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A fluorescence study of horse plasma gelsolin labelled with 6-acryloyl-2-dimethylaminonapthalene Reid, Scott William 1990

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A FLUORESCENCE STUDY OF HORSE PLASMA GELSOLIN LABELLED WITH 6-ACRYL0YL-2-DIMETHYLAMIN0NAPTHALENE By SCOTT WILLIAM REID B.Sc. Robert Gordon's I n s t i t u t e of Technology, 1988 A THESIS SUBMITTED IN OF THE REQUIREMENTS MASTER OF PARTIAL FULFILLMENT FOR THE DEGREE OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA NOVEMBER 1990 ©Scott W i l l i a m Reid In presenting this thesis in partial fulfilment of the requirements for an advanced « degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i Abstract G e l s o l i n was l a b e l l e d w i t h the s u l p h y d r y l - s p e c i f i c f l u o r e s c e n t reagent 6-acryloyl-2-dimethylaminonaphthalene ( a c r y l o d a n ) . The degree of l a b e l l i n g using non-denaturing c o n d i t i o n s was 1.9 ± 0.5 acrylodans per g e l s o l i n molecule. C i r c u l a r dichroism and v i s c o s i t y s t u d i e s showed no s i g n i f i c a n t e f f e c t on g e l s o l i n s t r u c t u r e and f u n c t i o n on i n c o r p o r a t i o n of the l a b e l . 2+ C i r c u l a r dichroism s t u d i e s d i d not detect Ca e f f e c t s on a e r y l o d a n - l a b e l l e d g e l s o l i n , but fl u o r e s c e n c e s t u d i e s 2+ detected s u b t l e changes i n the p r o t e i n . The presence of Ca causes a decrease and r e d - s h i f t i n fl u o r e s c e n c e emission, an increase i n s e n s i t i v i t y t o quenching by I" and a decrease i n f l u o r e s c e n c e p o l a r i s a t i o n of the a c r y l o d a n - l a b e l l e d g e l s o l i n . These i n d i c a t e an increased degree of exposure of the f l u o r e s c e n t l a b e l t o the s o l v e n t environment on i n t e r a c t i o n of g e l s o l i n with C a 2 + . A c t i n b i n d i n g t o g e l s o l i n was evident from a decrease i n fl u o r e s c e n c e i n t e n s i t y , an increase i n s e n s i t i v i t y t o quenching and an increase i n fl u o r e s c e n c e p o l a r i s a t i o n . A c t i n b i n d i n g i n c r e a s e s the exposure of the acrylodan l a b e l 2+ t o s o l v e n t , as does Ca b i n d i n g . i i i Table of Contents Page Abst r a c t i i L i s t of Tables v i L i s t of Fig u r e s v i i Acknowledgements i x Abbr e v i a t i o n s x 1= I n t r o d u c t i o n  P a r t 1= P r o t e i n s 1.1.1 I n t r o d u c t i o n t o A c t i n 1 1.1.2 P o l y m e r i s a t i o n of A c t i n 2 1.1.3 L o c a t i o n of A c t i n 3 1.1.4 A c t i n - b i n d i n g P r o t e i n s 6 1.1.5 I n t r o d u c t i o n t o G e l s o l i n 7 1.1.6 I n t e r a c t i o n s of G e l s o l i n w i t h A c t i n 8 1.1.7 G e l s o l i n and the Cytoskeleton 10 1.1.8 G e l s o l i n i n Plasma 12 Part 2: O p t i c a l Techniques 1.2.1 Fluorescence Spectroscopy 14 1.2.2 Fluorophores 16 iv 1.2.3 Environmental E f f e c t s 17 1.2.3.1 Solvent R e l a x a t i o n 17 1.2.3.2 Quenching 19 1.2.4 Fluorescence P o l a r i s a t i o n 19 1.2.5 C i r c u l a r Dichroism 21 2: M a t e r i a l s and Methods P a r t 1: P r o t e i n s 2.1 P u r i f i c a t i o n of A c t i n 24 2.2.1 P u r i f i c a t i o n of G e l s o l i n 25 2.2.2 Determination of P u r i t y 27 2.2.3 Determination of A c t i v i t y 27 2.2.4 Fluorescent L a b e l l i n g of G e l s o l i n 27 2.2.5 Degree of L a b e l l i n g 28 2.2.6 Summary of G e l s o l i n P u r i f i c a t i o n 30 Pa r t 2: O p t i c a l Techniques 2.3.1 Absorbance Measurements 31 2.3.2 Fluorescence Measurements 31 2.3.3 C i r c u l a r Dichroism 31 3: R e s u l t s and D i s c u s s i o n P a r t 1: E f f e c t of Ca 2 + on ACRG 3.1.1 L a b e l l i n g G e l s o l i n w i t h Acrylodan 33 3.1.2 CD Studies on ACRG 35 2+ 3.1.3 E f f e c t of Ca on the Emission Spectrum of ACRG 37 3.1.4 Fluorescence Quenching Studies on ACRG 44 V 3.1 5 Fluorescence P o l a r i s a t i o n S t udies on ACRG 47 Pa r t 2: A c t i n B i n d i n g t o ACRG 3.2.1 E f f e c t of A c t i n on Emission Maximum of ACRG 48 3.2.2 Quenching Studies of A c t i n - G e l s o l i n Complexes 51 3.2.3 A c t i n T i t r a t i o n P o l a r i s a t i o n S t u d i e s 53 Pa r t 3: Conclusion 3.3.1 Summary of R e s u l t s 55 3.3.2 Suggestion f o r Further Study 56 References 57 v i L i s t of Tables Table Page I V i s c o s i t y Measurements on ACRG 34 I I Iodide Quenching of ACRG-Actin Complexes 51 v i i L i s t of Figures 1. Schematic diagram of the h e l i c a l a r r a y of a c t i n monomers i n F - a c t i n 1 2. E l e c t r o n micrograph of f i l a m e n t s of F - a c t i n decorated w i t h p r o t e o l y t i c fragments of the c o n t r a c t i l e p r o t e i n myosin 2 3. Schematic diagram of the proposed model of a t h i n f i l a m e n t of v e r t e b r a t e s t r i a t e d muscle 4 4. Immunofluorescence micrograph of a c t i n f i l a m e n t s i n a r e s t i n g r a t f i b r o b l a s t 5 5. Summary of a c t i n - b i n d i n g p r o t e i n s and a schematic r e p r e s e n t a t i o n of t h e i r e f f e c t s on a c t i n f i l a m e n t s or a c t i n monomers 6 6. Schematic model t o show the i n t e r a c t i o n of human plasma g e l s o l i n w i t h a c t i n 9 7. Summary of d i f f e r e n t g e l s o l i n - a c t i n complexes as r e g u l a t e d by Ca and P I P 2 11 8. Schematic diagram f o r the process i n v o l v e d i n e l e c t r o n i c e x c i t a t i o n and d e - e x c i t a t i o n of f l u o r e s c e n t molecules 15 9. T h i o l - s p e c i f i c f l u o r e s c e n t probe, acrylodan 16 10. E x c i t a t i o n and emission s p e c t r a of acrylodan-l a b e l l e d g e l s o l i n 18 11. Diagramatic r e p r e s e n t a t i o n of e x c i t a t i o n of a f l u o r e s c e n t molecule w i t h v e r t i c a l l y p o l a r i s e d l i g h t and subsequent d e t e c t i o n of emission 20 12, CD s p e c t r a of ACRG ( l a b e l l e d i n the presence of excess EGTA) i n the presence and absence of Ca 36 13. Change i n normalized e l l i p t i c i t i e s of ACRG l a b e l l e d i n Ca , ACRG l a b e l l e d i n EGTA and u n l a b e l l e d g e l s o l i n . Ca present i n b u f f e r 38 14. Change i n normalized e l l i p t i c i t i e s of ACRG l a b e l l e d i n Ca , ACRG l a b e l l e d i n EGTA and u n l a b e l l e d g e l s o l i n . EGTA present i n b u f f e r 39 V l l l 15. Change i n normalized e l l i p t i c i t i e s of u n l a b e l l e d g e l s o l i n i n the presence and absence of Ca wi t h temperature 40 16. Change i n normalized e l l i p t i c i t i e s of ACRG ( l a b e l l e d i n the presence pf Ca ) i n the presence and absence of Ca w i t h temperature 41 17. M e l t i n g curve of ACRG ( l a b e l l e d i n absence of Ca 2 +) i n the presence and absence of Ca2+ 42 18. E f f e c t of calcium on the emission spectrum of ACRG 43 19. Stern-Volmer p l o t s f o r i o d i d e quenching of ACRG s o l u t i o n s . One sample i n the presence of Ca and another i n the absence of Ca 45 20. Stern-Volmer p l o t s f o r i o d i d e quenching of ACRG s o l u t i o n s . One sample has been denatured i n Gu-HCl and one sample i s i n t a c t ACRG 46 21. Fluorescence p o l a r i s a t i o n changes i n ACRG wit h temperature. One sample was i n the presence of Ca w h i l e the other was i n EGTA 47 22. Changes i n fluo r e s c e n c e i n t e n s i t y of ACRG on the b i n d i n g of a c t i n . One sample was i n a b u f f e r c o n t a i n i n g Ca , the other sample was i n EGTA. 49 23. Stern-Volmer p l o t s f o r i o d i d e quenching of a number of a c t i n - g e l s o l i n complexes. 52 24. Fluorescence p o l a r i s a t i o n of ACRG f o l l o w i n g the a d d i t i o n of monomeric a c t i n 54 ix Acknowledgement I would l i k e t o thank Dr L e s l i e D B u r t n i c k f o r h i s guidance and encouragement throughout t h i s p r o j e c t . I would a l s o l i k e t o thank Robert Robinson and Ian C l a r k f o r the i n t e r e s t they have shown i n t h i s p r o j e c t and f o r t h e i r p a r t i n the c o l l e c t i o n of horse plasma. X A b b r e v i a t i o n s Acrylodan 6-acryloyl-2-(dimethylamino)naphthalene ACRG Acrylodan l a b e l l e d G e l s o l i n ATP Adenosine 5'-Triphosphate CD C i r c u l a r Dichroism DEAE-Sephadex Diethylaminoethyl-Sephadex DBP Vitamin D-Binding P r o t e i n DMF A/,/'/-Dime thy I f ormamide DMSO Dimethylsulphoxide DTT D i t h i o t h r e i t o l EDTA E t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d (disodium s a l t ) EGTA Ethylenebis( o x y e t h y l e n e n i t r i l o ) -t e t r a a c e t i c a c i d Gu-HCl Guanidine Hydrochloride P I P 2 P h o s p h a t u d y l i n o s i t o l 4,5-bis-phosphate PMSF Phenylmethylsulphonyl F l u o r i d e SDS Sodium Dodecyl Sulphate TEMED N3N,N* ,W-Tetramethylethylenediamine T r i s Tris(hydroxymethyl)methylamine TM Tropomyosin TN Troponin B u f f e r A 2mM T r i s - H C l , 0.2mM CaCl2» 0.2mM ATP, l.OmM DTT, pH 8.0. Buf f e r I 25mM T r i s - H C l , ImM EGTA, ImM NaN 3, PH 8.0. 