UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Isolation and partial characterization of vesicles derived from the plasma membrane of the chicken gizzard.. 1979

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1979_A1 A93.pdf [ 8.04MB ]
Metadata
JSON: 1.0094743.json
JSON-LD: 1.0094743+ld.json
RDF/XML (Pretty): 1.0094743.xml
RDF/JSON: 1.0094743+rdf.json
Turtle: 1.0094743+rdf-turtle.txt
N-Triples: 1.0094743+rdf-ntriples.txt
Citation
1.0094743.ris

Full Text

ISOLATION AND PARTIAL CHARACTERIZATION OF VESICLES DERIVED FROM THE PLASMA MEMBRANE OF THE CHICKEN GIZZARD MUSCLE by ARISTOTLE AZAD B. Sc., The U n i v e r s i t y of B r i t i s h Columbia, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Anatomy We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1979 © A r i s t o t l e Azad, 1979 I n p r e s e n t i n g 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 o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e 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 c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r 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 g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f , . — The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V6T 1W5 ANATOMY V B P 75-51 1 E ABSTRACT Plasma membrane v e s i c l e s , i s o l a t e d from the chicken g i z z a r d using d i f f e r e n t i a l c e n t r i f u g a t i o n and sucrose gradient c e n t r i f u g a t i o n , were b i o c h e m i c a l l y c h a r a c t e r i z e d . Two f r a c t i o n s obtained from the sucrose g r a d i e n t , F r a c t i o n s 4 and 5 (32% and 34% sucrose r e s p e c t i v e l y ) , were judged to be the most pure i n plasma membranes as based on 5' n u c l e o t i d a s e and i o d i n a t i o n s t u d i e s . Both f r a c t i o n s had the same coomassie blue and PAS s t a i n i n g p r o f i l e when electrophoresed and under e l e c t r o n micro- scopy both f r a c t i o n s c o n s i s t e d of membrane v e s i c l e s of v a r y i n g s i z e . A Mg^ + stimulated ATPase a c t i v i t y was found to be present and highest i n F r a c t i o n 5 w h i l e F r a c t i o n 4 e x h i b i t e d l i t t l e a c t i v i t y . This enzyme was i n h i b i t e d i n the presence of high concentrations of ATP and Mg. A s i m i l a r ecto Mg2+ stim u l a t e d ATPase was observed i n i s o l a t e d smooth muscle c e l l s . Phosphorylation using [Y-^P] ATP was observed a t 205,000, 165,000 and 145,000 daltons i n F r a c t i o n 5 only. Mg promoted dephosphorylation of the 205,000 d a l t o n band w h i l e Ca promoted phosphorylation of the 165,000 d a l t o n band. A l l phosphorylated peaks were s e n s i t i v e to hydroxylamine treatment. These r e s u l t s would seem to i n d i c a t e that there i s a d i f f e r e n c e i n membrane o r i e n t a t i o n between F r a c t i o n 4 and F r a c t i o n 5. The membrane o r i e n t a t i o n i n F r a c t i o n s 4 and 5 was then examined using a c e t y l c h o l i n e s t e r a s e and s i a l i c a c i d was e x t e r n a l plasma membrane markers. F r a c t i o n 4 was found to c o n t a i n mainly i n s i d e - o u t v e s i c l e s i n co n t r a s t to F r a c t i o n 5, which was thought i i i to c o n s i s t mainly of r i g h t - s i d e - o u t plasma membrane v e s i c l e s . The o r i e n t a t i o n d i f f e r e n c e s were f u r t h e r examined using 125 lactoperoxidase c a t a l y z e d i o d i n a t i o n using I . I o d i n a t i o n of F r a c t i o n 4 r e s u l t e d i n the appearance of 125 I i n a band m i g r a t i n g i n SDS electrophoretigrams w i t h an apparent molecular weight of 100,000 d a l t o n s , and minor l a b e l l i n g was seen at 205,000 and 55,000 daltons. 0.05% T r i t o n X-100 s i g n i f i c a n t l y enhanced l a b e l l i n g of a l l three- bands. I o d i n a t i o n of F r a c t i o n 5 r e s u l t e d i n l a b e l l i n g of a l l three bands, but treatment of the membranes w i t h T r i t o n X-100 enhanced l a b e l l i n g only at 100,000 d a l t o n s . I o d i n a t i o n of i n t a c t s i n g l e c e l l s r e s u l t e d i n an i o d i n a t i o n p a t t e r n s i m i l a r to that of F r a c t i o n 5 i n the absence of T r i t o n X-100. Attempts were made to f u r t h e r p u r i f y the membranes using concanavalin A - Sepharose a f f i n i t y chromatography. A f t e r F r a c t i o n 4 was a p p l i e d to the column, four peaks of p r o t e i n could be e l u t e d . The f i r s t two peaks, e l u t e d i n the absence of a methyl-D-mannoside were thought to c o n s i s t of i n s i d e - o u t v e s i c l e s as judged by i o d i n a t i o n and a c e t y l c h o l i n e s t e r a s e sidedness s t u d i e s . The other two peaks, eluted i n the presence of a methyl-D-mannoside were thought to conta i n unsealed plasma membrane v e s i c l e s . Over 90% of the o r i g i n a l l y a p p l i e d p r o t e i n was e l u t e d , 20% being contained i n the two peaks eluted i n the presence of a methyl-D-mannoside. F r a c t i o n 5 behaved q u i t e d i f f e r e n t l y on the a f f i n i t y columns. Approximately 90% of the o r i g i n a l l y a p p l i e d p r o t e i n could not be eluted even i n the presence of a methyl-D-mannoside. iv:- iv. Of the two peaks e l u t e d , one peak obtained i n the absence of a methyl-D-mannoside, was thought to c o n s i s t of i n s i d e - o u t plasma membrane v e s i c l e s . The second peak, e l u t e d i n the presence of amethyl-D-mannoside was thought to c o n t a i n unsealed membrane v e s i c l e s as i n d i c a t e d by sidedness s t u d i e s . I t was concluded that F r a c t i o n s 4 and 5 represent plasma membrane preparations of d i f f e r i n g o r i e n t a t i o n , F r a c t i o n 5 being predominantly r i g h t - s i d e - o u t and F r a c t i o n 4 a f t e r a f f i n i t y chromatography mainly i n s i g h t - o u t . These two f r a c t i o n s may have some a p p l i c a b i l i t y i n i n v e s t i g a t i n g the asymmetry of v a r i o u s membrane transport systems. V •TABLE OF CONTENTS Page Abst r a c t i i Table of Contents v L i s t of Tables v i L i s t of Figures v i i Acknowledgements x I n t r o d u c t i o n 1 A. I s o l a t i o n of Plasma Membranes 3 B. The O r i e n t a t i o n Problem 10 C. Smooth Muscle Preparations 16 D. Ra t i o n a l e 21 M a t e r i a l s 27 Methods 27 A. Enzyme Assays • 27 B. Plasma Membrane I s o l a t i o n 37 C. C e l l Sheet and S i n g l e C e l l P r e p a r a t i o n 39 D. Gel E l e c t r o p h o r e s i s 41 E. I o d i n a t i o n Experiments 46 F. Membrane E x t r a c t i o n Procedure 51 G. A f f i n i t y Chromatography 53 H. Phosphorylation Studies 54 I. E l e c t r o n Microscopy 57 Results 58 A. General 58 B. Membrane Marker Studies 58 C. Plasma Membrane Mg2+ ATPase A c t i v i t i e s 66 D. O r i e n t a t i o n Studies Using 83 A c e t y l c h o l i n e s t e r a s e and S i a l i c A c i d E. I o d i n a t i o n Studies 83 F. E x t r a c t i o n Studies 108 G. A f f i n i t y Chromatography 131 H. Summary 140 Disc u s s i o n 141 L i t e r a t u r e C i t e d 150 v i LIST OF TABLES Table Page I Summary of smooth muscle pr e p a r a t i o n s . 23 I I Substrate s o l u t i o n s to t e s t Mg2+ ATPase 33 s e n s i t i v i t y to pH, Na, L i , K, and ouabain. I I I G e l l f o r m u l a t i o n s used f o r e l e c t r o p h o r e s i s . 42 IV E x t r a c t i o n media used i n membrane e x t r a c t i o n . 52 V Components used i n phosphorylation s t u d i e s . 55 VI T o t a l and s p e c i f i c a c t i v i t i e s of s e l e c t e d marker 60 enzymes at v a r i o u s stages of the f r a c t i o n a t i o n procedure. V i l a S p e c i f i c a c t i v i t i e s of marker enzymes. F r a c t i o n s 60 obtained from sucrose g r a d i e n t s . V l l b S p e c i f i c a c t i v i t i e s of marker enzymes. F r a c t i o n s 61 obtained from sucrose g r a d i e n t s . V I I I A c c e s s i b i l i t y of markers i n sucrose gradient 84 f r a c t i o n s . IX Molecular weight assignments of bands and peaks 85 depicted i n Figures 15 to 41. Xa A c c e s s i b i l i t y of F r a c t i o n s 4 and 5 to 107 lactoperoxidase c a t a l y z e d i o d i n a t i o n . Xb S e l f l a b e l l i n g of lactoperoxidase i n the 107 presence of T r i t o n X-100. XI P r o t e i n contained i n e x t r a c t i o n media and p e l l e t 114 f o l l o w i n g e x t r a c t i o n procedure. X I I C h a r a c t e r i z a t i o n of column f r a c t i o n s e l u t e d 136 from Con A - Sepharose a f f i n i t y columns. v i i LIST OF FIGURES Figure Page 1 Scheme f o r i s o l a t i o n of smooth muscle plasma 40 membranes from the chicken g i z z a r d . 2 E l e c t r o n micrograph of chicken g i z z a r d smooth 59 muscle. 3 E l e c t r o n micrograph of chicken gizzard, smooth 59 muscle c e l l . 4 E l e c t r o n micrograph of F r a c t i o n 4 plasma membranes 65 i s o l a t e d from the chicken g i z z a r d smooth muscle. 5 E l e c t r o n micrograph of F r a c t i o n 5 plasma membranes 65 i s o l a t e d from the chicken g i z z a r d smooth muscle. 6. Opti m i z a t i o n of Mg2+ ATPase a c t i v i t y i n 67 F r a c t i o n 5. 7. E f f e c t s of pH, Na, L i , K and ouabain on the Mg2+ 69 ATPase a c t i v i t y of F r a c t i o n 4 and F r a c t i o n 5. 8. P l o t of ra t e versus logarithm of the substrate 71 [MgATP]-2 f o r the Mg2+ ATPase observed i r i F r a c t i o n 5. 9. Phase c o n t r a s t micrograph of a suspension of 72 s i n g l e smooth muscle c e l l s r ( 8 0 x ) . 10 Phase c o n t r a s t micrograph of a s i n g l e i s o l a t e d 72 smooth muscle c e l l . 11 Phosphorylation patterns of F r a c t i o n 5 at va r i o u s 75 in c u b a t i o n times using [y- 32p] ATP. 12 Phosphorylation patt e r n s of F r a c t i o n 4 and 76 F r a c t i o n 5. 13 Phosphorylation patterns of F r a c t i o n 4 and 78 F r a c t i o n 5. 14 Phosphorylation patterns of F r a c t i o n 5. 80 15 Coomassie blue s t a i n i n g p a t t e r n (top) and PAS 86 p r o f i l e (bottom) of F r a c t i o n 4. 16 Coomassie blue s t a i n i n g p a t t e r n (top) and PAS 87 p r o f i l e (bottom) of F r a c t i o n 5. 125 17 L a b e l l i n g of muscle cubes w i t h I . 89 125 18 L a b e l l i n g of c e l l sheets w i t h I . 90 v i i i F igure Page 19 L a b e l l i n g of a suspension of i s o l ^ f r e d 91 s i n g l e smooth muscle c e l l s w i t h I . 20 I o d i n a t i o n patterns of F r a c t i o n 4 using 92 p r e l a b e l l e d muscle cubes. 21 I o d i n a t i o n patterns of F r a c t i o n 5 using 93 p r e l a b e l l e d muscle cubes. 22 I o d i n a t i o n patterns of F r a c t i o n 4 using 94 p r e l a b e l l e d c e l l sheets. 23 I o d i n a t i o n patterns of F r a c t i o n 5 using 95 p r e l a b e l l e d c e l l sheets. 24 I o d i n a t i o n of F r a c t i o n 4. 97 25 I o d i n a t i o n of F r a c t i o n 5. 98 26 I o d i n a t i o n of sucrose f r e e F r a c t i o n 4. 99 27 I o d i n a t i o n of sucrose free F r a c t i o n 5. 100 28 S e l f i o d i n a t i o n of lactoperoxidase. 102 29 A c c e s s i b i l i t y of F r a c t i o n 4 to i o d i n a t i o n 103 using 1251. 30 A c c e s s i b i l i t y of F r a c t i o n 5 to i o d i n a t i o n 105 using 1251. 31 E x t r a c t i o n of F r a c t i o n 4 using H2O, 115 Ethylenediamine t e t r a a c e t a t e and D i g i t o n i n . 32 E x t r a c t i o n of F r a c t i o n 5 using H20, 117 Ethylenediamine t e t r a a c e t a t e and D i g i t o n i n . 33 E x t r a c t i o n of F r a c t i o n 4 using Dimethyl maleic 119 anhydride (DMMA). 34 E x t r a c t i o n of F r a c t i o n 5 using Dimethyl maleic 121 anhydride (DMMA). 35 E x t r a c t i o n of F r a c t i o n 4 using p-Chloromercuri- 123 benzene sulphonic a c i d (pCMBS). 36 E x t r a c t i o n of F r a c t i o n 5 using p-Chloromercuri'- 125 benzene sulphonic a c i d (pCMBS). 37 E x t r a c t i o n of F r a c t i o n 4 using T r i t o n X-100 '" .127 (TX-100). i x Figure Page 38 E x t r a c t i o n of F r a c t i o n 5 using T r i t o n X-100 129 (TX-100). 39 Con A - Sepharose a f f i n i t y chromatography of 134 F r a c t i o n 4- (bottom) and F r a c t i o n 5 (t o p ) . 40a A n a l y s i s of peak f r a c t i o n s obtained by Con A - 137 Sepharose a f f i n i t y chromatography of F r a c t i o n 4. 40b A n a l y s i s of peak f r a c t i o n s obtained by Con A - 138 Sepharose a f f i n i t y chromatography of F r a c t i o n 4. 41 A n a l y s i s of peak f r a c t i o n s obtained by Con A - 139 Sepharose a f f i n i t y chromatography of F r a c t i o n 5. X ACKNOWLEDGEMENTS To Profes s o r V. P a l a t y , my humble and most s i n c e r e s u p e r v i s o r , I which to extend my s i n c e r e s t thanks, e s p e c i a l l y f o r h i s t o l e r a n c e and guidance. As w e l l , I would l i k e to thank Ms. Maryette Mar f o r her expert t e c h n i c a l a s s i s t a n c e and f r i e n d l y advice. The t e c h n i c a l e x p e r t i s e of Mrs. V i r g i n i a L a i and Ms. Susan Shinn was a l s o appreciated. The encouragement and immense tolerance d i s p l a y e d by the f a c u l t y and s t a f f of the Department of Anatomy were prime f a c t o r s i n the completion of t h i s t h e s i s . F i n a l l y I wish to thank Miss Judy Myar f o r typing the t h e s i s d e s p i t e a l l odds and I wish to thank the n i g h t people, L y l e , Fred, Lea, Peter and Don f o r p r o v i d i n g proof reading s k i l l s and tremendous encouragement. To Mike S i l v e r s t e i n f o r h i s a l l n i g h t d i s c u s s i o n s on membranes, my s i n c e r e s t thanks. - 1 - INTRODUCTION Although the stu d i e s w i t h i s o l a t e d preparations r i c h i n smooth muscle have provided v a l u a b l e i n f o r m a t i o n on many fe a t u r e s of membrane phenomena i n t h i s type of c e l l s , p e r u s a l of the l i t e r a t u r e r e v e a l s that some bas i c questions have not been answered s a t i s f a c t o r i l y as yet. For example, i t i s recognized that the mechanism of Ca e x t r u s i o n from the c e l l plays a key r o l e i n the c o n t r o l of te n s i o n , but the present knowledge of the mechanism i s r a t h e r incomplete. Indeed, i t has not even been e s t a b l i s h e d whether the energy r e q u i r e d f o r e x t r u s i o n of Ca against a steep gradient of the negative chemical p o t e n t i a l of t h i s i o n i s provided by h y d r o l y s i s of ATP, or spontaneous i n f l u x of Na (BLAUSTEIN, 1977). I t must be appreciated, however, that study of these and other problems i s complicated by f a c t o r s l a r g e l y unique to smooth muscle preparations;. The dimensions of smooth muscle c e l l s make i t rather u n l i k e l y that a number of the methods already a p p l i e d s u c c e s s f u l l y to s t u d i e s on other types of muscle c e l l s , such as the monitoring of the i n t r a c e l l u l a r c o n c e n t r a t i o n of 2+ Ca by measurement of the luminiscence of i n j e c t e d aequorin (ALLEN & BLINKS, 1978) can be used f o r smooth muscle s t u d i e s . Secondly, the l a r g e e x t r a c e l l u l a r space w i t h i t s high con- c e n t r a t i o n of f i x e d charged groups makes i t d i f f i c u l t , i f not impossible, to o b t a i n r e l i a b l e i n f o r m a t i o n on the transmembrane ion. f l u x e s from e v a l u a t i o n of isotope f l u x data. L a s t , but not l e a s t , c e l l s other than smooth muscle are f r e q u e n t l y present i n i s o l a t e d p r e p a r a t i o n s , and unless t h i s f a c t i s taken i n t o account, some of the observations may be i n t e r p r e t e d i n c o r r e c t l y . 2 - 2 - To e l i m i n a t e the l a s t two f a c t o r s , i s o l a t e d i n d i v i d u a l c e l l s have been introduced by the group of FAY (FAY, 1973; FAY, 1977; SCHEID et a l . , 1979), but the s t u d i e s have been r e s t r i c t e d almost e x c l u s i v e l y to c e l l s i s o l a t e d from the stomach muscularis of Bufo marinus by enzymic d i g e s t i o n . I t remains to be shown that t h i s approach would apply e q u a l l y w e l l to smooth muscle c e l l s from mammaliam t i s s u e s . An a t t r a c t i v e a l t e r n a t i v e i s to study membrane phenomena using the i s o l a t e d plasma membrane. The advantages that t h i s approach o f f e r s are obvious. U n f o r t u n a t e l y , r e l a t i v e l y l i t t l e a t t e n t i o n has been paid to the f a c t t h a t , i f meaningful r e s u l t s are to be obtained, the p r e p a r a t i o n must meet c e r t a i n c r i t e r i a . I f the p r e p a r a t i o n were to be used, e.g., f o r a study of. Ca transport., i t should be at l e a s t e s s e n t i a l l y f r e e of contamination by membranes of sarcoplasmic r e t i c u l u m . In other types of muscle 2+ t h i s c e l l u l a r component i s known to e x h i b i t a Ca - s t i m u l a t e d ATPase a c t i v i t y , which, i n c o n t r a s t to the m i t o c h o n d r i a l 2+ Ca - stimulated ATPase, cannot be i n h i b i t e d s e l e c t i v e l y (CARAFOLI &. CROMPTON, 1978). C l e a r l y , an i d e a l p r e p a r a t i o n should be f r e e of contamination by membranes derived from i n t r a c e l l u l a r o r g a n e l l e s . There are, however, other c r i t e r i a which an i d e a l p r e p a r a t i o n should meet. In order to remove the contaminating membranes and components of the cytoplasm, a m u l t i - s t e p f r a c t i o n a t i o n procedure i s u s u a l l y r e q u i r e d . The problem here 3 - 3 - i s t h a t , i n the course of f r a c t i o n a t i o n , some important, but l o o s e l y bound components of the plasma membrane may become l o s t , or, conversely, some components that are not associated w i t h the membrane i n s i t u may become f i r m l y attached to the f i n a l p r e p a r a t i o n . I d e a l l y the process of f r a c t i o n a t i o n should produce a preparation whose c o m p o i s i t i o n , conformation and other f e a t u r e s are i d e n t i c a l to those e x h i b i t e d by the plasma membrane i n s i t u . This problem i s much more complicated than i t may seem to be a t f i r s t sight., because the membrane i n s i t u i s t y p i c a l l y under the i n f l u e n c e of an e l e c t r i c a l f i e l d of appreciable s t r e n g t h and the l a t t e r may have a pronounced e f f e c t on the conformation and d i s p o s i t i o n i n the membrane of any component possessing at l e a s t a d i p o l e or a charged, group. The s i g n i f i c a n c e of the f a c t that the two surfaces of the membrane are t y p i c a l l y exposed to s o l u t i o n s of markedly d i f f e r e n t composition should not be underestimated. F i n a l l y , i f the membrane i s o l a t i o n procedure i s to be of any value, i t s y i e l d should be reasonably high, p a r t i c u l a r l y because the qu a n t i t y of t i s s u e s r i c h i n smooth muscle that can be obtained from t y p i c a l l a b o r a t o r y animals i s u s u a l l y q u i t e l i m i t e d . A. I s o l a t i o n of Plasma Membranes In ge n e r a l , the i n i t i a l step i n the pr e p a r a t i o n of i s o l a t e d plasma membranes i s homogenization, which i s u s u a l l y achieved by a p p l i c a t i o n of shear fo r c e s (BIRNIE, 1972). This i s followed by d i f f e r e n t i a l c e n t r i f u g a t i o n to separate the plasma . . . 4 0 - 4 - membranes from the c e l l d e b r i s , n u c l e i and mitochondria (GRAHAM, 1972; GRAHAM, 1975; NEVILLE, 1975; SCHAPIRA, 1975; SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). The r e s u l t i n g crude "microsomal" p r e p a r a t i o n can be f u r t h e r f r a c t i o n a t e d by d e n s i t y gradient c e n t r i f u g a t i o n . T y p i c a l l y , plasma membranes and endoplasmic r e t i c u l u m are found at lower d e n s i t i e s (1.14 - -3 1.15 g x cm ) wh i l e mitochondria are at higher d e n s i t i e s -3 (1.16 - 1.18 g x cm ) (PRICE, 1974; TOLBERT, 1974) . I f warranted, f u r t h e r p u r i f i c a t i o n of the p r e p a r a t i o n can be attempted using techniques such as a f f i n i t y chromatography (CUATRECASES, 1973; PHARMACIA, 1974; SHARON & LIS, 1975; HYNES, 1976; WALSH et a l . , 1976; BRUNNER e t . a l . , 1977). The homogenization technique i s of c r i t i c a l importance i n that i t a l s o determines the f i n a l y i e l d of plasma membranes. There are three b a s i c types of homogenization mechanical, l i q u i d and gaseous (BIRNIE, 1972; GRAHAM, 1975; WALLACH & SCHMIDT-ULLRICH, 1977). Mechanical p r e p a r a t i o n using mechanical shear employs two ba s i c techniques. The f i r s t , the fre e z e thaw technique, used l e s s f r e q u e n t l y , i n v o l v e s using c y c l e s of f r e e z i n g and thawing which r e s u l t i n the d i s r u p t i o n of c e l l s by i n t r a c e l l u l a r i c e c r y s t a l formation. In the second type of mechanical shear, employed by commercial u n i t s l i k e the P o l y t r o n and MSE homogenizers, the sample i s drawn i n t o a working head where i t i s mixed by r o t a t i n g blades and sheared during e x p u l s i o n from the working head. Membranes are u s u a l l y obtained i n the form of v e s i c l e s rather than c e l l sheets. U n t i l r e c e n t l y , i t was f e l t t hat the drawbacks of mechanical shear f a r outweighed the advantages. . . 5 - 5 - Drawbacks to t h i s method i n c l u d e the p o s s i b l e damage to the plasma and i n t r a c e l l u l a r membranes by the high l o c a l temperatures generated during homogenization and d i s r u p t i o n of most o r g a n e l l e s . On the other hand, mechanical shear of t h i s type i f q u i t e e f f e c t i v e even f o r homogenization of t i s s u e s r i c h i n c o l l a g e n and e l a s t i n . In l i q u i d shear homogenization, the d i s r u p t i v e f o r c e s are c o n s i d e r a b l y I : weaker than those of mechanical shear. Tissues are d i s r u p t e d by being forced through a narrow space between a moving p e s t l e and the w a l l of the c o n t a i n i n g v e s s e l . Using t h i s method, s o f t t i s s u e s are r e a d i l y homogenized without con- comitant d i s r u p t i o n .of c e l l u l a r o r g a n e l l e s . In the homogenate, the plasma membranes are u s u a l l y present i n the form of sheets r a t h e r than, v e s i c l e s , but spontaneous v e s i c l e formation occurs w i t h time. Commercially a v a i l a b l e homogenizers of t h i s type i n c l u d e the Potter-Elvehjem and the Dounce homogenizers. The t h i r d method of homogenization i s gaseous shear. Nitrogen c a v i t a t i o n using a Parr bomb i n v o l v e s e q u i l i b r a t i n g a s t i r r e d c e l l suspension w i t h oxygen-free n i t r o g e n at pressures 2 between 500 and 800 l b . / i n . f o r periods of 15-20 minutes (HUNTER & COMMERFORD, 1961). C e l l d i s r u p t i o n occurs upon sudden r e l e a s e of the pressure 1due to the gas expanding w i t h i n the c e l l or by the shearing f o r c e s of the r a p i d l y forming bubbles of gas i n the l i q u i d phase. Gaseous shear, however, r e q u i r e s that the t i s s u e be i n the form of i s o l a t e d s i n g l e c e l l s . The p r e p a r a t i o n of s i n g l e c e l l s i t s e l f i s a s s o c i a t e d w i t h drawbacks i n c l u d i n g a l t e r a t i o n of the plasma membranes by proteases found i n commercial collagenase preparations (R0DBELL, 1964; BAGBY et a l . , 1971; FAY & DELISE, 1973; 6 - 6 - RODBELL & KRISHNA, 1974; SMALL, 197.7). The plasma membranes, (PMs) , endoplasmic r e t i c u l u m and nuclear membranes form very small v e s i c l e s , which makes f u r t h e r d e n s i t y g r a d i e n t separations necessary. Other c e l l organelles are g e n e r a l l y maintained i n t a c t . Osmotic l y s i s (BARBER & JAMIESON, 1973) used i n f r e q u e n t l y , has been a p p l i e d to the i s o l a t i o n of membranes from s k e l e t a l muscle (McCOLLESTER, 1962). In t h i s method,, segments are incubated at high temperatures and then excess d i s t i l l e d water i s added. This leads to an abrupt d i s s o l u t i o n of i n t r a c e l l u l a r components, le a v i n g only muscle plasma membranes. At.the same time, however, p a r t i a l s o l u b i l i z a t i o n of p e r i p h e r a l PM p r o t e i n s occurs accompanied by an increase i n membrane p e r m e a b i l i t y . With few exceptions, none of the shear techniques provides p e r f e c t homogenization of the s t a r t i n g m a t e r i a l . I t i s o f t e n necessary, t h e r e f o r e , to remove the unhomogenized fragments by f i l t r a t i o n . The f i l t r a t e contains i n a d d i t i o n to plasma membranes, i n t a c t and d i s r u p t e d o r g a n e l l e s as w e l l as s o l u b l e a n d . i n s o l u b l e components of the cytoplasm. As mentioned e a r l i e r , the f i l t r a t e . i s then f r a c t i o n a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n , a technique based on d i f f e r e n c e s i n buoyant d e n s i t i e s of the v a r i o u s c e l l components. For example, n u c l e i sediment r a p i d l y i n a g r a v i t a t i o n a l f i e l d of 2000 g. A f t e r the c e l l d e b r i s , nuclear membranes, i n t a c t mitochondria and other o r g a n e l l e s have been removed by c e n t r i f u g a t i o n s a t lower g f o r c e s (2000 g - 17,000 g ) , the microsomal f r a c t i o n c o n t a i n i n g PM i s u s u a l l y p e l l e t e d by c e n t r i f u g a t i o n at 100,000 g (GRAHAM, 1975; 7 - 7 - SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). Many i n v e s t i g a t o r s consider the 100,000 g p e l l e t s u f f i c i e n t l y enriched i n plasma membranes to warrant i t s use i n v a r i o u s s t u d i e s . However, i t has c l e a r l y been demonstrated that t h i s enriched 100,000 g plasma membrane f r a c t i o n s t i l l contains contamination from fragmented o r g a n e l l e s . These fragments, o f t e n i n the form of v e s i c l e s , may s e r i o u s l y b i a s any r e s u l t s observed i n t h i s plasma membrane f r a c t i o n . The demonstrated presence of contamination warrants a t h i r d stage of membrane i s o l a t i o n such as the use of d e n s i t y gradient c e n t r i f u g a t i o n . This i n v o l v e s c e n t r i f u g a t i o n of the microsomal f r a c t i o n on a sucrose d e n s i t y gradient at h i g h g f o r c e s (120,000 g) f o r long periods of time (2-24 hours) which allow s the i n d i v i d u a l components of the microsomal f r a c t i o n to reach t h e i r own buoyant d e n s i t i e s . The gradient used must span the f u l l range of d e n s i t i e s e x h i b i t e d by the v a r i o u s membrane fragments present. The gradient m a t e r i a l must be water s o l u b l e , which requirement i s not met by e i t h e r dextran or F i c o l l . Separations that do-use the l a t t e r r e l y on d i f f e r e n c e s i n q u a n t i t a t i v e r i s e s i n buoyant d e n s i t y when f i x e d charges are 2+ n e u t r a l i z e d by Mg (STECK, 1974a; GRAHAM,, 1975) but i n g e n e r a l , sucrose g r a d i e n t s are more commonly used. The plasma membranes f r a c t i o n d e r i v e d from the sucrose gr a d i e n t i s q u i t e o f t e n sub- s t a n t i a l l y f r e e from contamination by i n t r a c e l l u l a r membranes, though not n e c e s s a r i l y 100% pure. This r a i s e s a number of important questions. Can or should.the membranes be f u r t h e r p u r i f i e d and how does one assess the increases i n plasma membranes not only i n a f o u r t h stage of membrane i s o l a t i o n but i n each stage? 