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A spectrin-like protein in bovine retinal rod photoreceptor outer segments as defined by monoclonal antibodies Wong, Simon Yuk Chun 1988

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A SPECTRIN-LIKE PROTEIN IN BOVINE RETINAL ROD PHOTORECEPTOR OUTER SEGMENTS AS DEFINED BY MONOCLONAL ANTIBODIES By SIMON YUK CHUN WONG B. Sc., The U n i v e r s i t y o f B r i t i s h Columbia, 1981, M. Sc., The U n i v e r s i t y o f B r i t i s h Columbia, 1984, A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA ^Simon Yuk Chun Wong, A p r i l 1988. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE -6n /8-H \ ABSTRACT Bio c h e m i c a l and immunological s t u d i e s i n d i c a t e t h a t rod o u t e r segments (ROS) of bovine p h o t o r e c e p t o r c e l l s c o n t a i n a M r 240,000 p o l y p e p t i d e r e l a t e d t o the oC-subunit o f r e d b l o o d c e l l (RBC) s p e c t r i n . With the use of sodium dodecy l s u l f a t e g e l e l e c t r o p h o r e s i s i n c o n j u n c t i o n w i t h the immunoblotting technique, monoclonal antibody 4B2 was found t o b i n d t o a M 240,000 p o l y p e p t i d e i n ROS t h a t i s d i s t i n c t from the prominent M r 220,000 c o n c a n a v a l i n A b i n d i n g g l y c o p r o t e i n . The M r 240,000 p o l y p e p t i d e i s h i g h l y s u s c e p t i b l e t o d e g r a d a t i o n by endogenous p r o t e a s e s . I t does not appear t o be an i n t e g r a l membrane p r o t e i n but i s t i g h t l y membrane a s s o c i a t e d s i n c e i t can be p a r t i a l l y e x t r a c t e d from ROS membranes with urea i n the absence of d e t e r g e n t . The 4B2 antibody c r o s s - r e a c t e d w i t h RBC ghost membranes and bovine b r a i n microsomal membranes. Radioimmune assays and immunoblotting a n a l y s i s of p u r i f i e d bovine RBC s p e c t r i n f u r t h e r r e v e a l e d t h a t the 4B2 antibody predominantly l a b e l l e d the o^-chain of RBC s p e c t r i n having an apparent M r of 240,000. Monoclonal antibody 3A6 was found t o b i n d t o a p o l y p e p t i d e w i t h a s l i g h t l y lower M r than the 4 B 2 - s p e c i f i c p o l y p e p t i d e . I t i s a l s o h i g h l y s u s c e p t i b l e t o d e g r a d a t i o n by endogenous p r o t e a s e s , but u n l i k e the 4B2 antibody, i t predominantly l a b e l l e d the ^ - c h a i n of RBC s p e c t r i n having an apparent M of 220,000. P o l y c l o n a l a n t i - s p e c t r i n a n t i b o d i e s t h a t bound t o both the o( - and ^ - c h a i n o f RBC s p e c t r i n predominantly l a b e l l e d a M r 240,000 p o l y p e p t i d e of ROS membranes. Two f a i n t l y l a b e l l e d bands i n the M r range of 210,000-220,000 were a l s o observed. These components may r e p r e s e n t v a r i a n t s of the ft - c h a i n of s p e c t r i n t h a t are weakly c r o s s - r e a c t i n g or p r e s e n t i n s m a l l e r q u a n t i t i e s than the (X-chain. Immunocytochemical l a b e l l i n g s t u d i e s u s i n g the 4B2 antibody and immunogold-dextran markers i n d i c a t e d t h a t the ROS s p e c t r i n - l i k e p r o t e i n i s p r e f e r e n t i a l l y l o c a l i z e d i n the r e g i o n where the d i s c s come i n c l o s e c o n t a c t t o the plasma membrane of ROS. Immunoblotting a n a l y s i s i n d i c a t e d t h a t r h o d o p s i n and p e r i p h e r i n which c o n s t i t u t e over 90% of t o t a l d i s c membrane p r o t e i n s were s e l e c t i v e l y s o l u b i l i z e d i n T r i t o n X-100, whereas a s e t of p o l y p e p t i d e s i n c l u d i n g the 4B2-s p e c i f i c p o l y p e p t i d e and the M r 220,000 c o n c a n a v a l i n A-b i n d i n g g l y c o p r o t e i n was o n l y p a r t i a l l y s o l u b l e . E l e c t r o n microscopy of a n e g a t i v e l y s t a i n e d T r i t o n - e x t r a c t e d ROS p e l l e t r e v e a l e d a f i l a m e n t o u s network. These s t u d i e s i n d i c a t e t h a t ROS c o n t a i n a p r o t e i n r e l a t e d t o RBC s p e c t r i n , which may c o n s t i t u t e a major component of a fi l a m e n t o u s network l i n i n g the i n n e r s u r f a c e of the ROS plasma membrane as p r e v i o u s l y seen by e l e c t r o n microscopy. T h i s membrane s k e l e t a l system may serve t o s t a b i l i z e the ordered ROS s t r u c t u r e and m a i n t a i n a con s t a n t d i s t a n c e between the rim r e g i o n of the d i s c s and the plasma membrane. - i v -TABLE OF CONTENTS Page I. TITLE PAGE i I I . ABSTRACT i i I I I . TABLE OF CONTENTS i v IV. LIST OF FIGURES v i i i V. LIST OF ABBREVIATIONS X VI. ACKNOWLEDGMENT • x i V I I . INTRODUCTION A. V e r t e b r a t e R e t i n a 1 B. Photoreceptor C e l l s and V i s u a l Pigments 1 C. Rod Photoreceptors 6 D. P h o t o t r a n s d u c t i o n Process 10 E. Proposed Mechanism of P h o t o t r a n s d u c t i o n 11 (1) Role o f cGMP (2) Role o f Ca (3) Rhodopsin F. Rod Outer Segment 20 (1) S t r u c t u r e (2) B i o g e n e s i s and Renewal (3) Maintenance G. C y t o s k e l e t a l Network i n Red Blood C e l l 24 H. S p e c t r i n - l i k e P r o t e i n s 27 I. High M o l e c u l a r Weight P r o t e i n s i n Rod Outer Segment 28 J . Monoclonal A n t i b o d i e s t o Rod Outer Segment P r o t e i n s 29 K. T h e s i s I n v e s t i g a t i o n 34 - V -Page V I I I . MATERIALS AND METHODS A. M a t e r i a l s 36 B. Hybridoma C e l l C u l t u r e C o n d i t i o n s • 37 C. Maintenance of Hybridoma C e l l L i n e s 38 D. C l o n i n g o f Hybridoma C e l l L i n e s 39 E. P r o d u c t i o n of Monoclonal Antibody 40 F. P r e p a r a t i o n of ROS and D i s c Membranes 41 G. P r e p a r a t i o n of RBC G h o s t s , S p e c t r i n and B r a i n Homogenate 43 H. Sources of A n t i b o d i e s 4 3 (1) Monoclonal A n t i b o d i e s (2) C e l l F u s i o n (3) P o l y c l o n a l A n t i b o d i e s (4) T r a c e r Second A n t i b o d i e s I. R a d i o i o d i n a t i o n of P r o t e i n s 48 J . S o l i d - P h a s e Radioimmune and Competitive I n h i b i t i o n Assays 48 K. Sodium Dodecyl S u l f a t e - P o l y a c r y l a m i d e Gel E l e c t r o p h o r e s i s and Gel T r a n s f e r 50 L. S i z e E x c l u s i o n Chromatography of ROS P r o t e i n s on a Sepharose-CL 2B Column 52 M. L o w i c r y l T h i n S e c t i o n L a b e l l i n g 53 N. E x t r a c t i o n of ROS Membranes 54 O. T r i t o n X-100 E x t r a c t i o n of ROS 55 P. Detergent S o l u b i l i z a t i o n of ROS Membranes 55 Q. Negative S t a i n i n g of T r i t o n X-100-Extracted ROS P e l l e t 56 Page RESULTS A. High M o l e c u l a r Weight P o l y p e p t i d e s o f ROS Membranes 57 B. Degradation of 4 B 2 - S p e c i f i c P r o t e i n by an Endogenous Protease 59 C. Urea E x t r a c t i o n o f the 4 B 2 - S p e c i f i c P r o t e i n 61 D. Immunological C r o s s - R e a c t i v i t y o f Monoclonal Antibody 4B2 wi t h P r o t e i n s from Other C e l l T y p e s — 6 3 E. I d e n t i f i c a t i o n o f S p e c t r i n as the RBC A n t i g e n f o r the 4B2 Monoclonal Antibody 66 F. C h a r a c t e r i z a t i o n o f 3A6 Monoclonal Antibody 69 G. B i n d i n g o f 3A6 Monoclonal Antibody t o RBC S p e c t r i n and ROS Membranes 69 H. B i n d i n g o f P o l y c l o n a l A n t i - S p e c t r i n A n t i b o d i e s t o ROS P r o t e i n s 71 I. E x t r a c t i o n o f 4 B 2 - S p e c i f i c P r o t e i n under Low I o n i c S t r e n g t h C o n d i t i o n s 76 J . A s s o c i a t i o n o f 4 B 2 - S p e c i f i c P r o t e i n w i t h ROS Membranes 78 K. Immunocytochemical L a b e l l i n g of Rod C e l l s w i t h the 4B2 Antibody and Immunogold-Dextran Markers 80 L. Detergent S o l u b i l i z a t i o n o f ROS Membrane Prote i n s - 8 4 M. E l e c t r o n Microscopy of T r i t o n X-100 E x t r a c t e d ROS P e l l e t 89 N. Antibody Probes f o r I d e n t i f y i n g P r o t e i n Components of ROS C y t o s k e l e t o n 91 (1) A n t i - A c t i n Monoclonal Antibody (2) P o l y c l o n a l A n t i - A n k y r i n A n t i b o d i e s (3) C h a r a c t e r i z a t i o n o f 1D1 Monoclonal Antibody (4) C h a r a c t e r i z a t i o n o f 1H5 and 2A4 Monoclonal A n t i b o d i e s - v i i -Page X. DISCUSSION A. D i f f e r e n c e s between 4 B 2 - S p e c i f i c P r o t e i n and ROS 1.2 100 B. Immunological C r o s s - R e a c t i v i t y o f 4B2 and 3A6 Monoclonal A n t i b o d i e s w i t h RBC S p e c t r i n and P o l y c l o n a l A n t i - S p e c t r i n A n t i b o d i e s w i t h ROS P r o t e i n s • 101 C. E x t r a c t i o n o f ROS S p e c t r i n 107 D. A s s o c i a t i o n o f 4 B 2 - S p e c i f i c P r o t e i n w i t h ROS Membranes 108 E. L o c a l i z a t i o n and P o s s i b l e F u n c t i o n o f ROS Spectrin-109 F. Detergent S o l u b i l i z a t i o n o f ROS Membranes 111 G. I d e n t i f i c a t i o n o f ROS C y t o s k e l e t a l Components 114 H. Rod Photoreceptor C y t o s k e l e t o n 116 XI. CONCLUSIONS 117 XI I . FUTURE DIRECTIONS 118 X I I I . REFERENCES 121 - v i i i -LIST OF FIGURES Page F i g u r e 1. Gross Anatomy of the V e r t e b r a t e Eye 2 F i g u r e 2. I n t e r r e l a t i o n s h i p s of R e t i n a l Neurons and G l i a -4 F i g u r e 3. Diagram of a V e r t e b r a t e Rod Photor e c e p t o r C e l l -8 F i g u r e 4. Enzyme Cascade of V i s i o n 14 F i g u r e 5. F i n e S t r u c t u r e of Rod Outer Segment 22 F i g u r e 6. The Red C e l l Membrane S k e l e t o n 26 F i g u r e 7. L o c a l i z a t i o n o f Anti-Rhodopsin Monoclonal A n t i b o d i e s 32 F i g u r e 8. SDS Gel E l e c t r o p h o r e s i s and Immunoblots o f ROS D i s c Membranes 58 F i g u r e 9. P r o t e o l y t i c Degradation of the 4 B 2 - S p e c i f i c P r o t e i n o f ROS by Endogenous Proteases 60 F i g u r e 10. S i z e E x c l u s i o n Chromatography of S D S - S o l u b i l i z e d ROS P r o t e i n s on a Sepharose 2B Column 62 F i g u r e 11. E x t r a c t i o n of the 4 B 2 - S p e c i f i c P r o t e i n from ROS Membranes by Urea 64 F i g u r e 12. Competitive I n h i b i t i o n o f 4B2 Antibody B i n d i n g t o ROS Membrane P r o t e i n s by RBC Membrane Ghosts and B r a i n Microsomal Membranes 65 F i g u r e 13. B i n d i n g of 4B2 and rho-lD4 A n t i b o d i e s t o RBC S p e c t r i n 67 F i g u r e 14. SDS Gel E l e c t r o p h o r e s i s and Immunoblots of Bovine RBC S p e c t r i n 68 F i g u r e 15. T i t r a t i o n o f 3A6 Hybridoma 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 70 F i g u r e 16. SDS Gel E l e c t r o p h o r e s i s and Immunoblots of ROS Membranes L a b e l l e d w i t h 3A6 and 4B2 Antibodies-72 F i g u r e 17. The B i n d i n g of Rabbit Anti-Human RBC S p e c t r i n A n t i b o d i e s t o P u r i f i e d Bovine S p e c t r i n or ROS Membrane P r o t e i n s 74 F i g u r e 18. Immunoblots of Bovine RBC S p e c t r i n and ROS Membrane P r o t e i n s L a b e l l e d w i t h P o l y c l o n a l Anti-Human S p e c t r i n A n t i b o d i e s 75 - i x-Page F i g u r e 19. Immunoblots of Bovine RBC S p e c t r i n and ROS Membrane P r o t e i n s L a b e l l e d w i t h P o l y c l o n a l A n t i - B o v i n e S p e c t r i n A n t i b o d i e s 77 F i g u r e 20. E x t r a c t i o n o f ROS Membranes under Low I o n i c S t r e n g t h C o n d i t i o n s 79 F i g u r e 21. Immunoblots of ROS and ROS D i s c Membrane P r o t e i n s L a b e l l e d w i t h 4B2 Antibody and Con A -81 F i g u r e 22. Competitive I n h i b i t i o n o f 4B2 and rho-lD4 A n t i b o d i e s B i n d i n g t o ROS Membranes by ROS and D i s c Membranes 82 F i g u r e 23. Tr a n s m i s s i o n E l e c t r o n Micrographs of ROS L a b e l l e d w i t h Immunogold-Dextran Markers 83 F i g u r e 24. Immunoblots of T r i t o n X-100 E x t r a c t i o n o f ROS Membranes 86 F i g u r e 25. SDS Gel E l e c t r o p h o r e s i s o f Detergent-Treated ROS P e l l e t s • 88 F i g u r e 26. Negative S t a i n i n g o f T r i t o n X-100 E x t r a c t e d ROS P e l l e t 90 F i g u r e 27. B i n d i n g o f an A n t i - A c t i n Monoclonal Antibody t o ROS Membranes and P u r i f i e d A c t i n 92 F i g u r e 28. Immunoblots of Bovine RBC Membrane Ghosts L a b e l l e d w i t h P o l y c l o n a l A n t i - B o v i n e A n k y r i n A n t i b o d i e s 94 F i g u r e 29. Immunoblot of Bovine ROS Membrane P r o t e i n s L a b e l l e d w i t h 1D1 Monoclonal Antibody 96 F i g u r e 30. Immunoblots of T r i t o n X-100 T r e a t e d ROS Membranes L a b e l l e d w i t h 1H5 and 2A4 Monoclonal A n t i b o d i e s 98 F i g u r e 31. Immunoblots of Bovine RBC Membrane Ghosts and T r i t o n X-100 E x t r a c t e d ROS P e l l e t L a b e l l e d w i t h 1H5 and 4B2 A n t i b o d i e s 99 F i g u r e 32. A S p e c u l a t i v e Model f o r the O r g a n i z a t i o n of P r o t e i n s i n ROS Membranes 112 -x-LIST OF ABBREVIATIONS ATP adenosine 5' t r i p h o s p h a t e BSA bovine serum albumin cGMP c y c l i c guanosine monophosphate Con A c o n c a n a v a l i n A DEAE d i e t h y l a m i n o e t h y l EDTA ethylenediamine t e t r a a c e t i c a c i d EGTA e t h y l e n e g l y c o l b i s (0 -amino e t h y l e t h y l ) - N , N , N 7 , N 7 - t e t r a -a c e t i c a c i d FCS f e t a l c a l f serum GMP guanos i n e monophosphate GDP guanosine diphosphate GTP guanosine t r i p h o s p h a t e HAT hypoxanthine a m i n o p t e r i n thymidine Ig immunoglobulin kDa k i l o d a l t o n m o l e c u l a r weight PBS phosphate b u f f e r e d s a l i n e PDE phosphodiesterase RBC red b l o o d c e l l RIA r a d i o immune assay ROS rod out e r segment SDS sodium dodecyl s u l f a t e T r i s t r i s (hydroxymethyl) amino-methane WGA wheat germ a g g l u t i n i n ACKNOWLEDGMENT Many people have helped me throughout my graduate s t u d i e s , but I w i l l o n l y name those people whose c o n t r i b u t i o n s I c o u l d not do without. S p e c i a l thanks t o Dr. Robert Molday, my s u p e r v i s o r o f many y e a r s , f o r h i s expert guidance and cons t a n t encouragement. H i s s u p e r v i s i o n was e x c e l l e n t , and h i s c h o i c e o f r e s t a u r a n t f o r a q u i e t d i n n e r was " b e l l y " good. Many thanks t o my c l o s e c o l l e a g u e s , Dale L a i r d and L a u r i e Molday, 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 , much a p p r e c i a t e d c o o p e r a t i o n s , and many v a l u a b l e d i s c u s s i o n s . I would l i k e t o thank Dr. Dave H i c k s who c a r r i e d out the e l e c t r o n m i c r o s c o p i c s t u d i e s on the l o c a l i z a t i o n o f the 4 B 2 - s p e c i f i c p r o t e i n i n the ROS, and Dr. De l y t h R e i d f o r her a s s i s t a n c e i n p r e p a r i n g t h i s t h e s i s . I would l i k e t o thank Drs. James Rich a r d s and P i e t e r C u l l i s f o r bei n g i n my s u p e r v i s o r y committee, and Dr. E v e r a r d T r i p f o r g i v i n g me the o p p o r t u n i t y t o te a c h the undergraduate l a b o r a t o r y course. A l s o , I would l i k e t o thank my many f e l l o w students f o r t h e i r good nature and companionships. F i n a l l y , I would l i k e t o d e d i c a t e t h i s t h e s i s t o my f a m i l y f o r t h e i r l o v e and support. -1-INTRODUCTION A. 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 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 l i n i n g the back o f the eye chamber ( F i g u r e 1). I t c o n s i s t s of neurons and g l i a . There are s i x types o f n e u r a l c e l l s : rod p h o t o r e c e p t o r s , cone p h o t o r e c e p t o r s , b i p o l a r c e l l s , h o r i z o n t a l c e l l s , amacrine c e l l s , and g a n g l i o n c e l l s . The g l i a l c e l l s are a l s o c a l l e d M i i l l e r c e l l s . These r e t i n a l elements are o r g a n i z e d i n t o d i s t i n c t l a y e r s o f c e l l b o d i es and c e l l p r o c e s s e s ( F i g u r e 2) t o perform the two f u n c t i o n s of the r e t i n a . The f i r s t f u n c t i o n , performed by the p h o t o r e c e p t o r s , i s t o t r a n s f o r m l i g h t energy i n t o nerve impulses. The second f u n c t i o n , performed by the e l a b o r a t e a r r a y of h i g h e r - o r d e r neurons, i s t o i n t e g r a t e the l a r g e number of 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 i n f o r m a t i o n about form, movement, and c o l o r t o the b r a i n v i a the o p t i c nerve (reviewed i n S h i c h i , 1983; F a r b e r and A d l e r , 1986). Given the key r o l e of the p h o t o r e c e p t o r c e l l s i n v i s i o n , i t i s not s u r p r i s i n g t h a t they have been the s u b j e c t of c o n s i d e r a b l e i n v e s t i g a t i o n i n v i s i o n r e s e a r c h . B. P h o t o r e c e p t o r C e l l s and V i s u a l Pigments. The p h o t o r e c e p t o r c e l l s are c l o s e l y packed i n the r e t i n a -2-Cornea F i g u r e 1. Gross Anatomy of the V e r t e b r a t e Eye. The major components o f the v e r t e b r a t e eye are shown i n t h i s diagram. The eye chamber i s e n c l o s e d i n t h r e e l a y e r s : the s c l e r a , the c h o r o i d , and the r e t i n a . The fovea i s the c e n t e r of the v i s u a l f i e l d and the o p t i c d i s c a l s o known as the b l i n d s p o t i s where the r e t i n a l nerve f i b e r s converge t o form the o p t i c nerve ( m o d i f i e d a f t e r W alls, 1942). -3-F i g u r e 2. I n t e r r e l a t i o n s h i p s o f R e t i n a l Neurons and G l i a . The r e t i n a l l a y e r s and t h e i r s y n a p t i c r e l a t i o n s h i p s are i l l u s t r a t e d i n t h i s diagram of the a d u l t v e r t e b r a t e r e t i n a . The r e t i n a l neurons are amacrine c e l l s (A), b i p o l a r c e l l s (B), g a n g l i o n c e l l s (G), h o r i z o n t a l c e l l s (H), and rod (R) and cone (C) ph o t o r e c e p t o r c e l l s . Only the predominant g l i a l c e l l s , the M i i l l e r c e l l s (M) , are i n d i c a t e d . These r e t i n a l c e l l s a re o r g a n i z e d i n t o d i s t i n c t l a y e r s o f c e l l b o d i e s and c e l l p r o c e s s e s : o u t e r segment l a y e r (OSL), i n n e r segment l a y e r ( I S L ) , o u t e r l i m i t i n g membrane (OLM), o u t e r n u c l e a r l a y e r (ONL), o u t e r p l e x i f o r m l a y e r (OPL), i n n e r n u c l e a r l a y e r (INL), i n n e r p l e x i f o r m l a y e r (INL), g a n g l i o n c e l l l a y e r (GCL), nerve f i b e r l a y e r (NFL), and i n n e r l i m i t i n g membrane (ILM). The l o c a t i o n o f the r e t i n a l pigment e p i t h e l i u m (RPE) and the c h o r o i d are a l s o i n d i c a t e d ( m o d i f i e d a f t e r Dowling, 1970). -4-- - ILM -5-and a x i a l l y o r i e n t e d t o the i n c i d e n t l i g h t . L i g h t 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 b e f o r e impinging 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 t h a t passes through the p h o t o r e c e p t o r s i s absorbed by the melanin g r a n u l e s o f the p o s t e r i o r r e t i n a l pigment e p i t h e l i u m . In some animals, such as the c a t , whose eyes are s p e c i a l i z e d f o r low l i g h t i n t e n s i t i e s , p a r t of the pigment e p i t h e l i u m i s t r a n s p a r e n t l i k e the r e s t of the r e t i n a . T h i s enables the l i g h t t o reach the d e e p e r - l y i n g tapetum and t o be r e f l e c t e d back through the p h o t o r e c e p t o r s g i v i n g them a second chance t o absorb the l i g h t (reviewed i n Rodieck, 1973). There are two types of p h o t o r e c e p t o r c e l l s i n the eyes of most v e r t e b r a t e s . They were o r i g i n a l l y c a l l e d rods and cones because of t h e i r shapes. However, i t i s now e v i d e n t t h a t few, i f any, of t h e i r m o r p h o l o g i c a l f e a t u r e s are unique t o e i t h e r type of c e l l . Thus, f u n c t i o n a l p r o p e r t i e s such as p h o t o s e n s i t i v i t y and s p a t i a l and temporal r e s o l u t i o n are a l s o r e q u i r e d t o p r o p e r l y c l a s s i f y p h o t o r e c e p t o r s as e i t h e r rods or cones ( F e i n and Szuts, 1982). 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 from s p e c i e s t o s p e c i e s . In humans t h e r e are about 6.5 m i l l i o n cones and about 120 m i l l i o n rods d i s t r i b u t e d u n e q u a l l y throughout the r e t i n a (Pirenne, 1967). The h i g h e s t c o n c e n t r a t i o n of cones i s found i n the c e n t e r of the v i s u a l f i e l d c a l l e d the fovea, whereas the frequency of rods i n c r e a s e s towards the p e r i p h e r y of the r e t i n a . No rods are found i n the fovea, the r e g i o n of most d i s t i n c t v i s i o n . N e i t h e r rods nor cones are found i n -6 -the o p t i c d i s c , the r e g i o n where the g a n g l i o n f i b e r s converge t o form the o p t i c nerve. Cones f u n c t i o n i n b r i g h t l i g h t and are r e s p o n s i b l e f o r c o l o r v i s i o n . Rods are much more s e n s i t i v e t o l i g h t than cones, so they become s a t u r a t e d and i n c a p a b l e of s i g n a l l i n g i n o r d i n a r y d a y l i g h t . But t h e i r s e n s i t i v i t y enables them t o f u n c t i o n i n dim l i g h t , so they are r e s p o n s i b l e f o r achromatic n i g h t v i s i o n . Both rod and cone v i s i o n are mediated by v i s u a l pigments. These l i g h t - a b s o r b i n g molecules c o n s i s t of an a p o p r o t e i n , o p s i n , t h a t i s c o v a l e n t l y l i n k e d t o 1 1 - c i s -r e t i n a l i n most cases (reviewed i n Wald, 1953). Cone pigments are b l u e - , green-, and r e d - s e n s i t i v e because of t h e i r a b s o r p t i o n maxima a t approximately 420 nm, 530 nm, and 560 nm, r e s p e c t i v e l y (reviewed i n M o l l o n and Sharpe, 1983). Rod pigment has an a b s o r p t i o n maximum a t 500 nm (Wald and Brown, 1958) and i s c a l l e d rhodopsin (Klihne, 1878) . C. Rod P h o t o r e c e p t o r s . The g r e a t e r s i z e and abundance of rods compared t o 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 l e n d e r and elongated and occur i n a v a r i e t y of s i z e s . For example, f r o g rods are t y p i c a l l y 6-7 ju i n diameter and 100-120 ja i n l e n g t h whereas bovine rods are t y p i c a l l y 1-2 p. i n diameter and 40-50 ja i n l e n g t h . However, rods have a b a s i c s t r u c t u r e 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 o u t e r segment, an i n n e r segment, a - 7 -nucleus, and a s y n a p t i c body. The two segments are connected by a t h i n non-motile c i l i u m ( F i g u r e 3 ) . The s y n a p t i c body i s the s i t e o f i n f o r m a t i o n t r a n s f e r t o the h i g h e r - o r d e r neurons. I t i s c h a r a c t e r i z e d by the presence o f many s y n a p t i c v e s i c l e s and the fo r m a t i o n o f chemical synapses w i t h b i p o l a r and h o r i z o n t a l c e l l s . The i n n e r segment c o n t a i n s a l l the m e t a b o l i c and s y n t h e t i c machinery o f the c e l l and i s m e t a b o l i c a l l y v e r y a c t i v e . I t c o n s i s t s o f 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 myoid. The e l l i p s o i d i s c h a r a c t e r i z e d by a ve r y dense accumulation of m i t o c h o n d r i a . The myoid i s c h a r a c t e r i z e d by f r e e ribosomes, rough endoplasmic r e t i c u l u m , G o l g i apparatus, and c o n t r a c t i l e p r o t e i n s . In most v e r t e b r a t e s , w i t h the e x c e p t i o n o f the r a t , t h e r e are c a l y c a l p r o c e s s e s (Cohen, 1963) t h a t a r i s e from the d i s t a l end of the i n n e r segment and pass outward about the out e r segment f o r about a t h i r d o f i t s l e n g t h . These f i n e processes are s p e c u l a t e d t o have a s u p p o r t i v e r o l e ; perhaps t o prevent the o u t e r segment from r o t a t i n g about the co n n e c t i n g c i l i u m . A second c y t o p l a s m i c b r i d g e may serve t h a t f u n c t i o n i n many s p e c i e s , i n c l u d i n g the r a t (reviewed i n Rodieck, 1973). The o u t e r segment i s a s p e c i a l i z e d o r g a n e l l e t h a t s e r v e s as 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 n v i s i o n . I t i s a h i g h l y o r g a n i z e d s t r u c t u r e c o n s i s t i n g o f hundreds o f stac k e d p h o t o r e c e p t o r d i s c membranes t h a t a re o r i e n t e d p e r p e n d i c u l a r t o i t s lo n g a x i s and en c l o s e d by a plasma membrane. The s t r u c t u r e and f u n c t i o n o f f r o g and bovine r o d o u t e r segment -8-D i s c Plasma Membrane Connecting Cilium Ellipsoid Myoid Synaptic Body Outer Segment Inner Segment F i g u r e 3. Diagram of a V e r t e b r a t e Rod Pho t o r e c e p t o r C e l l . Common f e a t u r e s among v e r t e b r a t e r od p h o t o r e c e p t o r c e l l s are the o u t e r segment, the i n n e r segment, the nucleus, and the s y n a p t i c body. The two segments are connected by a t h i n non-m o t i l e c i l i u m . The out e r segment c o n t a i n s a s t a c k o f ph o t o r e c e p t o r d i s c s e n c l o s e d by a plasma membrane. The i n n e r segment c o n t a i n s a m i t o c h o n d r i a - r i c h r e g i o n known as the e l l i p s o i d and a r e g i o n c h a r a c t e r i z e d by f r e e ribosomes, rough endoplasmic r e t i c u l u m , G o l g i apparatus, and c o n t r a c t i l e p r o t e i n s known as the myoid. The s y n a p t i c body c o n t a i n s s y n a p t i c v e s i c l e s . -9-(ROS) components have been i n v e s t i g a t e d e x t e n s i v e l y s i n c e ROS can be o b t a i n e d i n h i g h p u r i t y . I n t a c t ROS membranes w i t h e n c l o s e d c y t o p l a s m i c p r o t e i n s and s m a l l molecules can be r e l e a s e d from the r e t i n a by 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 or P e r c o l l g r a d i e n t . The d i s c membranes are i s o l a t e d by o s m o t i c a l l y shocking the plasma membrane and s e a l e d d i s c s are 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 (Smith e t a l . , 1975). The d i s c membranes are composed of l i p i d s and p r o t e i n s 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 t o 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 r e p r e s e n t more than 80% of the t o t a l l i p i d c ontent; c h o l e s t e r o l accounts f o r o n l y 3% (Anderson e t a l . , 1975). The major p h o s p h o l i p i d s , on the b a s i s of t o t a l l i p i d phosphorous, are p h o s p h a t i d y l e t h a n o l a m i n e ( 4 4 % ) , p h o s p h a t i d y l c h o l i n e (36%), and p h o s p h a t i d y l s e r i n e (15%). 