1 i _ INTRODUCTION PART l : PROTEINS JLU.JL Introduction to Actin A c t i n i s one of the most abundant p r o t e i n s i n nature, making up about 25% of a l l p r o t e i n i n muscle c e l l s and, t y p i c a l l y , 10 - 20% of a l l p r o t e i n i n e u k a r y o t i c non-muscle c e l l s . I t was f i r s t i s o l a t e d from muscle t i s s u e by Straub (1942). This p r o t e i n undergoes p o l y m e r i s a t i o n from the monomeric G-actin t o a lon g , h e l i c a l , double-stranded polymer, F - a c t i n ( f i g u r e 1 ) , under the i n f l u e n c e of p h y s i o l o g i c a l i o n i c s t r e n g t h (0.1M KC l ) or by the a d d i t i o n of d i v a l e n t c a t i o n s O x (2mM Mg ) (Tellam and Fr i e d e n , 1982). Each a c t i n monomer i s a 42 kDa gl o b u l a r p r o t e i n and filamentous F - a c t i n has a diameter of 70 A. The s t r u c t u r e repeats i t s e l f a t i n t e r v a l s of 360 A along the h e l i x a x i s . < —> 360 A F i g u r e 1: Schematic diagram of the h e l i c a l array of a c t i n monomers i n F - a c t i n ( S t r y e r , 1981). 2 An arrowhead p a t t e r n appears i n e l e c t r o n micrographs ( f i g u r e 2) of F - a c t i n f i l a m e n t s decorated with p r o t e o l y t i c fragments of the c o n t r a c t i l e p r o t e i n myosin, i n d i c a t i n g the p o l a r i t y of the f i l a m e n t . P B Figur e 2: E l e c t r o n micrograph of f i l a m e n t s of F - a c t i n decorated with p r o t e o l y t i c fragments of the c o n t r a c t i l e p r o t e i n myosin. The "pointed' and 'barbed' ends are represented by P and B, r e s p e c t i v e l y ( S t r y e r , 1981). 1.1.2 P o l y m e r i s a t i o n of A c t i n The p o l y m e r i s a t i o n of a c t i n p h y s i o l o g i c a l l y i s accompanied by, but does not r e q u i r e , h y d r o l y s i s of one molecule of bound ATP t o one molecule of bound ADP f o r every monomeric a c t i n s u b u n i t . There are two d i s t i n c t steps i n v o l v e d i n p o l y m e r i z a t i o n . F i r s t l y , n u c l e a t i o n of polymer growth r e q u i r e s formation of a t r i m e r ( K a s a i e t a l . , 1960). N u c l e a t i o n i s slow and i s the r a t e l i m i t i n g s t e p . Secondly, e l o n g a t i o n occurs when monomers bind t o the ends of the 3 n u c l e i t o form f i l a m e n t s . The a f f i n i t y and exchange r a t e of a c t i n monomers w i t h the "barbed' end are an order of magnitude higher than at the 'pointed' end. 1.1-3 L o c a t i o n of A c t i n Every c e l l i n our bodies possesses a c t i n f i l a m e n t s b u i l t i n t o a v a r i e t y of s u b c e l l u l a r machinery. The muscles w i t h which we move, the d e l i c a t e s t e r e o c i l i a we need t o hear and the m i c r o v i l l i t h a t absorb n u t r i e n t s from our l a s t meal a l l share the common element of the a c t i n f i l a m e n t (De R o s i e r , 1990). Vertebrate s t r i a t e d muscle i s composed of t h i n and t h i c k f i l a m e n t s which s l i d e past one another i n the process of muscle c o n t r a c t i o n . The t h i n f i l a m e n t ( f i g u r e 3) i s formed by a c t i n , tropomyosin (TM) and one each of the three t r o p o n i n components, (TN), w i t h a c t i n making up the backbone of the s t r u c t u r e . Along each of the two grooves i n the F-a c t i n h e l i x l i e s a TM f i l a m e n t , formed by h e a d - t o - t a i l p o l y m e r i z a t i o n of TM. Each TM spans seven a c t i n monomers and the TM f i l a m e n t extends the length of the t h i n f i l a m e n t . Troponin binds p e r i o d i c a l l y along the TM f i l a m e n t and i s a complex of three p o l y p e p t i d e s , TN-C ( c a l c i u m b i n d i n g ) , TN-I ( i n h i b i t o r y ) and TN-T (TM b i n d i n g ) . The TM + TN p r o t e i n s form the calcium s e n s i t i v e r e g u l a t o r y s w i t c h i n s t r i a t e d muscle. 4 TN-C ACTIN TM Figu r e 3= Schematic diagram of the proposed model of a t h i n f i l a m e n t of v e r t e b r a t e s t r i a t e d muscle. A c t i n makes up the h e l i c a l backbone of the s t r u c t u r e w i t h the TN complex c o n s i s t i n g of TN-C, TN-I and TN-T (Cote, 1983). The h i g h l y organised s t r u c t u r e of a c t i n i n s t r i a t e d muscle c o n t r a s t s w i t h the dynamic nature of a c t i n i n non-muscle c e l l s . A c t i n i s the major component of mic r o f i l a m e n t s found i n the c e l l u l a r c y t o s k e l e t o n . The c y t o s k e l e t o n i s a f i b r o u s matrix of p r o t e i n s which can span the cytoplasm of c e l l s between the nucleus and the inner s u r f a c e of the plasma membrane e s t a b l i s h i n g c e l l shape and m o t i l i t y . The c y t o s k e l e t o n o f t e n i s p a r t i c u l a r l y dense j u s t below and p a r a l l e l t o the c e l l ' s plasma membrane. There are three d i s t i n c t systems of f i l a m e n t s i n the c y t o s k e l e t o n : m i c r o f i l a m e n t s (6-7nm diameter), intermediate f i l a m e n t s ( 7 -5 llnm diameter) and microtubules (22nm diameter). Figure 4 shows an immunofluorescence micrograph of ac t i n filaments in a resting c e l l . By c o n t r o l l i n g the assembly, s t a b i l i t y and interactions of a c t i n filaments, c e l l s are able to regulate t h e i r shapes and positions, to shuttle t h e i r i n t e r n a l contents about and to exclude them from certain regions. Some of the diverse c e l l u l a r a c t i v i t i e s that involve a c t i n include cytoplasmic streaming, phagocytosis, c e l l d i v i s i o n , the regulation of the d i s t r i b u t i o n of certa i n membrane proteins and functional interconnection of the c e l l membrane with the nucleus (Korn, 1978). Figure A- Immunofluorescence micrograph of a c t i n filaments in a resting rat f i b r o b l a s t (Stryer, 1981) 6 Non-muscle and muscle a c t i n s c l o s e l y resemble each other i n chemical composition, p h y s i c a l p r o p e r t i e s and p h y s i o l o g i c a l f u n c t i o n (Korn, 1978). 1.1.4 A c t i n - b i n d i n g P r o t e i n s The formation of a c t i n assemblies and t h e i r d i v e r s i t y , which a l l o w many d i f f e r e n t f u n c t i o n s , depends on the a c t i o n s of a l a r g e r e p e r t o i r e of a c t i n - b i n d i n g p r o t e i n s . These p r o t e i n s are named according t o the a c t i o n they have on a c t i n and are summarised i n f i g u r e 5. Figur e 5= Summary of a c t i n - b i n d i n g p r o t e i n s and a schematic r e p r e s e n t a t i o n of t h e i r e f f e c t s on a c t i n f i l a m e n t s or a c t i n monomers ( P o l l a r d and C r a i g , 1982) 7 1.1.5. I n t r o d u c t i o n t o G e l s o l i n G e l s o l i n i s r e p r e s e n t a t i v e of a c l a s s of actin-modulating p r o t e i n s o c c u r r i n g w i d e l y , from lower eukaryotes t o mammals, which have powerful e f f e c t s on the length of a c t i n f i l a m e n t s ( S t o s s e l e t a l . , 1985). I t was f i r s t discovered i n r a b b i t lung macrophages and sera by Y i n and S t o s s e l i n 1979. I t was o r i g i n a l l y c a l l e d " a c t i n depolymerizing p r o t e i n ' (ADP) and 'b r e v i n ' (from the L a t i n brevis, s h o r t ) a f t e r i t was found t o shorten r a t h e r than depolymerize a c t i n f i l a m e n t s . The name g e l s o l i n comes from i t s a b i l i t y t o soJate a c t i n f i l a m e n t gels (Noberg et a l . , 1979). I t i s unusual among mammalian p r o t e i n s i n th a t one form i s produced f o r i n t r i n s i c i n t r a c e l l u l a r use and another i s made s p e c i f i c a l l y f o r s e c r e t i o n ( Y i n et a l . , 1984). Cytoplasmic and plasma g e l s o l i n s are both g l o b u l a r p r o t e i n s , w i t h apparent molecular masses of about 90 kDa and 93 kDa, r e s p e c t i v e l y , as assessed by polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence of sodium dodecyl sulphate (SDS-PAGE). The chemical s i m i l a r i t y between cytoplasmic and plasma g e l s o l i n s , i n i t i a l l y suggested on the b a s i s of N-terminal p r o t e i n sequence a n a l y s i s ( Y i n e t a l . , 1984), was comfirmed when the complete sequence of human g e l s o l i n cDNA showed t h a t cytoplasmic and plasma g e l s o l i n s were i d e n t i c a l except f o r a 25 amino a c i d extension on the amino-terminal of the plasma v a r i a n t . A s i n g l e gene encodes two d i f f e r e n t mRNAs t h a t s p e c i f y the cytoplasmic and plasma forms (Kwiatkowski et a l . , 1986). 8 Horse plasma g e l s o l i n resembles other plasma and cytoplasmic g e l s o l i n s i n i t s p h y s i c a l and chemical p r o p e r t i e s ( R u i z S i l v a and B u r t n i c k , 1990). Hydrodynamic c a l c u l a t i o n s suggest a molecular mass of 75kDa, lower than the 90 kDa estimated from i t s m o b i l i t y on SDS-PAGE and c l o s e r t o 80.9 kDa c a l c u l a t e d f o r p i g plasma g e l s o l i n from i t s amino a c i d sequence (Way and Weeds, 1988). 