8 - 8 - Further p u r i f i c a t i o n of the plasma membranes has inv o l v e d one b a s i c approach. That i s , by s e l e c t i v e l y a l t e r i n g the d e n s i t y of the PM i n stage 1 or stage 3, b e t t e r separation can be e f f e c t e d based on l a r g e r d i f f e r e n c e s i n the buoyant d e n s i t e s of plasma membranes as compared to i n t r a c e l l u l a r membranes. This has been achieved by a t t a c h i n g l e c t i n s to plasma membrane carbohydrate m o i e t i e s (CUATRECASES, 1973; NICHOLSON, 1974; SHARON & L I S , 1975; HYNES, 1976; BARCHI et a l . , 1977; WALLACH & SCHMIDT-ULLRICH, 1977). The l a b e l l i n g of the PM w i t h p l a s t i c microspheres a f f o r d s the same advantage (LIM et a l . , 1975). D i g i t o n i n , incubated w i t h the microsomal p e l l e t p r i o r to gradient c e n t r i f u g a t i o n , has been found to increase the d e n s i t y of PM over other i n t r a c e l l u l a r membranes (LEWIS et a l . , 1975; MAGARGAL et a l . , 1978). Not only i s i t p o s s i b l e to a l t e r the d e n s i t y of the plasma membrane, but also that of the contaminating components. A good example of t h i s i s seen i n the sep a r a t i o n of plasma membranes from sarcoplasmic r e t i c u l u m i n heart muscle (LEVITSKY et a l . , 1976). The SR was allowed to accumulate Ca i n the presence of ATP and oxalate and the SR then removed by gradient c e n t r i f u g a t i o n . The v a l i d i t y of the whole process of membrane i s o l a t i o n r e s t s on the a b i l i t y to assess the r e s u l t s of each step used. The degree of plasma membrane p u r i f i c a t i o n can be monitored i n two ways. Removal of the contaminants can be assessed by s e l e c t i v e l y measuring a f e a t u r e such as an enzymatic a c t i v i t y of the r e s p e c t i v e o r g a n e l l e i n the supernatant and p e l l e t . A few examples of enzymatic markers are: mitochondria (BONNER, 1955; DONALDSON et a l . , 1972; TOLBERT, 1974): succinate dehydrogenase, cytochrome ĉ  oxidase, fumarase and NADH cyt ^ r e d u c t a s e (rotenone s e n s i t i v e ) ; 9 - 9 - GOLGI APPARATUS (FLEISCHER & KERVINA, 1974): g a l a c t o s y l t r a n s f e r a s e ; LYSOZOMES (HUBSCHER & NEST, 1965; HODGES & LEONARD, 1974): a c i d phosphatase; ENDOPLASMIC RETICULUM (NORDLIE & ARION, 1966; TOLBERT, 1974): glucose-6-phosphatase, NADPH cy t £ reductase and p r o t e i n s y n t h e s i s . Some of the above markers are found w i t h a n o n - s p e c i f i c d i s t r i b u t i o n i n other o r g a n e l l e s , and th e r e f o r e have l i m i t e d value as markers. The above assessment may be supplemented by s e m i - q u a n t i t a t i v e examination of the f r a c t i o n under the e l e c t r o n microscope. The gradual enrichment of plasma membranes can be followed by markers s p e c i f i c f o r the plasma membrane (WALLACH & WINZLER, 1974). Such markers i n c l u d e endogenous chemical markers ( c h o l e s t e r o l / p h o s p h o l i p i d r a t i o s ) , enzyme markers (5' n u c l e o t i d a s e , adenylate c y c l a s e , ATPases) (RODBELL & KRISHNA, 1974; WIDNELL, 1974), v i r u s r e c e p t o r s , covalent l a b e l s and immunological markers. C l o s e l y r e l a t e d to the l a s t marker are l e c t i n b i n d i n g s i t e s . L e c t i n s are thought to bind to c e l l plasma membrane surfaces r e a c t i n g w i t h t e r m i n a l non reducing sugars i n g l y c o p r o t e i n s and/or l i p i d s . Inherent i n t h i s work i s the assumption that the l a b e l does not permeate the plasma membrane (SCHIMMEL & KENT, 1977; WALLACH & SCHMIDT-ULLRICH, 1977). Once i t has been e s t a b l i s h e d that the plasma membrane pre p a r a t i o n i s of acceptable p u r i t y , one must consider the s t a t e of the plasma membranes themselves. I t i s known that the plasma membranes can be i n the form of membrane sheets or v e s i c l e s . A l s o , the v e s i c l e s , may be r i g h t - s i d e - o u t (RO) or 10 - 10 - in s i d e - o u t (10). Therefore, before using the p r e p a r a t i o n to i n v e s t i g a t e plasma membrane p r o p e r t i e s we must know the o r i e n t a t i o n of the membrane. As w e l l , i t may be p o s s i b l e to invoke a f i f t h stage of membrane i s o l a t i o n to separate out the unsealed v e s i c l e s , RO v e s i c l e s and 10 v e s i c l e s . This would a l l o w one to examine s p e c i f i c phenomena as s o c i a t e d w i t h the cytoplasmic membrane surface and/or the e x t e r n a l membrane surface. B. The O r i e n t a t i o n Problem Each b i o l o g i c a l membrane operates d i f f e r e n t l y on the two compartments i t separates. Being a n i s o t r o p h i c i n i t s f u n c t i o n , the plasma membranes has been shown to be assymetric w i t h respect to the composition of the two surfaces (STECK, 1974b). I d e n t i f i c a t i o n of the components at each surface would do much to de f i n e the s t r u c t u r e . I n v e s t i g a t o r s have approached the problem of o r i e n t a t i o n from two d i r e c t i o n s . (STECK, 1974a; STECK & KANT, 1974). The i n i t i a l one i n v o l v e s the assessment of membrane sidedness using marker enzymes s p e c i f i c f o r e i t h e r the e x t e r n a l or cytoplasmic surface. Detergents can be used to make both membrane surfaces e q u a l l y a c c e s s i b l e to the enzyme sub s t r a t e s used. S e l e c t i v e p r o t e i n e x t r a c t i o n a l s o occurs during detergent treatment (HELENIUS & SIMONS, 1975; TANFORD & REYNOLDS, 1976). This must be appreci a t e d . R e s u l t s observed using preparations c o n t a i n i n g a mixture of r i g h t - s i d e - o u t , i n s i d e - o u t and unsealed plasma, membrane v e s i c l e s are thought to r e f l e c t the general o r i e n t a t i o n of the membranes. The second approach to e l u c i d a t i o n of o r i e n t a t i o n i n v o l v e s assessment as above, combined w i t h a f i f t h stage of membrane 11 - 11 - f r a c t i o n a t i o n i n which attempts are made.to separate the p u r i f i e d plasma membrane prep a r a t i o n f u r t h e r on the b a s i s of membrane o r i e n t a t i o n . In general there are three techniques a v a i l a b l e f o r a f i f t h stage of i s o l a t i o n . These are the use high polymer gradients (STECK, 1974a) combined w i t h aqueous p a r t i t i o n / c o u n t e r current d i s t r i b u t i o n (AP./CCD) (DODGE et a l . , 1963; ALBERTSSON, 1970; WALTER & KROB, 1976; WALTER, 1978), f r e e flow e l e c t r o p h o r e s i s (FFE) (HANNIG & HEIDRICH, 1974; HANNIG, 1975a; HANNIG, 1975b) and a f f i n i t y chromatography (MURTHY & HERCZ, 1973; PHARMACIA, 1974; HYNES, 1976; WALSH et a l . , 1976; BRUNNER et a l . , 1977). E'ach has been a p p l i e d w i t h v a r y i n g degrees of success. High d e n s i t y F i c o l l polymer g r a d i e n t s combined w i t h AP/CCD have proven i d e a l f o r separating membranes of d i f f e r i n g o r i e n t a t i o n from c e r t a i n t i s s u e s (STECK, 1974a). Sealed v e s i c l e s are f i r s t separated from unsealed ones using F i c o l l d e n s i t y g r a d i e n t s . The sep a r a t i o n depends on the f a c t that sealed v e s i c l e s are not c o l l a p s e d as on sucrose g r a d i e n t s , but i n s t e a d they expand. They ther e f o r e have buoyant d e n s i t i e s d i f f e r e n t from unsealed v e s i c l e s . I t i s the low s o l u b i l i t y and p e r m e a b i l i t y of F i c o l l which permits t h i s . G l y c e r o l , f o r example, i s not acceptable s i n c e i t r e a d i l y permeates sealed v e s i c l e s . Once the sealed membranes have been separated from the sealed v e s i c l e s , AP i s used to separate the two populations of v e s i c l e s remaining. Aqueous p a r t i t i o n separations r e l y on the e x p l o i t a t i o n of subtile., physicochemical d i f f e r e n c e s between the two d i f f e r e n t membrane surfa c e s . These surface p r o p e r t i e s are h i g h l y dependent on i o n i c c o n d i t i o n s and pH. One can f u r t h e r e f f e c t separations by modifying the polymers used i n the p a r t i t i o n . U t i l i z a t i o n of 12 - 12 - polymer l i g a n d s , such as d e r i v a t i v e s of dextrans and l e c t i n s , s p e c i f i c f o r membrane receptors shows great promise. The most w e l l known a p p l i c a t i o n of the above technique i s that of Steck (STECK, 1974a) using red blood c e l l ghosts (rbcg). In the i n i t i a l i n v e s t i g a t i o n he l o c a l i z e d marker enzymes on both membrane surfaces. L o c a l i z e d on the cytoplasmic surfaces of rbc were Na+/K+ ATPase, gylceraldehyde-3-phosphate dehydrogenase, adenylate c y c l a s e , p r o t e i n kinase and NADH cyt <: reductase. E x t e r n a l l y l o c a l i z e d were a c e t y l c h o l i n e s t e r a s e , s i a l i c a c i d residues and the ouabain bind i n g s i t e of Na+/K+ ATPase. These markers were used to check on the i s o l a t i o n o f v e s i c l e s w i t h d i f f e r e n t o r i e n t a t i o n s . Unsealed ghosts were removed from RO and 10 v e s i c l e s by F i c o l l g r a d i e n t s . A dextran T110 and g l y c o l 6000 p a r t i t i o n was then used to separate the RO v e s i c l e s from the 10 v e s i c l e s ; w i t h separation o c c u r r i n g only under c e r t a i n i o n i c c o n d i t i o n s and temperatures. I n t e r e s t i n g l y i t was observed t h a t h i g h i o n i c s t r e n g t h l e a d to accumulation of a l l the v e s i c l e s at the i n t e r f a c e . Halide i o n drove the v e s i c l e s i n t o the lower phase w h i l e phosphate reversed t h i s trend. Under i n v e s t i g a t i o n as a technique f o r the sep a r a t i o n of RO.vesicles i s f r e e f l o w e l e c t r o p h o r e s i s (FFE) (HANNIG & HEIDRICH, 1974; HANNIG, 1975a; HANNIG, 1975b). This method e x p l o i t s d i f f e r e n c e s i n surface charge, d e n s i t y and s i z e between R0 and 10 v e s i c l e s . The mixture of plasma membrane v e s i c l e s i s i n j e c t e d i n t o a continuously f l o w i n g b u f f e r w i t h an ap p l i e d e l e c t r i c f i e l d a t r i g h t angle to the f l o w d i r e c t i o n . 13 - 13 - The v e s i c l e s separate according to e l e c t r o p h o r e t i c m o b i l i t y . d u r i n g flow. Problems a r i s e s i n c e thermally undisturbed f l o w of the l i q u i d c u r t a i n can only be obtained at high flow v e l o c i t i e s which may r e s u l t i n turbulence. S u f f i c i e n t d e f l e c t i o n only occurs w i t h longer and smaller chambers and at high f i e l d s t rengths. The l a t t e r i s r u l e d out as the heat generated increases w i t h the f i e l d s t r e n g t h squared but by employing very low i o n i c s t r e n g t h media f o r the sep a r a t i o n b u f f e r the heat problem can be avoided. As w e l l , i t should be noted that higher l i q u i d c u r t a i n v e l o c i t i e s f u r t h e r reduce the heat problem, the p r o b a b i l i t y s t i l l e x i s t s that turbulence may occur. The l i m i t i n g f a c t o r at low i o n i c strengths appears to be the i n s t a b i l i t y of the v e s i c l e s (STECK, 1974a). Further e f f e c t s can occur w i t h contamination from DNA, RNA and c e l l n u c l e i which bind to the membrane surfaces screening charge. To date, RO arid 10 rbc v e s i c l e s , v i r u s e s , b a c t e r i a , p r o t e i n s and n u c l e i a c i d s have been separated. The l a s t major method of current p r a c t i c a l use i n the separation of p r e f e r e n t i a l l y o r i e n t e d v e s i c l e s i s a f f i n i t y chromatography and r e l a t e d techniques. L e c t i n s , such as, WGA, Con A, RGA, are c o v a l e n t l y l i n k e d to s o l i d supports l i k e dextran, agarose, sepharose or nylon f i b r e s . RO v e s i c l e s and unsealed v e s i c l e s are thought to bind to the c o v a l e n t l y l i n k e d l e c t i n by s p e c i f i c sugar m o i t i e s l o c a t e d on the e x t e r n a l plasma membrane surface w h i l e 10 v e s i c l e s are not absorbed and pass through the column. The bound membranes are then e l u t e d by adding a sugar which competes w i t h the membranes f o r the l e c t i n b i n d i n g s i t e s . 14 - 14 - As mentioned e a r l i e r a v a r i a t i o n on t h i s theme in c l u d e s adding l e c t i n to a mixture of RO and .10 v e s i c l e s followed by d e n s i t y gradient c e n t r i f u g a t i o n to separate the higher d e n s i t y RO v e s i c l e s from the 10 ones (CUATRESAS, 1973). There have been two major st u d i e s using Con A a f f i n i t y chromatography to separate populations of 10 and RO v e s i c l e s , but w i t h c o n f l i c t i n g r e s u l t s . Using porcine lymphocyte homogenates Walsh (WALSH et a l . . , 1976) i s o l a t e d what appeared to be 10 lymphocyte plasma membranes as judged by marker s t u d i e s , immunoprecipitation, and f e r r i t i n l i n k e d Con A experiments. 40% of the p r o t e i n a p p l i e d to the column was recovered. However, 50% could not be eluted under any c o n d i t i o n s and appeared to be due to high a f f i n i t y n o n - s p e c i f i c m u l t i v a l e n t b i n d i n g . The study a l s o revealed a number of drawbacks to t h i s type of membrane p u r i f i c a t i o n . L e c t i n s i n h i b i t c e r t a i n enzymes and a l s o cause capping of surface markers. The low o s m o l a r i t y of the b u f f e r s may lead to an increase i n membrane p e r m e a b i l i t y . In the second study (BRUNNER et a l . , 1977), anywhere from 60 to.70% of the a p p l i e d membranes remained bound to the l e c t i n l i n k e d sepharose beads. The membranes, however, could be e l u t e d i n the presence of a methyl-D-mannoside accompanied by mechanical s t i r r i n g of the Con A l i n k e d sepharose beads. Whether the e l u t e d p r o t e i n was Con A or membranes i s subject to conjecture as no e l e c t r o p h o r e t i c g e l s were run and no enzyme marker assays were done. As w e l l , mechanical s t i r r i n g of the beads may have been associated w i t h fragmentation. 15 - 15 - The above technique has the advantages of being r e l a t i v e l y inexpensive and easy to c a r r y out. However, the f a c t that 50% of the p r o t e i n i s oft e n not recoverable does not auger w e l l f o r the i s o l a t i o n of a RO set of membranes. A l l the previous methods o u t l i n e d i n v o l v e the use of a membrane prep a r a t i o n that has a v a r i a b l e r a t i o of RO:10:unsealed membrane v e s i c l e s which may be dependent upon the homogenzation method used. The separation procedures could be e l i m i n a t e d i f i t became p o s s i b l e to c o n t r o l the membrane o r i e n t a t i o n during p r e p a r a t i o n and p r e d i c t membrane o r i e n t a t i o n based on the method of prep a r a t i o n . To date both questions remain b a s i c a l l y unanswered. Only Steck (STECK, 1974a; STECK & KANT, 1974) has s u c c e s s f u l l y prepared 10 and RO v e s i c l e s from rbc ghosts, by va r y i n g the i o n i c m i l i e u . While i t i s s t i l l not c l e a r how the i o n i c m i l i e u determines v e s i c l e sidedness, one might speculate that the l a b e l l i n g of plasma membranes w i t h l e c t i n s p r i o r to homogenization may a l t e r the R0:I0 membrane v e s i c l e r a t i o . Despite the many methods a v a i l a b l e and the many t h e o r i e s on how to increase y i e l d s of p r e f e r e n t i a l l y o r i e n t e d v e s i c l e s , the attainment of t h i s goal i s s t i l l q u i t e f a r away. The 10 and RO plasma membranes i s o l a t e d from the rbc membrane were the r e s u l t of fortunate observation and rig o r o u s c h a r a c t e r i z a t i o n . I t i s only through the l a t t e r that we can begin to understand which f a c t o r s are re s p o n s i b l e f o r the o r i e n t a t i o n s seen i n plasma membrane pre p a r a t i o n s . 16 - 16 - There are many plasma membrane preparations from v a r i o u s t i s s u e s which s u f f e r from problems of'contamination and i n d e f i n i t e membrane o r i e n t a t i o n . These preparations o f t e n have f a i l e d to use more than 2 stages i n the membrane i s o l a t i o n w h i l e others.have f a i l e d to c a r r y out proper c h a r a c t e r i z a t i o n of the membrane pr e p a r a t i o n . These d e f i c i e n c i e s are no more apparent than i n the case of smooth muscle though more r e c e n t l y some good s t u d i e s have appeared. C. Smooth Muscle Preparations As shown i n Table I , plasma membrane f r a c t i o n s derived from smooth muscle have been prepared by l i q u i d and mechanical shear techniques. Many of the i n v e s t i g a t i o n s l a c k proper e v a l u a t i o n of the p u r i t y of t h e i r plasma membrane pre p a r a t i o n s . Most s t u d i e s i n the past simply used d i f f e r e n t i a l c e n t r i f u g a t i o n to o b t a i n a 100,000 g microsomal plasma membrane.preparation (PREISS & BANASCHAK, 1975; ZELCH et a l . , 1975; GODFRAIND et a l . , 1976; RANGACHARI et a l . , 1976; WEBB & BHALLA, 1976; NISHIKORI et a l . , 1977; BHALLA et a l . , 1978a, 1978b). Some have replaced the 100,000 g p e l l e t by a 40,000 g p e l l e t (SHIBATA & HOLLANDER, 1974; FITZPATRICK & SVENTIVANYI, 1977). More recent s t u d i e s combine d i f f e r e n t i a l c e n t r i f u g a t i o n w i t h simple four step g r a d i e n t s (WEI et a l . , 1976a, 1976b, 1976c; JANIS et a l . , . 1977; AKERMAN & WIKSTROM, 1978; VALLIERES et a l . , 1978). The smooth muscle used was obtained from a r t e r i e s , the uterus and the ileum. The simplest of a l l plasma membrane preparations would be to use the crude homogenate. This k i n d of approach serves no r e a l 17 - 17 - purpose under these c o n d i t i o n s because of the contamination by o r g a n e l l e s and problems w i t h v e s i c l e o r i e n t a t i o n . Measurement of membrane ATPase a c t i v i t i e s i s c o n t r a d i c t e d due to the presence of actinomyosin ATPase. Furthermore the lysozomal enzymes w i l l e v e n t u a l l y degrade a l l membrane systems. Microsomal preparations of smooth muscle have i n v o l v e d the use of a 4,000 g or 100,000 g p e l l e t . Many of these s t u d i e s were done using v a s c u l a r smooth muscle. The main emphasis of 2+ these s t u d i e s was Ca t r a n s p o r t and measurement of ATPase a c t i v i t i e s a s s o c i a t e d w i t h the membranes. The c h a r a c t e r i z a t i o n of the membranes'varied from the combination'of e l e c t r o n microscopy w i t h one marker such as s u c c i n a t e dehydrogenase (ZELCK et a l . , 1975; CYLMAN et a l . , 1976; NISHIKORI et a l . , 1977; KRALL et a l . , 1978) to more exhaustive marker s t u d i e s (CHATURVEDI et a l . , 1978; KUTSKY & GOODMAN, 1978; THORENS & HAUESLER, 1978; MATLIB et a l . , 1979; THORENS, 1979) - See Table 1. The l i m i t e d use of markers i n a microsomal p r e p a r a t i o n i s unacceptable. To measure Ca^uptake or r e l e a s e i n a membrane pre p a r a t i o n without knowing the v a r i o u s p o s s i b l e types of membranes present i s unadvisable. A good example of t h i s i s i n 100,000 g microsomal preparations considered by many to be sarcoplasmic r e t i c u l u m (SHIBATA & HOLLANDER, 1974; WEBB & BHALLA, 1976; FITZPATRICK & SZENTIVANYI, 1977; BHALLA et a l . , 1978a, 1978b). This hypothesis has been advanced on the b a s i s of the marker enzyme NADH cyt ĉ  reductase. While mitochondria are excluded by e l e c t r o n microscopy s t u d i e s the p r o b a b i l i t y that plasma membranes are present i s acknowledged but r a r e l y checked by using standard marker enzymes and no attempts have been made to determine the 18 - 18 - o r i e n t a t i o n and/or p e r m e a b i l i t y of the membrane, t h i s l a t t e r p o i n t being c r u c i a l i n the observation of e f f l u x or i n f l u x of v a r i o u s i o n s . The use of NADH cyt c_ reductase as a sarcoplasmic r e t i c u l u m marker i s now suspect as t h i s a c t i v i t y has a l s o been shown to be present i n the plasma membranes and other c e l l o r g a n e l l e s (SOTTOCASA, 1967; SOTTOCASA, 1971; KELBERG & CHRISTENSEN, 1979). E l e c t r o n microscopy i s at best only s e m i - q u a n t i t a t i v e . A l s o , the presence of plasma membranes i n the 100,000 g sarcoplasmic r e t i c u l u m p e l l e t has been unequivocally demonstrated (HURWITZ et a l . , 1973; MOORE et a l . , 1975; VALLIERES et a l . , 1978; WUYTACK et a l . , 1978; MATLIB et a l . , 1979; THORENS, 1979). I t has been shown th a t t h i s p e l l e t contains s u f f i c i e n t m i t o c h o n d r i a l 2+ and plasma membranes to effect Ca accumulation observed i n the 100,000 g p e l l e t s . Another drawback to s t u d i e s using 100,000 g p e l l e t s has sarcoplasmic r e t i c u l u m or plasma membranes preparations i s seen 2+ i n s t u d i e s comparing Ca uptake i n microsomes prepared from the aortas of hypertensive and normotensive r a t e s (BHALLA et a l . , 1978b). I t i s thought that c e r t a i n u l t r a s t r u c t u r a l changes occur i n smooth muscle c e l l s of aortas i n hypertensive animals. To t r e a t both aortas under i d e n t i c a l p r e p a r a t i o n c o n d i t i o n s i s questionable because i t i s by no means c e r t a i n t h a t both t i s s u e types w i l l behave i n the same manner. Furthermore, i t i s d i f f i c u l t to a s s i g n 2+ much s i g n i f i c a n c e to s t u d i e s of Ca uptake by v e s i c l e s whose o r i g i n , o r i e n t a t i o n and contamination by other membranes i s not known. Many i n v e s t i g a t o r s r e a l i z i n g the drawbacks of the microsomal preparations have f u r t h e r p u r i f i e d the plasma membranes gradients (KIDWAI, 1974; MAGARGAL et a l . , 1978; WUYTACK et a l . , 1978; MATLIB et a l . , 1979; THORENS, 1979). Most st u d i e s have 19 - 19 - u t i l i z e d v i s c e r a l smooth muscle. Several investigators have car r i e d out extensive c h a r a c t e r i z a t i o n of the f i n a l preparations. A t y p i c a l example i s seen i n the preparations of myometrial plasma membranes. The sarcoplasmic reticulum, p r i o r to 3 homogenization, was loaded with H-leucine and t h i s was used to check the v i a b i l i t y of NADH cyt _c reductase as a sarcoplasmic reticulum marker (MATLIB et a l . , 1979). Electron microscopy can be made semi-quantitative by attaching WGA L e c t i n to the plasma membranes p r i o r to homogenization (VALLIERES et a l . , 1978). It was f u l l y appreciated that t h i s may change membrane d e n s i t i e s . Invesitgations by various authors to f i n d s u i t a b l e markers fo r smooth muscle c e l l components have met with some success (MAGARGAL et a l . , 1978; VALLIERES et a l . , 1978; MATLIB et a l . , 1979). By changing the density of plasma membranes on the gradient using d i g i t o n i n , i t has been shown that o l e y l CoA: l y s o l e c i t h i n acetyltransferase i s a s p e c i f i c marker for the SR (MAGARGAL et a l . , 1978). Interesting r e s u l t s have been obtained, using gradient 2+ ce n t r i f u g a t i o n . The f i r s t of these i s a Mg - stimulated ATPase thought to be located i n the plasma membrane f r a c t i o n s 2+ which may be the Mg - stimulated ATPase seen.in the so c a l l e d 100,000 g sarcoplasmic reticulum preparations (MOORE et a l . , 1975; WEI et. a l . , 1976; JANIS et a l . , 1977; VALLEIRES et a l . , 2+ 1978; MATLIB. et a l . , 1979). This Mg - stimulated ATPase obscures any Na+/K+ ATPase a c t i v i t y that might be present i n the membrane preparation. 20 - 20 - The second i n t e r e s t i n g point- i s that the l o c a t i o n of the plasma membranes on the gr a d i e n t s appears to be v a r i a b l e . In some preparations using the r a t a o r t a , the plasma membranes are found at higher d e n s i t i e s i n the gradient than are the sarcoplasmic r e t i c u l u m membranes. This i s i n c o n t r a s t to the r e s u l t s of other s t u d i e s i n which the plasma membranes are found at lower d e n s i t i e s r e l a t i v e to the sarcoplasmic r e t i c u l u m (HURWITZ et a l . , 1973; MOORE et a l . , 1975; WEI et a l . , 1976a, 1976b; THORENS & HAEUSLER, 1978). I t may be that the plasma membranes are bi n d i n g to denser fragments of other membranes. Studies using gradient c e n t r i f u g a t i o n s to compare plasma membranes from a r t e r i s of hypertensive animals to those of non-hypertensive animals r a i s e s i m i l a r questions to those mentioned e a r l i e r i n the d i s c u s s i o n of microsomal preparations (MOORE et a l . , 1975; WEI et a l . , 1976c). What i s necessary i n these s t u d i e s are marker and sidedness assays of each f r a c t i o n obtained a f t e r each step i n the procedure. F o r t u n a t e l y , t h i s i s now being done (VALLIERES et a l . , 1978; MATLIB et a l . , 1979). I t would be very improper to assume that the drawbacks mentioned above apply u n i v e r s a l l y to a l l plasma membrane prep a r a t i o n s . This i s obviously i n c o r r e c t , s i n c e there have been some n i c e s t u d i e s on membrane c h a r a c t e r i z a t i o n of s k e l e t a l , c a r d i a c and even smooth muscle. I t i s apparent, however, t h a t , u n t i l r e c e n t l y , the m a j o r i t y of smooth muscle preparations have been inadequately c h a r a c t e r i z e d . 21 - 21 - D. R a t i o n a l e The b a s i c aim of the present study was to prepare and c h a r a c t e r i z e a w e l l defined p r e p a r a t i o n of plasma membranes from smooth muscle. Such a p r e p a r a t i o n r e q u i r e s a t i s s u e r i c h i n smooth muscle c e l l s and r e l a t i v e l y f r e e from f a t as w e l l as connective t i s s u e . The smooth muscle should be r e a d i l y a v a i l a b l e i n l a r g e q u a n t i t i e s as w e l l . I d e a l l y s u i t e d f o r these purposes i s the chicken stomach m u s c u l a r i s . This muscle, based on the r e s u l t s of v a r i o u s s t u d i e s (CALHOUN, 1954; McCLEOD et a l . , 1964; KING & McCLELLAND, 1975; SOBIESZEK.