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 composition i n d i c a t e s the h i g h l y f l u i d nature of these d i s c 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 c o r e i n d i s c membranes i s p o s t u l a t e d t o serve the purposes of p h o t o t r a n s d u c t i o n and i o n i c t r a n s l o c a t i o n (reviewed i n O l i v e , 1980). SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of f r o g and bovine ROS d i s c membrane p r o t e i n s demostrates the presence of r h o d o p s i n w i t h apparent M r 34,000, a l a r g e p r o t e i n w i t h apparent M^ . 220,000, and s e v e r a l o t h e r minor components wi t h apparent M ranges from 36,000 t o 300,000. Rhodopsin -10-accounts f o r 85-90% of the t o t a l d i s c membrane p r o t e i n s whereas the 220 kDa-protein accounts- f o r o n l y 1-3% (Paper-master and Dreyer, 1974; Godchaux and Zimmerman, 1979a; Molday and Molday, 1979; Kvihn, 1980; Kiihn, 1981; Hamm and Bownds, 1986). In c o n t r a s t , about 90% of the t o t a l ROS p r o t e i n s i s accounted f o r by t h r e e p r o t e i n s : rhodopsin (70%), G - p r o t e i n (17%), and cGMP-dependent phosp h o d i e s t e r a s e (1.5%) [Hamm and Bownds, 1986], The G - p r o t e i n (Baehr e t a l . , 1982) a l s o c a l l e d t r a n s d u c i n (Fung e t a l . , 1981) o r guanine n u c l e o t i d e b i n d i n g p r o t e i n (Godchaux and Zimmerman, 1979b) and the cGMP-dependent phosphodiesterase (Pannbacker e t a l . , 1972; Chader e t al.,1973; M i k i e t a l . , 1973) are two d i s c membrane a s s o c i a t e d p r o t e i n s i n v o l v e d i n cGMP h y d r o l y s i s . These two p r o t e i n s and rhodopsin p l a y c e n t r a l r o l e s i n the p h o t o t r a n s d u c t i o n p r o c e s s . D. P h o t o t r a n s d u c t i o n Process. In the dark the Na + i o n p e r m e a b i l i t y o f ROS plasma 9 + membrane i s h i g h , approximately 10 Na io n s p e r second (Korenbrot and Cone, 1972; Hagins e t a l . , 1976) . N a + i o n s r a p i d l y flow i n t o the out e r segment because o f the e x i s t e n c e o f a l a r g e Na + 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. These N a + i o n s then d i f f u s e through the co n n e c t i n g c i l i u m and/or the c y t o p l a s m i c b r i d g e t o the i n n e r segment and are extruded by Na +-K +-ATPase pumps i n the i n n e r segment plasma membrane t o m a i n t a i n the N a + c o n c e n t r a t i o n g r a d i e n t . A b s o r p t i o n o f a photon causes the chromophore of rhodopsin, 1 1 - c i s - r e t i n a l , t o undergo a s e r i e s o f t r a n s f o r m a t i o n s t h a t r e s u 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 t o y i e l d bleached r h odopsin o r o p s i n (Wald, 1968; Ebrey and Honig, 1975; Honig, 1978; B i r g e , 1981) . T h i s c o n f o r m a t i o n a l t r a n s i t i o n i n a s i n g l e rhodopsin molecule r e s u l t s i n the c l o s u r e o f N a + channels. Consequently, as many as a m i l l i o n N a + i o n s a re prevented from e n t e r i n g i n t o the ROS ( S t r y e r e t a l . , 1981). The 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 t o the s y n a p t i c t e r m i n a l and communicated t o othe r c e l l s o f the r e t i n a by s u p p r e s s i n g n e u r o t r a n s m i t t e r s e c r e t i o n i n t o the rod synapse. I t i s g e n e r a l l y accepted t h a t an i n t e r n a l messenger i s e s s e n t i a l t o communicate between rhodopsin molecules i n the d i s c membranes and the N a + channels o f the plasma membrane 2+ (Ba y l o r and Fu o r t e s , 1970). Both Ca and cGMP have been proposed as i n t r a c e l l u l a r messengers (Yoshikami and Hagins, 1971; M i l l e r , 1981, 1986), but r e c e n t p r o g r e s s i n the v i s i o n f i e l d has e s t a b l i s h e d cGMP as the l i k e l y messenger i n rod ( C a r e t t a and Cavaggioni, 1983; Fesenko e t a l . , 1985; Koch and Kaupp, 1985) and cone (Cobbs e t a l . , 1985; Haynes and Yau, 1985) c e l l s . E. Proposed Mechanism of P h o t o t r a n s d u c t i o n . (1) Role o f cGMP -12-The c u r r e n t h y p o t h e s i s o f the p h o t o t r a n s d u c t i o n mechanism i s t h a t the u n u s u a l l y h i g h cGMP c o n c e n t r a t i o n (30-60 juM) i n the ROS c y t o s o l (Woodruff and Bownds, 1979) i s r e q u i r e d t o m a i n t a i n the opening o f Na + channels i n the dark. T h i s i s supported by the f a c t t h a t cGMP d e p o l a r i z e s the ROS plasma membrane w i t h i n m i l l i s e c o n d s a f t e r being e x p e r i m e n t a l l y i n j e c t e d i n t o an i s o l a t e d 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 ( M i l l e r and N i c o l , 1979). Upon i l l u m i n a t i o n the cGMP l e v e l d ecreases markedly. About a m i l l i o n cGMP molecules are h y d r o l y z e d per second f o r each p h o t o l y z e d r h o d o p s i n molecule (Fung e t a l . , 1981) r e s u l t i n g i n the c l o s u r e o f N a + channels. The h i g h r a t e o f cGMP h y d r o l y s i s i s due t o the a c t i v a t i o n o f an enzyme cascade (Liebman and Pugh, 1981). P h o t o a c t i v a t e d r h o d o p s i n c a t a l y z e s the exchange of bound GDP f o r GTP on a r e g u l a t o r y p r o t e i n c a l l e d G - p r o t e i n . The G-p r o t e i n w i t h bound GTP i s a b l e t o a c t i v a t e a cGMP-dependent phosphodiesterase (PDE), probably by r e l e a s i n g an i n h i b i t o r y f a c t o r (Hurley and S t r y e r , 1982). The a c t i v a t e d PDE h y d r o l y z e s cGMP t o 5'-GMP. The h y d r o l y s i s o f cGMP i s h i g h l y a m p l i f i e d as one ph o t o l y z e d rhodopsin can a c t i v a t e as many as 500 G - p r o t e i n molecules (Fung e t a l . , 1981), which i n t u r n a c t i v a t e s 500 PDE molecules. Each a c t i v a t e d PDE molecule h y d r o l y z e s about 2000 cGMP molecules per second ( F i g u r e 4 ) . In o r d e r t o r e s t o r e PDE a c t i v i t y t o the p r e a c t i v a t i o n l e v e l , both bleached rhodopsin and G - p r o t e i n must be -13-F i g u r e 4. Enzyme Cascade of V i s i o n . The c u r r e n t h y p o t h e t i c a l model of s i g n a l t r a n s d u c t i o n i n the rod p h o t o r e c e p t o r c e l l + i s i l l u s t r a t e d i n t h i s schematic diagram. cGMP-dependent Na channels on the rod o u t e r segment plasma membrane are kept opened i n the dark by a h i g h c o n c e n t r a t i o n o f cGMP i n the c y t o s o l . Phosphodiesterase (PDE) and G-p r o t e i n (G), two d i s c membrane a s s o c i a t e d enzymes i n v o l v e d i n t h e h y d r o l y s i s of cGMP, are i n a c t i v e i n the dark. A b s o r p t i o n of l i g h t by rhodopsin (R), the photopigment i n rod d i s c membranes, a c t i v a t e s G - p r o t e i n which i n t u r n a c t i v a t e s PDE*^ The r e s u l t i n g decrease i n cGMP c o n c e n t r a t i o n c l o s e s the Na channels (obtained from R.S. Molday). Disc Membrane PDE GDP-G -14-Plasma Membrane cGMP //Hi'' •Na* cGMP cGMP • ^ g ^ N a * hv \ p PDE PDE GDP-G GTP-G CGMP V f i M P Na Na -15-i n a c t i v a t e d (Liebman and Pugh, 1979). G - p r o t e i n i s i n a c t i v a t e d by the h y d r o l y s i s of bound GTP t o GDP due t o i t s slow i n t r i n s i c GTPase a c t i v i t y (Kvihn, 1980) . Bleached r h o d o p s i n i s i n a c t i v a t e d by p h o s p h o r y l a t i o n (Sitaramayya and Liebman, 1983) due t o a rhodopsin k i n a s e a c t i v i t y (Wilden and Klihn, 1982) . Only 1-2 P 0 4 / r h o d o p s i n are r e q u i r e d f o r the r a p i d , ATP-dependent i n a c t i v a t i o n o f b l e a c h e d rhodopsin as i n d i c a t e d by a r e c e n t r e p o r t (Sitaramayya, 1986). How the p h o s p h o r y l a t i o n of rhodopsin r e s u l t s i n the d e a c t i v a t i o n of PDE has been the s u b j e c t o f s e v e r a l r e c e n t s t u d i e s . Klihn e t al.(1984) have suggested t h a t a s o l u b l e 48 k D a - p r o t e i n competes wi t h G - p r o t e i n f o r b i n d i n g s i t e s a t the r h o d o p s i n s u r f a c e f o l l o w i n g the p h o s p h o r y l a t i o n o f rhodopsin, thus p r e v e n t i n g the a c t i v a t i o n of G - p r o t e i n by bleached rhodopsin. T h i s p r o p o s a l i s supported by the s t u d i e s i n d i c a t i n g t h a t the 48 kDa-protein b i n d s much more r e a d i l y t o the p h o s p h o r y l a t e d form of rhodopsin than t o the unphosphorylated form (Kiihn e t a l . , 1984), and t h a t rhodopsin p h o s p h o r y l a t i o n i s r e q u i r e d f o r the 48 k D a - p r o t e i n dependent quenching o f PDE a c t i v a t i o n (Wilden e t a l . , 1986). Zuckerman e t a l . (1984, 1985) a l s o r e p o r t the 48 kDa-protein p a r t i c i p a t e s i n the r a p i d i n a c t i v a t i o n of bleached rhodopsin. However they proposed t h a t the 48 k D a - p r o t e i n i s f i r s t a c t i v a t e d by phosphorylated rhodopsin b e f o r e quenching PDE a c t i v a t i o n by d i r e c t i n t e r a c t i o n w i t h the G - p r o t e i n or PDE (Zuckerman e t a l . , 1985; Zuckerman and Cheasty, 1986). C o n t r a r y t o these r e p o r t s , M i l l e r e t a l . (1986) have -16-i n d i c a t e d t h a t rhodopsin p h o s p h o r y l a t i o n alone r a t h e r than the 48 k D a - p r o t e i n r e p r e s e n t s a major component o f the PDE i n a c t i v a t i o n p r o c e s s . U s i n g a p r e p a r a t i o n c o n s i s t i n g of p u r i f i e d p h o s p h o r y l a t e d rhodopsin s p e c i e s r e c o n s t i t u t e d i n t o p h o s p h a t i d y l c h o l i n e v e s i c l e s and an e x t r a c t c o n t a i n i n g G-p r o t e i n , PDE, and o n l y a t r a c e amount o f the 48 kDa-protein, they found t h a t 80-90% of the l i g h t - i n i t i a t e d PDE a c t i v a t i o n can be i n h i b i t e d by rhodopsin p h o s p h o r y l a t i o n . They have suggested t h a t p h o s p h o r y l a t i o n of rhodopsin r e s u l t s i n the r e d u c t i o n o f the b i n d i n g a f f i n i t y o f G - p r o t e i n f o r p h o t o a c t i v a t e d rhodopsin. T h i s p r o p o s a l i s supported by p r e l i m i n a r y evidence i n d i c a t i n g t h a t the G-protein-dependent enhancement of metarhodopsin I I formation (Emeis and Hoffman, 1981) i s reduced by as much as 80% i n h i g h P 0 4 / r h o d o p s i n v e s i c l e s r e l a t i v e t o 0 P 0 4 / r h o d o p s i n v e s i c l e s ( M i l l e r e t a l . , u n p u b l i s h e d o b s e r v a t i o n ) . Obviously, more experiments are r e q u i r e d t o c l a r i f y the r o l e of p h o s p h o r y l a t i o n o f rhodopsin and the importance of the 48 kDa-protein i n r o d c e l l t r a n s d u c t i o n mechanism. The p r e a c t i v a t i o n l e v e l of cGMP i s r e s t o r e d by guanyl c y c l a s e which c a t a l y z e s the c o n v e r s i o n of GTP t o cGMP. Both 2+ l i g h t and Ca may c o n t r o l guanyl c y c l a s e a c t i v i t y (Cohen, 1981; Goldberg e t a l . , 1983). (2) Role of C a — 2+ . 2+ . The Ca h y p o t h e s i s s t a t e s t h a t Ca i o n s are s t o r e d i n the i n t r a d i s c a l space i n the dark. L i g h t s t i m u l a t e s the 2+ . r e l e a s e of Ca i o n s i n t o the c y t o p l a s m i c space, and then 2+ . . + Ca i o n s d i f f u s e t o the plasma membrane t o b l o c k Na channels. T h i s h y p o t h e s i s has been supported by e l e c t r o p h y s i o l o g i c a l experiments i n which the a d d i t i o n of 2+ . . . Ca t o the r e t i n a mimics the e f f e c t o f l i g h t (Hagins and Yoshikami, 1974; Brown e t a l . , 1977; L i p t o n e t a l . , 1977) and the a d d i t i o n o f c a l c i u m 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 t v e t o l i g h t (Brown e t a l . , 1977; Hagins and 2+ Yoshikami, 1977). Furthermore, many Ca i o n s are extruded from an i l l u m i n a t e d ROS f o l l o w i n g a l i g h t p u l s e (Gold e t a l . , 2+ 1980; Yoshikami e t a l . , 1980), and s u f f i c i e n t Ca i s p r e s e n t i n ROS and i n the d i s c s f o r i t t o be a v a i l a b l e as a t r a n s m i t t e r (Liebman, 1974; Szuts and Cone, 1977). However, 2+ r e c e n t experiments have shown t h a t m i l l i m o l a r Ca has l i t t l e e f f e c t on the opening of the l i g h t - r e g u l a t e d channel by cGMP i n an i s o l a t e d p a tch of ROS plasma membrane (Fesenko e t a l . , 1985) o r i n i s o l a t e d d i s c s (Koch and Kaupp, 1985). Furthermore, N i c o l e t a l . (1987) have shown t h a t the normal f l a s h responses of i s o l a t e d rod p h o t o r e c e p t o r s p e r s i s t a f t e r 2+ the d e p l e t i o n of i n t r a c e l l u l a r c a l c i u m . T h e r e f o r e , the Ca h y p o t h e s i s needs t o be m o d i f i e d , and a change i n 2+ i n t r a c e l l u l a r Ca c o n c e n t r a t i o n i s not e s s e n t i a l f o r e x c i t a t i o n of p h o t o r e c e p t o r s by l i g h t . 2+ The p r e c i s e r o l e of Ca i n the p h o t o t r a n s d u c t i o n p r o c e s s remains t o be determined, but i t s e f f e c t on the rod photoresponse c o u l d be e x p l a i n e d by i t s a c t i o n on the cGMP -18-metabolism i n the p h o t o r e c e p t o r (reviewed by Kaupp and Koch, 2+ 1986) because cGMP l e v e l s i n l i v i n g rods are Ca -dependent (Cohen, 1981; a l s o reviewed by Korenbrot, 1985). Thus, 2+ . f u r t h e r i n s i g h t s i n t o the r o l e o f Ca i n the rods c o u l d be gained by s t u d y i n g the p r o p e r t i e s and r e g u l a t i o n of ROS guanyl c y c l a s e . In c o n t r a s t t o guanyl c y c l a s e , the key components o f the enzyme cascade, rhodopsin, G - p r o t e i n , and PDE are w e l l c h a r a c t e r i z e d (reviewed i n Applebury and Hargrave, 1986; S t r y e r , 1986). (3) Rhodopsin Rhodopsin, the v e r t e b r a t e photopigment, has been a major focus i n v i s i o n r e s e a r c h . S e v e r a l v e r t e b r a t e o p s i n s have been sequenced (Ovchinnikov e t a l . , 1982; Hargrave e t a l . , 1983; Pappin e t a l . , 1984), and the genes f o r bovine rod and human rod and cone o p s i n s (Nathans and Hogness, 1983,1984; Nathans e t a l . , 1986a & b) have a l s o been c h a r a c t e r i z e d . Rhodopsin i s a transmembrane g l y c o p r o t e i n w i t h i t s N-terminus f a c i n g the i n t r a d i s c a l space and i t s C-terminus f a c i n g the c y t o p l a s m i c space ( C l a r k and Molday, 1979), and i s e n v i s i o n e d t o t r a s v e r s e the d i s c membranes seven times i n h e l i c a l segments (Ovchinnikov e t a l . , 1982; Hargrave e t a l . , 1983). The N-terminus c o n t a i n s two carbohydrate c h a i n s which b i n d such l e c t i n s as Con A and WGA. The C-terminus c o n t a i n s p h o s p h o r y l a t i o n s i t e s f o r rhodopsin k i n a s e (Wilden and Kiihn, 1982). The c y t o p l a s m i c s u r f a c e c o n t a i n s s i t e s f o r l i g h t -- I n -dependent b i n d i n g of ROS p r o t e i n s such as G - p r o t e i n , rhodopsin k i n a s e and the 48 kDa-protein (Kuhn, 1981; Kuhn e t a l . , 1984; a l s o reviewed by Hargrave, 1982; Applebury and Hargrave, 1986). Rhodopsin appears t o be r e l a t e d t o some /^ - a d r e n e r g i c r e c e p t o r s (Dixon e t a l . , 1986; Yarden e t a l . , 1986; K o b i l k a e t a l . , 1987), a m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r (Kubo e t a l . , 1986), and a p r o t e i n encoded by a c e l l u l a r oncogene c a l l e d mas (Young e t a l . , 1986). S i n c e rhodopsin a c t s through a G - p r o t e i n b e l o n g i n g t o a f a m i l y o f guanine n u c l e o t i d e b i n d i n g p r o t e i n s (designated G Q, G^, and G g) whose f u n c t i o n s are t o r e g u l a t e the a c t i v a t i o n o r i n h i b i t i o n of a d e n y l a t e c y c l a s e by hormones and n e u r o t r a n s m i t t e r s (Gilman, 1984;1986), i t has been suggested t h a t the mechanism of p h o t o t r a n s d u c t i o n i n r e t i n a l rods may be a model f o r the e a r l y events of s i g n a l t r a n s d u c t i o n i n many c e l l t y p e s . T h i s i s f u r t h e r supported by the o b s e r v a t i o n t h a t the components o f some o f these systems are f u n c t i o n a l l y i n t e r c h a n g e a b l e ( B i t e n s k y e t a l . , 1982; Cerione e t a l . , 1985). I t i s a l s o i n t e r e s t i n g t h a t c y c l i c - n u c l e o t i d e - d e p e n d e n t channels are now being i d e n t i f i e d i n o t h e r sensory systems (Johnson e t a l . , 1986; Nakamura and Gold, 1987). These channels w i l l no doubt be compared t o the cGMP-dependent channel i n r e t i n a l rods which has o n l y r e c e n t l y been b i o c h e m i c a l l y i d e n t i f i e d (Cook e t a l . , 1987; M a t e s i c and Liebman, 1987). -20-F. Rod Outer Segment. In a d d i t i o n t o the p h o t o t r a n s d u c t i o n p r o c e s s , ROS have a l s o been i n v e s t i g a t e d t o f u r t h e r the understanding of the mechanism of b i o g e n e s i s and renewal of ROS membranes, and how the h i g h l y ordered p h o t o r e c e p t o r s t r u c t u r e i s maintained. (1) S t r u c t u r e The o u t e r segment c o n s i s t s of a s t a c k o f hundreds of c l o s e d double-membrane d i s c s e q u a l l y spaced a p a r t (Cohen, 1972) . Both X-ray d i f f r a c t i o n measurements on e x c i s e d r e t i n a s (Blaurock and W i l k i n s , 1969) and f r e e z e - f r a c t u r e s t u d i e s (Korenbrot e t a l . , 1973) i n d i c a t e t h a t the c e n t e r - t o -c e n t e r d i s t a n c e between ad j a c e n t d i s c s has a c o n s t a n t v a l u e of about 300 A. The i n t e r n a l membrane s u r f a c e s of a d i s c are s e p a r a t e d by a 20 A-wide aqueous space (Chabre and Cavaggioni, 1975), which i s maintained over the e n t i r e e x t e n t of the d i s c ( F i g u r e 5). These s p e c i a l i z e d d i s c membranes occupy a r e l a t i v e l y compact volume, but p r o v i d e a l a r g e s u r f a c e area f o r embedding a l a r g e number of p h o t o r e c e p t o r molecules, which i s r e q u i r e d f o r the h i g h s e n s i t i v i t y of rods ( F e i n and Szuts, 1982). The d i s c s are d i v i d e d i n t o l o b e s by deep i n v a g i n a t i o n s of the d i s c membrane termed i n c i s u r e s . The number of i n c i s u r e s and d i s c s i n a ROS v a r i e s among the v e r t e b r a t e s , but i n v a r i a b l y the assembly of d i s c s i s e n c l o s e d by a plasma membrane t h a t i s o s m o t i c a l l y (Korenbrot e t a l . , 1973) and e l e c t r i c a l l y (Hagins and Ruppel, 1971) s e p a r a t e d from the -21-d i s c s over most of the l e n g t h o f the ROS ( F i g u r e 5 ) . The d i s c s a re composed of two a p p a r e n t l y d i s t i n c t membrane domains, the f l a t l a m e l l a r domain and the h i g h l y curved rim r e g i o n ( F a l k and F a t t , 1969). (2) B i o g e n e s i s and Renewal The d i s c a re c o n t i n u o u s l y renewed (Young, 1967; Young and Droz, 1968). A u t o r a d i o g r a p h i c s t u d i e s have shown t h a t the new d i s c membrane components are s y n t h e s i z e d i n the i n n e r segment a t one or more s i t e s and are then added t o the plasma membrane near the ROS base where f i n a l membrane assembly takes p l a c e ( H a l l e t a l . , 1969; Young, 1973). The d i s c s are formed by i n v a g i n a t i o n o f the plasma membrane, but are pinched o f f t o produce c l o s e d d i s c s as they a re d i s p l a c e d a p i c a l l y ( S j b s t r a n d , 1959; Moody and Robertson, 1960; Cohen, 1961). However, i t has a l s o been proposed t h a t the d i s c s are formed by repeated e v a g i n a t i o n o f the plasma membrane i n the r e g i o n o f the co n n e c t i n g c i l i u m (Eakin, 1973). A r e c e n t model has been proposed f o r the d i f f e r e n t i a l b i o g e n e s i s o f r i m and l a m e l l a e o f d i s c s , i n a d d i t i o n t o the i n c i s u r e s ( S t e i n b e r g e t a l . , 1980). In t h i s model each of the two s u r f a c e s o f an e v a g i n a t i o n o f the c i l i a r y membrane form one o f the s u r f a c e s o f adj a c e n t d i s c s . The d i s c r i m i s i n i t i a l l y s p e c i f i e d as a r e g i o n o f c i l i a r y membrane between ad j a c e n t d i s c - s u r f a c e e v a g i n a t i o n s . T h i s r e g i o n grows b i l a t e r a l l y around the ci r c u m f e r e n c e s o f a d j a c e n t d i s c s , -22-F i g u r e 5. F i n e S t r u c t u r e o f Rod Outer Segment, (a) The out e r segment c o n s i s t s o f a s t a c k o f c l o s e d double-membrane d i s c s e q u a l l y spaced a p a r t and en c l o s e d by a plasma membrane t h a t i s s e p a r a t e d from the d i s c s over most of the l e n g t h o f the ou t e r segment (modified a f t e r Young, 1970). (b) The c e n t e r -t o - c e n t e r d i s t a n c e between adj a c e n t d i s c s i s approximately 300 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 c are separ a t e d by a 20 A-wide aqueous space. The d i s c s a re d i v i d e d i n t o l o b e s by the i n c i s u r e s . The d i s c s a re composed of two a p p a r e n t l y d i s t i n c t membrane domains, the f l a t l a m e l l a r domain and the h i g h l y curved rim r e g i o n ( m o d i f i e d a f t e r S t e i n b e r g e t a l . , 1980). - 2 3 -z i p p e r i n g t o g e t h e r the apposed s u r f a c e s t o form the r i m and completed d i s c . At the same time the plasma membrane edges o f the e v a g i n a t i o n s which have become detached from the s u r f a c e s are s e a l e d . The i n c i s u r e s form i n r o d d i s c s by i n f o l d i n g o f the r i m and s u r f a c e s t o g e t h e r . They b e g i n t o form b e f o r e the r i m i s completed around the d i s c p e r i m e t e r . The r a t e of d i s c s y n t h e s i s v a r i e s from s p e c i e s t o s p e c i e s . For f r o g s the r a t e i s 1.2 d i s c s per hour, a f d a l l 1700 d i s c s i n a f r o g ROS are renewed i n two months (Young and Bok, 1969). For Rhesus monkeys a l l the d i s c s i n a ROS are renewed i n about 10 days (Young, 1971). 