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Preparation and characterization of bovine retinal pigment epithelial cell plasma membrane Laird, Dale W. 1984

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PREPARATION  AND  CHARACTERIZATION  OF  BOVINE  PIGMENT E P I T H E L I A L C E L L PLASMA  RETINAL  MEMBRANE  by DALE  B.Sc.,University Charlottetown,  A THESIS THE  W.LAIRD  of P r i n c e  Prince  SUBMITTED  Edward  Edward  IN P A R T I A L  REQUIREMENTS MASTER  FOR OF  Island,  Island,  1982  FULFILLMENT  THE  DEGREE  OF  SCIENCE  in THE  FACULTY  OF  GRADUATE  DEPARTMENT OF FACULTY UNIVERSITY We  accept to  THE  this  BIOCHEMISTRY  OF  OF  MEDICINE  BRITISH  COLUMBIA  t h e s i s as  the required  UNIVERSITY  OF  STUDIES  conforming  standard  BRITISH  COLUMBIA  October,1984 © D a l e  W.  Laird,  1984  OF  In  presenting  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make it  f r e e l y a v a i l a b l e f o r reference  and  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may department/, or by h i s or her  be granted by the head o f representatives.  my  It i s  u n d e r s t o o d t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not be allowed without my  permission.  k o  '/A.  Department of  (^^>^^d^^  The U n i v e r s i t y of B r i t i s h CoMimbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6  (3/81)  written  -ii-  ABSTRACT A  7-9  f o l d enriched p r e p a r a t i o n of bovine  epithelial  c e l l plasma membrane was  enzymatic  analysis.  transmission  electron  epithelial  microscopy  rhodopsin  immunoaffinity  rhodopsin  antibodies  was  classified  immunoglobulin. determined  by  It  proteins  activated  paper. by  localized  removed by  r a i s e d a g a i n s t the  anti-  by  SDS-  silver.  retinal  was  pigment  as  kappa  an  Ic  J 3 G  Limited  proteolytic  17-39  did  for  chain  rhodopsin  to  of  CNBr-  rhodopsin  CNBr- a c t i v a t e d to  the  Competition  amino-acid segment of rhodopsin.  not bind to sealed ROS  paper  N-terminal assays  solubilization  The  dependence  for  with be  Rho-5A3  d i s c s or frozen-thawed  solubilized  as  segment  f u r t h e r d e f i n e d the a n t i g e n i c s i t e to  d i s c s but d i d bind to T r i t o n X-100  accessibility.  light  digestion  binding s i t e of t h i s antibody molecule.  rod  designated  electrophoretically transferred  polypeptides  the  antibody,  shown to be s p e c i f i c  of the rhodopsin  rhodopsin  A monoclonal  e l e c t r o p h o r e t i c t r a n s f e r to  the  two-thirds  detergent  photoreceptor  radioimmune l a b e l i n g of bovine rod outer  membrane  antibody  partially  outer  plasma membrane p r e p a r a t i o n c r o s s reacted with  Rho-5A3,  within  rod  and  retinal  chromatography as determined  segment p r e p a r a t i o n s .  followed  was  rod  to a t i g h t  g e l s s t a i n e d with coomassie blue or  Monoclonal  outer  c h a r a c t e r i z e d by  large  c e l l u l a r adhesion to the  contaminating  epithelial  revealed a  i n the p r e p a r a t i o n due  c e l l s . The  polyacrylamide  and  pigment  SDS-polyacrylamide g e l e l e c t r o p h o r e s i s  segment contamination pigment  prepared  retinal  ROS  discs indicating a antigenic  site  - i i i Cultures started  by  RPMI-1640  of  initial  vivo  of  in  doubling .  FITC-RCA  clear  dense  l a b e l s were  for  the 60  endocytosis  experiments  occuring  accessible  for  so  was  The  protein  actin,  rabbit  bovine tissue  paper.  rod  by  were  recovery  the  of  in cells  cells  many  reached  their  pigmentation  in  and  an  may  culture.  FITC-WGA,  labeling  patterns.  incubating Treatment that  result  central  spots  the  labeled  cells  by  the  cell.  to  appropriate  labeled  sites  Continuous and  dense  had  labeling  internalization  unlabeled a  the  receptors  pattern  of  is  become  label  on  maintained. with  apparent  antisera  proteins  M  the  play  an  labeling  biochemical RPE  important by  of  was  the  detected  RPE  plasma  electrophoreticallytransferred  bovine  segments  =46,000,  labeling  Immunofluorescent  of  outer  A,  redistribute  previously a  FITC-Con  surface  redistribution  a n t i s e r a confirmed  component  by  the  As  anti-actin  preparation  to  37°C.  by  that  surface  filaments  of  with  random  indicated  cell  major  at  labeling.  nitrocellulose  a  until  degree  cells  epithelial  maintained  labeled  and  indicated that  constantly  anti-actin  high  surface  minutes  undergone  membrane  a  pigment  hours  also  induced  cell  inhibitors  with  as  52  epithelial  followed  retinal  of  glycoproteins  saccharide  the  The  cells  such  showed  from  buffer  isolation  time  The  pigment  microvilli.  surface  Fluorescent and  enzymatic  characteristics  abundance  and  a  confluency  Cell  retinal  c u l t u r e medium.  established culture  bovine  studies  cell. role  retinal  in  The  that  the  rabbit  actin  a c t i v a t i o n of  the  pigment  using  was actin  phagocytosis  epithelial  to  cells  of in  -ivScanning  electron  microscopy  microscopy  have  shown  epithelial  cells  in vitro  and  engulf In  dark  summary  adapted  pigment  however,  the antibody  a  contaminating  monoclonal and  i t s  pigment model  antibody, antigenic  epithelial system  sealed  epithelial  ROS  week  c a n be  a monoclonal  retinal  2  that  raised  and  o l d bovine  induced  rod outer  antibody cell  was  t o be  found  cells  grown  for studying c e l l  segment  phagocytosis at the l e v e l s  electron  microscopy,  of  pigment  recognize,  against  membrane specific the was  attach,  bovine  preparation, for rhodopsin,  fully  components  The  characterized  bovine  culture  retinal  acted and r o d  fluorescence,  and t r a n s m i s s i o n e l e c t r o n  a  preparation.  Finally,  in tissue  surface  retinal  raised  in  determined.  electron  segments.  d e s i g n a t e d Rho-5A3, site  to  plasma  proved  protein  transmission  as  a  outer  scanning  microscopy.  -v-  ACKNOWLEDGEMENTS  I would l i k e to thank Dr. guidance  i n the s u p e r v i s i o n of t h i s work.  extend my admiration with  Mackenzie,  sections Simon  I would a l s o l i k e  and g r a t i t u d e t o Dr. Dave Hicks  a l l e l e c t r o n microscopic  Lowicryl  Robert Molday for h i s p a t i e n c e and  used. Wong,  techniques  Finally, and  f o r h i s help  and h i s donation  I would l i k e  to  L a u r i e Molday for t h e i r  t h e o r e t i c a l and t e c h n i c a l a d v i c e .  to  of the  thank  Don  excellent  -vi-  LIST  OF  ABBREVIATIONS  BSA  bovine  serum  albumin  CHAPS  3-[(3-cholamidopropyl)  dimethylammonio]  -1-propanesulfonate Con  A  concanavalin  A  DMSO  dimethyl  EDTA  ethylenediamine  FCS  fetal  FITC  fluorescein  HAT  hypoxanthine,  IMDM  Iscove's  Ig M  r  sulfoxide  calf  tetraacetate  serum isothiocyanate aminopterin,  Modified Dulbecco's  Medium  immunoglobulin molecular  weight  PBS  phosphate-buffered  PEG  polyethylene  glycol  PMSF  phenylmethyl  sulfonyl  RIA  radioimmune  RCA  Ricinius  ROS  rod outer  RPE  retinal  SDS  sodium  SEM  scanning  TCA  trichloroacetic  •TEM  thymidine  saline  assay  communis  agglutinin  segment  pigment dodecyl  epithelial sulfate  electron  microscopy  acid  transmission electron  Tris  tris  WGA  wheat  fluoride  (hydroxymethyl) germ  agglutinin  microscopy aminomethane  -vii-  TABLE OF CONTENTS PAGE ABSTRACT  i i  ACKNOWLEDGEMENTS  v  LIST OF ABBREVIATIONS  vi  TABLE OF CONTENTS  v i i  LIST OF TABLES  xi  LIST OF FIGURES  x i i  INTRODUCTION 1 . Retina  1  2. P h o t o r e c e p t o r c e l l s  1  3. L o c a t i o n of t h e r e t i n a l pigment e p i t h e l i u m  4  4. F u n c t i o n of t h e r e t i n a l pigment e p i t h e l i u m  4  5. P a t h o l o g y of t h e r e t i n a l pigment e p i t h e l i u m  5  6. P r o c e s s of p h a g o c y t o s i s  8  7. The c y t o s k e l e t a l system  i n phagocytosis  9  8. Energy source of p h a g o c y t o s i s  11  9. L a t e n c y p e r i o d i n p h a g o c y t o s i s  11  10. Shedding  or " p i n c h i n g o f f " of ROS d i s c p a c k e t s . .  11. RPE c e l l s i n t i s s u e c u l t u r e  12 13  12. E f f e c t of a g i n g on RPE c e l l morphology and enzyme a c t i v i t y 13. L e c t i n c e l l  surface labels  14. Immunological  approaches t o s t u d y i n g membranes..  15. T h e s i s i n v e s t i g a t i o n  14 15 16 17  -viiiEXPERIMENTAL  PROCEDURES  1. Materials  19  2.  Protein  20  3.  Isolation  4.  Preparaton  5. E n z y m e A.  assays of bovine  RPE  of bovine  RPE c e l l  +  ATPase  Succinate  c reductase  cytochrome  SDS-gel  7.  P r e p a r a t i o n of goat rabbit  electrophoresis  Ig antibody  9.  Silver  Monoclonal  reductase  25  and g e l t r a n s f e r Ig and goat  25 anti27  by a n t i b o d y  27  technique  antibody  28  techniques  A.  Immunization  B.  Preparation of t i s s u e  C.  Production  D.  Standard  E.  Hybridoma  F.  Storage  G.  Solid-Phase  H.  Lowicryl thin bovine  24  reagents  Polypeptide detection stain  c  anti-mouse  8.  A.  22  24  6.  Adult  membrane...  23  C . NADPH c y t o c h r o m e  11.  plasma  5±nucleotidase +  10.  20  assays  B. N a K  D.  cells  o f BALB/C  mice  28  culture  of hybridoma  myeloma  cells  cells  29  RIA  31  cloning..  of v i a b l e  RPE  Culturing  28  31  hybridoma  competition section  cells  32  labeling  33  RPE  B. F l u o r e s c e n t d e t e c t i o n  32  assays  in tissue  of bovine  cells  culture  cells  of a c t i n  34 i n RPE  cells  34  -ixC. F l u o r e s c e n t  lectin  D. D i s c o n t i n u o u s E. 12.  Continuous  Phagocytosis  SECTION  1  of bovine  Preparation  B.  Phagocytosis  C.  fluorescent  fluorescent  A.  cells  l a b e l e d bovine lectin  lectin  dark  35  labeling..  36  labeling  ROS b y b o v i n e  of sealed  RPE c e l l s  RPE  adapted  o f ROS b y a d u l t  36  cells ROS  bovine  37  RPE  in vitro. .  37  Preparation  o f samples  f o r TEM  38  D. P r e p a r a t i o n  of samples  f o r SEM  38  A N A L Y S I S OF B O V I N E  R P E PLASMA  MEMBRANE  RPE p l a s m a  membrane  PREPARATIONS RESULTS 1. P r e p a r a t i o n 2. D e t e c t i o n membrane  of bovine  of a c t i n  40  i n t h e RPE p l a s m a  preparation  3. T E M o b s e r v a t i o n  46  of subcellular  fractionation....  DISCUSSION SECTION  2  46 49  T H E PRODUCTION MONOCLONAL  AND  CHARACTERIZATION  OF  ANTIBODIES  RESULTS 1. P r o d u c t i o n  of monoclonal  antibodies  2. C h a r a c t e r i z a t i o n o f R h o - 5 A 3 3.  Subclassification  4.  Identification  o f Rho-5A3  o f Rho-5A3  antibody  52  antibody  antigen  of bovine  membrane 5. C o m p e t i t i v e  52  56 ROS 56  inhibition  o f Rho-5A3  antibody  59  -x6.  A n a l y s i s o f Rho-5A3 to  7.  rhodopsin  Lowicryl  antibody  binding  peptides  thin  section  59 labeling  62  DISCUSSION SECTION  3  65 ANALYSIS  OF  BOVINE  RPE  CELLS  IN  VITRO  RESULTS 1.  Isolation  of bovine  2. T h e m o r p h o l o g y 3. F l u o r e s c e n t  Discontinuous  5.  Continuous cells  of bovine  cells RPE  70 cells  in vitro  70  d e t e c t i o n of a c t i n  4.  RPE  RPE  lectin  labeling  fluorescent lectin  74 of bovine labeling  RPE of  bovine  in vitro  6.  Probing  RPE  7.  Phagocytosis  78  cultured cells o f ROS  74  with  by b o v i n e  Rho-1D4  RPE  cells  84 in vitro  84  DISCUSSION  91  CONCLUSIONS  95  BIBLIOGRAPHY  97  -xi-  LIST  OF  TABLES  TABLE  PAGE  1. D i s t r i b u t i o n fractions  recovered  2. C o m p a r i s o n binding rod 3.  of enzymatic  outer  subtype  membrane  monoclonal  X-100  in various  a n d SDS  isolation...  o f Rho~5A3  41  antibody solubilized  segments  Identification Ig  during  o f Rho-5A3  to Triton  markers  55 monoclonal  antibody 57  -xii-  L I S T OF FIGURES FIGURE  PAGE  1.  Diagram t o i l l u s t r a t e  2.  Diagram of a r o d p h o t o r e c e p t o r  3.  Coomassie b l u e and s i l v e r  stained  g e l s o f v a r i o u s membrane  fractions  4.  A RPE c e l l t  5.  30-50%  (w/w) c o n t i n u o u s  2  cell  homogenate f r a c t i o n a t i o n  D e t e c t i o n of a c t i n bovine  t h e l a y e r s of t h e r e t i n a  3 SDS-polyacrylamide 44 p r o f i l e of a  sucrose gradient  45  i n r o d o u t e r segments and  RPE p l a s m a membrane p r e p a r a t i o n by S D S - p o l y -  a c r y l a m i d e g e l e l e t r o p h o r e s i s and immunoblot 6.  Transmission  e l e c t r o n micrographs  of bovine  47 RPE  cell  p l a s m a membrane p r e p a r a t i o n s 7.  Antibody  titration  curve  f o r b l o o d f r o m a BALB/C  mouse i m m u n i z e d w i t h b o v i n e 8.  Titration against  9.  48  RPE p l a s m a membranes  c u r v e o f Rho-5A3 h y b r i d o m a c u l t u r e  solubilized  fluid  r o d o u t e r segments  A n a l y s i s of p o l y p e p t i d e s from digested  .... 53  54  u n t r e a t e d and p r o t e a s e  r o d o u t e r s e g m e n t s by m o n o c l o n a l  antibody  binding 10.  Inhibition rod  11.  58 o f Rho~5A3 a n t i b o d y b i n d i n g t o s o l u b i l i z e d  o u t e r s e g m e n t s by r h o d o p s i n  Inhibition  o f Rho-5A3 a n t i b o d y b i n d i n g t o s o l u b i l i z e d  ROS d i s k s by f r o z e n / t h a w e d , ROS d i s k s  60  s e a l e d , and s o l u b i l i z e d '  . . 61  - x i i i -  12.  I n h i b i t i o n of Rho~5A3 antibody b i n d i n g to s o l u b i l i z e d rod  13.  outer segments by rhodopsin p o l y p e p t i d e s  Transmission e l e c t r o n micrograph  63  of a Rho~5A3  antibody gold dextran l a b e l e d rod photoreceptor  cell  embedded i n L o w i c r y l r e s i n  64  14.  A diagrammatic model of rhodopsin  66  15.  Bovine  16.  Scanning  r e t i n a l pigment e p i t h e l i a l e l e c t r o n micrograph  r e t i n a l pigment e p i t h e l i a l 17.  Scanning  18.  Scanning  r e t i n a l pigment e p i t h e l i a l 19.  F l u o r e s c e n t micrographs cells  20.  72  of a 10 day o l d bovine  cell  e l e c t r o n micrograph  73  of a 10 day o l d bovine  cell  grown on g l a s s  of u n f i x e d RPE c e l l s  F l u o r e s c e n t micrographs  79 of u n f i x e d RPE c e l l s  labeled  with FITC-WGA 22.  F l u o r e s c e n t micrographs  80 of u n f i x e d RPE c e l l s  labeled  with FITC-RCA 23. ' F l u o r e s c e n t micrographs  76  labeled  with FITC-Con A 21.  75  of 2 week o l d bovine RPE  incubated with r a b b i t a n t i - a c t i n a n t i s e r a  F l u o r e s c e n t micrographs  71  of a 10 day o l d bovine  cell  e l e c t r o n micrograph  r e t i n a l pigment e p i t h e l i a l  cells in vitro  81 of u n f i x e d RPE c e l l s  labeled  c o n t i n u o u s l y with FITC-Con A, FITC-WGA, and FITC-RCA. 82 24.  Rho-1D4 monoclonal antibody b i n d i n g to bovine RPE cells  in tissue culture  85  -xiv25.  Scanning  electron  RPE  incubated  cell  micrograph  of a  f o r 5 h with  2 week  sealed  old  rod  bovine  outer  segments 26.  86  Transmission incubated for  27.  5 h  with  cell  outer  dark  micrographs  adapted  of  sealed  bovine  rod outer  RPE  cells  segments  in vitro  Transmission RPE  electron  88  electron  incubated  segments  micrograph  with  for 5 h  dark  of  adapted  in vitro  another  bovine  sealed rod 89  -1-  INTRODUCTION  RETINA  The  retina  represents  a  has been highly  extensively  complex  extension  Developmentally  and f u n c t i o n a l l y ,  of  the central  nervous  the  optic  nerve.  The  visual  posterior optic the  part  vesicle outer  layer  becomes  layer  the  retina  ganglion  cell  outer  plexiform  cells,  respectively  layer,  for  responsible slender about segment cilium  1.5  and cone  nuclear  the  and white  approximately  t o 2 um. and  cells  cells.  while  f o r black  portion (Fig.  outer  layer,  part  connected  layer  by  lining  the  development,  nerve inner  layer  the  optic  cup,  the  inner  and  passes  of the o p t i c  plexiform  isolated  a two-layered  Light  system.  through  the  fiber  layer,  nuclear  layer,  and  photoreceptor  (Fig. 1).  vision  cells  nervous  epithelium,  retina.  photoreceptor  rod cells color  i s an  as i t  CELLS  Vertebrate called  into  t h e pigment  inner  nervous  In embryonic  consisting  layer,  of the  the years  i t remains  a thin  transformed  neural  layers  PHOTORECEPTOR  t o which  comprises  forming  over  the retina  of the e y e b a l l .  becoming  neural  system  retina  studied  consist  The cone more vision.  60 um  long  of modified cells  numerous  i s divided  a  segment  2 ) . The r o d o u t e r  segment  responsible  rod  cells  into  a  connected  consists  are  rod c e l l s are  an a v e r a g e  The r o d c e l l r o d inner  are  The b o v i n e with  neurons  width rod  by  of stacks  of  outer  a  thin  of  disc  - 2 -  Figure  1.  Pigment  epithelial.  Diagram  limiting  membrane.  layer.  (6.)  (8.)  Ganglion  limiting Diagram  (2.) L a y e r s (4.) Outer  Inner cell  membrane. taken  to illustrate  from  nuclear  layer.  the layers of rods  nuclear layer.  (9.) O p t i c  The arrow reference  2.  of the r e t i n a .  and cones.  layer.  (5.) Outer  (7.) Inner fiber  indicates  (3.)  External plexiform  plexiform  layer.  (10.)  the d i r e c t i o n  (1.)  of  layer. Internal light.  -3-  Outer segment  -/  i  Cilium  Inner segment  Outer rod fiber  Cell body Inner rod fiber Rod spherule  Figure  2.  Diagram of a  electron  microscope.  membrane.  Diagram  taken  rod photoreceptor  Note from  cell  the large  portion  reference  2.  as seen  of  outer  by  the  segment  -4-  membrane.  The  organelles membrane segment  and  the  The  THE  the  the  outer  whole  serrata  and  to  melanin  the  pigment inner  layer  surface  of of  OF  cells of  cilium At  to  the  outer  form  same segment  the  eyeball  new  its cells.  on  a  basal  in  inner discs  the of  engulfed  form  phagosomes.  these  RPE  cells.  in  (RPE)  optic  vesicle,  retina. as  far  the  face  ora  owing of  the  represents  the  functions  choroid. has  as  RPE  species  basal  which  The  The  in  apical  microvilli  photoreceptor  the  cell  which (1).  EPITHELIUM  be  involved  in  outer  segments  (1).  pass  base  discs  and  degraded  the  the  segment  at  groups  of  to  rod  epithelium  structure  to  PIGMENT  shown  the  The  epithelium  segments  outer  i n most  lamina  This  epithelium  been  time,  to  in  in  RETINAL  the  dark-colored  membrane.  outer  segment  the  the  of  have  epithelium  of  part  THE  in  layer  posterior  pigment  t i p of  pigment  visual  membranes  synthesized  retinal  the  retinal  outer  epithelium.  of  rests  the  The  layer  pigment  the  of  cytoplasmic  EPITHELIUM  external  Bruch's  the  FUNCTION  renewal  PIGMENT  pigment  the  interdigitate  RPE  pigment  i s predominantly  of  90%  the  most  epithelium  adhesion  THE  the  while  a l l  rhodopsin.  monostratified  from  represents  the  RETINAL  cuboidal  originating  in  contains  machinery  photopigment  i s embedded  OF  segment  metabolic  is  LOCATION  covers  inner  the  of are  the  continuous The  through  the  the  outer  shed  rod at  cytoplasm  Finally,  the  protein  connecting segment.  the  apex  of  of  the  pigment  phagosomes  the  are  -5-  It eye,  i s  the  bleaching into  now  retinaldehyde i n the outer  t h e pigment  during play  established that  dark  a vital  epithelium  liberated  segment  role  Thus  f o r the formation  vitamin  (2).  A  There polarity. giving  exists  a  retina  pigmented segments,  site of  processes indicates  continuous  between  OF R E T I N A L  pathology  dysfunction dysfunction more a  of  deficiency  at  different  of  pigment  of" t h e r e t i n a , these  two  PIGMENT  Several  strong  cellular  mitochondria, in with  photoreceptor  of t h e pigment  since  infoldings  surface,  exchanges  the i t s  outer  epithelium  are  important  two  classes:  layers.  and pigmentation  ( 1 ) .  In t h i s  retinal  i n the phagocytosis  of  function,  the  into  of the  paper  pathologies  of the p h a g o c y t i c  retinopathies  releasing  transport  o f t h e RPE c a n b e s e p a r a t e d  of p h a g o c y t o s i s  stages  with  i s  EPITHELIUM  polarization  to the latter.  contact  the complementarity  and  The  apical  pigment and  i t s basal  and a c t i v e  The  in intimate  cells  with  the  and  and the neighbouring  mammals.  the visual  very  place  epithelium  light  by s t o r i n g  a  of the c e l l ,  of absorption  and  PATHOLOGY  RPE c e l l s  vesicles  Moreover  migrates  takes  o f t h e pigment  the  during  and  process  absorbing  of  pigment  to retinol  process. by  adaptation  visual  of rhodopsin  the  face  to coated  represents vascular  in  The s c l e r a l  rise  the c e l l s  reflection  essential  from  The r e v e r s e  i n the visual  prevents  light  i s reduced  (1).  epithelium  adaptation.  during  which  process. retinitis  cell;  attention  and  i s paid  are the result c a n be  of  expressed  The m a n i f e s t a t i o n pigmentosa  type  -6involves the  derrangement  r e c o g n i t i o n and a d s o r p t i o n  the  apical  processes.  informative  as  photoreceptor the  actual  Surgeons or  the  site  gene  i s acting  This  extensively t h e RCS i s  i n studying  accumulation  the  accumulation  This  consequent  decline in retinal  t h e RPE  case  cells  resulting  release  of l y t i c  Lavail  as  There segment all  of  the  pigment rat  enzymes as  invade  into  Essner  cell  is  has  shedding  occurs  Initially,  that  in  the  segments  cells  lysosomal  t o be d e f i c i e n t  of d e b r i s  which  and  i s accompanied  The  have  used  during  photoreceptor  Gorrin  cells  been  of l a m e l l a r bodies outer  of  structural  epithelial  the i n t e r c e l l u l a r  the layer  of a  line  sensitivity.  and  the  College  of phagocytosis.  individual  reported  i n t e r p h o t o r e c e p t o r space  progresses  result  i n the d e s t r u c t i o n of rod outer  well  macrophages RCS  has been  Royal  of l a m e l l a r m a t e r i a l  deterioration  to  epithelial  the  the photoreceptor  progressive  of  in  diseased  between  particularly  extrinsically  t o be a  photoreceptor  space  by  (3).  the mechanism  a concomitant  that i s ,  material  are  the pigment  in the r e t i n a l  extracellular RPE.  shown  important  r a t , normal  that  segment  Chimeras  action  (RCS) r a t has been  (3,4,5).  of rod outer  degeneration  defect  of phagocytosis;  rat  but a l s o  retinal  step  The  o f t h e gene  functional  there  rdy  cell,  Inherited  in  of the f i r s t  and a system  i n the disease  segments space.  by  accumulates of  the  Studies  demonstated  as the degeneration  by  that  in  the  by  the  retina  (6,7). are several hypotheses  phagocytosis pigment  as t o the cause  incapability  epithelial  may  i n t h e RCS  lack  some  of the r o d outer  rat (8).  factor  First  necessary  of for  -7-  phagocytosis  or contain an abnormal  phagocytic activity. Alternately both  factor  that  inhibits  the rod outer segments and  the  RPE may contain defects or complementary defects that are  only  expressed in connection with each other. Gery and O'Brien  demonstrated phagocytic  that  peritoneal macrophages from RCS rats  capability  exhibit  equal to that found in macrophages from  normal strains of rat (8). Thus the genetic defect in RCS pigment epithelium is not expressed in the macrophages. Both  normal  and dystropic retinal  pigment  epithelium are  ( 9 ) . However, when  capable  of phagocytizing carbon particles  specfic  photoreceptor packets are incubated with dystrophic RPE  cells,  no uptake occurs ( 4 ) .  ROS packets  from  normal  Muller and Lavail have shown that  or dystrophic animals  are equally  phagocytized by normal RPE indicating that the pigment epithelium is  the primary  site  of the phagocytic  defect  ( 3 ) . O'Brien  proposed  that RPE phagocytosis may be triggered by a change in  terminal  sugars such as fucose and galactose on plasma membrane  glycoproteins are  of ROS ( 1 0 ) . It is possible that terminal sugars  not recognized by defective RPE because membrane receptors  for certain sugars are lacking or masked. Williams-Seyfried and Mclaughlin's  results  indicate  that  sugar  coated  beads when  presented to retinal pigment epithelial explants show differences in  phagocytosis ( 1 1 ) . Fucose coated beads were not taken up by  either normal or dystrophic explants while twice as many mannose beads are phagocytized by the normal as opposed to the dystrophic t i ssue.  -8-  P R O C E S S OF  The steps;  process first,  segments  to  engulfment of  most  phagocytosis  RPE  cell;  phenomena with  of  the  surface  et  of  phagocytosis  initial  stages  microvilli  of  prior  doughnut  human  and  antiserum assaying  the  in  and  distinct rod  and  i n v o l v e s some  post fusion  pH,  recognition  specific  outer  finally,  phagolysosome The  more  in  with  and  process  i l l  defined  interaction  of  a  double  the  the  by  beads  a the  membrane  results  (13).  of  ROS  by  and  done  was  Hall  cultured rat  of  the  Following leaving  followed  with  labeling  and  microvilli  engulfed  comparable  immunofluorescent  morphological  replicas.  are  of  spreading  surface  study  Chaitin  and  overlapping  been  explants  sequential  similar  have  the  replicas  a  A  culture.  phagocytosis  be  on  surface  surface  (12).  may  allows  receptors  in  Surface  attachment  This  engulfed  replicating  epithelium  (12).  that  i n the  for  pigment  beads.  phagocytosis  tissue  method  show  f l y traps"  domains  on  a  beads  microvilli  RPE  studies  the  or  of  are  impression  bovine  and  latex  beads  "Venus  of  cells  latex  ingestion  membrane  Many RPE  the  The  like  flattened  It  retinal  interaction  resembles  shortening  study.  rat of  to  membranes.  that  drop  material.  phagocytosis  over  circumferential RPE  the  s e q u e n t i a l movement  a  a l . developed  surfaces  movement  this  fairly  of  engulfment;  include  interactions  during  of  attachment  phagosome,  to  in three  molecules.  Mclaughlin membrane  and  phagocytized  charge  occurs  second,  which  the  interesting  electrical cell  of  recognition  the  lysosomes  digestion is  PHAGOCYTOSIS  done  with  embryonic  used  a  procedure  RPE  by  cells  ROS for (14).  -9Following segments ROS  the  attached  antiserum  conjugated under  i n c u b a t i o n o f RPE  goat  anti-rabbit acetone  antiserum  isothiocyanate  conjugated  outer  undergone the  case  where were  control Chaitin  was  used  of  ROS  and a  THE  first  a  to  small  from  number that  classes  two  sets  (  (15),  CYTOSKELETAL  attached that  ingestion  Hall  allows  o f ROS  normal  o f most  may  ingested. ROS  occurs has  suggests exist  for rapid  would  to  rate  attachment  also  receptors  outer  conclusive  were  once  In  endocytosis.  at a ROS  to  have  filters.  no  from  of these  d y s t r o p h i c case of  fluorescein  ROS  s e t of r e c e p t o r s that  The  a  labeled  radiolabeled  occurred  set of r e c e p t o r s that slower  were  the attachment  (14,15). of  ROS  fluorescent  attachment that  with  were d i s r u p t e d  the rods  choline  cells  membrane  cells  IgG).  i n c u b a t i o n time  the conclusion that  defective  H  showed  1 hour  o f ROS.  THE  3  labeled  FITC-GARG  phagocytosis  suggests  two  second  of these  R O L E OF  With is  One  and  outer  tetramethylrhodamine  same  different  epithelial  that  phagocytosis  the  by  the rod  were  (  anti-rabbit  using  procedure  evidence  phagocytosis. of  by  t o the plasma  possibility  ROS,  the i n t e r n a l  to distinguish  only  10 m i n u t e s  made  goat  t o study  pigment  Experimental  been  and  followed  C mannose  used  However,  within  14  and Hall±s  dystrophic (14).  The  s u r f a c e c a n be d i s t i n g u i s h e d phagocytosis  segments  RH-GARG  IgG).  series  ROS  the  the  with  to the surface of the c e l l s  in conjunction with  a graded  with  cells  i s also appear  the for  uptake capable to  lack  receptors (14).  SYSTEM  IN  PHAGOCYTOSIS  the ingestion  i n the d y s t r o p h i c  pigment  phase  of  epithelial  phagocytosis cells,  i t  -10-  seemed or  possible  functioning  directly cells.  Haley to  protein  and  proteins  the in  that  distributed  the  RPE  ROS  phagocytosis  dystrophic  involving  not  the  The  ingestion  involve  actin  It  may  be  In  the  to  the  but  RPE  changes of  study  ROS  other  of  actin,  that  an  and  They  apparent  spreading  and  fully  unlikely  that  an  for  the  responsible  phagocytic  i s the  at  only  a  filaments  seen  in  of  ROS  be  filament the  since  by  inward  would  lateral from  a l l  expected arrays  cell  to (17).  periphery.  leukocytes,  impairment  microtubules  dystrophic  altered  actin  However,  activated  antibodies  (19).  spread  (17).  to  distinct  in  an  locomotion  (18).  of  no  that  polymorphonuclear  particles  found  in  cell  cytoskeletal  surrounded  filaments  distribution  cultures  major  extend  RPE  Other  show  packets  caused  immunofluorescence the  than  are  phagocytic  human  actin  is  electrophoresis  ingestion  that  disc  in actin  of  for  processes  gel  (16).  becomes  Actin  other  major  cells  array  mobilization  actin. in  unique  RPE  packets  neutrophilic  ingestion  the  actin  suited  of  actin  functioning  Indirect  is a  antibodies  The  disc  sides.  the  represent  the  protein  mechanism  actin  in  of  case  that  involve  2-dimensional  actin  is  at  the  with  with  protrusions  and  ingestion  attachment.  processes  abnormal  contractile  the  may  normally  of  might  i t i s a c t i v e l y synthesized  mechanism  sites  defect  demonstrated  Studies  ingestion  this  fluoragraphy  of  proteins.  RPE  of  involved  coupled  few  that  tubulin  in  microtubule RPE  distribution defect  to  in  cells, of  these  rat  were RPE  defect  cell in  rendering  microtubules cells.  use  both i t is  -11-  ENERGY  A  SOURCE  OF  bovine  PHAGOCYTOSIS  RPE  organ  culture  125  analysis  of the phagocytosis  of  particles  (20).  was  colchicine  and  cytoskeletal appeared were  to  ingested.  the  (21).  into  and Chader  t o measure  for  B  the  chick  indicating Also,  membrane  of c e l l s RPE c e l l s  the phagocytosis  altered  possible  membrane  was  endocytosis(20). .of p h a g o c y t o s i s  pigment  ROS  M  particles  the e f f e c t  of bovine  a  10  latex  during  in tissue  by  microvilli  many  of the  latex  apical  microvilli  investigated  quantitative  - globulin  significantly  i n r e g i o n s where  machinery  the  I-labeled  in phagocytosis.  the phagosomal  embryonic  used  cytochalasin  Presumably  metabolic  Using  ug/mL  be d e p l e t e d  Masterson  able  10  mechanism  incorporated  on  Phagocytosis  was  epithelium  culture  they  as measured  were  by t h e  3 uptake  of  inhibited  H-labeled by  the lack  tricarboxylic malonate. its  The  source  LATENCY  obvious  designed  conclusion  studies  When  injected  into  have  of  the  dinitrophenol  and  i s that  tricarboxylic  play  important  i n these  an  markedly  inhibitors  system,  been  acid  role  and  as  an  cells.  performed  the latency period  polystyrene  spheres  the subretinal  polystyrene  Holleyfield$s  systems  was  PHAGOCYTOSIS  t o observe  (22,23,24).  the  IN  Phagocytosis  a n d by two  cycle-cytochrome  for phagocytosis  PERIOD  Several  only  acid  segments.  of glucose  a s s o c i a t e d cytochrome  energy  were  outer  spheres  experiments  involved in  o f Rana  actively  concluded  and i n v i v o phagocytosis  and n a t i v e S a r c i n a  space  were  in vitro  that  pipiens  subflava tabpoles  phagocytized the  nature  (22). of  the  -Immaterial  presented  whether  or  not  Quantitative a  that  12-17  rabbit  pigment period  h  as  related  the  rate  the  are  ROS  are has  pinching  off a  insist  of  shed  off  package  Since  phagocytic  the  tip  packet  discs  that of  the  are  not  shed  that  a a  day  calf  and  showed  no  (23,24).  The  be  to  whether  peak  about (26).  the  RPE  the  directly  of  RPE  disc  25%  ROS,  of  the  pigment  space  to  (25).  must  to  be  occur  to  the  1 h  shed  Basinger  after  (25).  on  the  there 30  the  Further  photoreceptors  done  that  opposed  researchers  phagocytosis.  the  Studies  seemingly  recognize  burst  of  Recent  appears  shedding  ROS  tips  RPE.  by  change  to  the  many  cells  chemical  indicated  f e t u s e s as  the  subretinal  light-triggered  animals  i n the  the  initiate  to  of  However,  the  undergo  delay  beads  to  whether  into  allows  frogs  each  of  some  and  tips  or  phagocytized  into  response  their  phagocytic  hour  made a s  microvilli  discs.  foreign  fetal  latex  cells  first  evaginating  ROS  indicating  been  the  that  in  (22).  PACKETS  by  indicate  patterns  determine  cultured  appear  phagocytosis  experiments apical  not  DISCS  has  as  light  of  r a t RPE  i n the  does  ROS  shedding, ROS  in explant while  can  phagocytized  phagocytosis  to  being  this  of  of  showed onset  by  of  tips  before  triggered  OF  microvilli  ROS  be  cells  phagocytosis.  prior  epithelium the  cells  discussion  shown  that  the  occured  OFF"  pinched  evidence  on  processes  "PINCHING  Considerable fragments  done  ingestion  apical  epithelial  material will  epithelial  of  S H E D D I N G OR  pigment  latency period  abundance to  the  studies  showed  latent  to  shed  phagocytic  was  minutes  a  2-3 in  h the  -13-  adult,  upon  first  evidence  population short was  of  latent  a  correspond The  a  increased a  One and  to  retinal by  of  i n the  This  late  levels  cycle.  the  burst  rate  onset  prerequisite  of  of  recent RPE RPE  in culture  and  on  decrease  increasingly  common  with  as  many  in as  s i x or  morphological  pattern  of  light  to  some  degree  by  of  cell  growth  developed  spindle  shaped  cells  the  IN  i s by  grown  to  possibly may  i n the growth  study of  cells to  of  explant  culture  observed  present.  