Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The kinetics of Ca²⁺ transport and (Ca²⁺ + Mg²⁺)-ATPase activity in human erythrocyte inside-out vesicles Akyempon, Christian Kweku 1981

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1981_A6_7 A49.pdf [ 4.7MB ]
Metadata
JSON: 831-1.0095303.json
JSON-LD: 831-1.0095303-ld.json
RDF/XML (Pretty): 831-1.0095303-rdf.xml
RDF/JSON: 831-1.0095303-rdf.json
Turtle: 831-1.0095303-turtle.txt
N-Triples: 831-1.0095303-rdf-ntriples.txt
Original Record: 831-1.0095303-source.json
Full Text
831-1.0095303-fulltext.txt
Citation
831-1.0095303.ris

Full Text

THE KINETICS OF C a 2 + TRANSPORT AND ( C a 2 + + Mg 2 + ) -ATPase ACTIVITY IN HUMAN ERYTHROCYTE INSIDE-OUT .VESICLES CHRISTIAN -KWEKUjAKYEMPON B. S c . , The Un i v e r s i t y of Water loo, 1975. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES The Facu l t y o f Pharmaceut ica l Sc iences D i v i s i o n of Pharmaceut ica l Chemistry We accept t h i s t h e s i s as conforming to the requ i red s tandard THE UNIVERSITY OF BRITISH COLUMBIA MARCH 1981 (cT) C h r i s t i a n Kweku Akyempon In p resen t ing 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 the requirements f o r an advanced degree a t the Un i v e r s i t y o f B r i t i s h Columbia, I agree tha t the 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 re ference and s tudy. I f u r t h e r agree tha t permiss ion f o r ex tens ive copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head o f my Department or by h i s r ep r e s en t a t i v e s . I t i s understood tha t copying o r pub l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l lowed w i thout my w r i t t e n pe rm iss ion . D i v i s i o n of Pharmaceut ica l Chemistry Facu l t y o f Pharmaceut ica l Sc iences The Un i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P lace Vancouver, Canada V6T 1W5 Date: March 1981 ABSTRACT 2+ The k i n e t i c s and the s to i ch i omet ry o f the Ca pump i n human e ry th rocy te s was i n v e s t i g a t ed us ing i n s i d e - o u t v e s i c l e s as a model. I n s i de -ou t v e s i c l e s were prepared by a mod i f i ed procedure o f Steck and Kant (12) o u t l i n e d by Qu i s t and Rou foga l i s (9 ) , S ince ca lmodul in has been shown to regu la te the a c t i v i t e s o f the ( C a 2 + + Mg 2 + ) -ATPase , and the C a 2 + t r anspo r t system (49, 50 ) , i t s e f f e c t on the k i n e t i c s and s to i ch i omet r y was a l s o i n v e s t i g a t e d i n i n s i d e - ou t v e s i c l e s t r ea ted w i th EDTA to remove endogeneous ca lmodul in (78) . The ca lmodul in used i n t h i s study was p u r i f i e d from human e r y t h r o c y t e s . 2+ The s to i ch i omet r y of the Ca pump was found to 2+ 2+ vary w i th i n t r a c e l l u l a r Ca concen t r a t i on : i n a low Ca concent ra t i on range o f 0.71 uM to 24 u M, i t was es t imated to be 2+ c lose to 2 whereas i n the high Ca range -50 yM to 300 yM, i t was es t imated to be c lose to 1. Ca lmodu l in , ATP concen t ra t i ons 2+ from 0.1 - 3 mM and Mg concent ra t ions o f 3 mM and above d i d not a l t e r the s to i ch i omet ry wh i l e a s t o i ch i ome t r y o f 2 was 2+ 2+ obta ined a t 1 mM Mg concen t ra t i on a t a l l Ca concen t ra t i ons 2+ s t ud i e d . A model f o r Ca e f f l u x i s suggested to e xp l a i n these r e s u l t s . i i . Calmodul in was found to inc rease the V m a x and the a f f i n i t y o f the ( C a 2 + + Mg 2 + )-ATPase f o r C a 2 + . The high a f f i n i t y component was found to have a ^ ^ s s (K-|) o f 2 to 4 pM 2+ 2+ Ca whereas the low Ca a f f i n i t y component was found to have a . K d i s s ^ o f 2 0 0 t 0 3 0 0 y M C a 2 + * L o w e r i n 9 M 9 2 + concent ra t ions i n the presence o f ca lmodu l in r e s u l t e d i n the predominance o f the 2+ high a f f i n i t y component of the Ca - t r anspo r t system which was 2+ i n h i b i t e d a t high Ca concen t ra t i ons . Compet i t ion between 2+ 2+ Ca and Mg f o r an i n h i b i t o r y s i t e l oca ted on the h igh a f f i n i t y 2+ 2+ form, E-|P o f (Ca + Mg )-ATPase enzyme i s suggested to account 2+ 2+ f o r the Ca i n h i b i t i o n observed a t low Mg concen t ra t i ons . Dr. B.D. Roufoga l i s (Superv i so r ) P ro fe s so r D i v i s i o n o f Pharmaceut ica l Chemistry Facu l t y of Pharmaceut ica l Sc i ences . i i i TABLE OF CONTENTS Page Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS x i INTRODUCTION 1 1. Regu la t ion o f i n t r a c e l l u l a r ca l c i um 3 2. (Ca + Mg )-ATPase a c t i v i t y and Ca Transpor t 5 3. Calmodul in Regu la t ion o f Enzymatic a c t i v i t y and Ca2+ Transpor t 6 4. The React ion Scheme o f the ( C a 2 + + Mg 2 + ) -ATPase 8 2+ 5. S to i ch iomet ry o f the Ca pump . - 10 2+ 6. Energe t i c s o f the Ca pump and s to i ch i ome t r y 11 7. The Resea l ing Process . 13 OBJECTIVES OF THE STUDY 17 MATERIALS AND METHODS 20 Ma te r i a l s 2 0 Methods, 22 1. P repara t i on o f Calmodul in from Human red b lood c e l l s 22 2. P repara t ion o f Stepwise ghosts from human e ry th rocy te s 24 3. P repara t i on of I n s i de -ou t Ve s i c l e s 2b" Resea l ing o f i n s i d e - ou t v e s i c l e s 26 EDTA t r ea t ed i n s i d e - ou t v e s i c l e s 26 4. Cha r a c t e r i z a t i on of Sidedness 27 a) Determinat ion o f the p ropo r t i on of i n s i d e - o u t v e s i c l e s 27 i v Page b) Determinat ion o f the p ropor t i on of r i g h t s i d e - ou t v e s i c l e s 28 5. Cha r a c t e r i z a t i o n of Leakiness o f Ve s i c l e s 29 2+ 2+ 2+ 6. Time course o f Ca Uptake and (Ca + Mg ) -ATPase a c t i v i t y 30 2+ 2+ 7. Determinat ion of (Ca + Mg )-ATPase a c t i v i t y and Ca2+ t r anspo r t at s e ve r a l , Ca^ + concent ra t i ons 31 8. Calc ium e x t r a c t i o n and i t s de te rm ina t ion 32 9. Phosphate and P r o t e i n determinat ion 32 RESULTS 35 P u r i f i c a t i o n o f Calmodul in 35 2+ L i n e a r i t y o f ATPase a c t i v i t y and Ca - t r a n s p o r t 35 Cha r a c t e r i z a t i o n of Sidedness 45 Cha r a c t e r i z a t i o n o f l eak iness o f v e s i c l e s 55 E f f e c t o f i n c r e a s i n g ca lmodu l in concen t ra t i on on Ca2+ t r an spo r t and ( C a 2 + + Mg2+)-ATPase a c t i v i t y 55 E f f e c t o f i n c r e a s i ng ATP concen t ra t i on on Ca2+ t r anspo r t and (Ca2+ + Mg2+)-ATPase a c t i v i t y 62 2+ E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on Ca2+ t r anspo r t and (Ca2+ + Mg2+)-ATPase a c t i v i ty 62 2+ E f f e c t o f Mg concent ra t i on 83 Comparis ion of bu f f e r used f o r study 89 v Page DISCUSSION 97 2+ Measurement o f Mg -ATPase a c t i v i t y as a t e s t f o r Calc ium Contaminat ion 97 I n t e g r i t y o f V e s i c u l a r P repa ra t i on 97 a) Cha r a c t e r i z a t i o n o f Leakiness 97 b) Cha r a c t e r i z a t i o n o f s idedness 99 E x t r a c t i o n o f ca lmodul in from i n s i d e - o u t v e s i c l e s w i th EDTA 100 Homogeneity o f Prepared Calmodul in 102 2+ E f f e c t o f ATP on the Ca pump and S to i ch iomet ry 102 2+ E f f e c t o f Ca and Calmodul in on the S to i ch iomet ry o f the C a 2 + pump 103 2+ The E f f e c t o f Mg on the K i n e t i c s and S to i ch iomet ry of the Ca2+ pump. 107 Phy s i o l o g i c a l Role of the High and Low A f f i n i t y Components 110 In f luence o f the cho ice of b u f f e r on the K i n e t i c s o f the C a 2 + - t ransport -ATPase 111 2+ A Model o f Ca T rans l o ca t i on i n Human E ry th rocy tes 112 2+ a) Low Ca range: S to i ch iomet ry o f 2. 112 2+ b) High Ca range: S to i ch iomet ry o f 1. 113 SUMMARY AND CONCLUSIONS 117 REFERENCES 119 APPENDIX 129 vi LIST OF TABLES TABLE PAGE 2+ 1 Time course of Mg -ATPase a c t i v i t y i n e r y th ro cy t e v e s i c l e s i n the absence and presence of 2 yg/ml ca lmodu l in 40 2 P ropor t i on of i n s i d e - ou t v e s i c l e s , r i g h t - s i d e out v e s i c l e s and unsealed v e s i c l e s i n v e s i c u l a r p repara t ions 49 3 E f f e c t o f i n c r ea s i ng T r i t o n X - l 00 concen t ra t i on on Tota l ATPase a c t i v i t y (at 100 yM C a 2 + f r e e ; i n two sample de t e rm ina t i on s ) . 52 2+ 4 E f f e c t of i n c r ea s i ng Ca concen t ra t i ons on the s to i ch i omet r y of the C a 2 + pump i n non-EDTA t r ea t ed IOV 69 2+ 5 E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s to i ch i omet r y of the C a 2 + pump i n EDTA t r ea ted IOV i n the absence of Calmodul in 75 2+ 6 E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s t o i ch i ome t r y of the C a 2 + pump i n EDTA t r ea ted IOV i n the presence of 0.5 yg/ml ca lmodu l in 77 2+ 7 E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s t o i ch i ome t r y o f the C a 2 + pump i n EDTA t r ea t ed IOV i n the presence of 2 ym /m l ' c a lmodu l i n 80 2+ 8 E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s t o i ch i ome t r y of the C a 2 + pump i n the presence o f 1 mM M g 2 + and 2 yg /m l ca lmodu l i n . 87 9 Summary of the K i n e t i c parameters of the ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y and C a 2 + Transpor t i n human e ry th rocy te i n s i d e - ou t v e s i c l e s . 