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 are p e r i o d i c a l l y pinched o f f t o be p h a g o c y t i z e d and degraded by the r e t i n a l pigment e p i t h e l i u m . Each shedding event removes the d i s t a l 10-20% of the o u t e r segment (Kaplan e t a l . , 1987) and t h i s shedding process has been found t o f o l l o w a d i u r n a l p a t t e r n i n a l a r g e v a r i e t y of s p e c i e s (Basinger and Gordon, 1982). The o r d e r l y p rocess of d i s c s y n t h e s i s and removal appears t o be extremely c r u c i a l t o p h o t o r e c e p t o r f u n c t i o n . I f the r e t i n a l pigment e p i t h e l i u m i s i n c a p a b l e of p h a g o c y t i z i n g the shed d i s c s , the rods e v e n t u a l l y degenerate and d i e (Bok and H a l l , 1971). The p r e c i s e mechanisms o f d i s c renewal however remain t o be determined. (3) Maintenance The nature and morphology of the s t r u c t u r a l components r e s p o n s i b l e f o r m a i n t a i n i n g a c o n s t a n t d i s t a n c e between -24-a d j a c e n t d i s c s and d i s c s t o plasma membrane are l a r g e l y unknown. However, Usukura and Yamada (1981) and Roof and Heuser (1982) have observed t h i n f i l a m e n t s extending from the rims o f ROS d i s c s by e l e c t r o n microscopy. In the l a t t e r study, one type o f filam e n t o u s s t r u c t u r e was observed t o l i n k a d j a c e n t s t a c k e d d i s c s and another f i l a m e n t o u s s t r u c t u r e appeared t o l i n k the d i s c s t o the plasma membrane. Although the m o l e c u l a r composition of these f i l a m e n t s has not been determined, i t has been suggested t h a t t h e s e f i l a m e n t o u s c o n n e c t i o n s may serve as s t r u c t u r a l elements o f a c y t o s k e l e t a l network r e s p o n s i b l e f o r s t a b i l i z i n g t he h i g h l y o r g a n i z e d o u t e r segment s t r u c t u r e . G. C y t o s k e l e t a l Network i n Red Blood C e l l . To date the b e s t understood membrane c y t o s k e l e t o n i n terms of the o r g a n i z a t i o n and a s s o c i a t i o n s o f i t s p r o t e i n components i s t h a t o f the red b l o o d c e l l plasma membrane (reviewed i n Marchesi e t a l . , 1976; Branton e t a l . , 1981; Marchesi, 1983; Bennett, 1985). Mammalian r e d b l o o d c e l l s c o n t a i n on the c y t o p l a s m i c s u r f a c e o f t h e i r plasma membrane, a two-dimensional meshwork of s t r u c t u r a l p r o t e i n s t h a t s t a b i l i z e s t he o v e r l y i n g l i p i d b i l a y e r . T h i s meshwork prob a b l y f u n c t i o n s i n the maintenance of c e l l shape and i n the o r g a n i z a t i o n o f the i n t e g r a l membrane p r o t e i n s (reviewed i n Cohen, 1983). The p r i n c i p a l component o f t h i s meshwork i s the p r o t e i n s p e c t r i n (reviewed i n Marchesi, 1980). -25-S p e c t r i n i s a f l e x i b l e rod-shaped molecule composed of an c< s u b u n i t o f M =240.000 and a & s u b u n i t o f M =225,000. r ' ^ r ' The ck and s u b u n i t s a re a l i g n e d s i d e - t o - s i d e t o form heterodimers, and the dimers s e l f - a s s o c i a t e i n a head-to-head o r i e n t a t i o n t o form tetramers o f 200 nm i n l e n g t h (Shotton e t a l . , 1979; Branton e t a l . , 1981). S p e c t r i n tetramers form the meshwork by b i n d i n g o f t h e i r ends t o s h o r t a c t i n f i l a m e n t s and t o a 78,000 p r o t e i n named band 4.1 which modulates s p e c t r i n - a c t i n i n t e r a c t i o n . T h i s complex i s a t t a c h e d t o the membrane by a s s o c i a t i o n o f s p e c t r i n w i t h a n k y r i n , a membrane-associated p r o t e i n which a t t a c h e s t o a membrane-spanning anion t r a n s p o r t e r p r o t e i n known as band 3 (F i g u r e 6) [reviewed by Speicher, 1986]. A n k y r i n , o r i g i n a l l y d e s i g n a t e d as band 2.1 (Steck,1974),is a g l o b u l a r p r o t e i n w i t h apparent M r 200,000. A n k y r i n and i t s membrane-associated p r o t e o l y t i c fragments have been c o l l e c t i v e l y r e f e r r e d t o as syndeins (Yu and Goodman, 1979). A n k y r i n i s l o c a t e d a t the c y t o p l a s m i c membrane s u r f a c e and mediates the attachment of s p e c t r i n t o the membrane by b i n d i n g t o the j& c h a i n o f s p e c t r i n and band 3. T h e r e f o r e , a n k y r i n i s l a r g e l y r e s p o n s i b l e f o r m a i n t a i n i n g the c l o s e a s s o c i a t i o n of the membrane s k e l e t a l p r o t e i n s w i t h the l i p i d b i l a y e r . A c t i n , o r i g i n a l l y d e s i g n a t e d as band 5 (Sheetz e t a l . , 1976; T i l n e y and Detmers, 1975), has a monomer M r 42,000. I t i s a h i g h l y conserved p r o t e i n t h a t p o l y m e r i z e s i n t o -26-F i g u r e 6. The Red C e l l Membrane S k e l e t o n . The o r g a n i z a t i o n and a s s o c i a t i o n s o f c y t o s k e l e t a l p r o t e i n components on the c y t o p l a s m i c s u r f a c e of red b l o o d c e l l plasma membrane are i n d i c a t e d i n t h i s schematic diagram. S p e c t r i n - a c t i n - b a n d 4.1 and s p e c t r i n dimer-dimer i n t e r a c t i o n s are thought t o be important i n forming a two-dimensional meshwork. T h i s meshwork i s a t t a c h e d t o the plasma membrane by s p e c t r i n -ankyrin-band 3 i n t e r a c t i o n s (modified a f t e r Palek and Lux, 1980). -27-f i l a m e n t s . Short f i l a m e n t s of a c t i n (25 nm long) a s s o c i a t e w i t h s p e c t r i n t o form the c y t o s k e l e t a l meshwork i n e r y t h r o c y t e . T h e i r a s s o c i a t i o n i s promoted, strengthened, or s t a b i l i z e d by band 4.1 i n an undetermined manner. The r o l e o f band 4.1 has been suggested t o m a i n t a i n membrane e l a s t i c i t y and shape (reviewed i n Cohen, 1983). Analogs o f r e d b l o o d c e l l membrane p r o t e i n s i n c l u d i n g s p e c t r i n , a n k y r i n , band 3 , and band 4.1 have been i d e n t i f i e d i n o t h e r c e l l types u s i n g p o l y c l o n a l a n t i b o d i e s as probes (reviewed i n Baines, 1984; Bennett, 1985). The s p e c t r i n analogs have been most e x t e n s i v e l y c h a r a c t e r i z e d t o date. H. S p e c t r i n - l i k e P r o t e i n s . Two d i s t i n c t s p e c t r i n - l i k e p r o t e i n s have been i s o l a t e d from b r a i n and i n t e s t i n a l m i c r o v i l l i . The former i s known as f o d r i n (Levine and W i l l a r d , 1981) or c a l s p e c t i n (Sobue e t a l . , 1981) or b r a i n s p e c t r i n (Bennett e t a l . , 1982) , and the l a t t e r i s known as TW 260/240 (Glenny e t a l . , 1982a). These p r o t e i n s are c o n s i d e r e d s p e c t r i n - l i k e because not o n l y do they have o(. and s u b u n i t s w i t h M r s i m i l a r t o t h a t o f s p e c t r i n and s i m i l a r t e t r a m e r i c rod s t r u c t u r e , but they a l s o c r o s s l i n k a c t i n f i l a m e n t s and b i n d t o c a l m o d u l i n and a n k y r i n (Bennett e t a l . , 1982; B u r r i d g e e t a l . , 1982; Burns e t a l . , 1983). The cX s u b u n i t s i n a l l a v i a n s p e c t r i n s b i n d c a l m o d u l i n and appear t o be a widespread u n i t o f s t r u c t u r e as -28-immunological e q u i v a l e n t s are d e t e c t e d i n a v a r i e t y o f mammalian c e l l types (Repasky e t a l . , 1982). The /3 su b u n i t s o f a v i a n s p e c t r i n s d i f f e r from each o t h e r i n M r (260,000 f o r TW 260/240 , and 235,000 f o r f o d r i n compared t o 225,000 f o r s p e c t r i n ) , and they have d i s t i n c t a n t i g e n i c s i t e s and y i e l d d i f f e r e n t p e p t i d e maps ( Bennett e t a l . , 1982; Bu r r i d g e e t a l . , 1982; Glenny e t a l . , 1982a, 1982b). S i n c e many o f the f u n c t i o n a l p r o p e r t i e s o f r e d b l o o d c e l l s p e c t r i n such as p h o s p h o r y l a t i o n (Cohen, 1983) and a n k y r i n b i n d i n g (Bennett and Stenbuck, 1979) are c h a r a c t e r i s t i c f e a t u r e s o f the /? s u b u n i t , i t has been suggested t h a t the d i v e r s i t y o f the /3 s u b u n i t c o n t r i b u t e s t o the f u n c t i o n a l d i f f e r e n c e s between s p e c t r i n and s p e c t r i n - l i k e p r o t e i n s i n t h i s and oth e r s p e c i e s . However, almost a l l s p e c t r i n s of many a v i a n and mammalian c e l l types have been l o c a l i z e d by immunofluorescence t o the plasma membrane (Goodman e t a l . , 1981; Levine and W i l l a r d , 1981; B u r r i d g e e t a l . , 1982; Repasky e t a l . , 1982). I. High M o l e c u l a r Weight P r o t e i n s i n Rod Outer Segment. In view of the importance o f s p e c t r i n i n m a i n t a i n i n g membrane i n t e g r i t y i n the red b l o o d c e l l and the widespread occurrence o f s p e c t r i n - l i k e p r o t e i n s i n nature, i t i s of i n t e r e s t t o determine the e x i s t e n c e o f such a p r o t e i n i n the rod o u t e r segment. S e v e r a l h i g h m o l e c u l a r weight p r o t e i n s have been i d e n t i f i e d i n f r o g and bovine ROS p r e p a r a t i o n s , -29-however they have not been w e l l c h a r a c t e r i z e d . Papermaster e t al.(1978) have used immunocytochemical tech n i q u e s t o l o c a l i z e a 290 kDa d i s c membrane p r o t e i n , r e f e r r e d t o as the r i m p r o t e i n . Szuts (1985) has r e p o r t e d t h a t two p r o t e i n s o f 220 kDa and 240 kDa undergo a l i g h t -mediated p h o s p h o r y l a t i o n r e a c t i o n i n f r o g ROS. The 220 kDa-p r o t e i n appears t o be e q u i v a l e n t t o the r i m p r o t e i n . Molday and Molday (1979) have r e p o r t e d a 220 kDa transmembrane g l y c o p r o t e i n d e s i g n a t e d as ROS 1.2 i n bovine ROS d i s c membrane p r e p a r a t i o n s . Whether or not ROS 1.2 i s e q u i v a l e n t t o the r i m p r o t e i n remains t o be determined as i t s l o c a l i z a t i o n t o the rim r e g i o n s o f bovine ROS d i s c s has not been r e p o r t e d . The f u n c t i o n s o f the rim p r o t e i n and 240 kDa-p r o t e i n i n f r o g ROS and ROS 1.2 i n bovine ROS are as y e t unknown, although i t has been suggested t h a t the r i m p r o t e i n c o n s t i t u t e s the f i l a m e n t s t h a t l i n k d i s c s t o g e t h e r i n f r o g ROS (Roof and Heuser, 1982). J . Monoclonal A n t i b o d i e s t o ROS P r o t e i n s . In o r d e r t o d e f i n e i n more d e t a i l the r o l e o f these and ot h e r minor p r o t e i n s i n the maintenance of the h i g h l y o r g a n i z e d ROS s t r u c t u r e , i n the p h o t o t r a n s d u c t i o n p r o c e s s and i n the o t h e r s p e c i a l i z e d p r o c e s s e s such as d i s c renewal t h a t occur i n r e t i n a l r o d c e l l s , MacKenzie and Molday (1982) have used the technique o f Kohler and M i l s t e i n (1975) t o prepare monoclonal a n t i b o d i e s a g a i n s t bovine ROS p r o t e i n s . The -30-m a j o r i t y o f monoclonal a n t i b o d i e s generated were found t o be s p e c i f i c f o r rhodopsin (MacKenzie and Molday, 1982). Monoclonal a n t i b o d i e s t o r a t rhodopsin (Fekete and B a r n s t a b l e , 1983) and f r o g rhodopsin (Witt e t a l . , 1984) have a l s o been generated. These r h o d o p s i n - s p e c i f i c monoclonal a n t i b o d i e s have been used as immunological probes t o study the l o c a t i o n , s t r u c t u r e and f u n c t i o n of rhodopsin. P o l y c l o n a l a n t i - r h o d o p s i n a n t i s e r a have been used f o r the immunocytochemical l o c a l i z a t i o n of the p r o t e i n i n the ROS d i s c and plasma membrane and i n the i n n e r segment s u b c e l l u l a r o r g a n e l l e s by s e v e r a l l a b o r a t o r i e s (Dewey e t a l . , 1969; Jan and Revel, 1974; Papermaster e t a l . , 1978). However, the major advantage of u s i n g monoclonal a n t i b o d i e s r a t h e r than p o l y c l o n a l a n t i s e r a i n such s t u d i e s i s t h a t monoclonal a n t i b o d i e s do not b i n d t o m u l t i p l e e p i t o p e s on the rhodopsin molecule. T h i s i s e s p e c i a l l y important i n s t r u c t u r a l and f u n c t i o n a l s t u d i e s , s i n c e monoclonal a n t i b o d i e s a l l o w the immunological d i s s e c t i o n of p r o t e i n s . A p p l i c a t i o n of monoclonal a n t i b o d i e s as immunological probes f o r the s t r u c t u r e and f u n c t i o n of rhodopsin r e q u i r e s the l o c a l i z a t i o n of the e p i t o p e s f o r these a n t i b o d i e s . R e c e n t l y , Molday e t a l . have r e p o r t e d the p r o p e r t i e s of s e v e r a l monoclonal a n t i b o d i e s a g a i n s t v a r i o u s segments of rhodopsin (MacKenzie and Molday, 1982; MacKenzie e t a l . , 1984; L a i r d e t a l . , 1987). The b i n d i n g s i t e s f o r these monoclonal a n t i b o d i e s i n r e l a t i o n t o the proposed transmembrane h e l i c a l model of rhodopsin (Hargrave e t a l . , -31-1983) a re shown i n F i g u r e 7. The g e n e r a l l o c a t i o n o f the e p i t o p e s f o r these r h o d o p s i n - s p e c i f i c a n t i b o d i e s was determined by l i m i t e d p r o t e o l y s i s o f rhodopsin u s i n g t r y p s i n and S_^  aureus p r o t e a s e s and subsequent immunoblotting a n a l y s e s . More p r e c i s e l o c a l i z a t i o n o f antibody b i n d i n g s i t e s was determined by s o l i d - p h a s e radioimmune c o m p e t i t i v e i n h i b i t i o n s t u d i e s u s i n g s y n t h e t i c p e p t i d e s and s m a l l p e p t i d e s d e r i v e d from chemical o r p r o t e o l y t i c cleavage o f rhodopsin. Almost a l l o f these a n t i b o d i e s were found t o b i n d e i t h e r the N - t e r m i n a l o r the C-terminal segments o f rhodopsin, and on l y two a n t i b o d i e s were found t o b i n d t o o t h e r segments of rhodopsin. The reason f o r the h i g h degree of a n t i g e n i c i t y found f o r the N-terminal and C-t e r m i n a l r e g i o n s o f rhodopsin (Hargrave e t a l . , 1986; L a i r d e t a l . , 1987) as w e l l as many ot h e r p r o t e i n s (Anderer and Schlumberger, 1965; A l t s c h u h and Regenmortel, 1982; Quesnizux e t a l . , 1983.) i s unknown. I t has been h y p o t h e s i z e d t h a t the segmental m o b i l i t y o r f l e x i b i l i t y o f p e p t i d e r e g i o n s i s an e s s e n t i a l component of a n t i g e n i c i t y ( T a i n e r e t a l . , 1985). But i n the case o f s c o r p i o n n e u r o t o x i n , an e x c e p t i o n a l s u r f a c e exposure of r e l a t i v e l y s h o r t loop segments r a t h e r than segmental m o b i l i t y appears t o make the b e s t c o r r e l a t i o n (Novotny and Haber, 1986). One C-t e r m i n a l s p e c i f i c a n t i - r h o d o p s i n antibody r e f e r r e d t o as rho-lD4 has been c h a r a c t e r i z e d e x t e n s i v e l y . T h i s I g G 1 antibody binds t o the 341-348 ( l ' - 8 ' ) C - t e r m i n a l segment and r e q u i r e s the t e r m i n a l A l a amino a c i d f o r b i n d i n g C y t o p l a s m i c S u r t « c « - 3 2 -rho3D«  lho1P<2C1.3C2 rholCS rt>o4D2 ftio4A8 l n l r « d l « k « l 8 u r ( « c « F i g u r e 7. L o c a l i z a t i o n of Anti-Rhodopsin Monoclonal A n t i b o d i e s . The b i n d i n g s i t e s o f s e v e r a l monoclonal a n t i b o d i e s s p e c i f i c f o r rhodopsin are i l l u s t r a t e d i n t h i s model f o r the o r g a n i z a t i o n o f rhodopsin i n rod o u t e r segment d i s c membranes (modified a f t e r Hargrave e t a l . , 1983). In t h i s model, rhodopsin t r a s v e r s e s the d i s c membranes seven times i n h e l i c a l segments w i t h i t s N-terminus f a c i n g the i n t r a d i s c a l s u r f a c e , w h i l e i t s C-terminus f a c e s the c y t o p l a s m i c s u r f a c e . The N-terminus c o n t a i n s two carbohydrate c h a i n s ( s o l i d squares and unmarked c i r c l e s ) t h a t b i n d such l e c t i n s as Con A and WGA. The C- t e r m i n a l segments and c y t o p l a s m i c loop r e g i o n s o f rhodopsin are a c c e s s i b l e t o pr o t e a s e s such as S_. aureus and t r y p s i n . Monoclonal a n t i b o d i e s rho-2B2, -4A2, -4A3, -4D2, and -5A3 b i n d t o the N-t e r m i n a l segment of rhodopsin. The C-t e r m i n a l segment of rhodopsin c o n t a i n s b i n d i n g s i t e s f o r monoclonal a n t i b o d i e s rho-3D6, -1D4, -2C1, -3C2, -3A6, and -1C5. Only two monoclonal a n t i b o d i e s , rho-4B4 and rho-8A6, b i n d t o the loop r e g i o n s ( m o d i f i e d a f t e r L a i r d e t a l . , 1987). -33-t o r h o dopsin. Rho-lD4 has been s u c c e s s f u l l y used i n the p u r i f i c a t i o n o f the 318-348 p e p t i d e from cyanogen bromide c l e a v e d rhodopsin by immunoaffinity chromatography (MacKenzie e t a l . , 1984; L a i r d e t a l . , 1987). I t has a l s o been used wi t h the immunogold-dextran l a b e l l i n g method t o l o c a l i z e r h o d o p s i n a l o n g the c y t o p l a s m i c s u r f a c e o f d i s c s (Molday e t a l . , 1987) and t o study the p r o p e r t i e s o f l i g h t - d e p e n d e n t p h o s p h o r y l a t i o n o f rhodopsin (Molday and MacKenzie, 1985). Because i t has been w e l l c h a r a c t e r i z e d , rho-lD4 has been r o u t i n e l y used as a s p e c i f i c probe f o r rhodopsin i n many b i o c h e m i c a l and immunocytochemical l a b e l l i n g s t u d i e s . Molday e t a l . (1987) have a l s o r e p o r t e d monoclonal a n t i b o d i e s a g a i n s t a novel membrane p r o t e i n , termed p e r i p h e r i n , w i t h an apparent M r of 33,000. Two monoclonal a n t i b o d i e s d e s i g n a t e d as 2B6 and 3B6 were found t o b i n d t o p e r i p h e r i n . Immunogold l a b e l l i n g o f m o r p h o l o g i c a l l y i n t a c t i s o l a t e d d i s c s i n d i c a t e d t h a t p e r i p h e r i n i s l o c a l i z e d along the r i m r e g i o n o f d i s c s . P e r i p h e r i n appears d i s t i n c t from the M r 290,000 rim p r o t e i n i n f r o g ROS p r e v i o u s l y d e s c r i b e d by Papermaster e t a l . (1978), although both p r o t e i n s l o c a l i z e d a l o n g the r i m r e g i o n s o f d i s c s . In a d d i t i o n t o monoclonal a n t i b o d i e s a g a i n s t rhodopsin and p e r i p h e r i n , MacKenzie and Molday (1982) have a l s o r e p o r t e d a monoclonal antibody d e s i g n a t e d as 4B2 which bound t o a h i g h m o l e c u l a r weight p o l y p e p t i d e having a s i m i l a r m o b i l i t y t o ROS 1.2, a 220 kDa Con A - b i n d i n g g l y c o p r o t e i n , on SDS-polyacrylamide g e l s . T h i s antibody i s of i n t e r e s t -34-because v e r y l i t t l e i s known about the s t r u c t u r e and f u n c t i o n of h i g h m o l e c u l a r weight p r o t e i n s found i n v e r t e b r a t e ROS. Although the 4B2-binding p r o t e i n was shown t o be a s s o c i a t e d w i t h ROS d i s c membranes, i t s p r o p e r t i e s were not c h a r a c t e r i z e d i n d e t a i l . K. T h e s i s I n v e s t i g a t i o n . T h i s t h e s i s c h a r a c t e r i z e s f u r t h e r the 4B2-binding p r o t e i n i n bovine ROS u s i n g the 4B2 monoclonal antibody as a m o l e c u l a r probe. In p r e v i o u s s t u d i e s , the 4B2 antibody was shown t o be an IgM molecule w i t h l i g h t c h a i n s . Although the 4B2 hybridoma c e l l l i n e c o u l d be grown e i t h e r i n c u l t u r e medium o r as a s c i t e s tumors i n mice, the c o n c e n t r a t i o n o f 4B2 antibody i n a s c i t e s f l u i d was g e n e r a l l y 25- t o 4 0 - f o l d h i g h e r than i n c e l l c u l t u r e supernatant. In t h i s t h e s i s i n v e s t i g a t i o n , the 4B2 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 r a t h e r than p u r i f i e d 4B2 antibody were used f o r i d e n t i f y i n g and c h a r a c t e r i z i n g the 4 B 2 - s p e c i f i c p r o t e i n i n ROS membrane p r e p a r a t i o n s because i t was shown t h a t the 4B2 antibody a c t i v i t y decreased a t l e a s t 4 - f o l d a f t e r the p u r i f i c a t i o n and l y o p h i l i z a t i o n procedures. Moreover, t h i s a ntibody 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 from a s c i t e s f l u i d by DEAE-Sephacel column chromatography (Wong, 1984). There were f o u r major o b j e c t i v e s o f t h i s t h e s i s . The f i r s t o b j e c t i v e was t o determine whether the 4B2-binding p r o t e i n i s the same as the 220 k D a - g l y c o p r o t e i n d e s i g n a t e d as -35-ROS 1.2. The second o b j e c t i v e was t o use both monoclonal and p o l y c l o n a l a n t i b o d i e s as m o l e c u l a r probes t o determine whether the 4B2-binding p r o t e i n (or some o t h e r u n i d e n t i f i e d p r o t e i n ) i s immunologically r e l a t e d t o s p e c t r i n from r e d bl o o d c e l l s . The t h i r d o b j e c t i v e was t o use immunocytochemical techniques t o determine the l o c a t i o n o f the 4B2-binding p r o t e i n w i t h i n the rod c e l l . The f i n a l o b j e c t i v e was t o use b i o c h e m i c a l and immunoblotting t e c h n i q u e s t o d e v i s e a simple and e f f i c i e n t method f o r e x t r a c t i n g the 4B2-binding p r o t e i n from the ROS membranes. -36-MATERIALS AND METHODS A. MATERIALS. General l a b o r a t o r y chemicals o f reagent grade were o b t a i n e d from e i t h e r F i s h e r S c i e n t i f i c Company (Ottawa, O n t a r i o ) , Sigma Chemical Company (St. L o u i s , MO), or BDH Chemicals Canada L i m i t e d (Toronto, O n t a r i o ) . S p e c i a l i z e d m a t e r i a l s and s u p p l i e r s are l i s t e d below o r i n d i c a t e d i n the Methods S e c t i o n s . C e l l c u l t u r e media, f e t a l c a l f and h e a t - i n a c t i v a t e d horse s e r a , p e n i c i l l i n and s t r e p t o m y c i n , fungizone, and Freund's complete and incomplete adjuvants were o b t a i n e d from Grand I s l a n d B i o l o g i c a l Company (Grand I s l a n d , NY). D i s p o s a b l e and s t e r i l e c e n t r i f u g e tubes (50 mL) and p e t r i d i s h e s (100 x 20 mm) were o b t a i n e d from Corning L a b o r a t o r y S c i e n c e s Company (Oneonta, NY). D i s p o s a b l e and s t e r i l e needles and s y r i n g e s , 96-well p o l y s t y r e n e and f l e x i b l e v i n y l p l a t e s f o r s o l i d - p h a s e RIA assays, 1- and 2-mL s e r o l o g i c a l p i p e t s , and p e t r i d i s h e s (60 x 15 mm) were o b t a i n e d from F a l c o n Labware, Becton and Dickenson Canada L i m i t e d ( M i s s i s s a u g a , O n t a r i o ) . Bovine eyes and b r a i n f o r a n t i g e n p r e p a r a t i o n were o b t a i n e d from Olympic I n t e r c o n t i n e n t a l Packers L i m i t e d , a l o c a l s l a u g h t e r house. Bovine b l o o d samples were o b t a i n e d l o c a l l y e i t h e r from J.L. Meats (Surrey, B r i t i s h Columbia), or -37-the Animal S c i e n c e Farm of the U n i v e r s i t y o f B r i t i s h Columbia. BALB/c mice and New Zealand Whites r a b b i t s f o r monoclonal and p o l y c l o n a l antibody p r o d u c t i o n were o b t a i n e d from the Animal Care U n i t o f the U n i v e r s i t y o f B r i t i s h Columbia. Aery1amide, ammonium p e r s u l f a t e , N-N'methylene b i s a c r y l a m i d e , N, N, N #, N'- t e t r a m e t h y l e t h y l e n e d i a m i n e , Coomassie b l u e R-250, and a Protean Dual V e r t i c a l S l a b G e l E l e c t r o p h o r e s i s C e l l apparatus f o r SDS-polyacrylamide g e l e l e c t r o p h o r e s i s were o b t a i n e d from Bio-Rad (Richmond, CA). The power supply (Model ECPS 3000/150) used f o r g e l e l e c t r o p h o r e s i s was o b t a i n e d from Pharmacia (Uppsala, Sweden). For e l e c t r o p h o r e t i c t r a n s f e r s , a Bio-Rad power supply (Model 250/2.5) was used. L o w i c r y l K4M embedding medium and b a s i c s u p p l i e s f o r e l e c t r o n microscopy were o b t a i n e d from JB EM S e r v i c e s I n c o r p o r a t e d (Dorval, Quebec). U r a n y l a c e t a t e was o b t a i n e d from P o l y s c i e n c e s I n c o r p o r a t e d (Warrington, PA). A S o r v a l l U l t r a Microtome MT 5000 from DuPont Company (Wilmington, DE) was used t o c u t embedded specimens i n t o t h i n s e c t i o n s f o r e l e c t r o n microscopy. B. Hybridoma C e l l C u l t u r e C o n d i t i o n s . A l l c e l l c u l t u r e procedures were performed i n a s t e r i l e c u l t u r e hood (NuAire I n c . ) . The window and working s u r f a c e -38-area o f the hood were washed w i t h 70% e t h a n o l b e f o r e and a f t e r each usage and p e r i o d i c a l l y i r r a d i a t e d w i t h u l t r a v i o l e t l i g h t f o r 24 h t o keep the hood s t e r i l e . Glasswares, p i p e t s and p i p e t t i p s , v i a l s , and s o l u t i o n s were s t e r i l i z e d i n an a u t o c l a v e ( C o n s o l i d a t e d ) . Hybridoma c e l l s were grown i n e i t h e r RPMI 1640 o r IMDM c e l l c u l t u r e medium. The c u l t u r e media were s t e r i l i z e d by p a s s i n g them through a Sterivex-GS 0.22 jxm f i l t e r u n i t ( M i l l i p o r e ) under co n s t a n t p r e s s u r e of 15-20 p s i , d e l i v e r e d by a p e r i s t a l t i c pump from M i l l i p o r e (Bedford, MA), i n t o s t e r i l e 500-mL b o t t l e s . The RPMI 1640 c e l l c u l t u r e medium was supplemented wi t h 2 mM L-glutamine, 24 mM sodium b i c a r b o n a t e , 2 mM sodium pyruvate, 10% FCS, 5% heat-i n a c t i v a t e d horse serum, 100 units/mL p e n i c i l l i n , 100 ;ug/mL stre p t o m y c i n , and 1.25 jag/mL fungizone. The pH of t h i s medium was a d j u s t e d t o 7.2. The IMDM c e l l c u l t u r e medium was supplemented w i t h 3 6 mM sodium b i c a r b o n a t e , 10% FCS, 100 units/mL p e n i c i l i i n , 100 ;ug/mL streptomycin, and 1.25 ;ug/mL fungizone. The pH o f t h i s medium was 7.2. C. Maintenance of Hybridoma C e l l L i n e s . Hybridoma c e l l s i n c e l l c u l t u r e medium were grown a t 37°C i n an atmosphere of 5% C0 2 i n a h u m i d i f i e d i n c u b a t o r ( N a t i o n a l ) . Growing c u l t u r e s were maintained i n 10-mL c u l t u r e 5 p l a t e s a t c o n c e n t r a t i o n s r a n g i n g from 4-10X10 c e l l s / m L . The c o n c e n t r a t i o n of v i a b l e c e l l s was determined by s t a i n i n g c e l l -39-c u l t u r e s w i t h an equal volume of 0.4% Trypan b l u e and co u n t i n g u n s t a i n e d c e l l s w i t h a hemacytometer (American O p t i c a l ) . Hybridoma c e l l s were s t o r e d by c e n t r i f u g i n g 10 mL of a c e l l c u l t u r e a t 1,500 rpm f o r 5 min. Each c e l l p e l l e t was resuspended i n 5 mL o f 10% d i m e t h y l s u l f o x i d e i n supplemented