& SMALL, 1976) i s thought to c o n s i s t p r i n c i p a l l y of v i s c e r a l smooth muscle. A c e r t a i n degree of cauti o n i s r e q u i r e d as s k e l e t a l muscle i s present i n neighbouring regions of the d i g e s t i v e t r a c t . As much as 10 to 15 grams of smooth muscle can be obtained from one domestic chicken. The next stage i n the pr e p a r a t i o n of the plasma membranes ishomogenization. L i q u i d shear would r e q u i r e that the t i s s u e be dispersed i n t o s i n g l e c e l l s p r i o r to homogenization, a process that may e f f e c t the plasma membranes of the c e l l s . The best approach at f i r s t i s to use mechanical shear of muscle cubes by a P o l y t r o n homogenizer. I t was borne i n mind that the c o n d i t i o n s used could be optimized to give maximal plasma membrane y i e l d s at the v a r i o u s stages of the procedure. The crude homogenate contains a mix of c e l l components, many of which are fragmented. D i f f e r e n t i a l c e n t r i f u g a t i o n combined w i t h marker studies, a l l o w s removal of much of the contamination. However, d i f f e r e n t i a l c e n t r i f u g a t i o n i n i t s e l f 22 - 22 - would not y i e l d a s u f f i c i e n t l y p u r i f i e d plasma membrane preparation r e q u i r i n g a f o u r t h stage of i s o l a t i o n . Sucrose gradients are the e a s i e s t to use and al l o w f o r f i n e separations of contaminating o r g a n e l l e s . The gradients used would have to be s u f f i c i e n t l y long and l a r g e to a l l o w f o r t h i s s e p a r a t i o n . The f i n a l plasma membrane preparation obtained from the g r a d i e n t , once c h a r a c t e r i z e d f o r p u r i t y and sidedness, was expected to be b a s i c a l l y f r e e of contaminants. I t i s u n l i k e l y , however, that the v e s i c l e s would have uniform o r i e n t a t i o n of the membrane. I f the membranes are i l l defined w i t h regards to t h e i r o r i e n t a t i o n i t would become necessary to f u r t h e r p u r i f y the pr e p a r a t i o n . There wre three choices. Free f l o w e l e c t r o p h o r e s i s i s v e r y expensive, the r e s o l u t i o n i s poor and the experimental c o n d i t i o n s are q u i t e v a r i a l b e . AP/CCD i s dependent on many v a r i a b l e s and there are d i f f i c u l t i e s encountered i n removing p a r t i t i o n i n g compounds from the membranes. The e a s i e s t method to use seemed to be a f f i n i t y chromatography, and i f a s u i t a b l e l i g a n d can be found, the most e f f i c i e n t . I t s only drawback i s the danger of high a f f i n i t y non s p e c i f i c binding of the plasma membranes. As a p o s s i b l e method of f i r s t choice i t should be used. F a i l u r e of t h i s technique would mean r e s o r t i n g to FFE or AP/CCD. What i s extremely important i s that the membranes be c h a r a c t e r i z e d at each step i n the procedure w i t h respect to p u r i t y and i f at a l l p o s s i b l e w i t h respect to sidedness, the l a t t e r being very c r i t i c a l i n the l a t t e r stages of the i s o l a t i o n procedure. I t was hoped that t h i s approach would make i t p o s s i b l e to o b t a i n a s u i t a b l e plasma membrane pr e p a r a t i o n . 23 Table I Summary of smooth muscle p r e p a r a t i o n s . 1. Source of smooth muscle - a o r t a AUTHORS METHODS USED PREPARATION CHARACTERIZATION PETERS et a l . , Dounce S i n g l e c e l l s Cytochrome oxidase (1972) homogenization Lysozomes 5' Nucleotidase D i f f e r e n t i a l DNA c e n t r i f u g a t i o n Phosphatases Sucrose Glycosidases gradients N - a c e t y l - glucosaminidase Napthylamidase Cathepsin A-D Monamine oxidase HURWITZ et a l . (1973) Homogenization not i n d i c a t e d D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose g r a d i e n t s Plasma membranes Na+/K+ ATPase Ca^+ uptake 5" Nucleotidase NADH oxidase HESS & FORD (1974) Homog e n i za t i o n no t . ind i c a t ed D i f f e r e n t i a l c e n t r i f u g a t i o n Microsomal r.  2 + Ca uptake Cytochrome oxidase Na+/K+ ATPase 5' Nucleotidase Mg 2+ ATPase KIDWAI (1974) P o l y t r o n homogenization D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose g r a d i e n t s Plasma membranes Mitochondria Suecinate?.^ dehydrogenase 5' Nucleotidase NADH oxidase BHALLA et a l . , (1978a, 1978b) FITZPATRICK & SZENTTIVANYI (1977) MOORE et a l . , (1975) SHIBATA & HOLLANDER (1974) WEBB & BHALLA (1976) P o l y t r o n Microsomes homogenization Plasma membranes D i f f e r e n t i a l Sarcoplasmic c e n t r i f u g a t i o n r e t i c u l u m C a 2 + ATPase (N,~ i n s e n s i t i v e ) cAMP Cytochrome oxidase Succinate dehydrogenase Phosphodiesterase I - .24 - Table I (cont'd) 1. a o r t a (cont' d ) . . . AUTHORS METHODS USED PREPARATION CHARACTERIZATION WEI et a l . , P o l y t r o n Plasma membranes Phosphodiesterase I (1976a) homogenization Mitochondria 5* Nucleotidase D i f f e r e n t i a l Endoplasmic Cytochrome c reductase c e n t r i f u g a t i o n r e t i c u l u m K+ phosphatase Sucrose (ouabain s e n s i t i v e ) g r a dients ATPases Ca^+ accumulation E l e c t r o n microscopy CHATURVEDI et Po t t e r Elvehjem Microsomes Na+/K+ ATPase a l . , (1978) homogenization Plasma membranes 5' Nucleotidase THORENS & D i f f e r e n t i a l Glucose-6-phosphatase HAEUSLERQ978) c e n t r i f u g a t i o n NADH oxidase Sucrose Cytochrome oxidase gradients Ca2+ uptake KUTSKY & Po l y t r o n Microsomes 5'Nucleotidase GOODMAN (1978) homogenization M g 2 + ATPase D i f f e r e n t i a l Succinate-cytochrome c e n t r i f u g a t i o n c_ reductase NADPH cyt c reductase Ca2+ uptake MAGARGAL et a l . , Vortex, Dounce Plasma membranes NADH f e r r i c y a n i d e (1978) homogenization reductase D i f f e r e n t i a l NADH c y t c reductase c e n t r i f u g a t i o n (rotenone i n s e n s i t i v e ) Sucrose NADPH c y t c_ reductase gradients Cytochrome oxidase D i g i t o n i n 5' Nucleotidase treatment Monamine oxidase A l k a l i n e Phospho- d i e s t e r a s e I A c y l t r a n s f e r a s e WEI et a l . , P o l y t r o n , Plasma membranes 5' Nucleotidase (1976b,1976c) P o t t e r Elvehjem Mitochondria Na +/ K+ ATPase THORENS (1979) homogenization Endoplasmic A l k a l i n e phosphatase D i f f e r e n t i a l r e t i c u l u m Phosphodiesterase I Mesenteric c e n t r i f u g a t i o n Cytochrome c_ oxidase a r t e r y used. Sucrose Ca2+ uptake gradients CLYMAN et a l . , P o t t e r Elvehjem Microsomes None (1976) homogenization U m b i l i c a l D i f f e r e n t i a l a r t e r y used. c e n t r i f u g a t i o n - 25 - Table _I (cont'd) 1. aorta (cont'd)... AUTHORS METHODS USED PREPARATION CHARACTERIZATION PREISS & Homogenizer Microsomal Adenylate c y c l a s e BANASHEK(1975) not i n d i c a t e d 5' Nucleotidase Sucrose gradient C a r o t i d a r t e r y used. WUYTACK et a l . , P o t t e r Elvehjem Plasma membranes C a2+ uptake (1978) homogenization NADH cytochrome c D i f f e r e n t i a l reductase (rotenone Coronary c e n t r i f u g a t i o n i n s e n s i t i v e ) a r t e r y used. Sucrose Choline phosphotransf gradients erase Cytochrome oxidase 2. Smooth muscle source - ileum AUTHORS METHODS USED PREPARATION CHARACTERIZATION GODFRAIND et a l . , P o t t e r Elvehjem Microsomes (1973,1976,1977) homogenization Mitochondria D i f f e r e n t i a l c e n t r i f u g a t i o n C a i T uptake ATPases 5' Nucleotidase Cytochrome oxidase ZELCK et a l . , (1975) Homogenizer not i n d i c a t e d D i f f e r e n t i a l c e n t r i f u g a t i o n N u c l e i Mitochondria E l e c t r o n microscopy Succinate dehydrogenase NILSSON et a l . , (1977) Homogenizer not i n d i c a t e d D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose gr a d i e n t s Plasma membranes Mitochondria N u c l e i 5' Nucleotidase Phosphodiesterase I ^H-leucine uptake C h o l e s t e r o l / p h o s p h o l i p i d Cytochrome c_ oxidase % - o u a b a i n bind i n g RAEYMAEKERS et Po t t e r Elvehjem Mitochondria 0 2 consumption al.,(1977) homogenization Cytochrome £ oxidase D i f f e r e n t i a l Ca + uptake c e n t r i f u g a t i o n - 26 - Table I (cont'd) 3. Source of smooth muscle - myometrium AUTHORS METHODS USED PREPARATION CHARACTERIZATION JANIS et a l . (1976) P o l y t r o n homogenization D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose gradients Plasma membranes 5 1 Nucleotidase Mitochondria glucose-6-phosphatase Smooth endoplasmic Mg2+ ATPase r e t i c u l u m C a 2 + uptake Rough endoplasmic Ouabain s e n s i t i v e r e t i c u l u m phosphatase Cytochrome c oxidase RANGACHARI et a l . , (1976) P o t t e r Elvehjem homogenization Sucrose g r a d i e n t s Microsomes Adenyl c y c l a s e 5' Nucleotidase MATLIB et a l . (1979) P o l y t r o n homogenization D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose gradients Plasma membranes Succinate cytochrome ĉ reductase NADH cyt £ reductase (rotenone s e n s i t i v e and i n s e n s i t i v e ) Monamine oxidase 3H-WGA l a b e l l i n g of PM 3H-oxytocin binding s i t e s M g 2 + A T p a s e 5' Nucleotidase NADPH reductase NISHIKORI et a l . , (1977) P o l y t r o n Microsomal homogenization D i f f e r e n t i a l c e n t r i f u g a t i o n Succinate dehydrogenase KRALL et a l . (1978) P o l y t r o n Microsomes homogenization D i f f e r e n t i a l c e n t r i f u g a t i o n P r o t e i n Kinase VALLIERES et a l . , (1978) Parr bomb D i f f e r e n t i a l c e n t r i f u g a t i o n Sucrose gradients Plasma membranes Adenyl c y c l a s e 2 5' _Nucleotidase Mg*" ATPase Phosphodiesterase I Cytochrome ĉ  oxidase 1 25l-WGA l a b e l l i n g of plasma membranes (PM) E l e c t r o n microscopy - 27 - MATERIALS Concanavalin A - l i n k e d to Agarose (Con A-Agarose)-, amethyl-D-mannoside, T r i t o n X-100, l a c t o p e r o x i d a s e , acrylamide, ammonium persulphate, T r i s (hydroxymethyl) aminomethane (TRIS), 2 (N-Morpholino) ethane sulphonic a c i d (MES) and a l l enzyme substrates were obtained from Sigma Chemical Company, vanadium-free ATP from Boehringer Manheim, concanavalin A - l i n k e d to Sepharose (Con A-Sepharose) and Sepharose 4B from Pharmacia Fine Chemicals, collagenase (Grade A) from Cal Biochem, Coomassie Blue R-250, sodium dodecylsulphase (SDS), N,N,N',N' - Tetramethyl ethylenediamine (TEMED) from Biorad L a b o r a t o r i e s . 125 Radioactive c a r r i e r f r e e [ i ] Nal (100 mCi/ml) was obtained from Amersham and New England Nuclear. Adenosine 32 5'-triphosphate, t e t r a (triethylammonium) s a l t , [ y4 p] (20-40 Ci/mmol) was purchased from New England Nuclear as w e l l as ,0mnifluor. Unless otherwise s t a t e d , s c i n t i l l a t i o n grade toluene, MIRACLOTH and a l l common chemicals were re c e i v e d from North American Chemical Supply Li m i t e d . Domestic chickens (Rhode I s l a n d Reds, 18-24 months old) were obtained from the U.B.C. P o u l t r y Farm U n i t . Animals were k i l l e d by breaking t h e i r necks. Gizzards were immediately excised and used. METHODS A. Enzyme Assays 5'-nucleotidase The method used was that of WIDNELL (1974) and BURGER & LOWENSTEIN (1975). The r e l e a s e of i n o r g a n i c phsophate from 5'-AMP i s measured. To 0.9 ml of substrate s o l u t i o n (11.11 mM 5'-AMP, sodium s a l t ; 1.11 mM magnesium:chloride; 0.1 M TRIS-HCL, pH 8.5 a t 22°C) at 37°C, 50 1 of membrane suspension (10-20 yg p r o t e i n ) i s added. A f t e r i n c u b a t i o n f o r 20 minutes at 37°C, 50 V1 of 50% 28 - 28 - t r i c h l o r o a c e t i c acid (TCA) and 0.5 ml of Ames reagent (1 part 10% ascorbic acid and 6 parts 0.42% ammonium molybdate i n 0.5 M sulphuric acid) (AMES, 1966) are added with s t i r r i n g . The samples are then incubated at 37°C for a further one hour. Colour development i s then stopped by addit i o n of 0.5 ml i c e cold d i s - t i l l e d water. The samples are then centrifuged at 2000 g, 0°C, 3 minutes i n a S o r v a l l RC-2B centrifuge and the absorbance of the supernatant read at 660 nm. Blanks without either 5'-AMP or membranes are run with each sample. Nanomoles of phosphate released are calculated based on a standard curve obtained using inorganic phosphate and the procedure above leaving out membranes. Results are expressed as mole phosphate released per hour per mg protein. NADPH/NADH antimycin A - i n s e n s i t i v e cytochrome c_ reductase The methods used were those of TOLBERT (1974), HATEFI & RIESKE (1967) and HODGES & LEONARD (1974). To 2.80 ml of substrate s o l u t i o n (0.375 mg/ml cytochrome c; 45 mM sodium phosphate, .pH 7.0 at 20°C; 1.8 mM sodium cyanide) i s added 20 y l of antimycin A so l u t i o n i n ethanol (2 mg/|ml) . This i s followed by the addition of 100 to 200 yl of membrane preparation (40-80 yg protein) a f t e r which the rate of change i n absorbance at 550 nm i s recorded for 5 minutes. Once the i n i t i a l rate has been established, 100 yl of NADH or NADPH (3 mg/ml) i s added. The new rate of change i n absorbance at 550 nm i s recorded and substracted from the i n i t i a l rate to y i e l d the actual r a t e . Micromoles of cyt c_ consumed per unit time are calculated assuming the ex t i n c t i o n c o e f f i c i e n t of cyt ĉ  at 550 nm to be 29 - 29 - 18.5 mM -cm . , S p e c i f i c a c t i v i t i e s are expressed i n moles per hour per mg p r o t e i n . NADH antimycin A - s e n s i t i v e cytochrome £ reductase. This assay i s c a r r i e d out as the assay f o r NADPH/NADH antimycin A - i n s e n s i t i v e cytochrome ĉ  reductase except that 45 mM glycine-sodium hydroxide b u f f e r , pH 9.0 i s s u b s t i t u t e d f o r sodium phsophate b u f f e r and antimycin A omitted. a c i d p h o s p h a t a s e The method used was a m o d i f i c a t i o n of the methods of HODGES & LEONARD (1974) and HUBSCHER & WEST (1965). To 0.9 ml substrate s o l u t i o n (3.33 mM p-nitrophenol phosphate; 1.67 mM magnesium sulphate; 36.7 mM TRIS-MES, pH 5.5 at 25°C) i s added 100 1 of membrane suspension (20-30 yg p r o t e i n ) . For determination of K+ stimulated a c i d phosphatase the su b s t r a t e s o l u t i o n i s made to contain 55.5 mM potassium c h l o r i d e . This s o l u t i o n i s then incubated at 37°C f o r 20 minutes a f t e r which 50 y l of 50% TCA and 0.5 ml of Ames reagent (1 part 10% a s c o r b i c and 6 p a r t s 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c acid) (AMES, 1966). The samples are then incubated a t 37°C f o r one hour a f t e r which 0.5 ml of i c e c o l d d i s t i l l e d water i s added. Samples are next ce n t r i f u g e d at 2000 g, 3 min., 0°C i n a S a r v a l l RC-2B c e n t r i f u g e . The supernatants are withdrawn and the absorbance of the super- natant at 660 nm determined. Nanomoles of phosphate re l e a s e d are read from a 0-10 nanomole c a l i b r a t i o n curve using the above procedure. Blanks without e i t h e r p-nitrophenol phsophate or membranes are run f o r each sample. S p e c i f i c a c t i v i t i e s are expressed as moles phosphate released per hour per mg p r o t e i n . 30 - 30 - succinate dehydrogenase The method used was based on those of BONNER (1955) and TOLBERT (1974). To a 4.0 ml quartz cuvette are added 0.3 ml of 0.1 M potassium cyanide; 0.3 ml of 0.01 M potassium f e r r i c y a n i d e ; 0.2 ml of 0.2 M sodium succinate and 2.0 ml of 55 mM sodium phosphate b u f f e r , pH 7.2. At time zero, 0.20 ml of membrane suspension (40-80 yg pr o t e i n ) i s added and the change i n absorbance. at 400 nm recorded. C o n t r o l samples without e i t h e r the membrane p r e p a r a t i o n or suc c i n a t e were processed i n the same manner. S p e c i f i c a c t i v i t i e s are c a l c u l a t e d using an e x t i n c t i o n c o e f f i c i e n t a t 400 nm, at 20°C, of 0.892 x 10 3M _ 1cm - 1 f o r potassium f e r r i c y a n i d e . R e s ults are expressed i n moles succinate u t i l i z e d per hour per mg p r o t e i n . glucose-6-phosphatase The method used was that of NORDLIE & ARION (1966). To 0.90 ml of substrate s o l u t i o n (3.33 mM glucose-6-phsophate; 111 mM sodium cacodylate b u f f e r , pH6.5) at 37°C i s added 50 y l of membrane suspension (10-20 g p r o t e i n ) . This mixture i s then incubated at 37°C f o r 20 minutes a f t e r which 50 y l of 50% TCA and 0.5 ml of Ames Reagent (.1 part 10% a s c o r b i c a c i d to 6 pa r t s 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c acid) (AMES, 1966) are added. The samples are then incubated f o r 1 hour at 37°C a f t e r which 0.5 ml of i c e c o l d d i s t i l l e d water i s added. Samples are then c e n t r i f u g e d at 2000 g, 3 min. 0°C i n a S o r v a l l RC-2B c e n t r i f u g e , f o l l o w i n g which the supernatants are withdrawn and t h e i r absorbance a t 660 nm measured. Blanks without e i t h e r glucose-6-phosphate or membrane suspension were 31 - 31 - a l s o run w i t h each sample. The absorbance data are converted to micromoles of phosphate by use of a standard curve obtained by the above procedure using inorganic phosphate i n place of glucose-6-phosphate and membrane suspension. Results are expressed i n moles phosphate released per hour per mg p r o t e i n . Lowry P r o t e i n Assay The method i s based on the o r i g i n a l procedure of LOWRY et a l . (1951). The f o l l o w i n g stock s o l u t i o n s are prepared f r e s h and stored at room temperature p r i o r to use. Reagent A i s prepared i n the f o l l o w i n g order: 2% sodium carbonate i n 0.1 M sodium hydroxide, 49 ml; 2% potassium t a r t r a t e , 0.5 ml; 1% copper sulphate, 0.5 ml. Reagent B i s the F o l i n - C i o c a l t e a u 2 N d i l u t e d 1:1 w i t h d i s t i l l e d water. The sample to be assayed i s made up to 1.0 ml w i t h d i s - t i l l e d water. At time zero, 5.0 ml reagent A i s added and the mixsture s t i r r e d . 0.5 ml of reagent B i s added a f t e r 10 minutes w i t h s t i r r i n g and the absorbance at 750 nm read 20 minutes l a t e r . A standard curve i s obtained i n the same manner using BSA (0-250 yg/ml). I f TRIS b u f f e r or sucrose i s present i n the samples to be assayed appropriate c o n t r o l samples must be included (ROBSON et a l . , 1968; GERHARDT & BEEVERS, 1968). 2+ C h a r a c t e r i z a t i o n of Mg Stimulated,ATPase (a) Determination of Optimum Conditions TRIS-ATP (vanadium f r e e , see HUDGINS & BOND, 1977; BEAUGE & GLYNN, 1977; JOSEPHSON & CANTLY, 1977) concentrations of 0, 0.05, 0.10, 0.20, 0.40, 0.80, 1.20, 2.00, 3.00, 4.00 mM were used. The substrate s o l u t i o n s were buffered w i t h 50 mM TRIS-HC1, pH7.2. 32 - 32 - To 0.9 ml of each substrate s o l u t i o n at 37°C, 50 y l of membrane suspension (10-20 yg protein) are added. The samples are then incubated f o r 10 minutes at 37°C following which the reactions are terminated using 50 y l of 50% TCA and 0.5 ml of Ames Reagent (.1 part 10% ascorbic a c i d to 6 parts 0.42% ammonium molybdate i n 0.5 M sulphuric acid) (AMES, 1966). Colour develop- ment i s allowed to proceed for 20 minutes at 37°C a f t e r which 0.5 ml of ice cold water are added and the absorbance of each sample read at 660 nm. Blanks without enzyme were run with each sample, controls without ATP at frequent i n t e r v a l s . Micromoles phosphate released are calculated based on a phosphate standard curve using inorganic phosphate. S p e c i f i c a c t i v i t i e s are expressed i n umoles. phosphate l i b e r a t e d per hour per mg protein. 2+ Optimum Mg t o t a l and ATP t o t a l concentrations were then used for the next series of experiments. (b) pH Optima and Na +, Li" 1", K + and Ouabain S e n s i t i v i t y For determination of the pH optimum, the following pH's at 37°C were used: 6.00, 6.50, 7.00, 7.20, 7.40, 7.60, 7.80, 8.00, 8.50 and 9.00. For each pH value, substrate solutions were prepared as i n Table II to test f o r stimulation by ions and i n h i b i t i o n by ouabain. 33 ^-33 - Table I I Substrate S o l u t i o n s to Test Mg2+ - Stimulated ATPase + S e n s i t i v i t y to pH, Na', L i + , K + and Ouabain [ATP] max 30 mM Buf 1 2 [ATP] max 3 [ATP] max 4 [ATP] max 50 mM Buf 50 mM Buf 50 mM Buf [Mg2+] a [ M g2+] [Mg2+] max ° max a max 120 mM Na + 120 mM L i + 5 [ATP] max [Mg2+] max 6 [ATP] max 50 mM Buf 50 mM Buf [Mg2+] max 120 mM Na+ 120 mM Na + 20 mM K+ 20 mM K + 2 mM Ouabain optimal concentrations based on the r e s u l t s of part ( a ) . b:,pH at 37°C - 6.0, 6.5 Cacodylate B u f f e r , - 7.0-8.5 TRIS-HC1, - 9.0 Glycine-NaOH Buf f e r To 0.9 ml of each substrate s o l u t i o n at 37°C are added 50 y l of membrane suspension (10-20 jS-g p r o t e i n ) . The samples are then incubated at 37°C f o r 10 minutes a f t e r which 50 y l of 50% TCA s o l u t i o n and 0.50 ml of Ames reagent (1 par t a s c o r b i c a c i d to 6 parts 0.42% ammonium molybdate i n 0.5 M s u l p h u r i c acid) (AMES, 1966) are added with s t i r r i n g . Following f u r t h e r i n c u b a t i o n a t 37°C f o r 20 minutes, 0.50 ml of i c e c o l d d i s t i l l e d water are added. The absorbance at 660 nm of each sample i s read and recorded. Blanks were run f o r each sample d e l e t i n g the 50 y l of membrane suspension. Controls without ATP were a l s o run on a random spot .34 - 34 - check b a s i s . Micromoles phosphate r e l e a s e d were c a l c u l a t e d based on a standard curve of 0 to 50 umoles Inorganic phosphate. S p e c i f i c a c t i v i t i e s were expressed i n micromoles phosphate released per hour per mg p r o t e i n . (c) V e l o c i t y P r o f i l e Twenty ml of substrate s o l u t i o n were prepared using 2+ the optimal Mg t o t a l and ATP t o t a l concentrations derived from p a r t ( a ) , using 50 mM TRIS-HG1, pH7.2. For the time course 0.9 ml of substrate were used w i t h 50 y'JL membrane suspension (10-20 ug p r o t e i n ) f o r each time. The r e a c t i o n times used were 0, 3, 5, 10, 15 and 30 minutes at 37°C, a f t e r which the r e a c t i o n s were terminated by the a d d i t i o n of 50 y 1 of 50% TCA and 0.50 ml of Ames Reagent (see p a r t s (a) and ( b ) ) . Phosphate released was c a l c u l a t e d based on standard curves. a c e t y l c h o l i n e s t e r a s e The assay was based on the methods of STECK & KANT (1974) . The assay was done i n d u p l i c a t e w i t h 100 y1 of the membrane suspension per sample. The 100 y l a l i q u o t s of membrane suspension (50-60 yg p r o t e i n ) were p i p e t t e d i n t o the bottom of a 1 cm semi-micro quartz cuvette and mixed w i t h an equal volume of e i t h e r 5 mM sodium phosphate, pH 8.0 or (0.40% T r i t o n X-100 (v/v) i n 5 mM sodium phosphate, pH 8.0). The conc e n t r a t i o n of detergent was chosen so that a l l l a t e n t enzymatic a c t i v i t y would be released w i t h l i t t l e or no a c t i v a t i o n . The volume was made to 0.70 ml w i t h 100 mM sodium phosphate, pH 7.5, then 50 y l of 5, 5' - d i t h i o b i s ( 2 - n i t r o b e n z o i c acid) (DTNB) stock 35 - 35 - s o l u t i o n (10 mM DTNB; 100 mM sodium, phosphate, pH 7.5; sodium bicarbonate: DTNB, 3:8; stored at -5°C u n t i l used) i s added. F i n a l l y , 50 p i of a c e t y l c h o l i n e c h l o r i d e or bromide (12.5 mM i n H^O),stored frozen) i s added. The cuvette contents are then mixed by i n v e r s i o n and the r a t e of change i n absorbance at 412 nm recorded. An i n c r e a s e i n absorbance of 17.0 corresponds to 1 um of product based on an e x t i c t i o n c o e f f i c i e n t 4 - 1 - 1 of 13.6 x 10 M cm at 412 nm f o r DTNB. To e s t a b l i s h the b a s e l i n e , the absorbance was recorded a l s o p r i o r to a d d i t i o n of the s u b s t r a t e . F i n a l r e s u l t s are expressed i n nmoles per hour per mg p r o t e i n . measurement of s i a l i c a c i d The method used was a m o d i f i c a t i o n of the methods of WARREN (1955; 1959) and STECK & KANT (1974). A neuraminidase ( s i a l i d a s e ) s o l u t i o n was prepared by d i s s o l v i n g 1 mg of C l o s t r i d i u m P e r f r i n g e n s neuraminidase (type G from Sigma Chemical Co.) i n 1 ml of 0.03% aqueous s o l u t i o n of bovine serum albumin. The s o l u t i o n was stored at 4°C. For the assay proper, the stock s b l t u i o n i s d i l u t e d 10 f o l d w i t h 0.1 M TRIS- acetate buffer,. pH 5.7 + 0.40% T r i t o n X-100. To ensure that the enzyme i s a c t i v e w i t h respect to the s i a l o g l y c o p r o t e i n s and s i a l o g l y c o l i p i d s present i n the membrane p r e p a r a t i o n , the amount of N-acetyl neuramic a c i d (NANA) released by the enzyme was compared to that released by a 1 hour exposure of the p r e p a r a t i o n to 0.05 M s u l p h u r i c a c i d at 80°C. P r i o r to use membrane pr e p a r a t i o n are d i a l y z e d i n an Amicon ML-2 d i a f i l t r a t i o n u n i t to remove i n t e r f e r i n g chromophores 36 - 36 - as 2-deoxyribose. D u p l i c a t e 100 y 1 a l i q u o t s of membrane suspension (60-80 y g p r o t e i n ) are mixed w i t h or without the detergent. The sample i s incubated f o r 30 minutes at room temperature i n a t e s t tube w i t h a Tef l o n l i n e d screw cap. Released NANA i s then determined as f o l l o w s . A f t e r the 30 minute i n c u b a t i o n .100 y 1 of sodium metaperiodate (0.2 M i n 9 M phosphoric acid) was thoroughly mixed i n t o each sample and allowed to re a c t f o r at l e a s t 20 minutes. Following t h i s , 1.5 ml of sodium a r s e n i t e s o l u t i o n (10% w/v i n 0.5 M sodium phosphate) i s mixed i n t o each sample and allowed to r e a c t f o r at l e a s t 20 minutes.. F o l l o w i n g t h i s , 1.5 ml of sodium a r s e n i t e s o l u t i o n (10% w/v i n 0.5 M sodium phosphate) i s mixed i n v i g o r o u s l y w i t h the samples. A f t e r 2 minutes the mixing i s repeated, and i s followed by the a d d i t i o n of 3.0 ml of t h i o b a r b i t u a r i c a c i d s o l u t i o n (0.6% of w/v i n 0.5 M sodium sulphate). Next the t i g h t l y capped tubes are placed i n a b o i l i n g water bath f o r e x a c t l y 15 minutes and t h e r e a f t e r c o l l e d under tap water to room temperature. Two ml from each sample i s withdrawn and extr a c t e d w i t h 2 ml cyclohexanone. This was done by vigorous shaking f o r 15 seconds. The two phases were separated by c e n t r i f u g a t i o n of the samples at 2000 rpm/20°C/5 minutes i n an I n t e r n a t i o n a l SBV type c e n t r i f u g e . The rosy pink cyclohexanone (upper phase) l a y e r i s t r a n s f e r r e d to a 1 cm path length cuvette and the absorbances a t 549, 562, and 532 nm read. Micromoles NANA released were c a l c u l a t e d i n two ways. 37 - 37 - The f i r s t : moles NANA = v o l (ml) x A549 nm doesn't c o r r e c t f o r 2-deoxyribose contamination which i n t e r f e r e s . I f 2-deoxyribose contamination i s suspected, the NANA absorbance maxima (A562 nm) and the 2-deoxyribqse maxima (A532 nm) are used to c a l c u l a t e the micromoles of NANA re l e a s e d , which = , 133 A562 nm - 8 A532 nm v o l i m i ; ( 3 2 . 6 ) ( 1 3 3 ) - C 2 6 ) ( 8 ) (32.6) (133)-(26) (8) Other equations are a v a i l a b l e using the absorbance at 549 and 532 nm f o r other i n t e r f e r i n g chromophores. Results are expressed as nmoles s i a l i c a c i d released per mg of p r o t e i n . B. Plasma Membrane I s o l a t i o n The method used f o r i s o l a t i o n of smooth muscle plasma membrane; (PM) was modified from the procedure of KIDWAI (1974). The g i z z a r d s were exci s e d from f r e s h l y s a c r i f i c e d chicken, cleaned of f a t and placed i n i c e c o l d b u f f e r A (0.25 M sucrose; 3.0mMMg ; 1 mM TRIS-MES, pH 7.4, at 22°C). A l l subsequent operations were c a r r i e d out at 4°C. The stomach muscularis was removed and 2. grams of v i s c e r a l smooth muscle ( v i s i b l y f r e e of connective t i s s u e ) were minced w i t h s c i s s o r s and placed i n 30 ml b u f f e r A. The muscle (chunks 2 mm x 2 mm 3 mm) was then homogenized f o r 30 seconds at a s e t t i n g of 6.0 using a Brinkmann P o l y t r o n PT-20. A f t e r one minute the muscle was rehomogenized f o r 40 seconds. Homogenization times were s e l e c t e d on optimal y i e l d s of 5' - nu c l e o t i d a s e s p e c i f i c a c t i v i t y i n the f i r s t 5 gradi e n t f r a c t i o n s (see d i s c u s s i o n page 141 ) . 