 4B2 monoclonal antibody by ion 39 exchange chromatography on a DEAE-Sephacel column 5. P u r i f i c a t i o n of 4B2 monoclonal antibody by ge l 44 f i l t r a t i o n chromatography on a Sephacryl-S-500 column 6. P u r i f i c a t i o n of 4B2 monoclonal antibody by ion exchange 47 chromatography on a DEAE-Aff i -Gel -Blue column 7. Comparison between rho-lD4 and 4B2 hybridoma c e l l l i n e s 47 i n t h e i r a b i l i t y to produce monoclonal antibody 8. A c t i v i t y o f p a r t i a l l y - p u r i f i e d 4B2 monoclonal antibody 50 9. Detect ion of a c t i n i n ROS membrane preparat ions 50 10. C r o s s - r e a c t i v i t i e s of 4B2 monoclonal antibody 54 (a) Myosin (b) Red blood c e l l membranes and b r a i n homogenate 125 11. E l e c t r o t r a n s f e r o f 1 - l a b e l e d p r o t e i n s 61 (a) E f f e c t o f p r o t e i n molecular weight (b) E f f e c t of g e l pore s i ze 12. Cytoplasmic pool of 4B2 antigen i n ROS 64 13. Degradation and aggregation of ROS 1.2 68 14. P u r i f i c a t i o n of ROS 1.2 by g e l f i l t r a t i o n 72 chromatography on a Sephacryl-S-200 column 15. P u r i f i c a t i o n of ROS 1.2 by a f f i n i t y chromatography 72 on a 4B2 antibody-agarose column DISCUSSIONS 1. Product ion , c h a r a c t e r i z a t i o n ,and p u r i f i c a t i o n of 4B2 75 monoclonal antibody - x i -2. P u r i f i c a t i o n o f ROS 1.2 77 3. Immunological c r o s s - r e a c t i v i t i e s of 4B2 monoclonal 77 antibody to myosin, red blood c e l l membranes, and bra in homogenate 4. ROS 1.2 belongs to a f ami ly of immunologica l ly-re lated 79 pro te ins 5. Poss ib le s t r u c t u r a l r o l e of ROS 1.2 i n ROS 80 6. Propert ie s o f ROS 1.2 82 7. Future research on ROS 1.2 84 REFERENCES 86 - x i i -LIST OF TABLES I d e n t i f i c a t i o n of lg subtype of 4B2 monoclonal antibody P u r i f i c a t i o n of 4B2 monoclonal antibody from a s c i t e s f l u i d by DEAE-Sephacel column ch romatog raphy C r o s s - r e a c t i v i t i e s of 4B2 monoclonal antibody 125 E l e c t r o t r a n s f e r o f I - l abe led prote ins —xiii— LIST OF FIGURES Figure 1. I l l u s t r a t i o n of the l ayers of a ver tebrate r e t i n a . Figure 2. I l l u s t r a t i o n of a vertebrate rod photoreceptor . Figure 3. Schematic representat ion of the membrane topology o f rod outer segment. Figure 4. Schematic representat ion of the ca lc ium hypothesis for the mechanism of v i s u a l e x c i t a t i o n . Figure 5.' Schematic representat ion of the cGMP hypothesis for the mechanism of v i s u a l e x c i t a t i o n . Figure 6. Proposed l i g h t - a c t i v a t e d a m p l i f i c a t i o n c y c l e . Figure 7. Schematic representat ion of the extended ca lc ium hypothesis with a r o l e for cGMP model. Figure 8. T i t r a t i o n of 4B2 a s c i t e s f l u i d and c u l t u r e supernatant against ROS membranes. Figure 9 . /Analysis of ROS polypept ides which bind recloned 4B2 ant ibody. Figure 10. Chromatography of 4B2 a s c i t e s f l u i d on a DEAE-Sephacel column. Figure 11. Chromatography of 4B2 c u l t u r e supernatant on a DEAE-Sephacel column. - x i v -Figure 12. Chromatography of 4B2 a s c i t e s f l u i d on a Sephacryl-S-500 column. Figure 13. Chromatography of 4B2 a s c i t e s f l u i d on a DEAE-Af f i -Ge l -B lue column. Figure 14. E l e c t r o p h o r e t i c comparison of lg p u r i f i e d from 4B2 and rho-lD4 a s c i t e s f l u i d by DEAE-Sephacel column chromatography. Figure 15. T i t r a t i o n of p a r t i a l l y - p u r i f i e d 4B2 antibody against ROS membranes. Figure 16. T i t r a t i o n of a n t i - a c t i n antibody (2C2) against ROS membranes. Figure 17. T i t r a t i o n of 4B2 antibody against myosin. Figure 18. I n h i b i t i o n of 4B2 antibody binding to ROS membranes by myosin. Figure 19. T i t r a t i o n of 4B2 antibody against red blood c e l l s and b r a i n homogenate. Figure 20. I n h i b i t i o n of 4B2 antibody binding to ROS membranes by red blood c e l l s and b r a i n homogenate. Figure 21. Radioimmune l a b e l i n g of myosin, red blood c e l l s , and b r a i n homogenate with 4B2 ant ibody. Figure 22. E l e c t r o t r a n s f e r s of rad io- labe led myosin, BSA, and p r o l a c t i n . Figure 23. SDS-polyacrylamide ge l pore s i ze e f f ec t on the e l e c t r o t r a n s f e r s of r ad io- l abe led myosin and p r o l a c t i n . Figure 24. T i t r a t i o n of 4B2 and rho-lD4 ant ibodies against ROS and ROS d i sk membranes. -xv-I n h i b i t i o n of 4B2 and rho-lD4 ant ibodies b inding to ROS membranes by ROS and ROS d i sk membranes. Radioimmune l a b e l i n g of heat- treated ROS membranes with 4B2 ant ibody. Radioimmune l a b e l i n g of ROS membranes prepared i n the presence o f leupept in with 4B2 ant ibody. Chromatography of ROS prote ins on a Sephacryl-S-200 column. Chromatography of ROS prote ins on a 4B2 antibody-agarose column. Proposed ro le for ROS 1.2 i n ROS. INTRODUCTION 1. V e r t e b r a t e r e t i n a The v e r t e b r a t e r e t i n a has been the o b j e c t o f cons iderab le i n v e s t i g a t i o n f o r two main r e a s o n s . I t i s a r e a d i l y - a c c e s s i b l e por t ion of the c e n t r a l nervous system vvhich f a c i l i t a t e s the study of neurons , and i t i s the r e c e p t o r t i s s u e which i n i t i a t e s the complex process of v i s i o n . The r e t ina i s the innermos t l a y e r o f the e y e b a l l . I t i s a t h i n l a y e r o f n e u r a l t i s s u e c o n s i s t i n g o f pigment e p i t h e l i a l c e l l s , photoreceptor c e l l s , h igher-order neurons, and g l i a l c e l l s . These r e t i n a l elements are o r g a n i z e d into d i s t i n c t l ayer s of c e l l bodies and processes ( F i g . l ) . The r e t i n a has two func t ions . The f i r s t , performed by the p h o t o r e c e p t o r s , i s to t r a n s f o r m l i g h t energy i n t o n e r v e i m p u l s e s . The s e c o n d , p e r f o r m e d by the i n t r i c a t e a r r a y o f h i g h e r - o r d e r n e u r o n s , i s to i n t e g r a t e the l a rge number o f p h o t o r e c e p t o r s i g n a l s and t r a n s m i t the v i s u a l information on to the b r a i n v i a the o p t i c nerve. 2. Photo r e c e p t o r s The photoreceptor c e l l s are c l o s e l y packed i n the r e t i n a , and a x i a l l y or iented to the i n c i d e n t l i g h t . L ight must pass through almost the e n t i r e t h i c k n e s s o f the r e t i n a before impinging Figure 1. Diagram to i l l u s t r a t e the l a y e r s o f the r e t i n a (1) pigment e p i t h e l i u m (2) p h o t o r e c e p t o r s (3) e x t e r n a l l i m i t i n g membrane (4) o u t e r n u c l e a r l a y e r (5) o u t e r p l e x i f o r m l a y e r (6) inner nuclear l a y e r (7) i n n e r p l e x i f o r m l a y e r (8) g a n g l i o n c e l l l a y e r (9) o p t i c nerve f i b e r layer (10) i n t e r n a l l i m i t i n g membrane. Arrow indica tes the d i r e c t i o n of inc ident l i g h t ( as taken from reference 78) -3-upon the p h o t o r e c e p t o r c e l l s . L i g h t , not d e t e c t e d by the p h o t o r e c e p t o r s , i s a b s o r b e d b y t h e pigment p a r t i c l e s o f the adjacent pigment ep i the l ium. There are two types o f p h o t o r e c e p t o r c e l l s . They are c a l l e d rods and cones because o f t h e i r shape s . Cones funct ion i n b r i g h t l i g h t and are re spons ib le f o r c o l o r v i s i o n . Rods funct ion i n dim l i g h t and g ive achromat i c v i s i o n . The number o f rods and cones , and t h e i r d i s t r i b u t i o n i n a r e t i n a v a r i e s f r o m s p e c i e s to spec i e s . In humans, there are about three m i l l i o n cones and about a b i l l i o n rods d i s t r i b u t e d u n e g u a l l y throughout the r e t i n a . The highest c o n c e n t r a t i o n o f cones i s found i n the c e n t e r , whereas the highest c o n c e n t r a t i o n o f rods i s found away from the center of the r e t i n a . No rods are found i n the fovea r eg ion , and ne i ther rods nor cones are found i n the o p t i c d i s c o f the r e t i n a . 3 . R o d s The g r e a t e r s i z e and abundance o f rods compared to cones d i c t a t e t h e i r use i n many b i o c h e m i c a l s t u d i e s . Rods are s lender and e longated , and o c c u r i n a v a r i e t y o f shapes and s i z e s . For example, f rog rods are t y p i c a l l y 6-7p. i n diameter and 100-120u i n length whereas b o v i n e rods a re t y p i c a l l y l - 2 u i n d i amete r and 40-50>i i n l eng th . However, rods have a b a s i c s t ruc ture which i s s i m i l a r i n a l l v e r t e b r a t e s p e c i e s . Each rod c e l l i s composed of an outer segment connected to an inner segment by a t h i n c i l i u m , a nuc leus , and a synaptic body ( F i g . 2 ) . c i l i u m ELLIPSOID MYOID OUTER SEGMENT INNER SEGMENT NUCLEUS SYNAPTIC TERMINATION Figure 2. Schematic d iagram o f a v e r t e b r a t e rod photoreceptor . The o u t e r segment i s c o n n e c t e d to the i n n e r segment by a t h i n c i l i u m . E l l i p s o i d and myoid are c h a r a c t e r i s t i c r e g i o n s of the i n n e r segment. The n u c l e u s and s y n a p t i c t e r m i n a t i o n a r e a l s o shown ( as taken from reference 79 ). The inner segment c o n t a i n s a l l the m e t a b o l i c and synthe t i c machinery o f the c e l l . I t g e n e r a t e s ATP a t a very rap id rate and i s h i g h l y a c t i v e i n s y n t h e s i z i n g p r o t e i n s . I t c o n s i s t s of two d i s t i n c t r e g i o n s c a l l e d e l l i p s o i d and m y o i d . The e l l i p s o i d i s charac te r i zed by a v e r y dense a c c u m u l a t i o n o f mi tochondr ia . The myoid i s c h a r a c t e r i z e d b y f r e e r i b o s o m e s , rough endop la smic r e t i c u l u m , Golg i apparatus, and c o n t r a c t i l e p r o t e i n s . The synapt ic body i s the s i t e o f information t r ans fe r to the higher-order neurons. I t i s c h a r a c t e r i z e d by the presence o f many synapt ic v e s i c l e s , and the f o r m a t i o n of a chemical synapse with a b i p o l a r c e l l . 4. Rod o u t e r segment The outer segment i s the s i t e o f p h o t o t r a n s d u c t i o n . I t i s a h igh ly-ordered s t ructure c o n s i s t i n g o f hundreds o f stacked photoreceptor d i s k membranes t h a t a re o r i e n t e d perpendicular to i t s l o n g a x i s , and e n c l o s e d b y a p lasma membrane. Rod o u t e r segments have been the s u b j e c t o f numerous s t u d i e s dea l ing with the s t ruc ture o f b i o l o g i c a l membranes and the molecular bas i s o f v i s u a l e x c i t a t i o n . (a) S t r u c t u r e Both X-ray d i f f r a c t i o n measurements on e x c i s e d re t ina s (1) and f reeze- f rac ture s tudies (2) i n d i c a t e that the center - to-center - 6 -dis tance between adjacent d i s k s has a constant value of about 300 o A . The i n t e r n a l membrane s u r f a c e s o f a d i s k are separated by a 20 A^wide aqueous space ( 3 ) , which i s m a i n t a i n e d o v e r the e n t i r e e x t e n t o f the d i s k ( F i g . 3 ) . These s p e c i a l i z e d d i s k membranes occupy a r e l a t i v e l y compact vo lume , but p r o v i d e a l a rge surface area f o r embedding a l a r g e number o f p h o t o r e c e p t o r m o l e c u l e s , which i s r e g u i r e d f o r t h e h i g h s e n s i t i v i t y o f rods ( 4 ) . The mechanisms for the b i o g e n e s i s o f the rod outer segment and the maintenance o f i t s h i g h l y - o r d e r e d s t r u c t u r e a r e not f u l l y understood. (b) D i s k renewal Rod o u t e r segment (ROS) d i s k s h a v e b e e n f o u n d t o be cont inuous ly renewed (5,6) . A u t o r a d i o g r a p h i c s tud ie s have shown t h a t the new d i s k membrane components a re s y n t h e s i z e d i n the inner segment at one o r more s i t e s , then transported to the base of the outer segment where f i n a l membrane assembly takes p lace (7,8) . The d i sk s are formed by the invag ina t ion o f the ROS plasma membrane near the ROS b a s e , b u t a r e p i n c h e d o f f as t h e y a re d i s p l a c e d a p i c a l l y . C o n s e g u e n t l y , a l l the d i s k s except those c l o s e to the ROS base are c l o s e d , i n t r a c e l l u l a r v e s i c l e s , as i n d i c a t e d by e l e c t r o n m i c r o s c o p y (9) and e l e c t r o p h y s i o l o g i c a l o b s e r v a t i o n s ( 1 0 , 1 1 ) . The r a t e o f d i s k s y n t h e s i s v a r i e s from species to s p e c i e s . F o r f r o g s , the r a t e i s 1.2 d i s k s per hour , and a l l 1,700 d i sks i n a f rog ROS are renewed i n two months (12). -7-disk. plasma membrane c i l i a r y b r i d q e 3 disk repeat distance - 3 0 0 A disk thickness ~ 150 A calycal process f o l d i n g o f V p l a s m a m e m b r a n e Figure 3. Schematic representation of the membrane topology of rod outer segment. Disks are formed by the infolding of the plasma membrane at the basal end of the outer segment, and eventually pinch off the c e l l membrane and form isolated vesicles. Thickness of each disk and the repeat distance between disks are shown ( as taken from reference 4 ) . - 8 -For humans, a l l the d i s k s i n a ROS are renewed i n about 10 days (13). To m a i n t a i n the ROS a t a c o n s t a n t l e n g t h , the t i p s a re p e r i o d i c a l l y sloughed o f f to be p h a g o c y t i z e d and degraded by the pigment ep i the l ium. Thi s shedding process has been found to f o l l o w a d i u r n a l pa t tern i n a l a rge v a r i e t y of species (14). The o r d e r l y process of d i sk synthesis and des t ruc t ion appears to be extremely c r u c i a l to photoreceptor f u n c t i o n . I f the pigment ep i the l ium i s incapable of phagocytiz ing the shed d i s k s , the rods eventua l ly d i e o f f (15). The mechanism o f d i s k renewal remains to be determined. (c) C h e m i c a l c o m p o s i t i o n The s t r u c t u r e and f u n c t i o n o f ROS components have been inves t iga ted ex tens ive ly s ince ROS can be obtained i n high p u r i t y . Intact ROS membranes with e n c l o s e d cytoplasmic prote ins and small m o l e c u l e s c a n be r e l e a s e d f r o m t h e r e t i n a b y g e n t l e homogenization and p u r i f i e d by f l o t a t i o n on a sucrose g r a d i e n t . The d i s k membranes are i s o l a t e d by o smot i ca l ly shocking the plasma membrane and sealed d i s k s a re p u r i f i e d by f l o t a t i o n on 5% F i c o l l (16) . The d i sk membranes a re composed o f l i p i d s and pro te ins i n a b i l a y e r c o n f i g u r a t i o n . L i p i d to p r o t e i n r a t i o , on a dry weight b a s i s , i s about one. P h o s p h o l i p i d s represent more than 80% of the t o t a l l i p i d content ; c h o l e s t e r o l a c c o u n t s f o r only 3% (17) . The m a j o r p h o s p h o l i p i d s a r e p h o s p h a t i d y 1 e t h a n o l a m i n e , -9-phospha t idy lcho l ine , and phosphat idy l ser ine ; 44, 36, and 15% l i p i d phosphorous r e s p e c t i v e l y (18) . P o l y u n s a t u r a t i o n i n more than 50% of the t o t a l f a t t y a c i d c o m p o s i t i o n i n d i c a t e s the h i g h l y f l u i d nature o f these d i s k membranes. The e x i s t e n c e of a h i g h l y f l u i d l i p i d core i n d i sk membranes i s p o s t u l a t e d to serve the purposes o f phototransduct ion and i o n i c t r a n s l o c a t i o n , and a l low rhodopsin to be renewed r a p i d l y (18). SDS-polyacry lamide g e l e l e c t r o p h o r e s i s o f b o v i n e ROS d i s k membrane p r o t e i n s i n d i c a t e the p re sence o f a major component, rhodopsin, with an apparent 1^1^=34,000, a h i g h molecular weight p r o t e i n w i t h an apparent 1X1^=220,000, and s e v e r a l o t h e r minor components ranging i n M from 36,000 to 300,000 (16,19,20). 