IMDM c u l t u r e medium. C e l l s were then 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 (Nunc) 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 styrofoam. The box was l e f t i n a -70°C f r e e z e r ( K e l v i n a t o r ) . A f t e r 24 h the v i a l s were p l a c e d i n a l i q u i d n i t r o g e n tank ( C r y o g e n i c s ) . Frozen c e l l s were r e t r i e v e d by q u i c k l y thawing and washing the c e l l s w i t h 10 mL of non-supplemented IMDM c u l t u r e medium. The c e l l p e l l e t was then resuspended i n 10 mL of supplemented IMDM c u l t u r e medium. D. C l o n i n g o f Hybridoma C e l l L i n e s . A n t i b o d y - s e c r e t i n g 1D1, 3A6, and 4B2 hybridoma c e l l l i n e s were c l o n e d by l i m i t i n g d i l u t i o n . Approximately 230 l i v e hybridoma c e l l s were suspended i n 4.6 mL o f a c l o n i n g 7 medium c o n s i s t i n g o f 10 BALB/c mouse thymocytes p e r mL of supplemented IMDM c u l t u r e medium. A l i q u o t s (100 ;uL) of t h i s s uspension were used t o p l a t e 36 w e l l s o f a 96-well c u l t u r e p l a t e , such t h a t an average o f 5 c e l l s were seeded i n each w e l l . To the remaining 1 mL of c e l l suspension, 4 mL of c l o n i n g medium were added. A l i q u o t s (100 ;uL) of t h i s -40-s uspension were used t o 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 l i q u o t s (100 ;uL) of the c l o n i n g medium were added t o each w e l l o f the c l o n i n g p l a t e a t f i v e and a t twelve days l a t e r . The growth of c l o n e d c e l l s was monitored under a l i g h t microscope w i t h p h a s e - c o n t r a s t o p t i c s ( Z e i s s ) . Wells w i t h c e l l growth were then t e s t e d f o r antibody a c t i v i t y by standard RIA as d e s c r i b e d i n Methods S e c t i o n J . P o s i t i v e c l o n e s were expanded and s t o r e d as d e s c r i b e d i n Methods S e c t i o n C. E. P r o d u c t i o n of Monoclonal Antibody. C u l t u r e supernatants and a s c i t e s f l u i d s from 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 s were r o u t i n e l y used f o r antibody b i n d i n g a n a l y s e s . The 3A6 and 4B2 hybridoma c e l l s were grown and expanded c o n t i n u o u s l y f o r a 2-week p e r i o d . C u l t u r e supernatants were c o l l e c t e d by c e n t r i f u g i n g the c e l l s a t 1,500 rpm f o r 5 min and s t o r e d i n 0.1% sodium a z i d e a t 4°C. The 3A6 and 4B2 hybridoma c e l l s were a l s o grown as a s c i t e s tumors i n BALB/c mice t h a t 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 w i t h 0.5 mL of p r i s t a n e ( A l d r i c h ) 7 days g p r i o r t o 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 , a s c i t e s f l u i d was h a r v e s t e d . A s c i t e s f l u i d was separated from the contaminating b l o o d c e l l s by c e n t r i f u g i n g the mixture a t 12000 rpm f o r 20 min and s t o r e d i n 5 mL a l i q u o t s i n a -70°C f r e e z e r . -41-F. P r e p a r a t i o n o f ROS and D i s c Membranes. P u r i f i e d ROS membranes from f r o z e n r e t i n a s (Hormel) were prepared by a m o d i f i c a t i o n o f the procedure o f Papermaster and Dreyer (1974) as f o l l o w s : F i f t y bovine r e t i n a s i n 50 mL of a sucrose s o l u t i o n c o n t a i n i n g 0.15 M NaCl, 2 mM MgCl 2, and 10 mM T r i s - a c e t a t e , pH 7.4 ( B u f f e r B), and having a d e n s i t y of 1.16 g/mL, were s w i r l e d g e n t l y f o r 1 min and c e n t r i f u g e d a t 4,000 rpm f o r 4 min i n a SS-34 S o r v a l l r o t o r . P e l l e t was resuspended i n 30 mL of the same sucrose s o l u t i o n , and then c e n t r i f u g e d a t 8,000 rpm f o r 8 min. Supernatants were combined and passed through two l a y e r s o f c h e e s e c l o t h . Two volumes o f c o l d B u f f e r B were added t o the f i l t r a t e and the suspension was c e n t r i f u g e d a t 10,000 rpm f o r 10 min. P e l l e t was resuspended i n 15 mL of a sucrose s o l u t i o n c o n t a i n i n g B u f f e r B and having a d e n s i t y o f 1.10 g/mL, and the suspension was c e n t r i f u g e d a t 15,000 rpm f o r 30 min. P e l l e t was resuspended and homogenized twice i n 20 mL o f the same sucrose s o l u t i o n i n a t i g h t d u a l t i s s u e homogenizer (Wheaton). The homogenate (5 mL/gradient) was l a y e r e d on a d i s c o n t i n u o u s g r a d i e n t c o n s i s t i n g o f 4 mL of sucrose having a d e n s i t y o f 1.15 g/mL, 11 mL of sucrose having a d e n s i t y o f 1.13 g/mL, and 9 mL o f sucrose having a d e n s i t y o f 1.11 g/mL. A f t e r c e n t r i f u g a t i o n a t 25,000 rpm f o r 1 h i n a SW-27 r o t o r , the band o f p u r i f i e d ROS membranes was c o l l e c t e d a t the i n t e r f a c e between the 1.11 g/mL and 1.13 g/mL sucrose -42-s o l u t i o n s , d i l u t e d w i t h 30 mL of B u f f e r B, and washed twice by c e n t r i f u g a t i o n a t 15,000 rpm f o r 15 min i n a SS-34 S o r v a l l r o t o r . The p e l l e t was resuspended i n B u f f e r B. P u r i f i e d ROS membranes from f r e s h l y d i s s e c t e d r e t i n a s were prepared as p r e v i o u s l y d e s c r i b e d (Wong and Molday, 1986). F i f t y bovine r e t i n a s i n 15 mL o f homogenizing s o l u t i o n c o n t a i n i n g 20% sucrose, 0.25 mM MgCl 2, 10 mM t a u r i n e , 10 mM gl u c o s e , and 20 mM T r i s - a c e t a t e , pH 7.4, were s w i r l e d g e n t l y f o r 1 min and f i l t e r e d through one l a y e r o f c h e e s e c l o t h . R e t i n a s were then washed w i t h an a d d i t i o n a l 5 mL of homogenizing s o l u t i o n , s w i r l e d , and f i l t e r e d as above. F i l t r a t e s were combined, r e f i l t e r e d t w i ce through two l a y e r s of c h e e s e c l o t h , and p l a c e d on i c e f o r 5 min. F i l t r a t e (5 mL/gradient) was a p p l i e d t o f o u r 22 mL 25-60% (w/w) l i n e a r s u c r o s e g r a d i e n t s c o n t a i n i n g 10 mM t a u r i n e , 10 mM g l u c o s e , and 20 mM T r i s - a c e t a t e , pH 7.4. The g r a d i e n t s were c e n t r i f u g e d a t 25,000 rpm f o r 50 min i n a SW-27 r o t o r . P u r i f i e d ROS membranes were c o l l e c t e d as a s i n g l e band a t the upper r e g i o n o f the g r a d i e n t and washed i n 20 mM T r i s b u f f e r , pH 7.4. D i s c membranes were prepared from p u r i f i e d ROS by hy p o t o n i c l y s i s o f ROS f o l l o w e d by f l o t a t i o n on 5% F i c o l l a c c o r d i n g t o the method o f Smith e t a l . (1975). B r i e f l y , ROS were s l o w l y suspended i n seven volumes o f c o l d 7 mM T r i s -a c e t a t e , pH 7.4, and c e n t r i f u g e d a t 15,000 rpm f o r 15 min. P e l l e t was resuspended i n 10 mL of 7 mM T r i s - a c e t a t e , pH 7.4 -43-and kept i n the dark i n s i d e a SW-27 bucket a t 4°C f o r 30-60 min b e f o r e adding 10 mL of 10% F i c o l l 400 (Pharmacia). T h i s mixture was shaken and o v e r l a y e d w i t h 2 mL d e i o n i z e d water, and l e f t a t 4°C f o r 15 min. A f t e r c e n t r i f u g a t i o n a t 25,000 rpm f o r 1 h i n a SW-27 r o t o r , the d i s c membranes were c o l l e c t e d a t the i n t e r f a c e between water and F i c o l l and washed w i t h 20 mM T r i s - a c e t a t e (pH 7.4) by c e n t r i f u g a t i o n a t 15,000 rpm f o r 30 min i n SS-34 S o r v a l l r o t o r . Membranes were e i t h e r used immediately o r s t o r e d i n s m a l l a l i q u o t s i n l i g h t - t i g h t v i a l s a t -20°C. The 280- and 500-nm absorbance r a t i o s ( A28o/ A500^ f o r m e m b r a n e s s o l u b i l i z e d i n 50 mM c e t y l t r i m e t h y l ammonium bromide were t y p i c a l l y 2.4-3.0. G. P r e p a r a t i o n o f RBC Ghosts, S p e c t r i n , and B r a i n Homoqenate. RBC ghosts were prepared from bovine b l o o d by u s i n g the method o f Dodge e t a l . (1963). Bovine b r a i n microsomal membranes were prepared from bovine b r a i n t i s s u e by u s i n g the method o f Bu r r i d g e e t a l . (1982). S p e c t r i n was p u r i f i e d from RBC ghosts by u s i n g the method o f Marchesi and S t e e r s (1968), or more r e c e n t l y the method o f Sp e i c h e r e t a l . (1980) . P u r i f i e d r a b b i t s k e l e t a l muscle a c t i n was ob t a i n e d from Sigma Chemical Company. P r o t e i n c o n c e n t r a t i o n was measured by the method o f Lowry e t a l . (1951) w i t h bovine serum albumin as a standard. H. Sources o f A n t i b o d i e s . -44-(1) Monoclonal A n t i b o d i e s The hybridoma c e l l technique of K o h l e r and M i l s t e i n (1975) was used t o prepare monoclonal a n t i b o d i e s a g a i n s t ROS p r o t e i n s . Monoclonal a n t i b o d i e s d e s i g n a t e d as 4B2 (MacKenzie and Molday,1982), rho-lD4 (Molday and MacKenzie,1983), and 3A6 (MacKenzie and Molday, unpublished r e s u l t ) were ob t a i n e d from hybridoma c e l l l i n e s generated by f u s i o n o f NS-1 mouse myeloma c e l l s (a generous g i f t o f Drs. Robert McMaster and Mi c h a e l Weaver) w i t h lymphocytes from mice immunized w i t h ROS membranes. A monoclonal antibody d e s i g n a t e d as 2C2 (MacKenzie and Molday, un p u b l i s h e d r e s u l t ) was o b t a i n e d from a hybridoma c e l l l i n e generated by f u s i o n o f NS-1 mouse myeloma c e l l s w i t h lymphocytes from a mouse immunized w i t h a neuroblastoma c e l l membrane p r e p a r a t i o n . Monoclonal a n t i b o d i e s d e s i g n a t e d as 2A4 and 1H5 (Jarausch and Molday, un p u b l i s h e d r e s u l t s ) were o b t a i n e d from hybridoma c e l l l i n e s generated by f u s i o n o f NS-1 mouse myeloma c e l l s w i t h lymphocytes from a mouse immunized w i t h 1% T r i t o n X - 1 0 0 - i n s o l u b l e ROS p r o t e i n s . Monoclonal antibody d e s i g n a t e d as 1D1 was o b t a i n e d from a hybridoma c e l l l i n e generated by f u s i o n o f NS-1 mouse myeloma c e l l s w i t h lymphocytes from a mouse immunized w i t h an urea e x t r a c t o f ROS membrane p r e p a r a t i o n . The immunization and c e l l f u s i o n p r o t o c o l s are b r i e f l y d e s c r i b e d as f o l l o w s : A female BALB/c mouse was immunized 3 weeks a p a r t w i t h two i n t r a p e r i t o n e a l i n j e c t i o n s of 10 jug of urea e x t r a c t a b l e ROS -45-p r o t e i n s e m u l s i f i e d i n 0.2 mL of Freund's complete adjuvant. The mouse was boosted 30 days a f t e r immunization w i t h a s i m i l a r i n t r a p e r i t o n e a l i n j e c t i o n . The s p l e e n o f the immunized mouse was removed f o u r days a f t e r the b o o s t e r i n j e c t i o n and used f o r c e l l f u s i o n . (2) C e l l F u s i o n 7 . 7 Approximately 5X10 s p l e e n c e l l s were fuse d w i t h 1X10 NS-1 mouse myeloma c e l l s i n 1.0 mL o f 50% p o l y e t h y l e n e g l y c o l as d e s c r i b e d by G a l f r e e t a l . (1977). B r i e f l y , t he s p l e e n and NS-1 c e l l s were mixed i n a 50-mL c e n t r i f u g e tube and c e n t r i f u g e d a t 1,500 rpm f o r 5 min t o form a t i g h t p e l l e t . The c e l l p e l l e t was g e n t l y s t i r r e d w h i l e 1-mL o f warm p o l y e t h e l e n e g l y c o l was added over a 1-min p e r i o d . A f t e r s t i r r i n g the tube f o r an a d d i t i o n a l min, 1-mL of IMDM medium was s l o w l y added t o the mixture, f o l l o w e d by the a d d i t i o n o f 7-mL o f IMDM medium over a 2-3 min p e r i o d . F o l l o w i n g f u s i o n , t h e c e l l s were c e n t r i f u g e d a t 1,500 rpm f o r 5 min and suspended i n 50 mL of IMDM medium c o n t a i n i n g 100 juM hypoxanthine, 0.4 juM aminopterin, 16 thymidine, and 20% o FCS (HAT medium) and 2.5X10 BALB/c fe e d e r thymocytes. A l i q u o t s (100 juL) o f c e l l suspension were seeded i n t o 96-well c u l t u r e p l a t e s and maintained a t 37°C i n an atmosphere of 5% C0 2 i n a h u m i d i f i e d i n c u b a t o r . Approximately h a l f o f the HAT medium was c a r e f u l l y removed by a s p i r a t i o n and r e p l a c e d by f r e s h HAT medium s e v e r a l days l a t e r . Two weeks a f t e r the f u s i o n day, HAT -46-medium was r e p l a c e d by IMDM medium supplemented w i t h 100 AIM hypoxanthine, 16 jiM thymidine, and 20% FCS. F i f t y ;uL o f medium was removed from w e l l s w i t h almost c o n f l u e n t hybridoma c e l l growth and t e s t e d s e v e r a l times f o r antibody p r o d u c t i o n u s i n g the standar d RIA procedure as d e s c r i b e d i n Methods S e c t i o n J . A n t i b o d y - s e c r e t i n g hybridoma (1D1) was c l o n e d by l i m i t i n g d i l u t i o n , expanded and s t o r e d as d e s c r i b e d i n Methods S e c t i o n C. (3) P o l y c l o n a l A n t i b o d i e s R a b b i t anti-human RBC s p e c t r i n p o l y c l o n a l antiserum was a g i f t o f Dr. R i n e h a r t Reithmeier. Rabbit a n t i - b o v i n e RBC s p e c t r i n and r a b b i t a n t i - b o v i n e RBC a n k y r i n p o l y c l o n a l a n t i s e r a (Penz and Wong, unpublished r e s u l t s ) were o b t a i n e d from two New Zealand Whites r a b b i t s immunized w i t h 4 biweekly i n j e c t i o n s o f p u r i f i e d bovine RBC s p e c t r i n (100 jug P r o t e i n / i n j e c t i o n ) o r p a r t i a l l y p u r i f i e d RBC a n k y r i n (10 ;ug P r o t e i n / i n j e c t i o n ) e m u l s i f i e d i n Freund's adjuvant a c c o r d i n g t o the procedure of Cooper (1978). B r i e f l y , 20-200 ;ug of a n t i g e n i n 1 mL o f PBS was mixed w i t h equal volume of Freund's complete adjuvant and taken up i n a 2-mL g l a s s s y r i n g e . The mixture was r e p e a t e d l y passed through a s y r i n g e h a v ing a 20 G - l s t e r i l e needle and then through a 23 G - l s t e r i l e needle. The e m u l s i f i e d a n t i g e n s were i n j e c t e d i n t o r a b b i t s subcutaneously over 6-10 s i t e s on t h e i r backs. For the second and subsequent immunizations, Freund's incomplete adjuvant was used t o e m u l s i f y the a n t i g e n s . R a b b i t s were -47-b l e d b e f o r e immunization , and one week a f t e r the second and each subsequent immunizations. Preimmune and immune s e r a were o b t a i n e d by a l l o w i n g b l o o d c l o t s t o form a t room temperature f o r 1-2 h and then c e n t r i f u g i n g the b l o o d a t 15,000 rpm f o r 20 min. Sera were s t o r e d i n 3-mL a l i q u o t s a t -70°C. C u l t u r e f l u i d o b t a i n e d from hybridoma c e l l s c l o n e d a t l e a s t t w i c e and p o l y c l o n a l a n t i s e r a were passed through a 0.22 ;um f i l t e r ( M i l l i p o r e ) o r c e n t r i f u g e d a t 4,000 rpm f o r 5 min t o remove any c e l l u l a r d e b r i s and used i n i n d i r e c t immunochemical and immunocytochemical s t u d i e s as d e s c r i b e d i n Methods S e c t i o n s J-N. (4) T r a c e r Second A n t i b o d i e s Goat anti-mouse Ig was p a r t i a l l y p u r i f i e d from goat anti-mouse Ig a n t i s e r a ( A n t i b o d i e s Incorporated) by i o n exchange chromatography on a DEAE-Sephacel column u s i n g the procedure of Garvey e t a l . (1977). Goat a n t i - r a b b i t Ig 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 as f o l l o w s : Goat a n t i - r a b b i t Ig antiserum (Miles) was a p p l i e d t o a r a b b i t Ig-Sepharose 4B column prepared by the method o f Cuatrecass (1970). The column was washed wi t h 2 column volumes o f 0.01 M sodium phosphate b u f f e r , pH 7.0, t o remove unbound p r o t e i n s . Goat a n t i - r a b b i t Ig was e l u t e d by washing the column w i t h 3 M sodium t h i o c y a n a t e (Baker), d i a l y z e d a g a i n s t s e v e r a l changes of 0.01 M sodium phosphate -48-b u f f e r , pH 7.0, l y o p h i l i z e d and s t o r e d a t -20°C. I. R a d i o i o d i n a t i o n o f P r o t e i n s . Goat anti-mouse Ig, goat a n t i - r a b b i t Ig, RBC s p e c t r i n , Con A (Sigma Chemical Company), and P r o t e i n A (Pharmacia) 125 were l a b e l l e d w i t h c a r r i e r - f r e e I (New England Nuclear) by u s i n g the chloramine T (Eastman Organic Chemicals) method as d e s c r i b e d by Hunter and Greenwood (1962). The i o d i n a t e d g p r o t e i n s (sp. a c t . 1-2 X 10 dpm/jug) were se p a r a t e d from f r e e T125 L I ] i o d i d e and oth e r r e a c t a n t s as p r e v i o u s l y d e s c r i b e d by Molday and MacKenzie(1985). B r i e f l y , the r e a c t i o n mixture was d i l u t e d w i t h 0.5 mL of PBS and c e n t r i f u g e d through 0.5 g o f AG1X10-C1 i o n exchange r e s i n (Bio-Rad) p l a c e d i n the r e s e r v o i r o f a 0.2-;um m i c r o f i l t r a t i o n d e v i c e ( S c h l e i c h e r and S c h u e l l ) . J . S o l i d - P h a s e Radioimmune and Competitive I n h i b i t i o n Assays. The b i n d i n g o f monoclonal a n t i b o d i e s t o muscle a c t i n , ROS membranes and RBC s p e c t r i n was measured by u s i n g i n d i r e c t s o l i d - p h a s e RIA as p r e v i o u s l y d e s c r i b e d by MacKenzie e t a l . (1984). B r i e f l y , bleached ROS membranes, RBC s p e c t r i n , o r a c t i n were s o l u b i l i z e d w i t h 1% T r i t o n X-100, and 25 ;uL o f a 0.25 mg/mL s o l u t i o n was d r i e d onto f l e x i b l e v i n y l m i c r o t i t e r w e l l s a t 60°C. The w e l l s were r i n s e d w i t h water and incubated i n RIA b u f f e r (PBS c o n t a i n i n g 1% BSA, 1% FCS, and 0.1% sodium azide) f o r 60 min. The w e l l s were then r i n s e d i n -49-PBS and incubated w i t h 25 juL o f s e r i a l l y d i l u t e d hybridoma c u l t u r e f l u i d c o n t a i n i n g antibody f o r 60 min a t 23°C. F i n a l l y , the w e l l s were r i n s e d i n PBS and in c u b a t e d w i t h 25 125 J JL o f I - l a b e l l e d goat anti-mouse Ig [10-30 ;ug/mL; (1-2 ) X l O 6 dpm/jug] i n RIA b u f f e r f o r 30-60 min a t 23°C. The p l a t e s were then r i n s e d i n PBS and i n d i v i d u a l w e l l s were counted i n a Beckman 8000 gamma counter. The b i n d i n g o f p o l y c l o n a l r a b b i t anti-human RBC s p e c t r i n a n t i b o d i e s was measured by a s i m i l a r procedure. V a r i a b l e amounts o f T r i t o n X-100 t r e a t e d ROS membrane p r o t e i n s , bovine RBC s p e c t r i n , bovine RBC ghosts, o r BSA were d r i e d onto m i c r o t i t e r w e l l s . In the f i r s t s t e p the w e l l s were t r e a t e d w i t h 25 juL of p o l y c l o n a l r a b b i t anti-human RBC s p e c t r i n a n t i b o d i e s . A f t e r 30 min, the w e l l s were r i n s e d and 125 inc u b a t e d w i t h 25 juL of I - l a b e l l e d P r o t e i n A [30 jug/mL; (1-2)X10 6 dpm/ug]. The w e l l s were r i n s e d i n PBS, and the r a d i o a c t i v i t y was measured as d e s c r i b e d above. The e f f e c t o f ROS membranes, RBC ghosts, and b r a i n microsomal membranes on the b i n d i n g o f 4B2 monoclonal antibody t o T r i t o n X-100 s o l u b i l i z e d ROS was s t u d i e d by u s i n g RIA c o m p e t i t i v e i n h i b i t i o n assay p r e v i o u s l y d e s c r i b e d by Molday and MacKenzie (1983). B r i e f l y , 25 juL o f v a r y i n g c o n c e n t r a t i o n s o f 0.1% T r i t o n X-100 t r e a t e d bovine ROS membranes, bovine b r a i n microsomal membranes, o r bovine RBC ghosts i n RIA b u f f e r was incubated a t 23°C w i t h 25 juL of 4B2 hybridoma 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 t h a t gave -50-80-90% s a t u r a t i o n o f b i n d i n g by s o l i d - p h a s e RIA. A f t e r a 60-min i n c u b a t i o n a t 23°C, 25 ;uL of the mixture was removed and screened f o r remaining antibody a c t i v i t y by s o l i d - p h a s e RIA employing T r i t o n X-100 s o l u b i l i z e d ROS membranes as the 125 immobilized a n t i g e n and I - l a b e l l e d goat anti-mouse Ig as a t r a c e r second antibody. K. SDS-Polyacrylamide Gel E l e c t r o p h o r e s i s and Gel T r a n s f e r . Samples were s o l u b i l i z e d i n an equal volume of d e n a t u r i n g s o l u t i o n c o n t a i n i n g 5% SDS, 40% sucrose, 10 mM T r i s , pH 6.8, 10% 2-mercaptoethanol (Eastman Organic Chemicals), and 4% bromophenol b l u e . Samples (10 juL) were a p p l i e d t o a 6% p o l y a c r y l a m i d e (the r a t i o of acrylamide t o N-N'methylene b i s a c r y l a m i d e i s 3 0 t o 0.8) m i n i s l a b g e l (0.75 mm t h i c k n e s s X 3.0 cm length) 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 by u s i n g the b u f f e r system of Laemmli (1970). In one experiment, a 5-15% p o l y a c r y l a m i d e g r a d i e n t s l a b g e l (0.75 mm t h i c k n e s s X 12.0 cm length) w i t h 25% of r e g u l a r N-N'methylene b i s a c r y l a m i d e c o n c e n t r a t i o n was used t o i n c r e a s e the r e s o l u t i o n of p r o t e i n s i n the 200,000 t o 260,000 M r range. Gel s l i c e s were e i t h e r s t a i n e d w i t h Coomassie blu e (Fairbanks e t a l . , 1971) or s i l v e r (Wray e t a l . , 1981) or 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 . In the l a t t e r procedure u n s t a i n e d SDS-polyacrylamide g e l s were washed over 20 min w i t h two 50-mL changes of t r a n s f e r b u f f e r : 20 mM T r i s -a c e t a t e , pH 7.4, c o n t a i n i n g 2 mM Na EDTA and 0.01% SDS. -51-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 from SDS-p o l y a c r y l a m i d e g e l s t o n i t r o c e l l u l o s e paper (Towbin e t a l . , 1979) i n t r a n s f e r b u f f e r a t 400 mA f o r 2-4 h i n a Hoefer T r a n s b l o t (Model TE 22) apparatus. A f t e r g e l t r a n s f e r , n i t r o c e l l u l o s e paper was s t a i n e d w i t h 0.025% Amido b l a c k (Merck) i n 22.5% e t h a n o l and 7.5% a c e t i c a c i d i n o r d e r t o d e t e c t t r a n s f e r r e d p r o t e i n s . The n i t r o c e l l u l o s e paper was then incubated a t 23°C f o r 1 h i n immunoblot b u f f e r : 0.15 M NaCl, 10 mM sodium phosphate, 1 mM Na 2EDTA, 1 mM sodium a z i d e , 0.2% T r i t o n X-100, and 2% BSA. The quenched paper was incubated w i t h e i t h e r 5-10 mL of hybridoma c u l t u r e f l u i d o r p o l y c l o n a l r a b b i t anti-human or a n t i - b o v i n e RBC s p e c t r i n a n t i b o d i e s f o r 30 min a t 23°C and then r i n s e d 5 times w i t h immunoblot b u f f e r (without BSA), once w i t h 2 M urea, 0.1 M g l y c i n e , and 1% T r i t o n X-100, and once w i t h PBS. A f t e r b e i n g washed, the paper was incubated w i t h 5 mL o f I - l a b e l l e d goat anti-mouse Ig (1.8 X 10 dpm/^ig; 2.3 jag/mL) or 1 2 5 I - l a b e l l e d P r o t e i n A (0.8 X 1 0 5 dpm/ug) i n immunoblot b u f f e r f o r 30 min a t 23°C. F i n a l l y , t he paper was washed as d e s c r i b e d and d r i e d f o r autoradiography on Kodak Royal X-Omat f i l m w i t h an X-ray i n t e n s i f y i n g s c r e e n . For most experiments, exposure time was 125 1-3 days, but i n the case of I - l a b e l l e d P r o t e i n A b i n d i n g t o ROS membranes, exposure time was i n c r e a s e d t o 5-7 days. In some experiments, quenched n i t r o c e l l u l o s e papers were 125 . . . l a b e l l e d w i t h I - l a b e l l e d Con A ( s p e c i f i c a c t i v i t y = 7.9 X 5 10 dpm/ug). -52-In the case o f 1D1 l a b e l l i n g o f ROS membrane p r o t e i n s , an immunostaining method a c c o r d i n g t o the procedure o f O'Connor and Ashman (1982) was used. B r i e f l y , 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 e l e c t r o p h o r e t i c a l l y ROS membrane p r o t e i n s was quenched i n 5% g e l a t i n (Sigma) i n PBS f o r 1 h. The paper was washed s e v e r a l times w i t h 0.05% (v/v) Tween 20 i n PBS f o r a p e r i o d o f 20 min and incubated w i t h 1D1 c e l l c u l t u r e supernatant f o r 2 h. The paper was a g a i n washed s e v e r a l times w i t h 0.05% Tween 20 i n PBS and then i n c u b a t e d w i t h goat anti-mouse I g - a l k a l i n e phosphatase conjugates (Sigma) i n 0.2 M sodium b a r b i t a l b u f f e r , pH 9.5, f o r 2 h. The paper was washed s e v e r a l times w i t h 0.05% Tween 20 i n PBS f o r a p e r i o d of 20 min and once w i t h 0.2 M sodium b a r b i t a l b u f f e r , pH 9.5. Before s t a i n development, the paper was in c u b a t e d w i t h 5 mM of MgCl 2 i n 0.2 M sodium b a r b i t a l b u f f e r f o r 15 min. The s t a i n was developed by s e q u e n t i a l l y adding the s u b s t r a t e f o r a l k a l i n e phosphatase 5-bromo-4-chloro-3-indoxyl-phosphate (50 ;ug/mL) i n dimethylformamide and n i t r o b l u e t e t r a z o l i u m s a l t (100 Aig/mL) i n sodium b a r b i t a l b u f f e r , pH 9.5. S t a i n development was stopped by washing the paper w i t h 0.05% Tween 20 i n PBS. F i n a l l y , the paper was a i r d r i e d and photographed. L. S i z e E x c l u s i o n Chromatography of ROS P r o t e i n s on a Sepharose-2B Column. ROS membranes prepared from f r e s h l y - d i s s e c t e d bovine -53-r e t i n a as d e s c r i b e d i n Methods S e c t i o n F were c e n t r i f u g e d a t 15,000 rpm f o r 30 min. ROS p e l l e t (5 mg P r o t e i n ) was s o l u b i l i z e d i n 1% SDS, 10 mM T r i s - H C l , 0.5 mM 2-mercaptoethanol, 0.5 mM EDTA, 0.03 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e , and 0.02 % NaN^, pH 8.5 and a p p l i e d t o a Sepharose-CL 2B column (1 cm i