of  structure cells  (28). to  were  Mosaic, with  cells  were  increased  i n some c e l l s . to  be  The  The  influenced  mosaic explants  these  in  evolve  time  original  periphery  is  cultures  Multinucleate  the  ROS  light  these  observed  of  exposing  CULTURE  c o l o n i e s appeared  i n the  of  (25).  were  nuclei  adjacent  followed  that  s i x months  eight  of  the  ROS  phagocytize  to  pigment.  number  with  phagocytosis  indicating  as  up  patterns  the  activity  s t u d i e d by  ability  the  duration  were  was  TISSUE  the  of  "primed"  a  initial  correspond  phagocytosis  light RPE  for periods  in  An  phagocytic  of  the  phagocytosis.  The  cells  s p i n d l e growth  of  for phagocytosis  cells  indicates  phagocytosis  non-specific  developments  more  of  would  of  of  the  result  development.  phagocytosis  dependence  to  a  cells.  This  basal  pattern  CELLS  general  as  RPE  This  PIGMENT E P I T H E L I A L  maintained  a  followed  light-dark  culture.  tubular  during  (15).  prior  (27).  cyclic  decrease  the  function  tissue  light  in rate  ROS  definite  RETINAL  a  phagocytic  to  of  in chick  increase  "unprimed"  not  of  period  subsequent  rats  onset  fetuses  observed  initial by  the  pattern while  colonies.  In  -14-  areas  at  some  arrangements  distance  of  epitheloid  microenvironment expression  of  the  melanotic  that  time  was  components  activity,  and and  induced  mature  more  Berman,  moderate  the  density  task  as  red  to  obtain  the  of  the  highly  purified  EFFECT  OF  morphological  and  tubular  The  in  cellular  the  final  RPE  cells  as  cells, and  cells  CELL  done enzymatic  cells  in  ROS,  viable  and  and  Siakotos  have from  have  cells  with  succeeded bovine  in  eyes  by  Ficoll  detach  the  the  by  as  RPE  pigment gentle  cells  pigment  90%  granules  a  from and  pigment  epithelial  cells  must  placed  obtaining  subcellular  effects  pigmentation  as  membrane  isolate  RPE  Several  t i s s u e c u l t u r e with to  MORPHOLOGY AND  on  of  tyrosinase  epithelial  Jones  were  Emphasis for  in  c e n t r i f u g a t i o n in a  basement to  dilution  synthesis.  well  and  recovery goals  a  (30,31).  buffers,  by  intact  RPE  cell  embryonic  increase  epithelial  the  blood  RPE  studies  role  was  tyrosinase  Heller  free  procedure.  A G I N G ON  (28).  decay  p u r i f y i n g pigment  from  finally  Other  of  possible,  contaminating  There  Feeney  Primary  procedure  end  of  pigment  (34). cells  decisive  growth,  of  and  followed  epithelial  a  (29).  cell  type  in calcium  gradient  viability  by  h  (32,33).  retinal  dissociation  play  explants,  observed  d i b u t y r y l cyclic-AMP  success  purifying  36  Schwell,  undertaken  were  in cultured chick  cessation  prostaglandins a  to  original  differentiation.  found  doubling  the  cells  appears  cell  Whittaker  from  cells of  be  on  at  fractionation.  ENZYME  have  aging  ACTIVITY  looked  (35).  at  Lipofusion  the was  -15-  found  to  human  and  months  accumulate r a t RPE.  of  age  and  Apical  at  which  This  corresponds  with  cells  from  month  may  be  4  RPE  in  the  activity  cells  lysosomal  (35).  fraction  degradation rhodopsin  of  The  was  i s slowly  rats.  was  no  The  outer  in  thin  up  11  tubular  in  and  of  the  in  proteins  rat  RPE rate  to  an  In  terms  of  in the  young  and  old  a  RPE  phase  of  the  by  initial  (36).  degraded  form.  renewal  rhodopsin  after  to  response  RPE.  of  in  segment  perhaps  occur  completely  senescence  i n phagosomes  degradation to  long  difference  membrane  but  are  during  become more  animals,  found  other  they  capability  there  size  reduction  old  older  in  microvilli  point  in phagocytic  phosphatase  gel  32  slowed  impairment  rat  to  increase  as  acid  Subsequently, demonstrated  by  electrophoresis.  LECTIN  CELL  The may lack  be  lack the  of  thought  of  to  be  LABELS  ROS  result  such  surfaces. find  SURFACE  of  involved  (37).  that  molecules  phagocytosis  in  mannopyranoside, numbers mannose,  of  the and  packets  and  Heath  of  D-fructose,  and  might  'act  D-mannose disc  lectins  Basinger as  system.  receptors  on  molecules been  of  looked  at  markers  D-galactose,  to  performed  are cell to  surface hypothesis  for  ROS  RPE.  D-glucose,  on  the  the  L-fucose,  i n the  cells  or  cell  a l l significantly found  RPE  glycoproteins  have  arrangement  frog  ROS  surface  r e c o g n i t i o n of  studies using  carbohydrates  in dystrophic  Carbohydrates  i n the  distribution  sugar  found  defective cell  receptors.  Recently,  the  phagocytosis  and  disc  «< - m e t h y l - D reduced D-fucose, sucrose  the Lwere  - 1 6 -  without  significant  Ultrastructural effective  on  phagocytosis Nir complexes agglutinin cells  of  ROS  Ricinus  (WGA),  (38).  They  sites  somewhat  (ferritin-RCA) a  more  plasma RPE  clustered  cells  also  the  more  pattern which  showed  A  the  showed an  process  A  (Con  a l l three Fer-WGA  of  were  than  on  ferritin-lectin  (RCA), A)  to  bound  wheat  fixed to  germ  frog  RPE  the  pigment  (ferritin-WGA)  binding  than  A).  microvilli  irregular  rather  distributed  sparse  sugars  (37).  (ferritin-Con  to  concentration.  the  agglutinin  regularly  Fer-Con  that  binding  concanavalin that  same  packets  quite clearly.  and  membrane  disc at  found  cells  shedding  communis  and  epithelial were  shed  looked  the  indicated  disc  already  Hall  of  at  examination the  and  effect  the  Fer-RCA  separated  binding  Fer-RCA  of  the  distribution  of  by  bound  in  areas  of  lectin. the  Fer-Con  Rat A  (39). In both  both proximal  whereas, labeled Lens  normal and  RCA both  normal  s t a i n e d the  than  diseased  diminished  of rate  IMMUNOLOGICAL  membrane  primarily  and  RPE  RCS  certain of  TO  distal RPE  uniformly of  regions  microvilli  specific of  RPE  normal  microvilli  (40).  Con  and  mannose  and  more  intensely  accessibility  surface  be  i n RCS  STUDYING  s t u d i e s have  sugars  been  may  A  sparsely,  for rats  labeled  D i f f e r e n c e s i n the  phagocytosis  APPROACHES  Immunological  cell  WGA  surfaces  (LCA),  microvilli  rats.  rats,  the  dystrophic  agglutinin  glucose,  composition  dystrophic  distal  labeled  culinaris  the  and  related  or  to  the  site  of  retina.  MEMBRANES  concerned  with  the  -17-  antibody-antigen performed  action.  involved  antisera  Until  against  phagocytosis  in retinal  significant  advancement  fusion  of  of  monoclonal  specific  cell  provide  components  several  tissue. and and  To  retinal  studying  (14). A  research  was  the  using  cell  Milstein  (41,42).  by c o n t i n u o u s  exquisite  more  cultures  serological  for light  and  and  electron  the l o c a t i o n and d i s t r i b u t i o n  over  the past  antibodies  being  monoclonal  raised against fully  raised  segments  as t o t h e i r  epithelial  few. y e a r s  antibodies  rod outer  no m o n o c l o n a l  pigment  for  cells  and  produced  antibodies  a polyclonal  antibodies  by R o h l e r  into  studies  monoclonal  date  segments  (monoclonal)  provide  used  of  in cells.  characterized  (43).  outer  immunological  Labeled  Anti-rhodopsin  Rho-1D4  immunocytochemistry  Hall  epithelial  specificity  insight  Immunological in  in  lines  probes.  microscopy  pigment  developed  of defined  biochemical  rod  monospecific  techniques  Antibodies  a l l  polyclonal antibodies.  directed  production  recently  cell  resulted  against  retinal  designated have  antigenic  antibodies  have  site  specific  plasma  been  Rho-4A2 localized  of  action  f o r the  bovine  membrane  have  been  reported.  THESIS  INVESTIGATION  My bovine  thesis RPE  receptors first  i n v e s t i g a t i o n was  cell  involved  approached  preparation  and  plasma  designed  membrane  i n the phagocytosis used to  was  to obtain  characterize  and  to  characterize  possible  of r o d outer a bovine the  cell  surface  segments.  RPE p l a s m a  polypeptides  the  The  membrane by  SDS-  - 1 8 -  polyacrylamide  gel  electophoresis. was  antibody  technique  enriched  plasma  membrane  preparation  tissue  culture.  Finally,  bovine  culture  to assist  i n the i d e n t i f i c a t i o n of c e l l  as  well  as  of  bovine  to establish rod outer  employed  Secondly,  a new  segments.  to raise  assay  RPE  the  antibodies  a n d RPE cells  to  cells  were  for studying  monoclonal  grown  surface the  the  RPE  grown  in  in  tissue  receptors  phagocytosis  -19-  EXPERIMENTAL  1.  PROCEDURES  MATERIALS All  general  obtained  from  laboratory Sigma  chemicals of  Chemical,  BDH  reagent  Chemicals,  grade  were  or  Fisher  Scientific. Ficoll Dimethyl  400  sulfoxide  Scientific. (SDS) in  was  BDH  and cyanogen Chemicals  and t h e p o l y e t h y l e n e  SDS-gel  Chemicals  Cytochrome Co. s u p p l i e d  Molecular Laboratories inhibitor  M. = 21,500, bovine  r  M =92,500, Bovine  rhodopsin  bromide  Hargrave  liquid  digest  which  were  analysis Goat  were  purchased  from  Sigma.  used  Organic Baker  M  soybean  were  r  isolated  t o t h e method  by r e v e r s e - p h a s e h i g h  Ig antisera  b  M =200,000.  N-terminal  (HPLC) a n d c h a r a c t e r i z e d  trypsin  phosphorylase  and myosin  peptides  Rad  ovalbumin  r  =66,200,  by P i o t r  Bio  M =31,000,  T h e 2-39 a n d 2-16  Antibodies  sulphate  J.T.  from  r  of rhodopsin according  generously supplied  anti-mouse  by  Eastman  purchased  =116,250,  purified  chromatography  from  M =14,400,  albumin M  dodecyl  Fisher  B-mercaptoethanol  anhydrase  N-terminus  et al.(44).  peptides  lysozyme  serum  Chemicals.  nitrate.  standards  B-galactosidase  r  supplied  Fine  o b t a i n e d from  t h e sodium  was p u r c h a s e d  carbonic  r  were  (PEG).  the silver  weight  Pharmacia  bromide  glycol  was  included:  M = 45,000,  cyanogen  c  from  supplied  electrophoresis  Chemicals.  were  purchased  by  from of  rhodopsin preformance amino  acid  Czaykowski.  and r a b b i t  anti-actin  antisera  I n c o r p o r a t e d and M i l e s - Y e d a L t d . ,  respect ively. Iscove's  Modified  Dulbecco's  Medium  (IMDM)  purchased  from  -20-  GIBCO  Laboratories  with  the  medium (100  filter  units/mL),  ug/mL) a s w e l l  as  manner.  atmosphere trypsin  RPMI  collagenase  Inc; Ricinus Ltd.;  A  MgCl .6H 0,  0.11  2  pH  8.0  g NaCl,  of  distilled  2.  3  PROTEIN All  Lowry 3.  A  ( C o n A)  and  prepared  in  a t 37°C  in  a an  incubator.  The  from  Difco  from  Vector  respectively.  was  purchased  agglutinin  wheat  Buffer  germ  1 M^  =120,000  agglutinin  and  A.  g KCl,  7 mg  1.22  contained  g KH P0 2  7.4.  RIA  g  (RCA)  (WGA)  Tris,  PMSF p e r l i t e r one  o f PMSF  Phosphate-buffer  0.2  a t pH  2% F C S  B  NaCl,  the exception  as B u f f e r  water  g  and  2  with  0.2  0.58  g CaCl ,  8.5.  concentration  NaN ,  was  purchased  Corporation,  (1.25  a l l s u p p l i e d by  maintained  were  communis  contained  concentrations  0.1%  used  penicillin  fungizone  (FCS),  c u l t u r e medium  culture  from  Mannheim.  Buffer  water,  serum  and  water  The  with  90% a i r i n a h u m i d i f i e d  concanavalin  2  supplemented  c u l t u r e s were  Purified  Boehringer  1640  distilled  bicarbonate.  calf  2  Miles-Yeda  and  fetal  and M i l l i p o r e  from  g of sodium  with  10-20%  Laboratories  Laboratories  liter  (100 ug/mL),  10% C 0 ,  and  t o one  streptomycin  A l lcell  of  up  sterilized  Laboratories.  similar  made  a d d i t i o n o f 3.7  was  GIBCO  was  and  4  third  which  of  2  these  at  (PBS)  g Na HP0  4  buffer c o n s i s t e d of  g  distilled  was  saline 2.1  of  1.02  equal  included per  liter  1%  BSA,  i n PBS.  ASSAYS  protein concentrations  et al.(45)  using  ISOLATION  OF  BOVINE  Usually  20  t o 50  BSA  as a  RETINAL fresh  were  determined  by  t h e method  of  standard. PIGMENT  bovine  EPITHELIAL  eyes  ( no more  CELLS than  3  hours  post  mortem) were  were  cooled  to a  demonstrated to  the  transported temperature  that  on of  the neural  epithelial  layer  i c e to the laboratory 10-12°C.  retina  when  where  Feeney-Burns  proper  the bovine  adheres  eyes  and  they  Berman  more  loosely  a r e kept  at  this  of a  pair  temperature (46). The of  eye b a l l  curved  the  scissors  level  of  squeezing humor  was  initially  and  posterior  the anterior  the ora serrata.  the optic  fell  cup,  out l e a v i n g portion  completely  of  covering around  was  not to severe  After once pieces  2 mL  of  contamination. an  additional  calcium rod  outer  tissue,  2 mL  methods such  but  the  optic  cup  and  remove  I n some c a s e s of Buffer This  segments t h a t  tried  cup.  were  as,  Two  the melanotic  removed or s c l e r a  and  most  the optic  of cold 1 minute  attached cells  and d i s s e c t i n g method  was  Buffer  A  of g e n t l e RPE  in  the  retina cup  was  piece.  c u p was  removed  Care  EDTA  remove  again but  with  without  some  t o t h e RPE  of the  cells.  the o p t i c  the c e l l s  cup  from  the  the c e l l s o f f  added t o the  from  stray  erythrocyte  rinsed  from  rinsed  any  the  brushing  cells  vitreous  layers.  to brush  were  gently  retina  i n one  0.01%  helped  t h e RPE  and a m e l a n o t i c  and  the optic  c u p was  wash  c u t at.  translucent  of  A containing  loosely  and  the optic  treatment  effective  approximately  of  This  aspirating  mL  The  portion  and  second  the eye  the aqueous and  removed  f o r removing  t h e most  cup.  A.  tissue  and magnesium.  Several were  of B u f f e r  been  t h e e y e was  choroid,  t h e chorc-id had  retina  nerve  the point  of  tilting and  the s c l e r a ,  the optic  the retina  with  the lens  the posterior  with  portion  By  the optic  collected taken  punctured  was the  optic  used  to  Bruch's  -22-  membrane. a  The  chilled  cell  50  was  used  cell  suspension  and 4.  to  mL  plasma  twice  minutes  at  of  the  Wheaton  in a  30  10%  spun  gauge Buffer  of  in a  in  collected  needle. and  at  Both  of  cold RPE  cellular  to  Buffer  A  cells.  The  fractionation  mL  and  0.01%  with  was  at  25,000  The  Buffer  25,000 between  were  A  A  rpm  and  SS  40%  for  at  plasma and  rotor  was buffer  following chilled were  3 minutes.  An  3  and  sucrose  and  hours.  The  Ficoll/sucrose suspension  15,000  rpm  membrane one  SW  27.1  at  pellet 20%  gradient  rotor.  sucrose with  for  volume  sucrose  separately 34  eyes  a  membrane  (w/w)  10  homogenate  the  Beckman  for  nuclei  (w/w)  continuous  diluted  Sorvall  42%  spun  The  in a  34%  for  the  plasma  Buffer  (w/w)  The  at  and  rotor.  rpm  s t r o k e s of  of  collected  bovine  EDTA. The  to  rotor  20-50%  in a  added  lOOOg  was  hypotonic  480g  top  volume  50  grinder. at  MEMBRANE  suspension  at  of  5  PLASMA  cell  30  on  34  CELL  from  loaded  SS  bands  pelleted  400  volume  a  7.4,  needle.  one  onto  cells  in  was  Sorvall  overnight  future  centifugation  gauge  one  RPE  pH  Ficoll  SW  14  with  loaded  were  A  by  membrane a  transferred  remaining  centrifugation  tissue  27.0  with  any  epithelial  homogenized  Beckman  resuspended  bands  were  by  4°C  was  minutes  sucrose, and  at  suspension  diluted  was  A  pellet  pelleted  plasma  interface was  pigment  Potter-Elvehjem  total  of  was  1 mL  PIGMENT E P I T H E L I A L  Buffer The  of  cup  Another  ice for  0.007M T r i s ,  volume  cup  optic  isolation.  retinal  cells  subsequently  crude  on  RETINAL  in  i n the  tube.  optic  kept  overnight  morning  spun  OF  4°C.  consisting  the  was  bovine  swelled  the  membrane  washed  equal  centrifuge  rinse  PREPARATION The  suspension  with one  15,000  Two a  14  volume  rpm  for  -23-  30  minutes.  enrichment  based  activity.  and  described  order  to  remove  solubilized  Na R ATPase  specific  +  reductase,  1  in  and c  respectively  1%  solubilized  clinical  bench  top centrifuge  5.  ENZYME  A.  5'NUCLEOTIDASE  column. as  as  suspension  Inc.  1  were  a t 2,500  until  Rho-1D4  (1  mg/mL)  mL  of  was  Rho-1D4  temperature  beads  f o r 10  by  Cuatrecasas  unit.  The  while  the  by c e n t r i f u g a t i o n  rpm  was  Sepharose  microfiltration  eluted  s t o r e d a t -20°C  to  a t room  to the Sepharose  the  described  coupled  with  from  membrane  a s d e s c r i b e d by  f o r 30 m i n u t e s  bound  components  m a t e r i a l was  method  Systems  rhodopsin  membrane  covalently  and i n c u b a t e d  2BC1 b e a d s  remaining  affinity  o f membrane  CHAPS  rhodopsin  f o r rhodopsin  ( 4 3 ) [ was  mL  Bioanalytical  contaminating  detergent  activation  antibody-Sepharose  in  minutes.  