1.1.6. Interactions of G e l s o l i n with Actin G e l s o l i n has three major i n t e r a c t i o n s with a c t i n . F i r s t , g e l s o l i n a c c e l e r a t e s the i n i t i a l r a t e of s a l t - i n d u c e d a c t i n p o l y m e r i z a t i o n from a c t i n monomers ( n u c l e a t i o n a c t i v i t y ) . Second, g e l s o l i n binds t o the f a s t growing end of a c t i n f i l a m e n t s and i n h i b i t s f u r t h e r e l o n g a t i o n at t h a t end (capping a c t i v i t y ) . T h i r d , g e l s o l i n has the a b i l i t y t o sever a c t i n f i l a m e n t s by breaking the non-covalent bond between a c t i n monomers ( s e v e r i n g a c t i v i t y ) . The combined e f f e c t of these three i n t e r a c t i o n s i s t o promote the formation of a l a r g e number of s h o r t a c t i n f i l a m e n t s which are capped a t t h e i r 'barbed' ends ( H a r r i s and Weeds, 1984). These e f f e c t s are dependent on calcium c o n c e n t r a t i o n s (Bryan and Kur t h , 1984). G e l s o l i n has two calcium b i n d i n g s i t e s and two a c t i n b i n d i n g s i t e s and r e q u i r e s micromolar c o n c e n t r a t i o n s of calcium ions t o bind any a c t i n s . On the removal of ca l c i u m , by c h e l a t i o n w i t h EGTA, only one a c t i n i s removed from the te r n a r y complex. The 1=1 a c t i n / g e l s o l i n complex t h a t remains does not sever a c t i n f i l a m e n t s but i t does block the 9 'barbed' end of a c t i n f i l a m e n t s w i t h high a f f i n i t y . Once g e l s o l i n b l o c k s the 'barbed* end of a f i l a m e n t , i t can not be d i s s o c i a t e d from a c t i n by c h e l a t i o n of c a l c i u m . Way e t a l . (1989) c a r r i e d out s t u d i e s on a number of mutant g e l s o l i n s t o show tha t g e l s o l i n was composed of s i x la r g e segmental repeats of about 15,000 Mr and proposed a hypothesis t o account f o r the p r o p e r t i e s of g e l s o l i n ( f i g u r e 6 ) . F i g u r e 6: Schematic model t o show the i n t e r a c t i o n of human plasma g e l s o l i n w i t h a c t i n (Way et a l . , 1989). 10 This i s a schematic model t o show the i n t e r a c t i o n of g e l s o l i n w i t h a c t i n . In the absence of ca l c i u m , a c t i n b i n d i n g i s prevented by the c l o s e a s s o c i a t i o n of segment 1 and segments 4-6. A d d i t i o n of calcium f a c i l i t a t e s b i n d i n g of two G-actin monomers, one t o segments 2-3 and another i n i t i a l l y t o segments 4-6 but a l s o t o segment 1. ( T h i s t e r n a r y complex i s the n u c l e a t i n g s p e c i e s under po l y m e r i z i n g c o n d i t i o n s . ) On removal of ca l c i u m , b i n d i n g by segments 2-3 and 4-6 i s l o s t , l e a v i n g a s i n g l e monomer t i g h t l y a s s o c i a t e d w i t h segment 1. In the presence of F - a c t i n , g e l s o l i n binds t o the s i d e s of f i l a m e n t s v i a segments 2-3 and both s e v e r i n g and capping occur by the i n t e r a c t i o n of segment 1 and probably segments 4-6 with an adjacent a c t i n s u b u n i t . Way and coworkers a l s o found s i m i l a r segmental repeats i n three other a c t i n - b i n d i n g p r o t e i n s . The evidence suggests t h a t these p r o t e i n s c o n s t i t u t e a f a m i l y of p r o t e i n s t h a t has evolved from a precursor of about 130-150 amino a c i d s (Way and Weeds, 1988). 1.1.7. G e l s o l i n and the Cytoskeleton As g e l s o l i n - a c t i n i n t e r a c t i o n s cannot be reversed completely by removal of c a l c i u m , a second agent i s r e q u i r e d t o d i s s o c i a t e the g e l s o l i n - a c t i n complex. I t has been proposed t h a t the membrane p h o s p h o l i p i d metabolites p h o s p h a t i d y l i n o s i t o l 4,5-bis-phosphate ( P I P 2 ) and p h o s p h a t i d y l i n o s i t o l monophosphate ( P I P ) serve as such agents. They are ab l e t o d i s s o c i a t e g e l s o l i n from s m a l l 11 a c t i n complexes and capped f i l a m e n t ends (Janmey and S t o s s e l , 1987). Figu r e 7- Summary of d i f f e r e n t g e l s o l i n - a c t i n complexes as re g u l a t e d by Ca and PIP 2. The b i n d i n g of P I P 2 t o g e l s o l i n i s u n a f f e c t e d by Ca (Janmey e t a l . , 1987). F i g u r e 7 i s a summary of d i f f e r e n t g e l s o l i n - a c t i n complexes t h a t are r e g u l a t e d by calcium and PIP 2/PIP. G e l s o l i n (G) binds 2 a c t i n monomers (Aj and A 2) i n the presence of 2 calcium i o n s . C h e l a t i n g the calcium w i t h EGTA removes A 2, l e a v i n g a b i n a r y complex GAj. Aj can be d i s s o c i a t e d by the 12 b i n d i n g of P I P 2 or PIP t o g e l s o l i n , t o g i v e PG. The second r o u t e , b i n d i n g P I P 2 before c h e l a t i n g calcium w i t h EGTA, r e s u l t s i n PG a l s o . 1.1.8. G e l s o l i n i n Plasma The i o n i c c o n d i t i o n s of plasma are i d e a l f o r a c t i n p o l y m e r i z a t i o n , so t h a t v i r t u a l l y a l l a c t i n r e l e a s e d i n t o blood would be expected to polymerize. A c t i n may be r e l e a s e d i n t o blood as a r e s u l t of t i s s u e i n j u r y , d i s e a s e , trauma or simply during normal c e l l u l a r turnover. Even a s m a l l number of long a c t i n f i l a m e n t s i n plasma could have s e r i o u s consequences. An increase i n plasma v i s c o s i t y could s e r i o u s l y impede blood f l o w . A l s o , c e l l s c i r c u l a t i n g i n the blood could become entrapped i n the f i l a m e n t s ( L i n d et a l . , 1988). A c t i n f i l a m e n t s could themselves plug up c a p i l l a r i e s (Kwiatkowski e t a l . , 1988) and may i n t e r f e r e with other processes, such as blood c l o t t i n g ( L i n d et a l . , 1986). Moreover, each a c t i n monomer has a bound ADP, and ADP a c t i v a t e s p l a t e l e t s (Scarborough e t a l . , 1981), so e x t r a c e l l u l a r a c t i n could induce p l a t e l e t aggregation. Plasma g e l s o l i n and v i t a m i n D-binding p r o t e i n (DBP) are the two major a c t i n - b i n d i n g p r o t e i n s i n mammalian blood. They serve t o scavenge a c t i n from plasma. Removal of a c t i n proceeds i n 2 stages. F i r s t l y , plasma g e l s o l i n n o n p r o t e o l y t i c a l l y severs F - a c t i n i n t o short f i l a m e n t s t h a t c o n t r i b u t e l i t t l e t o plasma v i s c o s i t y . G e l s o l i n remains t i g h t l y bound t o one end of the shortened f i l a m e n t s . Secondly, DBP, which binds w i t h high a f f i n i t y t o a c t i n monomers, causes a slow depolymerization of the s h o r t , g e l s o l i n capped f i l a m e n t s (Janmey e t a l . , 1987). DBP-actin complexes are r a p i d l y removed from plasma by the l i v e r . 14 PART 2: OPTICAL TECHNIQUES 1.2.1. Fluorescence Spectroscopy The measurement most f r e q u e n t l y performed on b i o l o g i c a l molecules i s the a b s o r p t i o n of v i s i b l e or u l t r a v i o l e t l i g h t . Absorption occurs i n about 10"^ seconds and i s governed by s p i n s e l e c t i o n r u l e s which all o w only t r a n s i t i o n s between s t a t e s of l i k e m u l t i p l i c i t y . L i g h t emission, on the other hand, can r e v e a l molecular p r o p e r t i e s of b i o l o g i c a l molecules q u i t e d i f f e r e n t from those revealed by l i g h t a b s o r p t i o n . The process takes place on a much slower t i m e - s c a l e , t y p i c a l l y 10 seconds f o r aromatic f l u o r o p h o r e s , a l l o w i n g a much wider range of i n t e r a c t i o n s and p e r t u r b a t i o n s t o i n f l u e n c e the spectrum. Since most organic chromophores have an even number of ( p a i r e d ) e l e c t r o n s , the t r a n s i t i o n s are between the ground s t a t e ( S 0 ) and higher e l e c t r o n i c s t a t e s ( S j , S 2, e t c . ) . As v i b r a t i o n a l motion i s on the 10 " second t i m e s c a l e , the a b s o r p t i o n process leaves the molecule i n a v i b r a t i o n a l l y e x c i t e d s t a t e (Franck-Condon p r i n c i p l e ) . In s o l u t i o n , t h i s excess v i b r a t i o n a l energy i s d i s s i p a t e d r a p i d l y t o the surrounding s o l v e n t v i a c o l l i s i o n s , q u i c k l y r e l a x i n g the molecule t o the lowest v i b r a t i o n a l l e v e l of the f i r s t e x c i t e d s i n g l e t s t a t e ( S j ) . Further d e a c t i v a t i o n t o S 0 can occur by emission of a photon ( f l u o r e s c e n c e , r a t e constant kf) or by competing n o n - r a d i a t i v e processes. The non-r a d i a t i v e processes (and the r a t e constants t h a t c h a r a c t e r i s e them) i n c l u d e i n t e r n a l conversion ( k ; c ) , 15 intersystem c r o s s i n g ( k j s ) and quenching ( k q ) . A l l of these processes compete d i r e c t l y t o depopulate the e x c i t e d s i n g l e t s t a t e . Therefore, the f r a c t i o n of e x c i t e d s i n g l e t s t h a t become de-excited through fluorescence (quantum y i e l d , **f) i s simply ««f = kf / (kf + k i c + k i s + k q[Q] ) where [Q] i s the c o n c e n t r a t i o n of quencher molecules. The a b s o r p t i o n and emission of l i g h t can be i l l u s t r a t e d by a J a b l o n s k i diagram ( f i g u r e 8 ) : F i g u r e 8: Schematic diagram f o r the process i n v o l v e d i n e l e c t r o n i c e x c i t a t i o n and d e - e x c i t a t i o n of f l u o r e s c e n t molecules. The ground, f i r s t and second e l e c t r o n i c s t a t e s are d e p i c t e d by S 0, S j , and S 2 , r e s p e c t i v e l y . At each of these 16 e l e c t r o n i c energy l e v e l s , the fluorophore can e x i s t i n a number of v i b r a t i o n a l energy l e v e l s depicted by 0, 1, 2, 3, e t c . The fl o u r e s c e n c e i n t e n s i t y , I ( t ) of a sample of f l u o r e s c e n t molecules at time t a f t e r t u r n i n g o f f the source of e x c i t a t i o n i s given by where I 0 i s the fl u o r e s c e n c e i n t e n s i t y a t time zero and the fl u o r e s c e n c e l i f e t i m e , t f = ( kf + k j c + k j s + kq[Q]) _ 1. 