n diameter and 42 cm i n l e n g t h ) , p r e -e q u i l i b r a t e d w i t h the same b u f f e r w i t h 0.1% SDS. The column was e l u t e d w i t h the same b u f f e r c o n t a i n i n g 0.1% SDS a t a flow r a t e o f 0.5 mL/min. F r a c t i o n s (0.5 mL) were c o l l e c t e d and assayed f o r absorbance a t 280 nm. F r a c t i o n s numbered 18, 20, 22, 24, 26 and 28 were analyzed by SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and the immunoblotting technique b e f o r e and a f t e r s t o r a g e a t 4°C f o r 7 days. M. L o w i c r y l T h i n S e c t i o n L a b e l i n g . Bovine r e t i n a was f i x e d i n 1.25% g l u t a r a l d e h y d e (Merck) i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, and 0.2 M sucrose f o r 30-60 min a t 23°C. A f t e r washing i n the same b u f f e r f o r 1 h, 2 the t i s s u e was c u t i n t o 1-mm p i e c e s . The samples were s e r i a l l y dehydrated i n dimethylformamide (50%, 70%, 90%, and 100%) a t -35°C and embedded i n L o w i c r y l K4M medium a t -35°C. T i s s u e was c a r e f u l l y o r i e n t e d i n g e l a t i n c a p s u l e s and poly m e r i z e d under l o n g wavelength u l t r a v i o l e t i r r a d i a t i o n o v e r n i g h t a c c o r d i n g t o the method of Roth e t a l . (1981). S e c t i o n s were c u t , c o l l e c t e d on c l e a n copper g r i d s , and p r e i n c u b a t e d i n 50 ;uL of PBS w i t h 0.1% BSA f o r 10 min t o quench n o n s p e c i f i c b i n d i n g s i t e s . The g r i d s were then -54-incubated i n 50 juL o f 4B2 hybridoma c u l t u r e f l u i d f o r 30 min a t 23°C, f o l l o w e d by e x t e n s i v e washing i n PBS w i t h 0.1% BSA. F i n a l l y , the g r i d s were incubated i n 50 ;uL o f goat anti-mouse Ig g o l d - d e x t r a n (Hicks and Molday,1986) f o r 30 min a t 23°C. The g r i d s were washed e x t e n s i v e l y i n PBS, s t a i n e d w i t h s a t u r a t e d u r a n y l a c e t a t e and l e a d c i t r a t e and viewed under a P h i l i p s 200 o r a JEOL 1200 EX e l e c t r o n microscope. N. E x t r a c t i o n o f ROS Membranes. Four samples of dark adapted ROS membranes (6 mg p r o t e i n / t u b e ) , prepared from f r e s h r e t i n a s as d e s c r i b e d i n Methods S e c t i o n F, were washed twice i n 20 mM T r i s b u f f e r , pH 7.4, by c e n t r i f u g a t i o n a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. Each ROS p e l l e t sample was resuspended w i t h o n l y one o f the f o l l o w i n g s o l u t i o n s : 6 M u l t r a p u r e urea (Schwarz and Mann) i n 20 mM T r i s - a c e t a t e , pH 7.4 ; 20 mM T r i s -a c e t a t e , pH 7.4; 0.3 mM ATP and 50 mM 2-mercaptoethanol i n 0.3 mM sodium phosphate, pH 8.0; and 1 mM EDTA i n 0.3 mM sodium phosphate, pH 8.0. The former sample was l e f t f o r 24 h a t 4°C w h i l e the l a t t e r t h r e e samples were d i a l y z e d a g a i n s t t h e i r r e s p e c t i v e s o l u t i o n s f o r 24 h a t 4°C. The mixtures were then c e n t r i f u g e d a t 25,000 rpm i n a SW-27 r o t o r f o r 1 h. F o l l o w i n g c e n t r i f u g a t i o n , the supernatant and the resuspended ROS p e l l e t were d i a l y z e d a g a i n s t 20 mM T r i s b u f f e r , pH 7.4. The d i a l y z a t e s were s u b j e c t e d t o SDS-polyacrylamide g e l e l e c t r o p h o r e s i s and immunoblot a n a l y s i s as d e s c r i b e d i n Methods S e c t i o n K. -55-O. T r i t o n X-100 E x t r a c t i o n o f ROS. Dark adapted ROS membranes prepared from 45 f r e s h l y d i s s e c t e d r e t i n a s as d e s c r i b e d i n Methods S e c t i o n F were washed t w i c e w i t h 20 mM T r i s - a c e t a t e and 20 mM NaCl, pH 7.4, by c e n t r i f u g a t i o n a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. ROS p e l l e t (5 mg p r o t e i n ) was resuspended i n 5 mL of T r i s b u f f e r , pH 7.4, c o n t a i n i n g 75 mM KC1 and 1% T r i t o n X-100. Suspension was shaken c o n t i n u o u s l y f o r 24 h a t 23°C and then c e n t r i f u g e d a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. The T r i t o n X-100 p e l l e t was resuspended i n 0.5 mL of T r i s b u f f e r c o n t a i n i n g 75 mM KC1 and 1% T r i t o n X-100. P r o t e i n c o n c e n t r a t i o n s o f the supernatant and p e l l e t were determined by the method of Lowry e t a l . (1951). Equal volume o f both f r a c t i o n s were s u b j e c t e d t o SDS-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 and immunoblot a n a l y s i s as d e s c r i b e d i n Methods S e c t i o n K. P. Detergent S o l u b i l i z a t i o n o f ROS Membranes. Dark adapted ROS membranes, prepared from 100 f r o z e n r e t i n a s as d e s c r i b e d i n Methods S e c t i o n F, were washed t w i c e w i t h 20 mM T r i s - a c e t a t e and 20 mM NaCl, pH 7.4, by c e n t r i f u g a t i o n a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. ROS p e l l e t (5 mg p r o t e i n was resuspended i n e i t h e r 1 mL of 1% T r i t o n X-100, or 1% SDS, or 1% o c t y l g l u c o s i d e , o r 1% sodium c h o l a t e , o r 1% (3-[(3-chloamidopropyl)dimethylammonio] -56-1-propanesulfonate) a l s o known as CHAPS i n T r i s b u f f e r , pH 7.4, c o n t a i n i n g 75 mM K C l . Suspensions were shaken c o n t i n u o u s l y f o r 24 h a t 23 °C and then c e n t r i f u g e d a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. Supernatants were removed and p e l l e t s were s u b j e c t e d t o SDS-polyacrylamide 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 as d e s c r i b e d i n Methods S e c t i o n K. Q. Negative S t a i n i n g of T r i t o n X-100 E x t r a c t e d ROS P e l l e t . T r i t o n X-100 e x t r a c t e d ROS prepared as d e s c r i b e d i n Methods S e c t i o n 0 were resuspended i n 0.1 mL o f 75 mM KCl and 20 mM T r i s - a c e t a t e , pH 7.4. F i f t y ;uL o f each samples was f i x e d w i t h 100juL o f 1.25% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, and washed t h r e e times w i t h the same b u f f e r by c e n t r i f u g a t i o n a t 15,000 rpm i n a SS-34 S o r v a l l r o t o r f o r 30 min. P e l l e t was resuspended i n c a c o d y l a t e b u f f e r . Formvar g r i d s p r e v i o u s l y coated w i t h 50 juL o f 0.1% p o l y l y s i n e f o r 30 min and r i n s e d e x t e n s i v e l y i n water were t r e a t e d w i t h 50 jaL of the above sample s o l u t i o n f o r 1 h. A f t e r e x t e n s i v e washing i n water, the g r i d s were t r e a t e d w i t h 50 uL of 1% sodium phosphotungstate, pH 7.2, f o r 20-25 sec. Excess s t a i n was removed by b l o t t i n g the g r i d s on t h e i r edges w i t h f i l t e r paper. G r i d s were a i r d r i e d b e f o r e b e i n g viewed under a JEOL 1200 EX e l e c t r o n microscope. -57-RESULTS A. High M o l e c u l a r Weight P o l y p e p t i d e s o f ROS Membranes. When ROS membranes were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s and s t a i n e d w i t h Coomassie b l u e , rhodopsin h a v i n g an apparent M r 34,000 was observed as the major band and ROS 1.2 o f apparent M r 220,000 was the second most i n t e n s e band ( F i g . 8). Bands f a i n t l y v i s i b l e by Coomassie b l u e s t a i n i n g were i n t e n s i f i e d by u s i n g s i l v e r s t a i n i n g and s e v e r a l bands i n the m o l e c u l a r weight range o f 200,000-240,000 were seen. The b i n d i n g s p e c i f i c i t y o f ConA and 4B2 f o r ROS p o l y p e p t i d e s was determined by t r e a t i n g p o l y p e p t i d e s 125 t r a n s f e r r e d t o n i t r o c e l l u l o s e paper d i r e c t l y w i t h IE-l a b e l l e d ConA or i n d i r e c t l y w i t h 4B2 antibody f o l l o w e d by 125 . . 125 I - l a b e l l e d goat anti-mouse Ig. With I - l a b e l l e d 125 s p e c t r i n as an e x t e r n a l m o l e c u l a r weight r e f e r e n c e , I -l a b e l l e d ConA was found t o l a b e l rhodopsin and ROS 1.2 a t 220,000 as p r e v i o u s l y shown (MacKenzie and Molday, 1982). The 4B2 antibody, however, l a b e l l e d a p o l y p e p t i d e c h a i n of a s l i g h t l y h i g h e r m o l e c u l a r weight ( F i g . 8 ) . T h i s band had the same e l e c t r o p h o r e t i c m o b i l i t y as the s u b u n i t o f RBC s p e c t r i n and an apparent M r 240,000. A f a i n t l y l a b e l l e d band a t M r 220,000 was a l s o seen upon prolonged a u t o r a d i o g r a p h i c exposure. In some p r e p a r a t i o n s , bands i n the m o l e c u l a r weight range of 95,000-150,000 were a l s o l a b e l l e d w i t h the 4B2 antibody. These bands appear t o r e p r e s e n t p r o t e o l y t i c 5 8 -F i g u r e 8. SDS g e l e l e c t r o p h o r e s i s and immunoblots o f ROS d i s c membranes^ S D S - s o l u b i l i z e d ROS d i s c membranes (20 .jug/well) and I - l a b e l l e d bovine RBC s p e c t r i n (6 jag/well) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 6% p o l y a c r y l a m i d e s l a b g e l . Gels were e i t h e r s t a i n e d w i t h Coomassie b l u e (CB) or s i l v e r s t a i n (SS) o r t r a n s f e r r e d t o n i t r o c e l l u l o s e paper. T r a n s f e r papers were e i t h e r d i r e c t l y l a b e l l e d w i t h I - l a b e l l e d Con A or i n d i r e c t l y l a b e l l e d w i t h u n d i l u t e d 4B2-antibody c u l t u r e f l u i d and I - l a b e l l e d goat anti-mouse Ig f o r autoradiography. ROS membrane p r o t e i n s a re shown i n l a n e a, and I - l a b e l l e d s p e c t r i n used as an e x t e r n a l m o l e c u l a r weight marker f o r autora d i o g r a p h y i s shown i n l a n e b. - 5 9 -fragments of the M r 240,000 p o l y p e p t i d e (see below). B. Degradation o f 4 B 2 - S p e c i f i c P r o t e i n by an Endogenous  Protease. The s e n s i t i v i t y o f the 4 B 2 - s p e c i f i c p r o t e i n and ConA-s p e c i f i c g l y c o p r o t e i n s t o d e g r a d a t i o n by an endogenous pr o t e a s e i n ROS p r e p a r a t i o n s was d e t e c t e d by immunoblotting a n a l y s i s . As shown i n F i g u r e 9, the 4B2 antibody l a b e l l e d o n l y one major p o l y p e p t i d e of M r 240,000 i n f r e s h l y prepared ROS membranes. When the ROS membranes were maintained a t 4°C f o r 24 h, p a r t i a l d e g r a d a t i o n of the M r 240,000 p o l y p e p t i d e t o p o l y p e p t i d e s o f apparent M • 150,000, 120,000, and 95,000 was observed. In c o n t r a s t , no d e g r a d a t i o n of rhodopsin o r ROS 1.2 was d e t e c t e d by ConA l a b e l l i n g o r by Coomassie b l u e s t a i n i n g . ROS d i s c membranes prepared by h y p o t o n i c l y s i s of f r e s h l y prepared ROS and f l o t a t i o n on 5% F i c o l l were l e s s prone t o d e g r a d a t i o n under these c o n d i t i o n s . S i g n i f i c a n t d e g r a d a t i o n o f the 4 B 2 - s p e c i f i c p o l y p e p t i d e was g e n e r a l l y observed when ROS were prepared from f r o z e n r e t i n a . S e v e r a l p r o t e a s e i n h i b i t o r s such as a p r o t i n i n , l e u p e p t i n , a n d phenylmethy1sulphonyl f l u o r i d e , and a metal c h e l a t o r EGTA have been found t o be i n e f f e c t i v e i n p r e v e n t i n g the p r o t e o l y t i c d e g r a d a t i o n of the 4 B 2 - s p e c i f i c p o l y p e p t i d e s i n c e immunoblots of 4B2 l a b e l l i n g o f ROS membrane p r o t e i n s prepared i n the presence of these i n h i b i t o r s showed a band a t M r 150,000 (not shown). S i z e - e x c l u s i o n chromatography of S D S - s o l u b i l i z e d ROS -60-4B2 a b CON A - - -220 K Rho CB F i g u r e 9. P r o t e o l y t i c degradation of the 4B2-binding p r o t e i n of ROS by endogenous pro t e a s e s . ROS membranes prepared from f r e s h l y d i s s e c t e d r e t i n a s were e i t h e r d i r e c t l y s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s ( g el a) or s t o r e d a t 4 C f o r 24 h p r i o r t o SDS g e l e l e c t r o p h o r e s i s ( g e l b ) . The g e l s were e i t h e r s t a i n e d w i t h Coomassie blu e (CB) or t r a n s f e r r e d t o n i t r o c e l l u l o s e p a p e r ^ n d l a b e l l e d w i t h u n d i l u t e d 4B2-antibody c u l t u r e f l u i d and I - l a b e l l e d goat anti-mouse Ig or w i t h - " " ^ I - l a b e l l e d Con A. -61-p r o t e i n s on a Sepharose 2B column was attempted t o separate the 4 B 2 - s p e c i f i c p r o t e i n from the endogenous p r o t e a s e . The p r o t e i n components of the column f r a c t i o n s were an a l y z e d by SDS g e l e l e c t r o p h o r e s i s and immunoblotting. As shown i n F i g . 10, Coomassie b l u e s t a i n i n g o f e l u t a n t s a f t e r g e l e l e c t r o p h o r e s i s i n d i c a t e d the h i g h m o l e c u l a r weight g l y c o p r o t e i n o r ROS 1.2 w i t h apparent M r 220,000 was w e l l s e p a r a t e d from rhodopsin w i t h apparent M r 34,000. Two bands w i t h apparent M r o f 55-60,000 were most n o t i c e a b l e i n f r a c t i o n s numbered 18, 20, and 28. T h i s d o u b l e t appeared t o be an a r t e f a c t because i t was p r e s e n t even i n g e l l a n e s t h a t d i d not c o n t a i n any column f r a c t i o n sample. Immunoblots of the f r a c t i o n s b e f o r e and a f t e r s t o r a g e a t 4°C f o r 7 days showed the i n t e n s i t y of bands was h i g h e r i n immunoblot C than i n immunoblot B due t o d i f f e r e n c e s i n exposure time. However v e r y s i m i l a r 4B2 antibody l a b e l l i n g p a t t e r n s were observed. T h i s i n d i c a t e d t h a t the 4 B 2 - s p e c i f i c p o l y p e p t i d e w i t h apparent M r 240,000 and i t s p r o t e o l y t i c fragments were not f u r t h e r degraded t o s m a l l e r fragments t o any s i g n i f i c a n t e x t e n t d u r i n g the s t o r a g e p e r i o d . C. Urea E x t r a c t i o n of the 4 B 2 - S p e c i f i c P r o t e i n . The e x t r a c t a b i l i t y o f the 4 B 2 - s p e c i f i c p r o t e i n and ConA-s p e c i f i c membrane g l y c o p r o t e i n s w i t h urea was s t u d i e d t o determine t h e i r i n t e r a c t i o n w i t h the l i p i d b i l a y e r . ROS d i s c membranes were t r e a t e d w i t h 6 M urea o v e r n i g h t and -62-1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 A B Figure 10. Size exclusion Chromatography of SDS-solubilized ROS proteins on a Sepharose 2B column. ROS membranes prepared from freshly dissected bovine r e t i n a as described i n Methods Section F were centrifuged at 15,000 rpm f o r 30 min. ROS p e l l e t (5 mg Protein) was s o l u b i l i z e d i n 1% SDS, 10 mM Tris-HCl, 0.5 mM 2-mercaptoethanol, 0.03 mM phenylmethylsulfonylfluoride, 0.5 mM EDTA, and 0.02 % NaN , pH 8.5 and applied to a Sepharose-CL 2B column (1 cm In diameter and 42 cm i n length), pre-equilibrated with the same buffer with 0.1% SDS. The column was eluted with the same buffer with 0.1% SDS at a flow rate of 0.5 mL/min. Fractions (0.5 mL) were c o l l e c t e d and assayed f o r absorbance at 280 nm. Fractions numbered 18, 20, 22, 24, 26 and 28 were analyzed by SDS-polyacrylamide gel electrophoresis (Lanes 1-6 resp e c t i v e l y ) . Gels were either stained with Coomassie blue (gel A) or transferred to n i t r o c e l l u l o s e paper. Transfer papers were sequentially l a b e l l e d with 4B2 c e l l culture supernatant and I-lab e l l e d goat anti-mouse Ig for autoradiography (gel B). The same fr a c t i o n s were again analyzed by gel electrophoresis gnd immunoblotting technique a f t e r they have been stored at 4 C for 7 days (gel C). - 6 3 -subsequently separated i n t o a p e l l e t and supernatant f r a c t i o n by c e n t r i f u g a t i o n a t 25,000 rpm f o r 1 h. A n a l y s i s o f these f r a c t i o n s by SDS g e l e l e c t r o p h o r e s i s and immunoblotting i n d i c a t e d t h a t a s m a l l q u a n t i t y (5-10%) o f the 4 B 2 - s p e c i f i c M r 240,000 p o l y p e p t i d e and p r o t e o l y t i c fragments was e x t r a c t e d from the membrane ( F i g u r e 11). E x t r a c t i o n o f the 4B2-binding p r o t e i n by urea was r e p r o d u c i b l y observed i n t h r e e s e p a r a t e experiments. However, under these same c o n d i t i o n s , no e x t r a c t i o n o f rhodopsin o r ROS 1.2 i n t o the supernatant was d e t e c t e d by 125 I - l a b e l l e d ConA b l o t t i n g o r Coomassie b l u e s t a i n i n g . D. Immunological C r o s s - R e a c t i v i t y o f Monoclonal Antibody 4B2  w i t h P r o t e i n s from Other C e l l Types. The presence of 4B2-binding p r o t e i n s i n c e l l t y p e s o t h e r than the rod p h o t o r e c e p t o r s was i n v e s t i g a t e d by RIA c o m p e t i t i v e i n h i b i t i o n assays. As shown i n F i g u r e 12, both T r i t o n X-100 s o l u b i l i z e d bovine RBC ghosts and bovine b r a i n microsomal membranes i n a d d i t i o n t o bovine ROS were a b l e t o i n h i b i t the b i n d i n g of the 4B2 antibody t o T r i t o n X-100 s o l u b i l i z e d d i s c membranes immobilized on m i c r o t i t e r p l a t e s . F i f t y p e r c e n t i n h i b i t i o n o f 4B2 antibody b i n d i n g was a t t a i n e d a t t o t a l p r o t e i n c o n c e n t r a t i o n s of 300 and 400 ;ug/mL f o r b r a i n membranes and RBC ghosts, r e s p e c t i v e l y . ROS was a more e f f e c t i v e i n h i b i t o r , r e q u i r i n g a 3-4 f o l d lower c o n c e n t r a t i o n t o a c h i e v e 50% i n h i b i t i o n o f 4B2 antibody b i n d i n g . A c c u r a t e q u a n t i t a t i v e a n a l y s i s o f c r o s s - r e a c t i v i t y , however, c o u l d not -64-240K M f - ROS 12 2 Rho P S P S 4B2 CON A F i g u r e 11. E x t r a c t i o n of the 4B2-binding p r o t e i n from ROS membranes by urea. ROS membranes were t r e a t e d w i t h 6 M urea a t 4 C f o r 24 h. The membranes were then sedimented a t 25,000 rpm f o r 1 h. The p e l l e t was resuspended i n 20 mM T r i s - a c e t a t e , pH 7.4, and d i a l y z e d a g a i n s t the same b u f f e r f o r 24 h. The supernatant and p e l l e t were s o l u b i l i z e d i n SDS and s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on 6% p o l y a c r y l a m i d e g e l s and 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 . The supernatant ( g e l S) and the p e l l e t ( g el P) were l a b e l l e d w i t h e i t h e r 1 2 ^ B 2 antibody and I - l a b e l l e d goat anti-mouse Ig or I -l a b e l l e d Con A. -65-Protein Cone (mg/ml) F i g u r e 12. Competitive i n h i b i t i o n o f 4B2 antibody b i n d i n g t o ROS membrane p r o t e i n s by RBC membrane ghosts and b r a i n microsomal membranes. I n h i b i t i o n o f 4B2 antibody b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d , immobilized ROS p r o t e i n s by T r i t o n X-100 s o l u b i l i z e d bovine ROS ( s o l i d c i r c l e s ) , T r i t o n X-100 s o l u b i l i z e d bovine b r a i n microsomal membranes (open c i r c l e s ) , T r i t o n X-100 s o l u b i l i z e d bovine RBC membrane ghosts ( s o l i d t r i a n g l e s ) , and bovine serum albumin (open t r i a n g l e s ) . - 6 6 -be made s i n c e the q u a n t i t y of c r o s s - r e a c t i n g a n t i g e n i n these p r e p a r a t i o n s was not known. Bovine serum albumin s e r v i n g as a c o n t r o l d i d not i n h i b i t 4B2 antibody b i n d i n g . Competitive i n h i b i t i o n s t u d i e s a l s o i n d i c a t e d t h a t human RBC ghosts bound the 4B2 antibody, but w i t h a lower a f f i n i t y than bovine RBC ghosts (not shown). E. I d e n t i f i c a t i o n o f S p e c t r i n as the RBC A n t i g e n f o r t h e 4B2  Monoclonal Antibody. S o l i d - p h a s e RIA and immunoblot a n a l y s i s were used t o i d e n t i f y s p e c t r i n as the 4B2-binding p r o t e i n of RBC. F i g u r e 13 shows the e f f e c t o f 4B2 antibody and rho-lD4 antibody d i l u t i o n on the b i n d i n g o f these a n t i b o d i e s t o p u r i f i e d bovine RBC s p e c t r i n immobilized on m i c r o t i t e r p l a t e s . The 4B2 a n t i b o d y e x h i b i t e d s i g n i f i c a n t s a t u r a b l e b i n d i n g . In c o n t r a s t , rho-lD4 (an antibody t h a t b inds t o the C - t e r m i n a l r e g i o n o f rhodopsin) d i d not b i n d even a t h i g h antibody c o n c e n t r a t i o n s . The s u b u n i t of bovine RBC s p e c t r i n t h a t b i n d s the 4B2 antibody was determined by s e q u e n t i a l l y l a b e l l i n g s p e c t r i n s u b u n i t s separated by SDS g e l e l e c t r o p h o r e s i s w i t h the 4B2 125 antibody and I - l a b e l l e d goat anti-mouse Ig . As shown i n F i g u r e 14, the 4B2 antibody predominantly l a b e l l e d the o( s u b u n i t o f bovine s p e c t r i n having an apparent M r 240,000 (Branton e t a l . , 1981). In c o n t r a s t , rho-lD4 d i d not l a b e l e i t h e r s u b u n i t of bovine RBC s p e c t r i n . With prolonged exposure c o n d i t i o n s , the 4B2 antibody was found t o weakly -67-Reciprocal Dilution F i g u r e 13. B i n d i n g of 4B2 and rho-lD4 a n t i b o d i e s t o RBC s p e c t r i n . The e f f e c t of 4B2 and rho-lD4 antibody d i l u t i o n on b i n d i n g t o bovine s p e c t r i n . T r i t o n X-100 t r e a t e d bovine RBC s p e c t r i n immobilized i n m i c r o t i t e r w e l l s was i n c u b a t e d w i t h s e r i a l d i l u t i o n s of c u l t u r e f l u i d from eii^hgr 4B2 or rho-lD4 hybridoma c e l l s , r i n s e d and t r e a t e d w i t h I - l a b e l l e d goat anti-mouse Ig. -68-C B 4B2 1D4 Figure 14. SDS gel electrophoresis and immunoblots of bovine RBC spectrin. Bovine spectrin (2 jug/well) was subjected to electrophoresis on a 6% SDS-polyacrylamide slab gel and eithe r stained with Coomassie blue (CB) or transferred to n i t r o c e l l u l o s e paper. The transfer gapers were treated with eithe r 4B2 or rho-lD4 antibody and I - l a b e l l e d goat a n t i -mouse Ig and were subjected to autoradiography. The o( and A subunits of spectrin are indicated. -69-r e a c t w i t h a band a t M r 220,000 (not shown). F. C h a r a c t e r i z a t i o n o f 3A6 Monoclonal Antibody. A hybridoma c e l l l i n e r e f e r r e d t o as 3A6 was p r e v i o u s l y shown t o s e c r e t e a n t i b o d i e s s p e c i f i c f o r d i s c membrane p r e p a r a t i o n s by s o l i d - p h a s e RIA. I n i t i a l immunoblot a n a l y s i s i n d i c a t e d t h a t the 3A6 antibody l a b e l l e d s e v e r a l ROS p o l y p e p t i d e s i n the M r range of 100,000 t o 220,000 (MacKenzie and Molday, un p u b l i s h e d r e s u l t s ) . F u r t h e r c h a r a c t e r i z a t i o n o f t h i s a ntibody was c a r r i e d out t o determine whether t h i s a n tibody i s s i m i l a r t o the 4B2 monoclonal antibody. The 3A6 c e l l l i n e was r e c l o n e d and used t o produce a n t i b o d y - c o n t a i n i n g c e l l c u l t u r e supernatants and a s c i t e s f l u i d s . The e f f e c t o f d i l u t i o n o f the 3A6 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 on the b i n d i n g o f ant i b o d y t o T r i t o n X-100 s o l u b i l i z e d ROS membrane p r o t e i n s u s i n g an i n d i r e c t s o l i d - p h a s e RIA i s shown i n F i g u r e 15. T y p i c a l l y , half-maximum b i n d i n g o c c u r r e d a t r e c i p r o c a l d i l u t i o n s o f 8-16 f o r 3A6 c e l l c u l t u r e supernatants and 500-1000 f o r 3A6 a s c i t e s f l u i d s . G. B i n d i n g o f 3A6 Monoclonal Antibody t o RBC S p e c t r i n  and ROS Membranes. The p o l y p e p t i d e s i n bovine ROS and bovine RBC s p e c t r i n s e r v i n g as a n t i g e n s f o r the 3A6 monoclonal an t i b o d y were -70-1 4 16 64 256 1024 4096 Reciprocal Dilution F i g u r e 15. T i t r a t i o n o f 3A6 hybridoma c e l l a s c i t e s f l u i d ( s quares), 3A6 hybridoma c e l l c u l t u r e supernatant (diamonds), and c u l t u r e medium ( t r i a n g l e s ) a g a i n s t T r i t o n X-100-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 immobilized 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 supernatants or a s c i t e s f l u i d , r i n s e d and subsequently t r e a t e d w i t h I - l a b e l l e d goat anti-mouse Ig. i d e n t i f i e d by immunoblotting. As i l l u s t r a t e d i n F i g u r e 16, the 3A6 antibody o n l y l a b e l l e d the ft band of RBC s p e c t r i n w i t h apparent M r 220,000. In c o n t r a s t the 4B2 antibody o n l y l a b e l l e d the C*s band of RBC s p e c t r i n w i t h apparent M r 240,000 as p r e v i o u s l y i n d i c a t e d ( F i g . 