99 v i i LIST OF FIGURES FIGURE PAGE 1 0.1% SDS - 7.5% Po lyacry lamide ge l s o f p u r i f i e d human e r y th rocy te ca lmodu l in and standards of known molecu la r weight 36 2 P l o t o f Rf va lues aga ins t log of mo lecu la r weight of p r o t e i n standards 38 3 Time course of ATPase a c t i v i t y i n i n s i d e - ou t v e s i c l e s not t r ea t ed w i th EDTA at 100 yM f r ee C a 2 + i n the absence and presence of 2 yg/ml ca lmodu l in 41 2+ 2+ 4 Time course of (Ca + Mg )-ATPase a c t i v i t y i n non-EDTA t r ea t ed i n s i d e - ou t v e s i c l e s a t 100 yM f r ee Ca2+ i n the absence and presence of 2 yg/ml ca lmodu l in 43 2+ 2+ 5 Time course of Ca Transpor t a t 100 yM f r ee Ca i n non EDTA t r ea t ed i n s i d e - ou t v e s i c l e s . 45 6 Time course of ATPase a c t i v i t y at 100 yM and 2.34 yM Ca 2+ in i n s i d e - ou t v e s i c l e s t r ea t ed in EDTA. 47 7 E f f e c t o f i n c r ea s i ng T r i t o n X-100 concen t ra t i on on t o t a l ATPase a c t i v i t y at 100 yM f ree Ca?+ i n i n s i d e - ou t v e s i c l e s 50 8 Cha r a c t e r i z a t i o n of leakness of i n s i d e - ou t v e s i c l e s not t r ea ted w i th EDTA 53 9 Cha r a c t e r i z a t i o n of l e ak i ne s s o f i n s i d e - ou t v e s i c l e s t r ea t ed i n 1 mM EDTA f o r 30 min a t 37 UC 55 10 Cha r a c t e r i z a t i o n of the l eak i ne s s of EDTA t r ea t ed i n s i d e - ou t v e s i c l e s i n the presence of 2 yg/ml ca lmodu l in 57 11 E f f e c t o f i n c r ea s i ng ca lmodu l in concen t ra t i on on Ca2+ t r an spo r t and ( C a 2 + + Mg2+)-ATPase a c t i v i t y at 4 yM f r ee Ca2+ i n EDTA-treated i n s i d e - ou t v e s i c l e s 59 vi i i FIGURE PAGE 12 E f f e c t o f i n c r ea s i ng ca lmodu l in concen t ra t i on on C a 2 + t r anspo r t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y a t 100.uM f r ee C a 2 + i n EDTA t r ea ted i n s i d e - ou t v e s i c l e s , 61 13 E f f e c t o f i n c r ea s i ng ATP concen t ra t i on on C a 2 + Transpor t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y at 100 yM f r ee C a 2 + concen t ra t i on i n the presence of 2 yg/ml ca lmodu l in 63 2+ 14 E f f e c t o f i n c r ea s i ng Ca concen t ra t i ons on C a 2 + t r an spo r t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n non EDTA t r ea ted i n s i d e - ou t v e s i c l e s 65 2+ 15 E f f e c t of i n c r ea s i ng Ca concen t ra t i on on Ca t r anspo r t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n non-EDTA t r ea ted i n s i d e - ou t v e s i c l e s i n the presence of 2 yg/ml ca lmodu l in 67 16 E f f e c t o f i n c r ea s i ng C a 2 + concen t ra t i on on C a 2 + t r anspo r t and (Ca 2+ + Mg 2 + ) -ATPase a c t i v i t y i n EDTA t r ea ted v e s i c l e s i n the absence and presence of 0.2 yg/ml ca lmodu l in 70 2+ 17 E f f e c t of i n c r eas i ng Ca concen t ra t i ons on C a 2 + t r anspo r t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n the absence and presence of 0.5 yg/ml ca lmodu l in i n EDTA t r ea t ed i n s i d e - ou t v e s i c l e s 72 2+ 18 Per cent A c t i v a t i o n by ca lmodu l in of Ca Transpor t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n EDTA t r ea t ed i n s i d e - ou t v e s i c l e s 74 2+ 19 E f f e c t of i n c r ea s i ng Ca concen t ra t i on on C a 2 + t r anspo r t and (Ca 2+ + Mg 2 + ) -ATPase a c t i v i t y i n the absence and presence of 2 yg/ml ca lmodu l in i n EDTA t r ea ted I0V 78 20 Ead ie -Ho f f s tee p l o t o f ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y and C a 2 + Transport 81 i x FIGURE PAGE 2+ 21 E f f e c t of i n c r ea s i ng Ca concen t ra t i on on C a 2 + t r an spo r t and ( C a 2 + + Mg2^')-ATPase a c t i v i t y i n the presence o f 2 mM M g 2 + and i n the absence and presence of 2 yg/ml 83 2+ 22 E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on C a 2 + Transpor t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n the presence of 1 mM M g 2 + concen t ra t i on and i n the presence of 2 yg/ml ca lmodu l in 85 2+ 2+ 2+ 23 Comparison of Ca Transport and (Ca + Mg ) -ATPase a c t i v i t y i n T r i s bu f f e r and Hepes bu f f e r 88 x LIST OF ABBREVIATIONS A DP ATP ATPase °C ( C a 2 + -Mg 2 + ) -ATPase cAMP DEAE DiPI DTNB E EDTA EGTA EP e t a l F 9 xg IOV adenosine 5' -d iphosphate adenosine 5' - t r i phospha te adenosine t r iphosphatase degree cent ig rade 2+ Mg -dependent ca l c ium s t imu l a t ed ATPase c y c l i c adenosine 5' -monophosphate D ie thy l aminoethyl Di phosphati d y l i nosi t o l [5,5 - d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) ] enzyme ethy lene diamine t e t r a a c e t a t e , di sodium s a l t e t h y l e n e g l y c o l - b i s - ( g -aminoe thy l e the r ) N,N' - t e t r a - a c e t i c a c i d phosphory lated enzyme in te rmed ia te and others Faraday 's constant gram a c c e l e r a t i o n o f g r a v i t y i n s i d e - o u t v e s i c l e s d i s s o c i a t i o n constant M ichae l i s -Menten constant mil l i x i vi mi cro M molar mg mi 1 1 i gram min minute ml mi 1 1 i 1 i t e r p i m i c r o l i t e r mM m i l l i m o l a r concen t ra t i on viM rtiicromolar concen t r a t i on -MW molecu la r weight nmoles nanomoles P. i no rgan i c phosphate PI phosphat idy l i n o s i t o l PMSF p - /me thy l s u l f o n y l f l u o r i d e RBC red b lood c e l l ROV r i g h t s i d e - ou t v e s i c l e s SDS-PAGE sodium dodecyl su lpha te -po l yac ry l am ide gel e l e c t r opho r e s i s Z charge Rb rub id ium sec . second x i i x i i i ACKNOWLEDGEMENTS I wish to thank my supe r v i s o r , Dr. B.D. Roufoga l i s f o r h i s suppor t , encouragement and guidance throughout t h i s work. I am g ra te fu l to Dr. F.L. Larsen f o r h i s c on s t r u c t i v e c r i t i c i s m and a s s i s t ance i n the p u r i f i c a t i o n o f ca lmodu l i n . The help of Mr. Roland Burton i n the computer ana l y s i s o f the data was g r ea t l y app rec i a t ed . I am indebted to the Canadian Red Cross f o r the generous supply o f b lood f o r t h i s work and to the Medical Research Counc i l who prov ided the f i n a n c i a l a s s i s t ance throughout my study. My s i n ce re thanks to members i n our l abo ra to ry group f o r t h e i r moral suppor t , and the f a c u l t y and s t a f f and the graduate s tudents body f o r making my s tay a t UBC en joyab le and wor thwhi le . L a s t l y , to my committee, thank you f o r the f i n a l read ing and co r r e c t i on s suggested. xiv D E D I C A T I O N To my l o v i n g w i f e , C e c i l i a INTRODUCTION The con t ro l and r egu l a t i on of many c e l l u l a r processes i n mammalian c e l l s has been a t t r i b u t e d to the asymmetric d i s t r i b u t i o n o f ca l c ium in t i s s ue s and o r gane l l e s . The nerve a c t i on po t en t i a l f o r i n s t an ce , i s known to be t r i g g e r ed by an i n f l u x o f ca l c ium through the axonal membrane (1 ) . S i m i l a r l y , muscle c on t r a c t i on i s s a i d to be evoked by the re l ease o f ca l c ium from- the sa rcop lasmic r e t i cu l um (S .R . ) i n s k e l e t a l muscle or from membrane bound pools i n smooth muscle (2 ) . In many c e l l u l a r responses to d i f f e r e n t s t i m u l i , ca l c ium i s suggested to f unc t i on as a second messenger o r to a f f e c t adeny late cyc l a se or phosphodiesterase a c t i v i t i e s ( 3 ) . 2+ The ro l e s o f ca l c ium (Ca ) , such as these l i s t e d above, depend 2+ on the f a c t t ha t J t he i n t r a c e l l u l a r Ca concen t ra t i on i s very low. The normal f unc t i on o f the red c e l l i s a l s o dependent on main-2+ tenance o f a low i n t r a c e l l u l a r Ca concen t r a t i on . Schatzmann i n 1966 ( 4 ) , i n d i c a t e d tha t the maintenance o f the low i n t r a c e l l u l a r 2+ Ca concen t ra t i on observed i n the red b lood c e l l i s due i n pa r t to an ATP dependent ca l c ium pump now recogn ized to be coupled to the 2+ 2+ (Ca + Mg ) - ATPase enzyme. The ready a v a i l a b i l i t y o f human e r y t h r o c y t e s , i t s l ack of i n t r a c e l l u l a r o r g ane l l e s , i t s r e l a t i v e s t a b i l i t y and the f a c t t ha t they seem to lack a Na-Ca exchange (5 , 6) makes the human e r y th ro cy t e a unique model f o r d e f i n i n g 2 the mechanism and k i n e t i c s o f the ca l c ium pump. 2+ S ince the Ca t r an spo r t p r o t e i n i n the red c e l l i s found a t the i n t r a c e l l u l a r su r face o f the e r y th rocy te membrane, many d i ve r se procedures have been used to ob ta in e r y th rocy te ghosts which can then be loaded w i th s p e c i f i c agents ( 7 , 8 , 9 ) . 