1.2.2. Fluorophores Chromophores which d i s p l a y s i g n i f i c a n t f l u o r e s c e n c e ( f l u o r o p h o r e s ) g e n e r a l l y possess d e l o c a l i s e d e l e c t r o n s f o r m a l l y present i n conjugated double bonds. Fi g u r e 9: T h i o l - s p e c i f i c f l u o r e s c e n t probe, acrylodan (Prendergast e t a l . , 1983). The f l u o r e s c e n t reagent, a c r y l o d a n , r e a c t s s p e c i f i c a l l y w i t h t h i o l groups, such as occur on c y s t e i n e r e s i d u e s i n p r o t e i n s . I t i s h i g h l y s e n s i t i v e t o i t s environment I ( t ) = I 0 exp (-t/x f) O 17 (Pendergast e t a l . , 1983) and has proved u s e f u l i n s t u d i e s of tropomyosin ( C l a r k and B u r t n i c k , 1988). 1.2.3. Environmental E f f e c t s Flourescence i s g e n e r a l l y much more s e n s i t i v e t o the environment of a fluorophore than i s l i g h t a b s o r p t i o n . Therefore, f l u o r e s c e n c e i s an e f f e c t i v e technique f o r f o l l o w i n g i n t e r a c t i o n s among p r o t e i n s and conformational changes i n them. 1.2.3.1. Solvent R e l a x a t i o n The emission from fluorophores g e n e r a l l y occurs a t wavelengths longer than those of l i g h t a b s o r p t i o n ( f i g u r e 10). This l o s s of energy between absorption and emission of l i g h t , the Stokes' s h i f t , i s a r e s u l t of s e v e r a l dynamic processes, i n c l u d i n g v i b r a t i o n a l r e l a x a t i o n . The use of pol a r s o l v e n t s , such as water, w i l l cause c o n s i d e r a b l e r e l a x a t i o n of the e x c i t e d s t a t e of the f l u o r o p h o r e , which g e n e r a l l y has a higher d i p o l e moment than the ground s t a t e . This would reduce the energy gap between the e x c i t e d and ground s t a t e s , causing emission t o be r e d - s h i f t e d r e l a t i v e t o a b s o r p t i o n . The r a t e constants k i c and k j s are enhanced i n p o l a r s o l v e n t s , r e s u l t i n g i n a decrease i n fluo r e s c e n c e i n t e n s i t y . 18 400 O I I i i i i 1 i i i t 450 490 530 570 610 Wavelength /ran F i g u r e 10: E x c i t a t i o n ( a ) and emission ( b ) s p e c t r a of a c r y l o d a n - l a b e l l e d g e l s o l i n . Emission wavelength was 497nm i n ( a ) . E x c i t a t i o n wavelength was 390nm i n ( b ) . S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0 a t 25°C. 19 1.2.3.2. Quenching Flourescence quenching i n v o l v e s any process which decreases the f l u o r e s c e n c e quantum y i e l d , **f. The r a t e of c o l l i s i o n a l quenching of f l u o r e s c e n c e of a fluorophore bound t o a p r o t e i n by a quencher w i l l be l e s s than t h a t of the fluorophore f r e e i n s o l u t i o n and w i l l be r e f l e c t e d i n a change i n slope of a p l o t of Fq / F y s [Q] according t o the Stern-Volmer equation: FQ/F = 1 + k qx 0[Q] where F 0 and F are the f l u o r e s c e n c e i n t e n s i t i e s i n the absence and presence of quencher Q, x 0 i s the f l u o r e s c e n c e l i f e t i m e of the molecule i n the absence of Q and k q i s the bimolecular r a t e constant f o r quenching. A wide v a r i e t y of substances a c t as quenchers of f l u o r e s c e n c e , one of which, I", was employed i n s t u d i e s d e s c r i b e d i n t h i s t h e s i s . Quenching by t h i s halogen i s thought t o be a r e s u l t of intersystem c r o s s i n g t o an e x c i t e d t r i p l e t s t a t e , promoted by s p i n - o r b i t c o u p l i n g of the e x c i t e d f l u orophore and I" (Kasha, 1952). 1.2.4. Fluorescence p o l a r i s a t i o n I f p l a n e - p o l a r i s e d l i g h t i s used t o e x c i t e a f l u o r e s c e n t system, and i f l i n e a r l y p o l a r i s e d components of the emission are detected, i n f o r m a t i o n can be obtained about the s i z e , 20 shape and f l e x i b i l i t y of the macromolecule t o which the fluorophore i s attached. z x F i g u r e 11: Diagramatic r e p r e s e n t a t i o n of e x c i t a t i o n of a f l u o r e s c e n t molecule with v e r t i c a l l y p o l a r i z e d l i g h t and subsequent d e t e c t i o n of emission a t 90° (Cantor and Schimmel, 1979). T h i s f i g u r e shows a t y p i c a l arrangement f o r p o l a r i s a t i o n measurements where the i n c i d e n t l i g h t i s p a r a l l e l t o the z a x i s and p. i s the a b s o r p t i o n d i p o l e moment of the f l u o r e s c e n t s p e c i e s . The a b i l i t y of u. t o i n t e r a c t w i t h v e r t i c a l l y p o l a r i s e d l i g h t f a l l s o f f w i t h cos B, producing a s e t of e x c i t e d molecules t h a t i s c y l i n d r i c a l l y symmetrical about the z a x i s . P o l a r i s a t i o n ( P ) i s defined by: P = (I,, - / (I„ * I x ) where I„ and Ij_ , r e s p e c t i v e l y , are i n t e n s i t y of emission p a r a l l e l and perpendicular t o the plane of p o l a r i s a t i o n of the e x c i t a t i o n l i g h t . For a completely r i g i d system, P = 0.5. This value i s never found f o r fluorophores i n s o l u t i o n . The measured values are sma l l e r due t o r o t a t i o n a l d i f f u s i o n and long f l u o r e s c e n c e l i f e t i m e s . By monitoring p o l a r i s a t i o n v a l u e s , one can t e l l i f the bound f l u o r e s c e n t probe i s becoming more r i g i d or more mobile as a r e s u l t of some p e r t u r b a t i o n t o the system. 1.2.5. C i r c u l a r Dichroism Plane p o l a r i z e d l i g h t may be represented as the sum of two e q u a l l y i n t e n s e but o p p o s i t e l y handed c i r c u l a r l y p o l a r i z e d components. I f a sample absorbs one of the c i r c u l a r l y p o l a r i s e d components more s t r o n g l y than the o t h e r , the r e s u l t a n t l i g h t t h a t emerges from the sample i s e l l i p t i c a l l y p o l a r i s e d . T h i s d i f f e r e n c e i n abs o r p t i o n c o e f f i c i e n t of the sample f o r l e f t and r i g h t c i r c u l a r l y p o l a r i z e d beams d e f i n e s the c i r c u l a r d i c h r o i s m , A e , of the sample: 22 A e = e L - e R where e L a n c ' eR a r e , r e s p e c t i v e l y , e x t i n c t i o n c o e f f i c i e n t s f o r l e f t and r i g h t c i r c u l a r l y p o l a r i s e d l i g h t . The production of an e l l i p t i c a l l y p o l a r i s e d beam has lead t o c i r c u l a r d i chroism being expressed i n terms of the e l l i p t i c i t y , 0, of the e l l i p s e t h a t c h a r a c t e r i z e s the l i g h t , where tan 8 equals the r a t i o of the minor t o the major a x i s of the e l l i p s e . The molar e l l i p t i c i t y [©], expressed i n 2 -1 deg cm ditto 1 A, i s given by [0] = 3300 ( e L - e R) A CD spectrum c o n s i s t s of a p l o t of [0] ys. wavelength. Molar e l l i p t i c i t y , [ 0 ] , can be c a l c u l a t e d from observed e l l i p t i c i t y a t a s p e c i f i c wavelength: [0] = M0/lOdc where 0 i s the observed e l l i p t i c i t y ( d e g ), M i s the molar mass ( g mol' 1), d i s the c e l l pathlength (cm) and c i s the co n c e n t r a t i o n ( g mL*1). I f M. expresses the mean residue molar mass, then [0] i s c a l l e d the mean residue ( o r r e s i d u a l ) e l l i p t i c i t y . P r o t e i n c i r c u l a r dichroism i n the re g i o n of the spectrum between 190nm and 230nm i s dominated by c o n t r i b u t i o n s from the peptide backbone. One can consider a p r o t e i n t o c o n s i s t 23 of oc-helix, B-sheet and random c o i l (more c o r r e c t l y , undefined) s t r u c t u r e s . Estimates of the f r a c t i o n of amino a c i d r e s i d u e s i n each of these types of conformation can be obtained from measured CD of the p r o t e i n being s t u d i e d . To c a l c u l a t e the f r a c t i o n a l composition of secondary s t r u c t u r e t y p e s , one could consider the f o l l o w i n g equation a t 2 d i f f e r e n t wavelengths and use the c o n s t r a i n t t h a t f « + f B + f r = 1 [e] = f^a + f p X B + f r X r where X^, Xg and X r a r e , r e s p e c t i v e l y , the mean residue e l l i p t i c i t i e s of 100% pure oc-helix, ft-sheet and random c o i l p r o t e i n s . These values can be estimated using CD s p e c t r a of homopolypeptides of known s t r u c t u r e ( G r e e n f i e l d and Fasman, 1969) or by comparative a n a l y s i s of the CD s p e c t r a of p r o t e i n s of known three dimensional s t r u c t u r e (by X-ray c r y s t a l l o g r a p h y ) (Saxena and Wetlaufer, 1971). P r o t e i n denaturation can be f o l l o w e d using c i r c u l a r d i c h r o i s m . The l o s s of CD s i g n a l between 210nm and 230nm on thermal denaturation i s due t o u n f o l d i n g of cc-helix and &-sheet s t r u c t u r e s i n t o the random c o i l s t r u c t u r e , which has a low CD s i g n a l i n t h i s s p e c t r a l r e g i o n . 