5. V i s u a l e x c i t a t i o n In the d a r k , the s o d i u m i o n p e r m e a b i l i t y o f ROS plasma membrane i s h i g h , a b i l l i o n s o d i u m i o n s per second ( 2 1 , 2 2 ) . Sodium ions r a p i d l y f l o w i n t o the o u t e r segment because of the ex i s t ence o f a l a r g e sodium c o n c e n t r a t i o n g r a d i e n t a c r o s s the plasma membrane, then d i f f u s e t o t h e i n n e r segment, and a re e x t r u d e d by N a + - K + - A T P a s e pumps i n the i n n e r segment plasma membrane to maintain the sodium concentrat ion grad ient . Absorpt ion of a photon causes the chromophore of rhodops in , 1 1 - c i s - r e t i n a l , to undergo a s e r i e s o f transformations that r e su l t i n the d i s s o c i a t i o n of a l l - t r a n s - r e t i n a l from the p r o t e i n to y i e l d b l e a c h e d r h o d o p s i n o r o p s i n ( 2 3 - 2 5 ) . T h i s c o n f o r m a t i o n a l -10-t r a n s i t i o n i n a s i n g l e r h o d o p s i n m o l e c u l e b l o c k s the entry of a m i l l i o n s o d i u m i o n s i n t o t h e ROS ( 2 6 ) . T h e r e s u l t i n g h y p e r p o l a r i z a t i o n o f the plasma membrane i s t r a n s m i t t e d to the synapse a t the o t h e r end o f the rod and communicated t o other c e l l s o f the r e t i n a . I t i s g e n e r a l l y a c c e p t e d t h a t an i n t e r n a l t r a n s m i t t e r i s e s s e n t i a l to communicate between r h o d o p s i n molecules i n the d i sk membranes and the sodium c h a n n e l s o f the plasma membrane. Two 2+ a t t r a c t i v e t r a n s m i t t e r c a n d i d a t e s a re Ca and cGMP. 6. Proposed mechanisms f o r v i s u a l e x c i t a t i o n 2 + (a) C a h y p o t h e s i s 2+ 2+ A c c o r d i n g t o t h e Ca h y p o t h e s i s (27) , Ca i o n s a re s tored i n the i n t r a d i s k a l space i n the d a r k . When induced by 2+ l i g h t , Ca i o n s a re r e l e a s e d i n t o the c y t o p l a s m i c s p a c e , and d i f fu se to the plasma membrane to b l o c k sodium channels (Fig.4) . 2 + E v i d e n c e i n f a v o r o f a C a t r a n s m i t t e r c o m e s f r o m 2+ e l e c t r o p h y s i o l o g i c a l exper iment s where the a d d i t i o n of Ca to the r e t i n a mimics the e f f ec t o f l i g h t (28-30) and the a d d i t i o n o f ca lc ium c h e l a t o r s such as EGTA makes the rod l e s s s e n s i t i v e to 2+ l i g h t (30,31) . F u r t h e r m o r e , many Ca i o n s a re extruded from an i l luminated ROS f o l l o w i n g a l i g h t p u l s e ( 3 2 , 3 3 ) , and s u f f i c i e n t 2+ Ca i s present i n ROS and i n the d i s k s f o r i t to be a v a i l a b l e as a t r a n s m i t t e r ( 3 4 , 3 5 ) . However, t h e r e i s c o n t r o v e r s y as to 2+ whether the number o f Ca r e l e a s e d from d i s k s into the cy to so l -11-Outer segment Na+. Na+> Ca2+ closes the channels for Na+ Discs containing * Ca2+ ions Light • • . •Na+ ~Na+ -Na+ Figure 4. Schematic r e p r e s e n t a t i o n o f the Ca h y p o t h e s i s for the mechanism o f v i s u a l e x c i t a t i o n ( as taken from reference 80) . Upper: In the d a r k , sodium c h a n n e l s on the plasma membrane are open and ca lc ium ions are s tored in s ide the d i s k s . Lower: L ight a c t i v a t i o n of rhodops in r e l e a s e s c a l c i u m ions from the d i sks in to the c y t o s o l . Ca lc ium i o n s then d i f f u s e d to plasma membrane where they block sodium channels . -12-on i l l u m i n a t i o n i s s u f f i c i e n t f o r t r a n s m i t t e r a c t i o n . A l s o , i t has not been e s t a b l i s h e d t h a t , i n the d a r k , d i s k s a c t i v e l y take „ 2 + up Ca (b) cGMP h y p o t h e s i s A c c o r d i n g t o t h e cGMP h y p o t h e s i s (26) , a h i g h cGMP concentra t ion i n the c y t o s o l i s r e q u i r e d to maintain the opening o f sodium c h a n n e l s i n the d a r k . When induced by l i g h t , cGMP concentra t ion f a l l s , r e s u l t i n g i n the c l o s u r e o f sodium channels ( F i g . 5 ) . E v i d e n c e i n f a v o r o f a cGMP t r a n s m i t t e r comes from biochemical and e l e c t r o p h y s i o l o g i c a l f i n d i n g s . ROS conta in about 30 juM of cGMP, an unusual ly h i g h c o n c e n t r a t i o n of cGMP (36). Thi s 5 cGMP l e v e l d e c r e a s e s marked ly on i l l u m i n a t i o n as more than 10 cGMP molecules are hydrolyzed per second per photolyzed rhodopsin molecule (37). Th i s high r a t e o f h y d r o l y s i s of cGMP i s accounted f o r b y t h e c y c l i c i n t e r a c t i o n s o f t r a n s d u c i n and cGMP p h o s p h o d i e s t e r a s e , two p e r i p h e r a l enzymes found i n ROS, w i t h photolyzed rhodopsin ( F i g . 6 ) . F u r t h e r m o r e , cGMP depolar izes the ROS plasma membrane w i t h i n m i l l i s e c o n d s o f i t s b e i n g i n j e c t e d in to the c e l l , and i n c r e a s e s the l a t e n c y o f the l i g h t - i n d u c e d h y p e r p o l a r i z a t i o n (38 ) . However, i t i s unknown as to how cGMP concentra t ion might regulate sodium channels . -13-Outer segment Na+< Na+^ Na+> c N^a+ N^a+ Na+ ••/••• Cyclic GMP molecules in cytosol Light Na+ channels are closed \ by the lowered level^  of cyclic GMP c •R- ( / t t Light decreases the level of cyclic GMP Figure 5. Schematic r e p r e s e n t a t i o n o f the cGMP h y p o t h e s i s f o r the mechanism of v i s u a l e x c i t a t i o n ( as taken from reference 80). Upper: In the d a r k , sodium c h a n n e l s on the plasma membrane are open due to a high concentra t ion o f cGMP i n the c y t o s o l . Lower : L i g h t a c t i v a t i o n o f r h o d o p s i n c a u s e s a d e c r e a s e i n c y t o s o l i c cGMP c o n c e n t r a t i o n , r e s u l t i n g i n the c l o s u r e of sodium channels . -14-F i g u r e 6 . P r o p o s e d l i g h t - a c t i v a t e d a m p l i f i c a t i o n c y c l e . In format ion f lows from p h o t o l y z e d r h o d o p s i n (R*) to t r a n d u c i n (T-GTP) and then to the phosphodiesterase (PDE*) ( as taken from reference 3 7 ) . -15-2+ (c) Extended Ca h y p o t h e s i s w i t h a r o l e f o r cGMP 2+ According to t h i s h y p o t h e s i s (39) , Ca i o n s are s tored i n the i n t r a d i s k a l space i n the d a r k . When induced by l i g h t , 2+ . Ca ions are r e l e a s e d i n t o the c y t o p l a s m v i a pores composed of m u l t i m e r i c r h o d o p s i n , and d i f f u s e to the plasma membrane to 2+ block sodium c h a n n e l s . To r e s t o r e the i n t r a d i s k a l Ca l e v e l 2+ p r e s e n t i n the d a r k , c y t o p l a s m i c Ca ions a re removed by a 2+ Ca -b ind ing p r o t e i n whose b i n d i n g c a p a c i t y i s increased by the 2+ decrease o f cGMP which f o l l o w s r h o d o p s i n b l e a c h i n g . Ca ions 2+ are then t r a n s p o r t e d to the i n t r a d i s k a l space by Ca -ATPase pumps ( F i g . 7 ) . 2+ E s s e n t i a l f ea tures o f t h i s m o d e l , not i n c l u d e d i n the Ca h y p o t h e s i s , a re t h a t m u l t i m e r i c r h o d o p s i n form pore s f o r the 2+ 2+ r e l e a s e o f i n t r a d i s k a l l y s t o r e d Ca , t h a t c y t o p l a s m i c Ca 2+ i o n s are removed by a Ca - b i n d i n g p r o t e i n , and t h a t cGMP 2+ regulates cy top la smic Ca by a f f e c t i n g the b ind ing capac i ty of 2+ t h e Ca - b i n d i n g p r o t e i n . T h e s e f e a t u r e s a r e n o t w e l l e s tab l i shed y e t . F o r example , r h o d o p s i n has not been shown to be an i o n i c p o r e . However, r h o d o p s i n i s an i n t e g r a l p r o t e i n spanning the ROS d i s k membrane w i t h 7 o ^ - h e l i c a l segments (40) c o n s i s t i n g o f mostly nonpolar amino ac ids (39). For i t to be an i o n i c p o r e , s e v e r a l r h o d o p s i n m o l e c u l e s a re r e g u i r e d to provide a s u f f i c i e n t number o f p o l a r s i d e - g r o u p s to e s t a b l i s h a h y d r o p h i l i c r e g i o n p e n e t r a t i n g t h e membrane. C a l m o d u l i n , a 2+ Ca -b ind ing p r o t e i n , has been r e p o r t e d to be present i n bovine -16-'-F i g u r e 7. S c h e m a t i c r e p r e s e n t a t i o n o f t h e e x t e n d e d Ca h y p o t h e s i s w i t h a r o l e f o r cGMP f o r the mechanism o f v i s u a l 2+ e x c i t a t i o n . A c t i o n o f Ca as i n t r a c e l l u l a r t r a n s m i t t e r i s shown i n the s e c t i o n on the r i g h t (A) . P h o t o - a c t i v a t i o n o f cGMP p h o s p h o d i e s t e r a s e i s s h o w n i n t h e m i d d l e s e c t i o n ( B ) . 2+ 2+ c G M P - c o n t r o l l e d Ca b i n d i n g a n d A T P - d r i v e n Ca -pump a c t i v i t y a re shown i n t h e s e c t i o n o f t h e l e f t (C) . S h o r t double-headed arrows i n d i c a t e p r o t e i n - p r o t e i n i n t e r a c t i o n . Other a c t i v e s i t e s on p r o t e i n s a re i n d i c a t e d as f o l l o w s : (O) s i t e o f 2+ r e v e r s i b l e Ca b i n d i n g ; (•) s i t e o f r e v e r s i b l e cGMP b i n d i n g ; (•) s i t e of GDP and GTP binding and of GTP h y d r o l y s i s ; (•) s i t e o f cGMP h y d r o l y s i s ; (•) s i t e of ATP h y d r o l y s i s ( as taken from reference 39 ). -17-ROS (41), but t h e r e i s no e v i d e n c e f o r the i n t e r a c t i o n of cGMP w i t h c a l m o d u l i n i n ROS. A l s o , cGMP has been r e p o r t e d to 2+ s t i m u l a t e Ca u p t a k e by ROS d i s k s s u s p e n d e d i n m e d i a c o n t a i n i n g h i g h - e n e r g y p h o s p h a t e e s t e r s and e l e c t r o l y t e s approximating the cytoplasmic c o m p o s i t i o n of l i v e rod c e l l s (42), 2+ but the mechanism o f c o n t r o l o f Ca i n ROS by cGMP remains to be c l a r i f i e d . 7. ROS p r o t e i n s R h o d o p s i n ha s b e e n t h e f o c u s o f r e s e a r c h on v i s u a l e x c i t a t i o n , s i n c e i t i s the i n i t i a l s i t e o f i n t e r a c t i o n w i t h l i g h t . I t represents 80-90% of the t o t a l p r o t e i n content of ROS (18) . I t i s a transmembrane us g l y c o p r o t e i n with i t s N-terminus f a c i n g t h e i n t r a d i s k a l s p a c e a n d C - t e r m i n u s f a c i n g t h e cytoplasmic space (53), and transverses the d i s k membranes with 7 h e l i c e s (43). The complete amino a c i d seguence of rhodopsin has been e l u c i d a t e d , but whether i t i s a p h o t o s e n s i t i v e channel or a m o d u l a t o r o f t h e cGMP l e v e l i n ROS o r b o t h r e m a i n s to be determined (43). S e v e r a l r e c e n t l y - i d e n t i f i e d b o v i n e ROS m i n o r p r o t e i n components have been s tudied i n o r d e r to c l a r i f y the mechanism of v i s u a l e x c i t a t i o n . These i n c l u d e cGMP phosphodiesterase (44-46), t ransducin (37) or G - p r o t e i n (47) , o r guanine nuc leot ide-b ind ing p r o t e i n (48), o p s i n k i n a s e ( 4 9 , 5 0 ) , and a h i g h molecular weight p r o t e i n re ferred to as ROS 1.2 (20). -18-8. I n i t i a l c h a r a c t e r i z a t i o n o f ROS 1.2 ROS 1.2 c o n s t i t u t e s about 1-3% o f the t o t a l p r o t e i n content i n ROS. I t has been shown t o be a transmembranous g lycopro te in which binds the l e c t i n s Con A and WGA ( 2 0 , 5 1 ) . L e c t i n b inding s tudies on s e a l e d d i s k s (52,53) and Con A l a b e l i n g s tudies for e l e c t r o n m i c r o s c o p i c a n a l y s i s ( 5 3 - 5 6 ) i n d i c a t e t h a t l e c t i n - s p e c i f i c o l i g o s a c c h a r i d e c h a i n s a re o r i e n t e d toward the i n t r a d i s k a l s p a c e . P r o t e a s e - s e n s i t i v e segments (20,53) and ant igen ic s i t e s o f ROS 1.2 (57) a re exposed on the cytoplasmic surface of sealed d i s k s . A monoclonal antibody re ferred to as 4B2 has been shown to be s p e c i f i c f o r ROS 1.2 ( 5 7 ) . B i o c h e m i c a l , i m m u n o l o g i c a l and e l e c t r o n m i c r o s c o p i c l a b e l i n g s t u d i e s u s i n g 4B2 monoc lona l antibody have shown t h a t the 4B2 a n t i g e n i c s i t e o f ROS 1.2 i s r e a d i l y - a c c e s s i b l e on the c y t o p l a s m i c s ide o f sealed d i s k s . A l s o , ROS 1.2 can be c l e a v e d w i t h t r y p s i n i n t o a M r=140,000 fragment which b i n d s 4B2 monoc lona l a n t i b o d y and a M r =120,000 fragment which binds Con A (57). 