a  The  required.  ASSAYS  Nucleotidase  to  Solyam  in  0.5  M  was  et a l . (48). Tris  monophosphate, minutes  membrane  +  the contaminating  Jspecific  MacKenzie  Generally  5'  c  on a n a n t i - r h o d o p s i n  by t h e CNBr-  eluted  and  plasma  q u a n t i t a t e d by s u c c i n a t e c y t o c h r o m e  cytochrome  antibody  and  a  for  o f t h e p r e p a r a t i o n by m i t o c h o n d r i a  preparation,  monoclonal  in  assayed  5$ n u c l e o t i d a s e  NADH  chromatographed  (47).  were  below.  membrane  2BC1  on  r e t i c u l u m were  reductase  Molday  bands  Contamination  endoplasmic  In  Both  a t 37°C  assayed Membrane  buffer, pH  7.5,  10 mM in  the reaction  ,  with  protein MgCl  a total was  2  modifications, according (40-60  and  10  volume  stopped  of  u g ) was mM 1  by a d d i n g  incubated  adenosine mL. 1 mL  After of  5' 20 cold  -24-  10%  TCA  3000  rpm f o r 5 m i n u t e s .  under  and the p r e c i p i t a t e A heat  t h e same c o n d i t i o n s .  determined  by t h e F i s k e  volume  the assay  of  ammonium of was  molybdate  FeS0 .7H 0) 4  measured  +  K +  using  50  mM  ATPase  +  were  was r u n  To a  0.5  reagent,  The a b s o r b a n c e 15-30  from  a  minutes.  standard  mL  (0.1g  o f 0.5 M H ^ S O ^ w i t h  0.4  g  a t 700 nm Phosphate  curve  run  phosphate.  was a s s a y e d b y a m o d i f i c a t i o n et  incubated  Tris-Cl,  Tris-ATP  between  at  g e n e r a t e d was  (49).  of the assay  and vortexed.  calculated  control  phosphate  method  i n 10.0 mL  inorganic  Costantino-Ceccarin protein)  centrifugation  ATPASE  +  Na K  dissolved  were  simultaneously Na  0.5 mL  f o r a l l samples  concentrations  B.  The i n o r g a n i c  sample  by  d e n a t u r e d membrane  a n d Subbarow  was a d d e d  2  was p e l l e t e d  al.(50).  Thirty  uL  in a total  volume  o f 0.5 mL  pH 7.4,  f o r 15 m i n u t e s  of t h e method o f  100 mM  a t 37°C.  NaCl,  samples  a n d 3 mM  The r e a c t i o n  ug  consisting of  MgCl  was  (30-60  with  2  started  3  mM  by t h e - 4  addition M  o f 20 u L o f 0.5 M KC1 i n t h e p r e s e n c e o r a b s e n c e  ouabain.  The  terminated  reaction  by t h e a d d i t i o n  samples  were  at  rpm f o r 5 m i n u t e s  3000  was r u n f o r 30 m i n u t e s  chilled  o f 0.5 mL  on i c e f o r f i v e  aliquots  of  0.5 mL  from  phosphate  by t h e method  to pellet each  minutes  10%  and then  the precipitate.  sample  of Fiske  of i c ecold  prior  were  assayed  and Subbarrow  o f 10  to TCA.  being The  centrifuged Supernatant  for  (49) as  inorganic previously  described. C.  NADPH CYTOCHROME NADPH  the  method  C  REDUCTASE  cytochrome  of Ragnotti  c reductase  e t a l . (51). A  activity  50 u L s a m p l e  was a s s a y e d b y ( 50-100 ug  -25-  protein) umoles and  was  added  of potassium  0.075  started  umoles  activity  at  was  550 D.  X  of a 0.05  solution umoles  a t pH  KCN,  7.6.  30 u L o f 3 mM recorded  consisting  KC1,  reaction  was  The  f o r 4 minutes.  from the molar  extinction  increase i n  The  specific  coefficient  of  f o r cytochrome c  (reduced  minus  activity  was  oxidized)  at  nm. S U C C I N A T E CYTOCHROME C  method added  of  t o 0.92  cytochrome  (52).  mL  of a  2.5  c , a t pH  recorded  A  reductase  50 u L s a m p l e  solution  umoles  30 u L o f 0.6 M  was  REDUCTASE  cytochrome c  King  phosphate,  KCN,  succinate.  for 3 minutes.  ( 50-100 ug  consisting  of  0.3  EDTA,  umoles  7.4. T h e r e a c t i o n The  measured  was  increase  The s p e c i f i c  100 u m o l e s and  started  from  the molar  cytochrome  c  extinction  (reduced  minus  SDS-POLYACRYLAMIDE RPE  cell  by  mg/mL) w e r e  solution  consisting  10% were  oxidized)  of  membrane  activity  a t 550  o f 5% SDS,  i n an e q u a l 40% s u c r o s e ,  B - m e r c a p t o e t h a n o l a n d 4% b r o m o p h e n o l applied  gel  or a  or  0.75  according  to wells  10% c o n t i n u o u s mm to  X  18.7 X  or rod outer  solubilized  of a  potassium  0.1  umoles  the a d d i t i o n  was  12.0 cm)  and  the procedure  GEL  cm  for  TRANSFER  segment volume 0.01  preparations of  denaturing  M Tris,  Samples  (53).  pH  6.8,  (15-25  gradient  g e l ( 0 . 7 5 mm  electrophoresis of Laemmli  -1  nm.  blue.  slab  nm  calculated  10 M  6.5-15% p o l y a c r y l a m i d e  polyacrylamide  was  i n a b s o r b a n c e a t 550  G E L E L E C T R O P H O R E S I S AND  plasma  (1.0-5.0  coefficient  by t h e  protein)  3-1  6.  66  100 u m o l e s  The  NADPH.  of  -1 cm  Succinate  of  of was  calculated  10 M  mL  cytochrome c,  550 nm  3 -1  1.42  phosphate,  by t h e a d d i t i o n  absorbance  18.7  to  X  uL) slab  5.0  was  carried  Gel  slices  cm out  were  either  stained  al.(54)  or  stained  some  cases  In as  adapted  Briefly, mM  with  from  with gels  SDS-gels  washed  nitrocellulose  2  the  nitrocellulose for  in  2-16  consisting  of  mM  and  NaN , 3  0.15  immediately  to  a  changes  of  for  5  to  0.1  M  minutes  in  sodium  phosphate,  place  in  in  0.02  M  a  Bio  sodium  the  remaining  pH  9.0,  temperature.  The  was  gel  the  pH  Rad  100  0.06  in  the  at  19  V  water  and  transfer binding buffer  1 mM  EDTA,  1  pH  7.5.  and  incubated  the  gel  was  pH two  7.4,  groups and  morning,  for were 1% the  method washed  7.4,  The  mL  in  Clark  three  changes  each.  at  of  transfer  containing  100  5 minutes  apparatus  pH  electrophoretic  by  by  glycine  following  transblot  immunoblot  at  20  pure  remaining  BSA  distilled  of  Rad out  of  buffer).  Following  any  4%  underwent  for  transblot  M  with  al.(56).  antibody.  ,followed  reactive  h.  of  sodium  phosphate,  phosphate,  Bio  carried  12-16  et  sheet  phosphate,  mM  prepared  7.4,  wet  a  transfer  (transfer  10  transfer  each  a  et  al.(55).  changes  with  slices  sodium  SDS  in was  mL  temperature  rinsed  paper  CNBr-  with  room  for  et  Towbin  100  0.01%  quenched  monoclonal  CNBr-activated Prior  was  Triton-X  cases,  al.(57).  buffer  of  with  placed  Fairbanks  electrophoretic  against  transfer  NaCl,  paper  with  other  M  2%  nitrocellulose  at  and  to  Wray  procedure  sandwiched and  to  to  twice  NaEDTA,  paper  h  according  washed  transfer  according  subjected  Electrophoretic 4°C  blue  transfer  membrane  A  In  mM  were  0.5  sites  at  were  were  gels  apparatus.  silver  blotting  Tris-Acetate,  The  coomassie  35  2-4  h.  V  filter  0.1% in  0.02  The  transfer  and  1.5  After  quenched BSA  100  in  the  mL SDS M took  at  4°C  transfer  Tris  overnight papers  A  et  buffer, at  were  room rinsed  -27-  in  distilled  water  and  incubated  with  the appropriate  monoclonal  antibody. 7.  PREPARATION  ANTIBODY  GOAT ANTI-MOUSE  I g AND  GOAT  ANTI-RABBIT  Ig  REAGENTS  Goat  anti-mouse  affinity column,  OF  Ig and  chromatography respectively  goat  anti-rabbit  on a m o u s e  ( 5 8 ) . Both  I g were  Ig or rabbit  goat  anti-mouse  p u r i f i e d by  Ig Sepharose Ig and goat  4B  anti-  1 25 rabbit  Ig antibodies  method  (59) t o g i v e  mg  of .  were  was  dissolved  .  addition  of  addition  500  with  I by  specific activities  the antibody  .  labeled  of  i n 0.1  1-2 M  the chloramine  x  PBS  10^ d p m / u g . followed  T  One  by  the  125  u C i o f Na  I.  The  o f 5 uL o f c h l o r a m i n e  T  reaction  was  initiated  (4 mg/mL) a t r o o m  by t h e  temperature.  1 25 After  10 m i n u t e s  reaction  the free  mixture  at  Centrex  Microfilter  exchange  resin.  8.  POLYPEPTIDE Transfer  10  mL  (100  fold  for  1-2  diluted) h with  followed 0.1  M  by air  were  by  two  changes  with  Ig  e x t e n s i v e washing and  rinsed  for  with  i n RIA  fluid  10  0.5  centrifuging  minutes g o f AG  of  i n wash  10  The  buffer  goat  and  the  through 1—X10  a  anion  The  papers  buffer  papers  anti-mouse  The  were  consisting  dpm/mL) f o r 1-2  as b e f o r e .  incubated  anti-actin  immunoblot  X-100.  6  buffer  or r a b b i t  h a t 23°C.  changes  (1-2 x  r e m o v e d by  ANTIBODY  a n d 1% T r i t o n 125 I-labeled  anti-rabbit  dried  BY  f o r 1-2  rpm  loaded  culture  several  glycine,  incubated goat  unit  of hybridoma  I was  1500-2000  DETECTION  papers  Na  Ig or  .antisera  then  M  on  BSA urea,  subsequently 125 I-labeled  h a t 23°C  followed  n i t r o c e l l u l o s e papers  subjected to autoradiography  washed  without  of 2  were  with  preflashed  were Kodak  -28-  Royal at  X-Omat  23°C.  place 9.  SILVER  gels  silver  The  fresh  stain 38%  The  distilled  developed  by  immunized  with  cells  grown  complete  wash  in deionized  One  vial  containing  days  x  agitation water.  The  a n d 0.25 m l  of  than  10  the g e l i n  45%  t o normal  high  21 mL  The g e l  f o r less  T h e g e l was s w e l l e d  (41).  of monoclonal  Male  culture,  or female  contrast  size i n film.  emulsified  the spleen  after  the last  10^ c e l l s  nitrogen  mice  preparation 0.1  mL  injections myeloma  were were o r RPE  of Freund's were  carried  cell  fusion  immunization.  frozen  was t h a w e d  with  cell-  C U L T U R E MYELOMA  liquid  antibodies BALB/C  membrane  intraperitoneal  with  OF T I S S U E of  1  o f 500 mL  Kodak  al.(55).  nitrate  gentle  acid  acid  MICE  Three  apart  four  PREPARATION  volume  et  distilled  o f 1% c i t r i c  acetic  o f 100 mL.  constant  100 u g o f R P E p l a s m a  in tissue  place  with  the production  Milstein  weeks  took  TECHNIQUES  OF B A L B / C  a  0.8 g s i l v e r  was s t o p p e d b y p l a c i n g  acid.  ANTIBODY  adjuvant.  three  taking B.  minutes  in a total  for  h  sarcosine.  o f Wray  volume  and photographed with  IMMUNIZATION Technique  out  15  acetic  MONOCLONAL  f o r 12-48  i n 50% m e t h a n o l / 1 0 %  in a total  development  water  0.4% N - l a u r o y l  contained  was d e v e l o p e d i n 2.5 mL  methanol/10%  A.  hydroxide  formaldehyde  screen  papers, a l l washing  to the protocol  solution  by a 5 m i n u t e  minutes.  10.  for2 h  according  staining  for  activated  o f PBS w i t h  washed  stained  0.36% s o d i u m  followed  o f CNBr-  intensifying  TECHNIQUE  were  stained  an X - r a y  changes  STAINING  and  was  with  In t h e case  i n several  SDS  of  film  CELLS NS-1  i n a 37°C  (myeloma) water  bath.  cells The  -29-  cells 10%  were  washed  fetal  minutes.  calf The  medium.  A  trypan  blue  once  i n 10 mL  serum  cells  sample  (FCS) and p e l l e t e d  were  resuspended  of the c e l l s  and  o f IMDM m e d i u m  the  at  in 5  cells  1500  mL  was d i l u t e d  viable  supplemented rpm  of  for  5  supplemented  one t o one w i t h  were  with  counted  0.4% on  a  5 haemocytometer. per  mL  The c e l l s  i n 10 mL p e t r i  4 x  numbers  x  10 c e l l s  were  available  PRODUCTION The  the  dishes.  plated  out at 1 x  The c e l l s  were  10  viable  maintained  cells  between  5  6  10 - 5  C  were  tubes.  cells  f o r 7-10  fora cell  OF HYBRIDOMA  myeloma  10  p e r mL  before  sufficient  fusion.  CELLS  grown  in tissue  culture  mL  petri  d i s h e s and c o l l e c t e d  The  cells  were  collectively  days  spun  resuspended  down in  in  were  aspirated  polystyrene  a t 1500 rpm f o r 5  10  mL  of  serum  off  sterile  minutes free  and  medium.  7 Approximately A  thymocyte  which  as a  from  above  o f serum  sterile  cell  acted  excised mL  2 x 1 0  free  piece  through  a  viable  suspension  feeder  layer  the r a theart IMDM m e d i u m  of cheese  20.5 gauge  centrifuged  cells  was p r e p a r e d forcell  in  10  mL  The c e l l into  0.4 uM a m i n o p t e r i n ,  20%  stored  performed The  a  a t 37°C.  on t h e h a e m o c y t o m e t e r spleen c e l l  suspension  from  fusion.  one weaning r a t  growth.  The thymus broken  t h e thymus  suspension  was  The thymocyte supplemented  16 uM t h y m i d i n e  A viable  cell  above.  was p r e p a r e d  from  u p i n 10 a  dispersed tube  and  pellet  was  with  100  uM  (1 x H A T ) a n d  count  as described  was  through  10 mL p o l y s t y r e n e  o f IMDM m e d i u m  hypoxanthine, and  fora cell  and subsequently  a t 1500 rpm f o r 10 m i n u t e s .  resuspended  FCS  used  by s q u e e z i n g  cloth. needle  were  the  was  also  immunized  -30BALB/C  mouse.  conditions IMDM  and placed  medium.  the  cells.  needle  medium  The s p l e e n  a  pellet  al.(60) . 10  ) were mixed  5  minutes.  and  1 mL  period  minute.  i n t h e same  added.  cells  o f 2 mL  o f serum 7 mL  IMDM m e d i u m in  100  b u t i n 1 mL  to  and fresh The w e l l s  IMDM were  mL  by t h e s o l i d - p h a s e  of  mouse  Galfre cells  i n a 37°C  free  radioimmune  assay  over  1  a  minute  IMDM  over  the  free  medium  was cell  and the p e l l e t  was  1  Finally, the x  HAT per  medium mL.  dishes.  The After  was a s p i r a t e d o f f  supplemented  forpositive  bath  The  in multiwell  t h e o l d medium  x  and  water  2-3 m i n u t e s .  IMDM  -et  (2  IMDM  for  10^ t h y m o c y t e s  1 x HAT m e d i u m assayed  the  a t 1500 r p m f o r  and r e p e l l e t e d .  volumes  f o r o n e week,  IMDM  free  o f serum  t h e next  5  free  ( P E G ) 1500 was a d d e d  20% FCS a n d 2 x  a t 37°C  serum  a t 1500 r p m f o r 5 m i n u t e s  resuspended  were p l a t e d  glycol  suspension over  with  in  was p l a c e d  An a d d i t i o n a l  resuspended  was  according  washed  of c e l l s  the addition  was s p u n  wells  were  gauge  of the t i t r e .  T h e s u s p e n s i o n was s t i r r e d  suspension  the  from  and p e l l e t e d  to the c e l l  incubation  o f serum  tube  cells  free  to disperse  o f serum  assay  out  serum  a t 1500 rpm f o r  in a centrifuge  2 minutes.  cells  i n 5 mL  A sample  sterile  t h r o u g h a 20.5  10 ) a n d m y e l o m a  following  supplemented  i n order  a n d spun  radioimmune  o f 50% p o l y e t h y l e n e  was  containing  (2 x  by  pellet  tube  was c a r r i e d  The p e l l e t  1  dish  under  cells  followed  added  a t 37°C.  spleen  The  of  t h e mouse  chopped  was r e s u s p e n d e d  fusion  Immune  repelleted.  petri  was f i n e l y  f o ra solid-phase cell  from  s u s p e n s i o n was f o r c e d  and incubated  The  10 mL  10 mL p o l y s t y r e n e  The  saved  was r e m o v e d  in a  The c e l l  into  minutes.  was  The s p l e e n  with  antibody  method,  20% FCS  producing  10-14  days  -31-  after D.  the fusion.  STANDARD R I A A  standard  solid-phase  antibody  secreting  developed  by MacKenzie  membrane mg/mL to  hybridomas  preparation  protein)  or  down  on f l e x  vinyl  was  washed  indistilled  for  vinyl  plate  was r i n s e d  25  plate  was w a s h e d  with  water  uL o f c u l t u r e  Aliquots  extensively  with  procedure RPE  plasma  preparation  a t 60°C  for  2 h.  The  by i n c u b a t i o n binding  1 h at  prior  room  dried plate  with  RIA  sites.  The  to incubation  temperature.  PBS t o r e m o v e  (1-2  diluted  o f 25 u L w e r e  i n PBS b u f f e r  for  t o detect  the  The bovine  the non-specific  fluid  to  segment  followed  briefly  was u s e d  1% T r i t o n X - 1 0 0 a n d  water.  microtitre wells  1 h t o quench  with  (58).  a r o douter  distilled  assay  according  and Molday  was s o l u b i l i z e d  0.25 mg/mL w i t h  buffer  radioimmune  unbound  The  antibody  1 25 and  subsequently  mouse  Ig  (  In as  some c a s e s  HYBRIDOMA  off  the flat  and  spun  washed  flat-bottom  6  I-labeled  dpm/ug)  for  extensively  1 as  goat h  anti-  at  room  before  and  were  used  Counter. actin  antigen.  Fixed  or other bovine  proteins  RPE c e l l s ,  microtitre wells,  were  also  previously used  as an  assays.  CLONING cells  from  bottomed  positive microtitre wells  wells  a t 1500 rpm f o r  mL o f IMDM  was  rhodopsin,  phase  25 u L o f  1-2 x 1 0  8 0 0 0 Gamma  i n radioimmune  Hybridoma  1  plate  i n a Beckman  invinyl  antigen E.  The  the solid  grown  with  1 5 - 4 0 ug/mL;  temperature. counted  incubated  aspirated  and t r a n s f e r r e d t o a centrifuge  5 minutes.  1 x HT m e d i u m  were  The p e l l e t  supplemented  with  tube  was r e s u s p e n d e d i n 20% FCS a n d c o u n t e d  -32on  a  in  haemocytometer. 0.1  mL  cell/well cells  Approximately  multiwell with  were  plates  2x10^  pipeted  into  One  week  colony  The  cloned  RIA  and  the  virtually for  monoclonal  ascites F.  positive  a l l wells  VIABLE  Hybridoma  cells  dishes,  before.  The  to  insulated vials G.  avoid cell  were  specifically  23°  C  with  used  was  The  or  10-14  2  x  for  days  as  10 " 5  single later  before  were  then  in culture  1  uncloned  with  marked  cells  either  box  by  until  expanded  fluid  or  in  antibody  of  of  binding  the  activity  by  of  diluted by  the was  the  1.0  mL  of  DMSO.  The  by  site  placing  FCS  frozen  in a  24  foam  hours  the  nitrogen.  