24 2_ MATERIALS AND METHODS PART 1= PROTEINS 2_J^ P u r i f i c a t i o n of A c t i n A c t i n was p u r i f i e d from an acetone powder of r a b b i t muscle t i s s u e ( P e l - F r e e z B i o l o g i c a l s ) according t o a m o d i f i c a t i o n of the method of Spudich and Watt (1971). lOg of acetone powder was disp e r s e d ( a t 4°C) i n 200mL of 2mM T r i s - H C l , 0.2mM C a C l 2 , 0.2mM ATP, 1.OmM DTT, pH 8.0 ( B u f f e r A) f o r 30 minutes. The mixture was f i l t e r e d through a double l a y e r of cheesecloth. The residue was washed with lOOmL of b u f f e r A and f i l t e r e d again before being d i s c a r d e d . Both f i l t r a t e s were combined and f i l t e r e d through Whatman #1 f i l t e r paper and c e n t r i f u g e d a t 80,000 xg f o r 1 hour. A f t e r d i s c a r d i n g the p e l l e t , KCl ( t o 50mM) and MgCl 2 ( t o 2mM) were added t o the supernatant and the a c t i n was allowed t o polymerise (without s t i r r i n g ) a t room temperature f o r 2 hours. S o l i d KCl then was added t o 0.8M and the s o l u t i o n was s t i r r e d g e n t l y f o r 1.5 hours. I t then was c e n t r i f u g e d at 80,000 xg f o r 3 hours. The supernatant was d i s c a r d e d . The F-a c t i n p e l l e t was resuspended i n 30mL of b u f f e r A and converted t o G-actin by d i a l y s i n g a t 4°C f o r 3 days, changing t o f r e s h b u f f e r A every 24 hours. The G-actin was c l a r i f i e d by c e n t r i f u g a t i o n a t 80,000 xg f o r 3 hours and st o r e d i n b u f f e r A. 25 The c o n c e n t r a t i o n of G-actin was measured s p e c t r o p h o t o m e t r i c a l l y using an abso r p t i o n c o e f f i c i e n t at 290nm of 0.63 ml_ mg^cm-1 ( P o r t e and Harr i c a n e , 1986). 2.2.1 P u r i f i c a t i o n of G e l s o l i n G e l s o l i n was p u r i f i e d from f r o z e n horse blood plasma by a m o d i f i c a t i o n of the methods of Bryan (1988) and I t o , et a l . (1990). During the thawing process lOOuL each of l e u p e p t i n and p e p s t a t i n ( s t o c k s o l u t i o n s at 2 mg ml__1 i n water and DMSO, r e s p e c t i v e l y ) were added t o 1L of plasma along w i t h PMSF t o 0.2mM. The thawed plasma was d i a l y s e d at 4°C aga i n s t 4 changes, 10 volumes per change, of 25mM T r i s - H C l , 0.5mM C a C l 2 and ImM NaN 3, pH 7.5, and c e n t r i f u g e d at 10,000 xg f o r 10 minutes t o remove aggregated m a t e r i a l . S o l i d NaCl was added t o the supernatant t o 35mM. The plasma then was mixed w i t h 4L of DEAE Sephadex A-50 ion exchanger (Pharmacia) t h a t had been e q u i l i b r a t e d against 50mM NaCl, 25mM T r i s - H C l , 0.5mM C a C l 2 , ImM NaN 3, pH 7.5. This batch procedure was c a r r i e d out at 4°C with o c c a s i o n a l , g e n t l e s t i r r i n g f o r approximately 2 hours. The s l u r r y then was f i l t e r e d and the i o n exchanger was washed w i t h b u f f e r . At t h i s stage the plasma l o s t i t s c h a r a c t e r i s t i c yellow colour t o the i o n exchanger and the f i l t r a t e was c o l o u r l e s s . 26 To the f i l t r a t e ( t o t a l volume between 2.1L and 2.5L), EDTA and NaN 3 were added t o lOmM and ImM, r e s p e c t i v e l y , and the pH was adjusted t o 7.8. This s o l u t i o n was a p p l i e d t o a 36x6 cm column of DEAE-Sephadex A-50 i o n exchanger p r e v i o u s l y e q u i l i b r a t e d at 4°C i n 50mM NaCl, 25mM T r i s - H C l , ImM EDTA, ImM NaN 3, pH 7.8. A f t e r washing the column wi t h the e q u i l i b r a t i n g b u f f e r (2 bed volumes), p r o t e i n s were e l u t e d w i t h a 0.05M t o 0.3M NaCl gra d i e n t and c o l l e c t e d i n 25mL f r a c t i o n s . Those f r a c t i o n s c o n t a i n i n g g e l s o l i n were pooled and concentrated by u l t r a f i l t r a t i o n t o 2.5-3.0 mg ml__1 using an Amicon YM30 membrane. No p r e c i p i t a t i o n was observed, i n c o n t r a s t t o re p o r t s t h a t , at con c e n t r a t i o n s greater than 1 mg ml_ _1, p i g plasma g e l s o l i n was unstable i n s o l u t i o n (Weeds et a l . , 1986). These concentrated s o l u t i o n s were d i a l y s e d at 4°C again s t 2 changes of 25mM T r i s - H C l , ImM EDTA, ImM PMSF, pH 8.0 ( B u f f e r I ) f o r 24 hours. The d i a l y s e d s o l u t i o n was a p p l i e d a t 4°C t o a 2.5x17 cm A f f i - G e l Blue (immo b i l i s e d c i b a c r o n Blue F3GA g e l ) column e q u i l i b r a t e d w i t h b u f f e r I . The column then was washed wi t h 2 bed volumes of b u f f e r I and g e l s o l i n was e l u t e d w i t h b u f f e r I c o n t a i n i n g ImM ATP and c o l l e c t e d i n 2mL f r a c t i o n s . These f r a c t i o n s were concentrated by u l t r a c e n t r i f u g a t i o n using an Amicon YM30 membrane. Concentrations of horse plasma g e l s o l i n were measured s p e c t r o p h o t o m e t r i c a l l y using an absorption c o e f f i c i e n t at 280nm of 1.4 ml_ mg^cm"1 ( R u i z S i l v a and B u r t n i c k , 1990). 27 2_JL^2 Determination of P u r i t y The p u r i t y was checked at v a r i o u s stages of the pr e p a r a t i o n of g e l s o l i n by e l e c t o p h o r e s i s on 10% polyacrylamide g e l s (BioRad Mini-Protean I I System) i n the presence of sodium dodecyl sulphate (SDS) and 2-mercaptoethanol (LaemmLi, 1970). Phosphorylase b (Sigma) was used as a standard. Pure g e l s o l i n produced a s i n g l e band w i t h a r e l a t i v e molecular mass of 90,000. 2.2.3 Determination of A c t i v i t y A c t i n - s e v e r i n g a c t i v i t y of g e l s o l i n was measured by viscometry. A l i q u o t s of g e l s o l i n c o n t a i n i n g s o l u t i o n s were added t o 50uM a c t i n s o l u t i o n s i n 150mM K C l , 25mM T r i s - H C l , 2mM C a C l 2 , ImM MgCl 2, O.lmM ATP, and ImM DTT, pH 8.0. Measurements were made i n a Cannon-Manning semi-micro viscometer ( s i z e 100) at 27°C. A volume of lmL was r e q u i r e d t o f i l l the viscometer, which had a flow time of 102s f o r b u f f e r . 2.2.4 Fluor e s c e n t l a b e l l i n g of G e l s o l i n . G e l s o l i n i n i t i a l l y was d i a l y s e d a g a i n s t 1L of 150mM K C l , 25mM T r i s - H C l , ImM EDTA and ImM DTT, pH 8.0 at 4°C f o r 24 hours. DTT was removed p r i o r t o l a b e l l i n g by d i a l y s i s i n the above b u f f e r without DTT f o r a f u r t h e r 4 hours. Acrylodan (Molecular Probes) was d i s s o l v e d i n lOOuL of /V,W-dimethylformamide (DMF) and added d i r e c t l y t o the g e l s o l i n sample u n t i l an approximate 20x molar excess of 28 acrylodan t o p r o t e i n had been achieved. The r e a c t i o n was allowed t o proceed overnight i n the dark at 4°C on a mechanical r o c k e r . The g e l s o l i n / a c r y l o d a n mixture was d i a l y s e d a g a i n s t 1L of 150mM K C l , 30mM T r i s - H C l , ImM EGTA, and ImM DTT, pH 8.0 f o r 4 hours and c e n t r i f u g e d a t 15,000 xg f o r 15 minutes t o remove the unreacted acrylodan. The a e r y l o d a n - l a b e l l e d g e l s o l i n (ACRG) was d i a l y s e d f u r t h e r a g a i n s t the above b u f f e r f o r 3 days, changing the b u f f e r on the second and t h i r d days. ACRG produced a s i n g l e f l u o r e s c e n t band on a 10% SDS-PAGE g e l . 2.2.5 Degree of L a b e l l i n g The average number of acrylodan molecules per g e l s o l i n molecule (degree of l a b e l l i n g ) was measured by independent determination of acrylodan and p r o t e i n concentrations i n an ACRG s o l u t i o n . Acrylodan was analysed s p e c t r o p h o t o m e t r i c a l l y u s i n g a molar a b s o r p t i o n c o e f f i c i e n t of 12900 M^cm-1 at 360nm. The c o n c e n t r a t i o n of l a b e l l e d g e l s o l i n i n ACRG s o l u t i o n was measured using the BioRad microassay procedure based on the ob s e r v a t i o n t h a t the absorbance maximum of a dye (Coomassie B r i l l i a n t Blue G-250) s h i f t s from 465nm t o 595nm when b i n d i n g t o p r o t e i n occurs ( B r a d f o r d , 1976). U n l a b e l l e d g e l s o l i n ( o f known c o n c e n t r a t i o n ) i n the presence of dye was used t o p l o t a standard curve of c o n c e n t r a t i o n ys_ absorbance a t 595nm. The c o n c e n t r a t i o n of g e l s o l i n i n an ACRG s o l u t i o n was i n t e r p o l a t e d from the standard curve. For a given ACRG s o l u t i o n : Degree of _ TAcrvlodanl l a b e l l i n g = [ G e l s o l i n ] The degree of l a b e l l i n g was found t o be 1.9 ± 0.5 acrylodans per g e l s o l i n based on 6 l a b e l l i n g experiments. 