9. T h e s i s i n v e s t i g a t i o n The aim of t h i s p r e s e n t p r o j e c t i s to f u r t h e r charac ter i ze ROS 1.2 using the 4B2 monoc lona l a n t i b o d y and determine i t s ro le i n the ROS. T h i s t h e s i s r e p o r t s the r e s u l t s o f the produc t ion , -19-c h a r a c t e r i z a t i o n , and p u r i f i c a t i o n o f t h e 4B2 m o n o c l o n a l a n t i b o d y , t h e i m m u n o l o g i c a l c r o s s - r e a c t i v i t i e s o f the 4B2 monoclonal antibody to muscle m y o s i n , red blood c e l l membranes , and b r a i n homogenate, the d e t e c t i o n o f a c t i n i n ROS preparat ions , and t h e p u r i f i c a t i o n o f ROS 1 . 2 . The s i g n i f i c a n c e o f a c y t o s k e l e t a l network i n the ROS, and the pos s ib le ro le o f ROS 1.2 i n maintaining the h ighly-ordered ROS s t ructure are d i scus sed . -20-MATERIALS AND METHODS 1. C h e m i c a l r e a g e n t s A l l g e n e r a l l a b o r a t o r y c h e m i c a l s o f reagent grade were obtained from e i t h e r F i s h e r S c i e n t i f i c , Sigma C h e m i c a l , or BDH Chemicals . 2. P r o t e i n a s s ay P r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d by the method o f Lowry et a l . (58), using BSA as a s tandard. 3. T i s s u e c u l t u r e A l l t i s sue c u l t u r e media, s e r a , a n t i b i o t i c s , and accessor ies were obtained from Grand Island B i o l o g i c a l Company. (a) Maintenance o f hybr idoma c e l l l i n e s Hybridoma c e l l l i n e s were grown i n a RPMI 1640 cu l ture medium supplemented with 2mM L - g l u t a m i n e , 24mM sodium bicarbonate , 2mM sodium pyruvate , 10% f e t a l c a l f serum, 5% hea t - inac t iva ted horse serum, 100 u n i t s / m l p e n i c i l l i n , 100 jug/ml s treptomycin, and 1.25 >ig/ml f u n g i z o n e . The c e l l s were grown a t 3 7 ° C i n an atmosphere - 2 1 -o f 10% C 0 2 , 90% a i r i n a h u m i d i f i e d i n c u b a t o r . G r o w i n g c u l t u r e s were m a i n t a i n e d i n 1 0 - m l c u l t u r e d i s h e s a t 5 c o n c e n t r a t i o n s r a n g i n g f r o m 4 - 1 0 x 1 0 c e l l s / m l . T h e c o n c e n t r a t i o n o f v i a b l e c e l l s was de te rmined by s t a i n i n g c e l l c u l t u r e s w i t h e q u a l volume o f 0.4% t r y p a n b l u e and c o u n t i n g unstained c e l l s wi th a hemacytometer. Storage of hybridoma c e l l s was done by cent r i fug ing 10 ml of a growing c u l t u r e at 1 ,500 rpm f o r 5 m i n . C e l l p e l l e t was resuspended i n 5 ml of 10% DMSO i n supplemented RPMI 1640 c u l t u r e medium. C e l l s were t h e n t r a n s f e r r e d t o f r e e z i n g v i a l s and immediately p l a c e d i n a box i n s u l a t e d w i t h s t y r o f o a m . The box was l e f t i n a - 7 0 ° C f r e e z e r . The v i a l s were f i n a l l y placed i n a l i q u i d n i t rogen f reezer a f t e r 24h. Frozen c e l l s were r e t r e i v e d by g u i c k l y thawing and washing the c e l l s w i t h 10 ml o f n o n - s u p p l e m e n t e d RPMI 1640 c u l t u r e medium. The c e l l p e l l e t was t h e n r e s u s p e n d e d i n 10 ml o f supplemented RPMI 1640 c u l t u r e medium. (b) R e c l o n i n g o f e s t a b l i s h e d a n t i b o d y - s e c r e t i n g 4B2  hybr idoma c e l l l i n e Ant ibody-secre t ing 4B2 hybr idoma c e l l l i n e was recloned by l i m i t i n g d i l u t i o n . About 230 l i v e 4B2 hybridoma c e l l s were 7 suspended i n 4 . 6 ml o f a c l o n i n g medium c o n s i s t i n g o f 10 BALB/c mouse thymocytes per ml o f supplemented RPMI 1640 c u l t u r e medium. A l i g u o t s (100 jal) o f t h i s su spens ion were used to p la te 36 we l l s o f a 9 6 - w e l l c u l t u r e p l a t e , such t h a t an average of 5 c e l l s were seeded i n each w e l l . To the remain ing 1 ml of c e l l -22-suspension, 4 ml of c lon ing medium were added. A l i q u o t s (100>ul) o f t h i s suspension were used to p l a t e another 36 w e l l s , g i v i n g an average of one c e l l per w e l l . A t day 5 and a g a i n a t day 1 2 , a l i q u o t s (100 J J I ) o f the c lon ing medium were added to each w e l l o f the c lon ing p l a t e . The growth o f c l o n e d c e l l s was m o n i t o r e d under a l i g h t microscope. Wel l s with c e l l growth were then t e s t e d f o r a n t i b o d y a c t i v i t y . P o s i t i v e c lones were expanded and s tored as descr ibed above. 4. Product ion o f 4B2 monoc lona l antibody BALB/c mice were o b t a i n e d from the Animal Care Unit o f the U n i v e r s i t y o f B r i t i s h C o l u m b i a . P r i s t a n e was o b t a i n e d from A l d r i c h . (a) Mass c u l t u r e The 4B2 hybridoma c e l l s were grown and expanded cont inuous ly for a 2-week p e r i o d . C u l t u r e s u p e r n a t a n t s were c o l l e c t e d and s t o r e d i n 0.1% NaN_. -23-(b) A s c i t e s tumors The 4B2 hybr idoma c e l l s were grown as a s c i t e s tumors i n BALB/c mice that had been i n j e c t e d i n t r a p e r i t o n e a l l y with 0.5 ml g o f p r i s t ane 7 days p r i o r to i n j e c t i o n o f 5-10x10 c e l l s . When a s c i t e s tumors became n o t i c e a b l e , t u m o r - b e a r i n g mice were b l e d . A s c i t e s f l u i d was c o l l e c t e d from the blood by cen t r i fuga t ion at 12,000 rpm for 20 min and s t o r e d i n a - 7 0 ° C freezer u n t i l used. 5. C h a r a c t e r i z a t i o n o f 4B2 monoclonal antibody The l g sub type o f 4B2 monoc lona l a n t i b o d y was i d e n t i f i e d using a Mouse l g Subtype I d e n t i f i c a t i o n K i t (Boehringer Mannheim B i o c h e m i c a l s ) and t h e i n d i r e c t s o l i d - p h a s e r a d i o i m m u n e assay d e s c r i b e d i n 6(c) except the f o l l o w i n g : A f t e r incubat ion with 4B2 hybridoma c u l t u r e f l u i d , the w e l l s were incubated with 25 jal o f r a b b i t ant i -mouse l g s u b t y p e - s p e c i f i c a n t i b o d i e s or preimmune s e r u m i n RIA b u f f e r f o r 60 min a t 2 3 ° C . A f t e r w a s h i n g i n P B S , t h e w e l l s w e r e i n c u b a t e d w i t h 25 ju l o f 125 6 I - l a b e l e d g o a t - a n t i - r a b b i t l g (15-40 Mg/ml ; 1-2x10 dpm/ug) i n RIA b u f f e r f o r 30 min at 2 3 ° C . The molecular we ight s o f the heavy and l i g h t chains of 4B2 a n t i b o d y w e r e e s t i m a t e d f r o m t h e i r m o b i l i t i e s i n SDS-polyacrylamide g e l a f t e r g e l e l ec t rophore s i s . -24-6. P u r i f i c a t i o n o f 4B2 monoclona l antibody DEAE-Sephacel and S e p h a c r y l - S - 5 0 0 beads were obtained from Pharmacia. DEAE-Af f i -Ge l -B lue beads were obtained from Bio-Rad. (a) C h r o m a t o g r a p h y o f 4B2 a s c i t e s f l u i d and c u l t u r e  supernatant on a DEAE-Sephace l column One volume o f 4B2 a s c i t e s f l u i d o r c u l t u r e supernatant was d i l u t e d with t h r e e volumes of PBS and c e n t r i f u g e d at 10,000 rpm for 10 min. The s u p e r n a t a n t was p r e c i p i t a t e d with an equal volume o f s a t u r a t e d ammonium s u l f a t e s o l u t i o n a t 4 ° C , f o r at l e a s t an hour. I t was then c e n t r i f u g e d ' a t 10,000 rpm for 10 min. The p e l l e t was resuspended i n 20mM T r i s - a c e t a t e , pH 7.9 (Buffer A ) , c o n t a i n i n g 40mM N a C l , a n d d i a l y z e d a g a i n s t B u f f e r A c o n t a i n i n g 20mM N a C l , o v e r n i g h t a t 4 ° C . The d i a l y z a t e was a p p l i e d to a D E A E - S e p h a c e l column (5-6 mg p r o t e i n / m l column) p r e - e q u i l i b r a t e d w i t h B u f f e r A c o n t a i n i n g 20mM N a C l . Ant ibody m o l e c u l e s were e l u t e d w i t h a c o n t i n u o u s NaCl c o n c e n t r a t i o n gradient (20-400mM) i n B u f f e r A . The NaCl concentrat ion of each f r a c t i o n was determined by c o n d u c t i v i t y measurement. F rac t ions were tested for a n t i b o d y a c t i v i t y by the s tandard RIA procedure descr ibed below, and a s sayed f o r absorbance at 280 nm. Pro te in composition of f r a c t i o n s was a n a l y z e d by SDS-polyacrylamide g e l e l ec t rophores i s as d e s c r i b e d b e l o w . F r a c t i o n s conta ining a c t i v e 4B2 monoc lona l a n t i b o d y were p o o l e d and l y o p h i l i z e d . In some experiments, a n t i b o d y m o l e c u l e s were e l u t e d w i t h lOOmM NaCl i n Buffer A . The r e m a i n i n g p r o t e i n s were e l u t e d w i t h 1M NaCl i n -25-Buffer A . (b) Chromatography of 4B2 a s c i t e s f l u i d on a Sephacryl-S-500  c o l u m n One volume o f 4B2 a s c i t e s f l u i d was d i l u t e d w i t h t h r e e vo lumes o f PBS and c e n t r i f u g e d a t 1 0 , 0 0 0 rpm f o r 10 m i n . S u p e r n a t a n t was a p p l i e d t o a S e p h a c r y l - S - 5 0 0 c o l u m n p r e - e q u i l i b r a t e d w i t h PBS. F r a c t i o n s (1 ml) were treated as i n 4 (a ) , except that the p o o l e d f r a c t i o n s were not l y o p h i l i z e d , but s t o r e d i n 0.1% NaN^ a t 4 ° C . ( c ) C h r o m a t o g r a p h y o f 4 B 2 a s c i t e s f l u i d o n a  D E A E - A f f i - G e l - B l u e column One m l o f 4 B 2 a s c i t e s f l u i d was a p p l i e d t o a D E A E - A f f i - G e l - B l u e column (6 ml) p r e - e q u i l i b r a t e d w i t h 0.02M K 2 H P ° 4 ' ^ H 8 . 0 . A n t i b o d y m o l e c u l e s were o b t a i n e d i n the f i r s t f r a c t i o n s to flow through the c o l u m n , and the remaining prote ins were e l u t e d w i t h 1.4M N a C l . F r a c t i o n s (1 ml) were t r e a t e d as descr ibed i n 4 (b ) . (d) Comparison between r h o - l D 4 and 4B2 hybridoma c e l l l i n e s The 4B2 and r h o - l D 4 a n t i b o d i e s were p u r i f i e d from e q u a l volumes of 4B2 and r h o - l D 4 a s c i t e s f l u i d s by chromatography on a DEAE-Sephacel column a's d e s c r i b e d i n 4 ( a ) . The apparent M of the heavy and l i g h t c h a i n s o f b o t h a n t i b o d i e s , as w e l l as the amount of each antibody p u r i f i e d were compared. -26-7. Standard s o l i d - p h a s e radioimmune assay M i c r o t i t e r w e l l s were o b t a i n e d from F a l c o n . Chloramine-T was o b t a i n e d from Eastman O r g a n i c C h e m i c a l s and c a r r i e r - f r e e 125 I was o b t a i n e d from Amersham. Phosphate-buffered s a l i n e c o n t a i n s 8g of NaCl , 0.2g of KC1, 0 . 2 g K H 2 P 0 4 , a n d 2 . 1 g o f N a 2 H P 0 4 * 7 H 2 0 p e r l i t e r o f d i s t i l l e d H 2 0 . (a) P r e p a r a t i o n o f a n t i g e n s Intact ROS and s e a l e d ROS d i s k s were i s o l a t e d from frozen bovine r e t i n a s (Hormel) u s i n g the method o f Smith et a l . (16). Red blood c e l l g h o s t s were p r e p a r e d from human red blood c e l l s u s i n g the method o f Dodge e t a l . ( 5 9 ) . B r a i n homogenate was prepared from f r o z e n b o v i n e b r a i n s u s i n g the method of Burridge et a l . (60). P u r i f i e d r a b b i t s k e l e t a l musc le myos in and a c t i n were obtained from Sigma Chemicals . (b) P r e p a r a t i o n o f r a d i o l a b e l e d goat ant i -mouse and goat  a n t i - r a b b i t a n t i b o d y reagent's" G o a t a n t i - m o u s e l g was p a r t i a l l y p u r i f i e d f r o m g o a t anti-mouse a n t i s e r a ( A n t i b o d i e s I n c o r p o r a t e d ) by ion exchange chromatography on a DEAE-Sephacel column. Goat a n t i - r a b b i t lg was p u r i f i e d by a f f i n i t y chromatography on a r a b b i t Ig-Sepharose 4B column. Goat anti-mouse l g and goat a n t i - r a b b i t lg were labeled -27-125 6 w i t h I ( s p e c i f i c a c t i v i t y o f 1-2x10 dpm/iug) u s i n g the 125 Chloramine-T method (61) . F ree I was s e p a r a t e d from e i t h e r 125 125 I - l a b e l e d g o a t a n t i - m o u s e l g o r I - l a b e l e d g o a t a n t i - r a b b i t l g by g e l f i l t r a t i o n chromatography on a B i o - G e l P-6DG column ( Bio-Rad ) . (c) S t andard RIA procedure Ant ibody- secre t ing hybr idomas were d e t e c t e d by an i n d i r e c t s o l i d - p h a s e radioimmune a s s ay p r o c e d u r e o f Molday & MacKenzie (57). Bleached ROS or ROS d i s k membranes (2.5 mg/ml protein) were s o l u b i l i z e d with 1% (w/v) T r i t o n X-100 and d i l u t e d to 0.25 mg/ml with d i s t i l l e d H 2 0 . A l i q u o t s (25 / i l ) were d r i e d on f lex v i n y l o m i c r o t i t e r w e l l s a t 60 C f o r 2 h . The w e l l s were then r i n s e d with d i s t i l l e d H,,0 and n o n - s p e c i f i c b i n d i n g s i t e s were quenched with RIA b u f f e r c o n s i s t i n g o f 1% BSA (Sigma) , 1% FCS, and 0.1% NaN^ i n PBS. A f t e r the RIA b u f f e r was removed, the we l l s were incubated with 25 jal o f hybr idoma c u l t u r e f l u i d or a s c i t e s f l u i d for 60 min a t 2 3 ° C , washed i n PBS, and then i n c u b a t e d with 25 125 6 jj.1 o f I - l a b e l e d goat a n t i - m o u s e l g (15-40 jug/ml ; 1-2x10 dpm/ug) i n RIA b u f f e r f o r 30-60 min a t 2 3 ° C . The p l a t e s were then r i n s e d e x t e n s i v e l y i n PBS, c u t i n t o i n d i v i d u a l w e l l s and counted i n a Beckman 8000 gamma counter . In some exper iments , o t h e r a n t i g e n s such as a c t i n , myosin, b r a i n homogenate, and red b l o o d c e l l preparat ion were immobilized on m i c r o t i t e r w e l l s , and a n t i b o d y b i n d i n g was a s s a y e d as - 2 8 -described above. 