2+ Others have used i n t a c t c e l l s loaded w i th ca l c ium by a Ca s p e c i f i c ionophore A23187 f o r t h e i r s tud i e s (10) . A major l i m i -t a t i o n o f these procedures i s the d i f f i c u l t y i n knowing and 2+ 2+ manipu la t ing the i n t r a c e l l u l a r concent ra t ions o f Mg , Ca and ATP (5 ,11 ) . Hence, i t i s not s u r p r i s i n g t ha t many d i s c r epanc i e s e x i s t i n the l i t e r a t u r e regard ing the s t o i ch i ome t r y and the k i n e t i c parameters of the ca l c ium pump i n human red c e l l s . In t h i s s tudy , i n s i d e - ou t v e s i c l e s prepared from human e r y th rocy te ghosts by a mod i f i ed procedure o f Steck ;and Kant (12) was used. Whereas t h i s i s not n e c e s s a r i l y the best method, i t enables the i n v e s t i g a t o r to c a r e f u l l y con t ro l and manipulate the environment and ma te r i a l s under study s ince the enzyme -2+ 2+ (Ca + Mg )-ATPase i s now e x t e r n a l l y o r i e n t e d . Using human 2+ e r y th rocy te i n s i d e - ou t v e s i c l e s , the s t o i ch i ome t r y o f the Ca pump and the k i n e t i c parameters o f the enzyme was i n v e s t i g a t e d 2+ over a wide range o f f ree Ca concent ra t ions and a t d i f f e r e n t 2+ concent ra t i ons of Mg , ATP and p u r i f i e d ca lmodu l i n . 3 1. Regu la t ion o f i n t r a c e l l u l a r ca l c i um: 2+ Under normal p h y s i o l o g i c a l c ond i t i o n s , the f ree Ca concen t ra t i on i n s i d e the red c e l l i s low - between ICf^M to 10~^M , 2+ whereas the plasma or e x t r a c e l l u l a r Ca concen t ra t i on i s i n the m i l l i m o l a r range. I t has been demonstrated tha t the maintenance 2+ of t h i s low i n t r a c e l l u l a r Ca concen t ra t i on i s ach ieved by the 2+ opera t ion of an a c t i v e Ca pump, coupled to an i n t r a c e l l u l a r 2+ 2+ membrane bound enzyme - the (Ca + Mg )-ATPase - whose a c t i v i t y requ i res the expend i ture o f ATP as an energy source ( 4 ,7 ,8 ,13) .0 the r 2+ f a c t o r s se r v i ng to con t ro l i n t r a c e l l u l a r Ca concen t ra t i on i n c l ude : a) The ra the r low pe rmeab i l i t y o f f r e sh red c e l l s to 2+ ca lc ium (14, 15) and b) The b ind ing o f Ca to i n t r a c e l l u l a r h igh 2+ a f f i n i t y b ind ing s i t e s now recogn ized to be Ca b ind ing p ro t e i n s and i n t r a c e l l u l a r anions (16-19) . 2+ The low i n t r a c e l l u l a r Ca concen t ra t i on i n the e r y th rocy te i s necessary to support the b iconcave shape o f the red c e l l and a l so to ensure red c e l l d e f o rmab i l i t y . The b iconcave shape endows the red c e l l w i t h a h igh su r f a ce : volume r a t i o r equ i r ed f o r r ap i d d i f f u s i o n o f r e s p i r a t o r y gases. Red c e l l d e f o rmab i l i t y a l lows the passage o f red c e l l s through very smal l c a p i l l a r i e s , o f ten one h a l f the red c e l l d iameter (20) . I t has been observed t ha t i nc reased i n t r a c e l l u l a r 4 ca l c ium r e s u l t s i n i nc reased c rena t i on and r i g i d i t y o f red c e l l s (21,22) probably due to ca l c ium i n t e r a c t i o n s w i th c o n t r a c t i l e - l i k e p ro te i n s of the a c t i n - s p e c t r i n network which under l ines the i n t e r n a l su r face o f the red c e l l membrane (23, 24, 25) . This i n t e r a c t i o n may then a l t e r the c on f i gu r a t i on of t h i s network and hence the c e l l shape, as suggested independent ly by K i r k p a t r i c k e t a l . (26) and Pa lek , (27) . Weed e t a l . (28) and LaCe l l e e t a l . (29) a s soc i a t ed inc reased ca l c ium accumulat ion w i th decreased de fo rmab i1 i t y and inc reased v i s c o s i t y o f e r y t h r o c y t e s , , r e s p e c t i v e l y . Other workers , i n c l u d i n g A l l a n and M i c h e l l , have shown tha t ca l c ium accumulat ion 2+ may r e s u l t i n Ca - dependent changes i n the l i p i d compos i t ion o f the red c e l l membrane (30 ,31) , r e s u l t i n g i n accumulat ion o f 1, 2 2+ d i a c y l g l y c e r o l dur ing c r ena t i o n . Increases i n i n t r a c e l l u l a r Ca concent ra t i on a l s o lead:; to a s e l e c t i v e K + l o s s , a phenomenon r e f e r r e d to as the Gardos e f f e c t , ( 3 2 ) which r e s u l t s i n shr inkage o f the red c e l l (33-35) . Dunham and Glynn (36) have a l so demonstrated tha t Ca concent ra t ions above 0.1mM.'strongly i n h i b i t the ouabain s e n s i t i v e (Na + + K + ) - ATPase a c t i v i t y i n i s o l a t e d red c e l l membranes. Enzymatic changes imp l i c a t ed as a consequence o f ca l c ium accumulat ion i nc lude a c t i v a t i o n o f . a po lyphosphat idy l i n o s i t o l phosphodiesterase (37) and a c t i v a t i o n o f a transaminase enzyme (38) . I t i s hence ev iden t from the above tha t i n t r a c e l l u l a r ca l c ium accumulat ion leads to ser ious- de l e t e r i ou s e f f e c t s which e ven tua l l y 5 r e s u l t s i n a degenerat ive process o f ageing o f red c e l l s . 2+ 2+ 2+ 2. (Ca + Mg )-ATPase a c t i v i t y and Ca Transpor t : Dunham and Glynn (39) i n 1961 were the f i r s t to repor t 2+ 2+ the presence i n the red c e l l membrane o f a Mg dependent Ca a c t i v a t e d ATPase a c t i v i t y . S t rong exper imenta l ev idence a l so 2+ suggests t ha t the red c e l l membrane conta ins a Ca pump which 2+ a c t i v e l y extrudes Ca aga in s t an e l e c t rochem ica l g rad ien t (4 ,40 -42) . There i s now overwhelming ev idence tha t there i s an 2+ 2+ 2+ a s s o c i a t i o n between Ca pumping and (Ca + Mg )-ATPase a c t i v i t y , analogous to the a s s o c i a t i o n between the Na + pump and the (Na + + K +)-ATPase a c t i v i t y (40) . Evidence suppor t ing the 2+ 2+ 2+ a s s o c i a t i o n between Ca pumping and (Ca + Mg )-ATPase a c t i v i t y i n c l ude the f o l l o w i n g : a . Ca s t imu la tes the (Ca + Mg )-ATPase a c t i v i t y on ly at the i n t r a c e l l u l a r membrane sur face (40) ; and tha t 2+ ATP and Mg are requ i r ed w i t h i n the c e l l (41) . Inorgan ic phosphate re leased a l s o appears i n s i d e the c e l l (42) . b. Both the a c t i v e C a 2 + t r anspo r t and ( C a 2 + + Mg 2 + ) -ATPase 2+ a c t i v i t y r equ i r e Mg (39, 41) ; and both s p e c i f i c a l l y use up ATP over other nuc l eo t i des (43) . 2+ c. S t ront ium can s u b s t i t u t e f o r Ca i n s t i m u l a t i n g the 2+ 2+ (Ca + Mg )-ATPase i n red c e l l membranes (44,45) and 6 i s a l s o t r anspo r ted from resea led ghosts by a 2+ mechanism tha t requ i res Mg and ATP (7 ) . d. The ra te of ca l c ium t r an spo r t from resea led ghosts (9 , 47) or i n t a c t c e l l s (10) i s independent o f 2+ e x t r a c e l l u l a r Ca concen t r a t i on . e. E f f l u x o f ca l c i um does not appear to be coupled to the 2+ + inward movement o f Mg ( 7 , 56") or H ion (41) . 2+ 3. Calmodul in Regu la t ion o f Enzymatic A c t i v i t y and Ca Transpor t : 2+ 2+ I t is. now known tha t the a c t i v i t y o f the (Ca + Mg ) -2+ ATPase enzyme and Ca t r an spo r t are enhanced and regu la ted by a 16t-17,000 da l t o n , heat s t ab l e a c i d i c p r o t e i n (49, 50) now c a l l e d ca lmodul in (51) . Calmodul in i s present on the cy top lasmic su r face o f the e r y th rocy te membrane. I t i s ub i qu i t ou s l y d i s t r i b u t e d i n most eukaryotes and i t e x i s t s i n app rec i ab l e q u a n t i t i e s i n bovine b r a i n . Calmodul in has a l s o been found to l a ck t i s s u e o r spec ies 2+ 2+ s p e c i f i c i t y (52) . Apar t from the r egu l a t i o n o f (Ca + Mg )-ATPase 2+ a c t i v i t y and Ca t r a n spo r t , ca lmodul in a l s o regu la tes many fundamental c e l l u l a r a c t i v i t i e s summarized i n the diagram below as d e t a i l e d by Cheung (53) : 7 Myosin l i g h t cha in k inase Adenylate cyc l a se Phosphodiesterase Phosphor lyase k inase Phosphol ipase Guanylate <s -Cyc lase 2+ ^ Ca Dependen^-""^ p ro t e i n k inase NAD k inase Neurotransmi tter^ re l ease Calmodul in ^ C a 2 + -ATPase M ic ro tubu le d i sassembly Membrane Phsphory la t i on Others? ( po s t s ynap t i c , nuc lea r f un c t i on s ) Wins (54) i n 1969 was the f i r s t to r epo r t the ex i s t ence 2+ 2+ o f two forms o f the (Ca + Mg )-ATPase enzyme. The high a f f i n i t y form he i n d i c a t e d was sa tu ra ted at around 10 yM 2+ 2+ Ca whereas the low a f f i n i t y form was sa tu ra ted a t 100 yM Ca or more. This observa t ion by Wins has been conf i rmed by many i n v e s t i g a t o r s (54-58) a l though Schatzmann (5) a t t r i b u t e d the ex i s tence o f the two forms to an a r t i f a c t presumably formed dur ing membrane p repa ra t i on . 8 Gopinath and V incenz i (59) have determined the apparent values to be 2 yM and 17 yM i n the absence o f ca lmodu l i n . 2+ However, a s i n g l e apparent Km f o r Ca o f 4 yM was ob ta ined by them i n the presence o f near s a t u r a t i n g ca lmodul in concen t r a t i ons . They i n d i c a t ed tha t the h igher Km form d isappeared i n the presence o f s a t u r a t i n g concent ra t ions o f ca lmodu l i n . A l so Schar f f and Foder (58) Hanahan and Ekholm (60) , Mac lntyre (61) and Sakardi e t a l (62) 2+ have a l so shown tha t ca lmodu l in s t imu l a t i o n of Ca t r an spo r t i n i n s i d e - o u t e r y th rocy te v e s i c l e s i s a s soc i a t ed w i th a s h i f t i n a 2+ high Km Ca pump to a low Km form w i th a r e s u l t a n t i n c rease i n the t r an spo r t r a t e . 