30 2.2.6 Summary of G e l s o l i n P u r i f i c a t i o n FROZEN HORSE PLASMA thaw add a n t i - p r o t e a s e s d i a l y s e c e n t r i f u g e ADD TO ANION EXCHANGER (BATCH PROCEDURE) e q u i l i b r a t e d i n presence of calcium f i l t e r wash LOAD FILTRATE ONTO SECOND ANION EXCHANGER (COLUMN) e q u i l i b r a t e d i n presence of EDTA wash apply gradient pool g e l s o l i n f r a c t i o n s concentrate d i a l y s e . AFFI-GEL BLUE COLUMN - wash - e l u t e g e l s o l i n - pool f r a c t i o n s - concentrate DETERMINE PURITY J, DETERMINE ACTIVITY I LABEL WITH ACRYLODAN I DETERMINE ACTIVITY 31 PART 2 = OPTICAL TECHNIQUES 2.3.1 Absorbance Measurements A Lambda 4B UV/Vis Spectrophotometer (Perkin-Elmer) was used t o detect p r o t e i n s and measure con c e n t r a t i o n s a t va r i o u s stages d u r i n g and a f t e r the p u r i f i c a t i o n procedure. 2-3-2 Flourescence Measurements An LS-5B Luminescence Spectrometer equipped w i t h a 7500 S e r i e s computer (Perkin-Elmer) was used f o r f l u o r e s c e n c e s t u d i e s . The e x c i t a t i o n and emission wavelengths were 390nm and 497nm, r e s p e c t i v e l y , w i t h both e x c i t a t i o n and emission s l i t s s e t at lOnm band widths. P o l a r i s a t i o n values were obtained by i n c o r p o r a t i n g a semi-automatic p o l a r i s a t i o n accessory and using PTPOL software (Per kin-Elmer). The c e l l holder was t h e r m o s t a t i c a l l y c o n t r o l l e d f o r temperature s t u d i e s . 2.3-3 C i r c u l a r Dichroism A r e b u i l t J-20 Automatic Recording Spectro-Polarimeter ( J a s c o ) was used f o r r e c o r d i n g c i r c u l a r d i chroism measurements. The c e l l chamber was t h e r m o s t a t i c a l l y c o n t r o l l e d and purged w i t h n i t r o g e n . Thermal denaturation s t u d i e s were performed a t a f i x e d wavelength of 215nm. The output was standardized using D-Pantolactone (12mg i n lOOmL H 2 O ) , which has an e l l i p t i c i t y of -17.3 x 10 3 deg cm 2dmol _ 1 a t 220nm (Tuzimura et a l . , 1977). Thermal e q u i l i b r a t i o n was assessed t o take between 10 and 15 minutes when monitored w i t h a thermocouple i n a cuvet i n the sample chamber. C o r r e l a t i o n of sample and bath temperatures i n t h i s way-enabled a l l temperatures quoted i n the t e x t t h a t f o l l o w s be reported as sample temperatures. 33 3_ RESULTS AND DISCUSSION PART 1= EFFECT OF CALCIUM ON ACRG 3.1.1. L a b e l l i n g G e l s o l i n w i t h Acrylodan The degree of l a b e l l i n g of g e l s o l i n w i t h acrylodan was found t o be 1.9 ± 0.5 acrylodans per g e l s o l i n molecule, based on 6 l a b e l l i n g experiments. A l l ACRGs showed a s i n g l e f l u o r e s c e n t band and then a s i n g l e Coomassie b l u e - s t a i n e d band on SDS-PAGE g e l s . This demonstrates the covalent b i n d i n g of the acrylodan t o g e l s o l i n and t h a t the r e a c t i o n c o n d i t i o n s have not r e s u l t e d i n cleavage of peptide bonds. On e x c i t a t i o n a t 390nm, ACRG i s h i g h l y f l u o r e s c e n t , showing a broad emission band wi t h a maximum near 497nm ( f i g u r e 10). The emission maximum i s b l u e - s h i f t e d r e l a t i v e t o 540nm f o r an acrylodan-mercaptoethanol adduct (Prendergast et a l . , 1983), 518nm f o r a c r y l o d a n - l a b e l l e d tropomyosin ( C l a r k and B u r t n i c k , 1988) and 510nm f o r a c r y l o d a n - l a b e l l e d DBP (Robinson, 1990). The emission maximum of ACRG i s s i m i l a r t o those of a c r y l o d a n - l a b e l l e d papain (491nm), parvalbumin (498nm) and carbonic anhydrase (501nm) (Prendergast e t a l . , 1983) and suggests the environment of acrylodan on g e l s o l i n t o have co n s i d e r a b l e hydrophobic c h a r a c t e r . A v i s c o s i t y experiment was c a r r i e d out t o determine whether or not i n c o r p o r a t i o n of the acrylodan l a b e l s i g n i f i c a n t l y a f f e c t e d the f u n c t i o n of g e l s o l i n w i t h respect t o i t s a c t i n - s e v e r i n g a b i l i t y (Table I ) . 34 TABLE I : VISCOSITY MEASUREMENTS ON ACRG Sample R e l a t i v e V i s c o s i t y 1. B u f f e r A 1.00 2. F - a c t i n 1.53 3. G e l s o l i n - a c t i n 1.08 4. ACRG-actin 1.09 Each sample (lmL volume) contained 150mM K C l , 25mM T r i s - H C l , 2mM C a C l 2 , ImM MgCl 2, ImM DTT and O.lmM ATP, pH 8.0, at 27°C. A c t i n (1.2uM) was added t o samples 2, 3, 4 and allowed t o polymerize and e q u l i b r a t e f o r 20 minutes. G e l s o l i n and ACRG were added t o samples 3 and 4, r e s p e c t i v e l y , t o 0.92uM. R e l a t i v e v i s c o s i t i e s were c a l c u l a t e d from the flow time of sample d i v i d e d by the flow time of b u f f e r A. These values demonstrate t h a t , under the c o n d i t i o n s employed, ACRG i s j u s t as capable of seve r i n g a c t i n f i l a m e n t s as i s u n l a b e l l e d g e l s o l i n . An a d d i t i o n a l experiment was c a r r i e d out t o i n v e s t i g a t e the p o s s i b l e e f f e c t on the degree of l a b e l l i n g of calcium i o n s . I d e n t i c a l samples were prepared by d i a l y s i s a g a i n s t 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0. One sample subsequently was d i a l y s e d a g a i n s t the same bu f f e r c o n t a i n i n g ImM C a C l 2 i n s t e a d of EGTA. The l a b e l l i n g r e a c t i o n was c a r r i e d out as u s u a l . Although the degree of l a b e l l i n g was found t o be 2.3 i n the presence of calcium and 1.6 i n i t s absence, these values f e l l w i t h i n the range of values 35 obtained p r e v i o u s l y , so no s i g n i f i c a n c e could be attached t o the d i f f e r e n c e . 3_J^2 CD Studies on ACRG Comparison of the CD s p e c t r a of ACRG l a b e l l e d i n the presence or absence of calcium w i t h t h a t of u n l a b e l l e d g e l s o l i n over the re g i o n from 210 t o 245nm showed no s i g n i f i c a n t d i f f e r e n c e s ( t y p i c a l s p e c t r a are shown i n f i g u r e 12). This suggests t h a t n e i t h e r i n c o r p o r a t i o n of the l a b e l nor the presence or absence of Ca 2 + during l a b e l l i n g produced a change i n the conformation of the g e l s o l i n molecule. These s p e c t r a resemble i n shape and i n t e n s i t y those presented by Doi e t a l . (1990) f o r p i g plasma g e l s o l i n , except t h a t we were not able t o detect the small decrease i n e l l i p t i c i t y they reported t o be induced by 2+ a d d i t i o n of Ca t o t h e i r samples. 36 | I 210 220 230 240 Wavelength /nm Fig u r e 12: CD s p e c t r a of ACRG ( l a b e l l e d i n the presence of excess EGTA) i n the presence and absence of Ca . S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and e i t h e r ImM EGTA or ImM C a C l 2 , pH 8.0. ACRG was present i n each sample at luM i n a 0.1 mm pathlength c e l l and the temperature was 10°C. The top spectrum i s t h a t f o r the EGTA-c o n t a i n i n g b u f f e r . The lower s p e c t r a are not d i s t i n g u i s h a b l e s t a t i s t i c a l l y . 37 A comparison was made of the thermal s t a b i l i t i e s of ACRG l a b e l l e d i n the presence of Ca 2 +, ACRG l a b e l l e d i n EGTA and u n l a b e l l e d g e l s o l i n . The e l l i p t i c i t i e s a t 215nm f o r each of the three p r o t e i n s were observed a t v a r i o u s temperatures i n the presence and absence of Ca . In each case, the g e l s o l i n or ACRG was q u i t e s t a b l e up t o about 40°C. Subsequently, e l l i p t i c i t y values dropped d r a m a t i c a l l y . The EGTA-containing samples showed a melt i n g t r a n s i t i o n centred a t 48°C ( f i g u r e 13). The Ca c o n t a i n i n g samples were somewhat more s t a b l e , l o s i n g s t r u c t u r e near 52°C ( f i g u r e 14). At about 2°C above the m e l t i n g temperature, a v i s i b l e , i r r e v e r s i b l e p r e c i p i t a t e appeared i n a l l samples. Fi g u r e 15 compares the me l t i n g curves of g e l s o l i n i n the presence and absence of Ca . S i m i l a r comparisons are made f o r the l a b e l l e d p r o t e i n s i n 2+ f i g u r e s 16 and 17. The s l i g h t s t a b i l i z a t i o n a f f o r d e d by Ca i s evident i n each case. 3.1.3 E f f e c t of Calcium on the Emission Spectrum of ACRG Figur e 18 i l l u s t r a t e s the e f f e c t of calcium on the emission spectrum of ACRG. ACRG was d i a l y s e d a g a i n s t 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0. C a C l 2 (0.1M stock s o l u t i o n ) was added t o 2mM, so t h a t a ImM excess 2+ o f Ca over EGTA e x i s t e d i n the s o l u t i o n . The spectrum a t 25°C was recorded 5 minutes a f t e r the a d d i t i o n of C a C l 2 . To c o r r e c t f o r d i l u t i o n e f f e c t s , a c o n t r o l experiment was c a r r i e d out simultaneously i n which an equal volume of 1.2 1.0-H 0.8 H o Q. • _J 111 Q in 0.6-N oc o z 0.4-0.2-0.0-A 'ti A A A £ 1 ' 1 • 1 ' 1 ' 1 ' 1 1 — 10 20 30 40 50 60 70 temperature/°C Fig u r e 13 Change i n normalized e l l i p t i c i t i e s of ACRG l a b e l l e d i n Ca 2 + ( A ) , ACRG l a b e l l e d i n EGTA ( A ) , and u n l a b e l l e d g e l s o l i n ( n ) w i t h temperature. S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM CaClo, PH 8.0. CD was f o l l o w e d at 215nm. A 20 minute e q u i l i b r a t i o n p e r i o d was allowed subsequent t o each change i n temperature. 