8. Sol id-phase radioimmune compet i t ion assay Competition assays were used to s tudy the cross r e a c t i v i t i e s o f the 4B2 monoc lona l a n t i b o d y , and to d e t e c t the d i f f e r e n t concentrat ions of 4B2 a n t i g e n i n ROS and ROS d i s k s . B r i e f l y , 25 J J I o f varying c o n c e n t r a t i o n s o f r a b b i t muscle myosin, bovine ROS membranes, ROS d i sk membranes, b o v i n e b r a i n homogenate, or human red blood c e l l ghost p r e p a r a t i o n s , i n RIA buf fer was incubated at o 23 C w i t h 25 >ul o f h y b r i d o m a c u l t u r e f l u i d d i l u t e d t o a c o n c e n t r a t i o n which g i v e s 80-90% s a t u r a t i o n o f b i n d i n g by the standard s o l i d - p h a s e RIA. A f t e r l h , 25 jil o f the mixture was removed and s c r e e n e d f o r r e m a i n i n g a n t i b o d y a c t i v i t y by the standard sol id-phase RIA as descr ibed i n 6 ( c ) . 9. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and g e l t rans fer A l l c h e m i c a l s and a p p a r a t u s e s f o r S D S - p o l y a c r y l a m i d e ge l e l e c t r o p h o r e s i s and g e l t r a n s f e r were o b t a i n e d from B i o - R a d , except cyanogen bromide ( F i s h e r S c i e n t i f i c ) and^^-mercptoethanol (Eastman Organic Chemica l s ) . Samples with vary ing c o n c e n t r a t i o n s (0.3-8.0 irg/ml protein) were s o l u b i l i z e d i n an equal-volume mixture conta ining 5% SDS, 40% -29-s u c r o s e , lOmM T r i s , pH 6 . 8 , 10% ^ - m e r c a p t o e t h a n o l , and 4% bromophenol b l u e . Samples (20-25 ;ul) were a p p l i e d to a 6%, 7%, 8%, 9%, or 10% p o l y a c r y l a m i d e s l a b g e l ( 0 .15x5 .0 cm or 0.15x12 cm ) and e l e c t r o p h o r e s i s was c a r r i e d o u t a c c o r d i n g t o the procedure of Laemmli (62 ) . G e l s l i c e s were e i t h e r s ta ined with Coomassie blue (63) o r s u b j e c t e d to e l e c t r o p h o r e t i c t rans fer as adapted from the b l o t t i n g t r a n s f e r p r o c e d u r e o f B h u l l a r et a l . (64) . Unstained SDS ge l s were washed with three 100-ml changes of 0.1M sodium p h o s p h a t e , pH 7 . 4 , c o n t a i n i n g 0.1% SDS over 20 min and two 100-ml changes o f 20mM sodium p h o s p h a t e , pH 7.4 , over a 10-min p e r i o d . The washed g e l was sandwiched against a wet sheet of CNBr-activated paper , p r e p a r e d by the method of Clarke et a l . (65) , and p l a c e d i n a T r a n s b l o t a p p a r a t u s . E l e c t r o p h o r e t i c t rans fer was c a r r i e d out a t 35V and 1.5A a t 4 ° C i n 20mM sodium phosphate, pH 7 .4 , for 8-12h. F o l l o w i n g t r a n s f e r s , the remaining r e a c t i v e groups on the C N B r - a c t i v a t e d paper were quenched by incubat ion i n T r i s b u f f e r , pH 9 . 0 , conta in ing 60mM g l y c i n e and 1% BSA, o v e r n i g h t a t 2 3 ° C . The paper c o n t a i n i n g the t r a n s f e r r e d prote ins was r insed i n d i s t i l l e d H 2 0 , a i r - d r i e d , and stored i n a d e s i c c a t o r a t 4 ° C u n t i l used. R e c e n t l y , g e l s l i c e s were s u b j e c t e d to e l e c t r o p h o r e t i c t r a n s f e r as adapted from t h e b l o t t i n g t r a n s f e r p r o c e d u r e o f Towbin et a l . (66). SDS g e l s were washed with two 100-ml changes o f 20mM T r i s - a c e t a t e , pH 7 . 4 , c o n t a i n i n g 2mM EDTA and 0.01% SDS -30-over 20 min. . The washed g e l was sandwiched against a wet sheet of n i t r o c e l l u l o s e p a p e r ( B i o - R a d ) and p l a c e d i n a T r a n s b l o t apparatus. E l e c t r o p h o r e t i c t r a n s f e r was c a r r i e d out at 35V and 1.5A a t 4 ° C i n 20mM T r i s - a c e t a t e , pH 7 . 4 , c o n t a i n i n g 2mM EDTA and 0.01% SDS f o r 8-12h. F o l l o w i n g t r a n s f e r s , the remaining b i n d i n g s i t e s on t h e n i t r o c e l l u l o s e p a p e r were guenched by incubat ion i n immunoblot b u f f e r , pH 7 . 4 , c o n t a i n i n g 0.15M NaCl , lOmM sodium p h o s p h a t e , ImM E D T A , ImM NaN^/ and 0.2% T r i t o n X-100, with 4% BSA , o v e r n i g h t at 2 3 ° C . The paper conta ining the t r a n s f e r r e d p r o t e i n s was r i n s e d i n d i s t i l l e d H^O and used immediately. 125 10. S tudy on the e l e c t r o t r a n s f e r o f I - l a b e l e d p r o t e i n s Myosin (200K), BSA (66K) , and p r o l a c t i n (23K) were labeled 125 6 w i t h I ( s p e c i f i c a c t i v i t y o f 1-2x10 dpm/jug) u s i n g the 125 C h l o r a m i n e - T method ( 6 1 ) . S a m p l e s o f I - l a b e l e d myosin ( 1 . 5 x l 0 5 d p m / w e l l ) , 1 2 5 I - l a b e l e d BSA ( 2 . 2 x l 0 6 d p m / w e l l ) , and 125 5 I - l a b e l e d p r o l a c t i n (2.0x10 dpm/wel l ) were a p p l i e d to 8% SDS-polyacrylamide s l ab g e l s and e l e c t r o p h o r e s i s was c a r r i e d out as descr ibed p r e v i o u s l y . A f t e r e l e c t r o p h o r e s i s , unstained ge l s l i c e s w e r e s u b j e c t e d t o e l e c t r o p h o r e t i c t r a n s f e r t o n i t r o c e l l u l o s e paper s u s i n g the b l o t t i n g t r a n s f e r procedure of Towbin e t a l . ( 66 ) . A t d i f f e r e n t t imes a f t e r e l e c t r o p h o r e t i c t r a n s f e r , c o r r e s p o n d i n g s t r i p s o f g e l and paper s were removed, a i r - d r i e d , and exposed to X - r a y f i l m as d e s c r i b e d be low. The -31-polypeptide bands d e t e c t e d by a u t o r a d i o g r a p h y were excised and counted i n a Beckman 8000 gamma counter . S i m i l a r s tudies were done u s i n g 6% and 9% SDS-polyacrylamide 125 s l a b g e l s f o r the e l e c t r o t r a n s f e r o f I - l a b e l e d myosin and 125 I - l a b e l e d p r o l a c t i n , r e s p e c t i v e l y . 11. Detect ion of p o l y p e p t i d e s which b i n d monoclonal ant ibodies Transfer CNBr-act ivated papers were r insed i n RIA buffer and incubated with 10-ml o f hybr idoma c u l t u r e f l u i d conta ining mouse monoc lona l a n t i b o d y f o r l h a t 2 3 ° C . The p a p e r s were then w a s h e d w i t h s e v e r a l c h a n g e s o f PBS c o n t a i n i n g 0.4% N - l a u r o y l s a r c o s i n e o v e r a p e r i o d o f l - 2 h and s u b s e q u e n t l y 125 i n c u b a t e d w i t h 10-15 ml o f I - l a b e l e d goat ant i -mouse l g ( l - 2 x l 0 6 dpm/ml) i n RIA b u f f e r f o r l h at 2 3 ° C . F i n a l l y , the p a p e r s were washed e x t e n s i v e l y w i t h PBS c o n t a i n i n g 0.4% N - l a u r o y l s a r c o s i n e o v e r l - 2 h , a i r - d r i e d , and s u b j e c t e d to a u t o r a d i o g r a p h y on Kodak R o y a l X-Omat f i l m w i t h an X - r a y i n t e n s i f y i n g s c r e e n f o r 6-48h at 2 3 ° C . T r a n s f e r n i t r o c e l l u l o s e paper s were t r e a t e d as d e s c r i b e d above except that the paper s were washed with f i v e 50-ml changes o f immunoblot b u f f e r , pH 7 . 4 , c o n t a i n i n g 0.15M NaCl , lOmM sodium phosphate, ImM EDTA, ImM NaN^r and 0.2% T r i t o n X-100. Thi s was followed by one washing w i t h 0.1M g l y c i n e b u f f e r conta in ing 1% T r i t o n X-100, and 2M urea . -32-12. P u r i f i c a t i o n o f ROS 1.2" S e p h a c r y l - S - 2 0 0 beads were o b t a i n e d from Pharmacia and REACTI-GEL (6X) , a h i g h l y a c t i v a t e d imidazo ly l -ca rbamate matrix o f 6% c r o s s - l i n k e d agarose beads was obtained from P i e r c e . (a) G e l f i l t r a t i o n chromatography o f ROS pro te ins on a  S e p h a c r y l - S - 2 0 0 column ROS membranes (9 mg/ml p r o t e i n ) were s o l u b i l i z e d with 3-5% SDS, and a p p l i e d t o a S e p h a c r y l - S - 2 0 0 co lumn (2 .0x48 .0 cm) p r e - e q u i l i b r a t e d w i t h PBS c o n t a i n i n g 0.1% SDS and ImM EDTA. The column was e luted w i t h PBS c o n t a i n i n g 0.1% SDS and ImM EDTA and 30-drop f r a c t i o n s were c o l l e c t e d . F r a c t i o n s were assayed for absorbance at 280 nm and a n a l y z e d by S D S - p o l y a c r y l a m i d e g e l e lec t rophores i s as p r e v i o u s l y d e s c r i b e d . (b) A f f i n i t y chromatography o f ROS prote ins on a 4B2  a n t i b o d y - a g a r o s e column L y o p h i l i z e d and p a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y was resuspended i n 0.1M sodium b o r a t e b u f f e r , pH 9 .0 , and coupled to REACTI-GEL (6X) by the method of B e t h e l l et a l . (67). ROS membranes (9 mg/ml p r o t e i n ) were s o l u b i l i z e d with lOmM CTAB i n 0.1M sodium phosphate b u f f e r , pH 7.4, and appl ied to a 4B2 a n t i b o d y - a g a r o s e c o l u m n ( 0 . 5 mg a n t i b o d y / m l c o l u m n ) p r e - e q u i l i b r a t e d with the same b u f f e r . The column was washed with the same b u f f e r and e l u t e d w i t h 50mM d i e t h y l a m i n e i n the same b u f f e r . F r a c t i o n s (1 ml) w e r e c o l l e c t e d and a s s a y e d f o r absorbance at 280 nm and 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 g e l -33-e lec t rophores i s as p r e v i o u s l y de sc r ibed . -34-RESULTS 1. Product ion o f 4B2 monoc lona l antibody The 4B2 monoclonal antibody was obtained by two conventional methods: mass c u l t u r e of 4B2 hybr idoma c e l l l i n e and induct ion of a s c i t e s tumors i n mice. C u l t u r e supernatant (300 ml) and a s c i t e s f l u i d (50 ml) c o n t a i n i n g 4B2 m o n o c l o n a l a n t i b o d y were c o l l e c t e d a f t e r 2 and 5 weeks, r e s p e c t i v e l y . The volume o f a s c i t e s f l u i d obtained from a tumor-bearing mouse v a r i e d from 2 to 15 ml . Both the c e l l c u l t u r e s u p e r n a t a n t and a s c i t e s f l u i d were analyzed f o r t h e i r a n t i b o d y - b i n d i n g p r o p e r t i e s . The e f f e c t of d i l u t i o n of 4B2 c e l l c u l t u r e s u p e r n a t a n t and a s c i t e s f l u i d on the binding o f a n t i b o d y to T r i t o n X - 1 0 0 - s o l u b i l i z e d d i s k s using an i n d i r e c t sol id-phase radioimmune a s s ay i s shown i n F i g . 8. Both the c e l l c u l t u r e supernatant and a s c i t e s f l u i d contained ac t ive a n t i b o d y s p e c i f i c f o r d i s k m e m b r a n e s . However, half-maximum b i n d i n g o c c u r r e d a t a r e c i p r o c a l d i l u t i o n o f 16 f o r c u l t u r e supernatant and 512 f o r a s c i t e s f l u i d . N o n s p e c i f i c c e l l c u l t u r e s u p e r n a t a n t d i d n o t c o n t a i n a n y a n t i b o d y s p e c i f i c f o r d i s k membranes. 2. S p e c i f i c i t y of 4B2 m o n o c l o n a l a n t i b o d y from recloned  hybr idoma c e l l l i n e The once cloned 4B2 a n t i b o d y - s e c r e t i n g hybridoma c e l l l i n e -35-R e c i p r o c a l Di lut ion Figure 8. T i t r a t i o n of 4B2 hybr idoma c e l l a s c i t e s f l u i d ( • ) , 4B2 hybridoma c e l l c u l t u r e s u p e r n a t a n t (•) , and nonspec i f i c c u l t u r e supernatant (A) a g a i n s t T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes. S o l u b i l i z e d ROS membranes i m m o b i l i z e d i n m i c r o t i t e r w e l l s were t rea ted with s e r i a l d i l u t i o n s o f c u l t u r e supernatants and a s c i t e s 125 f l u i d , r i n s e d , and s u b s e q u e n t l y t r e a t e d w i t h I - l a b e l e d goat anti-mouse l g . -36-was rec loned . Antibody from c u l t u r e supernatant of recloned c e l l s was analyzed for i t s s p e c i f i c i t y against ROS membranes. CNBr-activated paper with t rans fe r red p r o t e i n s , s e q u e n t i a l l y l a b e l e d w i t h c u l t u r e s u p e r n a t a n t o f r e c l o n e d c e l l s and 125 I - l a b e l e d goat ant i -mouse l g i s shown i n F i g . 9. Ant ibody secreted by the recloned 4B2 c e l l c u l t u r e labe led a polypeptide of a p p a r e n t 1^=220,000 a n d a s m a l l e r f r a g m e n t o f a p p a r e n t M r =140,000 , as p r e v i o u s l y r e p o r t e d f o r t h e once c l o n e d 4B2 hybr idoma c e l l l i n e (57 ) . A n t i b o d y s e c r e t e d by r h o - l D 4 c e l l c u l t u r e l a b e l e d r h o d o p s i n , t h e r h o d o p s i n d i m e r , and a l a r g e a g g r e g a t e o f r h o d o p s i n . N o n s p e c i f i c s u p e r n a t a n t d i d n o t l a b e l any ROS po lypept ides . 3. C h a r a c t e r i z a t i o n o f 4B2 monoclonal antibody The l g s u b t y p e o f t h e 4B2 m o n o c l o n a l a n t i b o d y was de te rmined by b i n d i n g r a b b i t a n t i - m o u s e l g s u b t y p e - s p e c i f i c a n t i b o d i e s to 4B2 a n t i b o d y a l r e a d y bound to i m m o b i l i z e d and T r i t o n X-100- so lub i l i zed ROS d i s k membranes. As shown i n Table I , r a b b i t ant i -mouse ja and K c h a i n a n t i b o d i e s b i n d to the 4B2 a n t i b o d y 15-20 f o l d more t h a n t h e c o n t r o l w h i c h c o n t a i n e d preimmune serum. Other l g s u b t y p e - s p e c i f i c a n t i b o d i e s bind at most 2 f o l d more than the c o n t r o l . -37-T o p 2 2 0 K 1 4 0 K R h o 2 34K mm R h o i 4 B 2 1 D 4 c o n t r o l Figure 9 . A n a l y s i s o f ROS p o l y p e p t i d e s which b i n d monoclonal a n t i b o d y s e c r e t e d by r e c l o n e d 4B2 h y b r i d o m a c e l l l i n e . ROS m e m b r a n e s w e r e s u b j e c t e d t o S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (20 jug p r o t e i n / g e l ) . P r o t e i n s w e r e e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d from SDS g e l s to CNBr-act ivated papers . C N B r - a c t i v a t e d papers c o n t a i n i n g t r a n s f e r r e d pro te in s were l abe led with 4B2, 1D4, and n o n s p e c i f i c c u l t u r e supernatants , 125 fol lowed by I - l a b e l e d goat anti-mouse l g . -38-TABLE I . I d e n t i f i c a t i o n o f l g subtype o f 4B2 monoclonal antibody Rabbit anti-mouse Ig-subtype s p e c i f i c antibody b inding to 4B2 monoclonal antibody anti-(0() -(#,) - ( 7 ^ -(6^ -(# 3) -(/<-) -(/<) -(X) c o n t r o l DPM bound , 2.4 5.5 3.0 1.6 1.4 60.1 45.9 6.4 3.0 X 10 -39-4. P u r i f i c a t i o n of 4B2 m o n o c l o n a l a n t i b o d y by ion exchange  chromatography on DEAE-Sephace l The 4B2 monoc lona l a n t i b o d y from c u l t u r e supernatant and a s c i t e s f l u i d was p a r t i a l l y p u r i f i e d b y i o n e x c h a n g e chromatography on a D E A E - S e p h a c e l c o l u m n . The e l u t i o n p r o f i l e for the p u r i f i c a t i o n o f 4B2 a n t i b o d y from a s c i t e s f l u i d and SDS g e l s o f e l u t a n t s are shown i n F i g . 