38 - 38 - The homogenate was then f i l t e r e d through 2 crossed l a y e r s of M i r a c l o t h . The f i l t r a t e was made up to 38 ml and ce n t r i f u g e d a t 2000 g, 10 minutes, 0°C i n a SS-34 angle r o t o r i n a S o r v a l l RC-2B c e n t r i f u g e . The supernatant was c a r e f u l l y removed and saved. The p e l l e t was normally d i s c a r d e d , unless marker assays were to be done. The supernatant was then c e n t r i f u g e d as above except at 15,000 g , 15 minutes, 0°C. av The p e l l e t was discarded, and the supernatant c e n t r i f u g e d at 100,000 g , 75 minutes, 0°C, i n a SW-27 r o t o r i n a av L5-65 Beckman U l t r a c e n t r i f u g e . The r e s u l t i n g p e l l e t was saved and the supernatant discarded unless marker assays were to be done. The p e l l e t was then resuspended i n a 2.5 ml of 0.25 M sucrose and layer e d on a discontinuous sucrose g r a d i e n t c o n s i s t i n g of 2.5 ml each of 27.0, 30.0, 32.0, 34.0, 35.0, 36.5, 40.0, 43.0, 45.0 and 66.0% sucrose. The layered g r a d i e n t s were then c e n t r i f u g e d a t 12,000 g ,150 minutes, 0°C i n a av Beckman L5-65 u l t r a c e n t r i f u g e using a SW-27 swinging bucket r o t o r . F o l l o w i n g c e n t r i f u g a t i o n each l a y e r was c a r e f u l l y removed by a hypodermic syringe and e i t h e r used immediately or stored f r o z e n a t -20°C. Membrane enzymatic a c t i v i t y was s t a b l e f o r 2 weeks. To remove the bulk of sucrose from the f r a c t i o n s , which was necessary i n those s i t u a t i o n s when sucrose i n t e r f e r e s w i t h the enzyme assay, the. f r a c t i o n s were d i l u t e d 1:1 w i t h d i s t i l l e d water and c e n t r i f u g e d a t 140,000 g a v> 150 minutes, 0°C using a Type 65 Beckman angle r o t o r in. a L5-65 Beckman u l t r a c e n t r i f u g e . Recovery of membranes was greater than 85% using t h i s procedure. A d d i t i o n of 5.0 mM C a C ^ to the d i l u t i n g s o l u t i o n p r i o r to c e n t r i f u g a t i o n increased y i e l d s only 3%. 39 - 39 - Membranes can be stored f o r 3-4 weeks i n the p e l l e t form. I t should be added that an Amicon u l t r a f i l t r a t i o n Ml-2 u n i t was a l s o used to remove sucrose. Using t h i s procedure 40% of the membranes were l o s t by absorption to the f i l t e r . Sucrose was most e f f e c t i v e l y removed by using d i a l y s i s tubing and d i a l y z i n g a gainst 20 volumes of 20 mM TRIS-HC1, pH 7.0, 1 mM magnesium c h l o r i d e , 1 mM EDTA and 1 mM CaC^ f o r 2 days w i t h r e g u l a r changes of d i a l y z i n g b u f f e r . C. C e l l Sheet and S i n g l e C e l l P r e p a r a t i o n C e l l sheets (LEWIS et a l . , 1975) 0.25 mm t h i c k were obtained from chicken g i z z a r d smooth muscle using a S o r v a l l Tissue S l i c e r . The c e l l sheets were immediately washed w i t h TRIS-MES b u f f e r (1 mM TRIS-MES, pH 7.2; 0.25 M sucrose; 1 mM magnesium c h l o r i d e ) twice. I f the c e l l sheets were to be used f o r i s o l a t i o n of plasma membranes, they were suspended i n 35 ml of Buffer A (see e a r l i e r ) and t r e a t e d as described i n the plasma membrane i s o l a t i o n procedure. The only d i f f e r e n c e was that the P o l y t r o n homogenization was done i n two - 10 second b u r s t s ; one minute apart (again optimized on the b a s i s of 5' n u c l e o t i d a s e a c t i v i t y of the f i n a l p roduct). I f the c e l l sheets were to be used to prepare i n d i v i d u a l c e l l s (BAGBY et a l . , 1971; LEWIS et a l . , 1975; RODBELL et a l . , 1964; FAY & DELISE, 1973; FAY & SINGER, 1977; SMALL, 1977) they were suspended i n 9.0 ml of B u f f e r B- (100 mM sodium phosphate, pH 7.4; 0.25 M sucrose) c o n t a i n i n g 0.03% grade 40 - 40 - F r e s h l y excised g i z z a r d - 2.0 grams minced i n 30 mis 0.25 M sucrose + 1.0 mM TRIS-MES pH 7.4 + 3fl mM MgCl . ~ - homogenized w i t h a PT 20 P o l y t r o n probe f o r 30 + 45 seconds at 0°C. - homogenate f i l t e r e d through 2x l a y e r s of M i r a c l o t h Residue (D) ( c e l l d e b r i s ) F i l t r a t e (S) j volume made up to 38 mis •• c e n t r i f u g e d a t 2000 g, 10 min. 0°C P e l l e t (D) (mito, er) Supernatant (S) j c e n t r i f u g e d at 15,000 g, 15 min. 0 C P e l l e t (D) (mito, er) Supernatant (S) 7 centr: 75 min. i f u g e d at-100,000 g, .n . 0°C P e l l e t (S) (pm, mito) Supernatant (D) ( l y s o , m i t o , pm) j resuspended i n 2.5 mis 0.25 M sucrose - a p p l i e d to a discontinuous sucrose g r a d i e n t 27% - 45% - c e n t r i f u g e d at 120,000 g, 150 min. 0 C C o l l e c t and assay f r a c t i o n s Figure 1 Scheme f o r i s o l a t i o n of smooth muscle plasma membranes from the chicken g i z z a r d . A b b r e v i a t i o n s are as f o l l o w s : pm=plasma membranes;. lyso=lvsozomes; mito=mitochondria; er=endoplasmic r e t i c u l u m ; S=saved; D=discarded f r a c t i o n . 41 - 41 - A collagenase (CALBIOCHEM) f o r 30 minutes at 37°C w i t h a e r a t i o n by O^^CO^ (95:5). The r e s u l t i n g c e l l sheets and c e l l s were then p e l l e t e d by c e n t r i f u g a t i o n a t 1000 g , 5 minutes, c lV 0 C i n a S o r v a l l RC-2B c e n t r i f u g e . The supernatant was c a r e f u l l y removed and discarded. The p e l l e t was g e n t l y resuspended i n 9.0 ml Bu f f e r B, w i t h 0.15% grade A c o l l a - genase and aerated a t 37°C by 0^:CO^ (95:5) f o r 20 minutes. The c e l l suspension was then, r e c e n t r i f u g e d as above and the formed p e l l e t resuspended i n Bu f f e r A only. The s i n g l e c e l l s were immediately c e n t r i f u g e d as above. This procedure was repeated f i v e times taking great care to not d i s c a r d the p e l l e t each time. The f i n a l p e l l e t was resuspended i n Bu f f e r A and used. C e l l s were examined by phase c o n t r a s t microscopy (See Figures 9 and 10) and were t r e a t e d w i t h trypan blue to t e s t c e l l v i a b i l i t y . D. Gel E l e c t r o p h o r e s i s The o v e r a l l approach used f o r SDS (sodium dodecylsuphate) g e l e l e c t r o p h o r e s i s was that of BARTON (1978) . The m o d i f i c a t i o n s used by LAMELLI et a l . (1973) and FAIRBANKS et a l . (1971a) were a l s o incorporated. Gel formulations are given below i n Table I I I . Reservoir b u f f e r s were 0.1% SDS i n 0.1 M sodium phosphate. 42 - 42 - Table I I I Gel Formulations Used f o r E l e c t r o p h o r e s i s COMPONENT 5% separating g e l 3% s t a c k i n g g e l STOCK Ml Used STOCK Ml Used Acrylamide Methylene-bis- acrylamide 22.2% 0.60% Ammonium persulphate 15mg/ml TEMED - d i s t i l l e d H 20 Buf f e r 0.2 M sodium phosphate, pH 7.2; + 0.2% SDS 9.0 ml 2.0 ml 30. y l 9.0 ml 20.0 ml 30.0% 1.50% 10 mg/ml d i s t i l l e d 1.25 M TRIS- HG1, pH6.8; +1.0% SDS 2.5 ml 0.75 ml 10 y l 19.25 ml 2.50 ml A f t e r p r e p a r a t i o n of the separating g e l s o l u t i o n , 9 cm long g e l s were formed i n 0.4 cm x 12 cm a c i d washed f l i n t g l a s s tubes. Each column was overlayered w i t h 1.0 cm of water or i s o b u t a n o l . F o l l o w i n g p o l y m e r i z a t i o n , the overlay was removed and 1 cm st a c k i n g g e l s o l u t i o n aided atop the'separating g e l and overlayered w i t h water or i s o b u t a n o l u n t i l p o l y m e r i z a t i o n . The o v e r l a y was removed a f t e r p o l y m e r i z a t i o n and replaced by r e s e r v o i r b u f f e r . The g e l s were then placed i n a Pharmacia Ge-4 e l e c t r o p h o r e s i s apparatus and pre-run at 8 ma/gel and 8-13 volts/cm f o r 4 hours. As the g e l s were pre-running, membrane suspension i n 15.0% sucrose was prepared f o r e l e c t r o p h o r e s i s . To 150 y l of membrane suspension wae added 125 y l of sample b u f f e r 43 - 43 - (0.01 M sodium phosphate, pH 7.2; 1% SDS; 0.14 M mercaptoethanol, 10% v/v g l y c e r o l ; 0.002 M bromophenol b l u e ) . The samples were then capped and placed i n a b o i l i n g water bath f o r 5-10 minutes to reduce d i s u l p h i d e bonds and to promote s o l u b i l i z a t i o n by SDS. Molecular weight markers were incubated as above. Those used were BSA (MW=70,000), A l d o l a s e (MW=161,000) egg albumin (MW=43,000), c y t c (MW=12,398), E l a s t a s e (MW=25,900), a c t i n (MW=43,000) and myosin (MW=210,000). A f t e r sample p r e p a r a t i o n was complete, each sample was taken up i n a disposable m i c r o p i p e t t e and discharged g e n t l y onto the top of the s t a c k i n g g e l . E l e c t r o p h o r e s i s was performed w i t h the v o l t a g e a t 4-5v/cm and current a t 4-5mA/gel. The running time was about 10 hours. V a r i a t i o n s i n absolute m i g r a t i o n d i s t a n c e s were minimized by removing tubes i n d i v i d u a l l y from the e l e c t r o p h o r e s i s apparatus one by one as the bromphenol blue t r a c k i n g dye migrated 9.0 cm from the o r i g i n . Tracking dye p o s i t i o n s were marked by s l o t s i n the g e l s . The g e l s were normally stained f o r p r o t e i n w i t h Coomassie Blue R-250 (Bi o r a d ) . Gels were s t a i n e d as f o l l o w s . A f t e r r i n s i n g each g e l twice w i t h d i s t i l l e d water they were placed i n 16 x 150 mm capped c u l t u r e tubes. To each tube 30 mis of f i x i n g , s t a i n i n g and d i s t a i n i n g s o l u t i o n s were added as given below. The tubes were ge n t l y a g i t a t e d a t room temperature i n a Dubnoff shaker. 44 - 44 - (.1) 25% isopropanol; 10% a c e t i c a c i d ; 0.05% Coomassie Blue - 12 hours. (2) 10% isopropanol; 10% a c e t i c a c i d ; 0.002% Coomassie Blue - 10 hours. (3) 10% a c e t i c a c i d ; 0.001% Coomassie Blue - 8 hours. (4) 10% a c e t i c a c i d - 3 x 10 hours each. The t h i r d and f o u r t h steps are a b s o l u t e l y necessary i f the background s t a i n i n g i s to be reduced to minimum. When methanol was s u b s t i t u t e d f o r isopropanol the ge l s d i d not de s t a i n as w e l l . Gels s t a i n e d w i t h Coomassie Blue from Sigma could not be destained e i t h e r by d i f f u s i o n or e l e c t r o p h o r e t i c methods. The P e r i o d i c - A c i d S c h i f f ' s (PAS) procedure was used to s t a i n the g e l s f o r carbohydrate (NEVILLE & GLOSSMAN, 1974). Because high concentrations of SDS produce an int e n s e background, i t was necessary to remove the detergent by c a r r y i n g out the steps 1 - 4 described above, but without the presence of the p r o t e i n s t a i n . The f i x e d g e l s were then placed i n d i v i d u a l l y i n t o s l o t t e d p l e x i g l a s s tubes and suspended f o r the s p e c i f i e d period to the f o l l o w i n g sequence of s o l u t i o n s . Each g e l requ i r e d 100 ml. 45 - 45 - (1) 0.5% p e r i o d i c a c i d - 2 hours (.2) 0.5% sodium a r s e n i t e ; 5% a c e t i c a c i d - 1 hour (3) 0.1% sodium a r s e n i t e ; 5% a c e t i c a c i d - 2 x 20 minutes (4) 5% a c e t i c a c i d - 10-20 minutes Each s o l u t i o n was s t i r r e d v i g o r o u s l y at room temperature. The g e l s were then t r a n s f e r r e d to tubes c o n t a i n i n g 10 ml of S c h i f f ' s reagent (ACHARIUS & ZELL, 1969; NEVILLE & GLOSSMAN, 1974) and l e f t overnight. F i n a l l y , they were returned to the s l o t t e d tubes and incubated i n 0.1% sodium b i s u l p h i t e i n 0.01 M HC1 f o r s e v e r a l hours ( s o l u t i o n changed once every hour) u n t i l the r i n s e s o l u t i o n f a i l e d to turn pink upon a d d i t i o n of formaldehyde. Rose pink bands appeared a f t e r 2 hours i n the S c h i f f ' s reagent. No v a r i a b l e background abso r p t i o n was seen. The g e l s were scanned using a G i l f o r d spectrophotometer equipped w i t h a Model 2420 Linear Transport Accessory. The Coomassie Blue s t a i n e d g e l s were scanned at 530 nm, those sta i n e d by the PAS procedure at 560 nm. The s l i t width was 0.1 mm i n e i t h e r case. When samples c o n t a i n i n g r a d i o a c t i v e l y l a b e l l e d components were run, g e l s were not st a i n e d but r i n s e d i n water twice, f i x e d w i t h 8% TCA (15 minutes) and 32 then s l i c e d i n t o 2 mm sec t i o n s f o r counting (see [ y P ] ATP l a b e l l i n g experiments). D u p l i c a t e g e l s f o r s t a i n i n g only were a l s o run. 46 - 46 - E. I o d i n a t i o n Experiments (a) I o d i n a t i o n of Muscle Chunks, C e l l Sheets and Free C e l l s w i t h 1 2 5 I . Approximately 2 grams of c e l l sheets (or 2 x 2 x 2 mm muscle chunks) were washed w i t h 200 ml of i c e c o l d 0.25 M sucrose i n 50 mM sodium phosphate, pH 7.2 (Buffer C). The sheets were then placed i n t o a polypropylene c e n t r i f u g e tube c o n t a i n i n g 0.43 ml of 10 nM l a c t o p e r o x i d a s e , 9.025 ml of B u f f e r C and 12 y l of c a r r i e r f r e e [ 1 2 5 I ] Nal (.1.2 m C i ) (ROMBAUTS et a l . , 1967; Morrison, 1970, 1974; MORRISON & BAYSE, 1970; MORRISON & SCHONBAUM, 1976; Hynes, 1976). A t o t a l of 40 y l of 1.6 mM hydrogen p e r i o x i d e was added i n 1 y l p o r t i o n s every 30 seconds f o r 20 minutes. Fo l l o w i n g the l a s t a d d i t i o n , the c e l l sheets were washed on a Buchner funnel f i l t r a t i o n u n i t w i t h 1.5 l i t r e s of B u f f e r C c o n t a i n i n g 5 mM sodium i o d i d e . The c e l l sheets were then homogenized as described under the membrane i s o l a t i o n procedures. F o l l o w i n g membrane i s o l a t i o n , F r a c t i o n s 4 and 5 of the sucrose gradient were immediately c o l l e c t e d and samples withdrawn f o r SDS g e l e l e c t r o p h o r e s i s . Two g e l s were run f o r each sample, one f o r Coomassie Blue s t a i n i n g and the 125 other f o r I counting. The remaining F4 and F5 were then subjected to the i o d i n a t i o n procedure to be described i n Section B. Controls without l a c t o p e r o x i d a s e and/or hydrogen p e r i o x i d e were a l s o run. Samples of homogenized c e l l sheets 125 or muscle chunks were removed f o r g e l e l e c t r o p h o r e s i s . I was detected using a Nuclear Chicago gamma counter (see S e c t i o n c) (FAIRBANKS et a l . , 1967; B0GDAN0VE & STRASH, 1975; LEINEN & WITCLIFFE, 1978). . 47 - 47 - When f r e e c e l l s i s o l a t e d from 1.0 grams of muscle 125 were to be l a b e l l e d w i t h I , the procedure was s i m i l a r except that the volume was reduced to 3.0 ml because of the smaller q u a n t i t y of the c e l l s but the f i n a l concentrations of 125 l a c t o p e r o x i d a s e , I and hydrogen peroxide remained unalt e r e d . The r e a c t i o n was stopped by the a d d i t i o n of 0.75 ml of 0.1% sodium azide i n a 25 mM sodium i o d i d e s o l u t i o n . The c e l l s were then immediately p e l l e t e d by c e n t r i f u g a t i o n at lOOOg, 5 minutes, 0°C i n a S o r v a l l RC-2B c e n t r i f u g e . The p e l l e t was suspended i n 3.0 ml of B u f f e r C and r e c e n t r i f u g e d as above. This procedure was repeated 5x u n t i l the supernatant 125 was v i r t u a l l y f r e e of I . The p e l l e t was then resuspended i n 3.0 ml B u f f e r C and used immediately f o r e l e c t r o p h o r e s i s . C o n t r o l s were run as described p r e v i o u s l y and a c o n t r o l using 125 125 homogenized I l a b e l l e d c e l l s was a l s o run. I was determined as i n part c : and as described under g e l e l e c t r o p h o r e s i s . (b) I o d i n a t i o n of Sucrose Gradient F r a c t i o n s For reasons to be discussed l a t e r three v e r s i o n s of the i o d i n a t i o n procedure were employed to l a b e l i s o l a t e d membrane f r a c t i o n s . (1) Membranes were i o d i n a t e d i n 4 0 0 u 1 of 32% sucrose (.34% f o r F r a c t i o n 5) made 50 mM i n sodium phosphate, pH 7.2, 0.8 pM i n l a c t o p e r o x i d a s e and 0.15 mCi/ml 125 i n t I ] Nal ( c a r r i e r f r e e ) . At room temperature, 1 p i a l i q u o t s of 1.6 mM hydrogen peroxide were added ( t o t a l volume added 30 V1) at 30 second i n t e r v a l s f o r 15 minutes. Reactions were terminated w i t h the a d d i t i o n of 35 P 1 of 0.1% sodium azide i n 28 mM sodium i o d i d e or by adding 100 U1 of e l e c t r o p h o r e s i s sample b u f f e r . The membranes i n both cases were used 48 - 48 - immediately f o r SDS g e l e l e c t r o p h o r e s i s . In the experiment, l a c t o p e r o x i d e and/or hydrogen peroxide wre omitted as c o n t r o l s . (2) 400 y l of membranes i n sucrose (32-34) were d i s t i l l e d 1:1 w i t h d i s t i l l e d water. The i o d i n a t i o n was c a r r i e d out using 125 the f i n a l concentrations of l a c t o p e r o x i d a s e , [ I] N a l , hydrogen peroxide and soium phosphate as i n ( 1 ) . F o l l o w i n g the l a s t a d d i t i o n to hydrogen peroxide, 100 y l of 0.1% sodium azide i n 25 mM.sodium i o d i d e s o l u t i o n was added and the sample c e n t r i f u g e d at 140,000g , 150 minutes, 0°C i n a clV L5-65 Beckmann U l t r a c e n t r i f u g e using a Type 65 angle r o t o r . The r e s u l t i n g p e l l e t was immediately resuspended i n 0.25 M sucrose-50 mM sodium phosphate b u f f e r , pH 7.2 and used f o r e l e c t r o p h o r e s i s . Controls were run as i n (1). (3) Membranes i n 0.8 ml were p e l l e t e d or d i a l y z e d as described under membrane p r e p a r a t i o n to remove sucrose. The p e l l e t was resuspended i n .400 y l of 100 mM sodium phosphate, pH 7.2 using 125 a s y r i n g e . The f i n a l concentrations of l a c t o p e r o x i d a s e , I and hydrogen peroxide were as i n (1). Reactions were terminated and samples prepared as i n (1). For i o d i n a t i o n of sucrose f r a c t i o n s i n detergent the only changes from (1) are that the membranes were made 0.05% and 0.40% i n T r i t o n X-100 20 minutes p r i o r to the a d d i t i o n of hydrogen peroxide a l i q u o t s to s t a r t the r e a c t i o n . (c) I o d i n a t i o n of l a c t o p e r o x i d a s e - " i o d o l a c t o p e r o x i d a s e " F i v e y l a l i q u o t s of a 64 yM l a c t o p e r o x i d a s e stock s o l u t i o n ( f r e s h l y prepared s i n c e s e l f i o d i n a t i o n increases w i t h aging of the enzyme) were added to 10 d i f f e r e n t 400 y l p o r t i o n s of sodium i o d i d e s o l u t i o n s , containg 0.00, 0.10, 0.20, 0.30, 0.40, 0.50, 1.00, 1.50, 2.00, and 5.00 mM sodium i o d i d e r e s p e c t i v e l y . Each s o l u t i o n was aldo made to be . . 49 0.15 mCi/ml i n f I ] Nal and 50 mM i n sodium phosphate, pH 7.2. To s t a r t the r e a c t i o n 1 ul a l i q u o t s of a 1.6 mM hydrogen peroxide s o l u t i o n were added every 30 seconds f o r 15 minutes, a f t e r which 600 ul of 12.5% TCA was added. The suspension were then c e n t r i f u g e d at 5000g, 10 minutes, 0°C i n a S o r v a l l RC-2B c e n t r i f u g e using a SS-34 r o t o r l e a d . The supernatants were poured o f f and saved. The p e l l e t s were then counted i n a Nuclear Chicago Model 1020 Gamma counter. Supernatants were counted as a check to ensure a l l samples contained the same i n i t i a l r a d i o a c t i v e c a r r i e r 125 f r e e [ I ] N a l . Controls were run o m i t t i n g l a c t o p e r o x i d a s e and/or hydrogen peroxide. The supernatants and p e l l e t s were a l s o subjected to e l e c t r o p h o r e s i s , s t a i n e d and counted f o r 125 I to ensure that only l a c t o p e r o x i d a s e was l a b e l l e d and that equal amounts of the enzyme were used. A f t e r determination of the optimal sodium i o d i d e con- c e n t r a t i o n r e q u i r e d f o r optimal I - i n c o r p o r a t i o n by the l a c t o p e r o x i d a s e , the above experiment was repeated using a constant i o d i d e c o n c e n t r a t i o n of 0.3 mM but v a r y i n g the hydrogen peroxide c o n c e n t r a t i o n used. A f t e r determination of the optimum hydrogen peroxide concentration ( l u l / 3 0 seconds f o r 15 minutes), the f o l l o w i n g c o l d l a b e l l i n g of l a c t o p e r o x i d a s e was done. F i v e -5 y l of lactoperoxidase were l a b e l l e d w i t h 0.5 mM sodium i o d i d e (cold) and the optimum hydrogen peroxide c o n c e n t r a t i o n above. Following the l a s t hydrogen peroxide a d d i t i o n , the l o t s were pooled, d i a f i l t e r e d at 0°C to 250 V1 and the concentrate a p p l i e d to a 1 x 40 cm Sephadex G-100 column at 0°C. The l a c t o p e r o x i d a s e came o f f the column s h o r t l y - 5b - a f t e r the v o i d volume as i n d i c a t e d by UV absorption at 280 nm and 125 previous runs using I l a b e l l e d l a c t o p e r o x i d a s e on the column. The f r a c t i o n s w i t h the l a c t o p e r o x i d a s e were pooled and 'the n o n - r a d i o a c t i v e i o d i n e i o d i n a t e d l a c t o p e r o x i d a s e concentrated by d i a f i l t r a t i o n to 200 y l . This was immediately assayed f o r s p e c i f i c a c t i v i t y p r o t e i n and electrophoresed. The s p e c i f i c a c t i v i t y found was i d e n t i c a l to the u n l a b e l l e d enzyme. S e l f - i o d i n a t i n g a b i l i t y of the c o l d l a b e l l e d l a c t o p e r o x i d a s e was assessed by comparison w i t h non-iodinated l a c t o p e r o x i d a s e . The c o n d i t i o n s used were those above. I t was found that the non r a d i o a c t i v e l y l a b e l l e d i o dolactoperoxidase s e l f l a b e l l i n g w i t h I was l e s s than. 10% of that observed w i t h the non-iodinated v a r i e t y . (d) E f f e c t of T r i t o n X-100 on Lactoperoxidase S e l f L a b e l l i n g S i x 400 y l a l i q u o t s of a 0.25 M sucrose -50 mM sodium phosphate, pH 7.2. b u f f e r s o l u t i o n c o n t a i n i n g 0.00, 0.05, 0.10, 0.20 and 0.40% of TX-100 (v/v) r e s p e c t i v e l y were a l l 125 made 0.8 M i n l a c t o p e r o x i d a s e and 0.15 mCi/ml i n [ I ] N a l . To each tube f o r 15 minutes a t 30 second i n t e r v a l s , 1 y l a l i q u o t s of 1.6 mM hydrogen peroxide were added subsequently. Reactions were terminated by the a d d i t i o n of 50 y l of 0.1% sodium azide i n 25 mM sodium i o d i d e s o l u t i o n or by the a d d i t i o n of 100 y l g e l e l e c t r o p h o r e s i s sample b u f f e r proceeded by heating at 100°C f o r 10 minutes and a l l the samples subjected to g e l e l e c t r o p h o r e s i s . Gels were prepared as described f o r e i t h e r s t a i n i n g w i t h Coomassie Blue or counting. 5<1 - 5 1 - (e) Studies of Membrane I o d i n a t i o n Times and E f f e c t of [ I] Nal on S p e c i f i c A c t i v i t y Nine sets of membranes were i o d i n a t e d and the samples prepared as described i n s e c t i o n b ( l ) except f o r the f o l l o w i n g changes: a) For the f i r s t 6 sets of membranes, the 1.6 mM hydrogen peroxide a d d i t i o n s were 1 y l a l i q u o t s / 3 0 seconds f o r 0, 5, 10, 15, 25 and 30 minutes r e s p e c t i v e l y ; b) For the remaining 3 s e t s , hydrogen peroxide was added as i n b ( l ) but 125 using three d i f f e r e n t s p e c i f i c a c t i v i t i e s of [ I] Nal of 0.075 mCi/ml, 0.15 mCi/ml, and 0.30 mCi/ml. Controls were run as described i n b ( l ) . F. Membrane E x t r a c t i o n Procedure, \. To make the e x t r a c t i o n experiments f e a s i b l e , (FAIRBANKS et a l . , 1971; STECK & YU, 1973; STECK, 1974b; COLEMAN et a l . , 1976; KAHLENBURG, 1976) the membranes had to be a v a i l a b l e i n s u f f i c i e n t l y l a r g e q u a n t i t y so that the p r o t e i n s e x t r a c t e d during the e x t r - c t i o n procedures could be detected on SDS g e l e l e c t r o p h o r e s i s . To accomplish t h i s , s i x t e e n 0.8 ml f r a c t i o n s of membranes obtained d i r e c t l y from sucrose gradients F4 and F5 were d i l u t e d 1:1 w i t h d i s t i l l e d water and c e n t r i f u g e d as described i n the Membrane P r e p a r a t i o n s e c t i o n . The p e l l e t s were pooled and suspended i n 15 mM sodium phosphate pH 7.5. The suspension contained 1.0 mg of p r o t e i n / m l . The supernatants and p e l l e t suspension were subjected to g e l e l e c t r o p h o r e s i s as described e a r l i e r to see I f changes i n the p r o t e i n com- p o s i t i o n of the membranes occurred due to the water d i l u t i o n p r i o r to sedimentation. 5 . 2 - 52 - For the e x t r a c t i o n experiments, 50 ul of membrane suspension was used and extracted w i t h 250 ul e x t r a c t i n g media l i s t e d i n Table IV. Following e x t r a c t i o n , samples were c e n t r i f u g e d at 150,000g , 120 minutes, 0°C i n a Type 65 r o t o r using a Beckman L5-65 u l t r a c e n t r i f u g e . Table IV E x t r a c t i o n Media Used i n Membrane E x t r a c t i o n Media Concentrations Conditions H 20 sodium phosphate, pH 7.5 5 mM 20 min., 0 C 20 min. 0 C pCMBS i n 5 mM sodium phosphate, pH 7.5 0.01, 0.20, 2.00 mM TX-100 i n 5 mM sodium phosphate pH 7.5 b DMMA 0.01, 0.05, 0.50% (y/v) 25 min., 25 C 0.05 mg/ml, 2.0 mg/ml 5.0 mg/ml n e u t r a l i z e d to pH 7.5 wi t h sodium hydroxide EDTA Cin 5 mM sodium phosphate, pH 7.5 0.50 mM 25 min., 0 C D i g i t o n i n 0.36 mg/ml i n 5 mM sodium phosphate, pH 7.5 25 min., 0 C pCMBS r e f e r s to p-Chloromercuribenzene sulphonic a c i d :>DMMA r e f e r s to Dimethyl maleic anhhydride "EDTA r e f e r s to Ethylenediamine t e t r a a c e t a t e 5-3 - 53 - Following c e n t r i f u g a t i o n the p e l l e t s were suspended i n 150 y l b u f f e r (sodium phospaate, pH 8.0 and supernatant 300 y l ) were analyzed f o r p r o t e i n and then immediately electrophoresed as described under the g e l e l e c t r o p h o r e s i s s e c t i o n (using 150 y l supernatant and 75 y l p e l l e t suspension). Coomassie Blue s t a i n i n g was done and a l l g e l s scanned. Peaks were assigned values and compared. G. Column Chromatography on Con A-Agarose or Con A-Sepharose (a) One ml of membrane suspension (1-2 mg p r o t e i n ) i n 50 mM sodium phosphate, pH 6.8, was loaded a t room temperature onto a Con A-Agarose column ( 1 x 5 cm) (CUATRECASES, 1973; MURTHY & HENEZ, 1973; SHARON & LIS, 1975; WALSH et a l . , 1976; BRUNNER et a l . , 1977). The column had p r e v i o u s l y been prepared by washing w i t h (.1) 20 ml of acetate b u f f e r (0.2 M sodium c h l o r i d e ; 6.0 mM magnesium c h l o r i d e ; 5 mM sodium acetate, pH 6.5), (2) 60 ml of 0.20 M sodium c h l o r i d e i n 5 mM sodium ac e t a t e , pH 6.5, and f i n a l l y , (3) 20 ml of 50 mM sodium phosphate, pH 6.5 The ap p l i e d membranes were el u t e d i n i t i a l l y w i t h 50 mM sodium phosphate, pH 6.5, at a flow r a t e of 12 ml/hr. f o r 4 hours, a f t e r which the e l u t i n g b u f f e r was supplemented w i t h amethyl-D-mannoside i n the f i n a l c o n c e n t r a t i o n of 100 mM. The column was allowed to run f o r an a d d i t i o n a l 4 hours a t the above r a t e , a f t e r which the e l u t i n g b u f f e r , was replaced by (50 mM_ sodium borate, pH 7.5; 150 mM a-methyl-D-mannoside). The e l u t i o n r a t e increased to 24 ml/hr. and the column ran f o r 2 hours. 