2+ 2+ 4. The React ion Scheme o f the (Ca •+ Mg )-ATPase: The r eac t i on sequence suggested to c ha r a c t e r i z e the ( C a 2 + + Mg 2 + ) -ATPase (63-65) can be desc r ibed as f o l l o w s : 9 The above scheme an t i c i p a t e s t ha t there are two forms o f the enzyme - and E£. The E-| form o f the enzyme, which i s 2+ suggested to be the high a f f i n i t y form, i n i t i a l l y b inds Ca and ATP w i th a high a f f i n i t y , r e s u l t i n g i n the format ion of a high 2+ energy phosphoenzyme E-jP. In the presence o f Mg , E-jP undergoes a conformat iona l change and i s thus converted to E2P, which i s 2+ presumably a low energy phosphoenzyme from which Ca d i s s o c i a t e s and i s t r a n s l o c a t ed across the membrane i n t o the e x t r a c e l l u l a r space. E2P i s then r a p i d l y hydro lysed i n the presence o f water to E2, the low a f f i n i t y form of the enzyme. 2+ Mg i s thought t o promote both the phosphory la t i on step ( i . e . the format ion o f E-jP) (63, 65, 66) and the dephosphory la t ion step ( i . e . the format ion o f E2) (63, 67 ) , s i n ce the presence of 2+ Mg reduces the T ^ value f o r the format ion of E-jP (from 2+ 40-60 sees, i n the absence o f Mg to about 5 sees) and s i m i l a r l y reduces the T - ^ value f o r dephosphory la t ion so t ha t complete decomposit ion of E-jP to E2 i s ach ieved w i t h i n 4-5 sees. (68) . The r o l e o f ca lmodu l in i n the r ea c t i on mechanism i s summarized below: A. Calmodul in i s suggested to decrease the o f the ca l c i um pump ( i . e . i n c rease the a f f i n i t y o f the enzyme f o r ca lc ium) (66, 68, 69 ) . 10 2+ B. In the absence of Mg , ca lmodu l in i nc reases both the 2+ ra te of phosphory la t ion and the l e v e l o f the Ca dependent E-jP format ion (66, 68, 69 ) . 2+ C. In the presence of Mg , ca lmodul in does not a f f e c t the 2+ l e v e l o f Ca dependent phosphoenzyme (E-jP) format ion but s t imu l a te s i t s r a te o f format ion (66) . D. K a r l i s h (66) , proposes t ha t the £2 s t a t e o f the enzyme i s converted to E-| p r e f e r e n t i a l l y by ca lmodu l i n , hence making a v a i l a b l e tha t form of the enzyme (E-j) to which 2+ ATP and Ca must b ind p r i o r to phospho ry l a t i on . E. Rega and Garrahan (69) have a l so i n d i c a t e d t h a t i n the 2+ absence o f Mg , ca lmodul in s t imu l a te s dephosphory la t ion o f E^ P to 2+ F. J e f f e r y e t a l . ( 7 0 ) showed tha t both Mg and ca lmodu l in i nc reased the decomposit ion of phosphory la ted enzyme; the e f f e c t s o f these two a c t i v a t o r s was a n t a gon i s t i c i n d i c a t i n g a s i m i l a r mechanism of Eyp decompos i t ion . 2+ 5. S to i ch iomet ry o f the Ca pump: 2+ Whereas i t i s f i r m l y e s t ab l i s h ed tha t the r a t i o o f Ca t r anspo r ted per ATP u t i l i z e d i n the sa rcop lasmic r e t i c u l um i s 2:1 (71) e f f o r t s to a r r i v e at a s i m i l a r r e l a t i o n s h i p regard ing 11 ATP -dependent C a C T e x t r u s i on i n the human red b lood c e l l has y i e l d e d c o n f l i c t i n g r e s u l t s , and i s y e t to be r e so l v ed . Using 2+ Ca loaded resea l ed e r y th rocy te ghosts , Schatzmann and V incenz i (40) and Schatzmann (47) i n d i c a t e d a s t o i c h i o m e t r i c r a t i o o f 1:1 f o r the red b lood c e l l ca l c ium pump. S i m i l a r r e s u l t s were obta ined by Larsen e t a l . (72) . These i nves t i ga to r s , howeve r , have used most ly outdated red b lood c e l l s f o r t h e i r exper iments i n a r r i v i n g a t t h e i r c onc l u s i on . Qu i s t and Roufoga l i s (9) us ing ghosts prepared from f resh red b lood c e l l s but us ing lanthanum to i n h i b i t complete ly ca l c ium t r an spo r t observed t ha t on ly 50 per cent o f the ATPase a c t i v i t y was i n h i b i t e d by lanthanum when 2+ 100% o f the Ca t r an spo r t was b locked . By s ub t r a c t i n g t h i s ATPase a c t i v i t y from the t o t a l ATPase a c t i v i t y , Qu i s t and 2+ Roufoga l i s a r r i v e d a t an es t imated Ca to ATP r a t i o o f 2 :1 . The i r work was supported by Sakard i e t a l . ( 1 0 ) i n i n t a c t c e l l s 2+ loaded w i th ca l c ium by the a i d o f a Ca s p e c i f i c ionophore A23187. Using a s i m i l a r lanthanum i n h i b i t i o n procedure to Qu i s t and Roufoga l i s (9) they a s ce r t a i ned the s t o i ch i ome t r y to be 2 :1 . 2+ 6. Ene rge t i c s o f the Ca Pump and S to i ch i ome t r y : 2+ The o ve r a l l Gibbs energy change (AG^) f o r a c t i v e Ca t r anspo r t coupled to the h yd r o l y s i s o f ATP i n red c e l l s can be c a l c u l a t e d v i a the f o l l ow i ng equat ion (73): [ADP]. [P. ] [ C a 2 + ] Q AG. = AG 0 + RT In - ' — + nRT In s^- +.nZFE 1 " [ATP] . [Ca ' ] ' A ?•• B »»-where AG i s the s tandard f r ee energy o f ATP and i s c a l c u l a t e d 2+ to be - 7 . 2 Kcal per mole (47) ; n i s the number o f Ca ions t r anspo r ted per mole o f ATP hyd ro l y s ed , E represents the:: membrane po t en t i a l (ex te rna l minus i n t e r n a l ) . The term des ignated A represents the change of f ree energy o r chemical energy produced dur ing the h yd r o l y s i s o f ATP and has been determined by Benz inger e t a ! . (74 ) to be-13.038 Kcal/mole a t 37°C. The term B and C represent the osmot ic and e l e c t r i c a l work r equ i r ed to be done i n o rder to t r a n s l o c a t e n ca l c ium. R and T represent the gas constant and abso lu te t empe ra tu r e , r e spec t i v e l y . 2+ To support a Ca t r an spo r t ed : ATP used r a t i o o f 1 to 1 ( i e n= l ) , Schatzmann (47) c a l c u l a t e d the sum o f the osmot ic and e l e c t r i c work requ i r ed to be equal to 5.349Kcal per mole (assuming t h a t the ( [ C a 2 + ] i = 5 x 1 0 " 7 and [ C a 2 + ] Q = 1.5 x 1 0 " 3 M). Under these c ond i t i o n s , Schatzmann contends t ha t " there i s no ob j e c t i on aga ins t the t r an spo r t because the chemical energy a v a i l a b l e (-13.038 Kcal/mole) i s l a r g e r than the work done." Although Schatzmann i nd i c a t e s tha t a s t o i ch i ome t r y o f 2 to 1 i s 2+ thermodynamical ly imposs ib le i n the red c e l l Ca pump, i t seems 13 ev iden t from h i s own c a l c u l a t i o n s t ha t the system has more than 2+ enough chemical energy to support the t r a n s l o c a t i o n of 2 Ca "/ATP u t i l i z e d under the same c ond i t i o n s . Th is i s because the sum o f the osmotic and e l e c t r i c a l work requ i r ed to be done can now be computed to be 10.698 Kcal/mole (n = 2; t he re fo re 2x5.349 = 10.698 Kca l /mo le ) . 2+ Even a t ex te rna l ca l c ium concentrat ions of lOmM (Ca = lOmM)., Schatzmann c a l c u l a t e d the t o t a l work needed to be done to t r a n s l o c a t e 2 C a 2 + per ATP s p l i t to be 13.036 Kcal/mole at 37°C,meaning t ha t the net Gibbs energy change AG^ was equal to zero (chemical energy a v a i l a b l e i s equal to -13.038 Kcal/mole a t 37°C). Hence even a t tha t h igh e l e c t r ochem i ca l g rad ien t i t seems the t r a n s l o c a t i o n of':-'. 2+ 2 Ca /ATP s p l i t i s p o s s i b l e . I t i s extremely u n l i k e l y though t ha t 2+ ex te rna l Ca concen t ra t i on under p h y s i o l o g i c a l cond i t i ons exceeds lOmM, under which cond i t i ons the s t o i c h i o m e t r i c r a t i o of 2 i s imposs i b l e . I t shou ld be noted however, tha t Shatzmann 2+ showed tha t a c t i v e Ca t r an spo r t cou ld proceed aga ins t an ex te rna l CaC^ concen t ra t i on as h igh as 5 mM. 7. The Resea l ing Process: Observat ions o f Hoffman(75) suggest t ha t a f t e r hypoton ic l y s i s and subsequent r e s t o r a t i o n of i s o t o n i c i t y by the add i t i o n o f a l k a l i s a l t s , ghost popu la t ions obta ined from human e ry th rocy tes can be d i f f e r e n t i a t e d i n t o 3 types as f o l l ow s : 14 Type 1 Ghosts t h a t resea l immediate ly a f t e r hemolys is Type 2 Ghosts t ha t resea l a f t e r a dd i t i o n o f a l k a l i ions Type 3 Ghosts tha t remain leaky regard less o f the exper imenta l c o n d i t i o n . The obse rva t i on above have been conf i rmed by Bodeman and Passow (76) » who a l so i n d i c a t e t ha t whereas ghosts o f the type 1 group are incapab le o f i n c o r po r a t i n g a l k a l i ions added a f t e r hemo lys i s , ghosts o f the type 2 group are ab le to t rap a l k a l i ions added to the hemolysate i n the course o f the r e s e a l i n g process . Through a s e r i e s o f exper iments on the temperature dependence and time course o f Rb l o s s from e ry th rocy te ghosts Bodeman and Passow (76) showed tha t the p ropo r t i on o f the three types o f ghosts i n the t o t a l popu la t i on s t r ong l y depend on the temperature a t which hemolys is was performed. The y i e l d o f type 2 ghosts inc reases i f one lowers the temperature a t the time of hemolys is from 27°C to 0°C. A f t e r hemolys is a t 0°C, the authors i n d i c a t e t ha t about 60% o f the ghosts r e t a i n the capac i t y to resea l upon rewarming a t 37°C. For most pe rmeab i l i t y s t u d i e s , a h igh y i e l d o f these ghosts (Type 2) i s d e s i r a b l e s i n ce they can be loaded a t low temperature w i th s a l t s and subs t ra tes and subsequent ly be resea led by i n cuba t i on a t 37°C. At 0°C, type 1 ghosts are almost n e g l i g i b l e ; however i f hemolys is i s performed a t 25°C o r 37°C, type 1 ghosts w i l l c o n s t i t u t e between 15 - 20% of the t o t a l . 