10. F r a c t i o n s e l u t e d w i t h 80-120mM NaCl c o n c e n t r a t i o n c o n t a i n e d a n t i b o d y s p e c i f i c for ROS membranes as de te rmined by R I A . SDS g e l s o f the se f r a c t i o n s showed t h r e e p o l y p e p t i d e b a n d s . Two o f which have apparent M r=55,000 and 22 ,000 . They a re p re sumab ly the heavy and l i g h t chains of 4B2 antibody s ince the corresponding chains o f standard IgG molecu le have apparent M r =50,000 and M r =25 ,000 . The other p r o t e i n t h a t c o p u r i f i e d w i t h 4B2 a n t i b o d y h a s a p p a r e n t M r =85 ,000 . F r a c t i o n s e l u t e d w i t h h i g h e r NaCl c o n c e n t r a t i o n contained no a c t i v e a n t i b o d y s p e c i f i c f o r ROS membranes. These f r a c t i o n s c o n t a i n e d many p o l y p e p t i d e s , w i t h serum a lbumin o f apparent 14^=66,000 being the major p r o t e i n as shown by SDS g e l s . The e l u t i o n p r o f i l e f o r t h e p u r i f i c a t i o n o f 4B2 antibody from 4B2 cu l ture supernatant and SDS g e l s of e lutants are shown i n F i g . 11 . F r a c t i o n s e l u t e d w i t h 0 . 1 M N a C l c o n c e n t r a t i o n c o n t a i n e d a n t i b o d y a c t i v i t y s p e c i f i c f o r ROS membranes as determined by RIA. However, t h e s e f r a c t i o n s contained two major polypeptides w i t h apparent i d e n t i c a l to the heavy and l i g h t c h a i n s o f s t andard IgG m o l e c u l e a s shown by SDS g e l s . T h i s -40-F i g u r e 10. (a) C h r o m a t o g r a p h y o f 4B2 a s c i t e s f l u i d on a DEAE-Sephacel c o l u m n . One ml of 4B2 a s c i t e s f l u i d was d i l u t e d w i t h 3 ml o f P B S , and p r e c i p i t a t e d w i t h 4 ml o f s a t u r a t e d ammonium s u l f a t e . P r e c i p i t a t e d p r o t e i n s were r e d i s s o l v e d and d i a l y z e d a g a i n s t 20mM T r i s - a c e t a t e , pH 7 . 9 , c o n t a i n i n g 20mM N a C l . D ia lyza te was app l ied to a DEAE-Sephacel column (1.0x40cm), p r e - e q u i l i b r a t e d with the same b u f f e r . The column was eluted with a l i n e a r gradient o f NaCl c o n c e n t r a t i o n ( ) at a flow rate of 0.5 ml/min and 30-drop f r a c t i o n s were c o l l e c t e d . Fract ions were assayed f o r absorbance at 280 nm ( • ) and a n t i b o d y a c t i v i t y s p e c i f i c for ROS membranes ( O ) . F r a c t i o n s numbered 29 to 31, and 54 were a n a l y z e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . F r a c t i o n s c o n t a i n i n g a n t i b o d y a c t i v i t y were p o o l e d and l y o p h i l i z e d . (b) E l e c t r o p h o r e t i c a n a l y s i s of l g p u r i f i e d from 4B2 a s c i t e s f l u i d by DEAE-Sephace l column chromatography . Equa l volume (20pi) o f each f r a c t i o n i n d i c a t e d i n (a) was appl ied to a 8% SDS-polyacrylamide s l a b g e l . Lane 1. s tandard mouse IgG. Lane 2. f r a c t i o n #29. Lane 3. f r a c t i o n #30. Lane 4. f r a c t i o n #31. Lane 5. f r a c t i o n #54. -42-Figure 11. (a) Chromatography o f 4B2 c u l t u r e supernatant on a DEAE-Sephacel c o l u m n . One hundred ml of 4B2 c u l t u r e f l u i d was d i l u t e d w i t h 300 ml o f P B S , and p r e c i p i t a t e d w i t h 400 ml o f saturated ammonium s u l f a t e . P r e c i p i t a t e d pro te ins were redissolved and d i a lyzed aga ins t 20mM T r i s - a c e t a t e , pH 7 . 9 , conta ining 20mM N a C l . D ia lyza te was appl ied to a DEAE-Sephacel column (1.0x40 cm). The column was f i r s t e l u t e d w i t h 0.1M N a C l , then with 1M NaCl i n 20mM T r i s - a c e t a t e b u f f e r , pH 7 . 9 , a t a f l o w ra te of 0.5 ml/min, and 30-drop f r a c t i o n s were c o l l e c t e d . F r a c t i o n s were assayed for absorbance at 280 nm ( • ) and antibody a c t i v i t y s p e c i f i c for ROS membranes ( O ) . F rac t ions numbered 68, 70, and 131 were analyzed by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s . F rac t ions containing a n t i b o d y a c t i v i t y w e r e p o o l e d a n d l y o p h i l i z e d . (b) E l e c t r o p h o r e t i c a n a l y s i s o f l g p u r i f i e d f r o m 4B2 c u l t u r e supernatant by DEAE-Sephacel column chromatography. Equal volume (20>ul) of each f r a c t i o n i n d i c a t e d i n (a) was appl ied to a 10% SDS-polyacrylamide s l ab g e l . Lane 1. standard mouse IgG. Lane 2. f r a c t i o n #68. Lane 3. f r a c t i o n #70. Lane 4. f r a c t i o n #131. - i n d i c a t e s t h a t t h e l o w s a l t - e l u t e d 4B2 a n t i b o d y was c o n t a m i n a t e d by n o n s p e c i f i c IgG m o l e c u l e s i n t h e c u l t u r e supernatant, s ince the 4B2 a n t i b o d y was determined to be an IgM molecule. The horse serum i n the c u l t u r e supernatant might be the source of these nonspec i f i c IgG m o l e c u l e s . F rac t ions e luted with 1M NaCl c o n c e n t r a t i o n c o n t a i n e d no a c t i v e an t ibody s p e c i f i c for ROS membranes. These f r a c t i o n s c o n t a i n e d many polypept ides ,with serum albumin being the major p r o t e i n as shown by SDS g e l s . In order to improve the y i e l d o f p u r i f i e d 4B2 ant ibody, ge l f i l t r a t i o n chromatography on a S e p h a c r y l - S - 5 0 0 column and ion exchange chromatography on a D E A E - A f f i - G e l - B l u e column were a l so used to p u r i f y 4B2 antibody from a s c i t e s f l u i d . 5. P u r i f i c a t i o n o f 4B2 m o n o c l o n a l antibody by g e l  f i l t r a t i o n chromatography on Sephacryl-S-500 The 4B2 monoclonal a n t i b o d y was a l s o p a r t i a l l y p u r i f i e d from a s c i t e s f l u i d by g e l f i l t r a t i o n c h r o m a t o g r a p h y on a S e p h a c r y l - S - 5 0 0 c o l u m n . T h e e l u t i o n p r o f i l e f o r t h e p u r i f i c a t i o n of 4B2 a n t i b o d y from a s c i t e s f l u i d and SDS ge l s o f e lutants are shown i n F i g . 12. There was o n l y one A peak but two antibody a c t i v i t y peaks. The f i r s t antibody a c t i v i t y peak was a t t r i b u t e d to the 4B2 monoc lona l a n t i b o d y i n i t s pentameric IgM form (M r =900,000) , s i n c e i t e l u t e d i n the v o i d volume o f the column having an exc lus ion l i m i t o f 500,000. The second antibody peak was a t t r i b u t e d to the d i s s o c i a t e d forms of the 4B2 ant ibody, s ince i t e luted i n the i n c l u d e d v o l u m e . F rac t ions e luted i n the -45-F i g u r e 12 . (a) C h r o m a t o g r a p h y o f 4B2 a s c i t e s f l u i d on a Sephacryl-S-500 c o l u m n . One ml o f 4B2 a s c i t e s f l u i d was d i l u t e d w i t h 3 ml o f PBS and c e n t r i f u g e d a t 1 0 , 0 0 0 rpm f o r 10 m i n . Supernatant was a p p l i e d to a S e p h a c r y l - S - 5 0 0 column (1.0x40 cm) p r e - e q u i l i b r a t e d w i t h PBS. The column was e l u t e d with PBS at a flow rate of 0 .5 m l / m i n and 30-drop f r a c t i o n s were c o l l e c t e d . Void volume of the column was determined by the e l u t i o n volume of Blue Dextran. F r a c t i o n s were a s sayed f o r absorbance at 280 nm ( • ) and ant ibody a c t i v i t y s p e c i f i c f o r ROS membranes ( • ) . F r a c t i o n s numbered 22 t o 2 4 , and 29 to 31 were a n a l y z e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . F r a c t i o n s c o n t a i n i n g ant ibody a c t i v i t y were p o o l e d and s t o r e d i n 0.1% NaN^ a t 4°c. (b) E l e c t r o p h o r e t i c a n a l y s i s of l g p u r i f i e d from 4B2 a s c i t e s f l u i d by Sephacryl-S-500 column chromatography . Egual volume (20 J J I ) o f each f r a c t i o n i n d i c a t e d i n (a) was a p p l i e d t o a 8% S D S - p o l y a c r y l a m i d e s l a b g e l . Lane 1. s t a n d a r d p r o t e i n s : p h o s p h o r y l a s e b (92K) , b o v i n e serum a l b u m i n (66K) , ova lbumin (45K). Lane 2. f r a c t i o n #22. Lane 3. f r a c t i o n #23. Lane 4. f r a c t i o n #24. Lane 5. s tandard mouse IgG. Lane 6. f r a c t i o n #29. Lane 7. f r a c t i o n #30. Lane 8. f r a c t i o n #31. (a) - 1 * 6 -1 2 3 4 5 6 7 8 -47-vo id and i n c l u d e d volume c o n t a i n e d p o l y p e p t i d e s o ther than the heavy and l i g h t chains o f the 4B2 antibody as shown by SDS g e l s . 6. P u r i f i c a t i o n o f 4B2 monoc lona l a n t i b o d y by ion exchange  Chromatography on D E A E - A f f i - G e l - B l u e The 4B2 monoclonal ant ibody was p u r i f i e d from a s c i t e s f l u i d by ion exchange chromatography on a DEAE-Af f i - G e l - B l u e column. SDS g e l s o f e l u t a n t s are shown i n F i g . 13. F r a c t i o n s e luted w i t h 0 .1M N a C l c o n t a i n e d two p o l y p e p t i d e s o f a p p a r e n t M i d e n t i c a l to the heavy and l i g h t c h a i n s o f the 4B2 a n t i b o d y . Frac t ions e l u t e d w i t h 1M NaCl c o n t a i n e d a major po lypept ide of apparent i d e n t i c a l to serum a l b u m i n . O n l y low s a l t - e l u t e d f r a c t i o n s contained a n t i b o d y a c t i v i t y s p e c i f i c for ROS membranes (not shown). 7. Comparison between r h o - l D 4 and 4B2 hybridoma c e l l l i n e s  i n t h e i r a b i l i t y to produce monoclonal antibody The l g subtype and the y i e l d o f 4B2 monoc lona l antibody f r o m 4B2 a s c i t e s f l u i d were c o m p a r e d t o t h a t o f r h o - l D 4 monoclonal antibody from r h o - l D 4 a s c i t e s f l u i d . As shown i n F i g . 14, r h o - l D 4 a n t i b o d y was an IgG m o l e c u l e s i n c e the two major po lypept ides , p u r i f i e d from low s a l t e l u t i o n , have i d e n t i c a l M as the heavy and l i g h t c h a i n s o f s t a n d a r d IgG molecu le . A l s o , these f r a c t i o n s c o n t a i n e d a n t i b o d y a c t i v i t y s p e c i f i c f o r r h o d o p s i n i n ROS membranes (shown i n F i g . 2 1 ) . SDS g e l s a l s o indica ted that much more r h o - l D 4 a n t i b o d y than 4B2 antibody was -48-1 2 3 4 5 F i g u r e 13. E l e c t r o p h o r e t i c a n a l y s i s o f l g p u r i f i e d from 4B2 a s c i t e s f l u i d by D E A E - A f f i - G e l - B l u e chromatography. Equal volumes (20jul) of low s a l t - e l u t e d f r a c t i o n s numbered 22 t o 24 and one h i g h s a l t - e l u t e d f r a c t i o n numbered 51 were a p p l i e d to a 8% SDS-polyacrylamide s l ab g e l . Lane 1. s t a n d a r d mouse IgG. Lane 2. f r a c t i o n #22. Lane 3. f r a c t i o n #23. Lane 4. f r a c t i o n #24. Lane 5. f r a c t i o n #51. A -49-1 2 3 4 5 9 2 K 6 6 K 4 5 K — ~ 3 1 K 2 1 K B 2 3 4 5 0 K 2 5 K Figure 14. E l e c t r o p h o r e t i c a n a l y s i s of lg molecules p u r i f i e d from r h o - l D 4 and 4B2 a s c i t e s f l u i d s b y D E A E - S e p h a c e l c o l u m n chromatography. Equal volumes o f 4B2 and rho-lD4 low s a l t - e l u t e d l g f r a c t i o n s were a p p l i e d to 9% SDS-polyacrylamide s l a b g e l s . Gel A : Lane 1. s t a n d a r d p r o t e i n s c o n t a i n i n g p h o s p h o r y l a s e b (92K), bovine serum a lbumin (66K) , o v a l b u m i n (45K) , ca rbonic anhydrase (31K), and soybean t r y p s i n i n h i b i t o r (21K) . Lane 2. standard mouse IgG. Lanes 3-5 . l o w s a l t - e l u t e d f r a c t i o n s c o n t a i n i n g rho-lD4 a n t i b o d y as d e t e r m i n e d by RIA. G e l B: Lane 1. standard mouse IgG. Lanes 2 -4 . low s a l t - e l u t e d f r a c t i o n s conta in ing 4B2 antibody as determined by RIA. -50-p u r i f i e d . The y i e l d o f 4B2 a n t i b o d y ( 0 .5 mg/ml) was 8 f o l d l e s s than the y i e l d o f rho-lD4 ant ibody. 8. A c t i v i t y of p a r t i a l l y - p u r i f i e d 4B2 monoclonal antibody F r a c t i o n s c o n t a i n i n g 4B2 m o n o c l o n a l a n t i b o d y , from the chromatography of 4B2 a s c i t e s f l u i d on DEAE-Sephacel, were pooled, l y o p h i l i z e d , and resuspended i n PBS. The e f f e c t of d i l u t i o n of t h i s p a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y on the b i n d i n g o f antibody to T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes i s shown i n F i g . 15. P a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y was a c t i v e a g a i n s t ROS m e m b r a n e s . H a l f - m a x i m u m b i n d i n g o c c u r r e d a t p r o t e i n concentrat ion of 0.2 mg/ml . As shown i n T a b l e I I , half-maximum binding for a s c i t e s f l u i d o c c u r r e d a t p r o t e i n c o n c e n t r a t i o n of 0.05 mg/ml . 9. D e t e c t i o n o f a c t i n i n ROS The e f f ec t of d i l u t i o n of an a n t i - a c t i n monoclonal ant ibody, r e f e r red to as 2C2, on the b i n d i n g o f the a n t i b o d y to T r i t o n X-100-so lub i l i zed ROS membrane p r e p a r a t i o n and p u r i f i e d a c t i n i s shown i n F i g . 