54 - 54^- The 1 ml f r a c t i o n s c o l l e c t e d were immediately assayed f o r p r o t e i n using the Lowry assay and the B i u r e t method. Samples of peak f r a c t i o n s were then subjected to g e l e l e c t r o p h o r e s i s as described e a r l i e r . Peak f r a c t i o n s were a l s o used f o r enzyme assays and i o d i n a t i o n . (b) Using Con A-Sepharose the above mentioned procedure was repeated y i e l d i n g s i m i l a r r e s u l t s . Controls were run using Sepharose 4B and Agarose columns. (c) R a d i o a c t i v e l y l a b e l l e d membranes from F4 or F5 were prepared as described e a r l i e r and a l s o a p p l i e d to the a f f i n i t y column. Samples were tre a t e d as i n (a) except that no i o d i n a t i o n of the e f f l u e n t was c a r r i e d out, only Coomassie Blue s t a i n i n g and r a d i o a c t i v e counting of the g e l s . H. Phosphorylation Studies (a) General Membrane F r a c t i o n s 4 and 5 C25 - 50 ug of p r o t e i n i n the r e s p e c t i v e sucrose s o l u t i o n ) were made up to the f i n a l volume of I. 05 ml by a d d i t i o n of components l i s t e d i n Table V and mixed. This and a l l the subsequent steps were c a r r i e d out at 0°C (NAGANO et a l . , 1965; AVRUCH & FAIRBANKS, 1972; KNAUF et a l . , 1975; CHA & SOOLEE, 1976; LANE, 1976). 55- - 5f, - Table V Components Used i n Phosphorylation Studies Sample 1 2 3 4 0.1mMMgCl„ 0.1mMMgCl„ 0.1 mM MgCl 0.1 mM MgCl 0.5 mM C a C l 2 100 mM NaCl 100 mM L i C l 5 6 7 8 9 0.1 mM MgCl 0.1 mM MgCl 2 0.1 mM MgCl ? C o n t r o l Hydroxylamine a 100 mM Choline 100 mM NaCl 100 mM NaCl 25 mM KC1 0.5 mM CaCl„ a - Used 0.4 M hydroxylamine h y d r o c h l o r i d e f r e s h l y prepared. E x t r a c t i o n done as by Knauf et a l . (.1974) except that the sample was c e n t r i f u g e d at 150,000g ; 47,000 rpm, 2.5 h r . , i n a Beckman L5-65 u l t r a c e n t r i f u g e using a Type 65 r o t o r . 32 [ P] - ATP was added to give the f i n a l ATP con c e n t r a t i o n of 3.74 M and the f i n a l s p e c i f i c a c t i v i t y of 28 C i / mol. A f t e r 30 seconds, 100 y l of 50% TCA was added and the samples spun down at 150,000g a v, 60 minutes, 0°C i n a Beckman 25-65 u l t r a - c e n t r i f u g e using a Type 65 r o t o r . The TCA p r e c i p i t a t e d p e l l e t s were then washed once w i t h and resuspended i n 200 ul 10% sucrose - 50 mM TRIS-HC1, pH 7.2. Each sample was s o l u b i l i z e d i n 3% SDS sample b u f f e r at 37^C f o r 25 minutes. The t o t a l volume of the s o l u b i l i z e d samples, 350 ul now, was d i v i d e d i n t o two 175 ul a l i q u o t s that were electrophoresed. F o l l o w i n g e l e c t r o p h o r e s i s , one sample was used f o r counting and the other 5 6 - 5 6 - f o r Coomassie Blue s t a i n i n g . The counting of r a d i a t i o n was done by s l i c i n g the g e l i n t o 2 mm s l i c e s and p l a c i n g them i n t o s c i n t i L l a t i o n v i a l s w i t h 1 ml of 0.05% SDS. The v i a l s were then incubated f o r 24 hours a t 37°C. Next, 20 "1 of T r i t o n toluene s c i n t i l l a t i o n f l u i d (1 l i t r e TX-100; 2 l i t r e s toluene; 16.9. grams of OMNIFLUOR) were added to each v i a l . Samples were cooled to 0°C and then counted using a Mark I I Nuclear 32 Chicago S c i n t i l l a t i o n counter. P counting e f f i c i e n c y was 32 approximately 85% as determined by adding a known P dpm to the v a r i o u s v i a l s . Various c o n t r o l s were run throughout the experiments. To see whether phosphorylation was a f f e c t e d by tra c e i m p u r i t i e s 32 on the l a b e l l e d [y - P ] ATP, phosphorylation was measured 32 using [y. - P ] ATP at 25 f o l d lower s p e c i f i c a c t i v i t i e s . I t was found that the r e s u l t s wre unaffected by v a r i a t i o n s i n the s p e c i f i c a c t i v i t y . Reactions were terminated using 10% TCA w i t h 0.2 mM ATP and 1.0 mM orthophosphate or 10% TCA w i t h 0.2 mM ATP or 10% TCA w i t h 1.0 mM orthophosphate r a t h e r than TCA alone. (b) Time Course In order to determine optimum phosphorylation, membranes (100 y l p r o t e i n ) i n 1000 y 1 of 30% sucrose w i t h 20 mM TRIS ; HCl, pH 7.8 at 0°C, were phosphorylated f o r 0, 15, 30, and 45 seconds 32 using 3.7 yM ATP made 28 mCi/mmol w i t h [ y - P ]ATP. The r e a c t i o n s were terminated as described e a r l i e r , and the samples processed i n a s i m i l a r manner. (c) Time Course In order to determine more p r e c i s e l y the nature of 2+ 2+' teh Mg dephosphorylation and Ca " phosphorylation the membranes were phosphorylated as above f o r 15 seconds a f t e r 5 7 - 57 - which the sample was made 0.1 mM i n Mg or 0.5 mM i n Ca f o r a further.15 seconds. The r e a c t i o n s were then terminated as described i n part ( a ) . Controls were run checking 2+ phosphorylation w i t h Mg and ATP added at the same time f o r 15- 2+ 30 seconds. The same a p p l i e s to Ca . Samples were processed as us u a l . I . E l e c t r o n Microscopy F4 and F5 were obtained d i r e c t l y from the sucrose gradient and deposited on a M i l l i p o r e F i l t e r (0.22 u pore) by means of a Sweeney Syringe. The deposited membranes or 0.5 mm cubes of muscle were post f i x e d f o r 1 hour w i t h Karnovsky's f i x a t i v e (KARNOVSKY, 1969) i n 1% osmiumrtetroxide, s t a i n e d f o r 1 hour i n saturated aqueous u r a n y l a c e t a t e , dehydrated through a graded a l c h o l s e r i e s and embedded using standard procedure i n Epon A r a l d l t e r e s i n . Gold s e c t i o n s cut on a Re i c h a r t OMU 3 ultramicrotome, were s t a i n e d w i t h Reynolds lead c i t r a t e , and examined under P h i l l i p s 300 tr a n s m i s s i o n e l e c t r o n microscope. 58- - 58 - RESULTS A. General The muscle i n the chicken stomach muscularis has been shown to c o n s i s t p r i n c i p a l l y of v i s c e r a l smooth muscle (Figures 2 and 3) (CALHOUN, 1954). As seen i n the e l e c t r o n micrographs the c e l l s are of v a r y i n g s i z e and each c e l l contains a c e n t r a l nucleus. A l s o , present are mitochondria, however, there was l i t t l e evidence of sarcoplasmic r e t i c u l u m being present. This l a t t e r p o i n t being e s p e c i a l l y i n t e r e s t i n g i n view of the enzyme marker assays done f o r the sarcoplasmic r e t i c u l u m (SR). Using P o l y t r o n homogenization of the smooth muscle, d i f f e r e n t i a l c e n t r i f u g a t i o n , and gra d i e n t c e n t r i f u g a t i o n we obtained membrane f r a c t i o n s enriched i n plasma membranes (Figure 1, see page 40 ). The homogenization times and c e n t r i f u g a t i o n r a t e s were optimized to y i e l d maximal s p e c i f i c and t o t a l a c t i v i t i e s of 5' n u c l e o t i d a s e i n the f i r s t 5 l a y e r s of the sucrose gradients.(45 seconds: F4 - 0.50 um/mg/hr, 0.005 um/mg/hr; F5 - 0.61 um/mg/hr, 0.006 um/hr: 60 seconds: F4 - 0.61 um/mg/hr, 0.007 .um/mg/hr; F5 - 0.90 um/mg/hr, 0.009 .um/hr . : : 75 seconds: F4 - 0.58 um/mg/hr, 0.022 um/hr; F5 - 0.99 um/mg/hr, 0.025 um/hr: 90 seconds: F4 - 0.47 um/mg/hr, 0.007 um/hr; F5 - 0.73 um/mg/hr, 0.006 um/hr). B. Membrane Marker Studies The i s o l a t i o n of the plasma membranes (PMs) was monitored by measuring the s p e c i f i c a c t i v i t i e s of the enzymes l i s t e d i n Table V I . Based on 5' nu c l e o t i d a s e a c t i v i t i e s , the PMs were 59 - 5 9 - Figure 2. E l e c t r o n micrograph of chicken g i z z a r d smooth muscle. The f i e l d shows a number of c e l l s . M a g n i f i c a t i o n at 12,000x. P M N / NM Figure 3. E l e c t r o n micrograph of a chicken g i z z a r d smooth muscle c e l l . The f i e l d shows v a r i o u s o r g a n e l l e s (M - mitochondria, N - nucleus, NM - nuclear membrane, PM - plasma membrane). M a g n i f i c a t i o n a t 21,000x. Table V I T o t a l and s p e c i f i c a c t i v i t i e s of s e l e c t e d marker enzymes at v a r i o u s stages of the f r a c t i o n a t i o n procedure. The s p e c i f i c a c t i v i t i e s are i n umole / mg p r o t e i n / hour, the t o t a l a c t i v i t i e s are i n ymole / hour. Marker Enzyme a c i d phosphatase s p e c i f i c a c t i v i t y t o t a l a c t i v i t y % y i e l d 4- K - sti m u l a t e d a c i d phosphatase s p e c i f i c a c t i v i t y t o t a l a c t i v i t y s uccinate dehydrogenase s p e c i f i c a c t i v i t y t o t a l a c t i v i t y % y i e l d NADPH cyt c_ ; reductase s p e c i f i c a c t i v i t y t o t a l a c t i v i t y % y i e l d 5 1 n u c l e o t i d a s e s p e c i f i c a c t i v i t y t o t a l a c t i v i t y % y i e l d Residue F i l t r a t e 0.033 0.65 11.4 0.046 0.91 0.75 14.80 8.0 0.21 4.17 14.0 0.031 0.59 15.0 0.044 5.01 89.0 0.026 2.96 1.45 165.00 92.0 0.23 26.11 86.0 0.030 3.42 85.0 2,000 x g p e l l e t 0.026 0.24 4.0 0.10 0.94 8.59 80.70 45.0 0.37 3.43 11.0 0.072 0.68 17.0 2,000 x g supernatant 0.056 4.36 77.0 0.068 5.30 1.24 97.00 54.0 0.16 12.50 41.5 0.040 3.12 78.0 15,000 x g p e l l e t 0.036 0.15 2.6 0.051 0.18 2.88 14.40 8.0 0.72 3.60 12.0 0.077 0.27 6.7' 15,000 x g supernatant 0.056 4.62 81.0 0.017 1.40 0.96 79.04 41.6 0.07 10.20 34.0 0.028 2.26 56.0 100,000 x g p e l l e t 0.057 0.29 5.0 0.063 0.33 2.87 14.80 8.2 0.17 0.86 2.8 0.310 1.60 40.0 100,000 x g supernatant 0.032 3.99 70.0 0.026 3.24 0.53 66.50 37.0 0.05 6.25 21.0 0.005 0.62 16.0 Table V i l a S p e c i f i c a c t i v i t i e s of marker enzymes. F r a c t i o n s obtained from sucrose g r a d i e n t s . S p e c i f i c a c t i v i t i e s i n ymole/mg protein/hour. See Table V l l b a l s o . F r a c t i o n 5 ' Mg 2 + ATPase 3 1 2 5 I s u c c i n a t e NADH cyt c 1 5 P r o t e i n % sucro: Number nuc l e o t i d a s e c. p.m./yg p r o t e i n dehydrogenase reductase (yg) 1 0.05 0.33 420 1.20 1.09 563 8.0 2 0.86 8.06 703 0.34 0.63 775 27.0 3 0.93 17.25 714 n.d. n.d. 500 30.0 4 0.57 8.97 743 2.29 0.11 387 32.0 5 1.11 23.30 760 0.37 0.01 263 34.0 6 1.10 22.72 690 0.37 0.01 225 35.0 7 0.27 19.86 420 10.39 0.62 263 36.5 8 0.17 14.57 310 9.90 1.70 200 40.0 9 0.12 14.43 200 0.11 0.65 113 42.0 10 0.05 4.20 n.d. 4 .58 1.13 250 43.0 11 0.07 n.d. n.d. 0.09 0.28 463 44.0 12 0.04 n.d. n.d. n.d. n.d. 375 45.0 13 0.01 n.d. n.d. n.d. n.d. 100 66.0 a S p e c i f i c a c t i v i t i e s i n ymole/: mg protein/minute • b „ ^ Refers to Antimycin s e n s i t i v e NADH cyt* c reductase. I O N Table .VIlb S p e c i f i c a c t i v i t i e s of marker enzymes. F r a c t i o n s obtained from sucrose g r a d i e n t s . NADH c y t c reductase a c t i v i t y measured i n the presence of Antimycin A. S p e c i f i c a c t i v i t i e s i n umol/ mg protein/hour. The r e s u l t s of t h i s t a b l e can be compared d i r e c t l y to those of Table V i l a . F r a c t i o n NADH cyt c_ NADPH cyt c glucose-6- a c i d phosphatase P r o t e i n % sucrose Number reductase reductase phosphatase -K + rfc"1" (yg) 1 3.24 0.23 2 7.08 0.63 3 7.50 0.34 4 4.70 0.44 5 4.60 0.28 6 4.80 0.23 7 6.67 0.37 8 4.38 0.32 9 0.24 0.25 10 0.81 0.09 11 2.82 0.18 12 n.d. n.d. 13 n.d. n.d. 0.11 0.19 0.25 563 8.0 0.25 0.21 0.31 775 27.0 0.11 0.28 0.39 500 30.0 0.09 0.10 0.11 387 32.0 0.09 0.05 0.05 263 34.0 0.04 n.d. n.d. 225 35.0 0.07 n.d. n.d. 263 36.5 0.03 0.09 0.10 200 40.0 0.03 0.11 0.13 113 42.0 0.10 n.d. n.d. 250 43.0 0.12 n.d. n.d. 463 44.0 0.01 n.d. n.d. 375 45.0 n.d. n.d. n.d. 100 66.0 63 - p u r i f i e d ten f o l d during the f i r s t f i v e steps of the i s o l a t i o n procedure (Figure 1). In the process of the gradient c e n t r i f u g a t i o n , the PMs were p u r i f i e d another 2 to 4 f o l d based on 5' n u c l e o t i d a s e values (Tables V i l a and V l l b ) , the s p e c i f i c a c t i v i t y of 5' nucleotidase being highest i n F r a c t i o n 5. The v a l i d i t y of using 5' n u c l e o t i d a s e as a PM marker was checked by using lactoperoxidase c a t a l y z e d i o d i n a t i o n of the PM p r i o r to homogenization. Table V i l a shows that F r a c t i o n 4 (F4) and F r a c t i o n 5 (F5) contained 125 the highest s p e c i f i c a c t i v i t y of I . The d i f f e r e n c e between 125 the I l a b e l l i n g r e s u l t s and the 5' n u c l e o t i d a s e r e s u l t s gave us the f i r s t i n d i c a t i o n s that there may be a d i f f e r e n c e between the o r i e n t a t i o n of the membranes i n F4 compared to F5. The contamination of the v a r i o u s PM gradient f r a c t i o n s was followed by standard markers f o r the SR, mitochondria, and lysozomes. Mitochondria, assessed using succinate dehydrogenase and NADH -.v.. antimycin A s e n s i t i v e cyt ĉ  reductase a c t i v i t i e s , were removed mainly i n the 2,000g c e n t r i f u g a t i o n step. This r e s u l t was q u i t e s u r p r i s i n g s i n c e higher g f o r c e s are u s u a l l y r e q u i r e d to sediment out mitochondria. Despite t h i s r e s u l t , there was s t i l l a 2 to 4 f o l d i n c r e a s e i n the s p e c i f i c a c t i v i t y of succinate dehydrogenase i n the microsomal p e l l e t , followed by a f u r t h e r 4 to 5 f o l d i ncrease a f t e r gradient c e n t r i g u g a t i o n . This increase i n s p e c i f i c a c t i v i t y was found i n F r a c t i o n s 7 and 8 of the sucrose g r a d i e n t . K + stimulated a c i d phosphatase, thought to be found mainly i n lysozomes, showed l i t t l e i ncrease i n s p e c i f i c a c t i v i t y a t any stage of the PM i s o l a t i o n procedure. Maximal s p e c i f i c a c t i v i t i e s were found i n the f i r s t 3 l a y e r s of the g r a d i e n t . 64 - 64 - While dependable markers f or the SR are rather c o n t r o v e r s i a l , i t has been f e l t that NADH antimycin A i n s e n s i t i v e cyt c_ reductase was s p e c i f i c for the SR. Recently, some doubt has been cast on th i s hypothesis as PMs and other organelles are thought to contain the above mentioned enzyme as well (SOTTOCASA et a l . , 1967; SOTTOCASA, 1971; KELBERG & CHRISTENSEN, 1979). S i m i l a r i l y glucose-6-phosphatase i s no longer regarded as being s p e c i f i c for the SR. However, NADPH cyt ĉ  reductase, one of the more commonly used SR markers, i s thought to be s p e c i f i c for these membranes. Based on studies with t h i s enzyme, the SR was l o c a l i z e d to fr a c t i o n s 1 to 3, 7, 10 and 11. There was d e f i n i t e contamination of Fractions 4 and 5 but th i s represented less than a 2 f o l d increase i n s p e c i f i c a c t i v i t y over the f i l t r a t e . The d i s t r i b u t i o n s of glucose-6-phosphatase and NADH antimycin A i n s e n s i t i v e cyt c, reductase were not exactly the same as that of NADPH cyt ĉ reductase. The exact meaning of t h i s i s treated i n the Discussion section (see also Tables V i l a and V l l b ) . Based on the above r e s u l t s , F4 and F5 were judged to be the purest PM fr a c t i o n s obtained from the gradient. They were shown to consist of v e s i c l e s under the electron microscope (Figures 4 and 5) but no quantitative r e s u l t s were derived from the electron micrographs other than v e s i c l e s i z e (which appeared quite v a r i a b l e ) . Further ch a r a c t e r i z a t i o n of the membranes using g e l electrophoresis, showed the PAS and coomassie blue staini n g p r o f i l e s of F4 and F5 to be the same (Figures 15 and 16, Table IX). These r e s u l t s would :'. indicate that any differences observed i n membrane properties do not appear to a r i s e from s t r u c t u r a l heterogeneity. 65 - 6 5 - Figure 4. Electron micrograph of Fraction 4 plasma membranes i s o l a t e d from the chicken gizzard smooth muscle. Magnification at 47,000x. Figure 5. Electron micrograph of Fract i o n 5 plasma membranes i s o l a t e d from the chicken gizzard smooth muscle. Magnification at 60,000x. - 66 - 2+ C. Plasma Membrane Mg Stimulated ATPase A c t i v i t i e s (a) Optimization of Mg2+ stimulated ATPase a c t i v i t y - When the gradient f r a c t i o n s were being characterized with respect to contamination, attempts were made to observe some Na +/K + ATPase 2+ a c t i v i t y . As shown i n Table V i l a , only a very a c t i v e Mg stimulated ATPase was observed. Maximal s p e c i f i c a c t i v i t y was observed i n F5, t h i s i n contrast, to the rather low s p e c i f i c a c t i v i t y observed i n F4. These r e s u l t s when combined with the aforementioned PM marker r e s u l t s , provided further evidence for F4 having the opposite o r i e n t a t i o n to F5. I t was f e l t a t t h i s time that a thorough analysis 2+ should be done of the Mg stimulated ATPase a c t i v i t y observed i n F5 2+ since there have been many reports of s i m i l a r Mg stimulated ATPases i n other smooth muscle preparations. Some investigators now consider Mg stimu. VALLIERES et a l . , 1978) 2+ now consider Mg stimulated ATPase as a s p e c i f i c PM marker ( 2+ Using F5 , the s p e c i f i c a c t i v i t y of the Mg stimulated 2+ ATPase was optimized f o r Mg and ATP (Figure 6) and under conditions of optimal a c t i v i t y the enzyme was tested f o r Na +, L i + and K + s e n s i t i v i t y , ouabain i n h i b i t i o n and pH e f f e c t s (Figure 7). K + stimulation was only observed at very high pH values, while ouabain and the various other cations tested had l i t t l e e f f e c t . The pH optima of the enzyme was 7.6 at 37°C. When Ca^ + stimulation 2+ was t r i e d no e f f e c t was noted. The enzyme appeared to be a Mg stimulated ATPase. Assumingthat [MgATP] was the enzyme substrate, a pl o t of v e l o c i t y versus substrate concentration was done (Figure 8). 67 - 67 - Figure 6. Optimization of Mg ATPase a c t i v i t y i n Fract i o n 5. 2+ 2-f Plot of s p e c i f i c a c t i v i t y of Mg ATPase versus [ Mg ] at C O Ucl-L various [ ATP ]. ATPase assay was as described under " Materials and Methods " . A , [ ATP ] = 0.00 mM; • , [ ATP ] = 0.05 mM; • , [ ATP ] = 0.10 mM; O, [ ATP ] = 0.20 mM; [ ATP ] = 0.40 mM; O , [ ATP ] =0.80 mM;^, [ ATP ] = 1.20 mM; • , [ ATP ] = 2.00 mM; A , [ ATP ] = 4.00 mM. S p e c i f i c a c t i v i t i e s represent the mean of 5 values. SPECIFIC ACTIVITY in jjmoles Pj/minmg protein 01 o cn 8 S 8 - 69 - Figure 7. E f f e c t s of pH, Na, K, L i and ouabain on the Mg"' ATPase a c t i v i t y of F r a c t i o n 4 (bottom) and F r a c t i o n 5 ( t o p ) . F r a c t i o n 4: A , 0.40 mM ATP; #, 0.40 mM ATP + 0.20 mil Mg 2 +; O , 0.40 mM ATP + 0.20 mM Mg 2 + + 120 mM Na*; A , 0.40 mM ATP + 0.20 mM Mg 2* + 120 mM Na + + 20 mM K +; • , 0.40 mM ATP 4 0.20 mM Mg 2 + 4 120 mM L i + ; • , 0.40 mM ATP + 0.20 mM Mg 2 + + 120 mM Na + 4 20 mM K + 4 1 mM ouabain. F r a c t i o n 5 ( a l l concentrations as f o r F r a c t i o n 4 ) : A , ATP; • , ATP + Mg 2 +; O , ATP + Mg 2 + + Na +; A , ATP + Mg 2 + + Na + 4 K1"; • , ATP * Mg 2 + + L i + ; I , ATP 4 Mg 2 + 4 Na + 4 K + 4 ouabain. S p e c i f i c a c t i v i t i e s are the mean of 5 val u e s . SPECIFIC ACTIVITY in iimoles Pj/minmg protein o i o SPECIFIC ACTIVITY in jumoles Pj/minmg protein _ L _ L ro ro o i o cn o en 01 - 7 1 - 30h c « _ i i i i i t -6 -5 -4 -3 -2 -1 LOG [SUBSTRATE] Figure 8. Plot of rate versus logarithm of the substrate [ MgATP ] for +2 the Mg ATPase observed i n Fraction 5. The enzyme appears to be i n h i b i t e d by high substrate concentrations. - 7 2 - Figure 10. Phase contrast micrograph of an i s o l a t e d smooth muscle c e l l ( see arrows ). Magnification 160x. - 73 - The r e s u l t s are representative of an enzyme that i s i n h i b i t e d by high subsrtate concentrations. Based on the maxima, we might -5 -3 calc u l a t e a Km of 5 x 10 M, Ks' of 2 x 10 M and a Vmax of 2.5 x 10^ M/sec. As we s h a l l see l a t e r the assumption that [MgATP] 2 i s the substrate, has to be somewhat modified. The presence of the 2+ Mg stimulated ATPase on the external surface of F5's PM 2+ correlated well with the ecto Mg stimulated ATPase a c t i v i t y noted i n suspensions of sing l e smooth muscle c e l l s derived from the 2+ chicken gizzard (Figures 9 and 10). The ecto Mg stimulated 2+ ATPase on the c e l l s had s i m i l a r c h a r a c t e r i s t i c s to the Mg stimulated ATPase found i n F5. (b) Phosphorylation of the plasma membranes i n F4 and F5 - A l l the r e s u l t s i n t h i s section are d i r e c t l y comparable. The amounts of protein, as judged by gel electrophoresis, were i d e n t i c a l i n a l l runs. The ATPase was further characterized by phosphorylation 32 32 of F4 and F5 PMs with[Y- P] ATP. For F5 i n the presence of [ p] 32 ATP only, the P incorporated was maximized with respect to 3 2 incubation times used (Figure 11). In the presence of [ p] ATP, F5 exhibited three major peaks; Peak A - 205,000 daltons; Peak B - 165,000 daltons; Peak C - 145,000 daltons. When F4 was l a b e l l e d wi th [ p] ATP, only a small amount of P was incorporated when compared with F5 (Figures 12a, 12b and 12c) . These r e s u l t s point toward an ATP binding s i t e being accessible from the external PM surface only, while the f a c t that some F4 was l a b e l l e d may have been i n d i c a t i v e of some of the v e s i c l e s i n th i s f r a c t i o n being 3 2 either leaky, unsealed of of mixed RO/IO o r i e n t a t i o n . [ p] ATP 2+ 32 added i n the presence of Mg caused a decrease i n the P 74 - 74 - incorporation of Peak A i n F5 (Figure 12b) . A decrease of 35% was seen i n Peak B of F5, t h i s being ha l f of that noted f or Peak A (F5), whereas i n F4 no detectable changes were noted. When 2+ 2+ Ca was substituted for Mg i n F5 a s l i g h t decrease was noted i n the phosphorylation of Peak A, t h i s compared to a 50% increase i n 32 the P incorporation of Peak B (Figure 12c). Again, no r e a l e f f e c t was noted i n F4. 2+ To show that Mg was promoting dephosphorylation of the 32 2+ bound P i n F5, a time delay study was done i n which Mg was 32 added 15 seconds a f t e r the addit i o n of [ P] ATP. The reaction was terminated a f t e r an a d d i t i o n a l 15 seconds. The r e s u l t s 2+ (Figures 14a and 14b) show that Mg promotes dephosphorylation 2+ 2+ of Peak A. When Ca replaced Mg i n the time delay experiment 32 (Figure 14a), a 3 f o l d increase was noted i n the P incorporation of Peak B. Peak A showed l i t t l e or no increase. As i l l u s t r a t e d 32 i n Figure 14a, addition of hydroxlamine reduced the P a c t i v i t y 32 incorporated into F5 membranes ( l a b e l l e d wi th [ p] ATP) which indicates that the l a b e l l e d phosphoryl groups i n Peaks A, B and C were bound to a c y l moieties. Both the enzymes involved i n Peak A and B appear to have s i t e s l o c a l i z e d externally on the F5 membranes but these s i t e s are inac c e s s i b l e i n F4 due to an apparent difference i n o r i e n t a t i o n . A l l phosphorylated enzymes (Peaks A, B 32 and C) are binding P by a c y l moieties. The c o r r e l a t i o n of the Mg^+ stimulated ATPase a c t i v i t y at 37°C with the l a b e l l i n g by ̂ 2P at 205,000 daltons i s extremely tempting. Based on these r e s u l t s t h i s may be p a r t i a l l y j u s t i f i a b l e , but i t i s by no means c e r t a i n . 7-5 - 7 5 - • • • • • i i i i i i 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 GEL SLICE NUMBER Figure 11. Phosphorylation patterns of Fr a c t i o n 5 at various incubation 32 times using [ y- P ] ATP. Procedure i s as described under " Materials and Methods ".Peak assignments are as followsrA - 205,000 MW; B - 165,000 MW; C - 14 5,000 MW. Peaks i n Figures 12 to 14 are assigned s i m i l a r l y . Figure 12. Phosphorylation patterns of Fraction as described under " Materials and Methods ". (a): 3 3.74 uM ATP ( [ y-32V ] ATP, 40 Ci/nmol ) + 0.10 mM 2+ nmol ) + 0.50 mM Ca . 4 (•—•) and Fract i o n 5 (Q—O) . Conditions are .74 uM ATP ( [ y - 3 2 P ] ATP, 40 Ci/nmol ); (b) : Mg 2 +; (c) 3.74 uM ATP ( [ y - 3 2 ? ] ATP, 40 C i / [32P] DPM INCORPORATED [32P] DPM INCORPORATED Figure 13. Phosphorylation patterns of Fraction 4 (.#—•) and Fr a c t i o n 5 (O—O) • A l l media contained 3.74 uM ATP ( [ Y~ 3 2P ] ATP, 40 Ci/nmol ). (a): ATP + 0 t10 mM Mg2"*" + 120 mM Na + or L i + or choline; (b): ATP + 0.10 mM Mg 2 + +0.50 mM C a 2 + ; ( c ) : ATP +• 0.10 mM Mg 2 + + 0.50 mM 2+ + +• Ca + 120 mM Na or L i , These r e s u l t s are d i r e c t l y comparable to Figures 11 to 14. [32P] DPM INCORPORATED _ L ho co ^. o o o o o o o o [32P] DPM INCORPORATED N> CO T 1 I [32P] DPM INCORPORATED -i r o CO Js, -u- Figure 14. Phosphorylation patterns of F r a c t i o n 5. A l l media contained 3.74 uM ATP ( [ j- P ] ATP, 40 Ci/nmol ). (a): (O—O), ATP + 0.10 mM Mg 2 +; ( • — # ) , ATP + 0.50 mM C a 2 + (the l a t t e r 2+ being added 15 seconds af t e r the ad d i t i o n of the ATP), (b) : ( O — O ) , ATP + 0.10 mM Mg (the l a t t e r being added 15 seconds af t e r the ad d i t i o n of the ATP); ( # — • ) , hydroxylamine treatment of membranes phosphorylated with ATP f o r 15 seconds. [32P] DPM INCORPORATED - L hO CO .̂ O O O o O O O O • 1 1 I 1 [32P] DPM INCORPORATED -i N> CO ^. - I S - - 8-2 - 2+ 2+ 32 F i n a l l y F5, i n the presence of Mg , Ca and [ P ] ATP (Figure 13b), showed a major decrease i n Peak A phosphorylation. Peak B showed l i t t l e or no change. F4 as before showed no r e a l e f f e c t . These r e s u l t s f o r F5 may be due to competition of the two cations for a sin g l e binding s i t e i n Peak B of they are due to the e f f e c t s of more than one binding s i t e i . e . independent s i t e s 2+ 2+ 2+ for Mg and Ca . Mg i s promoting dephosphorylation of t h i s 2+ Peak B, while Ca i s promoting phosphorylation. When the 32 2~f" membranes were phosphorylated i n the presence of [ P ] ATP, Mg and 120 mM Na + or L i + or choline, no increase i n phosphorylation of any of the three peaks was noted i n F4 or F5 (Figure 13a). 