39 1.2 1.0-• ft * 4 * t 0.8 H u 0. Ul O iu 0.6-N w ° DC o 0.4-0.2-A A A 0.0' 10 20 30 —I 4 0 50 60 70 temperature/°C Fig u r e 14 Change i n normalized e l l i p t i c i t i e s of ACRG l a b e l l e d i n Ca 2 + ( A ) , ACRG l a b e l l e d i n EGTA ( A ) , and u n l a b e l l e d g e l s o l i n (•) w i t h temperature. S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0. CD was fo l l o w e d at 215nm. A 20 minute e q u i l i b r a t i o n p e r i o d was allowed subsequent t o each change i n temperature. 40 temperature j °C F i g u r e 15 Change i n normalized e l l i p t i c i t i e s of u n l a b e l l e d g e l s o l i n i n the presence and absence of Ca 2 + w i t h temperature. S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s -HCl, ImM DTT and e i t h e r ImM Ca 2 + ( A ) or ImM EGTA (A).pH 8.0. C i r c l e d p o i n t s i n d i c a t e onset of p r e c i p i t a t i o n . CD was fo l l o w e d a t 215nm. A 20 minute e q u i l i b r a t i o n p e r i o d was allowed subsequent t o each change i n temperature. 41 temperature / ° C F i g u r e 16 Change i n normalized e l l i p t i c i t i e s of ACRG ( l a b e l l e d i n presence of Ca ) i n the presence and absence of Ca w i t h temperature. S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and e i t h e r ImM Ca 2 + ( A ) or ImM EGTA ( A ) , pH 8.0. C i r c l e d p o i n t s i n d i c a t e the onset of p r e c i p i t a t i o n . CD was fo l l o w e d a t 215nm. 20 minute e q u i l i b r a t i o n p e r i o d was allowed subsequent t o each change i n temperature. 42 temperature/ °C F i g u r e 17 M e l t i n g curve of ACRG ( l a b e l l e d i n absence of Ca ) i n the presence and absence of Ca . S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and e i t h e r ImM Ca 2 + ( A ) or ImM EGTA ( A ) , pH 8.0. C i r c l e d p o i n t s i n d i c a t e onset of p r e c i p i t a t i o n . CD was foll o w e d a t 215nm. A 20 minute e q u i l i b r a t i o n p e r i o d was allowed subsequent t o each change i n temperature. 43 d i s t i l l e d water was added t o the p r o t e i n s o l u t i o n i n place of CaCl 2- There i s a decrease i n fluo r e s c e n c e w i t h a s l i g h t red s h i f t . T h i s suggests t h a t a conformational change i n the p r o t e i n , induced by calcium b i n d i n g , i s i n c r e a s i n g the exposure of the probe t o the s o l v e n t . This process i s f u l l y r e v e r s a b l e by c h e l a t i n g the calcium ions w i t h EGTA, i n d i c a t i n g t h a t the e f f e c t i s due s o l e l y t o the a c t i o n of calcium i o n s . 600 100 440 460 480 500 520 540 Wavelength /nm Figur e 18: E f f e c t of calcium on the emission spectrum of ACRG. The upper curve i s the emission spectrum of ACRG (luM) i n the presence of ImM EGTA. The lower curve i s the spectrum 5 minutes a f t e r the a d d i t i o n of Ca 2 + t o 2mM. The e x c i t a t i o n wavelength was 390nm. I n i t i a l s o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT, ImM EGTA, pH 8.0, a t 25°C. 44 3.1.4 Fluorescence Quenching S t u d i e s on ACRG Increased exposure of the acrylodan probe t o s o l v e n t i n the presence of Ca was confirmed by quenching s t u d i e s . F i g u r e 19 i s a Stern-Volmer p l o t f o r i o d i d e quenching of ACRG f l u o r e s c e n c e . ACRG ( l u t l ) was d i a l y s e d i n 150mM K C l , 25mM T r i s - H C l , ImM DTT, pH 8.0, and e i t h e r ImM Ca 2 + or ImM EGTA. Two i d e n t i c a l s o l u t i o n s were r e q u i r e d f o r each p l o t , one t o which KI was added and one t o which was added equal molar amounts of K C l . These s o l u t i o n s , r e s p e c t i v e l y , gave F and F 0 f o r each a d d i t i o n . This p r o t o c o l simultaneously served t o c o n t r o l f o r the e f f e c t s of d i l u t i o n and increased i o n i c s t r e n g t h . The steeper slope of the p l o t f o r the sample i n the presence of C a 2 + r e f l e c t s increased s u s c e p t i b i l i t y of the probe t o quenching under those c o n d i t i o n s . In an attempt t o increase the a c c e s s a b i l i t y t o I~ i n the s o l v e n t , ACRG was denatured u s i n g 6M Gu-HCl. F i g u r e 20 presents Stern-Volmer p l o t s f o r i o d i d e quenching of i n t a c t ACRG i n the presence of calcium and f o r ACRG denatured with 6M Gu-HCl. The slopes of these p l o t s are very s i m i l a r . This suggests t h a t the acrylodan probe i n the presence of calcium i s e s s e n t i a l l y as exposed t o s o l v e n t as i t i s i n the denatured p r o t e i n . 45 [Kl]/M F i g u r e 19: Stern-Volmer p l o t s f o r i o d i d e quenching of ACRG. One sample i n i t i a l l y contained 0.25uM ACRG i n 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0, a t 25°C ( A ) . The second sample was i d e n t i c a l but contained 2mM C a C l 2 as w e l l ( A ) . E x c i t a t i o n was 390nm and emission was 497nm i n both cases. 1.4 [Kl]/M F i g u r e 20= Stern-Volmer p l o t s f o r i o d i d e quenching of ACRG s o l u t i o n s . The Gu-HCl data (A) were c o l l e c t e d from samples t h a t i n i t i a l l y contained 0.25uM ACRG i n the presence of 6M Gu-HCl. The +Ca data ( A ) are same as presented i n f i g u r e 19. E x c i t a t i o n wavelength was 390nm and emission wavelength was 497nm i n both cases. 3.1.5 Fluorescence P o l a r i s a t i o n S t u dies on ACRG Fluorescence p o l a r i s a t i o n was measured during thermal denaturation of ACRG i n the presence and absence of calcium ( f i g u r e 21). Two s o l u t i o n s were d i a l y s e d a g a i n s t 150mM K C l , 25mM T r i s - H C l , ImM DTT, pH 8.0, and e i t h e r ImM Ca 2 + or ImM EGTA. 0.39 0.38-0.37 g 0.36-E < O 0.35 0.34 0.33-A A A A A A A ® 0.32 i i 1 1 1 1 1 1 1——i r 10 20 30 40 50 60 temperat ure / 70 F i g u r e 21: Fluorescence p o l a r i s a t i o n changes i n ACRG (luM) w i t h temperature. Each p o l a r i s a t i o n value was the average of 3 measurements and t y p i c a l standard d e v i a t i o n s were ± 0.003. S o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT, pH 8.0, and e i t h e r ImM EGTA (A) or ImM CaClo ( A ) . C i r c l e d p o i n t s i n d i c a t e onset of p r e c i p i t a t i o n . E x c i t a t i o n wavelength was a t 390nm and emission wavelength at 497nm. 48 The steady decrease i n p o l a r i s a t i o n value t o about 50°C suggests a l o s s of g e l s o l i n s t r u c t u r a l r i g i d i t y i n the reg i o n of the acrylodan probe. The r i g i d i t y of the reg i o n i s minimal a t 48°C i n ImM EGTA and near t o 50°C i n ImM Ca 2 +. The sharp upward t u r n i n each p l o t c o i n c i d e s w i t h the onset of aggregation l e a d i n g t o i r r e v e r s i b l e p r e c i p i t a t i o n . The c o n s i s t e n t l y lower p o l a r i s a t i o n values found f o r ACRG i n the 2+ presence of Ca r e l a t i v e t o i n i t s absence are c o n s i s t e n t 2+ w i t h a model i n which Ca bi n d i n g exposes the segment of polypeptide c h a i n c o n t a i n i n g the acrylodan l a b e l t o s o l v e n t . PART 2 = ACTIN BINDING TO ACRG 3.2.1 E f f e c t of A c t i n on Emission Maximum of ACRG Figur e 22 i l l u s t r a t e s the change i n i n t e n s i t y of the fluo r e s c e n c e at 497nm f o r ACRG on t i t r a t i o n w i t h a c t i n . Two samples were s t u d i e d each i n i t i a l l y c o n t a i n i n g ACRG ( l u M ) , 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM EGTA, pH 8.0 and one sample contained 2mM CaCl 2- D i l u t i o n e f f e c t s were c o r r e c t e d f o r by m u l t i p l i c a t i o n of the i n t e n s i t y of each sample by the r a t i o of the sample volume t o i t s i n i t i a l volume. Both samples show a steady drop i n emission i n t e n s i t y on the a d d i t i o n of a c t i n , suggesting an i n t e r a c t i o n between ACRG and a c t i n t h a t quenches acrylodan f l u o r e s c e n c e . The 2+ e f f e c t s of a c t i n are independent of Ca , i n apparent 49 1.2 1.0-1 Ul O z UJ o 0) 0.8' K O 9 O Ul El 0.6-CC o \ 0.4-0.2- T" 2 Iactln]/[ACRG] F i g u r e 22: Changes i n fl u o r e s c e n c e i n t e n s i t y of ACRG on the b i n d i n g of a c t i n . One sample i n i t i a l l y contained luM ACRG i n 150mM K C l , 25mM T r i s - H C l , ImM DTT, ImM EGTA, pH 8.0, a t 25°C ( d ) . The other sample was i d e n t i c a l but contained 2mM C a C l 2 as w e l l (•). E x c i t a t i o n wavelength was a t 390nm and emission was a t 497nm. No r m a l i z a t i o n was achieved by d i v i d i n g a l l readings by the app r o p r i a t e sample flu o r e s c e n c e i n t e n s i t y i n the absence of a c t i n . 