16. The 2C2 m o n o c l o n a l a n t i b o d y was ac t ive against a c t i n and s o l u b i l i z e d ROS membrane p r e p a r a t i o n . There was a higher degree o f b i n d i n g of 2C2 a n t i b o d y to a c t i n than to ROS membrane p r e p a r a t i o n . In c o n t r o l s t u d i e s , two m o n o c l o n a l -51-Figure 15. T i t r a t i o n o f l y o p h i l i z e d and p a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y a g a i n s t T r i t o n X - 1 0 0 - s o l u b i 1 i z e d ROS membranes . S o l u b i l i z e d ROS membranes i m m o b i l i z e d i n m i c r o t i t e r w e l l s were t rea ted with s e r i a l d i l u t i o n s of 4B2 a n t i b o d y p a r t i a l l y p u r i f i e d by DEAE-Sephacel column c h r o m a t o g r a p h y , r i n s e d , and subsequently 125 t rea ted with I - l a b e l e d goat anti-mouse l g . -.52-TABLE I I . P u r i f i c a t i o n o f 4B2 monoclonal antibody from a s c i t e s f l u i d by DEAE-Sephacel column chromatography * ic ic Sample B c . (mg/ml) B r „ r a t i o 5 0 50 4B2 a s c i t e s f l u i d 0.05 1.0 0.1M NaCl-eluted^ ^ 4 > Q l g f r a c t i o n s Protein concentration of sample at which half-maximum antibody binding occurred. ic ic R a t i o of B r . (sample) :B_n (4B2 a s c i t e s f l u i d ) . b U b 0 *** These f r a c t i o n s were p o o l e d , l y o p h i l i z e d , and resuspended in PBS, before being assayed f o r antibody a c t i v i t y . -53-< > ~# 4 16 6 4 2 5 6 1 0 2 4 4 0 9 6 Rec ip roca l D i lu t ion Figure 16 . T i t r a t i o n o f 2C2, 4B2, and r h o - l D 4 hybr idoma c e l l c u l t u r e supernatants a g a i n s t T r i t o n X-100-treated rabb i t a c t i n ( • ) and 2C2 hybridoma c e l l c u l t u r e supernatant against T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes ( • ) . S o l u b i l i z e d ROS membranes and a c t i n immobilized i n m i c r o t i t e r w e l l s were treated with s e r i a l d i l u t i o n s o f c e l l c u l t u r e superna tan t , - r i n s e d , and subsequently 125 t r ea ted wi th I - l a b e l e d goat anti-mouse l g . -54-ant ibodies (rho-lD4 and 4B2) , s p e c i f i c fo r rhodopsin and ROS 1.2 r e s p e c t i v e l y , were i n a c t i v e against a c t i n . 10. C r o s s - r e a c t i v i t i e s o f 4B2 monoclonal antibody (a) M y o s i n The e f f e c t o f d i l u t i o n o f 4B2 a s c i t e s f l u i d and r h o - l D 4 c u l t u r e supernatant on the b i n d i n g o f the a n t i b o d i e s to T r i t o n X-100-treated r a b b i t s k e l e t a l musc le myos in i s shown i n F i g . 17 (upper) . The 4B2 a s c i t e s f l u i d was a c t i v e a g a i n s t p u r i f i e d myosin, but the r h o - l D 4 c e l l c u l t u r e s u p e r n a t a n t was n o t . In c o n t r o l s t u d i e s , bo th t h e 4B2 a s c i t e s f l u i d and the r h o - l D 4 c u l t u r e supernatant were a c t i v e a g a i n s t T r i t o n X-100-so lub i l i zed ROS membranes as shown i n F i g . 17 ( lower) . The r e l a t i v e degree o f c r o s s - r e a c t i v i t y of 4B2 antibody to myos in was de te rmined by an i n d i r e c t s o l i d - p h a s e radioimmune c o m p e t i t i o n a s s a y . A s s h o w n i n F i g . 1 8 , b o t h T r i t o n X-100- so lub i l i zed ROS membranes and m y o s i n were able to i n h i b i t the b i n d i n g o f 4B2 a n t i b o d y t o T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes . (b) Red blood c e l l membranes and b r a i n homogenate The e f f e c t o f d i l u t i o n o f c u l t u r e s u p e r n a t a n t s (4B2 and -55-30F R e c i p r o c a l D i l u t i o n 1 4 1 6 6 4 2 5 6 1 0 2 4 R e c i p r o c a l D i l u t i o n Figure 17. T i t r a t i o n o f 4B2 a s c i t e s f l u i d ( • ) and rho-lD4 h y b r i d o m a c e l l c u l t u r e s u p e r n a t a n t (•) a g a i n s t T r i t o n X - 1 0 0 - t r e a t e d r a b b i t m y o s i n ( u p p e r ) and a g a i n s t T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes ( l o w e r ) . S o l u b i l i z e d ROS membranes and myosin immobi l ized i n m i c r o t i t e r w e l l s were t reated w i t h s e r i a l d i l u t i o n s o f c u l t u r e s u p e r n a t a n t , r i n s e d , and 125 subsequently t reated w i t h I - l a b e l e d goat anti-mouse l g . -56-0 . 0 1 0 .1 1 10 1 0 0 Pro te in Cone ( /xg /ml ) F i g u r e 18. I n h i b i t i o n o f 4B2 a n t i b o d y b i n d i n g to T r i t o n X-100- so lub i l i zed ROS membranes by T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes (O) and T r i t o n X - 1 0 0 - t r e a t e d r a b b i t myosin ( • ) using the sol id-phase i n d i r e c t radioimmune competi t ion assay. A l i q u o t s (25 >ul) of s e r i a l d i l u t i o n s o f T r i t o n X-100-treated rabbit myosin (0.1 mg/ml) or ROS membranes (0.1 mg/ml) were incubated with 25 >ul of 4B2 hybr idoma c u l t u r e f l u i d d i l u t e d to a c o n c e n t r a t i o n which g ive s 80-90% s a t u r a t i o n o f a n t i b o d y binding by radioimmune assay. A f t e r l h , 25jul o f the mixture was removed and screened for remaining antibody by. radioimmune assay. -57-r h o - l D 4 ) o n t h e b i n d i n g o f t h e a n t i b o d i e s t o T r i t o n X-100- so lub i l i zed human red b l o o d c e l l s (RBC) and bovine bra in homogenate (BBH) i s shown i n F i g . 19 . The 4B2 c e l l c u l t u r e supernatant was a c t i v e aga ins t b o t h p repara t ions , but rho-lD4 was n o t . There was a 5 f o l d h i g h e r degree of 4B2 antibody binding to BBH than RBC membranes. The r e l a t i v e degree o f c r o s s - r e a c t i v i t y of 4B2 antibody to BBH and RBC membranes was a g a i n d e t e r m i n e d by c o m p e t i t i o n assay. As shown i n F i g . 20, T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS , RBC, and BBH membranes were a l s o a b l e to i n h i b i t the b inding of 4B2 antibody to T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes. As shown i n Table I I I , the r e l a t i v e c r o s s - r e a c t i v i t y was i n a decrea s ing o r d e r o f ROS, m y o s i n , BBH, and RBC membranes. H o w e v e r , t h e t r u e r e l a t i v e c r o s s - r e a c t i v i t y c a n o n l y be determined by using p u r i f i e d competing ant igens . In c o n t r o l s t u d i e s , n o n s p e c i f i c p r o t e i n such as BSA was unable t o i n h i b i t t h e b i n d i n g o f 4B2 a n t i b o d y t o T r i t o n X-100- so lub i l i zed ROS membranes o v e r the concent ra t ions t e s t e d . S i m i l a r l y , RBC and BBH membranes were unab le t o i n h i b i t the b i n d i n g o f r h o - l D 4 a n t i b o d y t o T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes (not shown). In order to determine which p r o t e i n i n BBH and RBC membranes binds the 4B2 a n t i b o d y , ROS, m y o s i n , RBC and BBH pro te ins were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e paper fo l lowing g e l e l e c t r o p h o r e s i s . N i t r o c e l l u l o s e p a p e r w i t h t r a n s f e r r e d Figure 19. T i t r a t i o n of hybridoma c e l l c u l t u r e supernatants 4B2 ( • ) and rho-lD4 ( • ) against T r i t o n X-100- so lub i l i zed human red b l o o d c e l l membranes ( u p p e r ) and b o v i n e b r a i n homogenate , ( l o w e r ) . S o l u b i l i z e d r e d b l o o d c e l l membranes and b r a i n homogenate i m m o b i l i z e d i n m i c r o t i t e r w e l l s were t r e a t e d w i t h s e r i a l d i l u t i o n s o f c u l t u r e s u p e r n a t a n t , r i n s e d , and subsequently 125 t rea ted with I - l a b e l e d goat anti-mouse l g . - 5 9 -P r o t e i n C o n e ( j u g / m l ) F i g u r e 20. I n h i b i t i o n o f 4B2 a n t i b o d y b i n d i n g to T r i t o n X-100- so lub i l i zed rod o u t e r segment membranes ( ROS ) by T r i t o n . X - 1 0 0 - s o l u b i l i z e d ROS membranes, bovine b r a i n homogenate (BBH) , human red b l o o d c e l l membranes (RBC) , and bovine serum albumin' (BSA) using the s o l i d - p h a s e radioimmune competit ion assay as descr ibed p r e v i o u s l y . -60-TABLE I I I . C r o s s - r e a c t i v i t i e s o f 4B2 monoclonal antibody Competing a n t i g e n * I 5 Q Cug/mD ** I r a t i o x 5 0 Bovine ROS membranes 58 1.0 Rabbit s k e l e t a l muscle myosin 68 1.2 Bovine b r a i n homogenate 316 9.0 Human red blood c e l l ghosts 1995 57.0 * Prote in concentra t ion o f compet ing ant igen required to i n h i b i t 50% of 4B2 antibody b inding to T r i t o n X-100 s o l u b i l i z e d ROS membranes. R a t i o o f I c-. (competing a n t i g e n ) : I (bovine ROS membranes) -61-p r o t e i n s , s e q u e n t i a l l y l a b e l e d w i t h 4B2 a n t i b o d y and 125 I - labe led goat ant i -mouse l g i s shown i n F i g . 21. ROS 1.2, m y o s i n , and a p o l y p e p t i d e w i t h a p p a r e n t 14^=160,000 i n BBH membranes were l a b e l e d , but no p o l y p e p t i d e i n RBC membranes was l a b e l e d . In c o n t r o l s t u d i e s u s i n g r h o - l D 4 a n t i b o d y , o n l y r h o d o p s i n , t h e r h o d o p s i n d i m e r , and a l a r g e a g g r e g a t e o f rhodopsin i n ROS membranes were l a b e l e d . In o r d e r to determine whether the e l e c t r o p h o r e t i c t r a n s f e r s o f RBC s p e c t r i n and b r a i n s p e c t r i n i n BBH were e f f i c i e n t enough f o r radioimmune l a b e l i n g 125 with 4B2 ant ibody, the e l e c t r o t r a n s f e r o f I - l abe led prote ins was s tud ied . 125 11. E l e c t r o t r a n s f e r o f I - l a b e l e d p r o t e i n s (a) E f f ec t o f p r o t e i n m o l e c u l a r weight 125 I - l a b e l e d p r o t e i n s o f d i f f e r e n t m o l e c u l a r w e i g h t s , myosin (200K) , BSA (66K) , and p r o l a c t i n (23K) , were appl ied to a 8% SDS-polyacrylamide s l a b g e l . F o l l o w i n g g e l e l e c t r o p h o r e s i s , these p r o t e i n s were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e p a p e r s . Time c o u r s e o f the e l e c t r o t r a n s f e r of each o f these prote ins i s shown i n F i g . 22. The rate o f t rans fer was i n v e r s e l y p r o p o r t i o n a l t o p o l y p e p t i d e s i z e . A f t e r l h , 125 t r a n s f e r s o f I - l a b e l e d p r o l a c t i n and BSA were a lmost 125 completed, but o n l y 20% o f I - m y o s i n was t r a n s f e r r e d . The - 6 2 -1 2 3 4 1 2 3 4 220K mm T o p 140K 34K 4B2 1D4 Figure 21. Radioimmune l a b e l i n g o f ROS membranes, myos in , b r a i n homogenate, and red b l o o d c e l l membranes with 4B2 an t ibody . ROS membranes (20 / j g / g e l ) , r a b b i t s k e l e t a l muscle myosin ( 25 j u g / g e l ) , b o v i n e b r a i n homogenate (14 jug/gel-) , and human red b l o o d c e l l membranes (13 j u g / g e l ) w e r e a p p l i e d t o a 8% SDS-polyacrylamide s l a b g e l . F o l l o w i n g g e l e l e c t r o p h o r e s i s , t h e p r o t e i n s were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d t o C N B r - a c t i v a t e d p a p e r s . C N B r - a c t i v a t e d p a p e r s c o n t a i n i n g t r ans f e r red pro te in s were t r e a t e d w i t h 4B2 and rho-lD4 c u l t u r e 125 supernatants , f o l l o w e d by I - l a b e l e d g o a t anti-mouse l g . Lane 1. ROS m e m b r a n e s . Lane 2 . m y o s i n . L a n e 3 . b o v i n e b r a i n homogenate. Lane 4. red blood c e l l membranes. -63-Time (h) 125 Figure 22. Time courses f o r the e l e c t r o t r a n s f e r of I - labe led 125 125 myosin ( A ) , I - l a b e l e d BSA ( • ) , and I - l a b e l e d p r o l a c t i n 12 5 ( • ) . I - l a b e l e d p r o t e i n s w e r e a p p l i e d t o 8% SDS-polyacrylamide s l a b ge l s and e l e c t r o p h o r e s i s was c a r r i e d out . F o l l o w i n g e l e c t r o p h o r e s i s , g e l s l i c e s were s u b j e c t e d t o e l e c t r o p h o r e t i c t r a n s f e r to n i t r o c e l l u l o s e papers. At d i f f e r e n t times a f te r e l e c t r o p h o r e t i c t r a n s f e r , corresponding s t r i p s of ge l and papers were removed and s u b j e c t e d to a u t o r a d i o g r a p h y . The polypept ide bands d e t e c t e d by a u t o r a d i o g r a p h y were excised and counted i n a Beckman 8000 gamma counter . -64-time r e q u i r e d f o r the t r a n s f e r o f about 70% o f each o f these 125 I - labe led p r o t e i n s i s shown i n Table IV. (b) E f f e c t o f g e l pore s i z e The e f f e c t o f g e l p o r e s i z e o n t h e t i m e c o u r s e o f 125 e l e c t r o t r a n s f e r o f I - l a b e l e d myos in i s shown i n F i g . 23. The rate o f t r ans fe r was p r o p o r t i o n a l to g e l p o r e s i z e . The rate of 125 t r a n s f e r o f I - l a b e l e d myos in i n c r e a s e d w i t h i n c r e a s i n g g e l pore s i z e . S i m i l a r s t u d i e s i n d i c a t e d the r a t e o f t r a n s f e r o f 125 I - labe led p r o l a c t i n d e c r e a s e d w i t h d e c r e a s i n g g e l pore s i z e (not shown). 12. Cytoplasmic p o o l o f 4B2 an t igen i n ROS The d i f ference i n the c o n c e n t r a t i o n s o f 4B2 ant igen between ROS and ROS d i s k membranes was d e t e c t e d b y s o l i d - p h a s e radioimmune assays and was guant i ta ted by competi t ion assays. The e f f e c t o f d i l u t i o n o f c u l t u r e s u p e r n a t a n t s (4B2 and rho-lD4) on the binding of a n t i b o d i e s to T r i t o n X-100-so lub i l i zed ROS and ROS d i sk membranes i s shown i n F i g . 24. The degree of 4B2 antibody binding to ROS was about 2 f o l d higher than ROS d i sk membranes, but the degree o f r h o - l D 4 an t ibody binding was about the same for both the ROS and ROS d i sk membranes. The degree o f i n h i b i t i o n o f r h o - l D 4 and 4B2 a n t i b o d i e s -65-125 TABLE I V . E l e c t r o t r a n s f e r o f I - l a b e l e d p r o t e i n s 125 I - p r o t e i n M % SDS-polyacrylamide g e l 6 8 9 * T r a n s f e r t ime (h) Myosin BSA P r o l a c t i n 200K 66K 23K 1-2 14 0.7 0.1 0.2 The time r e g u i r e d f o r the t r a n s f e r of 125 70% o f I - l a b e l e d p r o t e i n s - 6 6 -Time (h) Figure 23. Time c o u r s e s f o r the e l e c t r o t r a n s f e r of I - l abe led m y o s i n f r o m 6% a n d 8% S D S - p o l y a c r y l a m i d e s l a b g e l s . 