2+ However when Ca was added to the above incubating media, non ->cic s p e c i f i c increases i n Peaks A and B phosphorylation were seen (Figure 13c). We can explain the increase in,Peak B since we 2+ know Ca increases i t s phosphorylation. However, the increase i n 2+ Peak A i s much more puzzling. Whether the Ca i s causing large conformational changes i n the membrane enzymes i s uncertain. For each of the above experiments exhaustive c o n t r o l 32 studies were done. To ensure that the P bound was not adsorbed r 3 2 32 3 [ P ] ATP or PO^ , reactions were stopped by means other than the a d d i t i o n of 5% TCA (see Methods s e c t i o n ) . To see whether 32 phosphorylation was effected by trace impurities i n the [y - P ] ATP, phosphorylation was measured using 20-30 f o l d lower s p e c i f i c wei 32 T 32 a c t i v i t i e s of [y - P ] ATP. The same r e s u l t s were obtained regardless of the s p e c i f i c a c t i v i t y of the[y - P ] ATP used. Our o r i g i n a l hypothesis that [ MgATP ] i s the substrate 2+ for the Mg stimulated a c t i v i t y observed has to be modified somewhat. It appears that ATP i s binding p r i o r to the binding of 24* 2— Mg . The intermediate may s t i l l be the[ MgATP] but t h i s i s purely conjecture. . .83 - 83 - D. Orientation Studies Using Acetylcholinesterase and S i a l i c Acid It was f e l t at t h i s stage that F4 and F5 were c e r t a i n l y of d i f f e r i n g o r i e n t a t i o n . F4 appeared to be enriched i n i n s i d e - out (10) v e s i c l e s while F5 was thought to contain predominantly right-side-out (RO) v e s i c l e s . This hypothesis was s t i l l f a r from conclusive at t h i s stage so i t was decided to characterize F4 and F5 with respect to the i r o r i e n t a t i o n using acetylcholinesterase (AchE) and s i a l i c acid sidedness assays, both being externally l o c a l i z e d on the c e l l PM. A 1.5 f o l d increase i n AchE s p e c i f i c a c t i v i t y was observed i n F4 when both membrane surfaces were made equally accessible to the substrate by the addition of 0.05% TX-100 pr i o r to the assay (Table V I I I ) . S i m i l a r i l y , after;cleavage of s i a l i c acid by neuraminadase there was a 3 f o l d increase i n accessible s i a l i c acid content of F4 with the ad d i t i o n of TX-100. F5 membranes showed l i t t l e or no increase i n AchE s p e c i f i c a c t i v i t y and s i a l i c acid content i n the presence of 0.05% TX-100. If one examines the actual increases i n the s p e c i f i c a c t i v i t y of AchE and s i a l i c a c i d content for F4 with the addi t i o n of TX-100, we f i n d that the r e l a t i v e increases of the two markers d i f f e r s . This appears to indic a t e that there i s not a homogeneous d i s t r i b u t i o n of these markers on the membrane surface. I t should also be pointed out that the AchE s p e c i f i c a c t i v i t y was highest i n Fractions 4 to 7. S i a l i c acid was found to be highest i n Fra c t i o n 8, the mitochondrial enriched f r a c t i o n . E. Iodination Studies Iodination was the next step i n the i n v e s t i g a t i o n of membrane or i e n t a t i o n . The i n v e s t i g a t i o n began with the l a b e l l i n g . . . 84 Table VIII A c c e s s i b i l i t y of markers i n sucrose gradient f r a c t i o n s . Sulphuric a c i d heading ref e r s to t o t a l nanomoles of N - acetylneuramic acid / mg protein present i n each assayed : f r a c t i o n . The concentration of detergent used was 0.05% T r i t o n X-100 v/v. Sucrose gradient a c e t y l c h o l i n e s t e r a s e 3 s i a l i c a c i d ^ f r a c t i o n number - T r i t o n X-100 + T r i t o n X-100 - T r i t o n X-100 +• T r i t o n X-100 + Sulphuric Acid 1 1.21 3.36 3.82 3.80 3.90 2 11.22 9.13 3.22 2.90 3.15 3 9.05 10.80 9.99 10.16 10.01 4 8.00 13.60 3.01 10.38 10.50 5 9.17 9.69 10.19 8.90 9.86 6 10.08 11.24 9.01 9.32 9.30 7 8.05 12.06 5.51 7.50 8.00 8 0.84 3.68 10.19 26.16 28.67 9 3.70 5.51 2.43 3.76 3.63 10 4.12 6.28 10.50 16.34 17.17 11 1.06 1.07 n.d. n.d. 1.03 12 n.d. n.d. n.d. n.d. n.d. 13 n.d. n.d. n.d. n.d. n.d. Expressed as nanomoles of product per milligram protein per minute. Expressed as nanomoles of N - acetylneuramic acid per milligram p r o t e i n . - 85 - Table IX Molecular weight assignments of bands and peaks depicted in' Figures 15 to 41. Assignments are based on standard molecular weight markers as described under " Materials and Methods ". Band Peak Molecular Weight 1 A, r 207,000 2 3 - 205,000 - 210,000 4 - 191,000 5 B 165,000 6 C 136,000 - 145,000 7 - 130,000 8 s 100,000 9 - 82,000 10 t 55,000 11 - 45,000 12 - Tracking Dye 13 - _ 14 - 31,000 15 - 93,000 114' r ' 207,000 115* s' 100,000 : 16' t* 55,000 -86- 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 15. Coomassie blue s t a i n i n g pattern (top) and PAS p r o f i l e (bottom) of Fraction 4. Protein used for coomassie blue s t a i n i n g (15 yg) was h a l f that used for PAS s t a i n i n g (30 yg). -87- 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 16. Coomassie blue s t a i n i n g pattern (top) and PAS p r o f i l e (bottom) of Fra c t i o n 5. Protein used for coomassie blue staini n g (15 yg) was h a l f that used for PAS s t a i n i n g (30 yg). - 88 - of the PM e x t e r n a l l y using 2 mm cubes of muscle, c e l l sheets, and s i n g l e c e l l suspensions (Figures 17,18, and 19). When muscle 125 cubes (Figure 17) were l a b e l l e d w i t h I , l a b e l l i n g was seen at Band 16' (55,000 MW, peak t ' ) . C e l l sheets were l a b e l l e d at Band 14' (205,000 MW, peak r') and Band 16'. There was no prominent l a b e l l i n g i n the 100,000 MW r e g i o n . The l a b e l l i n g of s i n g l e c e l l suspensions (Figure 19) y i e l d e d s i m i l a r r e s u l t s , however, there was a notable increase i n the l a b e l l i n g of the 100,000 MW r e g i o n (Band 15', peak s ' ) . I t should be pointed out that i f c e r t a i n 125 regions of the p r o t e i n p r o f i l e have a low I s p e c i f i c a c t i v i t y , i t does not imply that the PM p r o t e i n s have been l a b e l l e d w i t h a low s p e c i f i c a c t i v i t y . These g e l s (Figures 17 to 19) i n c l u d e the PM p r o t e i n s and a l l other c e l l u l a r p r o t e i n s . Based on the above data we can conclude that a t l e a s t two regions of the PM are a c c e s s i b l e to the l a b e l l i n g species e x t e r n a l l y . Next, the l a b e l l i n g of the muscle cubes and c e l l sheets was repeated. However, t h i s time the muscle t i s s u e was homogenized and processed to y i e l d F4 and F5. The c o l l e c t e d F4 and F5 were 125 then examined f o r I i n c o r p o r a t i o n , F4 showing l a b e l incorporated at 205,000 MW (Band 1, peak r ) , 100,000 MW (Band 8, peak s) and 55,000 MW (Band 10, peak t) (Figures 20 and 22). Upon r e - l a b e l l i n g 125 of t h i s f r a c t i o n w i t h I , l a b e l l i n g was only noted at 100,000 MW. F5 showed a r a t h e r d i f f e r e n t behavior. Examination of F5 f o r i n i t i a l i n c o r p o r a t i o n showed the l a b e l at 205,000 MW, 100,000 MW and 55,000 MW (Figures 21 and 23). R e - l a b e l l i n g increased the loading i n these regions. Not only do these p r e l i m i n a r y r e s u l t s provide f u r t h e r evidence f o r the p r e f e r r e d o r i e n t a t i o n s of F4 and F5 but they a l s o provide us w i t h i n f o r m a t i o n concerning the d i s p o s i t i o n of c e r t a i n p r o t e i n s i n the membrane. 89 - 8 9 - 0.4 r CO I D < 0.2 0"- 1,000 CL Q H500 LO CM 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) 125 Figure 17. L a b e l l i n g of muscle cubes with I. The procedure used i s described under " Materials and Methods ". (• ), coomassie blue s t a i n i n g 125 pattern of iodinated muscle; (•-#), i o d i n a t i o n pattern of I l a b e l l e d muscle cubes. -90 - Figure 18. L a b e l l i n g of c e l l sheets with ± i J I . The procedure used i s described under " Materials and Methods ". ( ), coomassie blue s t a i n i n g 125 pattern of iodinated c e l l sheets; ()•—•) , i o d i n a t i o n pattern of I l a b e l l e d c e l l sheets. - 9 1 - 0.4 £ c O CO < 0.2 0 n 1,000 H500 1 2 3 4 5 6 7 8 9 GEL L E N G T H (cm) CL Q i _ _ i m CM i 1 - i Figure 19. L a b e l l i n g of a suspension of i s o l a t e d single smooth muscle 125 c e l l s with I. The procedure used i s as described under '' Materials and Methods ". ( ), coomassie blue staini n g pattern of iodinated s i n g l e 125. c e l l s ; ( • — • ) , i o d i n a t i o n pattern of I l a b e l l e d s i n g l e smooth muscle c e l l s . - 9 2 - E c O CO to < -.1,000 Q 500 m C M 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 20. Iodination patterns of Fr a c t i o n 4 using p r e l a b e l l e d muscle cubes. (-—•—), coomassie blue s t a i n i n g pattern of Fraction 4; (O-O), i o d i n a t i o n pattern of Fract i o n 4 obtained using muscle cubes labeled with 125 I p r i o r to homogenization: (•-#), i o d i n a t i o n pattern of Fraction 4 observed upon r e l a b e l l i n g of Fract i o n 4 prepared from iodinated muscle cubes. For the exact procedure see under " Materials and Methods ". - 9 3 - 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 21. Iodination patterns of F r a c t i o n 5 using p r e l a b e l l e d muscle cubes. ( ), coomassie blue s t a i n i n g pattern of Frac t i o n 5; (O—O), i o d i n a t i o n pattern of Fraction 5 obtained using muscle cubes l a b e l l e d with 125 _ I p r i o r to homogenization; ( • - • ) , i o d i n a t i o n pattern of F r a c t i o n 5 observed upon r e l a b e l l i n g of Frac t i o n 5 prepared from iodinated muscle cubes. For the exact procedure see under " Materials and Methods ". 0.4 r CO I D < 0.2 0 -,1,000 Q_ Q H500 r-=i I D CM Jo 1 2 3 4 5 6 7 8 9 GEL L E N G T H (cm) Figure 22. Iodination patterns of Frac t i o n 4 using p r e l a b e l l e d c e l l sheets. ( ) s coomassie blue s t a i n i n g pattern of Frac t i o n 4; (O—O), i o d i n a t i o n pattern of Frac t i o n 4 obtained using c e l l sheets l a b e l l e d with 1 2 5 I p r i o r to homogenization; ( • — • ) , i o d i n a t i o n pattern of Frac t i o n 4 observed upon r e l a b e l l i n g of Frac t i o n 4 prepared from iodinated c e l l sheets. For the exact procedure see under " Materials and Methods ". - 9 5 - F5 cs I I 1 I I I I • • • • 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 23. Iodination patterns of Frac t i o n 5 using p r e l a b e l l e d c e l l sheets. ( ), coomassie blue s t a i n i n g pattern of F r a c t i o n 5; (O—O), i o d i n a t i o n 125 pattern of Fraction 5 obtained using c e l l sheets l a b e l l e d with I p r i o r to homogenization; ( • — # ) , io d i n a t i o n pattern of F r a c t i o n 5 observed upon r e l a b e l l i n g of Frac t i o n 5 prepared from iodinated c e l l sheets. For the exact procedure see under " Materials and Methods ". - 96 - Similar r e s u l t s were obtained when F4 and F5 were l a b e l l e d 125 with I using the three d i f f e r e n t methods outlined i n the Methods section (the r e s u l t s of two of the methods are presented). The l a b e l l i n g of F4, d i r e c t l y from the gradient, shows that the l a b e l was incorporated at 100,000 MWwith minor l a b e l l i n g of bands at 205,000 MW and 55,000 MW (Figures 24 and 26). As F4 was thought to consist predominantly of 10 v e s i c l e s t t h e l a b e l l i n g at 205,000 MW and 55,000 MW appears due to e i t h e r leaky v e s i c l e s , unsealed 125 v e s i c l e s or right-side-out v e s i c l e s . Incorporation of I was noted at 205,000 MW, 100,000 MW and 55,000 MW for F5 (Figures 25 and 27). Some l a b e l was also seen to migrate with the tracking 125 - 125 dye, representing possibly free I or I - l a b e l l e d phospholipids. Based on the above we can conclude that F4 contains a 100,000 MW protein that i s accessible to io d i n a t i o n from the external surface of F4. F5 contains 3 proteins that are accessible to the iodinating species externally. These are the 205,000 MW, 100,000 MW and 55,000 MW bands. If the 100,000 MW band i s a s i n g l e protein, i t could be postulated that i t spans the membrane. I t cannot, however, be said that the 205,000 MW and 55,000 MW proteins do not span the membrane, because the cytoplasmic s i t e s of l a b e l l i n g may not be accessible or may not contain any iodinatable residues. I t i s worth noting.that the 205,000 MW band i s thought to be the s i t e 2+ of the ecto Mg stimulated ATPase described e a r l i e r . Using the above information i t should be possible to show i n a co n t r o l l e d study that F4 i s indeed 10 and that F5 consists of mainly RO oriented membrane v e s i c l e s . 97 - 9 7 - 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) i Figure 24. Iodination of Frac t i o n 4. 0 ), coomassie blue staini n g p r o f i l e of iodinated F r a c t i o n 4; ( • — • ) , i o d i n a t i o n p r o f i l e of Fraction 4. Fract i o n 4 was prepared f o r i o d i n a t i o n as described under " Materials and Methods, io d i n a t i o n studies, section B2 ". -98- 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 25. Iodination of Fract i o n 5. ( ), coomassie blue s t a i n i n g p r o f i l e of iodinated Fraction 5; (#-#), io d i n a t i o n p r o f i l e of Fraction 5. Fracti o n 5 was prepared for i o d i n a t i o n as described under " Materials and Methods, io d i n a t i o n studies, section B2 ". 0.4 r CO LO < 0.2 OL 1 2 3 4 5 6 7 8 9 GEL L E N G T H (cm) Figure 26. Iodination of sucrose free Fraction 4. ( ), coomassie blue s t a i n i n g p r o f i l e of sucrose free iodinated Fraction 4; (•—#), io d i n a t i o n p r o f i l e of sucrose free F r a c t i o n 4, The procedure used i s described under " Materials and Methods, io d i n a t i o n studies, section B3 ". - 1 0 0 - 0.4 r E c o c o < 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 27. Iodination of sucrose free Fraction 5. ( ), coomassie blue staining profile of sucrose free iodinated Fraction 5; (•—•), iodination profile of sucrose free Fraction 5. The procedure used is described under " Materials and Methods, iodination studies, section B3 ". - 101 - It can be argued that the l a b e l l i n g seen at 205,000 MW and 100,000 MW i s due to s e l f l a b e l l i n g of.lactoperoxidase and l a b e l l i n g of a 205,000 MW contaminant i n commercial lactoperoxidase preparations. To deal with t h i s p o t e n t i a l l y serious problem, the following experiments were done. I t i s well known lactoperoxidase w i l l l a b e l i t s e l f , e s p e c i a l l y as the age of the enzyme increases (Figure 28). Fresh lactoperoxidase was iodinated to saturation with non-radioactive iodine, p u r i f i e d and then examined for s p e c i f i c a c t i v i t y , s e l f l a b e l l i n g and p u r i t y . I t s s e l f l a b e l l i n g a b i l i t y was reduced to les s than 10% of that found f o r the fresh enzyme, whereas the s p e c i f i c a c t i v i t y of the enzyme was e q u i v i l a n t to that found f o r the fresh enzyme. The enzyme, on SDS gels, ran as a s i n g l e band at 98,000 MW. Using equal amounts of F4 membranes and i d e n t i c a l i o d i n a t i o n conditions, F4 was l a b e l l e d with f r e s h l y made lactoperoxidase and cold l a b e l l e d lactoperoxidase (Figure 29, r i g h t and l e f t ) . As w e l l , F4 was l a b e l l e d with fresh lactoperoxidase i n the presence of 0.05% TX-100. The r e s u l t s , tabulated i n Table Xa, show that l a b e l l i n g with the two enzymes was i d e n t i c a l . The 100,000 MW was mainly l a b e l l e d . There was also some l a b e l l i n g of the 55,000 MW band. This l a t t e r l a b e l l i n g may be due to contamination of F4 by unsealed, RO or leaky membrane v e s i c l e s . In the presence of 0.05% TX-100, large increases i n the l a b e l l i n g of the 100,000 MW and 55,000 MW bands (Bands 8 and 10 res p e c t i v e l y ) were noted. We can add here that s e l f — l a b e l l i n g ' b y lactoperoxidase i n the presence of 0.05% TX-100 i s 50% of that found i n the absence 102 o Q.I O O o E E o 1.0 2.0 3.0 4.0 5.0 [I-1] in mM Figure 28, Self i o d i n a t i o n of lactoperoxidase. (•—•),, three week old enzyme stored frozen; ( • — • ) , f r e s h l y prepared enzyme; (A~A), previously l a b e l l e d lactoperoxidase. For preparation of l a b e l l e d lactoperoxidase see under " Materials and Methods 125 Figure 29. A c c e s s i b i l i t y of Fraction 4 to iodination using I. RIGHT: ( ), coomassie blue s t a i n i n g p r o f i l e of Fraction 4; ( • — • ) , iodination p r o f i l e of F r a c t i o n 4 i n the absence of 0.05% T r i t o n X-100. LEFT: (O—O), iodination pattern of F r a c t i o n 4 using lactoperoxidase previously l a b e l l e d with cold iodine; ( • — • ) , iodination of Fr a c t i o n 4 i n the presence of 0.05% T r i t o n X-100. GEL LENGTH (cm) 40,000 H 20,000 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) Figure 30. Accessibity of Fraction 5 to iodination using X ^ J I . RIGHT: (- ), coomassie blue s t a i n i n g p r o f i l e of Fraction 5; (•—• iodination p r o f i l e of F r a c t i o n 5 i n the absence of 0.05% T r i t o n X-100. LEFT: ( O — O ) , iodination pattern of Fr a c t i o n 5 using lactoperoxidase previously l a b e l l e d with cold iodine; (•—•) , iodination of Fract i o n 5 the presence of 0.05% T r i t o n X-100. 1 2 3 4 5 6 7 8 9 GEL LENGTH (cm) GEL LENGTH (cm) - 107 - Table 'Xa Ac c e s s l b i t y of Fractions, 4 and 5 to lactoperoxidase catalyzed i o d i n a t i o n . ^ 125 Sucrose gradient I c.p.m./12 yg protein (55% e f f i c i e n c y ) f r a c t i o n number Band 8 - 100,000 daltons Band 10 -55,000 daltons 4 iodolactoperoxidase 83,000 42,000 - T r i t o n X-100 85,000 45,000 + T r i t o n X-100 149,000 145,000 5 iodolactoperoxidase 61,000 143,000 - T r i t o n X-100 70,000 153,000 + T r i t o n X-100 140,000 155,000 ~* Please see Figures 29 and 30 al s o . Table Xb Self l a b e l l i n g of lactoperoxidase i n the presence of" T r i t o n X-100. Lactoperoxidase used was one day o l d . 125 [ T r i t o n X-100 ] I c.p.m./25'yg enzyme 0.00% 1.00 x 10 5 0.05% 0.50 x 10 5 0.10% 0.50 x 10 5 0.20% 0.40 x 10 5 0.40% 0.30 x 10 5 - 108 - of detergent (Table Xb)• On the basis of the preceeding r e s u l t s , f a l s e peaks due to s e l f - l a b e l l i n g of commercial lactoperoxidase preparations, can be rul e d out. The r e s u l t s f o r F5 are j u s t as revealing.. In the absence of detergent, using fresh or cold l a b e l l e d lactoperoxidase, l a b e l l i n g was observed at 100,000 MW and 55,000 MW mainly. The r e s u l t s using the two d i f f e r e n t enzymes were i d e n t i c a l , thus again r u l i n g out lactoperoxidase s e l f - l a b e l l i n g giving f a l s e peaks. L a b e l l i n g i n the presence of TX-100 was shown to be increased only at 100,000 MW. There was no change i n the l a b e l l i n g at 55,000 MW. These r e s u l t s provide extremely strong arguements i n favour of the hypothesis that F5 PMs are mainly RO oriented and that F4 contains a predominantly 10 PM v e s i c l e population with some contamination. F. Extraction Studies At t h i s stage, given the indicated orientations of F4 and F5 we had hoped to corroborate t h i s d i f f e r e n c e i n o r i e n t a t i o n using extraction studies. As we l l , we had hoped to extract peripheral membrane proteins to si m p l i f y the coomassie blue stainin g p r o f i l e . Examination of the r e s u l t s y i e l d s a rather confusing picture indeed (Figures 31 to 38, Table XI). The agents used were Dimethylmaleic anhydride (DMMA), Ethylenediamine tetraacetate (EDTA), H 20, Didit o n i n (DT), T r i t o n X-100 (TX-100), and p-Chloromercuribenzene sulphonic acid (pCMBS). Their use was based on studies by Fairbanks (FAIRBANKS et a l . , 1971), Steck (STECK & YU, 1973) and Kahlenberg (KAHLENBERG, 1976). DMMA and pCMBS either denature or covalently modify proteins, pCMBS by 109 - 109 - breaking disulphide linkages. In the human red blood c e l l , these agents s e l e c t i v e l y s o l u b i l i z e a c e r t a i n group of membrane polypeptides, leaving the remainder s t i l l associated with a l l the l i p i d and cabohydrate i n the membrane residue. The s p e c i f i c a ction of the non-ionic detergent TX-100, a Type A amphiphile, i s nearly r e c i p r o c a l to that seen with perturbants such as DMMA. Polypeptides anchored i n the memnrane through apolar associations with l i p i d s can be s o l u b i l i z e d by TX-100 and hopefully the l i p i d s can be d i s - placed from the hydophobic proteins without denaturation. Digitonin, though classed as a Type B amphile, acts by a d i f f e r e n t mechanism compared to TX-100 but the end r e s u l t i s the same. EDTA i s thought to release membrane f i b r i l l a r proteins by chelating membrane bound divalent. The r e s u l t s are presented band by band. I t i s important to r e a l i x e that F4 may not be e n t i r e l y homogeneous with respect to 10 ori e n t a t i o n while F5 i s thought to be mainly RO. The behavior 2+ of Band 1 (205,000 MW), which may be associated with an ecto Mg stimulated ATPase, was quite i n t e r e s t i n g . We already knew that 125 Band 1 was accessible externally based on the I studies. When the membranes were extracted with ^ 0 , a small amount of the Band 1 protein was extracted from F5 (The appearance of Band 1 i n the supernatant of F4 extracted with 1^0 i s not s e l e c t i v e . We are seeing v e s i c l e s with a buoyant density s u f f i c i e n t to not allow sedimentation under the conditions used.). DMMA removed Band 1 t o t a l l y i n F4 while 25% was only removed i n F5. High concentrations of pCMBS removed 50% of Band 1 i n F5 but the band was not touched 110 - 110 - i n F4. In t e r e s t i n g l y Band 1 was t o t a l l y removed by TX-100 i n F4, but, i n F5 the band was not disturbed. D i g i t o n i n and EDTA had no e f f e c t on Band 1 i n F4 or F5. Based on the above we can summarize that Band 1 contains more than one protein, part of the band i s per i p h e r a l l y located and part i s embedded i n the membrane. There i s also disulphide bond character i n the band proteins. A rather unusual feature of our membrane preparations was Band 2-3. As a shoulder on Band 1 i t appeared at 210,000 daltons or 200,000 daltons. The positon could not be predicted, for example i n the TX-100 extraction of F4 (Figure 37) i t appeared at 200,000 daltons, whereas i n the presence of EDTA and K^O (Figure 31) i t migrated at 210,000 MW. Even when a s e r i e s of samples from F4 and F5 were subjected to g e l electrophoresis under i d e n t i c a l conditions, t h i s band appeared randomly at one p o s i t i o n or the other. Under treatment with the various extracting media t h i s band behaved l i k e Band 1. Band 4 was to small to be followed i n these studies. Band 5 was p a r t i a l l y removed by DT i n F4, i n contrast Band 5 was extracted by EDTA from F5 only. Using DMMA Band 5 was removed from both F4 and F5. However, the concentration used i n the extraction of Band 5 from F5 was lower than for F4. Band 5 i n F5 was p a r t i a l l y removed at low pCMBS concentrations of 0.01 mM but i t was not r e a d i l y apparent whether Band 5 was extracted i n F4 by pCMBS, as there was a large shoulder on Band 1 i n which Band 5 may have been present. T r i t o n X-100 t o t a l l y extracted Band 5 i n F4 whereas only a small amount of Band 5 was removed i n F5 under the same conditions. I l l - I l l - There might also have been some Band 5 hidden under the shoulder of Band 1 i n F5 extracted with 0.05% TX-100. I t should be noted 2+ that a portion of t h i s band displayed Ca dependent increases 2+ i n phosphorylation and that the binding s i t e s for ATP and Ca were accessible from the external surface only. We can thus conclude that Band 5 contains disulphide linkages, may consist of more than one protein component and i t l i e s p a r t i a l l y embedded i n the hydrophobic region of the membrane. That there must be a peripheral component can be shown by the EDTA extraction of Band 5. Bands 6.1 and 6.2 were apparently p a r t i a l l y removed by H^O extraction of F4 and F5 but i t appeared that there was no actual extraction of 6.1 and 6.2 i n F4. As pointed out e a r l i e r the gel p r o f i l e of the supernatant obtained from the Ĥ O extraction of F4 represents small v e s i c l e s that could not be sedimented. In some cases such as the EDTA extraction of F4, the Bands 6.1 and 6.2 co- uld not be i d e n t i f i e d . DT removed neither 6.1 or 6.2 from F4 or F5 while low concentrations of DMMA p a r t i a l l y extracted these bands i n F4 and F5. Treatment with pCMBS yie l d e d no extraction of 6.1 and 6.2 but TX-100 removed the bands p a r t i a l l y i n both F4 and F5. We can therefore conclude that Bands 6.1 and 6.2 also consist of more than one protein and are p a r t l y embedded i n the hydrophobic part of the membrane. Band 7 was a very minor band, at times d i f f i c u l t to detect. The only observation that can be made i s that TX-100 appeared to s e l e c t i v e l y remove Band 7 from F5 while Band 7 was only p a r t i a l l y removed from F4 by t h i s treatment. I t therefore appears that Band 7 may be found i n the hydrophobic regions of the membrane. 112 - 112 - Band 8, i f a sing l e protein was thought to span the membrane based on the iod i n a t i o n studies. EDTA and DT removed the band from F4 and F5. High concentrations of DMMA removed 100% of Band 8 i n F4 but less than 50% i n F5. The band was completely extracted by TX-100 i n both f r a c t i o n s . Using pCMBS Band 8 was not affected i n F4 but appears to have been extracted from F5 ( i n a broad peak on the shoulder of Band 9). We can summarize that Band 8 appears to span the membrane based on the io d i n a t i o n studies. This i s v e r i f i e d by the TX-100 removal of the band i n F4 and F5. There may be disulphide character and more than one protein component i n t h i s band. Of a l l the bands, Band 9 displayed the most v a r i a b l e behavior. EDTA and DT p a r t i a l l y removed Band 9 from F4 and F5. DMMA t o t a l l y removed Band 9 i n F4 but only 50% of t h i s band was extracted i n F5 using high concentrations of DMMA. The behavior of t h i s band i n the presence of pCMBS was again quite d i f f e r e n t i n F4 and F5. This treatment t o t a l l y removed the band from F5 whereas i t was v i r t u a l l y unaffected i n F4. Int e r e s t i n g l y , the TX-100 extraction of F4 appeared to r e s u l t i n some kind of modification of the Band 9 protein as i t became the predominant peak i n the ge l pattern, possibly at the expense of Band 10. Band 9 i n F5 was 75% removed by treatment with TX-100. We can te n t a t i v e l y conclude that Band 9 appears to be one protein with disulphide bond character, and i s embedded p a r t l y i n the hydrophobic regions of the membrane. I t can also be added that Bands 9 and 11 were iodinated only from the external membrane surface (Figures 25 to 28), but the extraction studies seem to indicate that Band 9 does have cytoplasmic s i t e s which may not be accessible to i o d i n a t i o n . 