50 c o n t r a d i c t i o n of the model of Way et a l . (1988). The model suggests t h a t a c t i n should not i n t e r a c t w i t h g e l s o l i n i n the 2+ absence of Ca . One ex p l a n a t i o n of a c t i n b i n d i n g i n the 2+ absence of Ca might be t h a t horse plasma g e l s o l i n does not 2+ d i s p l a y Ca - s e n s i t i v e a c t i n - b i n d i n g . This compares w i t h p i g plasma g e l s o l i n , which has been shown t o i n t e r a c t w i t h a c t i n 2+ both i n the presence and absence of Ca ( H a r r i s and Weeds, 1983). Indeed, Ca 2 +-dependent and Ca 2 +-independent forms of t h i s g e l s o l i n have been separated by a f f i n i t y chromatography on actin-Sepharose columns (Pope and Weeds, 1986). Plasma 2+ has a c o n s i s t e n t l y high f r e e Ca c o n c e n t r a t i o n (about ImM) 2+ and Ca - s e n s i t i v i t y of a plasma p r o t e i n f u n c t i o n would be i r r e l e v a n t . Another p o s s i b l e e x p l a n a t i o n comes from work by Pope et a l . (1989). Their r e s u l t s showed i r r e v e r s i b l e l o s s 2+ of Ca - s e n s i t i v i t y of g e l s o l i n w i t h respect t o a c t i n f i l a m e n t s e v e r i n g and a c t i n monomer b i n d i n g may be a s s o c i a t e d w i t h the presence of Ca 2 + during p r e p a r a t i o n and storage. The f i r s t stage of our p u r i f i c a t i o n of horse plasma g e l s o l i n i n v o l v e s d i a l y s i s i n a b u f f e r c o n t a i n i n g 0.5mM 2+ Ca . At t h i s s t age, one or more as yet u n i d e n t i f i e d plasma 2+ components may a c t t o reduce the Ca - s e n s i t i v i t y of g e l s o l i n by some unknown mechanism. In a d d i t i o n t o i n d i c a t i n g a c t i n b i n d i n g , the drop i n fl u o r e s c e n c e i n f i g u r e 22 suggests t h a t acrylodan i s becoming more exposed t o the s o l v e n t on i n t e r a c t i o n of ACRG w i t h a c t i n . 51 2^*2 Quenching Studies of A c t i n - G e l s o l i n Complexes Quenching s t u d i e s were c a r r i e d out on v a r i o u s actin-ACRG complexes t o determine the exposure of the acrylodan probe t o the s o l v e n t on the bi n d i n g of a c t i n . Two i d e n t i c a l s o l u t i o n s were r e q u i r e d f o r each p l o t , one t o which was added a l i q u o t s of KI and the other t o which was added equal amounts of K C l . These s o l u t i o n s , r e s p e c t i v e l y , gave F and F 0 f o r each a d d i t i o n . F i g u r e 23 presents a comparison of the i o d i d e quenching of 11:1, 2:1 and 1:1 actin:ACRG complexes w i t h ACRG alone. Linear r e g r e s s i o n a n a l y s i s of the curves i n f i g u r e 25 gave the slopes and standard d e v i a t i o n s shown i n Table I I : TABLE I I : IODIDE QUENCHING OF ACRG-ACTIN COMPLEXES Actin:ACRG Complex Slope/M - 1 S.D. 0:1 0.781 + 0.015 1:1 0.892 + 0.042 2:1 0.846 + 0.024 11:1 0.840 + 0.022 These r e s u l t s show t h a t the slopes f o r the actin-ACRG complexes are higher than t h a t f o r ACRG alone, but are s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e from each other . This suggests t h a t the b i n d i n g of a c t i n t o ACRG causes a s l i g h t l y 52 increased exposure of the probe t o I~ i n the s o l v e n t , i n agreement wi t h the fluorescence drop induced by a c t i n -b i n d i n g . 1.4-i : 1 1.3' 6 o • o 1.2-A • O A • O A 1.1 -1.0' . 8 A A © • D A A O O A • A Q O • A O A 0.9' 0.0 0.1 - l — 0.2 — I — 0.3 0.4 [Kl]/M Figure 23: Stern-Volmer p l o t s f o r i o d i d e quenching of 11:1 (•), 2:1 (O), and H I (A) actin:ACRG complexes with ACRG (A) S o l u t i o n c o n d i t i o n s were ACRG ( l u M ) , 150mM K C l , 25mM T r i s -HCl, ImM DTT and ImM C a C l 2 , pH 8.0. E x c i t a t i o n wavelength was 390nm and emission wavelength was 497nm. 53 3.2.3 A c t i n T i t r a t i o n P o l a r i s a t i o n studies The environment of the acrylodan probe was monitered by p o l a r i s a t i o n measurements duri n g t i t r a t i o n w i t h a c t i n ( f i g u r e 24). The experiment i n v o l v e d a d d i t i o n of a c t i n (7uM f i n a l c o n c e n t r a t i o n ) t o ACRG (lu.M i n i t i a l c o n c e n t r a t i o n ) and a d d i t i o n of equal volumes of a c t i n b u f f e r ( b u f f e r A) t o a c o n t r o l sample. The i n i t i a l s o l u t i o n c o n d i t i o n s were 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM Ca 2 +, pH 8.0, a t 25°C. The p o l a r i s a t i o n value i n c r e a s e s on the a d d i t i o n of a c t i n . Such a change confirms complex formation and i s l i k e l y t o be the r e s u l t of an increase i n the s i z e , and so, r o t a t i o n a l c o r r e l a t i o n time f o r the p r o t e i n complex t o which acrylodan i s attached. P o l a r i s a t i o n continues t o inc r e a s e w i t h added a c t i n because the excess a c t i n w i l l polymerize onto the nucleus provided by the GA2 complex formed. 54 0.37 0.36-< N < _l o a. 0.35-0.34 [actln]/[ACRG] F i g u r e 24: Fluorescence p o l a r i s a t i o n of ACRG f o l l o w i n g the a d d i t i o n of monomeric a c t i n . The p l o t r e q u i r e d a p a i r of i d e n t i c a l samples of ACRG (luM) i n 150mM K C l , 25mM T r i s - H C l , ImM DTT and ImM C a C l 2 , PH 8.0, a t 25°C. To one sample a l i q u o t s of a c t i n were added (A) and t o the other equal volumes of a c t i n b u f f e r were added (A). Each p o l a r i s a t i o n value was the average of 3 measurements and t y p i c a l standard d e v i a t i o n s were ± 0.003. E x c i t a t i o n ,-was at 390nm and emission was at 497nm i n both cases. 55 PART 3= CONCLUSIONS 3.3.1. Summary of R e s u l t s L a b e l l i n g of plasma g e l s o l i n w i t h acrylodan was s u c c e s s f u l , w i t h an average of about 2 c y s t e i n e s l a b e l l e d per g e l s o l i n . The degree of l a b e l l i n g was not s i g n i f i c a n t l y i n f l u e n c e d by the presence or absence of Ca 2 +. V i s c o s i t y and CD data showed t h a t i n c o r p o r a t i o n of the l a b e l d i d not a f f e c t g e l s o l i n * s a b i l i t y t o bind a c t i n or the f o l d i n g of the g e l s o l i n polypeptide chain i n a s i g n i f i c a n t manner. An i n v e s t i g a t i o n of the e f f e c t of Ca 2 + on horse plasma g e l s o l i n was undertaken. No e f f e c t was seen by CD, however s u b t l e changes c o u l d be detected by f l u o r e s c e n c e . The 2+ presence of Ca decreases the fluo r e s c e n c e of ACRG and causes a s l i g h t red s h i f t i n emission, an increase i n s e n s i t i v i t y t o quenching and a decreased f l u o r e s c e n c e p o l a r i s a t i o n . These three r e s u l t s are c o n s i s t e n t w i t h an increase i n exposure of the acrylodan l a b e l t o s o l v e n t i n the presence of Ca and demonstrate the "opening* of the 2+ g e l s o l i n s t r u c t u r e on bi n d i n g Ca' t h a t i s p r e d i c t e d by the model of Way et a l . (1989 ). A c t i n - b i n d i n g t o ACRG was revealed by flu o r e s c e n c e p o l a r i s a t i o n s t u d i e s . This i n t e r a c t i o n was shown t o be 2+ i n s e n s i t i v e t o Ca by f l u o r e s c e n t i n t e n s i t y t i t r a t i o n s t u d i e s . The decrease i n the fluo r e s c e n c e i n t e n s i t y on the bi n d i n g of a c t i n shown i n these t i t r a t i o n s i s c o n s i s t a n t w i t h an observed increase i n i o d i d e quenching of actin-ACRG 56 complexes r e l a t i v e t o ACRG alone. This suggests t h a t , as i n the case of having f r e e Ca present with ACRG, i n t e r a c t i o n w i t h a c t i n r e s u l t s i n increased exposure of the bound acrylodan t o the s o l v e n t . Such a c o n c l u s i o n would r e q u i r e the a c r y l o d a n - l a b e l l e d s i t e on g e l s o l i n t o be d i s t i n c t from an a c t i n i n t e r a c t i o n s i t e . 3.3.2. Suggestions f o r Further Study Most of the l i t e r a t u r e on g e l s o l i n , p u r i f i e d from a 2+ v a r i e t y of sources, r e p o r t s the Ca s e n s i t i v i t y of a c t i n b i n d i n g . An exception t o t h i s has been reported f o r p i g plasma g e l s o l i n ( H a r r i s and Weeds, 1983). Studies undertaken i n t h i s t h e s i s show a c t i n b i n d i n g by horse plasma g e l s o l i n 2+ t o be i n s e n s i t i v e t o Ca a l s o . The suggestion by Pope et 2+ a l . (1989) t h a t Ca' i n s e n s i t i v i t y i n g e l s o l i n may be the 2+ r e s u l t of the presence of Ca i n the p u r i f i c a t i o n procedure leads t o the question of whether or not a c t i n b i n d i n g with 2+ horse plasma g e l s o l i n would be Ca s e n s i t i v e i f p u r i f i e d 2+ u s i n g a method t h a t d i d not i n v o l v e Ca . Such a method might be developed t o take advantage of s e l e c t i v e b i n d i n g and e l u t i o n of g e l s o l i n using A f f i - G e l Blue, as i n the f i n a l stage of our p r e p a r a t i o n s . In a d d i t i o n , s p e c i f i c e l u t i o n of g e l s o l i n from an A f f i -Gel Blue column w i t h ImM ATP suggests a p o s s i b l e , as yet unstudied, r o l e f o r ATP i n the f u n c t i o n s of g e l s o l i n . 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