15 a) E f f e c t o f complexing agents and d i v a l e n t ca t i ons on the r e s e a l i n g process : Complexing agents ( l i k e EDTA and EGTA) have been shown to prevent the r e s e a l i n g o f the ghost membrane a t e l eva ted temperatures when present i n the medium at the i n s t a n t o f hemolys is (76) . Neve r the less , a t 0°C, the presence o f these agents i n the . . . medium had no e f f e c t on the subsequent r e s e a l i n g process a t 37°C. Magnesium and ca l c ium have been found to f a c i l i t a t e the r e s e a l i n g process by Bodeman and Passow (76),who a l so pos tu l a t e tha t one o f these ions may be the membrane cons t i t u en t which normal ly p a r t i c i p a t e s i n the maintenance o f the i n t e g r i t y o f the red b lood c e l l membrane. Johnson and Kirkwood (77) have i n d i c a t e d the e f f e c t o f d i v a l e n t ca t i ons to c on s i s t o f two d i s t i n g u i s h a b l e p rocesses : a) they inc rease the ra te of r e s ea l i ng b) they prevent the onset o f i r r e v e r s i b l e pe rmeab i l i t y probably by s t a b i l i z i n g the membrane. 2+ 2+ The h a l f maximal e f f e c t i v e Ca concen t ra t i on and Mg concen t ra t i on requ i red to induce the two processes have been determined to be 40 yM and 200 yM , r e spec t i v e l y (77) . Johnson and Kirkwood (77) a l so suggest t ha t an i n t a c t s p e c t r i n network i s r equ i r ed f o r r e s e a l i n g to macromolecules, and tha t d i v a l e n t ca t i ons s t a b i l i z e t h i s network. They a t t r i b u t e d t h e i l o s s o f r e s ea l i n g a b i l i t y to the re l ease o f s p e c t r i n po lypept ides from the i nne r sur face o f the membrane. 16 b) Dependence o f Resea l ing Process on Temperature and I on i c S t r eng th : Johnson and Kirkwood (77) a l so determined t ha t a t 30°C ghosts e i t h e r resea l o r denature depending on the i o n i c compos i t ion o f the medium.,When the i o n i c s t r eng th was low (7mM N a d ) , ghosts d i d not resea l a t 30°C and were i r r e v e r s i b l y l eaky . However,when the i o n i c s t r eng th was h igh - say 80mM NaC l , or when d i v a l e n t ca t i ons were p resen t , the ghosts d i d resea l when incubated at 30°C. No r e s e a l i n g was observed a t temperatures o f 10°C and l e s s . The authors suggest t h a t ghosts undergo a s t r u c t u r a l a l t e r a t i o n a t a t r a n s i t i o n temperature o f approx imate ly 15°C,such t ha t they e i t h e r recover t h e i r impermeab i l i t y to macromolecules o r become i r r e v e r s i b l y leaky depending on the i o n i c s t r eng t h . They a l s o i n d i c a t e tha t the ex ten t o f r e s e a l i n g i n c reases w i t h temperatures above 20°C up to 40°C above which membranes undergo f ragmenta t ion . 17 OBJECTIVES OF THE STUDY: The pr imary ob j e c t i v e o f t h i s study was to i n v e s t i g a t e 2+ the k i n e t i c s o f the Ca pump o f human red c e l l s and to determine i t s s t o i ch i omet r y us ing i n s i d e - o u t v e s i c l e s as a model. The f o l l ow i ng summarizes the approach undertaken to e l u c i d a t e the s t o i ch i ome t r y 2+ and the k i n e t i c s o f the Ca pump:, 2+ 2+ 1. The i n i t i a l study was to determine the time course o f (Ca +Mg )-2+ ATPase a c t i v i t y and Ca t r an spo r t i n i n s i d e - o u t v e s i c l e s . This was necessary to enable the experiments done to be terminated w i t h i n 2+ the l i n e a r range o f ATPase o r Ca t r an spo r t a c t i v i t i e s . S ince e ry th rocy tes are known to con ta in app rec i ab l e q u a n t i t i e s o f ca lmodu l in i n t h e i r c y t o s o l , i t was impera t i ve to determine whether our p repa ra t i on o f i n s i d e - o u t v e s i c l e s are devo id o f ca lmodu l i n . The quest ion was addressed by determin ing enzyme 2+ and Ca t r an spo r t ra tes i n non-EDTA t r ea t ed and EDTA t r ea t ed i n s i d e - o u t v e s i c l e s . EDTA t reatment supposedly removes endogeneous ca lmodu l in t ha t may s t i l l be bound to i n s i d e - ou t v e s i c l e membranes (78) . 2. I t was intended i n t h i s study to c ha r a c t e r i z e the i n s i d e - o u t v e s i c l e p repa ra t i on and to determine the r e l a t i v e p ropo r t i on o f i n s i d e - o u t v e s i c l e s (IOV), r i g h t - s i d e out v e s i c l e s (ROV) and fragments. Fragments i n p a r t i c u l a r w i l l a l s o con t r i bu t e to ATPase 2+ 2+ a c t i v i t y and hence r e s u l t i n ove r - e s t ima t i on o f the (Ca +Mg ) -18 2+ ATPase a c t i v i t y a s soc i a t ed w i th the t r an spo r t o f Ca' . The p ropo r t i on o f IOV was determined by measuring l a t e n t a ce t y l cho l i ne s -terase a c t i v i t y i n the presence and absence o f 0.2% T r i t o n X-100. The p ropo r t i on of ROV was determined by measuring t o t a l ATPase a c t i v i t y i n the absence and presence o f a c l ose range o f T r i t o n X-100 (Tx-100) concen t ra t i ons . 3. Most o f the c r i t i c i s m i n v o l v i n g the use o f e r y th ro cy t e ghosts and IOV to study k i n e t i c s have centered on the l eak iness and the r e s e a l i n g mechanism o f the ghost or IOV p r epa r a t i on . In t h i s study e f f o r t was made to cha r a c t e r i z e the l eak i ne s s o f IOV (both EDTA t r ea t ed or non-EDTA t rea ted) by measuring the time 2+ course o f Ca leak from loaded IOV a f t e r the add i t i o n o f l. iuM ( f i n a l concen t ra t i on ) ionophore A23187 or 4.12. mM EGTA. 4. The e f f e c t o f var ious concent ra t i ons o f p u r i f i e d human 2+ 2+ e r y th rocy te ca lmodu l in on the (Ca + Mg )-ATPase a c t i v i t y and 2+ Ca t r an spo r t was s tud i ed a t a h i gh ( - 100 uM)and low( - 4 u.M) 2+ f ree Ca concen t ra t i on to v e r i f y whether changing ca lmodu l in concent ra t i on a f f e c t ed the s t o i c h i o m e t r i c r e l a t i o n s h i p a t a 2+ high o r low Ca concen t ra t i ons . 2+ 5. The enzyme a c t i v i t y and Ca t r anspo r t was a l s o i n v e s t i g a t e d 2+ 2+ at var ious ATP and Mg concent ra t i ons a t low and h igh f ree Ca 2+ concent ra t i ons (2.5 uM and 100 yM f r ee Ca ) i n the absence and presence o f p u r i f i e d ca lmodu l i n , the r a t i o n a l be ing t ha t the 19 s t o i ch i ome t r y o f the Ca pump may be modulated by s ub s t r a t e , 2+ 2+ Mg and ca lmodu l i n , known e f f e c t o r s o f the Ca pump. 2+ 6. The ATPase a c t i v i t y and Ca t r an spo r t were s t ud i ed over a 2+ wide range o f f r ee Ca concent ra t i ons i n : a) Non-EDTA t r ea t ed IOV and b) EDTA t r ea ted IOV i n the absence and presence o f va r ious concent ra t i ons o f p u r i f i e d ca lmodu l in . From the r e s u l t s obta ined values o f the s t o i ch i ome t r y 2+ 2+ of the Ca pump at each Ca f ree concen t r a t i on , and the K^ .. and V m a x o f the high and low a f f i n i t y components o f the enzyme were made. 7. S ince i t has been shown i n our l abo ra to r y t ha t the s t a t e o f the 2+ Ca pump ATPase cou ld d i f f e r depending on whether c a t i o n i c or an i on i c bu f f e r s were used, i t was deemed necessary to i nves t i ga te . 2+ the k i n e t i c s o f the Ca pump i n both T r i s and Hepes bu f f e r . I t was a n t i c i p a t e d tha t through these procedures o u t l i n e d above, a c r i t i c a l e va l ua t i on o f the k i n e t i c s and mechanism o f 2+ Ca t r an spo r t system i n the red c e l l cou ld be made. 20 •MATERIALS AND METHODS: Ma te r i a l s: The f o l l ow i ng chemical were purchased from 1. SIGMA CHEMICAL COMPANY: A c e t y l t h i o c h o l i n e i od i de Acry lami de A s co rb i c a c i d DTNB EDTA EGTA Hepes Imidazole Mai e t c ; a c i d Ouabain PMSF Tr i s -ATP T r i t on X-100 Tn'zma base : 2. J . T . BAKER CHEMICAL COMPANY: Magnesium ch l o r i d e (MgCl^) Sodium ch l o r i d e (NaCl) Sodium hydroxide (NaOH) Sodium Phosphate (NaH2P0^) TCA 3. PHARMACIA FINE CHEMICALS: DEAE Sephadex (A-50) Sephadex G-75 21 4. FISHER SCIENTIFIC COMPANY: Calc ium ch l o r i d e (CaC^) Carbowax (PEG 20,000) Lanthanum ch l o r i d e (LaClg) 5. ALLIED CHEMICAL: Hyd roch l o r i c a c i d (HC1) G l a c i a l A c e t i c a c i d Su l phu r i c A c i d (HgSO^) Potass ium phosphate (I^PO^) 6. MALLINKRODT: Ammonium molybdate 7. MATHESON COLEMAN AND BELL: Sodium carbonate ^ 2 ^ 2 ) Ionophore A23187 was a g i f t from E l i L i l l y . 