113 - 113 - Band 10, which had been shown to have externally iodinated s i t e s only, could be extracted by EDTA and DT from F4 and to a lesser degree i n F5. DMMA appeared to have modified the band i n F4 and extracted 50% of the band i n F5. There appeared to have been an increase i n Band 9 corresponding to a decrease i n Band 10 and possibly implying some type of chemical modification. PCMBS removed part of Band 10 i n both F4 and F5 (50%). The only anomalous r e s u l t was noted i n the extraction of F4 by 0.05% TX-100. In t h i s e x traction the decrease i n Band 10 was again accompanied by an increase i n Band 9. Based on io d i n a t i o n studies and these r e s u l t s Band 10 has disulphide bond character, may consist of more than one protein and possesses external s i t e s that can be iodinated but i s thought to extend into the hydrophobic region. Band 11 i n both F4 and F5 was not affected by DT, but i t was, however, removed from F4 by EDTA. In F5, only 50% of Band 10 was removed by EDTA. DMMA also removed a l l of Band 10 from E4 but only 50% of the band i n F5. Low concentrations of pCMBS t o t a l l y removed Band 11 i n F4 while, i n contrast, much higher concentrations of pCMBS were required to elute the band i n F5. Again TX-100 s e l e c t i v e l y removed Band 11 from F4, whereas, i n F5 only 50% was removed. Iodination studies (Figures 25 to 28) showed Band 11 could only be iodinated externally. Our r e s u l t s i n d i c a t e that Band 11 may have disulphide bonds, consist of more than one protein and possesses some cytoplasmic s i t e s i n addition to the known externally accessible s i t e s . There were various anomalies recorded i n the extraction 114 - 114 - Table XI Protein (yg) contained in extraction media (300 yl) and pellet (resuspeded in 150 yl) following extraction procedure. For gel electrophoresis 150 y l of supernatant and 75 y l of pellet were were used unless otherwise indicated. Please see Figures 31 to 38 also. Extracting Agent Fraction 4 Fraction 5 pCMBS pellet supernatant pellet supernatant 0.01 mM 32 12 38 16 0.10 mM 40 6 32 14 2.00 mM 32 10 20 20 Triton X-100 0.0]% 24 38& 40 30 0.05% 22 60 a 30 40 0.50% 16 56 a 42 40 DMMA 0.1 mg/ml 6 < 20 26 0.4 mg/ml - *°a 20 26 1.0 mg/ml 4 40 16 44 Digitonin 0.36 mg/ml 24 14 34 10 EDTA 0.50 mM 30 a 16 a 44 32 H20 48 12 62 8 a Refers to 38 y l used for gel electrophoresis. - 115 - Figure 31. E x t r a c t i o n of F r a c t i o n 4 u s i n g H^O, Ethylenediamine t e t r a a c e t a t e and D i g i t o n i n . For yg p r o t e i n i n supernatant (S) and p e l l e t (P) see Table X. ( R i g h t ) , p e l l e t obtained a f t e r e x t r a c t i o n procedure, ( L e f t ), supernatant obtained a f t e r e x t r a c t i o n procedure. GEL LENGTH (cm) - 117 - Figure 32. Extraction of F r a c t i o n 5 using H^O, Ethylenediamine tetraacetate and D i g i t o n i n . For yg protein i n the supernatant (S) p e l l e t (S) see Table X. (Right), p e l l e t obtained a f t e r extraction procedure. (Left ), supernatant obtained a f t e r extraction procedure. GEL LENGTH (cm) - 119 - Figure 33. Extraction of F r a c t i o n 4 using Dimethyl maleic anhydride (DMMA). For yg protein i n supernatant (S) and p e l l e t (P) see Table X. (Right), p e l l e t obtained a f t e r extraction procedure. (Left ), supernatant obtained a f t e r e xtraction procedure. -120- i i 1 • ' • • • • • • 0 1 2 3 4 5 6 7 8 9 10 GEL LENGTH (cm) - 121 - Figure 34. Extraction of Fra c t i o n 5 using Dimethyl maleic anhydride (DMMA). For yg protein i n supernatant (S) and p e l l e t (P) see Table X. (Right), p e l l e t obtained a f t e r e xtraction procedure. (Left ), supernatant obtained a f t e r extraction procedure. -122~ r 1 0.4mg/ml 10 I 6.1,6.2 9 10 2-3 J 61,6.2 9 u 11 1 ! 8 5 A 7 / II 14 \ A 1 2 I 1 ' I 1 1 1 I I • • 0 1 2 3 4 5 6 7 8 9 10 GEL LENGTH (cm) - 123 - Figure 35. E x t r a c t i o n of F r a c t i o n 4 using p-Chloromercuribenzene sulphonic a c i d (pCMBS). For yg p r o t e i n i n supernatant (S) and p e l l e t (P) see Table X. ( R i g h t ) , p e l l e t obtained a f t e r e x t r a c t i o n procedure. ( L e f t ), supernatant obtained a f t e r e x t r a c t i o n procedure. -124 - ' I — I 1 — I 1 0 1 2 3 4 5 6 7 8 9 10 GEL LENGTH (cm) - 125 - Figure 36. Extraction of Fra c t i o n 5 using p-Chloromercuribenzene sulphonic acid (pCMBS) . For yg protein i n supernatant (S) and p e l l e t (P) see Table X. (Right), p e l l e t obtained a f t e r extraction procedure. (Left ), supernatant obtained a f t e r extraction procedure. ' 1 2 6 - F5 pCMBS S 0.01 mM 10 I 2.00 mM 9 10 1 10 1 | • 11 2-3 A J LJ J 5 9 I m A 1 1 1 1 1 0 1 2 3 4 5 6 7 8 9 10 GEL LENGTH (cm) - 127 - Figure 37. E x t r a c t i o n of F r a c t i o n 4 using T r i t o n X-100 (TX-100). For yg p r o t e i n i n supernatant (S) and p e l l e t (P) see Table X. ( R i g h t ) , p e l l e t obtained a f t e r e x t r a c t i o n procedure. ( L e f t ), supernatant obtained a f t e r e x t r a c t i o n procedure. - m - F4 TX-100 s p 0.01 % 10 10 9 9 . u 11 11 1 8 —< / A 1 2 0.05% 9 6.1 2,-3 1/ i 5 6 . 2 8 10 11 12 0 1 2 3 4 5 6 7 8 9 10 GEL LENGTH (cm) - 129 - F i g u r e 38. E x t r a c t i o n o f F r a c t i o n 5 u s i n g T r i t o n X-100 (TX-100). F o r yg p r o t e i n i n s u p e r n a t a n t (S) and p e l l e t (P) see T a b l e X. ( R i g h t ) , p e l l e t o b t a i n e d a f t e r e x t r a c t i o n p r o c e d u r e . ( L e f t ) , s u p e r n a t a n t o b t a i n e d a f t e r e x t r a c t i o n p r o c e d u r e . - 1 3 0 - F5 TX-100 0.01% 1 1 0 n i 6.1,6.2 ? 1° 6 1 ; 6.2 i 1 2 ^3 Hi V 1 2 UL 0.05% 1 6.1,6.2 9 i \ ! 1 5 : i I 1 0 i 1 0 6.1,62 II ' 7 a | 2 - 3 ! 9/I ; I I I 1 4 Ijjj*/ \J\ 1 2 GEL LENGTH (cm) - 131 - studies; two of these being Bands 14 and 15. Band 14 (31,000 MW) appeared both i n the DMMA and TX-100 extractions of F4 (Figure 33). Whether t h i s band represents chemical modification i s unclear at this, point but since i t i s present i t must be reported. S i m i l a r i l y , treatment of F5 with 0.05% and 0.50% TX-100 resulted i n the formation of Band 15 (93,000 MW). I t can be argued that Band 15's assignment should r e a l l y be designated as Band 8 and as a r e s u l t the designated Bands 7 and 8 should be designated 6.2 and 7 r e s p e c t i v e l y . Based on Rf values however, t h i s designation though desirable could not be made. Even though the r e s u l t s of the extraction studies were biased by unsealed v e s i c l e s i n F4 some information was gained by these studies. The r e s u l t s at times appeared to be i n d i c a t i v e of differences i n membrane o r i e n t a t i o n of the two f r a c t i o n s F4 and F5. Further pursuit of the extraction studies, however, was terminated as the a d d i t i o n a l information to be gained was questionable. G. A f f i n i t y Chromatography I t seemed desirable to determine as accurately as possible how pure F4 and F5 were with respect to t h e i r o r i e n t a t i o n s . We also intended to further p u r i f y the membranes as we were aware of the possible contamination of F4 by v e s i c l e s of d i f f e r i n g o r i e n t a t i o n . Con A - sepharose a f f i n i t y chromatography was c a r r i e d out using F4 and F5 i n the hopes of further p u r i f y i n g and characterizing the f r a c t i o n s . When F4 membranes, l a b e l l e d 125 with I, were applied to the a f f i n i t y column, 2 peaks were 132 - 132 - obtained. An a d d i t i o n a l 2 peaks being obtained with e l u t i n g buffer containing a methyl-D-mannoside (Figure 39). When the f i r s t 2 125 peaks were analyzed f or I p r o f i l e s (Figures 40a and 40b), l a b e l l i n g was observed ex c l u s i v e l y at 100,000 daltons, t h i s f a c t being consistent only with 10 v e s i c l e s but, an increase i n AchE a c c e s s i b i l t y was s u r p r i s i n g l y not noted, though there was an increase i n the s p e c i f i c a c t i v i t y (see Discussion). The peak at f r a c t i o n 17 was i n d i c a t i v e of E4 being a population of heterogeneous v e s i c l e s with respect to t h i e r s i z e . Analysis of column f r a c t i o n 35 for i o d i n a t i o n and AchE s p e c i f i c a c t i v i t i e s showed rather 125 i n t e r e s t i n g r e s u l t s . The I p r o f i l e showed the same number of counts i n Band 8 as i n Band 10, t h i s f a c t being consistent with unsealed v e s i c l e s (Table Xa). The AchE a c c e s s i b i l t y r e s u l t s agree with the above i n t e r p r e t a t i o n (Table XII). F r a c t i o n 52, eluted with higher a methyl-D-mannoside concentrations, yielded r e s u l t s s i m i l a r to f r a c t i o n 35, again i n d i c a t i n g unsealed v e s i c l e s . In a l l , 90% of the F4 protein applied to the column could be eluted from the column. F5 when applied to the a f f i n i t y column yielded only two elutable peaks and these peaks represented only 5-10% of the t o t a l protein o r i g i n a l l y applied to the column. A f u l l 90% could not be eluted even i n the presence of high concentrations of e l u t i n g sugar 125 and borate buffer. Column f r a c t i o n 6 when examined f o r I p r o f i l e and AchE s p e c i f i c a c t i v i t i e s yielded r e s u l t s consistent only with t h i s f r a c t i o n being mainly 10 v e s i c l e s (Figure 41). Fracti o n 73 studies i n d i c a t e that t h i s f r a c t i o n consisted of unsealed membranes. 133 - 133 - It should be pointed out that the r e s u l t s presented on pages 134 to 139 for the a f f i n i t y chromatography experiments represent the mean values for three to four experiments. Similar data was obtained for Con A - Agarose a f f i n i t y chromatography. This r e s u l t s are not presented here for that reason. Extensive controls were run with each column used. These are dealt with at length i n the Discussion section. 134 - 134 - Figure 39. Con A - Sepharose 4B a f f i n i t y chromatography of F r a c t i o n 125 4 (top) and Fraction 5 (bottom). (.#—#), I d.p.m. of l a b e l l e d membranes eluted from the column; (O—O), protein p r o f i l e of column e f f l u e n t using Lowry protein assay, a MM r e f e r s to a -methyl - D - mannoside. Borate r e f e r s to the buffer used i n place of the normal e l u t i n g b uffer. - 1 3 5 - 10 20 30 40 50 60 70 80 F R A C T I O N N U M B E R F 5 +100 mM oc MM 10 2 0 3 0 4 0 50 60 70 80 F R A C T I O N N U M B E R - 136 - Table . XII Characterization of column f r a c t i o n s eluted from Con A - Sepharose a f f i n i t y columns. Acetylcholinesterase sp. a c t i v i t y expressed as nm/mg protein/min. F r a c t i o n 4 F r a c t i o n 5 125 Tot a l I d.p.m. applied 3.6 x.10 7 4.0 x 10 7 Recovery 90 - 95% 10 - 15% Total protein on column 0.735 mg 0.850 mg Recovery 90 - 95% 8 - 1 0 % Fra c t i o n Column Fr a c t i o n acetylcholinesterase Number Number - T r i t o n X-100 + T r i t o n X- 4 stock 2.72 15.43 5 8.61 26.11 17 22.96 42.13 35 30.64 30.64 52 29.01 30.10 5 stock 14.16 13.50 6 7.00 22.50 28 - - 73 26.70 27.09 - 1 3 7 - F 4 s S T O C K 1 2 3 4 5 6 7 8 9 G E L L E N G T H (cm) Figure 40a. Analysis of peak fractions obtained by Con A r- Sepharose aff i n i t y chromatography of Fraction 4. ( ), coomassie blue staining profile of peak fractions; (O—O), iodination pattern of protein eluted in peak fractions. Fraction 4 stock refers to the membranes originally applied to the column. Greater than 85% of the protein applied to the column was eluted. See also Figure 40b for peak fractions 17,37 and 54. - 1 3 8 - J I I I I 1 I I L 1 2 3 4 5 6 7 8 9 G E L L E N G T H (cm) Figure 40b. Analysis of peak fractions obtained by Con A - Sepharose aff i n i t y chromatography of Fraction 4. ( ), coomassie blue staining profile of peak fractions; (O—O) , iodination pattern of protein eluted in peak fractions. Greater than 85% of the protein applied to the column was eluted. See also Figure 40a for peak fraction 5 and Fraction 4 stock membranes originally applied to the column. - 1 3 9 - 1 2 3 4 5 6 7 8 9 G E L L E N G T H (cm) Figure 41. Analysis of peak f r a c t i o n s obtained by Con A - Sepharose a f f i n i t y chromatography of Fraction 5. ( ), coomassie blue staining p r o f i l e of peak f r a c t i o n s ; ( P — O ), i o d i n a t i o n pattern of protein eluted i n peak f r a c t i o n s . Fraction 5 stock r e f e r s to the membranes o r i g i n a l l y applied to the column. Less than 10% of the protein applied to the column was eluted. - 140- - H. Summary In summary we can say that F4 was found to contain 20-25% unsealed membrane v e s i c l e s and that these were the contaminating components i n the preparations used for sidedness studies. F5 represents a membrane preparation already 90% right-side-out. Any attempt to improve on t h i s would be u n l i k e l y to succeed. The pro- blem of el u t i n g the 90% of protein bound ( i n F5) was not solved. The binding to the a f f i n i t y column appears to be non-specific adsorption. Perhaps conditions could be modified f o r the e l u t i o n of these membranes but further work would be required. In conclusion, we have prepared.two sets of membrane v e s i c l e s with enriched o r i e n t a t i o n s . At the same time we have demonstrated the nature of some of the proteins i n the membrane. Methods usable for the i d e n t i f i c a t i o n of RO and 10 membranes were also presented and i n d i c a t i o n s were given of a possible technique f o r preparing PM v e s i c l e s of pure defined o r i e n t a t i o n . 141 "141 " Discussion The chicken gizzard, although known to be r i c h i n smooth muscle, has not been used extensively i n membrane work. However i t was f e l t that t h i s was an i d e a l source of smooth muscle for an i s o l a t e d pure plasma membrane preparation. A Polytron was used for the i n i t i a l homogenization since the tissue i s d i f f i c u l t to homogenize by other methods. Homogenization times chosen for muscle cubes and c e l l sheets were optimized to y i e l d maximal s p e c i f i c a c t i v i t i e s and t o t a l a c t i v i t i e s of 5' nucleotidase, a plasma membrane marker. No other marker studies were performed during t h i s procedure- The Polytron, the most commonly used homogenizer i n membrane preparations from v i s c e r a l smooth muscle, was found to be quite successful i n our work. D i f f e r e n t i a l c e n t r i f u g a t i o n was quite successful i n enrichming the 100,000 g p e l l e t with plasma membranes, as 40% of the 5' nucleotidase a c t i v i t y was retained from the residue and crude f i l t r a t e . There was a 10 f o l d p u r i f i c a t i o n of the plasma membranes based on the marker.5' nucleotidase. This l a t t e r r e s u l t being s i m i l a r to those noted elsewhere i n the l i t e r a t u r e (see Table I, gradient preparations). No increase i n the s p e c i f i c a c t i v i t y of NADPH cytochrome c_ reductase was noted, t h i s r e s u l t being i n contrast to other studies which show increases i n the s p e c i f i c a c t i v i t i e s of markers for the SR i n 100,000 g p e l l e t s . S l i g h t increases i n the s p e c i f i c a c t i v i t i e s of acid phosphatase and succinic dehydrogenase. What i s more important i s to note that there was a decrease i n the t o t a l units of these enzymes. At the same time i t must be pointed 142 - 142 " out that no o r i e n t a t i o n s t u d i e s were done on the p e l l e t s and supernatants p r i o r to sucrose gradient c e n t r i f u g a t i o n . In r e t r o s p e c t t h i s should have been done to check i f the values f o r t o t a l u n i t s of a c t i v i t y were indeed c o r r e c t . I t must be remembered, however, that r e s u l t s a p p l i c a b l e , to one type of smooth need not n e c e s s a r i l y apply to smooth muscle obtained from another source. The f r a c t i o n a t i o n of the v a r i o u s c e l l membrane types achieved by the use of the sucrose gradient c e n t r i f u g a t i o n was s i m i l a r to that observed i n v a r i o u s other plasma membrane preparations but the d i s t r i b u t i o n of the d i f f e r e n t membranes was i n disagreement w i t h these other s t u d i e s . In our case, mitochondria were found i n the r e g i o n between 36 and 40% sucrose, which was i n agreement w i t h the l i t e r a t u r e . The SR was d i s t r i b u t e d throughout the g r a d i e n t w i t h the s p e c i f i c a c t i v i t y of marker enzymes f o r the SR being highest i n F r a c t i o n 2 (27% sucrose). Three peaks of a c t i v i t y were observed using the marker NADPH cyt c_ reductase, but these r e s u l t s d i d not agree w i t h the d i s t r i b u t i o n of the markers glucose-6-phosphatase and NADH cyt c_ reductase. The r e s u l t s of MATLIB (MATLIB et a l . , 1979) showed NADH cyt c_ reductase to be an unacceptable s p e c i f i c marker of the SR. Our r e s u l t s seem to bear t h i s out. I t may be that we are seeing the e f f e c t s of d i f f e r i n g o r i e n t a t i o n or a non s p e c i f i c enzyme d i s t r i b u t i o n of glucose-6-phosphatase or NADH c y t c_ reductase. The plasma membranes were g e n e r a l l y 143 - 143 " found at lower d e n s i t i e s , however, SR was also present i n cer t a i n f r a c t i o n s . The d i s t r i b u t i o n observed was i n agreement with the r e s u l t s of Moore (MOORE et a l . , 1975) and Hurwitz (1974). There were no i n d i c a t i o n s that the. plasma membranes were binding higher density membrane fragments.. On the other hand, most separations on gradients y i e l d r e s u l t s d i s s i m i l a r to these above as the SR i s found at higher d e n s i t i e s than plasma membrane i n these studies. A rather i n t e r e s t i n g feature of our plasma membrane 2+ f r a c t i o n s was the Mg stimulated ATPase observed on the external 3 2 membrane surface of F5 and sing l e c e l l s . L a b e l l i n g with [ p] ATP seemed to in d i c a t e that the ATPase was l o c a l i z e d to an apparent M.W. 2+ of 205,000 daltons. Mg stimulated ATPases have been found associated with the plasma membrane of a o r t i c smooth muscle, myometrial and i n t e s t i n a l smooth muscle, but t h e i r function i s 2+ curre n t l y under considerable dispute. A Mg stimulated ATPase s i m i l a r to that observed here has reportedly been found on the external surface of the red blood c e l l plasma membrane. This enzyme exhibits high substrate i n h i b i t i o n and other s i m i l a r properties to our enzyme (SMOLEN & WEISSMAN, 1978). The 2+ authors speculate that Mg stimulated ATPases may have some r o l e i n chemotaxis, phagocytosis and superoxide anion generation but i t remains to be seen whether these are r e a l l y true. When 2+ inv e s t i g a t i n g any Mg stimulated ATPase i n muscle there i s the added danger of myosin ATPase contaminating the preparation, as myosin may be absorbed to the membrane surface during homogenization. 2+ However, we f e e l that we can r u l e t h i s out as the Mg stimulated 144 - 144 - ATPase a c t i v i t y noted i n F5 was the same as that found i n free 2+ c e l l s . Another problem with the high Mg stimulated ATPase a c t i v i t y observed i n our Fra c t i o n 5 was that i t prohibited 2+ the measurement of any Na+/K+ ATPase or Ca ATPase a c t i v i t y . It was only through [ 32p ] ATP l a b e l l i n g that we became aware of the p o s s i b i l i t y that more than one ATPase may be present. It i s were to be demonstrated convincingly that the 165,000 MW 2+ peak represents a Ca ATPase, s e l e c t i v e extraction of these 2+ bands or s e l e c t i v e removal of the Mg stimulated ATPase would have to be achieved f i r s t (JORGENSON, 1974). I t might be further added that the phosphorylation conditions used by other authors (see Methods section) were not found to work i n our studies whatsoever, and our conditions were chosen as the r e s u l t of many d i f f e r e n t v a r i a t i o n s . Not only did the phosphorylation experiments r e s u l t s y i e l d information of possible ATPases but they provided further evidence for the d i f f e r e n t orientations of F4 and F5. Perhaps the most i n t e r e s t i n g r e s u l t s were obtained from the i o d i n a t i o n experiments for the l a t t e r provided another means for determining o r i e n t a t i o n and degree of p u r i f i c a t i o n of plasma membranes. The inherent assumption i n a l l such studies i s that the l a b e l l i n g species w i l l not permeate the membrane (MORRISON & SCHONBAUM, 1976). Though the exact mechanism of lactoperoxidase catalyzed i o d i n a t i o n i s not f u l l y known, i t i s f e l t that the iodinating species s a t i s f i e s t h i s condition. We might further point out that extensive controls must be run to account for 125 any inherent peroxidase a c t i v i t y and non s p e c i f i c I binding. 145 - 145 - A simple washing of the membranes a f t e r i o d i n a t i o n i s quite inadequate, therefore, controls deleting HO or lactoperoxidase 125 _ must be run. Free I can be removed by gel electrophoresis, i f the l a t t e r i s done properly. Our r e s u l t s have taken a l l these 125 factors into account and they represent the I incorporated a f t e r background controls have been subtracted'.. The treatment of membranes with T r i t o n X-100 to increase a c c e s s i b i l i t y of the substrate to enzymes located on the inner membrane surface was reported by Steck (1974a) but u n t i l recently t h i s has not been applied to the l a b e l l i n g of membranes (HARTIG & RAFTERY, 1977). Hartig and Raftery used Emulphogene as the detergent since TX-100 was found to i n h i b i t the enzyme, but, our experiments showed contrary r e s u l t s . Our i n v e s t i g a t i o n showed that TX-100 i n h i b i t e d s e l f l a b e l l i n g of the enzyme but, not l a b e l l i n g of the membranes, much to our surprise.. It was only at concentrations above 0.40% TX-100 that we observed decreased l a b e l l i n g of the membranes. The use of s e l f - l a b e l l e d lactoperoxidase could now be e a s i l y avoided by use of lactoperoxidase covalently linked to sepharose beads. This would greatly f a c i l i t a t e dealing with contamination due t o s e l f l a b e l l i n g . A l l i n a l l , the i o d i n a t i o n studies unequivocally demonstrated the p r e f e r e n t i a l o r i e n t a t i o n of the two sets of membranes. They also provided a convenient means of following the two sets of membranes i n any further p u r i f i c a t i o n procedure. The extraction r e s u l t s were quite unsuccessful i n many ways. We had hoped to s e l e c t i v e l y remove c e r t a i n bands and investigate them as well as the r e s i d u a l bands but there were no c l e a r cut r e s u l t s as were seen i n the extraction studies performed on red 146 - 146 " blood c e l l s (rbc). Our r e s u l t s , however, seemed to r e f l e c t differences i n membrane o r i e n t a t i o n between F4 and F5. This was not seen i n studies done on rbc by Fairbanks (FAIRBANKS et a l . , 1971 b) and Steck (STECK & YU, 1973). These studies using the red blood c e l l s have shown: that TX-100 removes glycoproteins embedded p a r t i a l l y i n the hydrophobic region of the membrane and that protein perturbants remove the inner cytoplasmic proteins. However, whether these agents act i n the same way on membranes other than the rbc i s questionable based on our r e s u l t s . A good example of t h i s i s Band 1. Band 1 has been shown to be externally l o c a l i z e d , but i t was removed t o t a l l y by TX-100 i n F4 and remained unchanged i n F5. pCMBS on the other hand, removed Band 1 i n F5 but not i n F4. I t would seem then that a c c e s s i b i l i t y i s the l i m i t i n g f a c t o r i n extraction. The r e s u l t s are further complicated by the lack of knowledge of the exact mechanism of extraction of the various agents used. A desirable a d d i t i o n to these experiments would be to measure the ATPase a c t i v i t i e s of the extracted membranes i n conjunction with electron microscopy and marker assay studies. This however, was beyond the scope of our work. The f i n a l step i n our membrane i s o l a t i o n procedure was p u r i f i c a t i o n of the membranes using a f f i n i t y chromatography. The r e s u l t s , presented f or Fractions 4 and 5 i n Table XII and Figures 39-41, are s i m i l a r to those obtained by Walsh (WALSH et a l . , 1976). Based on the i o d i n a t i o n studies alone, the l a b e l l i n g pattern observed f or the f i r s t two peaks eluted when F4 was applied to the column i s consistent only with the membranes 147 - 147 - being inside-out. As a c o n t r o l , non-iodinated membranes (F4) eluted from the column were iodinated. The pattern observed was s i m i l a r to that i n Figures 40a and 40b. However, l a b e l l i n g to a minor degree was also seen at 205,000 and 55,000 daltons. These con t r o l experiments would seem to indicate that the membranes, though i n i t i a l l y pure, the 10 v e s i c l e s , may have become leaky or due to t h e i r unstable nature, R0 or unsealed. The l a t t e r two peaks, eluted i n the presence of amethyl-D-mannoside, based on the i o d i n a t i o n studies appeared to consist of unsealed membrane v e s i c l e s . Control studies using non-iodinated membranes yielded s i m i l a r r e s u l t s . The above r e s u l t s wre confirmed using AchE sidedness assays of the various eluted membrane f r a c t i o n s . There was no increase i n a c c e s s i b i l i t y of column f r a c t i o n s 5 and 17 when compared to the i n i t i a l l y applied F4 stock membranes. In a c t u a l i t y , there was a decrease i n the a c c e s s i b i l i t y . What was noted was a 1-2 f o l d increase i n the s p e c i f i c a c t i v i t y of AchE. This may have been due to removal of mitochondrial contamination or contamination from the SR. This would be p l a u s i b l e as the s i a l i c acid determinations showed the presence of sialoglycoproteins i n f r a c t i o n s enriched with the SR and mitochondria (see Tables V i l a , V l l b and V I I I ) . If these contaminating organelle membranes were RO oriented they may have been bound to the column. I t i s also p l a u s i b l e that there may have been some modification of the membranes due to capping which i s known to occur i n the presence of l e c t i n s . The decrease i n AchE a c c e s s i b i l i t y may be a t t r i b u t e d to e i t h e r 148 - 148 - leaky 10 v e s i c l e s or 10 v e s i c l e s becoming RO and/or unsealed. The high osmolarities of the buffers used would seem to ru l e out osmotic e f f e c t s being responsible f o r the increase i n permeability. What would be necessar i s to do further marker assays, l e c t i n binding studies and maybe some c r o s s - l i n k i n g studies to determine the exact cause of the apparent increase i n membrane permeability. Based on the AchE assays the l a t t e r two eluted peaks from the F4 column appeared to contain unsealed membrane v e s i c l e s . These l a t t e r r e s u l t s being i n agreement with the iod i n a t i o n studies. The increase i n the s p e c i f i c a c t i v i t y of the AchE may have been due to ,the reasons discussed e a r l i e r . On the basis of the increase i n s p e c i f i c a c t i v i t y of AchE, i t may be stated that the membranes i n F4 were p u r i f i e d 1-2 f o l d . This, when combined with the p u r i f i c a t i o n of F4 based on 5'-nucleotidase studies, would make an o v e r a l l p u r i f i c a t i o n of 40-60 f o l d of the plasma membranes. Though t h i s i s considerably higher than most plasma membrane preparations, a c e r t a i n degree of caution i s required as the f i n a l "10" membrane preparation appears to be very unstable or permeable. These l a t t e r issues may be resolved before using these membranes for membrane transport i n v e s t i g a t i o n s . I t should also be pointed out that y i e l d s of membrane protein using the e a r l i e r described procedure are low and would require a batch preparation of plasma membranes for proper i n v e s t i g a t i o n . The behaviour of F5 membranes was rather expected based on the r e s u l t s of Walsh (WALSH et a l . , 1976). Only two peaks 149 - 149 " were eluted, these, representing 8-10% of the t o t a l applied protein. The i n i t i a l peak was assessed to consist of 10 plasma membrane vesicles., while the second peak, eluted i n the presence of amethyl-D-mannoside, found to contain unsealed membrane v e s i c l e s . These conclusions were based on the i o d i n a t i o n and AchE sidedness studies. As seen using F4, the 10 f r a c t i o n was found to be either permeable or contain unstable 10 v e s i c l e s . Since 90% of the applied protein could not be eluted the a p p l i c a b i l i t y of t h i s method for further p u r i f i c a t i o n of a set of RO membrane v e s i c l e s must be suspected. Further, i t must be questioned as to why a set of RO plasma membrane vesicles., already 90% pure, would require further p u r i f i c a t i o n . The two sets of membranes i s o l a t e d (F4 and F5) possess many desirable features f o r the i n v e s t i g a t i o n of membrane transport but as i s apparent, further work i s required to a s c e r t a i n the exact c h a r a c t e r i s t i c s of F5 and more importantly F4. 