14). In ROS membranes, the 3A6 antibody l a b e l l e d f i v e ROS p o l y p e p t i d e s w i t h apparent M r 240,000, 220,000, 160,000, 150,000, and 80,000. The M"r 240,000 p o l y p e p t i d e was the most i n t e n s e l y l a b e l l e d band. The s m a l l e r p o l y p e p t i d e s are b e l i e v e d t o be the degraded fragments of the M r 240,000 p o l y p e p t i d e s i n c e the s m a l l e r p o l y p e p t i d e s were more i n t e n s e l y l a b e l l e d than the M r 240,000 p o l y p e p t i d e i n some experiments. The 4B2 antibody a l s o l a b e l l e d s e v e r a l p o l y p e p t i d e s w i t h s i m i l a r but not i d e n t i c a l M r > I n t e r e s t i n g l y the l a r g e s t p o l y p e p t i d e i n ROS l a b e l l e d by the 4B2 antibody had an apparent M r 260,000 i n s t e a d of 240,000 as p r e v i o u s l y determined. T h i s d i f f e r e n c e i n M r i s most l i k e l y due t o the h i g h e r r e s o l v i n g power o f the more porous or l e s s c r o s s l i n k e d p o l y a c r y l a m i d e g r a d i e n t g e l used i n t h i s study. T h i s r e s u l t suggests t h a t 3A6 and 4B2 a n t i b o d i e s are s p e c i f i c f o r two d i f f e r e n t ROS p r o t e i n s . H. B i n d i n g of P o l y c l o n a l A n t i - S p e c t r i n A n t i b o d i e s  t o ROS P r o t e i n s . In o r d e r t o s u b s t a n t i a t e the f i n d i n g s t h a t i n d i c a t e an immunological e q u i v a l e n t of RBC s p e c t r i n e x i s t s i n bovine ROS, p o l y c l o n a l r a b b i t anti-human and a n t i - b o v i n e RBC s p e c t r i n 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 t o ROS membrane -72-Figure 16. SDS gel electrophoresis and immunoblots of ROS membranes. SDS-solubilized ROS membrane proteins (40 jag/well) and bovine RBC spectrin (6 jug/well) were subjected to SDS gel electrophoresis on a 5-15% polyacrylamide (acrylamide :bisacrylamide r a t i o was 150) gradient slab g e l . Gels were eithe r stained with Coomassie blue (CB) or transferred to n i t r o c e l l u l o s e paper. Transfer papers were i n d i r e c t l y l a b e l l e d either wjijji undiluted 3A6 antibody or 4B2 antibody culture f l u i d and I- l a b e l l e d goat anti-mouse Ig for autoradiography. Lane a- ROS membrane proteins. Lane b-spectrin. Arrows indicate the bands of highest apparent M that were l a b e l l e d with 3A6 and 4B2 antibodies. p r o t e i n s . The b i n d i n g of p o l y c l o n a l r a b b i t anti-human RBC s p e c t r i n t o bovine RBC s p e c t r i n and bovine ROS was determined 125 by the s o l i d - p h a s e RIA u s i n g I - l a b e l l e d P r o t e i n A f o r d e t e c t i o n . As shown i n F i g u r e 17, a l i n e a r i n c r e a s e i n b i n d i n g o f the a n t i - s p e c t r i n antibody was observed when i n c r e a s i n g c o n c e n t r a t i o n s of bovine s p e c t r i n o r ROS membranes were d r i e d onto the assay p l a t e s . T h i s i n d i c a t e d t h a t 125 n e i t h e r the primary antibody nor the I - l a b e l l e d p r o t e i n A was l i m i t i n g under the c o n d i t i o n s o f t h i s assay. For a g i v e n amount o f p r o t e i n , the a n t i - s p e c t r i n antibody was found t o b i n d over 100 times g r e a t e r t o p u r i f i e d s p e c t r i n compared t o t o t a l ROS membrane p r o t e i n . T h i s suggests t h a t the ROS s p e c t r i n makes up l e s s than 1% of the ROS membrane p r o t e i n . The immunoblotting technique was used t o i d e n t i f y the p o l y p e p t i d e s s e r v i n g as a n t i g e n s f o r the a n t i - s p e c t r i n a n t i b o d y . As i l l u s t r a t e d i n F i g u r e 18, the a n t i - s p e c t r i n a n tibody l a b e l l e d w i t h equal i n t e n s i t y the and j£ bands of bovine RBC s p e c t r i n . F a i n t l a b e l l i n g of a band near the top o f the g e l c o u l d a l s o be seen. T h i s may be u n d i s s o c i a t e d cA - ft dimer. In ROS membranes, the a n t i - s p e c t r i n antibody predominantly l a b e l l e d a M r 240,000 p o l y p e p t i d e h aving the same m o b i l i t y as the (X c h a i n o f s p e c t r i n . Two l e s s i n t e n s e bands were observed i n the m o l e c u l a r weight range o f 210,000-220,000. In a d d i t i o n , a band a t M r 150,000 was a l s o l a b e l l e d w i t h the a n t i - s p e c t r i n antibody. T h i s same band i s a l s o l a b e l l e d when ROS membranes are l a b e l l e d w i t h the 4B2 antibody and most l i k e l y r e p r e s e n t s the major p r o t e o l y t i c -74-F i g u r e 17. The b i n d i n g o f r a b b i t anti-human RBC s p e c t r i n a n t i b o d i e s t o p u r i f i e d bovine s p e c t r i n o r ROS membrane p r o t e i n s . (A) V a r i a b l e amounts of RBC s p e c t r i n (squares) and (B) v a r i a b l e amounts of ROS membrane p r o t e i n ( s o l i d c i r c l e s ) or BSA (open c i r c l e s ) were d r i e d onto m i c r o t i t e r p l a t e s and s e q u e n t i a l l y l a b e l l e d w i t h r a b b i t a n t i - s p e c t r i n a n t i b o d i e s and I - l a b e l l e d P r o t e i n A. -75-OL— 240 K 150 K dye a F i g u r e 18. Immunoblots of bovine RBC s p e c t r i n and ROS membrane p r o t e i n s l a b e l l e d w i t h p o l y c l o n a l anti-human s p e c t r i n a n t i b o d i e s . P u r i f i e d bovine RBC s p e c t r i n (6 ;ug) and ROS membrane p r o t e i n s (30 ,ug) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on 6% g e l s and t r a n s f e r r e d t o n i t r o c e l l u l o s e paper. The papers were s e q u e n t i a l l y ^ g b e l l e d w i t h r a b b i t anti-human RBC s p e c t r i n a n t i b o d i e s and I - l a b e l l e d p r o t e i n A. (Gel a) Autoradiograph of bovine RBC s p e c t r i n exposed f o r 5 h. (Gel b) Autoradiograph o f ROS membrane p r o t e i n s exposed f o r 7 days. - 7 6 -fragment o f the M r 240,000 p o l y p e p t i d e . An almost i d e n t i c a l l a b e l l i n g p a t t e r n was o b t a i n e d when p o l y c l o n a l r a b b i t a n t i - b o v i n e RBC s p e c t r i n was used i n s t e a d of p o l y c l o n a l r a b b i t anti-human RBC s p e c t r i n ( F i g . 19). The o n l y d i f f e r e n c e was t h a t a band a t M 150,000 i n the ROS r membranes was not l a b e l l e d w i t h t h i s a n t i - s p e c t r i n antibody. T h i s f u r t h e r supports the s u g g e s t i o n t h a t the M r 150,000 p o l y p e p t i d e may be the degraded fragment o f the M r 240,000 p o l y p e p t i d e . I. E x t r a c t i o n o f 4 B 2 - S p e c i f i c P r o t e i n under Low I o n i c  S t r e n g t h C o n d i t i o n s . S p e c t r i n a l o n g w i t h a c t i n and band 4.1 can be s e l e c t i v e l y e l u t e d from e r y t h r o c y t e membranes i n low i o n i c s t r e n g t h b u f f e r as d e s c r i b e d by Marchesi and S t e e r s (1968) and Furthmayr and Timpl (1970). Approximately 70-90% of t o t a l s p e c t r i n c o u l d be e x t r a c t e d u s i n g low i o n i c s t r e n g t h b u f f e r s ( G r a t z e r , 1982). T h e r e f o r e the methods used f o r e x t r a c t i n g s p e c t r i n from RBC membrane ghosts were t e s t e d f o r t h e i r e f f i c i e n c y i n e x t r a c t i n g the 4 B 2 - s p e c i f i c p r o t e i n from ROS membranes. ROS membranes were d i a l y z e d a g a i n s t 0.3 mM sodium phosphate (pH 8) c o n t a i n i n g e i t h e r 0.3 mM ATP and 50 mM 2-mercaptoethanol or 1 mM EDTA , f o r 24 h a t 4°C and subsequently separated i n t o a p e l l e t and supernatant f r a c t i o n by c e n t r i f u g a t i o n a t 25,000 rpm f o r 1 h. In c o n t r o l s t u d i e s , ROS membranes were d i a l y z e d a g a i n s t 20 mM T r i s - a c e t a t e , pH 7.4. -77--240K B F i g u r e 19. Immunoblots of bovine RBC s p e c t r i n and ROS membrane p r o t e i n s l a b e l l e d with p o l y c l o n a l a n t i - b o v i n e s p e c t r i n a n t i b o d i e s . P u r i f i e d bovine RBC s p e c t r i n (6 jag) and ROS membrane p r o t e i n s (30 oag) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on 6% g e l s and t r a n s f e r r e d t o n i t r o c e l l u l o s e paper. The papers were s e q u e n t i a l l y l a ^ g l l e d w i t h r a b b i t a n t i - b o v i n e RBC s p e c t r i n a n t i b o d i e s and I - l a b e l l e d p r o t e i n A. (Gel A) Autoradiograph o f bovine RBC s p e c t r i n exposed f o r 1 day. (Gel B) Autoradiograph o f ROS membrane p r o t e i n s exposed f o r 5 days. -78-A n a l y s i s o f these f r a c t i o n s by SDS g e l e l e c t r o p h o r e s i s and immunoblotting i n d i c a t e d t h a t no s i g n i f i c a n t q u a n t i t y o f the 4 B 2 - s p e c i f i c p r o t e i n was e x t r a c t e d from the ROS membranes under low i o n i c s t r e n g t h c o n d i t i o n s as compared t o the e x t r a c t i o n by 6M urea. As p r e v i o u s l y i n d i c a t e d ( F i g . 10) a s m a l l q u a n t i t y (5-10%) o f the 4 B 2 - s p e c i f i c p r o t e i n w i t h apparent M r 240,000 was e x t r a c t e d by urea. S i m i l a r l y , under these same c o n d i t i o n s , no e x t r a c t i o n o f ROS 1.2 (M r 220,000) 125 i n t o the supernatant was d e t e c t e d by I - l a b e l l e d Con A b l o t t i n g ( F i g . 20). In c o n t r o l s t u d i e s , 4B2 antibody and Con A l a b e l l i n g o f the p e l l e t f r a c t i o n o f u n t r e a t e d ROS membranes showed the presence of 4 B 2 - s p e c i f i c p r o t e i n and i t s degraded fragments and ROS 1.2, r e s p e c t i v e l y . J . A s s o c i a t i o n o f 4 B 2 - S p e c i f i c P r o t e i n w i t h ROS Membranes. The r e l a t i v e amount of 4 B 2 - s p e c i f i c p r o t e i n t h a t remains w i t h the d i s c membranes a f t e r h y p o t o n i c l y s i s o f ROS was i n v e s t i g a t e d t o determine the i n t e r a c t i o n o f the 4 B 2 - s p e c i f i c p r o t e i n w i t h the d i s c and plasma membranes. The 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 4B2 a n t i g e n between ROS and ROS d i s c membranes was d e t e c t e d by immunoblotting a n a l y s i s and q u a n t i f i e d by c o m p e t i t i v e i n h i b i t i o n assays. When equal amounts of ROS and ROS d i s c membrane p r o t e i n were separated by SDS g e l e l e c t r o p h o r e s i s and l a b e l l e d w i t h 125 4B2 antibody f o l l o w e d by I - l a b e l l e d goat anti-mouse Ig, -79-1 2 3 4 5 1 2 3 4 5 -Top 240K-4B2 Con A F i g u r e 20. E x t r a c t i o n of ROS membranes under low i o n i c s t r e n g t h c o n d i t i o n s . ROS membranes were t r e a t e d w i t h (2) water, (3) 0.3 mM sodium phosphate ,pH 8, c o n t a i n i n g 0.3 mM ATP and 50 mM 2-mercaptoethanol, (4) 0.3 mM sodium phosphate ,pH 8, c o n t a i n i n g 1 mM EDTA, and (5) 6M urea a t 4 C f o r 24 h. The membranes were then sedimented a t 25,000 rpm f o r 1 h. The supernatants (lanes 2-5) and the p e l l e t from the water e x t r a c t i o n (lane 1) were s o l u b i l i z e d i n SDS and s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 6% p o l y a c r y l a m i d e s l a b g e l . The bromophenol b l u e dye marker was allowed t o run o f f t h e g e l i n ord e r t o i n c r e a s e the s e p a r a t i o n o f p r o t e i n s w i t h h i g h apparent m o l e c u l a r weights. A f t e r e l e c t r o p h o r e s i s , the samples 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 n i t r o c e l l u l o s e paper. 5The t r a n s f e r papers were l a b e l l e d e i t h e r d i r e c t l y w i t h I - l a b e l l e d Con A c - r 5 i n d i r e c t l y w i t h u n d i l u t e d 4B2-antibody c u l t u r e f l u i d and I - l a b e l l e d goat anti-mouse Ig. -80-the i n t e n s i t y o f l a b e l l i n g o f the 240 kDa p o l y p e p t i d e i n ROS was g r e a t e r than t h a t i n ROS d i s c membranes ( F i g . 21). The degree of i n h i b i t i o n o f rho-lD4 and 4B2 a n t i b o d i e s b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d ROS membranes by T r i t o n X-100 s o l u b i l i z e d ROS and ROS d i s c membranes i s shown i n F i g u r e 22. Half-maximum i n h i b i t i o n o f an a n t i - r h o d o p s i n antibody (rho-lD4) b i n d i n g o c c u r r e d a t 0.5 jug/mL f o r both ROS and ROS d i s c membranes, but half-maximum i n h i b i t i o n o f 4B2 antibody b i n d i n g o c c u r r e d a t 60 jug/mL f o r ROS and a t 110 jag/mL f o r ROS d i s c membranes. T h i s suggests t h a t the c o n c e n t r a t i o n of 4 B 2 - s p e c i f i c p r o t e i n i n ROS i s about 2 - f o l d g r e a t e r than i n ROS d i s c membranes p r e p a r a t i o n . K. Immunocytochemical L a b e l l i n g o f Rod C e l l s w i t h the 4B2  Antibody and Immunoqold-Dextran Markers. L o c a l i z a t i o n o f the 4 B 2 - s p e c i f i c p r o t e i n i n ROS was determined by s e q u e n t i a l l y l a b e l l i n g L o w i c r y l t h i n s e c t i o n s o f bovine r e t i n a t i s s u e w i t h the 4B2 antibody and goat a n t i -mouse Ig - g o l d - d e x t r a n conjugates. As shown i n F i g u r e 23, the g o l d p a r t i c l e s were p r e f e r e n t i a l l y d i s t r i b u t e d a l o n g the p e r i p h e r y o f the ou t e r segments where the rims of the d i s c s are a d j a c e n t t o the plasma membrane. Gold p a r t i c l e s were o f t e n observed t o extend inward from the plasma membrane up t o 50 nm. In the b a s a l p o r t i o n o f the rod o u t e r segment, g o l d p a r t i c l e s were a l s o observed t o l i n e up i n the r e g i o n where the rims of the d i s c s are ad j a c e n t t o the c i l i a r y s p i n e extending i n t o the ou t e r segment from the c o n n e c t i n g c i l i u m -81-2 4 0 K - mg§ 2 2 0 K -3 4 K -4B2 R D ConA F i g u r e 21. Immunoblots of ROS and ROS d i s c membrane p r o t e i n s l a b e l l e d w i t h 4B2 and Con A. ROS and ROS d i s c membrane p r o t e i n s (20 jug/well) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 6% p o l y a c r y l a m i d e s l a b g e l and t r a n s f e r r e d t o n i t r o c e l l u l o s e paper. The papers were s e q u e n t i a l l y . ^ ^Labelled with u n d i l u t e d 4B2 a n t i b o d y c u l t u r e f l u i d and I - l a b e l l e d goat anti-mouse Ig o r d i r e c t l y l a b e l l e d w i t h I - l a b e l l e d Con A. (Lane R) ROS membrane p r o t e i n s . (Lane D) d i s c membrane p r o t e i n s . -82-0.1 1 10 100 1000 P r o t e i n C o n c e n t r a t i o n ^ t g / m l ) 0.1 1 10 100 1000 P r o t e i n C o n c e n t r a t i o n ( /Ug /ml) F i g u r e 22. I n h i b i t i o n o f 4B2 antibody (A) and rho-lD4 antibody (B) 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 ( s o l i d c i r c l e s ) and ROS d i s c membranes ( s o l i d squares) u s i n g the s o l i d - p h a s e i n d i r e c t radioimmune c o m p e t i t i o n assay. 1D4 4B2 F i g u r e 23. Tr a n s m i s s i o n e l e c t r o n micrographs o f ROS l a b e l l e d w i t h immunogold-dextran markers. T h i n s e c t i o n s o f L o w i c r y l -embedded ROS of bovine r e t i n a were s e q u e n t i a l l y l a b e l l e d w i t h e i t h e r rho-lD4 o r 4B2 antibody and goat anti-mouse I g - g o l d dextran p a r t i c l e s (diameter of g o l d , 10-15 nm). For 4B2 antibody l a b e l l i n g , the g o l d p a r t i c l e s a re p r i m a r i l y d i s t r i b u t e d a l o n g the p e r i p h e r y of the ROS where the rims o f the d i s c s come i n c l o s e p r o x i m i t y t o the c i l i a r y s p i n e (CS) and plasma membrane ( m a g n i f i c a t i o n 25,000 X). F o r rho-lD4 antibody l a b e l l i n g , the g o l d p a r t i c l e s a r e d i s t r i b u t e d u n i f o r m l y throughout the ROS ( m a g n i f i c a t i o n 22,000 X). -84-(Hicks and Molday, 1985). A few g o l d p a r t i c l e s can be found i n the c e n t r a l area o f the ROS. I t i s not c l e a r i f t h i s r e p r e s e n t s a low c o n c e n t r a t i o n o f the 4B2-binding p r o t e i n i n t h i s r e g i o n o r, a l t e r n a t i v e l y , r e s i d u a l n o n s p e c i f i c b i n d i n g o f the 4B2 antibody, an IgM immunoglobulin. In c o n t r a s t , o u t e r segments l a b e l l e d w i t h a n t i - r h o d o p s i n monoclonal a n t i b o d i e s were densely and u n i f o r m l y d i s t r i b u t e d over the e n t i r e ROS ( F i g . 23) as p r e v i o u s l y shown by H i c k s and Molday (1986). No l a b e l l i n g was observed when a n o n r e a c t i v e monoclonal antibody was used i n the f i r s t l a b e l l i n g s t e p . L. Detergent S o l u b i l i z a t i o n o f ROS Membrane P r o t e i n s . The s o l u b i l i t y of ROS membrane p r o t e i n s i n T r i t o n X-100 was i n v e s t i g a t e d because T r i t o n X-100 has been used t o o b t a i n the r e d c e l l c y t o s k e l e t o n from RBC ghosts membrane p r e p a r a t i o n (Yu e t a l . , 1973). T h i s m i l d n o n i o n i c d e t e r g e n t s e l e c t i v e l y s o l u b i l i z e d a l l the membrane g l y c o p r o t e i n s ( p r i n c i p a l l y bands 3 and PAS 1-3). The d e t e r g e n t - i n s o l u b l e f r a c t i o n c o n t a i n s n o n g l y c o s y l a t e d p o l y p e p t i d e s and c y t o s k e l e t a l p r o t e i n s ( p r i n c i p a l l y s p e c t r i n , a c t i n , and band 4.1). Dark adapted ROS membrane p r o t e i n s were t r e a t e d w i t h 1% T r i t o n X-100 and subsequently s e p a r a t e d i n t o a supernatant and p e l l e t f r a c t i o n by c e n t r i f u g a t i o n a t 15,000 rpm f o r 30 min. The supernatant changed from r e d t o y e l l o w i n c o l o r upon exposure t o l i g h t due t o b l e a c h i n g o f photopigment rhodopsin, w h i l e the p e l l e t remained white. -85-P r o t e i n assays of both f r a c t i o n s i n d i c a t e d t h a t approximately 95% o f the t o t a l ROS membrane p r o t e i n was s o l u b i l i z e d by T r i t o n X-100 and the remaining 5% o f the t o t a l ROS membrane p r o t e i n c o n s t i t u t e d the p e l l e t f r a c t i o n . P r o t e i n components i n both f r a c t i o n s were analyzed by SDS g e l e l e c t r o p h o r e s i s and immunoblotting. As shown i n F i g u r e 24, 125 Coomassie b l u e s t a i n i n g and I - l a b e l l e d Con A l a b e l l i n g o f p o l y p e p t i d e s from both f r a c t i o n s i n d i c a t e d r h o d o p s i n w i t h apparent M r 34,000 and i t s aggregated forms w i t h apparent M^ 68K, 102K, 136K, and 170K are the major p o l y p e p t i d e s i n the supernatant f r a c t i o n . ROS 1.2 w i t h apparent M r 220,000 r e p r e s e n t s a minor p r o t e i n component i n t h i s f r a c t i o n . In c o n t r a s t Coomassie b l u e s t a i n i n g o f p o l y p e p t i d e s i n the p e l l e t f r a c t i o n showed a s e r i e s o f e q u a l l y i n t e n s e bands w i t h M r ranges from 34,000 t o 220,000. A l s o the i n t e n s i t y o f 125 l a b e l l i n g o f rhodopsin and ROS 1.2 by I - l a b e l l e d Con A i s about the same i n the p e l l e t f r a c t i o n . These f r a c t i o n s were f u r t h e r a n alyzed by immuno-l a b e l l i n g w i t h 4B2 antibody and a monoclonal antibody (2B6) a g a i n s t p e r i p h e r i n , a r i m - s p e c i f i c p r o t e i n p r e v i o u s l y d e s c r i b e d by Molday e t a l . (1987). The 4 B 2 - s p e c i f i c p r o t e i n and i t s major degraded fragment were found i n the supernatant f r a c t i o n as shown by the l a b e l l i n g o f a M r 240,000 p o l y p e p t i d e and a M r 150,000 p o l y p e p t i d e w i t h the 4B2 antibody. However, o n l y the undegraded 4 B 2 - s p e c i f i c p o l y p e p t i d e was p r e s e n t i n the p e l l e t f r a c t i o n . The 2B6 antibody i n t e n s e l y l a b e l l e d a band a t about 34,000, the -86-a b a b a b a b CB ConA 4B2 2B6 F i g u r e 24. Immunoblots of T r i t o n X-100 e x t r a c t i o n o f ROS membranes. ROS membranes (1 mg protein/mL) prepared from f r e s h l y - d i s s e c t e d r e t i n a s were s o l u b i l i z e d w i t h 1% T r i t o n X-100 f o r 24 h a t 4 C. ROS membranes were then sedimented a t 15,000 rpm f o r 30 min. Supernatant (45 ;ug p r o t e i n / w e l l ) and p e l l e t (5 jug p r o t e i n / w e l l ) were s u b j e c t e d t o SDS e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e s l a b g e l ( l a n e s a and b, r e s p e c t i v e l y ) . A f t e r 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 i t h e r s t a i n e d w i t h Coomassie b l u e (CB) or 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 n i t r o c e l l u l o s e paggr. T r a n s f e r papers were e i t h e r d i r e c t l y l a b e l l e d w i t h I -l a b e l l e d C o n 1 2 A or i n d i r e c t l y l a b e l l e d w i t h 4B2 o r 2B6 antibody and I - l a b e l l e d goat anti-mouse I g . -87-approximate M r o f p e r i p h e r i n (Molday e t a l . , 1987), i n the supernatant f r a c t i o n . A v e r y f a i n t band was l a b e l l e d i n the p e l l e t f r a c t i o n ( F i g . 24). These r e s u l t s suggested t h a t p e r i p h e r i n and rhodopsin are almost completely s o l u b i l i z e d by T r i t o n X-100 w h i l e a s e t of p o l y p e p t i d e s i n c l u d i n g t h e 4B2-s p e c i f i c p r o t e i n and ROS 1.2 are o n l y p a r t i a l l y s o l u b l e . Thus they are p r e s e n t i n both the supernatant and p e l l e t f r a c t i o n . The s o l u b i l i t y o f ROS membrane p r o t e i n s i n z w i t t e r i o n i c and i o n i c d e t e r g e n t s was compared t o t h a t o f T r i t o n X-100 i n o r d e r t o determine whether the s e l e c t i v e s o l u b i l i z a t i o n o f ROS membrane p r o t e i n s ( p r i n c i p a l l y rhodopsin) c o u l d be f u r t h e r improved. The p e l l e t f r a c t i o n s o b t a i n e d from c e n t r i f u g i n g the dete r g e n t s o l u b i l i z e d ROS membranes a t 15,000 rpm f o r 3 0 min were analyzed by SDS g e l e l e c t r o p h o r e s i s . As shown i n F i g . 25, Coomassie b l u e s t a i n i n g o f p o l y p e p t i d e s i n the p e l l e t f r a c t i o n o b t a i n e d from e i t h e r CHAPS, or o c t y l g l u c o s i d e , o r c h o l a t e s o l u b i l i z a t i o n o f ROS membranes showed a s e r i e s o f bands w i t h M r ranges from 34,000 t o 220,000. T h i s p o l y p e p t i d e s t a i n i n g p a t t e r n i s a l s o observed i n the p e l l e t f r a c t i o n o b t a i n e d from T r i t o n X-100 s o l u b i l i z a t i o n o f ROS membranes and i n the p e l l e t f r a c t i o n o f n o n - s o l u b i l i z e d ROS membranes. However, the i n t e n s i t y o f the rhodopsin band a t apparent M r 34,000 was r e l a t i v e l y l e s s i n the T r i t o n X-100 p e l l e t than the o t h e r p e l l e t f r a c t i o n s w i t h the e x c e p t i o n of the SDS p e l l e t f r a c t i o n . In the case of SDS s o l u b i l i z a t i o n o f ROS membrane p r o t e i n s , no band i n the -88-F i g u r e 25. SDS g e l e l e c t r o p h o r e s i s o f d e t e r g e n t - t r e a t e d ROS p e l l e t . ROS membranes (5 mg p r o t e i n ) prepared from f r o z e n r e t i n a s were s o l u b i l i z e d with (1) 1% T r i t o n X-100, (2) 1% CHAPS, (3) 1% o c t y l g l u c o s i d e , (4) 1% sodium c h o l a t e , and (5) 1% SDS f o r 24 h a t 2 3 C. ROS membranes were then sedimented a t 15,000 rpm f o r 30 min. The p e l l e t s ( l a n e s 1-5 r e s p e c t i v e l y ) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e 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 , the g e l was s t a i n e d w i t h Coomassie b l u e . Lane 6- p e l l e t o b t a i n e d from u n t r e a t e d ROS membranes. -89-p e l l e t f r a c t i o n was observed w i t h Coomassie b l u e s t a i n i n g . These r e s u l t s i n d i c a t e t h a t SDS does not s o l u b i l i z e ROS membrane p r o t e i n s s e l e c t i v e l y , and T r i t o n X-100 i s b e t t e r than the o t h e r d e t e r g e n t s t e s t e d f o r s e l e c t i v e s o l u b i l i z a t i o n o f ROS membrane p r o t e i n s ( p r i n c i p a l l y r h o d o p s i n ) . M. E l e c t r o n Microscopy o f T r i t o n X-100 E x t r a c t e d ROS P e l l e t . E l e c t r o n microscopy of the RBC c y t o s k e l e t a l s t r u c t u r e t h a t remains a f t e r the ghost membrane i s e x t r a c t e d w i t h T r i t o n X-100 showed a fi l a m e n t o u s meshwork (Yu e t a l . , 1973; H a i n f e l d and Steck, 1977). R e c e n t l y , t h i s same approach was attempted u s i n g ROS membranes t o determine whether the T r i t o n - e x t r a c t e d p e l l e t c o n s i s t e d o f a f i l a m e n t o u s meshwork and c o u l d be d e t e c t e d by e l e c t r o n microscopy. T r i t o n -e x t r a c t e d ROS p e l l e t was f i x e d i n g l u t a r a l d e h y d e and adsorbed onto p o l y l y s i n e - c o a t e d formvar g r i d s . The p e l l e t was then n e g a t i v e l y s t a i n e d and viewed under an e l e c t r o n microscope. At low m a g n i f i c a t i o n , the e l e c t r o n micrographs from two sep a r a t e experiments showed a network of f i n e f i l a m e n t o u s m a t e r i a l . At h i g h m a g n i f i c a t i o n , the network appeared t o c o n s i s t o f i n t e r c o n n e c t i n g f i l a m e n t s o f v a r i a b l e l e n g t h and a sheet o f m a t e r i a l surrounding the f i l a m e n t s ( F i g . 