8 ; BIO RAD LABORATORIES •y, SDS A l l the chemica ls l i s t e d above and reagents used were of a n a l y t i c a l grade. 22 METHODS: 1. P repara t i on of Calmodul in from Human Red Blood C e l l s : One un i t o f f r e sh human b lood obta ined from the Red Cross was washed three times i n i s o t o n i c s a l i n e a t 2,000 x g to remove the buf fy coat and the washed c e l l s poured i n t o a 1 - l i t r e graduated c y l i n d e r ( t o t a l volume of red c e l l s was 150 m l ) . The c e l l s were l y sed i n 650 ml o f a s o l u t i o n t ha t conta ined ( i n mM) 20 im i da zo l e , 1 EDTA and 0.2 PMSF and shaken v i go rous l y f o r 5 min. . The hemolysate was c en t r i f uged a t 27,000 x g f o r 30 m i n ; , and the supernatant c a r e f u l l y decanted i n t o a 2 - l i t r e f l a s k . Sodium ch l o r i d e ( f i n a l concen t ra t i on o f 70 mM) was added to b r i ng hemolysate to the osmo la r i t y o f bu f f e r A ( bu f f e r A conta ined i n mM, 100 NaC l , 20 im idazo le and 1 EDTA at pH 6 . 8 ) . Approx imate ly 200 ml o f DEAE - Sephadex (A-50) was then added. Th is Sephadex had been p r ev i ou s l y soaked f o r 2 days i n bu f f e r A and washed three t imes i n t h i s bu f f e r to remove the f i n e s . The suspens ion was a l lowed to e q u i l i b r a t e a t room temperature w i th slow constant s t i r r i n g f o r a t l e a s t one and h a l f hours. The suspension was then f i l t e r e d through a ny lon n e t t i n g t ha t r e t a i ned the Sephadex beads. The beads were washed w i th bu f f e r A. 23 The beads were poured gent l y i n t o a column (50 x 3 cm) and then washed w i th a t l e a s t two bed volumes o f bu f f e r A to remove the remaining haemoglobin. Calmodul in was e l u t ed from the column w i th a s a l t g rad ien t (0.1M NaCl to 1M NaCl ; 250 ml o f each) . The f r a c t i o n s con ta i n i ng ca lmodul in were determined by s t i m u l a t i o n o f 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y i n ghosts . These f r a c t i o n s were pooled and heated to 70°C f o r 30 min, and the p r e c i p i t a t e d p ro t e i n s removed by c e n t r i f u g a t i o n a t 27,000 x g f o r 30 min. The supernatant , r ep resen t i ng the ca lmodu l in so lu t i on ,was concent ra ted by u l t r a - f i l t r a t i o n on an Amicon UM 10 f i l t e r and f u r t h e r concentrated to 10 ml w i th Spec t ra -Por 1 (6000 - 8000 M.Wt cut o f f ) us ing c r y s t a l y s ed po lye thy lene g l y co l (20,000 M Wt cut o f f ) . The p ro t e i n was then d i a l y s ed overn ight aga ins t bu f fe r A a t 0-4°C. Next, the 10 ml sample was p laced on a Sephadex G-75 gel f i l t r a t i o n column (100 x 3 cm) and e l u t ed w i th bu f f e r A. The f r a c t i o n s con ta i n i ng ca lmodu l in were pooled and concent ra ted w i th an Amicon UM 10 f i l t e r . The sample was then d i a l y s ed aga ins t bu f f e r B a t 0-4°C. ( bu f f e r B conta ined i n mM 100 NaC l , 20 im idazo le and 0.1 C a C l 2 a t pH 6 . 8 ) . In the f i n a l p u r i f i c a t i o n s t ep , the sample was p laced on a DEAE - Sephadex column (50 x 3 cm) tha t had been p r e v i ou s l y e q u i l i b r a t e d w i th at l e a s t 2-bed volumes o f bu f f e r B. The ca lmodul in was e l u t ed w i th a l i n e a r s a l t g rad ien t 24 (0.1M to 1M NaCl ; 150 ml o f each) tha t was made up i n bu f f e r B. 2+ 2+ F rac t i ons t ha t showed ca lmodul in s t imu l a t i o n o f (Ca •• + Mg ) -ATPase a c t i v i t y were pooled and d i a l y s ed overn igh t aga in s t 1 mM T r i s -HC l pH 7.1 a t 0 - 4°C. V e r i f i c a t i o n o f the p u r i t y o f ca lmodu l in was by 0.1% SDS gel e l e c t r opho r e s i s on 7.5% po lyacry lamide ge ls s t a i n ed w i th Coomasie b l ue . A s i n g l e dark band w i th a mo lecu la r weight o f 16,430 was ob ta ined . 2. P repa ra t i on o f ' S t epw i se Ghosts"from Human E r y t h r o c y t e s : Red b lood c e l l ghosts were prepared from f resh whole human b lood (not more than 4-5 days o ld ) t ha t had been s to red i n a c i d - c i t r a t e - d e x t r o s e s o l u t i o n and obta ined from the l o c a l Red Cross . Ghosts were prepared by the procedure o f s tepwise hemolys is o u t l i n e d by S h r i e r (79) and mod i f i ed and adopted by Qu i s t and Roufoga l i s ( 9 ) . Red b lood c e l l s were i n i t i a l l y washed three times i n i s o t o n i c s a l i n e a t 2,000 x g f o r s i x m in . . The supernatant and the bu f f y coat toge ther w i th the upper t h i r d o f packed c e l l s were removed by s u c t i o n . About 5 ml each o f the . washed c e l l s was pi petted i n t o s i x 50 ml po lypropy lene c en t r i f uge tubes and then su c ce s s i v e l y hemolysed f o r 10 min.\ i n 7 volumes o f i c e co ld 0.08M NaC l , 0.06M NaCl and 0.04M NaCl a t 8,000 x g , 25 13,000 x g and 15,000 x g r e s p e c t i v e l y , i n a Sorva l RC 2B c en t r i f uge at 0-4°C. The c e l l s were then hemolysed twice i n a c o l d s o l u t i o n con ta i n i ng ( i n mM) 15 NaCl , 5 T r i s -ma lea te a t pH 7 . 1 . Th is mixture was c en t r i f uged a t 20,000 x g f o r 10 m in .a t 0-4°C. Care was taken to remove most of the supernate haemoglobin and the red button u sua l l y observed at the bottom of the c en t r i f u ge tube. 3. P repara t i on of Ins ide -ou t V e s i c l e s : Ins ide -ou t v e s i c l e s were prepared immediate ly on the same day f rom"stepwise ghos ts "obta ined above by a method mod i f i ed from tha t o f Steck e t a l . (12) . The ghosts were resuspended to the o r i g i n a l volume of packed c e l l s (about 5 ml) i n the f i n a l hemolysing s o l u t i o n and then recombined -in a 500 ml Erlenmeyer f l a s k . Th is was d i l u t e d w i th 5 volumes (25 ml) o f a s o l u t i o n con ta i n i ng ( i n mM) 0.1 EGTA and 1 T r i s -ma l ea te a t pH 8.0 The mixture o f ghost suspension was incubated a t 37°C f o r 30 min . w i th constant shak ing . The mix ture was a l lowed to cool f o r 5 min. . i n i c e and then sub -d i v i ded equa l l y among s i x 50 ml c en t r i f uge tubes . This was then cen t r i f uged a t 20,000 x g a t 4°C f o r 15 m in . . The supernatant was d i sca rded by a s p i r a t i o n and the p e l l e t s o f v e s i c l e s resuspended to about 5 ml w i th a s o l u t i o n con ta i n i ng 26 ( i n mM) 15 NaCl and 5 T r i s -ma l ea te a t PH 7.1 Resea l i ng o f Ir ts ide-6ut V e s i c l e s : One volume (5 ml) o f v e s i c l e suspension was d i l u t e d w i th three volumes (15 ml) o f a s o l u t i o n made up of 10 mM T r i s -ma l e a t e , 4 mM MgCl2 and 0.5 mM CaC^ at pH 7 . 1 . The mixture was a l lowed to e q u i l i b r a t e f o r 5 min. a t 0-4°C and then 1 ml of 2.9 M NaCl added to r e s t o r e i s o t o n i c i t y . The i n s i d e - ou t v e s i c l e mixtures were incubated a t 30°C f o r 10 min. to promote r e s e a l i n g . V e s i c l e s were then p e l l e t e d a t 20,000 x g f o r 10 min. P e l l e t s were resuspended and washed tw ice in a s o l u t i o n con ta i n i ng ( i n mM) 66 NaCl ,55 T r i s -ma lea te at pH 7.1 The f i n a l p e l l e t s were recombined and suspended to a t o t a l volume of about 20 ml i n the same f i n a l washing medium. EDTA Treated i n s i d e - ou t V e s i c l e s : A f t e r the r e s e a l i n g procedure and the subsequent c e n t r i f u g a t i o n s t ep , the v e s i c l e s were resuspended to the o r i g i n a l volume o f packed c e l l s (5 ml) w i t h the washing medium. These were pooled i n t o a 500 ml Erlenmeyer f l a s k and then d i l u t e d w i th 5 volumes (25 ml) o f a s o l u t i o n made up of 10 mM T r i s -ma l ea te and 1 mM EDTA at pH 8 .0 . The mixture was incubated f o r 30 min. at 37°C, a l lowed to cool i n i c e and d i v i ded equa l l y i n t o s i x 50 ml c en t r i f u ge tubes . The suspension was cen t r i f uged a t 27 20,000 x g f o r 10 min, and the p e l l e t s washed twice and resuspended to a t o t a l volume o f 20 ml i n the washing medium. 4. Cha r a c t e r i z a t i o n of S idedness: a) Determinat ion of the P ropo r t i on of Ins ide -ou t V e s i c l e s by Measurement o f A c e t y l c ho l i n e s t e r a s e A c t i v i t y : The p ropo r t i on of i n s i d e - ou t v e s i c l e s present i n the v e s i c l e p repa ra t i on was assessed by measuring the i n a c c e s s i b i l i t y o f a c e t y l c h o l i n e s t e r a s e a c t i v i t y found on the e x t e r i o r o f the i n t a c t e r y th rocy te membrane. The method employed was analogous to t ha t desc r ibed by Steck and Kant (12) . E igh ty m i c r o l i t r e s of the v e s i c l e suspension was d i l u t e d w i th 4 ml o f bu f f e r con ta i n i ng 55 mM T r i s -ma l e a t e , 66 mM NaCl and 6.4 mM MgC l 2 a t p H 7 . 