150 - 150 - LITERATURE CITED Akerman, K.E.O. and Wikstrom, M.K.F. 1979. ( C a ^ + Mg"') - Stimulated ATPase a c t i v i t y of rabbi t myometrium plasma membrane i s blocked by oxytocin. FEBS L e t t e r s , 97_, 283-287. Albertsson, P-A. 1970. P a r t i t i o n of c e l l p a r t i c l e s and macromolecules i n polymer two phase systems. Adv. Protein Chem., 24, 309-341. A l l e n , D.G. and Bli n k s , J.R. 1978. Calcium transients i n aequorin-injected frog cardiac muscle. Nature (London), 273, 509-513. Ames, B. 1966. Assay of inorganic phosphate, t o t a l phosphate and phosphatases. In " Methods In Enzymology. " Vol. 8. Edited by E.F.Neufield and V.Ginsburg. Academic Press Inc., New York. pp. 115-118. Avruch, J . and Fairbanks, G. 1972. Demonstration of a phosphopeptide intermediate i n the Mg+2 dependent Na +- and K + - stimulated adenosine triphosphatase r e a c t i o n of the erythrocyte membrane. Proc. natn. Acad. S c i . U.S.A., 6>9, 1216-1220. Bagby, R.M., Young, A.M., Doison, R.S., Fischer, B.A. and McKinnon, K. 1971. Contraction of sing l e smooth muscle c e l l s from bufo marinus stomach. Nature (London), 234, 351-352. Barber, A.J. and Jamieson, G.A. 1970. I s o l a t i o n and ch a r a c t e r i z a t i o n of plasma membranes from human blood p l a t e l a t s . J . b i o l . Chem., 245, 6357-6365. Barchi, R.L., B o n i l l a , E. and Wong, M. 1977. I s o l a t i o n and char a c t e r i z a t i o n of muscle membranes using surface s p e c i f i c l a b e l s . Proc. natn. Acad. S c i . U.S.A., 74_, 34-38. Barton, R.E. 1978. Biochemical research techniques. Department of Biochemistry, Un i v e r s i t y of B r i t i s h Columbia, Vancouver. Beauge, L.A. and Glynn, I.M. 1977. A modifier of (Na + + K+) ATPase i n commercial ATP. Nature (London), 268, 355-356. 151 - 151 - Bhalla, R.C., Webb, R.C, Singh, D. and Brock, T. 1978a. Role of c y c l i c AMP i n . r a t a o r t i c microsomal phosphorylation and calcium,uptake. Am. J. P h y s i o l . , 234, H508-514. Bhalla, R.C., Webb, R.C, Singh, D., Brock, T. and Ashley, T. 1978b. Calcium fluxes, calcium binding, and adenosine c y c l i c 3', 5'-monophosphate-dependent protein kinase a c t i v i t y i n the aorta of spontaneously hypertensive and Kyoto Wistar normo- tensive r a t s . Molecular Pharmacology, 14_, 468-477. B i r n i e , G.D. 1972. Subcellular components-preparation and f r a c t i o n a t i o n . University Park Press Inc., Baltimore. Blaustein, M.P. 1977. Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis. Am. J . P h y s i o l . , 232, C165-173. Bogdanove, E.M. and Strash, A.M. 1975. Radioiodine escape i s an unexpected source of radioimmunoassay error and chronic low l e v e l environmental contamination. Nature (London), 257, 426-427. Bonner, W.D. 1955. Succinic dehydrogenase. In " Methods In Enzymology. " Vol. 1. Edited by S.P.Colowick and N.O.Kaplan. Academic Press Inc., New York. pp. 722-729. Brunner, G., Ferber, E. and Resch, K. 1977. F r a c t i o n a t i o n of membrane v e s i c l e s . Analyt. Biochem., 80, 420-429. Burger, R.M. and Lowenstein, J.M. 1975. 5' Nucleotidase from smooth muscle of the small i n t e s t i n e and from brain. I n h i b i t i o n by nucleotides. Biochemistry, 14,:'.2362-2366. Calhoun, M.L. 1954. Microscopic anatomy of the digestive system of the chicken. Iowa State College Press, Ames, Iowa. Cantley J r . , L.C., Josephson, L., Warner, R., Yanagisawa, M., Lechine, C. and G u i d a t t i , G. 1977. Vanadate i s a potent (Na, K)-ATPase i n h i b i t o r found i n ATP derived from muscle. J. b i o l . Chem., 252, 7421-7423. C a r a f o l i , E. and Crompton, M. 1978.. The regulation of i n t r a c e l l u l a r calcium. In " Current topics i n membranes and transport. " V o l . 10. Edited by F.Bonner and A . K l e i n z e l l e r . Academic Press Inc., New York. pp. 151-216. 152 - 152 - Cha, Y.N. and Soo-Lee, K. 1976. Phosphorylation of the red blood c e l l membrane during the act i v e transport of C a + 2 , j . gen. Physio., 67, 251-261. Chaturvedi, A.K., Landon, E.J. and Sastry, B.V.R. 1978. Influence of 6- (N,N-«diethylamino) hexyl 3, 4, 5-trimethoxybenzoate on the responsiveness of aorta to norepinephrine and calcium movements i n microsomes from spontaneously hypertensive and normotensive r a t s . Pharmacology, 17, 315-322. Clyman, R.I., Manganiello, V.C., Lovell-Smith, C.J. and Vaughan, M. 1976. Calcium uptake by s u b c e l l u l a r f r a c t i o n s of human umb i l i c a l artery. Am. J . Physiol., 231, 1074-1081. Coleman, R., Holdsworth, G. and Finean, J.B. 1976. Detergent extraction of erythrocyte ghosts. Comparison of residues a f t e r cholate and t r i t o n X-100 treatments. Biochim. biophys. Acta, 436, 38-44. Cuatrecasas, P. 1973. Interaction of wheat germ a g g l u t i n i n and concanavalin A w i t h l i s o l a t e d f a t c e l l s . Biochemistry, 1_2, 1312-1322. Daniel, E.E. and Janis, R.A. 1975. Calcium regulation i n the uterus. Pharmac. Therap. B., 1^, 695-729. Dodge, J.T., M i t c h e l l , C. and Hanahan, D.J. 1963. The preparation and chemical c h a r a c t e r i s t i c s of hemoglobin-free ghosts of human erythrocytes. Archs. Bioc. Biophys., 100, 119-130. Donaldson, R.P., Tolbert, N.E. and Schnarrenberger, C. 1972. A comparison of microbody membranes with microsomes and mitochondria from plant and animal t i s s u e . Archs. Bioc. Biophys., 152, 199-215. Fairbanks, G., Steck, T.L. and Wallach, D.F.H. 1971a. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry, 10, 2606-2617. Fairbanks, G., Steck, T.L. and Wallach, D.F.H. 1971b. D i s p o s i t i o n of the major proteins i n the i s o l a t e d erythrocyte membrane. P r o t e o l y t i c digestion. Biochemistry, 1_0, 2617-2624. Fairbanks J r . , G. Levinthal, C. and Reider, R.H. 1965. Analysis of l^C l a b e l l e d proteins by disc electrophoresis. Biochem. biophs. Res. Commun. , 2JD, 393-399. 153 - 153 - Fay, F.S. and Delise, CM. 1973. Contraction of i s o l a t e d smooth muscle c e l l s - s t r u c t u r a l changes. Proc. natn. Acad. S c i . U.S.A., 70, 641-645. Fay, F.S. and Singer, J . J . 1977. C h a r a c t e r i s t i c s of response of is o l a t e d smooth muscle c e l l s to cho l i n e r g i c drugs. Am. J. Phys i o l . , 232, C144- 154. F i t z p a t r i c k , D.F. and Szentivanyi, A. 1977. Stimulation of calcium uptake into a o r t i c microsomes by c y c l i c AMP and c y c l i c AMP dependent protein kinase. Naunyn-Schmiedeberg's Arch. Pharmac, 298, 255-257. F l e i s c h e r , S. and F l e i s c h e r , B. 1967. Removal and binding of polar l i p i d s i n mitochondria and other membrane systems. In " Methods In Enzymology. " Vol. 10. Edited by R.W.Estabrook and M.E.Pullman. Academic Press Inc., New York. pp. 406-433. F l e i s c h e r , S. and Kervina, M. 1974. Subcellular f r a c t i o n a t i o n of r a t l i v e r . In " Methods In Enzymology. " V o l . 31. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York, pp. 6-41. Gerhardt, B. and Beevers, H. 1968. Influence of sucrose on protein determination by the Lowry procedure. Arialyt. Biochem. , 24_, 337-339. Godfraind, T and Verbeke, N. 1973. The action of adrenalin on calcium pump and adenosine triphosphatases a c t i v i t i e s of i n t e s t i n a l smooth muscle microsomes. Arch. i n t . Pharmacodyn., 204, 187-190. Godfraind, T., Morel , N. and Verbeke, N. 1977. Microsomal and mitochondrial calcium pumps i n smooth muscle. In " E x c i t a t i o n contraction coupling i n smooth muscle. " Edited by R.Casteels, T.Godfraind and L.C.Rugg. E l s e v i e r , Amsterdam, pp. 219-224. Godfraind, T., Verbeke, N. and Sturbois, X. 1976. Calcium incorporation by smooth muscle microsomes. Biochim. biophys. Acta, 455, 254-268. Graham, J.M. 1972. I s o l a t i o n and ch a r a c t e r i z a t i o n of membranes from normal and transformed t i s s u e - c u l t u r e c e l l s . Biochem. J. , 130, 1113-1124. 154 - 154 - Graham, J.M. 1975. C e l l u l a r membrane f r a c t i o n a t i o n . In " New techniques i n biophysics of c e l l biology. " V o l . 2. Edited by R.M.Pain and B.J.Smith. J. Wiley and Sons Ltd., New York, pp. 1-42. Hannig, K. and Heidrich, H-G. 1974. The use of continuous prepara- t i v e free flow electrophoresis for d i s s o c i a t i n g c e l l f r a c t i o n s , and i s o l a t i o n of membrane components. In " Methods In Enzymology. " Vol. 31. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 746-761. Hannig, K., Wirth,H., Meyer, B-H. and Z e i l l e r , K. 1975a. Free flow electrophoresis I. Hoppe-Seylers Z. p h y s i o l . Chem., 356, S1209-1223. Hannig, K., Wirth, H., Meyer, B-H. and Z e i l l e r , K. 1975b. Free flow electrophoresis I I . Hoppe-Seylers Z. p h y s i o l . Chem., 356, S1225-1244. Hartig, P.R. and Raftery, M.A. 1977. Lactoperoxidase catalyzed membrane surface l a b e l l i n g of the acetylcholine receptor from Torpedo C a l i f o r n i c a . Biochem. biophys. Res. Commun., 78, 16-22. Ha t e f i , Y. and Rieske, J.S. 1967. The preparation and properties of DPNH-cytochrome\c reductase (ComplexI-III of the res p i r a t o r y chain). In " Methods In Enzymology. " V o l . 10. Edited, by R.N.Estabrook and M.E.Pullman. Academic Press Inc., New York. pp. 225-231. Helenius, A. and Simons, K. 1975. S o l u b i l i z a t i o n of membranes by detergents. Bichim. biophys. Acta, 415, 29-79. Hess, M.L. and Ford, G.D. 1974. Calcium accumulation by subcel l u l a r f r a c t i o n s from vascular smooth muscle. J . molec. c e l l , c a r d i o l . , 6_, 275-282. Hodges, T.K. and Leonard, R.T. 1974. P u r i f i c a t i o n of a plasma membrane bound adeosine triphosphatase from plant roots. In " Methods In Enzymology. " V o l . 32. Edited by s.Fleischer and L.Packer. Academic Press Inc., New York. pp. 392-406. Hubscher, G. and West, G.R. 1965. S p e c i f i c assays of some phosphatases i n su b c e l l u l a r f r a c t i o n s of small i n t e s t i n a l mucosa. Nature (London), 205, 799-800. 155 - 155 - Hudglns, P.M. and Bond, G.H. 1977. (Mg2+ + K+) - Dependent i n h i b i t i o n of Na,K-ATpase due to a contaminant i n equine muscle ATP. Biochem. biophys. Res. Commun. , _77_> 1024-1029. Hunter, M.J. and Commerford, S.L. 1961. Pressure homogenization of mammalian ti s s u e s . Biochim. biophys. Acta., 47, 580-589. Hurwitz,L., F i t z p a t r i c k , D.F. , Debbas, G. and Landon, E.J. 1973. L o c a l i z a t i o n of calcium pump a c t i v i t y i n smooth muscle. Science, 179, 384-386. Hynes, R.O. 1976. Su r f a c e . l a b e l l i n g techniques for eukaryotic c e l l s . In "New techniques i n biophysics and c e l l biology. " Vol. 3. Edited by R.H.Pain and B.H.Smith. J . Wiley and Sons Ltd., New York. pp. 147-212. Janis, R.A. , Crankshaw, D.J. and Daniel, E!.E. 1977. Control of ' ' • 2+ i n t r a c e l l u l a r Ca a c t i v i t y i n r a t myometrium. Am. J. Phy s i o l . , 232, C50-58. Jorgensen, P.L. 1974. P u r i f i c a t i o n and ch a r a c t e r i z a t i o n of (Na +/K* ATPase) I I I . Biochim. biophys. Acta., 356, 36-52. Josephson, L. and.Cantley J r . , L.C. 1977. I s o l a t i o n of a potent (Na-K) ATPase i n h i b i t o r from s t r i a t e d muscle. Biochemistry, '16, 4572-4578. Kahlenberg, A. 1976. P u r i f i c a t i o n of a membrane protein from human erythrocytes involved i n glucose transport. J . b i o l . Chem., 251, 1582-1590. Karnovsky, M.J. 1969. Morphology of c a p i l l a r i e s with s p e c i a l reference to muscle:;capillaries. In " Proceedings of the A l f r e d Bensen Symposium II". " Edited by C.Crone and N.Lassen. Copenhagen, pp. 1'341-350. 2+ Katz, S. and Bl o s t e i n , R. 1975. Ca Stimulated membrane phosphorylat- ion of the human erythrocyte. Biochim. biophys. Acta., 389, 314-324. Kelberg, M.S. and Christensen, H.N. 1979. Electron t r a n s f e r r i n g enzymes i n the plasma membranes of the E h r l i c h a s c i t e s tumour c e l l . Biochemistry, 18^, 1525-1535. Kidwai, A.M. 1974. I s o l a t i o n of plasma membranes from smooth, s k e l e t a l and heart muscle. In " Methods In Enzymology. " Vol. 31. Edited by S.Fleischer and L.Packer. Academic Press Inc,, New York. pp. 134-144. 156 - 156 - King, A.S. and McLelland, J . 1975. Outlines of avian anatomy. B a l l i e r e T i n d a l l , London. Knauf, P.A., Proverbio, F. and Hoffman, J.F. 1974. Electrophoretic separation of d i f f e r e n t phosphoproteins associated with Ca- ATPase and Na,K-ATPase i n human red c e l l ghosts. J . gen. P h y s i o l . , 63, 324-336. K r a l l , J.F., Schindler, A.M. and Korenman, S.G. 1978. Myometrial protein kinase .'hormone stimulated t r a n s l o c a t i o n and membrane binding of the soluble enzyme. Archs. Bioc. Biophys., 187, 1-11. Kutsky, P. and Goodman, F.R. 1978. Calcium incorporation by canine a o r t i c smooth muscle microsomes. Arch. Int. Pharmacodyn., 231, 4-20. Laemmli, U.K. and Favre, M. 1973. Maturation of the head of bacteriophage T4. J . molec. B i o l . , 80, 575-599. Lane, L.K. 1976. Gel chromatography and g e l electrophoresis of cardiac glycoside - Na,K-ATpase complexes i n sodium dodecyl sulphate:a caution. FEBS L e t t e r s , 64, 375-379. Leinen, J.G. and W i t t l i f f , J.L. 1978. Determination of r a d i o a c t i v i t y i n polyacrylamide.gel s l i c e s mounted on s o l i d supports. Analyt. Biochem., 86, 279-286. Levitsky, D.O., A l i e v , M.K., Kuzmin, A.V., Levenko, T.S., Smirnov, V.N. and Chazov, E.I. 1976. I s o l a t i o n of calcium pump system and p u r i f i c a t i o n of calcium ion-dependent ATPase from heart muscle. Biochim. biophys. Acta., 443, 468-484. Lewis, B.A., Elken, A., M i c h e l l , R.H. and Coleman, R. 1975. I d e n t i f i c a t i o n by lactoperoxidase catalyzed i o d i n a t i o n and i s o l a t i o n of plasma membranes a f t e r d e s i t y perturbation with d i g i t o n i n . Biochem. J . , 152, 71-84. Lim, R.W., Molday, R.S., Huang, H.V. and Yen, S.P.S. 1975. A p p l i c a t i o n of latex microspheres i n the i s o l a t i o n of plasma membranes. A f f i n i t y density perturbation of erythrocyte membranes. Biochim. biophys. Acta., 394, 377- 387. 157 - 157 - Lowry, O.H., Rosebrough, N.J. , Farr, A.L. and Randall, R.J. 1951. Protein measurement with the f o l i n phenol reagent. J. b i o l . Chem., 193, 265-275. McCollester, D.L. 1962. A method f o r i s o l a t i n g s k e l e t a l muscle c e l l membrane components. Biochim. biophys. Acta., 57, 427-437. McLeod, W.M., T r o t t e r , D.M. and Lumb, J.W. 1964. Avian anatomy. Burgess Publishing Company, Minneapolis. Magargal, W.W., Dickinson, E.S. and Slakey, L.L. 1978. D i s t r i b u t i o n of membrane marker enzymes i n cultured a r t e r i a l e ndothelial and smooth muscle c e l l s . J . b i o l . Chem., 253, 8311-8318. Matlib, M.A., Crankshaw, J . , G a r f i e l d , R.E., Crankshaw, D.J., Kwan, C-Y., Branda, L.A. and Daniel, E.E. 1979. Characterization of membrane f r a c t i o n s and i s o l a t i o n of p u r i f i e d plasma membranes from r a t myometrium. J. b i o l . Chem., 254, 1834- 1840. Moore, L., Hurwitz, L., Davenport, G.R. and Landon, E.J. 1975. Energy dependent calcium uptake a c t i v i t y of microsomes from the aorta of normal and hypertensive r a t s . Biochim. biophys. Acta.., 413, 432-443. Morrison, M. 1970. Iodination of tyrosine. I s o l a t i o n of lactoperoxidase (bovine). In " Methods In Enzymology. " Vol. 17. Edited by H.Tabour and C.W.Tabour. Academic Press Inc., New York. pp. 653-657. Morrison, M. 1974. The dtermination of the exposed proteins on membranes by the use of lactoperoxidase. In " Methods In Enzymology. " Vol. 32. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 103-109. Morrison, M. and Bayse, G.S. 1970. C a t a l y s i s of i o d i n a t i o n by lactoperoxidase. Biochemistry, 9_, 2995-3000. Morrison, M. and Schonbaum, G.R. 1976. Peroxidase-catalyzed halogenation. A. Rev. Biochem., 4_5, 861-888. Murthy, R.J. and Hercz, A. 1973. P u r i f i c a t i o n of human a n t i t r y p s i n by a f f i n i t y chromatography on Sepharose bound concanavalin A. FEBS L e t t e r s , 3_2, 243-246. 158 - 158 - Nagano, K., Kanazawa, T., Mizuno, N., Tashlma, Y., Nakao, T. and Nakao, M. 1965. Some ac y l phosphate l i k e properties of 32p-labelled sodium - potassium - activated adenosine triphosphatase. Biochem. biophys. Res. Gommun., 19, 759- 764. N e v i l l e , D.M. 1975. I s o l a t i o n of c e l l surface membrane f r a c t i o n s from.mammalian c e l l s and organs. In " Plasma membranes. " Vol. 3. Edited by E.D.Korn. Plenum Press Inc., New York, pp. 1-49. N e v i l l e J r . , D.M. and Glossman, H. 1974. Molecular weight determination of membrane protein and glycoprotein subunits by discontinuous gel.electrophoresis i n dodecyl sulphate. In " Methods In Enzymology. " V o l . 32. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 92-102. Nicholson, G.L. 1974. The i n t e r a c t i o n s of l e c t i n s with animal c e l l surfaces. Int. Review Cyt o l . , 39., 89-190. Nilsson, K.B., Andersson, R.G.G., Borjesson, S., Mackerlova, L., Mohme-Lundholm, E. and Lundholm, L. 1977. Ca2+ binding, c y c l i c nucleotide metabolism and membrane phosphorylation of s u b c e l l u l a r structures from rabbit i n t e s t i n e . In " Exc i t a t i o n - c o n t r a c t i o n coupling i n smooth muscle. " Edited by R.Casteels, T.Godfraind and L-C.Ruegg. E l s e v i e r , Amsterdam, pp. 189-197. N i s h i k o r i , K., Takenaka, T. and Maeno, H. 1977. Stimulation of microsomal calcium uptake and protein phosphorylation by adenosine c y c l i c 315'-monophosphate i n r a t uterus. Molecular Pharmacology, 13, 671-678. Nordlie, R.C. and Arion, W.J. 1966. Glucose-6-phosphatase. In " Methods In Enzymology. " Vol. 9. Edited by W.A.Woods. Academic Press Inc., New York. pp. 619-625. Peters, T.J., Muller, M. and deDuvre, C. 1972. Lysosmoes of the a r t e r i a l w a l l . J. Exp. Med., 136, 1117-1128. Pharmacia Ltd. 1974. Pharmacia a f f i n i t y chromatography c i r c u l a r . Upsula, Sweden. Pre i s s , R. and Banaschak, H. 1975. Demonstration of a Mg-ATPase and a Na,K-ATPase from the a r t e r i a c a r o t i s communis of the sheep. Acta. b i o l . med. germ., 34, 37-44. 159 - 159 - Price , C A . 1974. Plant c e l l f r a c t i o n a t i o n . In " Methods In Enzymology. " V o l . .31. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 501-519. Raeymaekers,. L., Wuytack, F. and Casteels, R. 1977 . Comparison of the calcium accumulation by d i f f e r e n t s u b c e l l u l a r f r a c t i o n s of smooth muscle. In " Exc i t a t i o n - c o n t r a c t i o n coupling i n smooth muscle. " Edited by R.Casteels, T.Godfraind and L-C.Ruegg. E l s e v i e r , Amsterdam, pp. 233- 238. Rangachari, P.K., Pernollet, M-G. and Worcel, M. 1976. Calcium uptake by myometrial membranes. E f f e c t of A-23187, a calcium lonophore. Europ. J. Pharmacol., 40, 291-294. Robson, R.M., G o l l , D.E. and Temple, M.J. 1968. Determination of proteins i n " t r i s " buffer by the biuret reaction. Analyt. Biochem., _24, 339-341. Rodbell, M. 1964. Metabolism of i s o l a t e d f a t c e l l s . J. b i o l . Chem., 239, 375-380. Rodbell, M. and Krishna, G. 1974. Preparation of i s o l a t e d f a t c e l l s and f a t c e l l ghosts. In " Methods In Enzymology. " Vol. 31. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 103-114. * Rombauts, W.A., Schroeder, W.A. and Morrison, M. 1967. Bovine lactoperoxidase. P a r t i a l c h a r a c t e r i z a t i o n of the further p u r i f i e d p r o t e i n . Biochemistry, 6_, 2965-2977. Schapira, G. , Dobocz, I., Piau, J..P. and Delain, E. 1974. An improved technique for preparation of s k e l e t a l muscle c e l l plasma membrane. Biochim. biophys. Acta., 345, 348-358. Schied, C.R., Honeyman, T.W. and Fay, F.S. 1979. Mechanism of - adrenergic r e l a x a t i o n of smooth muscle. Nature ( London), 277, 32-36. Schimmel, S.D. and Kent, C. 1977. I s o l a t i o n of plasma membranes from cultured muscle c e l l s . In " Methods In C e l l Biology. " Vol. 15. Edited by D.M.Prescott. Academic Press Inc., New York. pp. 290-302. Sharon, N. and L i s , H. 1975. Use of l e c t i n s f o r the study of membranes. In "Plasma membranes. " V o l . 3. Edited by E.D.Korn. Plenum Press Inc., New York. pp. 147-200. 160 - 160 - Shibata, N. and Hollander, W. 1974. Studies on the r o l e of a r t e r i a l microsomes i n the c o n t r a c t i l e function of the a r t e r i e s . Expl. Molec. Path., 21_, 1-15. Small, J.V. 1977. Studies on i s o l a t e d smooth muscle c e l l s : the c o n t r a c t i l e apparatus. J . C e l l Science, 24, 327-349. 2+ Smolen, J.E. and Weissmann, G. 1978. Mg - ATPase as-a membrane ecto enzyme of human i n h i b i t o r s , a c t i v a t o r s and response to phagocytosis. Biochim. biophys. Acta., 512, 525-538. Sobieszek, A. and Small, J.V. 1976. Myosin linked calcium regulation i n vertebrate smooth nuscle. J. molec. B i o l . , 101, 75-92. Sottocasa, G.L. 1971. S i m i l a r i t i e s and d i s - s i m i l a r i t i e s between outer mitochondrial membrane and endoplasmic reticulum. Adv. explt. Med. B i o l . , 14-, 229-244. Sottocasa, G.L., Kuylenstierna, B., Ernster, L. and Bergstrand, A. 1967. Separations and enzymatic properties of the inner and outer membranes of rat. l i v e r mitochondria. In " Methods In Enzymology. " Vol. 10. Edited by R.W.Estabrook and M.E. Pullman. Academic Press Inc., New York. pp. 448-463. Steck, T.L. 1974a. Preparation of impermeable 10 and RO v e s i c l e s from erythrocyte membranes. In " Methods In Membrane Biology. " V o l . 3. Edited by E.D.Korn. Plenum Press Inc. , New York. pp. 245-282. Steck, T.L. 1974b. The organization of proteins i n the human red blood c e l l membrane. J. c e l l B i o l . , 62̂ , 1-19. Steck, T.L. and Kant, J.A. 1974. Preparation of impermeable ghosts and inside-out v e s i c l e s from human erythrocyte membranes. In " Methods In Enzymology. " Vol. 31. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 172-180. Steck, T.L. and Yu, J . 1973. S e l e c t i v e s o l u b i l i z a t i o n of proteins from red blood c e l l membranes by protein perturbants. J . Supramolecular Structure, 220-248. Tada, M., Kirchberger, M.A. and Katz, A.M. 1975. Phosphorylation of a 22,000-dalton component of the cardie sarcoplasmic reticulum by adenosine.3':5' - monophosphate dependent protein kinase. J . b i o l . Chem., 250, 2640-2647. 161 - 161 - Tanford, C. and Reynolds, J.A. 1976. Characterization of membrane proteins i n detergent s o l u t i o n s . Biochim. biophys. Acta., 457, 133-170. 2+ Thorens, S. 1979. Ca ATPase and Ca uptake without requirement for Mg2+ i n membrane f r a c t i o n s of vascular smooth muscle. FEBS L e t t e r s , 98, 177-180. Thorens, S. and Haeusler, G. 1978. E f f e c t s of adenosine 3':5'- monophosphate and quanosine 3':5'-monophosphate on calcium ". '.: uptake, and phosphorylation i n membrane f r a c t i o n s of vascular smooth muscle. Biochim. biophys. Acta., 512, 415-428. Tolbert, N.E. 1974. I s o l a t i o n of su b c e l l u l a r organelles of metabolism on isopycnic sucrose gradients. In " Methods In Enzymology. " V o l . 31. Edited by S.Fleischer and L. Packer. Academic Press Inc., New York. pp. 734-746. V a l l i e r e s , J . , F o r t i e r , M., Somlyo, A.V. and Somlyo, A.P. 1978. I s o l a t i o n of plasma membranes from rabbit myometrium. Int. J . Biochem., 9_, 487-498. Wallach, D.F.H. and Schmidt-Ullrich, R. 1977. I s o l a t i o n of plasma membrane v e s i c l e s from animal c e l l s . In " Methods In C e l l Biology. " Vol. 15. Edited by D.M.Prescott. Academic Press Inc., New York. pp. 235-276. Wallach, D.F.H. and. Winzler, R.J. 1974. Evoloving s t r a t i g i e s and t a c t i c s i n membrane research. Springer-Verlag New York Inc., New York. Walsh, F.S. Barber, B.H. and Crumpton, M.J. 1976. Preparation of inside-out v e s i c l e s of pig lymphocyte plasma membrane. Biochemistry, 1_5, 3557-3562. Walter, H. 1978. C e l l p a r t i t i o n i n g i n two-polymer aqueous phase systems. Trends In Biochem. S c i . , 3_, 97-100. Walter, H. and Krob, E.J. 1976. Hydrophobic a f f i n i t y p a r t i t i o n i n aqueous two phase systems containing glycol-palmitate of right-side-out and inside-out v e s i c l e s from human erythrocyte membranes. FEBS Let t e r s , 61_, 290-293. Warren, L. 1955. Thiobarbituric acid assay of s i a l i c acids. In " Methods In Enzymology. " V o l . 1. Edited by S.P.Colowick and N.O.Kaplan. Academic Press Inc., New York. pp. 463- 465. 162 - 162 - Warren, L. 1959. The thiobarburic a c i d assay of s i a l i c acids. J . b i o l . Chem., 234, 1971-1975. Webb,, R.C. and Bhalla, R.C. 1976. Calcium sequestration by subcellular, f r a c t i o n s . i s o l a t e d from vascular smooth muscle: e f f e c t of c y c l i c nucleotides and prostaglandins. J . molec. c e l l . C a r d i o l . , 8_,' 145-157. Wei, J-W., Janis, R.A. and Daniel, E.E. 1976a. Calcium accumulation and enzymatic a c t i v i t i e s , s u b c e l l u l a r f r a c t i o n s from aortas and v e n t r i c l e s of g e n e t i c a l l y hypertensive r a t s . C i r c u l a t i o n Res., 39, 133-140. Wei, J-W., Janis, R.A. and Daniel, E.E. 1976b. I s o l a t i o n and c h a r a c t e r i z a t i o n of plasma membrane from rat mesenteric a r t e r i e s . Blood Vessels, JU3, 279-292. Wei, J-W., Janis, R.A. and Daniel, E.E. 1976c. Studies on s u b c e l l u l a r f r a c t i o n s from mesenteric a r t e r i e s of spontaneously hypertensive r a t s a l t e r a t i o n s i n both calcium uptake and enzyme a c t i v i t i e s . Blood Vessels, 13_, 293-308. Widnell, C.C. 1974. P u r i f i c a t i o n of f a t l i v e r 5' nucleotidase as a complex with sphingomyelin. In " Methods In Enzymology. " V o l . 32. Edited by S.Fleischer and L.Packer. Academic Press Inc., New York. pp. 368-374. Wuttack, F., Landon, E., F l e i s c h e r , S. and Hardman, J.G. 1978. Calcium accumulation i n a microsomal f r a c t i o n from porcine coronary artery smooth muscle. Biochim. biophys. Acta., 540, 253-269. Zacharius, R.M. and Z e l l , T.E. 1969. Glycoprotein s t a i n i n g following electrophoresis on acrylamide gels. Analyt. Biochem., 30, 148-152. Zelck, U., Karnstedt, U. and Albrecht, E. 1975. Calcium uptake and calcium, release by s u b c e l l u a l r f r a c t i o n s of smooth muscle.. Acta. bio. med. germ., 34_, 981-991.

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
Japan 5 0
United States 1 0
City Views Downloads
Tokyo 5 0
Ashburn 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items