125 I - l a b e l e d m y o s i n was a p p l i e d t o 6% ( • ) and 8% (A.) SDS-polyacrylamide s l ab g e l s and e l e c t r o p h o r e s i s was c a r r i e d o u t . F o l l o w i n g e l e c t r o p h o r e s i s , g e l s l i c e s were s u b j e c t e d t o e l e c t r o p h o r e t i c t r a n s f e r t o n i t r o c e l l u l o s e papers. At d i f f e r e n t times a f t e r e l e c t r o p h o r e t i c t r a n s f e r , corresponding s t r i p s of g e l and papers were removed and s u b j e c t e d to a u t o r a d i o g r a p h y . The polypept ide bands d e t e c t e d by a u t o r a d i o g r a p h y were excised and counted i n a Beckman 8000 gamma counter . -67-4 1 6 6 4 2 5 6 R e c i p r o c a l D i l u t i o n 4 1 6 6 4 2 5 6 R e c i p r o c a l D i l u t i o n F i g u r e 24. T i t r a t i o n o f r h o - l D 4 (upper) and 4B2 ( lower) h y b r i d o m a c e l l c u l t u r e s u p e r n a t a n t s a g a i n s t T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes ( • ) and ROS d i sk membranes ( O ) . S o l u b i l i z e d ROS and ROS d i s k membranes immobil ized i n m i c r o t i t e r we l l s were t rea ted with s e r i a l d i l u t i o n s o f c u l t u r e supernatant , 125 r i n s e d , and s u b s e q u e n t l y t r e a t e d w i t h I - l a b e l e d g o a t anti-mouse l g . -68-b i n d i n g to T r i t o n X - 1 0 0 - s o l u b i 1 i zed ROS membranes by T r i t o n X-100-so lub i l i zed ROS and ROS d i s k membranes i s shown i n F i g . 25. Half-maximum i n h i b i t i o n o f r h o - l D 4 a n t i b o d y b ind ing occurred at 0.5 jag/ml for both ROS and ROS d i s k membranes , but half-maximum i n h i b i t i o n of 4B2 a n t i b o d y b i n d i n g o c c u r r e d a t 60>ug/ml for ROS and at 110 jug/ml for ROS d i sk membranes. 13. Degradat ion and a g g r e g a t i o n of ROS 1.2 M o l e c u l a r p r o p e r t i e s o f ROS 1.2 were o b s e r v e d i n some radioimmune l a b e l i n g s t u d i e s . Hea t - t rea ted ROS membrane prote ins were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to C N B r - a c t i v a t e d paper f o l l o w i n g g e l e l e c t r o p h o r e s i s . C N B r - a c t i v a t e d p a p e r w i t h t r a n s f e r r e d ROS p r o t e i n s were s e q u e n t i a l l y l a b e l e d w i t h 4B2 125 ant ibody and I - l a b e l e d goat ant i -mouse l g i s shown i n F i g . 26. Only a 140,000 M^ p o l y p e p t i d e was l a b e l e d . ROS membrane p r o t e i n s p r e p a r e d i n the presence o f 2+ leupeptin( an i n h i b i t o r o f Ca - a c t i v a t e d proteases ) were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e paper fo l lowing g e l e l e c t r o p h o r e s i s . . N i t r o c e l l u l o s e paper w i t h t r a n s f e r r e d ROS p r o t e i n s were s e g u e n t i a l l y l a b e l e d w i t h 4B2 a n t i b o d y and 125 I- labeled goat anti-mouse l g i s shown i n F i g . 2 7 . ROS 1.2 and two higher M p o l y p e p t i d e s were l a b e l e d . -69-C 3 100* o m >. •o o JO ** c < Protein Concentration Protein Concentration F i g u r e 25. I n h i b i t i o n o f r h o - l D 4 a n t i b o d y (upper) and 4B2 a n t i b o d y ( l o w e r ) b i n d i n g t o T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes by T r i t o n X - 1 0 0 - s o l u b i l i z e d ROS membranes ( • ) and ROS d i sk membranes ( o ) u s i n g the s o l i d - p h a s e i n d i r e c t radioirrmune competit ion assay as descr ibed p r e v i o u s l y . - 7 0 -a b 1 4 0 K ^ g f e t Figure 26. Radioimmune l a b e l i n g o f h e a t - t r e a t e d ROS with 4B2 a n t i b o d y . ROS membrane p r e p a r a t i o n (8 mg/ml) was d i l u t e d with equal volume of sample c o c k t a i l c o n t a i n i n g 5% SDS, 40% sucro se , lOmM T r i s , pH 6.8 , 10% y^-mercaptoethanol, and 4% bromophenol b l u e , and hea ted a t 8 0 ° C f o r 5 m i n . S a m p l e was a p p l i e d to a 8% SDS-polyacrylamide s l a b g e l and s u b j e c t e d to g e l e l e c t r o p h o r e s i s . Fol lowing g e l e l e c t r o p h o r e s i s , p r o t e i n s were e l e c t r o p h o r e t i c a l l y t r ans fe r red to C N B r - a c t i v a t e d p a p e r s . C N B r - a c t i v a t e d papers wi th t r ans f e r red pro te ins was l a b e l e d w i t h 4B2 (3) and n o n - s p e c i f i c 125 ( b) c u l t u r e s u p e r n a t a n t s , f o l l o w e d b y I - l a b e l e d goat anti-mouse l g . -71-ROS 1 .2-Figure 27. Radioimmune l a b e l i n g o f ROS prepared i n the presence o f l eupept in with 4B2 antibody. ROS membranes (8 mg/ml) prepared 2+ i n the p r e s e n c e o f l e u p e p t i n , an i n h i b i t o r o f Ca - a c t i v a t e d p r o t e a s e s , were m i x e d w i t h e q u a l v o l u m e o f sample c o c k t a i l c o n t a i n i n g 5% S D S , 40% s u c r o s e , lOmM T r i s , pH 6 . 8 , 10% ^ - m e r c a p t o e t h a n o l , and 4% bromophenol b l u e and a p p l i e d to a 8% SDS-polyacrylamide s l ab g e l . F o l l o w i n g g e l e l e c t r o p h o r e s i s , the p r o t e i n s were e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d to n i t r o c e l l u l o s e paper . N i t r o c e l l u l o s e paper c o n t a i n i n g t r a n s f e r r e d p r o t e i n s was s e q u e n t i a l l y t r e a t e d w i t h 4B2 c u l t u r e s u p e r n a t a n t and 125 I - l a b e l e d goa t ant i -mouse l g . -72-1 4 . P u r i f i c a t i o n o f ROS 1 . 2 b y g e l f i l t r a t i o n  chromatography on a Sephacry l -S -200 column "* ROS 1.2 was p a r t i a l l y p u r i f i e d b y g e l f i l t r a t i o n chromatography on a S e p h a c r y l - S - 2 0 0 c o l u m n . S D S - p l y a c r y l a m i d e g e l s o f e lutants i n the v o i d volume and i n the included volume of the column were shown i n F i g . 28. E l u t a n t s i n the vo id volume contained ROS 1.2, other ROS minor p ro te in s , and a r e s i d u a l amount of rhodopsin whereas e lutants i n the included volume contained mainly rhodopsin. 15. P u r i f i c a t i o n o f ROS 1.2 by a f f i n i t y chromatography  on a 4B2 a n t i b o d y - a g a r o s e column ROS 1.2 was p a r t i a l l y p u r i f i e d by a f f i n i t y chromatography on a 4B2 an t ibody-agarose c o l u m n . As shown i n F i g . 29, the washed f r a c t i o n s contained no ROS 1.2 and s p e c i f i c a l l y - e l u t e d f r a c t i o n s contained ROS 1.2 and a smal l amount o f rhodopsin. -73-R0S1.2 R H O-41 m m m Figure 28. Chromatography o f ROS pro te in s on a Sephacryl-S-200 c o l u m n . ROS membrane p r e p a r a t i o n ( 4 . 5 mg p r o t e i n ) was s o l u b i l i z e d wi th 3% SDS, and a p p l i e d to a Sephacryl-S-200 column (2.0x48.0 cm) p r e - e q u i l i b r a t e d w i t h PBS c o n t a i n i n g 0.1% SDS and ImM EDTA. Void volume o f the column was determined by the e l u t i o n volume o f B l u e D e x t r a n . P r o t e i n c o m p o s i t i o n s o f a f r a c t i o n i n the v o i d volume (lane 2) and two f r a c t i o n s i n the included volume ( l a n e s 3 and 4) were a n a l y z e d by g e l e l e c t r o p h o r e s i s on a 8% S D S - p o l y a c r y l a m i d e s l a b g e l . Lane 1 c o n t a i n e d S D S - t r e a t e d ROS p r o t e i n s not a p p l i e d to the c o l u m n . ROS 1 . 2 and r h o d o p s i n are i n d i c a t e d . -74-ROS 1.2 21K F i g u r e 2 9 . C h r o m a t o g r a p h y o f ROS p r o t e i n s on a 4B2 a n t i b o d y - a g a r o s e c o l u m n . ROS membrane p r e p a r a t i o n (4 .5 mg prote in) was s o l u b i l i z e d w i t h 1% CTAB i n 0.1M sod ium phosphate b u f f e r , pH 7 .4 , and app l ied t o a 2 ml 4B2 antibody-agarose column (0.5 mg antibody/ml column) p r e - e q u i l i b r a t e d with the 0.1M sodium phosphate bu f fe r , pH 7 . 4 , c o n t a i n i n g lOmM CTAB. The column was washed w i t h 8 ml o f the same b u f f e r c o n t a i n i n g 10 mM CTAB and e l u t e d w i t h 50mM d i e t h y l a m i n e , pH 1 1 . 5 , i n the same b u f f e r c o n t a i n i n g 10 mM CTAB. P r o t e i n compos i t ions o f the buffer-washed f r a c t i o n s (lanes 2 and 3) and the d i e t h y l a m i n e - e l u t e d f r a c t i o n s ( lanes 4 and 5) were d e t e r m i n e d by g e l e l e c t r o p h o r e s i s on a 8% SDS-polyacrylamide s l a b g e l . Lane 1 contained standard p r o t e i n s : p h o s p h o r y l a s e b (92K) , BSA (66K) , o v a l b u m i n (45K) , c a r b o n i c anhydrase (31K) , soybean t r y p s i n i n h i b i t o r (21K) , and lysozyme (14K). -75-DISCUSSION The method o f a s c i t e s t u m o r s i n d u c t i o n i n mice f o r the product ion of 4B2 monoclonal a n t i b o d y was found to be ea s i e r and more economical than the mass c u l t u r e method. A l s o , a s c i t e s f l u i d c o n t a i n e d a much h i g h e r c o n c e n t r a t i o n o f 4B2 a n t i b o d y than c u l t u r e supernatant as de te rmined by s o l i d - p h a s e RIA, and i n contras t to c u l t u r e s u p e r n a t a n t i t c o n t a i n e d no nonspec i f i c IgG m o l e c u l e s d e t e c t a b l e b y S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . However, a s c i t e s tumors induct ion was not always s u c c e s s f u l and the v o l u m e o f a s c i t e s f l u i d o b t a i n e d from a tumor-bearing mouse v a r i e d cons iderab ly . The monoclonal c h a r a c t e r i s t i c o f 4B2 ant ibody was supported by the f a c t t h a t r e c l o n i n g o f 4B2 hybr idoma c e l l l i n e d i d not change the s p e c i f i c i t y o f t h e 4B2 a n t i b o d y , as i n d i c a t e d by radioimmune l a b e l i n g o f ROS p r o t e i n s w i t h 4B2 antibody secreted by r e c l o n e d 4B2 hybr idoma c e l l l i n e . The i d e n t i f i c a t i o n of lg subtype of 4B2 ant ibody was n e c e s s a r y f o r i t s use i n biochemical s tudies and i t s p u r i f i c a t i o n . The 4B2 a n t i b o d y was i d e n t i f i e d , by s o l i d - p h a s e RIA and S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c a n a l y s i s , to be an IgM m o l e c u l e w i t h )i heavy cha ins o f apparent M^=55,000 and K l i g h t c h a i n s o f apparent M^=22,000. The 4B2 ant ibody was p a r t i a l l y p u r i f i e d from a s c i t e s f l u i d by D E A E - S e p h a c e l i o n e x c h a n g e c o l u m n c h r o m a t o g r a p h y . The p a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y was shown by RIA to be a c t i v e -76-a g a i n s t ROS m e m b r a n e s . H o w e v e r , t h e c o n c e n t r a t i o n o f p a r t i a l l y - p u r i f i e d 4B2 a n t i b o d y r e g u i r e d f o r half-maximum antibody "binding to ROS membranes was 4 f o l d higher than that of the o r i g i n a l 4B2 a s c i t e s f l u i d . T h i s i n d i c a t e d that some o f the 4B2 a n t i b o d i e s was dena tured d u r i n g the p u r i f i c a t i o n p roce s s . S u b s e q u e n t l y , t h e two p u r i f i c a t i o n s t e p s l i k e l y t o cause denaturat ion of 4B2 ant ibody, l y o p h i l i z a t i o n and ammonium su l f a t e p r e c i p i t a t i o n , were e l i m i n a t e d from the p u r i f i c a t i o n procedure. In a d d i t i o n , g e l f i l t r a t i o n chromatography on a Sephacryl-S-500 column and i o n exchange chromatography on a D E A E - A f f i - G e l - B l u e column were used to p u r i f y the 4B2 antibody from a s c i t e s f l u i d . Inmunological ly a c t i v e 4B2 m o n o c l o n a l ant ibody was obtained by i o n e x c h a n g e c h r o m a t o g r a p h y on a D E A E - S e p h a c e l o r a DEAE-Af f i -Ge l -Blue column, and g e l e x c l u s i o n chromatography on a Sephacryl-S-500 column. As d e t e r m i n e d by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s , 4B2 a n t i b o d y of higher p u r i t y was obtained using the former method, but the l a t t e r method provided the s e p a r a t i o n o f p e n t a m e r i c and d i s s o c i a t e d forms of 4B2 a n t i b o d y . T h e d i f f e r e n c e b e t w e e n D E A E - S e p h a c e l a n d D E A E - A f f i - G e l - B l u e media was t h a t a p o l y p e p t i d e o f apparent M r =85,000 always c o p u r i f i e d w i t h 4B2 a n t i b o d y , o n l y when the former medium was used. The i d e n t i t y o f t h i s polypept ide was not determined. I t may be t r a n s f e r r i n , s i n c e t r a n s f e r r i n i s a serum p r o t e i n o f s i m i l a r M ^ , and has been found to c o p u r i f y wi th lg m o l e c u l e s (68) . The a d v a n t a g e o f u s i n g D E A E - S e p h a c e l over -77-DEAE-Aff i -Gel -Blue medium for the p u r i f i c a t i o n of 4B2 antibody i s the h i g h r e p r o d u c i b i l i t y o f t h e r e s u l t s . The y i e l d of 4B2 ant ibody from 4B2 a s c i t e s f l u i d was much lower than the y i e l d of rho-lD4 antibody from e q u a l volume o f rho- lD4 a s c i t e s f l u i d . The d i f f e rence may be due to the d i f f e r e n t i n t r i n s i c p r o p e r t i e s of I g M - s e c r e t i n g h y b r i d o m a (4B2) and I g G - s e c r e t i n g hybr idoma (rho-lD4) c e l l l i n e s . A 4B2 antibody-agarose a f f i n i t y column and a Sephacryl-S-500 g e l f i l t r a t i o n column were o n l y a b l e to p u r i f y ROS 1.2 to 30-40% homogene i ty , as d e t e r m i n e d b y S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c a n a l y s i s . Whether the c o e l u t i o n o f a r e s i d u a l amount o f r h o d o p s i n and ROS 1.2 was due t o t h e i n c o m p l e t e s o l u b i l i z a t i o n of ROS membranes o r to the strong p r o t e i n - p r o t e i n i n t e r a c t i o n s b e t w e e n ROS 1.2 and r h o d o p s i n r e m a i n s to be d e t e r m i n e d . S i n c e ROS 1.2 was not p u r i f i e d to homogene i ty , amino a c i d composition o f ROS 1.2 could not be determined. The molecular p r o p e r t i e s o f ROS 1.2 were probed by using the 4B2 a n t i b o d y to de te rmine whether c r o s s - r e a c t i v i t y ex i s t s between ROS 1.2 and some o t h e r l a r g e p r o t e i n s i n c e l l t ype s other than the rod c e l l , namely , myos in i n m u s c l e , s p e c t r i n i n red b l o o d c e l l s , and b r a i n s p e c t r i n i n b r a i n c e l l s . These p ro te in s were t e s t e d f o r c r o s s - r e a c t i v i t y w i t h the 4B2 antibody b e c a u s e t