1 . Exac t l y 50 y l a l i q u o t s o f each of the above was p re - incubated f o r 1 min.. .. w i th an equal volume of 100 mM sodium phosphate bu f f e r (pH 7.5) i n the absence and presence of 0.2% T r i t o n X-100 i n two 2 ml micro c u v e t t e s , r e s p e c t i v e l y , a f t e r which 0.6 ml o f the 100 mM sodium phosphate bu f f e r was added to b r i ng the volume to 0.7 m l . F i f t y m i c r o l i t r e s of 10 mM DTNB was added to each cuvet te fo l l owed by the add i t i o n o f 50 y l o f a 12.5 mM s o l u t i o n of a c e t y l t h i o c h o l i n e i od ide ( f i n a l concen t ra t i on = 0.78 mM) Contents i n both cuvet tes were thorough ly mixed and the r e a c t i on i n both fo l l owed s imu l taneous ly a t 412 nm in a Beckman Model 25 za spectrophotometer w i th an at tached recorder f o r about 3 m i n . The percentage o f i n s i d e - ou t v e s i c l e s present was determined from the d i f f e r e n c e in the ra tes o f r e a c t i on observed i n the absence and presence o f the T r i t o n X-100. V e s i c l e preparat ions were found to con ta in 55-60% i n s i d e - ou t v e s i c l e s . b) Determinat ion of the P ropor t i on of R i gh t - s i d e Out V e s i c l e s U t i l i z i n g Tota l ATPase A c t i v i t y .  Measurement of t o t a l ATPase ( ( C a 2 + + Mg 2 + ) -ATPase and 2+ Mg - ATPase) a c t i v i t y i n the absence and presence of T r i t o n X-100 was u t i l i z e d to determine the p ropor t i on of r i g h t s i de -ou t v e s i c l e s i n the p r epa ra t i on . 80 y l o f the v e s i c l e p repa ra t i on was incubated i n the absence and presence o f a smal l range of T r i t o n X-100 concent ra t i ons (between 0.1 mg/ml - 0.8 mg/ml) i n a 2 ml volume at 37°C. The bu f f e r used conta ined 100 yM f r ee C a 2 + , 55 mM Tr is:-maleate,66 mM NaC l , 6.4 mM M g C l 2 , 0.1 mM ouabain and 0.1 mM EGTA. The r ea c t i on was begun by the add i t i o n o f ATP (2 mM f i n a l concen t ra t i on ) and then quenched a f t e r 30 min. 1 w i th 1 ml o f 6% SDS. The amount o f P- re leased was determined by the F i ske and SubbaRow (80) method as desc r ibed in the Appendix. From the ra te of the peak a c t i v i t y observed i n the presence of T r i t o n X-100 ( u sua l l y observed between a T r i t o n X-100/Protein(w/wO r a t i o o f 2 .34-2.86) 29 and the ra te of P.. r e l ease i n the absence of T r i t o n X-100, the p ropo r t i on o f r i g h t s i de -ou t v e s i c l e s was assessed to be about 30% i n each p r epa r a t i on . By determin ing the percentage of IOV and ROV as desc r i bed the p ropo r t i on o f unsealed v e s i c l e s and fragments present i n the p repara t i on was obta ined by s imple s u b t r a c t i o n , as shown i n the f o l l ow i ng example: % fragments and unsealed v e s i c l e s = 100 - (55 + 30) where 55 = the % of IOV and 30 = % o f ROV. Hence the % of fragments and unsealed v e s i c l e s = 15% 2+ 2+ The c o n t r i b u t i o n by these fragments to (Ca + Mg )-ATPase a c t i v i t y was subt rac ted from t o t a l a c t i v i t i e s to ob ta in 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y due on ly to i n s i d e - ou t v e s i c l e s present i n the p r epa ra t i on . 5. Cha r a c t e r i z a t i o n of Leak iness o f V e s i c l e s : About 4.0 ml o f v e s i c u l a r suspension was p re - incubated 2+ i n an assay medium con ta i n i ng 100 yM f r ee Ca ( t o t a l volume was 10 ml) a t 37°C f o r h a l f an hour. (Assay medium conta ined 66 mM NaC l , 55 mM T r i s -ma l e a t e , 6..4 mM MgC l 2 0.1 mM EGTA and 0.1 mM Ouabain at pH 6 . 9 ) . Calc ium uptake i n t o i n s i d e - ou t v e s i c l e s was begun by the add i t i o n of 2 mM ATP ( f i n a l c on cen t r a t i o n ) . 30 1.0 ml a l i q u o t s were sampled at pre-determined time i n t e r v a l s of 0 . 5 , 2, 4, 6 , 8 , 10 and 12 minute time pe r i ods . The samples were p laced immediately i n t o 8 ml o f a co l d s o l u t i o n con ta i n i ng 0.5 mM L a C l 3 and 110 mM NaCl to stop the uptake p rocess . In two other r e a c t i on v e s s e l s , ionophore A23187 of f i n a l concen t ra t i on 1 yM in 33% e t hano l , and EGTA - f i n a l concen t ra t i on 4.12 mM were added, r e s p e c t i v e l y , immediately a f t e r the 6th minute a l i q u o t s had been sampled. The a b i l i t y o f the v e s i c l e s to leak or r e t a i n t h e i r ca l c ium content was determined by sampling a t the 7 t h , 9th 3+ and 11th minute and p laced immediate ly i n t o s i m i l a r La /NaCl s o l u t i o n . A l l the . tubes were c en t r i f uged at 20,000 x g f o r 10 mins. 3+ and washed once more i n s im i l a r La /NaCl s o l u t i o n . The time 2+ course of Ca uptake i n the absence of ATP was a l s o determined. The 2+ Ca content of p e l l e t s was ex t r a c t ed and determined as desc r i bed below (see ca l c ium e x t r a c t i o n ) . 6. Time Course o f C a 2 + Uptake and ( C a 2 + + Mg 2 + ) -ATPase A c t i v i t y : 2+ Procedure to determine the time course o f Ca uptake were s i m i l a r to t ha t p r e v i ou s l y desc r i bed above (see c h a r a c t e r i z a t i o n of l e a k i n e s s ) . The time course of t o t a l ATPase a c t i v i t y was a l s o performed by a s i m i l a r procedure except t ha t 1.0 ml a l i q u o t s sampled were quenched in 0.5 ml o f 6% S.D.S . i n de ion i zed water . 2+ The t ime course exper iments were performed a t 100 pM f r ee Ca or 31 2+ 2.34 yM f ree Ca , as i n d i c a t ed i n the f i g u r e s . The time course 2+ o f Mg -ATPase a c t i v i t y was a l s o determined w i th each exper iment , and t h i s was subt rac ted from t o t a l ATPase a c t i v i t y to ob ta in the 2+ 2+ t ime course of (Ca + Mg )-ATPase a c t i v i t y . 7. Determinat ion of ( C a 2 + + Mg 2 + ) -ATPase A c t i v i t y and C a 2 + Transpor t a t severa l f r ee Ca concen t r a t i on s : In both assays , 0.3 ml o f i n s i d e - ou t v e s i c l e suspension con ta i n i ng between 0.3 to 0.5 mg p r o t e i n was p i pe t t ed i n t o a f i n a l assay volume of 1.5 ml con ta i n i ng in (mM) 66 NaCl, 55 T r i s -ma l e a t e , 6.4 MgClg. 0.1 ouabain and 0.1 E6TA a t pH 6 .9 . Calc ium concen t ra t i on was 2+ va r i ed i n t h i s medium to a t t a i n a f r ee Ca concen t ra t i on as determined by a computer program accord ing to the equat ion o f 2_ Katz e t a l . (81) and t ak i ng i n t o account of Ca-EGTA , Mg-ATP 2 " , and Ca-ATP 2 " (pH o f 6 . 9 ) . (Appendix 2).The bu f f e r - c on t a i n i n g C a 2 + was i n i t i a l l y p i pe t t ed i n t o the r e a c t i on tubes which were kept on i c e . Calmodul in was then added when requ i red before v e s i c l e s were added. The mixture was p re - incubated a t 37°C f o r 30 min . and the r e a c t i on s t a r t e d w i th the add i t i o n of 0.075 ml of 40 mM ATP, r ep resen t i ng a f i n a l ATP concen t r a t i on of 2 mM. ATPase a c t i v i t i e s were measured by t e rm ina t i ng the r e a c t i on a f t e r 8 min. = by quenching w i th 0.75 ml of 6% SDS, b r i ng i ng the t o t a l volume i n the r e a c t i on vesse l to 2.25 m l . In some exper iments , ATPase 32 a c t i v i t i e s were terminated a f t e r 20 min. (see f i g u r e l egends ) . 2+ 2+ Mg - ATPase a c t i v i t i e s determined i n the absence of Ca and the ATPase a c t i v i t i e s con t r i bu ted by fragments and unsealed v e s i c l e s were subt rac ted from t o t a l ATPase a c t i v i t i e s to ob ta i n 2+ 2+ the (Ca + Mg )-ATPase a c t i v i t i e s due s o l e l y to sea led i n s i d e - ou t v e s i c l e s . ATPase a c t i v i t i e s were measured by determin ing the P^  re l eased by an automated F i ske and SubbaRow procedure (80) desc r ibed i n the appendix. For t r anspo r t determinat ions the uptake was terminated a f t e r 8 min by quenching w i th 6.0 ml of, i c e co ld lanthanum ch l o r i d e (0.5 mM)-NaCl(110 mM) s o l u t i o n and the tubes immediately p laced i n i c e . The L a C l 3 i s i nc luded to d i s p l a c e l o o s e l y bound 2+ 2+ ex te rna l Ca and a l s o i n h i b i t the a c t i v e Ca t r an spo r t system (9 ) . The tubes were c en t r i f uged at 20,000 x g f o r 10 min. and the p e l l e t s were washed once more i n the same s o l u t i o n . The f i n a l 2+ p e l l e t s were saved f o r Ca e x t r a c t i o n and measurement. 