of  in  antigen fluid.  concentration  and  order  action.  standard-solid  standard  10%  were  vials  as  ASSAYS  culture  removed  cells  freezer. After  liquid  a  the  in  competing  to  off  pelleted  performed  antibody  aspirated and  -70°C in  hybridoma  mixture  10%  were  concentrations  were  tube  COMPETITION  assays  uL  phase  centrifuge  plus  in a  RADIOIMMUNE  50  a  indefinitely  the  log  formation  located  define  of  in  resuspended  medium  previously  saturation uL  was  ice crystal  stored  varying  growing  growth  Competition  50  were  out  CELLS  t r a n s f e r r e d to  SOLID-PHASE  of  production  cells/well  recloned  positive.  plated  remaining  assayed  were  were  dish  wells  were  5  The  petri  the  wells  HYBRIDOMA  pellet  supplemented slowly  /mL.  5 mL  cells  in  cells  fluid.  STORAGE OF  petri  a later  were  antibody  resulting  thymocytes  thymocytes/mL. growth.  750-1000  Briefly, was  The  screened  more 50  uL  incubated culture  which  phase  solid-phase  to  RIA. for  gave  at fluid  80-90%  After  1  h  remaining  radioimmune  assay  -33described H.  above.  LOWICRYL THIN Retina  cacodylate at  4°C.  (61).  tissue buffer,  After  embedded  pH  fixed  in  changes  of  50%  100%  volume  to  Lowicryl DMF  a  K4M  1 volume  incubating  the  tissue  35°C,  Lowicryl  K4M  overnight  at  the  irradiation  Following the  blocks  were  and  collected  cut  incubated  in  non-specific of  10  gold grids  50  uL  fold  for  30  100%  of  50  method in  a  the of  at  Roth  of  100%  -35°C  series  for  1  h  K4M  at  resin  polymerized  and  The  by  2  tissue and  1  1  volume  -35°C.  After  overnight by  of  lower  DMF  followed  was  et a l .  -35°C,  2 volumes  K4M  minutes  progressively 90%  M  tissue  graded  each.  at  0.1  30-60  in  was  of  trimmed clean  PBS  with  sites.  PBS  by  copper 0.1%  at  ultraviolet  grids  culture  extensive  fold  diluted  minutes  at  room  BSA.  were  sections  for  10  were  then  fluid  for  washing  goat  stained  (60-70  grids  were  30  the  block  After  in  and  a  grids Ig  50 at 10  were  dextran-  washing  saturated  nm) pre-  minutes  i n PBS  with  to  incubated  anti-mouse  (62).  embedded  minutes  Finally,  temperature  they  resin  g r i d s . The  BSA  0.1%  5  Lowicryl  ultathin  with  of  i n PBS,  the  and  The  Rho-5A3  uL  extensively  1 h,  in  -35°C.  followed in  for  minutes  Lowicryl  resin  on  diluted  incubation in  1 h  Lowicryl  were  binding  temperature  incubated  for  the  -20°C,  30  of  polymerization  tissue,  to  for  at  at  a l l for  in  buffer  (DMF) 70%  resin  sucrose  dehydrated  mixture  and  minute  same  glutaraldehyde M  according  4°C,  -35°C  1.25% 0.25  t i s s u e was  at  at  transferred  room  the  resin  the  in  plus  dimethylformamide  temperatures:  uL  LABELING  7.2,  washing  Briefly,  100%  was  in Lowicryl  aqueous  was  SECTION  the  uranyl  -34-  acetate  for  minutes  at  Philips  200  11. A.  5-7  minutes  room  BOVINE RETINAL  Bovine  OF RPE  the  method  as  previously  was  trypsin  and  agitation  The  cell  suspension  were  petri  alcohol  sterile  Buffer  with  out  at  the c e l l s  The  and  or  glass  or u n t i l  B.  FLUORESCENT DETECTION RPE  stored  cells  The  sterile  RPE  a  with  100  with  f o r 5 minutes.  repelleted  plastic  for future  constant  gentle  subsequently  10 mM  10%  petri  The  cells  growth  was  The  pH  cell  pellet The  was  RPE  cells  i n 10 mL  Kimax  dishes were  medium  streptomycin  The  cells/mL)  tube.  Hepes,  FCS.  as b e f o r e .  5 x 1 0  i n 0.2%  was 0.25%  units/mL,  rpm  confluent  cup  3 volumes of c u l t u r e  supplemented  coverslips.  cup  to a centrifuge  ug/mL) a n d  10 mL  optic  with  were  50  and  PBS  to  dissected  optic  with  cells  diluted  fixed  harvested  A.  transferred  1500  or  Bovine  conditions.  a t 37°C  The  ( approximately  were  on  CULTURE  were  cup and  medium  dishes  days  viewed  eyes  of  collagenase  fungizone  sterilized  7-14  2 mL  minutes.  was  spun  1.5-2  by m o d i f i c a t i o n s  bovine  antibiotics (penicillin  in culture  glass  point  60-80  Fresh  sterile  units/mL  was  plated  for  in  and  were  for  IN T I S S U E  in vitro  under  o f RPMI-1640 medium  ug/mL,  washed  et a l . (63).  suspension  buffer,  100  cultured  were  o f f the optic  consisting 7.2,  cells  incubated  for  aspirated  CELLS  described  70  citrate  sections  EPITHELIAL CELLS  B O V I N E RPE  once  subsequently  The  lead  microscope.  PIGMENT  of Basu  rinsed  saturated  temperature.  electron  CULTURING  and  containing  grown  reached  glutaraldehyde,  at  at  37°C which  sub-cultured,  use.  OF  ACTIN  I N RPE  were  grown  on  CELLS  glass  coverslips  in  tissue  -35culture  f o r 2 weeks  coverslips mM  were  CaCl2  buffer with  Mg  prior  and C a  + +  RPE  were cells  antisera  for  40  incubated  goat  anti-rabbit  mounted  with  controls primary  100 C.  run with  antibody control,  was o m i t t e d  ug o f p u r i f i e d  actin  FLUORESCENT L E C T I N  Molday  (64,65)  lectin)  i n 0.02 M  by  sodium  lectins  appropriate  affinity  were  typically  acetone  prior  anti-actin  + +  The  to being  cells  viewed  microscope.  Two  I n one c o n t r o l  the  step;  i nt h e  incubated  with  step. IN VITRO  according with  .  washed  as before and  labeling  i n the f i r s t  The  FITC-labeled  washed  antibody  to  FITC  Maher (0.05  and mg/mg  pH 9.0, f o r 2 h a t 2 5 ° C . T h e  separated  from  free  dyes  ovomucoid-Sepharose,  4 B , f o r RCA: a n d S e p h a d e x G - 2 0 0 ,  preparations  i n 50%  were  and C a  + +  experiment.  the l e c t i n  column:  rabbit  fluorescent  carbonate,  were  treated  PBS  f o r 30 m i n u t e s .  BOVINE RPE C E L L S  reacting  were  same  in  i n absolute  +  slide,  are prepared  the  washed  100 u L o f  of the primary  LABELED  +  Mg  i n the f i r s t  was u s e d  lectins  fluorescent  Sepharose  Leitz  the test  a solution  Fluorescent  The c e l l s  in  coverslips  a t 4°C w i t h  on a g l a s s a  MgSO^ a n d 1.27  were  diluted,  The  The  a l la t 4°C. Again the  and C a  + +  o f PBS w i t h  Ig antibody.  50% g l y c e r o l  were  Mg  at 4°C.  changes  with  by 5 m i n u t e s  f o r 3 minutes,  f o r 40 m i n u t e s  photographed  The c e l l s  i n 100 t i m e s  minutes  0.81 mM  The c o v e r s l i p s  followed  i n PBS w i t h  i n three  were  second  a t 4°C.  incubated  was a c h i e v e d .  i n 1% p a r a f o r m a l d e h y d e  f o r 30 m i n u t e s .  +  washed  extensively  and  +  growth  i n PBS b u f f e r w i t h  i n 50% a c e t o n e  were  confluent  to fixation  f o r 3 minutes  replaced  cells  rinsed  f o r 30 m i n u t e s  acetone and  or u n t i l  f o r Con A.  h a d 4 9 5 nm/280 nm a b s o r b a n c e  on  the  for  WGA:  FITC-lectin ratios  of  -360.5-1.0. Coverslips  o f 1-2 week  1% p a r a f o r m a l d e h y d e M  sucrose  f o r 20 m i n u t e s  rinsed  37°C  f o r 5 minutes  a t 4°C.  i n PBS w i t h  The excess  washes  i n t h e same  bound  lectin  Mg  with  buffer.  lectin  Controls  15 m i n u t e s ,  for  WGA,  cells  fluorescent  were  observed  FLUORESCENT  bovine  rinsed  in  RPE c e l l s PBS w i t h  ug/mL  of the appropriate  37°C.  The  followed  coverslips  Subsequently  buffer  or incubated  E.  appropriate  the  weeks  were  37°C,  at  o f f by s u c c e s s i v e  with or  0.2  off  M  0.2 M  Fluorescent  the  methyl  D-galactose  lectin  under  lectin  bovine labeled  LECTIN LABELING  OF B O V I N E  grown  culture  + +  in tissue  and C a  with  +  treated  a  Leitz  rinsed  i n PBS w i t h  incubation period cells  were  cells  either  15 m i n u t e s , prior  that  the cells  were  continuously  at  The c o v e r s l i p s  were  washed  with  + +  in  and  100 at Ca  buffer the  a t 37°C,  +  +  at same with  to observation.  RPE C E L L S  in tissue  fluorescent lectins  weeks  minutes,  Mg  rinsed  OF B O V I N E  grown  5  i n t h e same  and rinsed  LABELING  for  RPE C E L L S  for2  and incubated  fluorescent lectin  inhibitor  RPE  +  exception 37°C.  cells  by i n c u b a t i o n  photographed  f o r an a d d i t i o n a l  CONTINUOUS F I T C - L E C T I N Unfixed  0.18  RPE  r u n by c o m p e t i n g  chibitose,  and  Mg  were  b y a 60 m i n u t e  37°C.  the  with  microscope.  D. D I S C O N T I N U O U S Unfixed  were  o r RCA, r e s p e c t i v e l y .  were  or unfixed  followed  +  fixed  pH 7 . 2 , w i t h  was w a s h e d  at  0.02 M N - N - d i a c e t y l  RPE  +  were  100 ug/mL o f t h e a p p r o p r i a t e f l u o r e s c e n t  or unbound  for  buffer,  The f i x e d  and C a  + +  mannoside, C o n A,  RPE c e l l s  i n 0.1 M c a c o d y l a t e  were  lectin.  o l d bovine  culture  as before  labeled  f o r two with  f o r 60  the  minutes  e x t e n s i v e l y i n PBS w i t h  Mg  + +  -37-  and  Ca  11.  P H A G O C Y T O S I S OF  A.  prior  to  PREPARATION  OF  Twenty-five slaughter  of  Tris, ROS  The  pH  7.2,  retinas  0.25  cheese  additional  on  Beckman of  was  SW  the  mL  solution  and  MgCl , 2  was was  27-60% 27.0  from  with  of  B.  P H A G O C Y T O S I S OF week  5 mM the  taurine, retina  at  1000  25,000  ( a p p r o x i m a t e l y 30%)  at  twice was  medium ROS  5000  Tris  rpm  at  repeated.  The  supplemented BY  o l d bovine  with  ADULT BOVINE RPE  cells  ROS  grown  ++ initially with h  at  dark 37°C  washed adapted  i n PBS ROS.  followed  by  w i t h Mg  The  cells  h.  the  filtration with  an  solution  for 5  was  discarded. once  The  and  more, spun  in  at  the  band  and  1  pH  7.4.  The  minutes.  The  Solution  volume  (HBSS)  resuspended  in  1  FCS.  CELLS  on  The  cloth  were  RPE  glucose.  pellet  Salt  10%  mM  by  buffer,  Balance  10-15  inverting  removed  4°C  in  30  20  gradient  1.5  was  M  i n Hank's  mM  The  cheese  for  for  repeated  the  sucrose  rpm  0.01  10  was  local  sucrose,  gently  and  through  continuous at  20%  solution. rpm  the  placed  followed  procedure  VITRO  from  and  and  by  IN  temperature  removed of  microscope.  ROS  room  times,  filtered  (w/w)  at  consisting  homogenizing  2 volumes  washed  mL  Two  CELLS  collected  adapted  The  centrifuged  1640  fluorescent  B O V I N E RPE  eyes  thirty  rotor  centrifugation RPMI  of  was  gradient  diluted  pellet  mM  f o r 2 minutes  a  BY  the  carefully  solution  loose  supernatant  top  were  cloth.  1-2  centrifuged  layered  dark  approximately  through  ROS  bovine  were  broken  solution  a  fresh  homogenizing  were  The  BOVINE  under  S E A L E D DARK A D A P T E D B O V I N E  house  minutes. mL  observation  IN  glass  VITRO  coverslips  were  ++ and  were  Ca  prior  to  i n c u b a t e d i n the  e x t e n s i v e washing  in the  same  incubation dark  for 5  buffer.  In  -38order ROS  t o enhance suspension  glutaraldehyde for C.  minutes to  M  fixing  dehydrated  changes  of  absolute  ethanol  oxide.  and  1-2  h  placed  in a  Seventy  stained  electron  and  S200  with  temperature. citrate  oven  h  copper  f o r 1.5-2  grids minutes  f o r 15  were  also  before  prior 4°C.  for 1  h,  they  at  OF  SAMPLES  FOR  SCANNING  room  mixture  in  of  at  room  propylene  and  embedded f o r 24  by resin  samples  h.  S200  f o r 5-7 with  observed  copper  ELECTRON  5000 grids  the grids  minutes  were  at  room  saturated  lead  on  a Philips  microscope.  PREPARATION  two  c u t on a S o r v a l l  coated  stained  and  each  to viewing,  being  buffer  oxide  The  M  30-60  facilitated  t o harden  acetate  0.1  for  minutes  was  resin.  formvar  in  at  incubation  of propylene  Prior  saturated uranyl The  oxide  in a  s e c t i o n s were  grids.  prepared  minutes  50:50  on  and  sucrose  continued  i n 100%  supported  M  in  and a l l o w e d  thick  0.2%-1%  MICROSCOPY  f o r 15 m i n u t e s  mix  in  (50%,70%,90%,  penetration  50:50  24-48  nanometer  Microtome  uncoated  60°C  ELECTRON  f o r 30-60  15 m i n u t e  resin  in a  f o l l o w e d by  a  7.2,  i n t h e same  series  propylene  Araldite/Epon samples  was  to the  fixed  i n t h e same b u f f e r  ethanol  by  pH  added  glutaraldehyde  washed  tetroxide  ethanol)  followed  incubating  Ultra  an  also  (TEM).  i n 2.5%  were  washed  Dehydration  temperature  were  in  buffer,  c o n t a i n i n g 0.25  samples  were  was were  TRANSMISSION  fixed  7.2,  absolute  temperature.  FOR  i n 1% o s m i u m  the samples  were  for  The  cells  microscopy  were pH  glucose  The  cacodylate  SAMPLES  buffer,  a t 4°C.  post  After  OF  samples  cacodylate  D.  i n 0.1  PREPARATION  10 mM  i n some c a s e s .  transmission electron  Tissue  or  phagocytosis  MICROSCOPY  200  -39Unlabeled scanning first h  e l e c t r o n microscopy  fixed  a t 4°C  cells  or labeled c e l l s  followed were  each  step.  by w a s h i n g  stained  with  The  with  extensive  cells  were  temperature.  nm  of  Samples Electron  critical  2 changes  point  tetroxide  osmium  in a  of a b s o l u t e The  drying  cells  photographed  (SEM).  f o r 30  buffer  ethanol)  f o r 15  dried  V  RPE 1%  minutes  between  from  each  series minutes  CO^  (67) and c o a t e d  a  for 1  and  ethanol  Hummer on  (0)  for were  buffer  graded  were  apparatus  cells  f o r 1 h. The  0:T:0:T:0  in cacodylate  prepared  RPE  M cacodylate  1%  were  Microscope  The  buffer  in a Technics  and  and  i n t h e same  gold-palladium viewed  i n PBS  (66).  i n 0.1  dehydrated  each  Polaron  follows  washing  and  room  washed  (T) i n t h e s e q u e n c e  (50%,70%,90% , at  as  i n 1% g l u t a r a l d e h y d e  thiocarbohydrazide in  were  in  with  a 200  Sputter-Coater.  Cambridge  250  Scanning  -40-  SECTION 1  ANALYSIS  OF  BOVINE RETINAL  PLASMA  MEMBRANE  PIGMENT  EPITHELIAL  PREPARATIONS  RESULTS 1. P R E P A R A T I O N The by  OF B O V I N E R P E PLASMA  fractionation  following  subcellular  the  (48,70),  (51,69)  for  cytochrome  c reductase activity  enzymes  as well  the  homogenate.  cell After  were  membrane  The respect NADPH  as t h e i r  of  the  bands,  the lower  plasma  membrane  to  the c e l l  cytochrome  homogenate c  reductase  fold  based  approximately  2-5  cytochrome  reductase  Ficoll/  c  sucrose  each  of  and recovery  with  a  480g Ficoll/ fold  in  two  h a d a more  42%  plasma  a  20-50% membrane  appearance.  b y 7-9 f o l d of  about  with 18%.  activity  decreased  homogenate.  Succinate  activity  but lower  on  plasma  a recovery  the  the  sucrose  enriched  turbid  enriched  specific on  respect t o  supernatant.  centrifugation  were  specific  interface  on  these  o f t h e RPE c e l l s ,  the  with  c  for  resulted  markers  cytochrome  1 follows  3.5 t o 5.0  of which  5$  Table  yielding  gradient  NADPH  and  succinate  activity  Subsequent  different  and  disruption  a  monitored  for  reticulum;  supernatant  was  (50,68,69)  membrane;  enrichment  gradient yielded  sucrose  markers  formitochrondia.  hypotonic  preparation.  continuous enriched  (52,69)  membranes  ATPase  +  endoplasmic  pelleted  Centrifugation discontinuous  +  and s p e c i f i c  the i n i t i a l  nuclei  Na K  of  f o r the plasma  reductase  total  RPE c e l l  distribution  components;  nucleotidase  the  of bovine  MEMBRANE  was  i n t h e bands  higher  at  obtained  the from  -41-  TABLE 1 D I S T R I B U T I O N OF MARKERS I N V A R I O U S F R A C T I O N S R E C O V E R E D DURING MEMBRANE I S O L A T I O N  + +  5'Nucleotidase Fraction TA  %R  SA  PF  TA  Na  K  ATPase  %R  SA  PF  Homogenate  936  100  15.6  1  852  1 00  14.2  1  480 g supernatant  915  97.0  18.3  1.2  841  98.0  16.8  1 .2  Interface  360  38.5  60.0  3.8  451  53.0  76.2  5.3  2 5 , 0 0 0 rpm Top Band  100  10.7  125  8.0  101  11.8  1 27  8.9  25,000 Bottom  72.1  7.8  90.0  5.7  71.4-  8.3  88.8  6.3  Cyto  C  rpm Band  NADPH C y t o  C  Reductase  Succinate  Reductase  Fraction TA  %R  SA  Homogenate  1632  100  34.3  480g Supernatant  1497  91.7  39.0  197  12.0  2 5 , 0 0 0 rpm Top Band  27.2  25,000  rpm  17.6  Bottom  Band  Interface  PF  %R  SA  912  100  19.0  1.13  916  100  23.5  1.2  47.0  1.14  182  20.0  43.3  2.27  1.6  16.0  0.46  15.7  1.7  9.1  0.47  1. 1  8.0  0.23  54.3  5.9  24.7  1.30  1  TA  PF  1  -42-  Table during  1.  Distribution  membrane  suspension  the c e l l  is  the  total  band  The 25,000 from  based  (nmoles/min/mg). homogenate.  disruption,  from  the  on t h e c e l l  %R  =  sucrose  PF = p u r i f i c a t i o n  SA fold  The  interface sucrose  a r e t h e two  represents  homogenate.  supernatant  Ficoll/42%  bands  continuous  recovered  the c e l l u l a r  t h e 480g  the n u c l e i .  rpm t o p a n d b o t t o m (w/w)  fractions  represents  while  pelleting  collected  (nmoles/min).  and  in various  The homogenate  after  t h e 20-50%  activity  recovery  hypotonic  suspension  membrane  interface. collected  isolation.  