h e y n o t o n l y h a v e s i m i l a r , b u t a l s o h a v e s i m i l a r s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s . For example, a l l three prote ins i n t e r a c t with a c t i n , a c y t o s k e l e t a l element, found - 7 8 -present i n ROS membrane p r e p a r a t i o n s by RIA us ing an a n t i - a c t i n monoclonal ant ibody. S o l i d - p h a s e RIA and competit ion assays have shown t h a t 4B2 a n t i b o d y c r o s s - r e a c t e d w i t h r a b b i t s k e l e t a l muscle myosin, human red b l o o d c e l l membranes, and bovine b r a i n homogenate to d i f f e r i n g d e g r e e s . Immunoblot a n a l y s i s has shown t h a t myos in and a 14^=160,000 p o l y p e p t i d e i n b o v i n e b r a i n homogenate c r o s s - r e a c t w i t h the 4B2 a n t i b o d y . T h i s a n a l y s i s , however, f a i l e d to demonstrate which polypeptide i n red b l o o d c e l l membranes c r o s s - r e a c t w i t h the 4B2 a n t i b o d y . The i d e n t i t y o f t h e 14^=160 , 000 p o l y p e p t i d e i n b o v i n e b r a i n homogenate has not been d e t e r m i n e d , but i t i s be l ieved to be the p r o t e o l y t i c breakdown p r o d u c t o f b r a i n s p e c t r i n . Burridge et a l . (60) have found t h a t p a r t i a l d i g e s t i o n o f n a t i v e b r a i n s p e c t r i n with Staphylococca l V8 p r o t e a s e g e n e r a t e d a po lypept ide with an apparent 14^=160,000. T h i s p o l y p e p t i d e c r o s s - r e a c t e d with a n t i -b r a i n s p e c t r i n a n t i b o d i e s . The immunolog ica l c r o s s - r e a c t i v i t y of na t ive b r a i n s p e c t r i n and red b l o o d c e l l s p e c t r i n with the 4B2 antibody was no t d e t e c t e d by immunoblot a n a l y s i s . T h i s may be due to the i n e f f i c i e n t e l e c t r o p h o r e t i c t r a n s f e r o f these large p r o t e i n s a n d / o r t h e i r weak c r o s s - r e a c t i v i t i e s w i t h the 4B2 ant ibody. The oC s u b u n i t s i n b o t h p r o t e i n s have 14^=240,000. The ^ s u b u n i t of b r a i n s p e c t r i n has 14^=235,000 and ^ s u b u n i t of red blood c e l l s p e c t r i n has 14^=220,000 (69) . As shown i n the study 125 on t h e e l e c t r o t r a n s f e r o f I - l a b e l e d p r o t e i n s , t h e e l e c t r o p h o r e t i c t r a n s f e r o f s m a l l p o l y p e p t i d e s such as BSA and -79-p r o l a c t i n , w i t h apparent M r =66,000 and 23,000 r e s p e c t i v e l y , was completed w i t h i n 30 min whereas l a r g e po lypept ides such as myosin, w i t h an apparent M r =200,000 r e q u i r e d 18-20h. Thus the amount o f e r y t h r o c y t e and b r a i n s p e c t r i n s t r a n s f e r r e d to n i t r o c e l l u l o s e papers might have been l e s s than adequate f o r immunoblot de tec t ion o f c r o s s - r e a c t i v i t i e s of these prote ins with the 4B2 a n t i b o d y . T h e i r weak c r o s s - r e a c t i v i t i e s w i th the 4B2 antibody are due to the d i f f e r e n t s p e c i e s and c e l l type o r i g i n s o f the se 4B2 a n t i g e n s . T h i s i s n o t s u r p r i s i n g s i n c e i n i t i a l immunologica l e v i d e n c e p r e s e n t e d was a g a i n s t the e x i s t e n c e o f s p e c t r i n i n o t h e r c e l l t y p e s (68) , y e t r e c e n t f i n d i n g s support the i d e a t h a t e r y t h r o c y t e s p e c t r i n , b r a i n s p e c t r i n , and i t s i m m u n o l o g i c a l l y r e l a t e d e q u i v a l e n t s i n o t h e r c e l l types a l l belong to a f ami ly o f r e l a t e d p r o t e i n s c a l l e d the spectr ins (60) . Whether ROS 1.2 be long t o t h e s p e c t r i n f a m i l y remains to be determined. The ro le of s p e c t r i n i n the submembranous cytoske le ton of mammalian e r y t h r o c y t e s i s t o m e d i a t e t h e c o u p l i n g o f a c t i n f i laments to the plasma membrane and t o m a i n t a i n the shape and de formab i l i ty o f the c e l l . A p a r a l l e l funct ion o f a c t i n attachment to membranes i n o t h e r c e l l s c o n t a i n i n g n o n e r y t h r o i d s p e c t r i n i s supported by the l o c a l i z a t i o n o f n o n e r y t h r o i d s p e c t r i n c lo se to the plasma membrane o f many c e l l t y p e s (71) . S i m i l a r l y , ROS 1.2 i s l o c a l i z e d c l o s e to the ROS plasma membrane o v e r the e n t i r e - 8 0 -l ength of the outer segment (72 ) . T h u s , the r o l e of ROS 1.2 i s suggested to be s i m i l a r to t h a t o f s p e c t r i n i n maintaining c e l l shape. The maintenance o f ROS s t r u c t u r e must be accomplished by two types o f connect ions : d i s k - t o - d i s k connect ions , s ince i t has been known that d i sks do ho ld t o g e t h e r even i n the absence o f the plasma membrane (73) , d i s k i n c i s u r e s a re somehow kept i n p rec i s e r e g i s t e r o v e r the e n t i r e l e n g t h o f the ROS, and the d i s k s are separated from each other by a c o n s t a n t 100 % space along the rod ax i s (74); and d i s k - t o - p l a s m a membrane c o n n e c t i o n s , s ince d i sks adhered s t rong ly to the plasma membrane when rods are d i s rupted ( 7 3 ) . Roof & Heuser (74) h a v e i n d i c a t e d t h a t f i l a m e n t l i k e s p e c i a l i z a t i o n s a s s o c i a t e d e x c l u s i v e l y w i t h d i s k r ims are respons ib le for connect ing d i s k r ims to each other and to plasma membrane. The m o l e c u l a r n a t u r e . o f t h e s e f i l a m e n t s i s unknown. However , i t has been sugges ted to be the 290,000 d a l t o n " r i m p r o t e i n " i n f r o g ROS d e s c r i b e d by Papermaster e t a l . (75) for two r e a s o n s . The l o c a t i o n o f r i m p r o t e i n and f i l a m e n t s i s s i m i l a r l y c o n f i n e d to r ims and i n c i s u r e s , and the s i z e o f rim p r o t e i n and f i l a m e n t s i s c o m p a r a b l e . ROS 1.2 i s proposed to be the " r i m p r o t e i n " i n b o v i n e ROS because i t i s s l i g h t l y smaller than the rim p r o t e i n i n f r o g ROS, i t extends some di s tance from the d i s k membrane (57 ) , and i t i s l o c a l i z e d i n d i s k rims (72). The s t r u c t u r a l r o l e o f ROS 1.2 i n l i n k i n g d i s k s to d i s k s and d i s k s to plasma membrane i s schemat ica l ly i l l u s t r a t e d i n F i g . 30. -81-ROD OUTER SEGMENT Figure 30. Proposed s t r u c t u r a l r o l e o f ROS 1.2. ROS 1.2 i s a myos in- l ike p r o t e i n l o c a l i z e d i n the r i m regions of d i s k s and i n conjunct ion with other c y t o s k e l e t a l elements serves to l i n k d i s k s to each other and to plasma membrane f o r the maintenance o f the h igh ly-ordered ROS s t r u c t u r e . -82-Radioimmune l a b e l i n g o f h e a t - t r e a t e d ROS membranes and ROS membranes prepared i n the p r e s e n c e o f l e u p e p t i n , with the 4B2 a n t i b o d y r e v e a l e d two p r o p e r t i e s o f ROS 1.2 : ROS 1.2 can be degraded to produce a 1^=140, 000 f r a g m e n t t h a t r e t a i n s 4B2 a n t i g e n i c s i t e and ROS 1.2 i s a b l e to aggregate with each other to form m u l t i m e r s t h a t r e t a i n 4B2 a n t i g e n i c s i t e . A l t h o u g h no c o n t r o l s tudies were done c o n c o m i t a n t l y , the degradation o f ROS 1.2 was l i k e l y induced by heat s i n c e s i m i l a r intense l a b e l i n g of t h e M r = 1 4 0 , 0 0 0 p o l y p e p t i d e was n o t d e t e c t e d i n many r a d i o i m m u n e l a b e l i n g o f ROS m e m b r a n e s e x p e r i m e n t s . The a g g r e g a t i o n o f ROS 1.2 was l i k e l y induced by the p r e s e n c e o f l e u p e p t i n d u r i n g t h e p r e p a r a t i o n o f ROS membranes s i n c e no l a b e l i n g o f p o l y p e p t i d e s w i t h g r e a t e r than ROS 1.2 has ever been d e t e c t e d p r e v i o u s l y u s i n g ROS membranes p r e p a r e d i n the absence of l e u p e p t i n . In t h i s r e g a r d , i t s h o u l d be noted that l e u p e p t i n has been u s e d t o show t h e a c c u m u l a t i o n o f n e u r a l f i l a m e n t s i n a x o n t e r m i n a l s ( 7 6 ) , b e c a u s e i t i n h i b i t s 2+ Ca - a c t i v a t e d p r o t e a s e s w h i c h a r e r e s p o n s i b l e f o r t h e 2+ d i s a g g r e g a t i o n o f n e u r a l f i l a m e n t s . However, Ca - a c t i v a t e d proteases have not been found i n ROS. The a b i l i t y of ROS 1.2 to s e l f - a s s o c i a t e may be important for i t s l o c a l i z a t i o n i n d i s k r i m s , because l a r g e aggregates o f ROS 1.2 can r e s t r i c t i t s m o b i l i t y i n h i g h l y f l u i d d i sk membranes. The degradation o f ROS 1.2 may be impor tan t i n the d i s k renewal p r o c e s s . The d i s r u p t i o n o f d i s k - t o - d i s k a n d / o r d i s k - t o - p l a s m a -83-membrane connections i s most l i k e l y an e a r l y event i n the renewal p r o c e s s . P r o t e o l y t i c d e g r a d a t i o n o f ROS 1.2 i s a way o f d i s r u p t i n g these c o n n e c t i o n s . A l s o , i f the degraded product of ROS 1.2 i s released in to the c y t o p l a s m i c space, and re ta ins 4B2 a n t i g e n i c s i t e , then t h e r e would be a c y t o p l a s m i c p o o l of 4B2 an t i gen . A c y t o p l a s m i c p o o l of 4B2 a n t i g e n i s suggested by a 2 f o l d d i f f e rence i n the c o n c e n t r a t i o n o f 4B2 ant igen between ROS and ROS d i s k m e m b r a n e s , a s i n d i c a t e d b y RIA and radioimmune c o m p e t i t i o n a s s a y s . In c o n t r a s t , t h e r e was no d i f f e r e n c e i n the c o n c e n t r a t i o n o f rhodopsin between ROS and ROS d i s k membranes. These f ind ings lead to a w o r k i n g model on the s t r u c t u r a l and f u n c t i o n a l ro le s of ROS 1.2 i n b o v i n e ROS. In t h i s model, ROS 1.2 s e l f - a s s o c i a t e to form d i sk r i m r e g i o n s o f high curvature dur ing d i s k assembly, and i n conjunct ion with c y t o s k e l e t a l elements such as a c t i n s e r v e s to anchor d i s k r ims i n c l o s e p r o x i m i t y o f the plasma membrane. In a d d i t i o n , p r o t e i n - p r o t e i n i n t e r a c t i o n s between ROS 1.2 o f a d j a c e n t d i s k s s e r v e t o s e p a r a t e d i s k s 100 % apar t . The d i s k - t o - d i s k and d i s k - t o - p l a s m a membrane connections are removed dur ing d i sk renewal by the p r o t e o l y t i c degradation o f ROS 1 .2 . The a c t i v a t i o n o f p r o t e a s e s p e c i f i c f o r ROS 1.2 may be ^dependent on the e x t e n t o f p h o t o b l e a c h i n g o f d i s k s , s ince only 8-10 d i s k s are shed at a t i m e . The exi s tence of c r o s s - r e a c t i v i t y between ROS 1.2 with myos in and p o l y p e p t i d e s i n b r a i n homogenate and red blood c e l l membranes i n d i c a t e s t h a t ROS 1.2 belongs to a -84-f ami ly of immunological ly r e l a t e d p r o t e i n s , perhaps the s p e c t r i n f a m i l y , because o f t h e i r s i m i l a r M and l o c a t i o n c l o s e to the plasma membrane. In t h i s v i e w , i t i s o f i n t e r e s t to note that members o f the s p e c t r i n f a m i l y have been found , i n many c e l l types , to be h igh m o l e c u l a r we ight a c t i n - and calmodul in-binding pro te ins which are both s t r u c t u r a l l y and f u n c t i o n a l l y homologous to erythrocyte s p e c t r i n (77). Since a c t i n and calmodulin have been found to be p r e s e n t i n b o v i n e ROS, ROS 1.2 i s a p o t e n t i a l c a n d i d a t e f o r b i n d i n g a c t i n and c a l m o d u l i n . I n mammalian e r y t h r o c y t e s , s p e c t r i n b i n d s t o o t h e r c y t o s k e l e t a l p r o t e i n s i n c l u d i n g a c t i n f i l a m e n t s . Band 2.1 ( a n k y r i n , syndeins) and Band 4 .1 , and through the l a t t e r two p r o t e i n s , i t i s attached to the cytoplasmic sur face o f the e r y t h r o c y t e membrane. S p e c t r i n binds 2+ c a l m o d u l i n i n a Ca -dependent manner, but i t remains unknown as to whether s p e c t r i n - c a l m o d u l i n i n t e r a c t i o n a f f ec t s the binding o f s p e c t r i n t o a c t i n , Band 2 . 1 , and Band 4 . 1 . I t w i l l be i n t e r e s t i n g to see i f an analogous c y t o s k e l e t a l network e x i s t s i n 2+ bovine ROS s i n c e the r o l e o f Ca i o n s appears to be important i n the event o f v i s u a l e x c i t a t i o n i n ROS. Thus i n f u t u r e work , t h e l o c a l i z a t i o n o f c y t o s k e l e t a l molecules c lo se to the ROS plasma membrane and the determination o f the f u n c t i o n o f ROS 1.2 may p r o v i d e new i n s i g h t s i n t o the mechanisms o f the m a i n t e n a n c e o f t h e h i g h l y o r d e r e d ROS s t r u c t u r e , d i sk renewal , and v i s u a l e x c i t a t i o n . Future research w i l l be d i r e c t e d t o w a r d 1) o b t a i n i n g an IgG a n t i - R O S 1.2 -85-monoc lona l a n t i b o d y i n o r d e r t o o b t a i n a l a r g e r q u a n t i t y o f immunological ly a c t i v e a n t i b o d y from a s c i t e s f l u i d , 2) improving the p u r i f i c a t i o n o f ROS 1.2 by u s i n g a c o m b i n a t i o n o f g e l f i l t r a t i o n chromatography on a Sephacryl-S-200 column ( s i n c e r h o d o p s i n can be p a r t i a l l y s e p a r a t e d from ROS 1.2 u s i n g t h i s method) and a f f i n i t y chromaography on an ant i -ROS 1.2 antibody column (since other minor p r o t e i n s can be separated from ROS 1.2 u s i n g t h i s method) , and 3) i n v e s t i g a t i n g the s t r u c t u r e and funct ion of ROS 1.2 by amino a c i d s eguenc ing and r e c o n s t i t u t i o n s tud ie s . ^86-REFERENCES 1. Blaurock, A . E . , and W i l k i n s , M . H . F . (1969) Nature (London) 223. 906-909. 2. Korenbrot, J . 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