26). The fi l a m e n t o u s m a t e r i a l was not observed when no sample was a p p l i e d t o g r i d s . T h i s r e s u l t suggests t h a t T r i t o n - e x t r a c t e d ROS p e l l e t may c o n t a i n f i b r o u s p r o t e i n s . -90-F i g u r e 26. Negative s t a i n i n g o f T r i t o n X-100 ROS p e l l e t . ROS membranes prepared from f r e s h l y - d i s s e c t e d r e t i n a s were s o l u b i l i z e d w i t h 1% T r i t o n X-100 f o r 24 h a t 4°C. ROS membranes were then sedimented a t 15,000 rpm f o r 30 min. The p e l l e t was f i x e d w i t h 0.8% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2. P e l l e t was washed t h r e e times by c e n t r i f u g a t i o n a t 15,000 rpm f o r 30 min and resuspended i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2. Sample s o l u t i o n was p l a c e d on p o l y l y s i n e - c o a t e d formvar g r i d s . The g r i d s were washed e x t e n s i v e l y i n water and s t a i n e d w i t h 1% sodium phosphotungstate, pH 7.2. E l e c t r o n micrographs o f n e g a t i v e l y s t a i n e d T r i t o n X-100 ROS p e l l e t from two s e p a r a t e experiments (A) and (C). (B) and (D) are h i g h e r m a g n i f i c a t i o n o f (A) and (C) r e s p e c t i v e l y . Bars r e p r e s e n t 2, 0.5, 2, 0.2 jam f o r (A)-(D) r e s p e c t i v e l y . - g i -l l . A ntibody Probes f o r I d e n t i f y i n g P r o t e i n Components  of ROS C y t o s k e l e t o n . In o r d e r t o i d e n t i f y the p r o t e i n components which comprise the ROS c y t o s k e l e t o n as observed by e l e c t r o n microscopy, s e v e r a l monoclonal and p o l y c l o n a l a n t i b o d i e s were generated a g a i n s t a c t i n and a n k y r i n (two components o f the RBC c y t o s k e l e t o n ) , u r e a - e x t r a c t a b l e and T r i t o n X-100 i n s o l u b l e ROS membrane p r o t e i n s . (1) A n t i - A c t i n Monoclonal Antibody The presence of a c t i n i n ROS membrane p r e p a r a t i o n s was d e t e c t e d by s o l i d - p h a s e RIA. The e f f e c t o f d i l u t i o n o f an a n t i - a c t i n monoclonal antibody, r e f e r r e d t o as 2C2 (MacKenzie and Molday, unpublished r e s u l t ) , on the b i n d i n g o f the antibody t o T r i t o n X-100 s o l u b i l i z e d 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 u r e 27. The 2C2 antibody was a c t i v e a g a i n s t 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 h i g h e r degree of b i n d i n g o f 2C2 antibody t o a c t i n than t o 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 , rho-lD4 and 4B2 which are s p e c i f i c f o r rhodopsin and a M r 240,000 p o l y p e p t i d e r e s p e c t i v e l y were i n a c t i v e a g a i n s t a c t i n . (2) P o l y c l o n a l A n t i - A n k y r i n A n t i b o d i e s The b i n d i n g s p e c i f i c i t y o f p o l y c l o n a l r a b b i t a n t i - b o v i n e RBC a n k y r i n a n t i b o d i e s t o bovine RBC membrane ghosts - 9 2 -1 4 16 64 256 1024 4096 Reciprocal Dilution F i g u r e 27. B i n d i n g o f an a n t i - a c t i n monoclonal antibody t o ROS membranes and a c t i n . T i t r a t i o n o f 2C2, 4B2, and rho-lD4 hybridoma 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 t r e a t e d r a b b i t muscle a c t i n ( s o l i d c i r c l e s ) and 2C2 hybridoma c e l l c u l t u r e supernatant a g a i n s t T r i t o n X-100 s o l u b i l i z e d ROS membranes ( s o l i d s q u a r e s ) . 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 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 e l l c u l t u r e supernatant, r i n s e d , and subsequently t r e a t e d w i t h I - l a b e l l e d goat anti-mouse Ig. -93-p r e p a r a t i o n was determined by immune-blotting. As shown i n F i g u r e 28 (Gel C), the a n t i - a n k y r i n a n t i b o d i e s i n t e n s e l y l a b e l l e d two p o l y p e p t i d e s w i t h apparent M r 240,000 and 210,000. Two f a i n t l y l a b e l l e d p o l y p e p t i d e s w i t h apparent M r 220,000 and 180,000 c o u l d a l s o be seen. In c o n t r a s t p o l y c l o n a l r a b b i t a n t i - b o v i n e RBC s p e c t r i n a n t i b o d i e s l a b e l l e d two p o l y p e p t i d e s w i t h apparent M r 240,000 and 220,000 (Gel B). These r e s u l t s i n d i c a t e p o l y c l o n a l a n t i -a n k y r i n and a n t i - s p e c t r i n a n t i b o d i e s have d i f f e r e n t b i n d i n g s p e c i f i c i t y . The p o l y c l o n a l a n t i - s p e c t r i n a n t i b o d i e s have been shown t o be s p e c i f i c f o r the C?( and /£> c h a i n s o f p u r i f i e d s p e c t r i n ( F i g . 18). However, the p o l y c l o n a l a n t i -a n k y r i n a n t i b o d i e s have not been t e s t e d a g a i n s t p u r i f i e d a n k y r i n . When the p o l y c l o n a l a n t i - a n k y r i n a n t i b o d i e s were t e s t e d a g a i n s t ROS membrane p r o t e i n s by immunoblotting, no bands were l a b e l l e d even a f t e r prolonged exposure. (3) C h a r a c t e r i z a t i o n o f 1D1 Monoclonal Antibody The 1D1 hybridoma c e l l l i n e was generated from a f u s i o n of NS-1 mouse myeloma c e l l s w i t h s p l e e n c e l l s from a mouse immunized w i t h urea e x t r a c t a b l e ROS membrane p r o t e i n s . T h i s c e l l l i n e s e c r e t e d a n t i b o d i e s s p e c i f i c f o r ROS membrane p r e p a r a t i o n s as determined by s o l i d - p h a s e RIA (not shown). The b i n d i n g s p e c i f i c i t y o f 1D1 antibody f o r ROS p o l y p e p t i d e s was determined by t r e a t i n g p o l y p e p t i d e s t r a n s f e r r e d t o n i t r o c e l l u l o s e paper i n d i r e c t l y w i t h 1D1 antibody f o l l o w e d by goat anti-mouse I g - a l k a l i n e phosphatase -94-F i g u r e 28. Immunoblots of bovine RBC membrane ghosts l a b e l l e d w i t h p o l y c l o n a l a n t i - b o v i n e a n k y r i n a n t i b o d i e s . RBC ghosts (20 jug p r o t e i n / w e l l ) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e s l a b g e l . A f t e r 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 i t h e r s t a i n e d w i t h Coomassie b l u e ( g e l A) or 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 n i t r o c e l l u l o s e paper. T r a n s f e r papers were s e q u e n t i a l l y l a b e l l l e d w i t h e i t h e r p o l y c l o n a l a n t i - b o v i n e s p e c t r i n ( g e l B) (j>r5 p o l y c l o n a l a n t i - b o v i n e a n k y r i n ( g e l C) a n t i b o d i e s and I - l a b e l l e d P r o t e i n A. -95-conjugates. Immunostaining of the paper showed t h r e e bands a t 100,000, 60,000, and 40,000 ( F i g . 29). These t h r e e p o l y p e p t i d e s have not been c h a r a c t e r i z e d p r e v i o u s l y . The M r 60,000 and 40,000 p o l y p e p t i d e s c o u l d be e i t h e r degraded fragments o f the M r 100,000 p o l y p e p t i d e o r immunologically r e l a t e d t o i t . The 1D1 antibody l a b e l l e d these p o l y p e p t i d e s weakly by both immunostaining and immunoblotting t e c h n i q u e s . Due t o i t s low a f f i n i t y f o r ROS membrane p r o t e i n s , the 1D1 anti b o d y was not used t o d e t e c t c r o s s - r e a c t i n g p r o t e i n i n the RBC system. (4) C h a r a c t e r i z a t i o n of 1H5 and 2A4 Monoclonal A n t i b o d i e s The 1H5 and 2A4 hybridoma c e l l l i n e were generated from a f u s i o n of NS-1 mouse myeloma c e l l s w i t h s p l e e n c e l l s from a mouse immunized wi t h T r i t o n X-100 i n s o l u b l e ROS membrane p r o t e i n s (Jarausch and Molday, unpublished r e s u l t s ) . The b i n d i n g s p e c i f i c i t y o f these a n t i b o d i e s f o r ROS p o l y p e p t i d e s was determined by immunoblotting. As shown i n F i g u r e 30, 1H5 antibody i n t e n s e l y l a b e l l e d a M^ 240,000 p o l y p e p t i d e and a M r 150,000 p o l y p e p t i d e i n the supernatant f r a c t i o n o b t a i n e d from the s o l u b i l i z a t i o n o f ROS membrane p r o t e i n s by T r i t o n X-100 and a M r 240,000 p o l y p e p t i d e i n the p e l l e t f r a c t i o n . T h i s l a b e l l i n g p a t t e r n i s almost i d e n t i c a l t o t h a t observed f o r the 4B2 antibody l a b e l l i n g o f these f r a c t i o n s ( F i g . 24). The 2A4 antibody l a b e l l e d s e v e r a l p o l y p e p t i d e s w i t h M r ranges from 68,000 t o 270,000 i n the supernatant f r a c t i o n , but o n l y -96-F i g u r e 29. Immunoblot of bovine ROS membrane p r o t e i n s l a b e l l e d w i t h 1D1 monoclonal antibody. SDS s o l u b i l i z e d ROS membrane p r o t e i n s (20 ;ug/well) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e s l a b g e l . G e l s were e i t h e r s t a i n e d w i t h Coomassie bl u e ( g e l a) or t r a n s f e r r e d t o n i t r o c e l l u l o s e paper. T r a n s f e r paper was s e q u e n t i a l l y l a b e l l e d w i t h once cloned 1D1 c e l l c u l t u r e supernatant and goat anti-mouse Ig conjugated t o a l k a l i n e phosphatase. T r a n s f e r paper was then incubated w i t h s u b s t r a t e 5-bromo-4-chlo r o - 3 - i n d o x y l - p h o s p h a t e and s t a i n e d by n i t r o b l u e t e t r a z o l i u m s a l t ( g e l b ) . -97-two p o l y p e p t i d e s w i t h apparent M R 270,000 and 240,000 were l a b e l l e d i n the p e l l e t f r a c t i o n ( F i g . 30). S i n c e the 1H5 antibody appears t o be s i m i l a r t o the 4B2 antibody i n b i n d i n g s p e c i f i c i t y , i t was t e s t e d f o r immunological c r o s s - r e a c t i v i t y w i t h RBC p r o t e i n s . Immunoblots shown i n F i g . 31 i n d i c a t e d t h a t 1H5 c r o s s - r e a c t s somewhat b e t t e r w i t h the than the ft band o f RBC s p e c t r i n , whereas 4B2 o n l y c r o s s - r e a c t s w i t h t h e band under the same c o n d i t i o n s . T h i s suggests t h a t 1H5 antibody r e c o g n i z e s a d i f f e r e n t e p i t o p e on the oC s u b u n i t o f RBC s p e c t r i n than the 4B2 antibody and t h i s e p i t o p e shares homology w i t h an e p i t o p e on the ft s u b u n i t . As was p r e v i o u l y shown i n F i g u r e s 24 and 30, both a n t i b o d i e s l a b e l l e d a M r 240,000 p o l y p e p t i d e i n the p e l l e t f r a c t i o n . -98-F i g u r e 30. Immunoblots of T r i t o n X-100-treated ROS membranes l a b e l l e d w i t h 1H5 and 2A4 monoclonal a n t i b o d i e s . ROS membranes were s o l u b i l i z e d w i t h 1% T r i t o n X-100 f o r 24 h a t 4 C. ROS membranes were then sedimented a t 15,000 rpm f o r 30 min. Supernatant (45 >ug p r o t e i n / w e l l ) and p e l l e t (5 jag p r o t e i n / w e l l ) were s u b j e c t e d t o SDS e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e s l a b g e l . A f t e r 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 i t h e r s t a i n e d w i t h Coomassie b l u e (CB) or 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 n i t r o c e l l u l o s e paper. T r a n s f e r papers w e r e ^ n d i r e c t l y l a b e l l e d w i t h e i t h e r 1H5 or 2A4 antibody and I - l a b e l l e d goat anti-mouse Ig f o r autoradiography. (Lane a) Supernatant. (Lane b) P e l l e t . -99-F i g u r e 31. Immunoblots of bovine RBC membrane ghosts and T r i t o n X-100 ROS p e l l e t l a b e l l e d w i t h 1H5 and 4B2. RBC membrane ghosts (20 jug p r o t e i n / w e l l ) and T r i t o n X-100 ROS p e l l e t (5 jug p r o t e i n / w e l l ) were s u b j e c t e d t o SDS g e l e l e c t r o p h o r e s i s on a 8% p o l y a c r y l a m i d e s l a b g e l . A f t e r 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 i t h e r s t a i n e d w i t h Coomassie b l u e (CB) or 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 n i t r o c e l l u l o s e paper. T r a n s f e r papers w e r e 1 2 ; L n d i r e c t r l y l a b e l l e d w i t h e i t h e r 1H5 or 4B2 antibody and I - l a b e l l e d goat anti-mouse Ig f o r autoradiography. (Lane a) RBC membrane ghosts. (Lane b) T r i t o n X-100 ROS p e l l e t . -100-DISCUSSION A. D i f f e r e n c e s between 4 B 2 - S p e c i f i c P r o t e i n and ROS 1.2. R e s u l t s o f t h i s study i n d i c a t e t h a t 4 B 2 - s p e c i f i c p r o t e i n of apparent M r 240,000 i s d i s t i n c t from ROS 1.2 based on t h e i r e l e c t r o p h o r e t i c m o b i l i t i e s on SDS-polyacrylamide g e l , s e n s i t i v i t y t o endogenous p r o t e a s e s , and e x t r a c t a b i l i t y by u r e a . The 240K p o l y p e p t i d e s p e c i f i c a l l y b i n d s the 4B2 monoclonal antibody, but not Con A, comigrates w i t h the oC c h a i n of bovine RBC s p e c t r i n on 6% p o l y a c r y l a m i d e g e l s , i s h i g h l y s u s c e p t i b l e t o d e g r a d a t i o n by endogenous p r o t e a s e s , and can be e x t r a c t e d from the membrane, although t o a l i m i t e d degree, w i t h urea i n the absence of d e t e r g e n t . In c o n t r a s t , ROS 1.2 b i n d s Con A but not the 4B2 antibody, m i g r a t e s w i t h a s l i g h t l y g r e a t e r m o b i l i t y i n d i c a t i v e of an apparent M r 210-22OK, i s not r e a d i l y degraded by endogenous p r o t e a s e s , and l i k e r hodopsin, i s not e x t r a c t e d by urea, but r e q u i r e s d e t e r g e n t f o r s o l u b i l i z a t i o n . The 240K p o l y p e p t i d e i s not simply a contaminant of the ROS p r e p a r a t i o n s i n c e immunocytochemical l a b e l l i n g s t u d i e s u s i n g immunogold-dextran markers c l e a r l y show t h a t t h i s p r o t e i n i s l o c a l i z e d w i t h i n the ROS o r g a n e l l e . I t i s important t o show the d i f f e r e n c e s between the 4B2-s p e c i f i c p r o t e i n and ROS 1.2, because ROS 1.2 has been proposed t o be i n v o l v e d i n d i s c - d i s c i n t e r a c t i o n (Roof and Heuser, 1982; C o r l e s s e t a l . , 1987) whereas the 4 B 2 - s p e c i f i c p r o t e i n has not been c h a r a c t e r i z e d p r e v i o u s l y . -101-B. Immunological C r o s s - R e a c t i v i t y o f 4B2 and 3A6 Monoclonal  A n t i b o d i e s w i t h RBC S p e c t r i n and P o l y c l o n a l  A n t i - S p e c t r i n w i t h ROS P r o t e i n s . The 4B2-binding p r o t e i n o f bovine ROS appears t o be r e l a t e d t o s p e c t r i n o f RBC. The apparent m o l e c u l a r weight of the 4B2-binding p o l y p e p t i d e i s s i m i l a r t o t h a t o f the o( s u b u n i t o f bovine RBC s p e c t r i n . More i m p o r t a n t l y , the 4B2 monoclonal antibody c r o s s - r e a c t s w i t h p u r i f i e d bovine RBC s p e c t r i n and s p e c i f i c a l l y w i t h i t s s u b u n i t as shown by RIA and immunoblotting s t u d i e s . I t a l s o c r o s s - r e a c t s w i t h b r a i n microsomal membranes which are known t o c o n t a i n a s p e c t r i n -r e l a t e d p r o t e i n c a l l e d f o d r i n (Levine and W i l l a r d , 1981). L i k e the 4B2-binding p r o t e i n , f o d r i n i s known t o be h i g h l y s u s c e p t i b l e t o d e g r a d a t i o n by p r o t e a s e s d u r i n g i t s p u r i f i c a t i o n . The major p r o t e o l y t i c fragments of f o d r i n have apparent M r of 150,000 and 160,000 (Burridge e t a l . , 1982) which are comparable i n s i z e t o the major p r o t e o l y t i c fragment o f 4B2-binding p r o t e i n o f apparent M r 150,000 . The i d e n t i t y o f the endogenous p r o t e a s e r e s p o n s i b l e f o r the d e g r a d a t i o n o f f o d r i n i s not known, but i t has been 2+ suggested t h a t a Ca - a c t i v a t e d p r o t e a s e i s i n v o l v e d 2+ (Burridge e t al.,1982). A f a m i l y of Ca -dependent t h i o l -p r o t e a s e s known c o l l e c t i v e l y as c a l p a i n I and I I (reviewed by Murachi, 1983) have been found t o be s p e c i f i c f o r f o d r i n as w e l l as s p e c t r i n (Siman e t a l . , 1984). In neurons c a l p a i n -mediated d e g r a d a t i o n of f o d r i n has been suggested t o p l a y a -102-r o l e i n c o n t r o l l i n g c e l l shape and the d i s p o s i t i o n of c e l l s u r f a c e p r o t e i n s on the b a s i s of the r e s u l t s i n d i c a t i n g both f o d r i n (Levine and W i l l a r d , 1981) and c a l p a i n I (Siman e t a l . , 1983) are p a r t o f the submembraneous c y t o s k e l e t o n and the f a c t t h a t s p e c t r i n s e r v e s such a f u n c t i o n i n e r y t h r o c y t e s . Whether o r not c a l p a i n e x i s t s i n ROS remains t o be determined. L i k e c a l p a i n w i t h s u b u n i t s o f apparent M r 72-82,000 and 25-30,000, the endogenous p r o t e a s e i n ROS appears t o be o f s m a l l m o l e c u l a r weight s i n c e s i z e - e x c l u s i o n chromatography of S D S - s o l u b i l i z e d ROS membrane p r o t e i n s on a Sepharose CL-2B column was shown t o be e f f e c t i v e i n s e p a r a t i n g the 4 B 2 - s p e c i f i c p r o t e i n and i t s degraded fragments from the p r o t e a s e . However, i t i s a l s o p o s s i b l e t h a t the p r o t e a s e was denatured by SDS treatment. Future s t u d i e s are needed t o c l a r i f y t h i s p o i n t . P r e l i m i n a r y s t u d i e s 2+ i n d i c a t e t h a t EGTA, a Ca c h e l a t i n g agent, and l e u p e p t m , an i n h i b i t o r o f c a l p a i n (Siman e t a l . , 1984), are i n e f f e c t i v e i n p r e v e n t i n g the p r o t e o l y t i c d e g r a d a t i o n of 4B2-binding p r o t e i n s i n c e immunoblots of 4B2 l a b e l l i n g of ROS membrane p r o t e i n s prepared i n the absence o r presence o f these i n h i b i t o r s showed a band a t M r=150,000, the major p r o t e o l y t i c fragment o f the 4B2-binding p r o t e i n (not shown). However, the e f f e c t i v e n e s s of these two i n h i b i t o r s c o u l d be b e t t e r a s s e s s e d i f the p u r i f i e d 4B2-binding p r o t e i n and i t s s p e c i f i c p r o t e a s e i n ROS were used, because the 4B2-binding p r o t e i n (240 kDa) c o u l d have been degraded b e f o r e ROS membrane -103-p r e p a r a t i o n . The 3A6-binding p r o t e i n has a s i m i l a r e l e c t r o p h o r e t i c m o b i l i t y on 6% SDS-polyacrylamide g e l s as the 4B2-binding p r o t e i n . In o r d e r t o show t h e i r d i f f e r e n c e i n e l e c t r o p h o r e t i c m o b i l i t y , a 5-15% g r a d i e n t p o l y a c r y l a m i d e g e l w i t h 25% o f normal c r o s s l i n k e r c o n c e n t r a t i o n was prepared f o r SDS g e l e l e c t r o p h o r e s i s . In t h i s g e l system, the 4B2-binding p r o t e i n has an apparent Mr=260,000 and the 3A6-binding p r o t e i n has an apparent M r=240,000. L i k e t h e 4B2-binding p r o t e i n , the 3A6-binding p r o t e i n i s a l s o h i g h l y s u s c e p t i b l e t o d e g r a d a t i o n by endogenous p r o t e a s e s . More i m p o r t a n t l y , the 3A6 monoclonal antibody c r o s s - r e a c t s s p e c i f i c a l l y w i t h the ft c h a i n o f RBC s p e c t r i n as determined by immunoblotting. Although monoclonal a n t i b o d i e s are more s u i t a b l e than p o l y c l o n a l a n t i b o d i e s as s p e c i f i c probes of p r o t e i n f i n e s t r u c t u r e and f u n c t i o n , they are l e s s u s e f u l i n d e t e r m i n i n g the e x t e n t of homology between two p r o t e i n s , because they b i n d t o s i n g l e a n t i g e n i c determinants which comprise o n l y a few amino a c i d r e s i d u e s . In t h i s study, i t was shown t h a t p o l y c l o n a l anti-RBC s p e c t r i n a n t i b o d i e s c r o s s - r e a c t e d w i t h p o l y p e p t i d e s o f ROS having m o l e c u l a r weights e q u i v a l e n t t o the cX and c h a i n s of RBC s p e c t r i n . The oC s u b u n i t of M r 240,000 was the major u n i t of ROS s p e c t r i n d e t e c t e d by these p o l y c l o n a l a n t i b o d i e s i n t h i s study. S e v e r a l bands i n the m o l e c u l a r weight range of 215,000-220,000 t h a t l a b e l l e d -104-w i t h p o l y c l o n a l a n t i - s p e c t r i n antibody may r e p r e s e n t v a r i a n t s o f the /& s u b u n i t of s p e c t r i n (Nelson and L a z a r i d e s , 1983) t h a t are weakly c r o s s - r e a c t i v e , more s u s c e p t i b l e t o p r o t e o l y t i c d e g r a d a t i o n , or p r e s e n t i n lower q u a n t i t i e s than the oC s u b u n i t i n ROS. A number o f s p e c t r i n - l i k e p r o t e i n s has been i d e n t i f i e d on the b a s i s o f a n t i g e n i c c r o s s - r e a c t i v i t y . For example, a p a i r of p o l y p e p t i d e s of M r 240,000 and 230,000 found i n embryonic c h i c k c a r d i a c myocytes, 3T3 f i b r o b l a s t s , and r a t hepatoma c e l l have been shown t o c r o s s - r e a c t w i t h a n t i -s p e c t r i n a n t i b o d i e s (Goodman e t a l . , 1981). S i m i l a r l y , a n t i b o d i e s s p e c i f i c f o r the c h a i n o f a v i a n e r y t h r o c y t e s p e c t r i n have been shown t o c r o s s ^ r e a c t w i t h p r o t e i n s of n e a r l y i d e n t i c a l M r (240,000) i n c e l l s from such d i v e r s e t i s s u e s as s m a l l i n t e s t i n e , l i v e r , l e n s , b r a i n , c i l i a , and s t r i a t e d muscle (Repasky e t a l . , 1982). Based on a n t i g e n i c c r o s s - r e a c t i v i t y , i t i s apparent t h a t ROS c o n t a i n a s p e c t r i n - l i k e p r o t e i n and t h a t ROS s p e c t r i n may a l s o c o n s i s t of an and a ft c h a i n . However i t i s d i f f i c u l t t o assess how the ROS s p e c t r i n i s s i m i l a r t o RBC s p e c t r i n i n s t r u c t u r e or f u n c t i o n based s o l e l y on a n t i g e n i c c r o s s - r e a c t i v i t y . The degree o f c r o s s - r e a c t i v i t y of both monoclonal and p o l y c l o n a l a n t i b o d i e s does not n e c e s s a r i l y c o r r e l a t e w i t h e i t h e r s t r u c t u r a l or f u n c t i o n a l homology among the s p e c t r i n f a m i l y of p r o t e i n s . For example, b r a i n and RBC s p e c t r i n s are s i m i l a r i n t h e i r amino a c i d composition, t h e i r t e t r a m e r i c s t r u c t u r e , and t h e i r a b i l i t y t o c r o s s - l i n k a c t i n -105-f i l a m e n t s and b i n d c a l m o d u l i n and a n k y r i n , y e t o n l y about 1% o f e p i t o p e s i n the two p r o t e i n s were s t r o n g l y c r o s s - r e a c t i v e as measured i n a study by H a r r i s e t a l . (1985). S i m i l a r l y , immunological and b i o l o g i c a l data i n d i c a t e t h a t c h i c k e n n o n e r y t h r o i d and human e r y t h r o i d c<-spectrin are two o f the more w i d e l y d i v e r g e d members o f the group o f s p e c t r i n p r o t e i n s . However, comparison of the amino a c i d sequence of human e r y t h r o i d 0 < - s p e c t r i n w i t h the d e r i v e d amino a c i d sequence o f the c h i c k e n n o n e r y t h r o i d c< - s p e c t r i n cDNA c l o n e r e v e a l e d a s t r i k i n g s i m i l a r i t y between these two molecules (Birkenmeier e t a l . , 1985). T h e r e f o r e , i t i s not s u r p r i s i n g t o f i n d t h a t p o l y c l o n a l a n t i - b o v i n e RBC s p e c t r i n a n t i b o d i e s r e a c t e d more s t r o n g l y w i t h bovine RBC s p e c t r i n than w i t h ROS s p e c t r i n from the same s p e c i e s . Furthermore, the 4B2 monoclonal antibody r a i s e d a g a i n s t the ROS a n t i g e n , r e a c t e d more i n t e n s e l y w i t h ROS s p e c t r i n than w i t h t h e RBC p r o t e i n . A low l e v e l o f c r o s s - r e a c t i v i t y between ROS s p e c t r i n and RBC s p e c t r i n does not n e c e s s a r i l y imply l i m i t e d s t r u c t u r a l or f u n c t i o n a l homology. For example, d e t a i l e d s t u d i e s on the a n t i g e n i c v a r i a t i o n of the i n f l u e n z a v i r u s hemagglutinin p r o t e i n i n d i c a t e t h a t marked r e d u c t i o n s i n monoclonal antibody b i n d i n g are caused by even a s i n g l e c o n s e r v a t i v e amino a c i d s u b s t i t u t i o n (Wiley e t a l . , 1981). I t i s a l s o noteworthy t o f i n d t h a t the e r y t h r o c y t e s p e c t r i n i s not r e l a t e d t o any o t h e r p r o t e i n s whose sequence was known (Speicher e t a l . , 1983a; 1983b; S p e i c h e r and Marchesi, 1984). -106-The primary s t r u c t u r e o f e r y t h r o c y t e s p e c t r i n has been e x t e n s i v e l y i n v e s t i g a t e d . The sequence o f 1,435 amino a c i d r e s i d u e s or 35% o f the t o t a l molecule has been determined ( S p e i c h e r e t a l . , 1983a; 1983b; and S p e i c h e r and Marchesi, 1984). Analyses o f the amino a c i d sequences o b t a i n e d t o date i n d i c a t e the presence of 22 homologous r e p e a t u n i t s throughout both s u b u n i t s of s p e c t r i n . I t was suggested t h a t each repeat u n i t , which c o n t a i n s 106 amino a c i d r e s i d u e s (M r 12,000) may be f o l d e d i n t o a t r i p l e h e l i c a l s t r u c t u r e w i t h a s h o r t n o n - h e l i c a l r e g i o n c o n n e c t i n g a d j a c e n t r e p e a t u n i t s . Another 16 r e p e a t u n i t s are expected t o be p r e s e n t i f most o f the s p e c t r i n molecule i s composed of t h i s s i n g l e type of r e p e t i t i v e s t r u c t u r e . T h i s has t o be confirmed by f u r t h e r sequencing s t u d i e s . The l o c a t i o n o f the 3A6 and 4B2 antibody b i n d i n g s i t e s on the e r y t h r o c y t e s p e c t r i n molecule has not been i d e n t i f i e d . But the f a c t the 3A6 antibody c r o s s - r e a c t s o n l y w i t h the c h a i n and the 4B2 antibody c r o s s - r e a c t s o n l y w i t h the o< s u b u n i t o f RBC s p e c t r i n , suggests t h a t these a n t i b o d i e s may perhaps b i n d t o r e g i o n s of f u n c t i o n a l s p e c i a l i z a t i o n . I t has been known t h a t the s u b u n i t of a v i a n s p e c t r i n b i nds c a l m o d u l i n (Glenney e t a l . , 1982) w h i l e the 0 s u b u n i t b i n d s a n k y