after  was  of markers  g r a d i e n t . TA = the  = specific based  bands  on  enzymatic activity the  cell  -43-  the  continuous  higher the of  sucrose  succinate  homogenate this  stained  apparent  of  bands  membrane  pattern  However,  from  a  fractionation  study  (anti-rhodopsin fraction  indicated sucrose  1%  had  activity  a  than  contamination  beads.  The  shown  by a  Once  affinity  column  below  =45,000  band  resulted  antibody)  an a t t e m p t  through  antibody  stained  from  (Fig.3  large  in lane  rhodopsin  M =34,000. r  spun in a  on  a  high level  at a  remove  Rho-1D4 the  (Fig.4).  some  most  column 4B  of the rhodopsin  as  passed  an a p p a r e n t  by c o o m a s s i e  the  t o Sepharose  SDS-polyacrylamide  preparation  of  in  solubilized  immunoaffinity linked  A  30-50%  at  peak  p r e p a r a t i o n was an  the p o l y p e p t i d e s above  n o t seen  to  removed  Fig.3  membranes c o m i g r a t e  covalently  column  t h e membrane  were  was  In  an  considerably  binding  activity  of  from  collected  at apparent  homogenate  t h e membrane  blue  b and c ) . bands  blue  diversity  preparation  t h e R P E a n d ROS In  immunoaffinity coomassie  wide  differ  segment  gradient  passed  o f Rho-1D4  a).  M  both  contamination,  consisting  which  o f a RPE c e l l  5'nucleotidase  and  the  coomassie  p r e p a r a t i o n s had a  by a m a j o r  gradient.  CHAPS  3,lane  rod outer  sucrose  that  by  (Fig.3 lanes  gradient  monoclonal  where  rhodopsin  indicated  t h e RPE membrane  continuous  preparations  the t o p and bottom  sucrose  as seen  in  a mitochondria  t o 200,000  contamination  the  specific  band  i n t h e RPE p r e p a r a t i o n s r a n g i n g  represent  continuous  This  t h e lower  reductase  gels  found  r  b and c  (w/w)  c  t h e membrane  M =20,000  staining d).  indicating  polyacrylamide  polypeptide  the  cytochrome  However,  band.  Analysis  lanes  gradient.  blue  gel (Fig. through  the  1^=100,000 a n d  staining  due  to  s  —dye a  b  c  d  e  C B  SILVER  Figure gels  3. of  Coomassie various  solubilized  applied to  5-16%  gradient  i n the  each  coomassie  bovine  retinal  blue  through  an  column.  of  were  eludent  collected  presence  or  The  loaded  centrifuged  i n an  Beckman  SW  h.  of  lower  band  uL  the 25  gradient,  d)  molecular  weight  stained  with  uL  to  silver.  of  the  (w/w)  a  standards.  silver  Gels a)  gel  rotor  collected  rod  outer  Lanes  identify  the  b-e  25  out  uL  on  stained  a  with  solubilized  membrane  preparation  (Rho-1D4)  Sepharose  c e n t r i f u g e d and  25  uL  continuous  27.1  were  CHAPS  was b)  and  carried  were  1%  plasma  suspension on  samples  2-mercaptoethanol  anti-rhodopsin  20-50%  25  Membrane  gel.  epithelial  a  c)  from  of  SDS-polyacrylamide  E l e c t r o p h o r e s i s was  (CB)  pigment  stained  fractions.  well.  immunoaffinity the  silver  SDS-polyacrylamide  either  passed  and  membrane  i n SDS  were  blue  STAIN  at  of  sucrose  25,000  from  the  segment were  the  same  top  for  band  14-16  continuous  developed  bands  uL  gradient  preparation.  over  polypeptide  rpm  25  in lane  e) when a.  -45-  1  3  2  5  4  6  7  8  9  10 11  12  Fraction Number  Figure 50%  4.  A RPE  (w/w)  continuous  25,000  rpm.  in  volumes.  2 ml  cell  sucrose  Fractions  nmoles/min/mg.  homogenate  were  gradient  collected  5 nucleotidase  anti-rhodopsin  profile  centrifuged with  specific  1  Mouse  fractionation  a  f o r 14  fraction  activity  Rho-1D4  (•  of a h  30at  collector •)  hybridoma  was i n ascites  125 fluid used  in  conjunction  to determine  standard  RIA  Procedures. refractive  I-labeled  t h e amount  method Sucrose  index  with  (•  of b i n d i n g •)  densities  of each  goat  anti-mouse  to each  as  described  were  determined  fraction.  fraction in  Ig  was  by  the  Experimental  by m e a s u r i n g  the  -46-  polypeptide  dilution.  Using  method  most  through  the immunoaffinity  was  silver  the 2.  of t h e g e l bands  stained  polypeptide  bands  i t  was  i n lane  that  expected  component  due t o t h e r o l e  rod  segments.  outer  on  a  antisera  to  the  bovine  significance 3.  where  actin  of a c t i n  Rhodopsin  SDS-polyacrylamide possibility rod like  outer  that  segment  material from  fragments  appeared  i s an actin  passing  The g e l t h a t  i n order  to  identify  important would in  the  5).  i n t h e ROS  membrane cells  cell  phagocytosis  of  was r u n  transferred rabbit I t was  preparation.  to  anti-actin also  preparation  will  RPE  preparation  by a  (Fig.  r  cytoskeletal  be a m a j o r  membrane  detected  detected  PREPARATION  subsequently  M =46,000  as  The  be d i s c u s s e d  noted opposed  possible later.  SUBCELLULAR FRACTIONATION  detected  i n the plasma  gel  electrophoresis  t h e membrane disc  was  collected  was  i n these  OF  was  actin  g e l and  RPE p l a s m a  stain  after  a).  MEMBRANE  o f RPE c e l l s  actin  was  TEM OBSERVATION  3 lane  developed  T h e RPE p l a s m a  a t an a p p a r e n t  little  (Fig.  silver  a.  that  SDS-polyacrylamide  nitrocellulose  that  column  sensitive  conserved  I N T H E R P E PLASMA  t h e knowledge  protein,  more  had been  h a d t o be o v e r  D E T E C T I O N OF A C T I N With  a much  preparation  material.  found  the sucrose  which was  At the l e v e l  i n the plasma gradient  as v e s i c l e s  membrane  preparation led  contaminated o f TEM,  membrane  size.  ROS  enriched  ( F i g . 6 ) . The p l a s m a  of varying  to  by the  with discbands  membrane  -47-  Fiqure membrane  5.  Detection  preparation  conjunction  with  RPE  cell  plasma  in  the  presence  subjected  to  polyacrylamide  of  actin  in  ROS  and b o v i n e  by S D S - p o l y a c r y l a m i d e  immunoblot.  membrane of  Rod o u t e r  preparations  electrophoresis g e l . The  gels  on were  a  plasma  gel eletrophoresis  segments  (b) were  2-mercaptoethanol  RPE  bovine  solubilized  a n d 25 u L 5-16%  stained  (a) and  with  of  in  i n SDS  each  was  gradient  SDS-  coomassie  blue  (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 to n i t r o c e l l u l o s e  paper.  The  nitrocellulose  rabbit  paper  anti-actin  antisera,  for  1 hour  in  10^  dpm/mL).  was  washed  incubated  f o r two h o u r s  i n immunoblot  buffer  with  and  incubated  12 5  autoradiography.  I-labeled The  paper  goat  was  a n t i - r a b b i t Ig antibody  washed  as before  and  (1-2  subjected  X to  -48-  Figure  6.  pigment from  Transmission epithelial  t h e t o p band  Arrows  denote  isolated membrane.  with  cell  of a  areas  electron plasma  20-50%  membrane  (w/w)  of p o s s i b l e  the bovine  micrographs  retinal  bovine  preparations  continuous  contaminating pigment  of  sucrose ROS  collected gradient.  disc  epithelial  retinal  cell  material plasma  -49-  DISCUSSION  The  RPE  based  on  plasma the  membrane  cellular  5'nucleotidase  plasma  purification.  Similar  activities  plasma  several  of  other  (68,71).  A  minor  mitochondrial primary rod  cell  outer In  junction  prevent  lines  adult  The  the clean  removal  with  Buffer  tightly  bound  ROS  continuous  organelles plasma on  like"  collecting pelleted residual  a crude  nuclei,  removing  of  found  and numerous a l l ROS  preparation  indigitated cells  is  the  RPE  of  the retina. to  Subsequent  release  to separate o f ROS  membrane  i n 10% F i c o l l  and  were  ROS  Ficoll  band  approximately  was  T h i s method half  of  the  at the interface.  centrifugation  and pigment.  top  the  unbroken proved  disc  from  RPE  layered  to float  on  the  subcellular  a n d c e n t r i f u g e d i n an a t t e m p t discs  was  cells.  A n a l y s i s of the f r a c t i o n s  the comigration  the  preparation  the  failed  was  However  microvilli  with  with  purification  band.  that  fold specific  and the p h o t o r e c e p t o r  gradient designed  membrane  this  in  of  photoreceptor  D i s r u p t e d RPE c e l l s  sealed  through  rod  t h e RPE.  demonstrated  t o p o f 42% s u c r o s e  "sack  in  sucrose  membrane.  sucrose  and  similar  been  methods  A c o n t a i n i n g EDTA  from  have  enriched  ATPase  +  increases  eye i t appears  long  +  o f t h e membrane  t h e RPE c e l l s  tight.  Na K  i n t h e R P E membrane  from  bovine  and  similar  i n the lower  membrane  washing  the  with  fold  showed  markers  contamination  between  extremely  membrane  7-9  Both  markers  recoveries  of impurities  segment the  homogenate.  membrane  membrane  source  p r e p a r a t i o n was  the while  The m a t e r i a l mitochondria,  t o be  material  successful consisting  -50-  primarily  of  rhodopsin  Considerable when as  plasma  t h e membrane much  pelleted  Plasma primary The in  SDS bands  due  conserved Most  polyacrylamide ranging  from  coomassie  gel  to  Actin  reported  the  rhodopsin membrane  immunoaffinity fold  with  PBS  was  lost  sucrose  the pigment  and  i n both  the  that  many  was  One  actin  t o be a n  of  membrane  have  important  by  purification this  SDS  apparent  'M  band  identified apparent  and  polypeptide  in  primary  with  membrane  gradient  the  seen  and  further  and the upper  sucrose  system  gels  gradient.  migrated  membrane  polypeptides  SDS  sucrose  of  mitochondrial  out the plasma  possible  membrane  contamination was  plasma  examination  indicated  enclosed  gels-  seen by  on the  M =  46,000.  cytoskeletal  protein  r  (16,17).  Ultrastructural preparations  found  probably  with  200,000.  stained  RPE c e l l s  band  electrophoretic  technique  human  was  the continuous  revealed  the  immunoblotting  in  from  through  20,000  has been  (50-60%)  with  o f f of the continuous  of  blue  activity  the crude  collected  procedure.  stained  on t o p o f t h e 42%  got trapped  i t s aggregation  electrophoresis  were  activity  spun  i n t h e lower  Analyzing  bands gel  marker  collected  membrane  fragments. lower  membrane  blue  the sucrose.  bands  the sucrose  marker  p r e p a r a t i o n was  membrane  plasma  on c o o m a s s i e  membrane  of the plasma through  based  which  ROS  vesicles.  the  disc  RPE  plasma  material located  In an a t t e m p t  i n the plasma  solubilized column.  of  and passed  membrane through  to  amongst  remove  the  preparation,  the  an  anti-rhodopsin  The s o l u b i l i z e d  membrane  resulted  requirement  in a  membrane  was  diluted for  a  2-3 more  sensitive  silver  polyacrylamide polypeptide  stain gels.  bands  with  for polypeptide  band  This  retained  procedure  a considerable  identification  reduction  the  on  SDS  majority  of  in the  rhodopsin  contamination. The  primary  fractionation membrane antibody  study  preparation production.  gradient were  goal  and  used  monoclonal  f o r performing  was  to obtain  for  use as  to  immunize  antibodies  BALB/C  against  RPE  RPE  cell  reasonably  an  Consequently,  the anti-rhodopsin  a  a  enriched  immunogen  t h e t o p band  immunoaffinity mice plasma  for  the  membrane  subcellular  in from  plasma  monoclonal the  column  sucrose eludent  production components.  of  -52SECTION  THE  PRODUCTION  AND  2  CHARACTERIZATION  OF  A MONOCLONAL  ANTIBODY  RESULTS 1. PRODUCTION Several against  OF MONOCLONAL attempts  bovine  w e r e made  RPE  plasma  purified  plasma  screened  f o r antibody  was  found  plasma  to  membrane  have  membrane  indicating  that  (Fig.  spleen  lymphocytes obtained  X-100  solubilized  antibodies SDS  a half  and  found  t o be a g a i n s t  a half  60  fold  RIA  maximum  dilution  i n which  microtiter  antibodies  preparations.  Partially  immunized  into  RIA.  T h e mouse  antibody diluted was  with  were  RPE  indicated that preparations  the in  mouse  fused cell  plasma  of T r i t o n  RPE  buffer  towards  these  and titre  to the  hybridoma  reactive  solubilized  testing  cells  mouse,  to  times  antigenic  mice  blood  binding 350  myeloma  antibodies  BALB/C  with lines  Triton membrane  monoclonal X-100  and  ROS.  particular  had  when  SDS  cross-reacted  2. C H A R A C T E R I Z A T I O N OF One  maximum  secreted  Further  solubilized  by  preparation  an  monoclonal  injected  7 ) . When m o u s e  from  which  preparations.  was  production  membrane  system  to raise  membrane  preparation  immune  were  ANTIBODIES  monoclonal  antibody  rhodopsin. binding  (Fig.  detergent  wells  RHQ-5A3  were  designated  Rho-5A3  hybridoma  to solubilized  8).  Table  solubilized sequentially  a s Rho-5A3 culture  rod outer  2 shows t y p i c a l ROS  membranes  treated  with  was  fluid  segments results  at a for a  immobilized hybridoma  in cell  125 supernatant antibody  and  showed a  I-labeled  goat  26% r e d u c t i o n  anti-mouse  in activity  Ig antibody. t o ROS  Rho-5A3  solubilized  in  -53-  Fiqure  7.  Antibody  immunized pigment in  Triton  with in  with  bovine  epithelial X-100  serial  titration  was  cell  After  plasma  serum  I-labeled  goat  anti-mouse  Ig  antibody.  Bovine  wells  initially  the wells  125  a BALB/C mouse  preparation  on m i c r o t i t e r  i n PBS  from  membranes.  membrane  of the blood washing  f o r blood  plasma  immobilized  dilutions  RIA b u f f e r .  RPE  curve  were  retinal  solubilized and  incubated 5  fold  incubated  with  diluted  -54-  00  I  c o  CQ  CL  O  16  64  256  1024  4096  Reciprocal Dilution  Figure culture outer  8.  Titration  fluid  segments.  against  curve  of  immobilized  Solubilized  Rho-5A3 Triton  rod outer  microtiter  w e l l s and incubated  monoclonal  antibody  with  culture fluid.  monoclonal  X-100  antibody  solubilized  segments were d r i e d serial After  dilutions washing  of  with  rod  down o n Rho-5A3  PBS, t h e  125 microtiter Ig  w e l l s were  antibody.  incubated  with  I - l a b e l e d goat  anti-mouse  -55-  TABLE SOLID-PHASE  RADIOIMMUNE  ASSAY  2 FOR A ROS S P E C I F I C A N T I B O D Y  1 25 I - L A B E L E D GOAT A N T I MOUSE I g BOUND (DPM)  HYBRIDOMA  CELL  TRITON X-100  SUPERNATANT  RHO-5A3  38,246  CONTROL  2,940  Control=  Table Triton Rod  uL  non-specific  2. A c o m p a r i s o n  SOLUBILIZED  ROS  segments  1 , 450  antibody  of Rho-5A3 monoclonal  ( 2 . 5 mg/mL) w e r e  X - 1 0 0 o r 1% S D S , d i l u t e d  were  dried  specific hybridoma  down  sites  in microtiter  i n RIA b u f f e r ,  culture  fluid,  ROS  28,244  antibody  binding  X - 1 0 0 s o l u b i l i z e d a n d SDS s o l u b i l i z e d r o d o u t e r  outer  Triton  SOLUBILIZED  SDS  in distilled  wells.  the wells  followed  segments.  solubilized in  10 f o l d  After were  to  either  1%  w a t e r a n d 25  blocking  incubated  the  non-  i n Rho~5A3  by w a s h i n g a n d i n c u b a t i o n  with  1 25  in  I-labeled  goat  anti-mouse  a similar  manner  using  Ig antibody.  a non-specific  The c o n t r o l antibody.  was  treated  -56-  SDS  as opposed to ROS  used was  solubilized  i n T r i t o n X-100.  a primary n o n - s p e c i f i c antibody to  The  control  ROS.  3. SUBCLASSIFICATION OF RHQ-5A3 MONOCLONAL ANTIBODY Solid-phase radioimmune assays were performed, described,  using  microtiter  wells.  Triton  X-100  s o l u b i l i z e d ROS  as p r e v i o u s l y  immobilized  on  The w e l l s were incubated with Rho-5A3 c u l t u r e  f l u i d with an a d d i t i o n a l s u b c l a s s Ig antibody  i n c u b a t i o n followed  1 25  by  detection  Results  in  with  I - l a b e l e d goat  anti-rabbit  Ig  antibody.  Table 3 i n d i c a t e d that the Rho-5A3 monoclonal i s  IgG^ kappa l i g h t chain antibody. 4. IDENTIFICATION OF RHQ-5A3 ANTIGEN OF BOVINE ROS  MEMBRANES  In order to i d e n t i f y the a n t i g e n i c s i t e f o r Rho-5A3, ROS subjected  to  separation  t r y p s i n and S.Aureus V-8 of  the  proteolytic  protease  treatment  fragments  an  by  SDS  were with gel  e l e c t r o p h o r e s i s followed by t r a n s f e r of the p o l y p e p t i d e s to CNBra c t i v a t e d paper. The paper s t r i p s were then d i r e c t l y t r e a t e d with Rho-5A3  or  Rho-1D4 a n t i b o d i e s and d e t e c t i o n was  made  by  using  1 25 I - l a b e l e d goat anti-mouse Ig as a second antibody. R e s u l t s are shown i n F i g . 9 , along with the coomassie blue s t a i n i n g p a t t e r n . Both major  a n t i b o d i e s Rho-1D4 and Rho-5A3 bind to  coomassie  34,000. rhodopsin  When ROS  blue  staining  glycoprotein  of  bands were l o c a t e d at apparent  M=  rhodopsin When  apparent  M^=  33,000 ,25,000  f  r  bands  the  were t r e a t e d with S.Aureus protease three major  12,000 (72,73). Rho-5A3 bound to the M =33,000 and rhodopsin  rhodopsin,  while  Rho-1D4 only bound  and  25,000 c l e a v e d  residual  undigested  (74). ROS  were  s u b j e c t e d to  proteolysis  by  trypsin,  .one  -57-  TABLE  3  I D E N T I F I C A T I O N OF I g S U B T Y P E OF MONOCLONAL ANTIBODY R a b b i t anti-mouse Ig antibody binding  Ant i -  DPM  subtype specific t o Rho-5A3  -(X  -<**> - * >  ) -(y,)  (  )  )  control  bound  X  10~  Table  2.2  3  3.  antibody with  («.  RHQ-5A3  3.0  Immunoassay subtypes.  Triton  microtiter incubated  2.7  X-100 wells.  with  for  Rho-5A3  2.5  the  screening  hybridoma  solubilized After  subclass  2.2  6.0  rod  washing  specific  of  i n PBS  rabbit  2.6  Rho-5A3  culture  outer  9.6  monoclonal  fluid  segments buffer  was  Ig  incubated  dried  the  anti-mouse  2.9  down  wells  on were  immunoglobulin 1 25  followed goat  by  washing  anti-rabbit  Ig  as  before  antibody.  and  incubation  with  I-labeled  -58-  F i g u r e 9. digested  A n a l y s i s of p o l y p e p t i d e s from u n t r e a t e d and  protease  rod o u t e r segments which b i n d Rho-1D4 and Rho-5A3 a n t i -  rhodopsin monoclonal a n t i b o d i e s . Rod o u t e r segments were d i g e s t e d w i t h S.aureus  protease  centrifugation, polyacrylamide coomassie blue latter  was  ( l a n e b) and t r y p s i n  ( l a n e c ) , washed by  s o l u b i l i z e d and e l e c t r o p h o r e s i z e d on a 10% g e l . The  p o l y p e p t i d e s were  either  SDS-  stained  (CB) or t r a n s f e r r e d t o CNBr- a c t i v a t e d paper.  treated  with  Rho-1D4  or  Rho-5A3  culture  by The  fluid.  1 25 Identification  was  made u s i n g  I - l a b e l e d goat  a n t i b o d y . Lane a was u n t r e a t e d r o d o u t e r segments.  anti-mouse  Ig  -59-  fragment  migrated  j u s t ahead of the l e a d i n g edge of  undigested  rhodopsin. The Rho-5A3 monoclonal antibody binds s t r o n g l y to t h i s large  (apparent  M =32,000) fragment while  Rho-1D4  r  residual  undigested  rhodopsin.  fragment  corresponds  It  has been  only  that  this  shown  to rhodopsin with a 9 amino  removed from the c a r b o x y l terminus  binds  acid  segment  (74).  5. COMPETITIVE INHIBITION OF RHQ-5A3 MONOCLONAL ANTIBODY A  competition  immunoaffinity approximate maximum 10)  assay  was  purified  performed  rhodopsin  concentration  that  half  (43)  of rhodopsin  Rho~5A3 antibody b i n d i n g .  indicated  with  Rho-1D4-Sepharose  to  determine  required  The  to  the  give  half  inhibition profile  maximum b i n d i n g  was  (Fig.  achieved  at  a  c o n c e n t r a t i o n of 5 ug/mL. The  effectiveness  d i s c s , and T r i t o n X-100 to  studied  i n order to determine  (Fig.  competition X-100  11).  X-100  treated,  frozen-thawed  d i s c s to i n h i b i t  immobilized  ROS  antibody  discs  frozen-thawed  d i s c s showed  f o r the antibody even at high c o n c e n t r a t i o n s .  to  ROS  was  the a c c e s s i b i l i t y of the a n t i g e n i c  Sealed d i s c s and  solubilized  binding  discs,  s o l u b i l i z e d ROS  binding  site  Triton  of sealed ROS  d i s c s were found to i n h i b i t Rho-5A3  immobilized and T r i t o n X-100  no  Triton antibody  solubilized  discs  at  c o n c e n t r a t i o n s above 0.1 mg/mL. 6. ANALYSIS OF RHQ-5A3 ANTIBODY BINDING PEPTIDE Rhodopsin  peptides  were a l s o used  Rho-5A3 b i n d i n g to T r i t o n X-100 segments. polypeptide  When from  Rho-5A3  was  rhodopsin,  i n an attempt  solubilized competed with  to  immobilized a  2-39  i n h i b i t i o n of Rho-5A3  inhibit rod outer  N-terminal binding  to  -60-  «-5  10  10 ~  r4 10  IO'3  r2  10  -1  10  Rhodopsin (mg/ml)  Figure Triton  10. X-100  rhodopsin. rhodopsin wells. down  Inhibition  o f Rho-5A3 m o n o c l o n a l a n t i b o d y  solubilized  Serial  rod outer  dilutions  s e g m e n t s by i n c u b a t i o n  o f Rho-1D4 i m m u n o a f f i n i t y  were i n c u b a t e d w i t h Rho-5A3 c u l t u r e  Twenty-five solubilized  uL f r o m e a c h w e l l  rod outer  binding to  segments.  was  fluid  incubated  in  purified microtiter  with  F o l l o w i n g washing  with  in  dried PBS,  125 antibody antibody.  d e t e c t i o n was  made u s i n g  I - l a b e l e d goat anti-mouse  Ig  - 6 1 -  Fiqure  11.  Inhibition  X-100  Triton  frozen/thawed X-100 were  wells. with  solubilized discs  (A  solubilized discs incubated  o f Rho~5A3 rod A), (•  monoclonal  outer sealed  •  ).  Rho-5A3  hybridoma  Twenty-five  uL were  removed  X-100  microtiter  wells  solubilized previously  segment discs  disc  (•  culture  from  disc  each  fluid well  proteins  incubated  in  binding protein  • ) , and  Serial dilutions  with  Triton  antibody  by  Triton  of i n h i b i t o r in microtiter  and dried  RIA  to  incubated down  buffer.  i n  After  1 25 washing anti-mouse  i n PBS Ig  the wells  antibody.  were  incubated  with  I-labeled  goat  -62-  immobilized observed  Triton  X-100  solubilized  a t c o n c e n t r a t i o n s a b o v e 0.5  observed  with  the  2-16  rod  uM,  N-terminal  outer b u t no  segments inhibition  rhodopsin  was was  polypeptide  (Fig.12). 7.  LOWICRYL Thin  THIN  sections  Lowicryl  resin,  followed  by  examinaton heavily very  SECTION  and  little  by  of bovine were  goat  randomly  photoreceptor  labeled  anti-mouse  TEM,  label  LABELING  revealed labeled  (Fig.13).  with Ig  Rho-5A3  cells,  monoclonal  gold-dextran.  that while  embedded  the rod outer the rod inner  in  antibody  Ultrastructural segments segments  were had  -63-  Figure  12.  Inhibition  Triton  X-100  solubilized  terminus  polypeptide  polypeptide incubated wells. 100  ( with  #  previously  rod  rod  segments ) and  Serial  outer  each  segments  i n RIA  culture  well  was  dried  buffer.  antibody  by  of  incubated  After  2-39  2-16  to N-  N-terminus  polypeptides  fluid  down  binding  rhodopsin  rhodopsin  dilutions  hybridoma  from  incubated  monoclonal  outer •  ).  Rho-5A3 uL  Rho-5A3  ( •  #  Twenty-five  solubilized  of  in  were  microtiter  with  Triton  in microtiter washing  in  anti-mouse  Ig  PBS  X-  wells the  125 wells  were  incubated  in  I - l a b e l e d goat  antibody.  -64-  Figure  13,  fixed  bovine  labeled followed labeling inner  Transmission rod  with by on  electron  photoreceptor  Rho-5A3 m o n o c l o n a l goat  the  segment.  anti-mouse  rod  outer  micrograph  cell  antibody  Ig-Au  segment  embedded  2 n  a in  hybridoma  dextran.  with  of  little  glutaraldehyde Lowicryl culture  Notice labeling  the on  and fluid heavy  the  rod  -65-  DISCUSSION  Several BALB/C  cell  mice  fusions  previously  preparations  for  the  hybridoma  cell  membrane  preparation  hybridoma  result  probably  injected  into  injected  production  were  reflects  the  lack  antigenic  to  cells  fusion  of  with  RPE  plasma  membrane  continuous  in  a  sucrose  hybridoma  specific  for  designated  cell  the  rhodopsin  as  site  the  and  by  from  competition  N-terminus  studies  IgG  antibodies  serological specific  are  the  labeling  and  binding  tends  to  most  quite  monoclonal  useful  low.  This  initially proteins  the  mouse. immunized  collected cells  from  resulted  monoclonal  antibody  The  monoclonal  antibody,  to  be  to  of  indicated light type studies  specific  of  localized  kappa  immunological be  of  no  a  region  IgG^  an  plasma  mice,  mouse  band  gels  5A3  was  BALB/C  labeling  SDS  intradiskal  system  top  found  subtypes  antibody  RPE  preparations.  (myeloma)  m o l e c u l e ( F i g . 14). A n a l y s i s of i m m u n o g l o b u l i n monoclonal  a  NS-1  radioimmune from  secreting  solubilized  immune  secreted  was  antibody  secreted  the  from  membrane  sufficient protein  preparation.  transferred  Radioimmune to  which  however  determined  the  plasma  BALB/C  membrane  preparation  gradient  membrane  electrophoretically paper.  spleen  line  Rho-5A3,  that  cells  purified  immunize  Alternatively  the  the  of  spleen  RPE  monoclonal  produced plasma  not  with  to  RPE  mouse.  with  immunoaffinity  used  However, the  of  for  the  were  the  was  lines  specific  performed  immunized  l i n e s . When  cell  antibodies  were  ROS  for  proteins  CNBr-  activated  the  antigenic  the  rhodopsin  that  the  chain of  antibody.  antibody as  Rho-  their  for non-  -66-  Figure  14.  Hargrave variety of  (75), of  the  t o be  diagrammatic and  the  monoclonal  Rho-5A3  rhodopsin shown  A  location  antibodies.  monoclonal  molecule. located  The on  model  the  of of  rhodopsin, the  Note  a n t i b o d y on  antigenic  the the  terminus  sites  approximate  Rho-1D4 m o n o c l o n a l carboxyl  modified  N-terminus antibody (43).  from for  a  location of has  the been  - 6 7 -  The along  antigenic site the  first  rhodopsin.(43) digestion action.  seven  With  amino  Rho-1D4  which  o f ROS  removed  of rhodopsin  while  antigenic site  the  (Fig.9).  In  monoclonal  the case antibody  fragment  (M =25,000)  contain  the  Rho-5A3  antibody  terminal  two-thirds  With  these  accessibility not  material well to  cause  it  appears  to  the  native may  cause In  5A3  was  or  acid  V-8  protease  was  fragment results  competition  site.  the  Rho~5A3  proteolytic  which  i s known  indicate along  to  ROS  in Triton  changes.  that  the  to the  amino-  explore  disc  X-100  Based  i s i n the  identify were  polypeptides.  the  binding  The Rho~5A3  disc  rhodopsin bound  which  on  these  very  i s known results  i s inaccessible membrane.  The  bilayer  determinant.  the antigenic site  performed  the  antibody  or the phospholipid  o f t h e Rho-5A3  assays  or  maintained.  rhodopsin  interesting  conformation  to further  from  o f Rho-1D4  a n t i g e n i c determinant  rhodopsin  Rho-5A3  peptide  digestion,  frozen-thawed  structural  protein  rhodopsin  of  S.aureus  the  binding  the i n a c c e s s i b i l i t y  action,  the s i t e  (Fig.14).  solubilized  when  an a t t e m p t  prepared  i t  t h e Rho-5A3  rhodopsin  proteolytic  c o n c e n t r a t i o n s . However, Rho~5A3  when  antibody  limited  t o an a n t i g e n i c s i t e  to sealed  that  to  These  of rhodopsin  irreversible  of  binding  V-8  the F  (73).  o f t h e Rho-5A3  to rhodopsin  terminus  i n the loss  bound  designated  at high  the carboxyl  amino  f o r Rho-5A3  results,  bind even  or nine  of S.aureus  binds  is  to localize  resulted  N-terminus  Rho-1D4,  material with  also  r  on  antibody,  as a c o n t r o l ,  a seven  C-terminus  did  acids  s t u d i e s were p e r f o r m e d Digestion  trypsin  f o r the monoclonal  with  P r e v i o u s l y , Rho-5A3  of  Rho-  specifically antibody  had  -68-  been  shown  t o compete w i t h  binding  when  similar  to  the  rhodopsin  rhodopsin  (another  monoclonal  antibody)  (43).  demonstrated  Rho-5A3  However,  no  terminal  polypeptide  competition  preliminary located  labeled  ROS,  after  (Fig.13).  rod  The  and  used  to  that  with  t h e 2-39  t h e 2-16  N-terminal  of  a n d embedded by  goat  labeling  techniques.  0.5  uM.  rhodopsin  10  uM.  N-  These  antigenic site  peptide  of the s e c t i o n s with  i s  region.  in Lowicryl,  were  anti-mouse  Ig  t h e Rho-5A3  antibody  o f Rho-5A3  Rhodopsin  and not found  produce  was  which had  injected cell  as to the  f o r the bovine  Bovine into  fusions  RPE  monoclonal  to introduce  immunization. and  ROS  compete w i t h  t h e Rho-5A3  randomly  remained  to  cells  myeloma  neither  to  specific  RPE  lines  assays  sections the antigenic sites  question  culture  and  binding  gold  action  was  found  at a l l in  the  segments.  approach  prior  that  i n the r o d outer, segments  antibodies  bovine  to  quite  competition  to a concentration  rhodopsin  treatment  accessible  inner  observed  indicate  ug/mL,  a t a c o n c e n t r a t i o n above  previously fixed  In these  abundantly  was  up  extensively  dextran  are  studies  i n t h e 17-38  Bovine  peptide  5  antibody  rhodopsin  Solid-phase ability  maximum  was  N-terminal  antibody's  N-terminal  half  concentration  Rho-4A2  rhodopsin  with  resulted  plasma  cells  mice. i n two  secreted antibodies against rhodopsin  and  residual  as t h e i r ROS  in  into  tissue  Subsequent more  Another  specific  were grown  ROS  obtaining  membrane.  antibodies  the c e l l s  RPE  BALB/C  difficulty  for  culture  in  tissue  spleen  cell  hybridoma  cell  preparations  a n t i g e n i c determinant.  proteins associated with  It  but  appears  t h e RPE  cells  -69are  highly  possibly  due  antigenic to  a  while  blood-brain  RPE  plasma  barrier.  membranes  are  not,  -70-  SECTION  ANALYSIS  OF  BOVINE  3  RPE  CELLS  IN  VITRO  RESULTS 1.  ISOLATION Several  eye  optic  cells  OF  BOVINE  procedures  c u p were  from  membrane  yields  and  RPE  was  excellent 2.  THE  plated  support  bovine  shape  bovine  RPE  amelanotic  1  cells  RPE  ( Fig. more  the c e l l u l a r  growth  became  days  the pigmentation the level  in  tissue  approximately cells  also surface  one  enzymatic  had  cells  results  and  RPE  from  due  produced  the  to  poor  the  best  collagenase.  culture  with  in  After  The  provided  tissue  an  became  appeared  several  the  less  melanosomes  after  (Fig.  and 15c).  time  of  after  10  apparent  on  dense.  flattened  (Fig.  tissue  melanotic  a doubling  urn w i d e v e s i c l e s  ( F i g . 17). U n d e r l y i n g  in  culture  microvilli  surface  cuboidal  ( F i g . 1 5 a ) . The  in appearance  confluent with  cells  were  4 days  shape  15b) a n d b o t h  of the c e l l 1-2  treatment  a cuboidal  of the c e l l s RPE  scattered  bovine  VITRO  in size.  dispersed  o f SEM,  half  IN  established  became  At  trypsin  the  scraping the  o u t t h e RPE  minimal  CELLS  retained  cells  hours  as  method which  with  after  became week  such  from  growth.  BOVINE  still  cells  approximately  cell  The  and c o n s i d e r a b l y reduced the c e l l s  52  with  cells  o u t on a g l a s s s u b s t r a t e w h i c h  for cell  RPE  culture  As  performed  digestion  MORPHOLOGY OF  The in  were  RPE  Techniques  breakage.  enzymatic  cells  for isolating  cup or d i s s e c t i n g  were  cell  CELLS  explored.  the optic  Bruch's  yield  RPE  16).  attached bulged  The  RPE  to  the  out a g a i n s t  -71-  Figure  15.  RPMI-1640  medium  microscopy. (1600X).  a)  Bovine  enzymatic collagenase o 37"C  Bovine on  retinal glass  4 days RPE  treatment dissolved  ( approximately  containing  glass  pigment  coverslips  (1000X).  cells  were  with  x  coverslips.  5 10  as  seen  7 days  isolated  0.25%  in Buffer 5  b)  epithelial  A.  from  cells  cells/mL)  grown  phase  (1460X).  trypsin The  by  cells  contrast c)  the optic and were  i n 10 mL  in  70  18  days  cup  units/mL  incubated petri  by  at  dishes  -72-  Figure pigment tissue  16.  Scanning  epithelial culture  microvilli  while  cell  medium. dark  electron grown Clear  arrow  micrograph  on g l a s s arrow  indicates  of a  bovine  f o r 10 d a y s indicates area  with  in  area dense  retinal RPMI-1640 with  few  microvilli.  -73-  Figure  17.  Scanning  pigment  epithelial  medium  f o r 10 d a y s .  segments  electron  cell  grown  Arrows  attached to the c e l l  micrograph on  denote  glass  of a in  vesicles,  surface.  bovine  retinal  RPMI-1640  culture  possibly  rod outer  -74-  the  plasma  drying  of  features Extreme RPE  membrane  the  samples  of  the  caution  cells  due  quite  for  cell, had  i n the  3.  FLUORESENT DETECTION  fragile  of  throughout plasma  the  the  membrane.  minimal  OF  were  f o r SEM  IN  When  actin  fluorescence  point  the  point  morphological after  drying.  drying  plasma  of  membrane  B O V I N E C U L T U R E D RPE RPE  incubated  FITC-labeled  with  general  maintained  as  bovine  RPE  critical  the tends  disrupted.  ACTIN  were  bovine  during  in c r i t i c a l  biochemical  cell  purified  used  fixed  by  shrinkage  ( F i g . 1 8 ) . The  easily  cells  confirmed  component  with  the  followed  Results  be  and  Paraformaldehyde  antisera  SEM  preparation  be  and  cell  however,  to  to  acetone  to  goat  were  with  rabbit  heavy  treated  anti-rabbit  studies that  cell.  rabbit  cells  antibody.  was  was  with  anti-actin  Ig  actin  Fluorescence  CELLS  a  major  concentrated  fluorescence associated with anti-actin  was  used  as  was  detected  the in  antisera primary  this  the  preincubated  antibody,  control  sample  only (Fig.  19). 4.  DISCONTINUOUS  FLUORESCENT  Various  fluorescent  arrangement,  distribution,  on  the  Con  surface  A,  specific  specific  for  specific  f o r D-  Studies 37°C, surface  of  with  N-  bovine for  LABELING  lectins and RPE  were  OF use  redistribution cells.  *s-D-mannose  acetylglucosamine  Such  and  BOVINE to of  CELLS  study sugar  lectins  and  the  residues  include  °<-D-glucose;  oligomers;  RPE  FITC-  FITC-WGA, FITC-RCA,  galactose.  with  2 week  FITC-Con  (Fig.  of  LECTIN  20a).  A  o l d RPE showed  Similar  cells  incubated  extensive  results  were  for  labeling found  5 minutes on  with  at  the  cell  cells  fixed  -75-  Figure  18.  Scanning  epithelial  cell  texture  the c e l l  of  grown  electron micrograph on  surface  glass due  f o r 10  of a  days.  to the abundance  bovine Note of  the  retinal rough  melanosomes.  -77-  Figure week  19.  Fluorescent  o l d bovine  rabbit  treatment and  Ca  +  antisera labeled the with  retinal  anti-actin  coverslips  were  the  ug  in  After  coverslips  of  that  the  purified  Ig  RPE  actin.  cells  cells  i n PBS  incubated as  before  grown  (2000x).  on  followed  buffer containing  with and  was  rabbit  b)  As  with  previously  2  with glass by Mg  + +  anti-actin  incubation with  (2000x)  antibody  fixed  incubated  paraformaldehyde  antibody.  primary  paraformaldehyde  epithelial  washing  were  of  a)  1%  washing  anti-rabbit  exception 100  fixed  f o l l o w e d by goat  pigment  antisera.  in acetone.  +  micrographs  a),  FITCwith  incubated  -78with  1% p a r a f o r m a l d e h y d e .  was c o m p e t e d (Fig.  o f f the c e l l  20b).  followed patchy Con  Labeling  A  was  achieved  with  with  incubation  (Fig.  <* m e t h y l  lectin  a possible  for  in buffer  at the l e v e l  t o compete  mannoside  which  proved  study  performed  A  5  minutes  resulted  the  receptor  fluorescence,  o f f the FITC-Con t o be  in  of  of of  lectin  mannoside  internalization  Verification  and i n t e r n a l i z a t i o n  methyl  A  period  20c).  by an a t t e m p t  the FITC-Con  0.25 M  FITC-Con  indicating  receptors  redistribution  surface  with  by a 60 m i n u t e  fluorescence  As a c o n t r o l ,  A  lectin  unsuccessful  (Fig.  20d) . A  similar  Labeling and  of u n f i x e d  intense  with  cell  M  21b).  N-N  galactose WGA  diacetyl  FITC-RCA  followed  patchy  with  fluorescence  due  RPE  competed  treated  to  receptor  off  the  inhibitor  b y 0.2 M with  in partial  saccharide  In t h e  minutes ( F i g .  manner  internalization  when  labeling  22a).  f o r 5 minutes  resulted  uniform  but the  f o r 15  similar  RPE c e l l s  D-  FITC-  receptor  due t o t h e l a c k inhibitor  i n t h e same way  (Fig. yielded  redistribution  and  (Fig.22c).  CONTINUOUS F L U O R E S C E N T L E C T I N Bovine  FITC-RCA.  a very  (Fig. was  and  the saccharide  a t 37°C,  the appropriate FITC-RCA  intense  with  in buffer  gave  (Fig.21a),  off in a  some a p p a r e n t  with  Similarly,  internalization 5.  chitobiose competed  FITC-WGA  FITC-WGA  the c e l l s  by 60 m i n u t e s  competition  21c-d).  was  less  with  ( F i g . 22b). Labeling  redistribution of  f o r FITC-WGA  labeling  by t r e a t i n g  using  f o r 5 minutes  was c o n s i d e r a b l y  experiments  surface  0.02  RPE c e l l s  fluorescence  FITC-RCA  control  was  cells  were  LABELING  labeled  OF  RPE CELLS  continuously  for  IN V I T R O 60  minutes  Figure  21 .  Figure 2 2 . a.  b.  c.  -83-  Fiqure with  20.  Fluorescent  FITC-Con  A  in  by  wash  buffer  as  in  c)  of  a)  5 min.;  5 min.  M  *<-  methyl  and  60  for:  incubation in  micrographs  0.25  followed  by  a  min.  15  min.  b)  unfixed  RPE  cells  followed  mannoside;  c)  incubation  in  incubation  with  5-  by  labeled  a  15  min.  buffer  followed  at  0.25  min.  37°C; M  d)  ^-methyl  mannoside.  Figure with  21.  Fluorescent  FITC-WGA  for:  a)  incubation  with  followed  wash and  in  c)  for  15  by  followed  labeled  0.2  Figure minutes  0.020  by  M  60  b)  unfixed  5 min.  N-N-diacetyl  min.  incubation  Fluorescent FITC-RCA  incubation  and M  22. with  min.  wash  5 min.;  of  cells  followed  by  chitobiose;  incubation in  RPE  in buffer  0.020 M  labeled  a  c)  at  15  min.  5  min.  37°C;  N-N-diacetyl  d)  as  chitobiose  min.  Figure  15  micrographs  60  min.  in  for: 0.2  M  incubation  micrographs a)  of  5 min.;  b)  D-galactose; in buffer  c)  at  unfixed 5 min.  cells  followed  5 min.  37°C,  RPE  plus  by  a  followed  by  15  in  min.  D-galactose.  23. at  Fluorescent 37°C  with;  a)  micrographs FITC-Con  A  of b)  RPE  cells  FITC-WGA  labeled c)  for  FITC-RCA.  60  -84-  with  FITC-lectins,  extensive with  and  some  resulting Continuous  a  labeling  fluorescence patches  on  (Figs.  6. PROBING Due  was  interesting cells  grown  X-100  and assayed  a  Presumably, RPE  the  cells  cells, cell  SEM  bound high either  cells  bovine  cell  were  induced  became  on . t h e  cell  apparent  These to  available surface.  resulted  i n heavy  fluorescent  whether  rhodopsin  level  f o r over  10 d a y s  observation  RPE c e l l s  segments  Results  are s t i l l  has n o t been  bovine  i n 0.1%  vitro  RPE  Triton  indicated  i n the  RPE  ( F i g .24).  associated  completely  in  monoclonal  ,  component in  i t  with  degraded  by  culture.  ROS  (Fig.  RIA method.  residual  eyes,  Rho-1D4  solubilized  in  be d e t e c t e d  of rhodopsin  to a  rod outer  could  Using  were  of rhodopsin  f r e s h bovine  rhodopsin  f o r the C terminus  OF ROS  bovine  from  culture.  by t h e s t a n d a r d  RHQ-1D4  concentration  preparation  or r h o d o p s i n  in tissue  surface  WITH  in microtitre wells  7. P H A G O C Y T O S I S When  CELLS  of a high  in tissue  cells  at  some  the  out  23a).  and FITC-RCA  with  was  (Fig.  receptors  label  FITC-WGA  to explore  specific  Rho-1D4  new  of  membrane  membrane  grown  antibody,  receptors  time  coat  to the presence  the  A  BOVINE RPE CULTURED  RPE p l a s m a  cells  Con  with  the  through  there  23b-c)  the  that  that  heavy  found  o f C o n A,  fluorescent patches  b u t a t t h e same in  In t h e case  fluorescence  large  indicated  internalize  37°C.  intense  prominent  results  RPE  at  BY B O V I N E RPE C E L L S were  revealed  25).  were  incubated that  for 5 h  t h e ROS  Preliminary  recognizing  IN VITRO with  were  results  and a t t a c h i n g  bovine  attached  RPE  to the  indicated that the to the rod  outer  -85-  Figure  24.  solubilized RPE in  cells  Rho-1D4 bovine  grown  0.1% T r i t o n  fluid.  monoclonal  RPE c e l l s  in flat  antibody  grown  bottom  Following  washing  in microtiter  microtiter  X-100 a n d i n c u b a t e d in buffer,  with  goat  anti-mouse  Ig  wells  Rho-1D4  the wells  125 I-labeled  binding  antibody.  to Triton  X-100  wells.  Bovine  were  solubilized  hybridoma  were  culture  incubated  with  -86-  Fiqure RPE  cell  25. S c a n n i n g incubated  Arrows  indicate  amongst  t h e mat  f o r 5 h with some  of  electron  micrograph sealed  o f t h e numerous  microvilli.  of a  bovine bound  2 week  rod outer rod  outer  o l d bovine segments. segments  -87-  segments. of  An assay c o n d i t i o n was e s t a b l i s h e d  for the phagocytosis  dark adapted ROS by 2 week o l d bovine RPE c e l l s .  were c o l l e c t e d from a sucrose g r a d i e n t 37°C,  with  RPE  c e l l s grown on  Sealed  and incubated for 5 h , at  glass  coverslips.  Qualitative  o b s e r v a t i o n s made by TEM i n d i c a t e d that some,  but not a l l ,  bovine  phagocytizing  RPE  (Figs.26,27)  cells  in v i t r o were capable  These r e s u l t s  ROS  of  adult ROS  e s t a b l i s h e d a new system for studying  the phagocytosis of rod outer  segments.  -90-  Figure bovine  26. RPE  segments  cells  for  membrane.  Note  completely along  with  Figure old  27.  bovine  segments  phagosomes  5  electron  incubated h.  Arrow  that  inside  with  Transmission RPE c e l l  of  further stacks  (PH).  The n u c l e u s  week rod  of disc  (N) i s  the  old outer  plasma  membrane  quite  is  evident  (M).  micrographs  with  illustrates o f ROS  2  sealed  ( P H ) o f ROS  The n u c l e u s  electron  incubated  adapted  of  the p o s i t i o n  t h e phagosome  the c e l l .  micrographs  dark  denotes  s e v e r a l melanosomes  which  arrangement  evident.  Transmission  dark ROS  disc  of another  adapted  sealed  phagocytosis.  membrane  forming  (N) a n d m e l a n o s o m e s  (M)  2  week  rod outer  Notice the are  the large also  - 9 1 -  DISCUSSION  The  introducing of adult  opened  up a whole  RPE-ROS  enzymatic  cuboidal  as  with  developed  a  confluent microscopy  ROS  after  were  grown  antibody  protein, slowly  This  of degradation  52  the  level  seen  by  patterns h  )  until  of  light  found  nature  over  a  large  interspersed attached  surface  have  the appearance  associated with  a n d SDS  RPE c e l l s  culture  gel electrophoresis  there  was  t h e RPE c e l l s . terminal  still  Probing  even  the bovine  RPE c e l l s  or at least  t h e Rho-1D4  culture  with  of other  idea  fractions membrane  rhodopsin in  the  tissue  membrane  antigenic site,  that  occurs  RPE  t h e Rho-1D4  a possible initial  the proposed  lysosomal  with  that  of  considerable  of rhodopsin),  I t appears  support  b y RPE  with  less  multinucleated  time  a s 2 weeks.  in tissue  would  as  growth  were  the  culture.  for carboxyl  rhodopsin,  rhodopsin  phase  be t i g h t l y  tissue  as long  degraded  period.  may  on t h e c e l l  from  h i g h l y pigmented  surface  t o be a s s o c i a t e d w i t h  for  flat  RPE c e l l  in tissue  (specific  a  At  Microvilli  associated with  found  culture  obtained.  tissue.  vesicles  in  (doubling  demonstrated  in intact  still  rate  the fractionation  rhodopsin  of  cultures  which  From  was  growth  spindle  and  became  in vitro and  culture  surfaces  recovered  normal  and  tissue  morphology  growth  The  mosaic  hardy  10 d a y s  cells  primarily  These  cell  into  RPE c e l l  RPE c e l l s  their  (28,30,31).  of the bovine  vesicles.  the  established rapid  the c e l l s  observed  section  of  they  Once  RPE c e l l s  in studying  treatment  researchers  cells,  as  approach  interactions.  traumatic  other  new  bovine  i s  latent  the degradation  after  proteins (36).  the  initial  -92Preliminary surface  studies  glycoproteins  interactions. distribution  of  sugar  and  unfixed  vitro  with  FITOCon  uniform  labeling  Labeling minutes  label,  high  label.  most  cells on  indicating  that  internalize RCA.  membrane a l l the  redistribute  RCA  peroxidase by  were  shown  TEM  binding  sites  continuously  rearrangement  for  a l l  fluorescence,  a l l  three  of  concentrations  their of  internalization unlabeled a  dense  is  receptors  pattern  of  after  l a b e l e d WGA seems  to  labeled  of  sites  the  with  parts  cell  for  to  labeled  in  buffer  induced  with  when  Con  induced  level  surface  being  of  redistribute subsaturating  that  labeling  A  surface  Redistribution so  to  embryonal  37°C  the to  since  specific  Cell  at  The  have  were  At  appear  occuring  accessible  the  (76).  used.  label  cultured  used.  counter  the  case  lectins  60  intense  cell.  minutes  on  receptors  were  of  remove  60  be  dense  by  as  the  appeared  to  in a  in  redistribution  appeared  to  cells  followed  receptors  lectins  the  both  observation.  dependent  internalize  constantly  on  of  lectins  failed  even  lectin  become  resulted  region  still  unlabeled  label  level  WGA  three  lectins  37°C  energy  labeled  neurons  independently  at  fluorescence  as  the  RPE  internalization  with  at  bovine  inhibitor  treated  not  or  of  an  look  to  amelanotic  extranuclear  reflect  the  the  used  cell  ROS-RPE  Labeling  RCA  the  in  cells.  fluorescent  in  of  the  concentration  receptors  and  as  patterns  The  with  certain  RPE  fluorescent  resulted  centralized in  staining a  cells  in buffer  the  spots  RPE  and  of  important  were  bovine  and  WGA,  the  on  arrangement be  lectins  residues  A,  the  may  melanotic  at  at  that  Fluorescent  fixed  of  looked  and  previously resulting  maintained.  in  -93Further  experiments  conclusively  predict  Differences  in  role  t h e mode o f l e c t i n  turnover  further  redistribution plasma  rates  define  studies  are  role  segments  and  receptor  arrangement  packets found  on  (37).  frog  system  TEM  are required  actin  the  was  cells  also tends  fluorescence  was  may  region  much  membrane  to  many  outer  and  membrane  i t was  D-mannose in  lectins  noted treated  of  cells  the of on  that RPE  preventing system.  but  pigmentation a  good  Anti-actin  more c o n c e n t r a t e d  microvilli.  ROS  fluorescence  of the  degree  cytoskeletal  brighter  of  at the level  anti-actin  The h i g h  sugar  Carbohydrates  Acetone  of the f l u o r e s c e n t  surface  rod  r a t RPE.  techniques.  with  their  that  various  preparation  being  to  phagocytized  studies  localize  on t h e p l a s m a  interspersed  as  possible  packets  specific  non-uniform  paper  L-fucose  and d y s t r o p h i c  the i n t e r n a l  represent  binds  segments.  further  out the c e l l .  obscure  receptor  i n the recognition  as  disc  immunoblot  primarily  of  which  such  demonstrated  observation  nuclear  by  is  the rod outer  of normal  through  to  involved  t h e ROS  RPE p l a s m a  also  found  from  It  to quantitatively  detected  concentrated  be  In the f u t u r e ,  surfaces  From  may  reduced  itself  a  experiments  i n t e r a c t i o n between  surfaces.  glycoproteins  significantly  cell  cell  released  play  multiple  interesting in this  in receptor-receptor RPE  may  labeling  of  to  internalization.  receptors  Double  action  required  (77).  proteins  possible  disc  receptor  of l a b e l e d  the  are  and i n t e r n a l i z a t i o n a s Con A  membrane  Lectin  the  lectins  i n the r e d i s t r i b u t i o n patterns.  would  as  using  fluorescent  near  the  patches  -94-  It  appears  phagocytosis cells  is  (3,9,14).  mobilization  actin  of  case,  attached In  suitable  repeatedly. explants  non-specific cells  in  ROS,  attach segments  the level  also  in  possibly  to a  loss  of  needed  opens  up a w h o l e  used  to  needed f o rthe  ROS  have  series  become  been  were  by t h e  bovine  failed  sites  ROS or  through  new  RPE  but bovine the  observed RPE  adapted,  outer  (Fig.  25).  attached  quantitative  phagocytize  future experiments  assay for  Some  ROS  divisions  phagocytosis  and  (Figs.  phagocytized.  cell  to  Rod  cells  any  of  not only  ROS.  of RPE  study  dark  b y SEM  to provide of  used  embryonic  shown,  same  packets  be  a  the phagocytosis  t o RPE c e l l s  This of  rat  prepared  these  to recognize  microvilli.  could  previously in  o f t h e number  of receptor  that  and  specially  attached  b y TEM  failed  new  the  interaction  (4,5,14,15),  to phagocytize  l a r g e ROS  terms  loss  when  receptor  t o study  a s ROS  o l d RPE c e l l s  endocytosed  vitro  from  periphery  few s i t e s  chick  i n the past  as w e l l  seen  o f TEM,  in  in a  using  By u s i n g  Observations  information  the  (17).Possibly, i n the dystrophic  t h e ROS-RPE  n o t been  but a l s o been  involve  points  to the c e l l  of  epithelial  Evidence  h a d t o be d e v e l o p e d  used  have  completely  26,27).  cells  been  two week  have  study  systems  o f ROS.  ROS,  actin.  i n RPE c e l l s  activated  system  vitro  o f ROS  defect  phase  surface.  particles  phagocytosis sealed  only  Assay  have  of  filaments  to further  assay  this  and rearrangement  to the c e l l  order  ingestion  may  actin  is  the  Consequently,  phagocytosis  actin  that  pigment  functioning  activation  mechanistic  literature  d e f e c t i v e i n the dystrophic  or  arrangement  At  from  due or a  system studying  -95ROS  receptor-RPE  receptor  interactions.  CONCLUSIONS  A  7-9  prepared  fold  was  was  found  partially  monoclonal RPE  RPE p l a s m a  membrane  a n d c h a r a c t e r i z e d by e n z y m a t i c  preparation which  enriched  rhodopsin. segments  removed  antibody,  membrane  t o have by  and  large  b u t i t was  localized  bound  The  rhodopsin  Rho-5A3,  Rho-5A3 a n t i b o d y was  analysis.  immunoaffinity  designated  preparation,  The  a  preparation  was  membrane  contamination  chromatography.  was  found  raised  against the  t o be s p e c i f i c  to solubilized  to the N-terminus  of  A  rod  the  for outer  rhodopsin  molecule. In  order  introduced  into  their  in vivo  WGA,  and  induced dense  to further  RCA  sites  pattern  culture  RPE p l a s m a  new  assay  of  ROS  by b o v i n e  system  Future all,  the  obtained  designated  with  terminus  RPE c e l l s  rhodopsin ability  surface  of  were  many  labeled  bovine  .of  C o n A,  RPE  were  and i n t e r n a l i z e  maintaining  surface.  was  Actin  identified  protein. the  a  Finally  phagocytosis  in vitro. can take  RPE p l a s m a  RPE  monoclonal to  lectin  and c y t o s k e l e t a l  RPE-3A6 c o u l d  i t s bovine  the c e l l s  maintained  established for studying  investigation  a newly  antibody  was  they  Fluorescent  on t h e c e l l  membrane  RPE c e l l s ,  where  o f l a b e l , on t h e c e l l  a  has  bovine  to constantly redistribute  a major  along  tissue  characteristics.  as  of  study  bind  several membrane  be  to sealed  specific  completely  determinant. antibody',  approaches.  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