r i n (Bennett and Stenbuck, 1979) and p r o b a b l y band 4.1 (Coleman e t a l . , submitted). However, i t i s a l s o p o s s i b l e t h a t these two monoclonal a n t i b o d i e s simply r e c o g n i z e two n o n - i d e n t i c a l sequences l o c a t e d i n n o n - f u n c t i o n a l r e g i o n s of the s p e c t r i n c h a i n s . -107-C. E x t r a c t i o n o f ROS S p e c t r i n . S p e c t r i n from RBC (Marchesi e t a l . , 1976) and s p e c t r i n -r e l a t e d p r o t e i n s from ot h e r c e l l types (Glenney and Glenney, 1983) a re n o n i n t e g r a l , membrane-associated p r o t e i n s c o n s i s t i n g o f an elongated o(~ ft heterodimer h a v i n g a l e n g t h of about 100 nm (Branton e t a l . , 1981; Shotton e t a l . , 1979). Two heterodimers f u r t h e r a s s o c i a t e end t o end t o form a f l e x i b l e 200-nm-long c h a i n as p a r t o f a f i b r o u s s k e l e t a l network l i n i n g the c y t o p l a s m i c s u r f a c e o f the plasma membrane. In the case of RBC t h i s f i b r o u s network i n t e r a c t s w i t h the ani o n t r a n s p o r t p r o t e i n "band 3" p r i m a r i l y through i n t e r a c t i o n w i t h a n k y r i n (Bennett and Davis, 1982). T h i s membrane s k e l e t a l system appears t o s t a b i l i z e t he plasma membrane and ma i n t a i n the shape of RBC. ROS s p e c t r i n a l s o appears t o be a n o n i n t e g r a l , membrane-a s s o c i a t e d p r o t e i n t h a t can be p a r t i a l l y e x t r a c t e d from ROS membranes w i t h urea under c o n d i t i o n s i n which the i n t e g r a l ROS membrane p r o t e i n s are not e x t r a c t e d . A s t r o n g a s s o c i a t i o n o f ROS s p e c t r i n w i t h d i s c and plasma membranes e x i s t s , however, s i n c e r e l a t i v e l y s m a l l amounts o f t h i s p r o t e i n are e x t r a c t e d under the c o n d i t i o n s used i n t h i s study. A l s o ROS s p e c t r i n was not e x t r a c t e d i n low i o n i c s t r e n g t h b u f f e r i n the presence of c h e l a t i n g agents, i . e . , c o n d i t i o n s used t o e x t r a c t RBC s p e c t r i n . In t h i s r e s p e c t ROS s p e c t r i n i s d i f f e r e n t from RBC s p e c t r i n . -108-D. A s s o c i a t i o n o f 4 B 2 - S p e c i f i c P r o t e i n w i t h ROS Membranes. The s t r o n g a s s o c i a t i o n o f ROS s p e c t r i n w i t h ROS membranes was a l s o suggested by the f a c t t h a t d i s c membranes prepared by hyp o t o n i c l y s i s o f ROS membranes c o n t a i n e d ROS s p e c t r i n as determined by immunoblotting. Competitive i n h i b i t i o n assays i n d i c a t e d a 2 - f o l d d i f f e r e n c e i n 4B2 a n t i g e n c o n c e n t r a t i o n between ROS and d i s c membrane p r e p a r a t i o n s . T h i s i s unexpected because i t suggests t h a t 50% o f t o t a l ROS s p e c t r i n was l o s t when d i s c membranes were prepared. S i n c e ROS s p e c t r i n was not e x t r a c t e d i n low i o n i c s t r e n g t h b u f f e r , i t would appear t h a t the l o s t ROS s p e c t r i n c o u l d be found i n the p e l l e t e d membrane f r a c t i o n c o n s i s t i n g o f unsealed plasma and d i s c membranes and p o s s i b l y o t h e r contaminating r e t i n a l d e b r i s . However, t h i s membrane f r a c t i o n o n l y r e p r e s e n t s about 15% of the t o t a l ROS membranes (Molday and Molday, unpublished r e s u l t s ) . A c c u r a t e q u a n t i t a t i v e d e t e r m i n a t i o n o f 4B2 a n t i g e n c o n c e n t r a t i o n 125 . . . . u s i n g I - l a b e l l e d 4B2 antibody i n a d i r e c t radioimmune assay has not been attempted because 4B2 antibody was never p u r i f i e d t o homogeneity. The a s s o c i a t i o n o f ROS s p e c t r i n w i t h ROS d i s c membrane fragments has been p r e v i o u s l y shown by immunocytochemical l a b e l l i n g o f d i s c membrane p r e p a r a t i o n w i t h the 4B2 antibody (MacKenzie and Molday, 1982). But i t i s s t i l l u n c e r t a i n whether ROS s p e c t r i n a s s o c i a t e s w i t h the d i s c and/or plasma membrane s i n c e Molday and Molday (1987) have shown t h a t d i s c membranes prepared from hypotonic l y s i s o f ROS c o n t a i n e d -109-i n s i d e out or i n v e r t e d plasma membrane v e s i c l e s . In o r d e r t o show the a s s o c i a t i o n o f ROS s p e c t r i n w i t h plasma membrane, a r e l i a b l e procedure f o r p u r i f y i n g ROS plasma membrane must be a v a i l a b l e . Although a method f o r i s o l a t i n g t he plasma membrane has been d e s c r i b e d (Kamps e t a l . , 1982), t h e p u r i t y o f the plasma membrane p r e p a r a t i o n c o u l d not be asse s s e d due t o the l a c k o f a plasma membrane s p e c i f i c marker. Plasma membrane s p e c i f i c markers (Molday and Molday, 1987) and a new method f o r i s o l a t i n g the plasma membrane (Molday and Molday, i n press) have r e c e n t l y been d e s c r i b e d . Thus, i t i s p o s s i b l e i n t he f u t u r e t o i n v e s t i g a t e the a s s o c i a t i o n o f 4 B 2 - s p e c i f i c p r o t e i n w i t h the ROS plasma membrane. E. L o c a l i z a t i o n and P o s s i b l e F u n c t i o n o f ROS S p e c t r i n . The m o l e c u l a r s t r u c t u r e and f u n c t i o n o f ROS s p e c t r i n i s not y e t known. Immunocytochemical l a b e l l i n g s t u d i e s , however, i n d i c a t e t h a t t h i s p r o t e i n i s l o c a l i z e d a l o n g the p e r i p h e r y o f the ROS o r g a n e l l e i n the r e g i o n where the d i s c s come i n c l o s e c o n t a c t t o the plasma membrane and c i l i a r y s p i n e . In the same r e g i o n , f i l a m e n t o u s s t r u c t u r e s l i n k i n g d i s c s t o the plasma membrane have been seen i n f r o g ROS under the e l e c t r o n microscope by Usukura and Yamada (1981), u s i n g freeze-deep etched r e p l i c a t e c h n i q u e s . Roof and Heuser (1982) have a l s o observed s p e c i a l i z e d f i l a m e n t s c o n n e c t i n g the d i s c rims t o each o t h e r and d i f f e r e n t f i l a m e n t s l i n k i n g the d i s c s t o the plasma membranes. - l l O -F i l a m e n t s c o n n e c t i n g the d i s c s t o each o t h e r a r e more numerous and have been suggested t o be composed o f the r i m p r o t e i n f i r s t c h a r a c t e r i z e d by Papermaster e t a l . (1978) i n f r o g ROS. T h i s p r o t e i n may be r e l a t e d t o ROS 1.2 g l y c o p r o t e i n o f bovine ROS (Molday and Molday, 1979) although d i r e c t evidence f o r t h i s i s l a c k i n g . The m o l e c u l a r composition of the l e s s numerous f i l a m e n t s c o n n e c t i n g the d i s c s t o the plasma membrane t h a t may be r e l a t e d t o the f i l a m e n t o u s network observed by Usukura and Yamada (1981), i s not known. R e s u l t s of t h i s study i n d i c a t e t h a t the M r 240,000 s p e c t r i n - l i k e p r o t e i n i s p r e s e n t i n s i g n i f i c a n t l y lower amounts than the M r 220,000 g l y c o p r o t e i n ROS 1.2 as observed by Coomassie b l u e and s i l v e r s t a i n i n g o f ROS p r o t e i n s s e p a r a t e d by SDS g e l e l e c t r o p h o r e s i s ( F i g u r e 8 ) . RIA s t u d i e s comparing the b i n d i n g of a n t i - s p e c t r i n a n t i b o d i e s t o RBC s p e c t r i n and ROS f u r t h e r suggest t h a t ROS c o n t a i n s a p r o t e i n t h a t i s immunochemically r e l a t e d t o s p e c t r i n , and t h i s s p e c t r i n - l i k e p r o t e i n c o n s t i t u t e s l e s s than 1% o f the ROS p r o t e i n ( F i g u r e 17). I t would appear t h a t the 24OK p o l y p e p t i d e s and fragments r e c o g n i z e d by p o l y c l o n a l anti-RBC a n t i b o d i e s are i d e n t i c a l w i t h those r e c o g n i z e d by the 4B2 monoclonal antibody. T h i s can be t e s t e d by i mmunoprecipitating ROS p r o t e i n s w i t h the p o l y c l o n a l a n t i b o d i e s and measuring b i n d i n g of monoclonal a n t i b o d i e s t o the complex. On the b a s i s of these s t u d i e s and analogy w i t h RBC s p e c t r i n , the ROS s p e c t r i n p r o t e i n i s suggested t o be a major - I l l -c o n s t i t u e n t o f the fil a m e n t o u s network l i n i n g t h e c y t o p l a s m i c s u r f a c e o f the ROS plasma membrane as observed by Usukura and Yamada (1981), and more s p e c i f i c a l l y , may comprise the f i l a m e n t s extending from the d i s c s t o the plasma membrane observed by Roof and Heuser (1982). T h i s f i l a m e n t o u s network, which probably i n t e r a c t s w i t h i n t e g r a l membrane p r o t e i n s , would serve t o m a i n t a i n a co n s t a n t d i s t a n c e between the plasma membrane and d i s c s as seen by e l e c t r o n microscopy and would g e n e r a l l y s t a b i l i z e the ROS s t r u c t u r e . The o b s e r v a t i o n t h a t g o l d l a b e l l i n g w i t h the 4B2 ant i b o d y extends inward from the plasma membrane up t o 50 nm may i n d i c a t e t h a t the s p e c t r i n network i n t e r d i g i t a t e s between the rims of the d i s c s . A s p e c u l a t i v e model on the o r g a n i z a t i o n o f ROS s p e c t r i n and oth e r ROS p r o t e i n s (R.S. Molday, p e r s o n a l communication) i s i l l u s t r a t e d i n F i g u r e 32. In t h i s model, rhodopsin i s mainly d i s t r i b u t e d i n the l a m e l l a r r e g i o n o f d i s c membranes whereas p e r i p h e r i n (33 kDa r i m - s p e c i f i c p r o t e i n ) and ROS 1.2 (220 kDa g l y c o p r o t e i n ) r e s i d e i n the rim r e g i o n s . A s p e c t r i n - l i k e p r o t e i n (240 kDa) i n the c y t o p l a s m i c space e i t h e r d i r e c t l y o r i n d i r e c t l y a s s o c i a t e w i t h both the d i s c and plasma membrane. The plasma membrane c o n t a i n s two g l y c o p r o t e i n s o f M r 230,000 and 110,000 as w e l l as rhodopsin. F. Detergent S o l u b i l i z a t i o n o f ROS Membrane P r o t e i n s . T r i t o n X-100 has been found t o be b e t t e r than some i o n i c -112-Specu la t i ve M o d e l Fo r Organ i za t i on o l P ro te i ns in R O S Memorai F i g u r e 32. A s p e c u l a t i v e model f o r o r g a n i z a t i o n o f p r o t e i n s i n ROS membranes i s i l l u s t r a t e d i n t h i s schematic diagram. Rhodopsin molecules are mainly d i s t r i b u t e d i n the l a m e l l a r r e g i o n s whereas p e r i p h e r i n and a 220 kDa g l y c o p r o t e i n are d i s t r i b u t e d i n the r i m r e g i o n s o f d i s c membranes. A s p e c t r i n - l i k e p r o t e i n i n the c y t o p l a s m i c space e i t h e r d i r e c t l y o r i n d i r e c t l y a s s o c i a t e w i t h both the d i s c and plasma membrane. Two g l y c o p r o t e i n s o f M 230,000 and 110,000 as w e l l as rhodopsin are found i n the plasma membrane (obtained from R.S. Molday). -113-and z w i t t e r i o n i c d e t e r g e n t s i n s e l e c t i v e l y s o l u b i l i z i n g ROS membrane p r o t e i n s . R e s u l t s o f the study on T r i t o n X-100 e x t r a c t i o n o f ROS membrane p r o t e i n s i n d i c a t e t h a t rhodopsin and p e r i p h e r i n which c o n s t i t u t e over 90% of t o t a l d i s c membrane p r o t e i n s (Molday e t a l . , 1987) are almost completely s o l u b i l i z e d by t h i s d e t e r g e n t . In c o n t r a s t , ROS 1.2 g l y c o p r o t e i n which c o n s t i t u t e s 1-3% of t o t a l d i s c membrane p r o t e i n (Molday and Molday, 1979) i s r e l a t i v e l y i n s o l u b l e as determined by SDS g e l e l e c t r o p h o r e s i s and immunoblotting a n a l y s e s . These r e s u l t s suggest t h a t r h o d o p s i n and p e r i p h e r i n may be more hydrophobic than ROS 1.2 and hence have s t r o n g e r a s s o c i a t i o n w i t h the l i p i d c o r e i n d i s c membranes. When the hydrophobic l i p i d c o r e i s d i s r u p t e d by T r i t o n X-100, rhodopsin and p e r i p h e r i n become s o l u b l e . ROS 1.2 g l y c o p r o t e i n , however, may have s t r o n g p o l a r a s s o c i a t i o n s w i t h o t h e r ROS p r o t e i n s . S i n c e p o l a r p r o t e i n - p r o t e i n a s s o c i a t i o n s are not d i s r u p t e d by T r i t o n X-100, ROS 1.2 remains i n s o l u b l e . The i d e n t i t y o f t h e remaining ROS p o l y p e p t i d e s i n the T r i t o n - i n s o l u b l e f r a c t i o n as observed by Coomassie b l u e s t a i n i n g o f these p o l y p e p t i d e s s e p a r a t e d by SDS g e l e l e c t r o p h o r e s i s has not been i n v e s t i g a t e d mainly due t o the l a c k o f p r e v i o u s l y c h a r a c t e r i z e d a ntibody probes. Only the 4B2-binding p r o t e i n (ROS s p e c t r i n ) has been i d e n t i f i e d . E l e c t r o n microscopy of a n e g a t i v e l y s t a i n e d T r i t o n -e x t r a c t e d ROS p e l l e t r e v e a l e d a f i l a m e n t o u s network. The mo l e c u l a r composition o f t h i s f i l a m e n t o u s network i s as y e t -114-unknown, but the 4B2-binding p r o t e i n i s l i k e l y a component of t h i s network s i n c e i t i s p r e s e n t i n the T r i t o n - e x t r a c t e d ROS p e l l e t . Moreover, s p e c t r i n and s p e c t r i n - l i k e p r o t e i n s are known t o be components of c y t o s k e l e t a l system i n o t h e r c e l l t y p e s . G. I d e n t i f i c a t i o n of ROS C y t o s k e l e t a l Components. S t u d i e s have been i n i t i a t e d t o i d e n t i f y the components t h a t c o n s t i t u t e the ROS c y t o s k e l e t o n . A c t i n and a n k y r i n are two p r o t e i n s t h a t i n t e r a c t w i t h s p e c t r i n i n the RBC c y t o s k e l e t a l system. S i n c e a s p e c t r i n - l i k e p r o t e i n has been i d e n t i f i e d i n ROS, i t i s of i n t e r e s t t o determine whether a c t i n - and/or a n k y r i n - l i k e p r o t e i n s a l s o e x i s t i n t h i s o r g a n e l l e . A monoclonal antibody s p e c i f i c f o r a c t i n and p o l y c l o n a l antiserum r a i s e d a g a i n s t a p a r t i a l l y p u r i f i e d e r y t h r o c y t e a n k y r i n p r e p a r a t i o n were t e s t e d f o r immunological c r o s s - r e a c t i v i t y w i t h ROS p r o t e i n s . A c t i n was d e t e c t e d i n some ROS membrane p r e p a r a t i o n s as determined by s o l i d - p h a s e RIA. However, on the b a s i s of immunocytochemical l a b e l l i n g experiments which l o c a l i z e d a c t i n o n l y t o the d i s t a l c o n n e c t i n g c i l i u m i n p h o t o r e c e p t o r s of many s p e c i e s i n c l u d i n g the cow ( C h a i t i n e t a l . , 1984; C h a i t i n and Bok, 1986), i t i s u n l i k e l y t h a t a c t i n i s a component of the f i l a m e n t o u s network l i n i n g the c y t o p l a s m i c s u r f a c e of the o u t e r segment plasma membrane. N e v e r t h e l e s s , t h e r e remains the p o s s i b l i t y t h a t the c o n c e n t r a t i o n o f a c t i n -115-i n the rod o u t e r segments i s too low t o be d e t e c t e d by the immunocytochemical l a b e l l i n g technique. A n k y r i n i s a l i k e l y c a n d i d a t e f o r an attachment p r o t e i n t h a t l i n k s ROS s p e c t r i n t o ROS plasma and/or d i s c membranes, s i n c e i t s e r v e s such a f u n c t i o n i n e r y t h r o c y t e s . However, i n i t i a l attempts t o d e t e c t an a n k y r i n - l i k e molecule i n ROS membrane p r e p a r a t i o n s u s i n g p o l y c l o n a l anti-RBC a n k y r i n a n t i b o d i e s were not s u c c e s s f u l . T h i s suggests t h a t ROS may not c o n t a i n an a n k y r i n - l i k e molecule or t h e r e i s l i m i t e d c r o s s - r e a c t i v i t y between e r y t h r o c y t e and ROS a n k y r i n s . S i n c e immunologically r e l a t e d forms o f a n k y r i n have been d e t e c t e d i n a wide v a r i e t y of n o n - e r y t h r o i d c e l l s , i n c l u d i n g p l a t e l e t s (Bennett, 1979; Bennett and Davis, 1981), a n k y r i n would a l s o be expected t o be found i n ROS. Thus, i t i s necessary t o use s e v e r a l p o l y c l o n a l a n t i - a n k y r i n a n t i b o d i e s t o f u r t h e r t e s t f o r c r o s s - r e a c t i v i t y w i t h ROS p r o t e i n s . F u r t h e r c h a r a c t e r i z a t i o n of the T r i t o n - e x t r a c t e d ROS p e l l e t by both monoclonal and p o l y c l o n a l a n t i b o d i e s s p e c i f i c f o r c y t o s k e l e t a l elements w i l l determine the s i m i l a r i t i e s and d i f f e r e n c e s between the e r y t h r o c y t e c y t o s k e l e t a l network and t h a t found i n ROS. S e v e r a l a n t i b o d i e s i n c l u d i n g 1H5 and 2A4 p r e s e n t e d i n t h i s t h e s i s , were generated a g a i n s t T r i t o n -e x t r a c t e d ROS p e l l e t ( J . Jarausch, p e r s o n a l communication). One antibody (1D1) was r a i s e d t o u r e a - e x t r a c t a b l e ROS membrane p r o t e i n s , and p o l y c l o n a l anti-RBC a n k y r i n a n t i b o d i e s were generated a g a i n s t p a r t i a l l y p u r i f i e d RBC a n k y r i n . However, f u r t h e r c h a r a c t e r i z a t i o n of these a n t i b o d i e s i s -116-r e q u i r e d p r i o r t o t h e i r use as mo l e c u l a r probes. H. Rod Photor e c e p t o r C y t o s k e l e t o n . S e v e r a l c y t o s k e l e t a l elements have been i d e n t i f i e d i n the r od i n n e r segment and i n the non-motile c i l i u m t h a t connects the i n n e r segment t o the o u t e r segment. The c i l i u m c o n s i s t s o f ni n e m i c r o t u b u l e d o u b l e t s p r i m a r i l y composed of a p r o t e i n known as t u b u l i n . A c t i n has been l o c a l i z e d i n the p e r i c i l i a r y r i d g e complex (Peters e t a l . , 1983), a s p e c i a l i z e d c e l l u l a r s t r u c t u r e t h a t surrounds the c i l i u m a t the apex o f the i n n e r segment. T h i s p r o t e i n has a l s o been l o c a l i z e d i n the b a s a l d i s c s , and the d i s t a l end of the c i l i a r y p r o c e s s ( C h a i t i n e t a l . , 1984). Myosin, another c o n t r a c t i l e p r o t e i n , has been l o c a l i z e d throughout the c i l i a r y e x t e n s i o n ( C h a i t i n and Bok, 1984). Furthermore, a bundle of m i c r o t u b u l e - l i k e elements has been found t o be en c l o s e d by the d i s c i n c i s u r e s ( F i g . 5 ) . On the b a s i s o f i t s l o c a l i z a t i o n a t the s i t e o f d i s c formation, a c t i n has been suggested t o p l a y a r o l e i n o p s i n t r a n s p o r t and i n c o r p o r a t i o n i n t o the o u t e r segment membrane d u r i n g d i s c formation ( C h a i t i n e t a l . , 1984). I t has a l s o been suggested t h a t i n lower v e r t e b r a t e rods, a c t i n may p l a y a s i m i l a r r o l e i n the assembly of d i s c s (Burnside and Dearry, 1986). I n t e r e s t i n g l y , the c y t o s k e l e t o n s o b t a i n e d from T r i t o n e x t r a c t i o n o f whole t e l e o s t r e t i n a were found t o c o n t a i n a c t i n f i l a m e n t s , m i c r o t u b u l e s , the c o n n e c t i n g c i l i u m , and a -117-240 kDa c a l m o d u l i n - b i n d i n g p r o t e i n (Nagle and Burnside, 1984). T h i s p r o t e i n appears t o be s i m i l a r t o the ROS s p e c t r i n d e s c r i b e d i n t h i s t h e s i s . I t comigrates w i t h the c h a i n d of b r a i n s p e c t r i n and c r o s s - r e a c t s w i t h a n t i b o d i e s s p e c i f i c f o r r a t b r a i n s p e c t r i n (Nagle and Burnside, 1984). T h i s s p e c t r i n - l i k e p r o t e i n and a c t i n a r e thought t o p r o v i d e s c a f f o l d i n g f o r the l a r g e membrane s u r f a c e o f the lo n g d e l i c a t e t e l e o s t rods (Burnside and Dearry, 1986). In c o n t r a s t , the bovine ROS s p e c t r i n - l i k e p r o t e i n i d e n t i f i e d i n t h i s i n v e s t i g a t i o n i s proposed t o be a c o n s t i t u e n t o f a c y t o s k e l e t a l network t h a t s t a b i l i z e s the h i g h l y - o r d e r e d ROS s t r u c t u r e . E l u c i d a t i o n o f the p r e c i s e f u n c t i o n s o f ROS s p e c t r i n and o t h e r c y t o s k e l e t a l elements r e q u i r e s f u r t h e r i n v e s t i g a t i o n . CONCLUSIONS T h i s t h e s i s i n v e s t i g a t i o n i n d i c a t e s bovine rod ph o t o r e c e p t o r o u t e r segments c o n t a i n a 240 kDa s p e c t r i n - l i k e p r o t e i n t h a t i s d i s t i n c t from the p r e v i o u s l y d e s c r i b e d ROS 1.2 g l y c o p r o t e i n . T h i s s p e c t r i n - l i k e p r o t e i n i s h i g h l y s e n s i t i v e t o p r o t e o l y t i c d e g r a d a t i o n by an endogenous p r o t e a s e and l o c a l i z e s along the p r e i p h e r y o f the ROS o r g a n e l l e i n the r e g i o n where the d i s c s come i n c l o s e c o n t a c t t o the plasma membrane. I t a s s o c i a t e s s t r o n g l y w i t h the plasma membrane and/or d i s c membrane and due t o these s t r o n g i n t e r a c t i o n s w i t h the membranes, i t i s not e a s i l y e x t r a c t e d . -118-However, t h i s s p e c t r i n - l i k e p r o t e i n i s e n r i c h e d i n T r i t o n -e x t r a c t e d ROS p e l l e t because i t i s a c o n s t i t u e n t o f a f i b r o u s m a t r i x t h a t i s i n s o l u b l e i n T r i t o n X-100 whereas the major component rhodopsin i s s o l u b l e . FUTURE DIRECTIONS Future s t u d i e s should be d i r e c t e d toward the u n d erstanding of the m o l e c u l a r s t r u c t u r e and p r o p e r t i e s of ROS s p e c t r i n . T h i s w i l l r e q u i r e the p u r i f i c a t i o n o f the 4B2-s p e c i f i c p r o t e i n from ROS membrane p r e p a r a t i o n s . S e v e r a l d i f f i c u l t i e s i n t h i s r e g a r d are suggested by t h i s t h e s i s i n v e s t i g a t i o n . F i r s t l y , the 4 B 2 - s p e c i f i c p r o t e i n c o n s t i t u t e s l e s s than 1% of the t o t a l ROS p r o t e i n . Secondly, i t i s h i g h l y s e n s i t i v e t o p r o t e o l y t i c d e g r a d a t i o n by an endogenous p r o t e a s e whose i d e n t i t y and s p e c i f i c i n h i b i t o r are y e t unknown. F i n a l l y , i t a s s o c i a t e s s t r o n g l y w i t h ROS membranes, presumably due t o s t r o n g i n t e r a c t i o n s w i t h o t h e r ROS p r o t e i n s . N e v e r t h e l e s s , a p u r i f i c a t i o n method can be d e v i s e d t o t a ke advantage of the f a c t t h a t the undegraded 4 B 2 - s p e c i f i c p r o t e i n i s e n r i c h e d i n T r i t o n X-100-extracted ROS p e l l e t . In t h i s method, ROS membranes are e x t r a c t e d w i t h T r i t o n X-100 and c e n t r i f u g e d a t h i g h speed. The r e s u l t i n g p e l l e t f r a c t i o n i s then s o l u b i l i z e d and a p p l i e d t o an a n t i - s p e c t r i n antibody column. The 4B2 monoclonal antibody i s not s u i t a b l e f o r t h i s purpose because i t has been found t o be u n s t a b l e and -119-d i f f i c u l t t o p u r i f y from a s c i t e s f l u i d . P u r i f i e d p o l y c l o n a l a n t i - s p e c t r i n a n t i b o d i e s and IgG monoclonal a n t i b o d i e s s p e c i f i c f o r the 4B2-binding p r o t e i n are more s u i t a b l e f o r t h i s purpose. Another approach t o study ROS s p e c t r i n would be t o determine i t s gene sequence. An o l i g o n u c l e o t i d e probe c o r r e s p o n d i n g t o a p a r t i a l amino a c i d sequence of RBC s p e c t r i n c o u l d be used t o s c r e e n a bovine r e t i n a l genomic or cDNA l i b r a r y ; a l t e r n a t i v e l y , the 4B2 monoclonal antibody o r p o l y c l o n a l a n t i - s p e c t r i n a n t i s e r a c o u l d be used t o s c r e e n a bovine r e t i n a l cDNA e x p r e s s i o n l i b r a r y . The i n t e r a c t i o n o f the 4 B 2 - s p e c i f i c p r o t e i n w i t h o t h e r ROS p r o t e i n s s hould a l s o be i n v e s t i g a t e d i n f u t u r e s t u d i e s i n or d e r t o understand i t s p o s s i b l e f u n c t i o n i n the maintenance ROS s t r u c t u r e , p h o t o t r a n s d u c t i o n , and o t h e r s p e c i a l i z e d p r o c e s s e s such as d i s c renewal t h a t occur i n the ROS o r g a n e l l e . S e v e r a l major q u e s t i o n s t o ask are : (1) How does the ROS s p e c t r i n a t t a c h t o the plasma membrane and/or d i s c membrane? (2) I f ROS s p e c t r i n i s p a r t o f a c y t o s k e l e t a l network t h a t maintains the shape of the rod o u t e r segment, what happens t o t h i s network when d i s c s a re shed d u r i n g the d i s c renewal process? (3) Does ROS s p e c t r i n o r o t h e r ROS c y t o s k e l e t a l components serve any r o l e i n the opening and c l o s i n g o f sodium channels d u r i n g the p h o t o t r a n s d u c t i o n p r o c e s s o r i n the o r g a n i z a t i o n o f the i n t e g r a l membrane p r o t e i n s o f the plasma and d i s c membranes? (4) How do the c y t o s k e l e t a l elements assemble and migrate from the rod -120-i n n e r segment t o the ou t e r segment ? 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