8. Calc ium Ex t r a c t i o n and I t s Determinat ion: Calc ium was e x t r a c t ed from pe l l e t e d v e s i c l e s by a mod i f i c a t i o n o f the method of Sparrow and Johnstone (82) . The p e l l e t s were i n i t i a l l y d i s s o l v ed i n 1.0 ml o f 3M TCA-G l a c i a l A c e t i c a c i d mixture (1:1) w i th v igorous v o r t e x i n g . Two ml o f d i s t i l l e d water was added, and the content made up to a 33 f i n a l volume o f 5 ml w i th 30 mM L a C l 3 . The mixture was Vortex mixed and and cen t r i f uged a t 20,000 x g f o r 30 min. a t room temperature. The supernatant was decanted i n t o a 10 ml t e s t - t ube ( a l l t e s t tubes had been p r ev i ou s l y washed i n 10% HC1) and i t s ca l c ium content determined by atomic absorp t ion spectrophotometry w i th a Techtron model AA5 inst rument a t a wavelength o f 422.9 nm» us ing n i t r ou s ox ide -ace ty l ene gas mix ture . Calc ium standards conta ined I d en t i c a l concent ra t ions o f a 3M TCA- G l a c i a l a c e t i c a c i d mixture and LaC lg and was l i n e a r between 0 and 20 pM CaClg standard used. The zero s tandard was read and used to ad jus t the AA before each sample was read. 9. Phosphate and P ro te i n Determinat ion: P.j determinat ions were performed us ing ;an automated mod i f i ed F i ske and SubbaRow procedure desc r ibed by Raess e t a l . (83) A phosphate s t anda rd i z a t i o n curve ranging from 0-400 nmoles/ml KF^PO^ was run w i th each experiment and was found to be l i n e a r over the range of concent ra t ions used, (see Append ix ) . P r o t e i n content of i n s i d e - ou t v e s i c l e s prepared was determined by an automated Lowry procedure (84) w i th the Technicon Autoana lyser . S ince T r i s has been shown to i n t e r f e r e w i th the Lowry reagent (85) , p r o t e i n assay was performed on 34 samples tha t had been d i l u t e d 80 f o l d i n de ion i zed water . Ins i de -ou t v e s i c l e s were determined to con ta in 2-3 mg p r o t e i n 2+ per m l . In the Ca t r anspo r t de te rm ina t i ons , i t was necessary to determine the p r o t e i n content of p e l l e t e d v e s i c l e s because a s p i r a t i o n s o f supernatant r e su l t e d i n the l o s s of some v e s i c l e s . 3+ In t h i s i n s t an ce , a f t e r the f i n a l wash i n La -NaCl s o l u t i o n , the p e l l e t s were d i s s o l v ed i n 0.1 ml o f IN NaOH by vo r t ex i ng v i g o r ou s l y . The volume was made up to 1.0 ml w i th deionzed water and the p r o t e i n content of 50 y l of t h i s mixture d i l u t e d to 1 ml was determined. P ro t e i n standards prepared from BSA in de ion i zed water was l i n e a r between 0 and 150 yg/ml . In f i gu r e s 17, 18, 19 and 20 i n the r e s u l t s s e c t i o n , ...the graphs are t y p i c a l r ep resen ta t i ons o f the r e s u l t s ob ta ined from a number o f s i m i l a r exper iments , s i nce the l e v e l s o f ATPase a c t i v i t y a f t e r EDTA treatment were v a r i a b l e , from experiment to exper iment. The tabu la ted data shown i n t he -Tab l e s , however, represent the means p lus or minus the s tandard dev i a t i ons o f the means. 35 RESULTS P u r i f i c a t i o n o f Calmodul in The p u r i t y o f the prepared ca lmodu l in was determined by 0.1% SDS - 7.5% po lyacry lamide gel e l e c t r o p h o r e s i s , as shown i n F igure 1. From the p l o t o f R^  va lues of known standards aga ins t mo lecu la r wt. (F igure 2 ) , the mo lecu la r weight of the ca lmodu l in was est imated to be 16430 2+ L i n e a r i t y o f ATPase a c t i v i t y and Ca - t r a n spo r t : I t was necessary i n t h i s study to determine the l i n e a r i t y o f 2+ ATPase a c t i v i t y and Ca t r an spo r t i n i n s i d e - ou t v e s i c l e s prepared, i n order to determine the most app rop r i a te t ime to quench the r ea c t i on s w i t h i n the l i n e a r range. Tota l ATPase 2+ a c t i v i t y at 100 pM Ca was l i n e a r f o r a t l e a s t 15 min i n non-EDTA t r ea ted v e s i c l e s i n the absence or presence o f 2 pg/ml 2+ ca lmodu l in (F igure 3 ) . Mg - ATPase a c t i v i t y was the same i n 2+ the presence o r absence of ca lmodu l in (Table 1 ) . Mg - ATPase a c t i v i t i e s were subt rac ted from t o t a l ATPase a c t i v i t i e s to 2+ 2+ g ive the a c t i v i t i e s due to (Ca + Mg )-ATPase enzyme, which were a l s o l i n e a r f o r a t l e a s t 10 min , as shown i n F igure 4. 2+ 2+ I t was a l s o apparent from F igure 6A and 6B tha t (Ca + Mg ) -ATPase a c t i v i t y was l i n e a r f o r at l e a s t 10 min i n EDTA-treated 2+ I0V. At a low f ree Ca concen t r a t i on o f 2.34 pM, l i n e a r i t y 36 F igure 1 : 0.1% SDS -7.5% po lyacry lamide ge l s o f p u r i f i e d human e ry th rocy te ca lmodu l in ( l e f t ) and standards of known molecu lar weights ( r i g h t ) . Only a s i n g l e p ro t e i n band was observed i n the p u r i f i e d sample of ca lmodu l i n . 37 F igure 1 CALMODULIN PHOSPHORYLASE b (94,000) Bovine Serum Albumin (67,000) Ovalbumin (43,000) Carbonic Anhydrase (30,000 Soybean T ryps in i n h i b i t o r (20,100) a -Lacta lbumin (14,400 ) 38 F igure 2: P l o t o f R f va lues aga ins t log o f mo lecu la r weight o f p r o t e i n standards as determined from F igure 1. R.^  va lue was c a l c u l a t e d as . . f d i s tance t r a v e l l e d by standards  tne r a t i o OT d i s t a n c e t r a v e l l e d by so l ven t dye f r o n t From t h i s p l o t , the molecu lar weight o f the p u r i f i e d e ry th rocy te c a lmodu l i n , shown by the arrow,was determined to be 16,430. 39 F igure 2 10 | -0.8 a-Lacta lbumin ^ Calmodul in Soybean T ryps in i n h i b i t o r 0.61 Rf 0.4 0.2 Carbonic Anhydrase # Ovalbumin Bovine Serum Albumin Phosphorylase B 4.2 4-6 Log M.Wt. 5.0 40 Table 1: Time course of Mg - ATPase a c t i v i t y i n e r y th ro cy t e v e s i c l e s i n the absence and presence o f 2 yg/ml ca lmodu l i n . These r e s u l t s represent the means of a t l e a s t 3 exper iments . Mg - ATPase a c t i v i t y ( nmoles per mg p ro t e i n ) Time (Min) No Calmodul in 2 yg/ml Calmodul in 3 28.14±3.34 2 8 . 2 U 3 . 2 5 6 37.69±1.61 38.38±3.26 9 46.81±2.56 50.88±2.85 12 58.46±2.54 58.46±2.69 15 68.98±5.62 69.80±5.80 41 F igure 3: Time course of ATPase a c t i v i t y i n i n s i d e - ou t 2+ v e s i c l e s not t rea ted w i th EDTA at 100 yM f r ee Ca A. Tota l ATPase a c t i v i t y i n the absence of ca lmodu l in (# - — # ) ; 2+ Mg - ATPase a c t i v i t y in the absence or presence o f 2 ug/ml ca lmodu l in (o o) ( data here was pooled from both c o n d i t i o n s . ) B. Tota l ATPase a c t i v i t y i n the presence of 2 yg/ml ca lmodu l in (0 <>) Each po in t represents the mean of at l e a s t d u p l i c a t e determi na t i ons . 43 2+ 2+ F igure 4: Time course of (Ca + Mg )-ATPase a c t i v i t y in non-EDTA t r ea ted i n s i d e - ou t v e s i c l e s at 2+ 100 yM f ree Ca in the absence (A A); and in the presence of 2 yg/ml ca lmodu l in ( • • ) • Th is data was obta ined from f i g u r e 3 by s ub t r a c t i n g 2+ Mg - ATPase a c t i v i t i e s from t o t a l ATPase a c t i v i t i e s i n the absence and presence of 2 yg/ml ca lmodu l i n . (Ca 2 ++ Mg 2 + )-ATPase Activity umoles Pi released/mg protein 45 was observed f o r a t l e a s t 60 min. S i m i l a r to the i n i t i a l t ime course exper iments of enzyme 2+ a c t i v i t y , i t was demonstrated tha t the time course of Ca t r an spo r t 2+ i n non-EDTA t r ea ted v e s i c l e s a t 100 uM f r ee Ca was a l s o l i n e a r f o r a t l e a s t 10 min, as shown i n F igure 5. Th is l i n e a r i t y o f 2+ Ca.. t r an spo r t was a l s o ev iden t i n EDTA t r ea ted i n s i d e - ou t v e s i c l e s ( r e s u l t s not shown). On the bas i s o f these s tud i e s i t 2+ was decided to terminate a l l Ca t r an spo r t exper iments a f t e r 8 min. ATPase a c t i v i t i e s were a l s o terminated a f t e r 8 min , except where i n d i c a t ed i n the f i g u r e legends, when ATPase a c t i v i t i e s were terminated a f t e r 20 min. Cha r a c t e r i z a t i o n of s idedness: Measurement of t o t a l ATPase a c t i v i t y i n the absence and presence of a narrow range of T r i t o n X-100 concen t ra t i ons was performed w i th each i n s i d e - ou t v e s i c l e p r epa r a t i o n , i n o rder to determine the percentage o f r i g h t - s i d e out v e s i c l e s . The data shown i n F igure 7 represent a t y p i c a l exper iment. The peak a c t i v i t y was always obta ined a t a T r i t o n X-100 to p r o t e i n r a t i o o f 2.34 to 2.86. The assumption made here i s t ha t the peak a c t i v i t y observed i n the presence of T r i t o n X-100 represents the t o t a l a c t i v i t y o f the v e s i c l e popu l a t i o n , due to the e f f e c t o f T r i t o n X-100 which makes the sea led v e s i c l e s 46 F igure 5: Time course of Ca Transport at 100 yM f r ee Ca in non-EDTA t r ea ted i n s i d e - ou t v e s i c l e s . (• A), i n the absence of ATP. (•• • ) , i n the presence of 2 mM ATP. ; ( • • ) , i n the presence of 2 mM ATP; and 2 yg/ml ca lmodu l i n . Each data po in t represents the mean of dup l i c a t e 2+ exper iments . Ca uptake data in the absence or presence of 2 yg/ml ca lmodu l i n ,bu t i n the absence 2+ of ATP,-,were s i m i l a r . Values rep resen t ing Ca uptake were d i v i d ed by t o t a l p r o t e i n . 47 F igure 5 4 6 8 TIME (min.) 10 48 F igure 6A: Time course of ATPase a c t i v i t y at 100 yM f r ee Ca in i n s i d e - ou t v e s i c l e s t r ea ted i n EDTA as desc r ibed in Methods s e c t i o n . 2+ (A A ) , Mg -ATPase a c t i v i t y . -( • • ) , Tota l ATPase a c t i v i t y i n the absence of 2 yg/ml c a lmodu l i n . ; ( • - — • ) , Tota l ATPase a c t i v i t y i n the presence of 2 yg/ml ca lmodu l i n . F igure 6B: Time course of Tota l ATPase a c t i v i t y at 2.34 yM 2+ f ree Ca i n i n s i d e - ou t v e s i c l e s t r ea ted in EDTA. Po in t s represent the means of dup l i c a t e exper iments . ATPase Activity nmoles Pi released/mg protein Total ATPase Activity pmoles Pi released/mg protein 617 50 Figure 7: Effect of increasing Triton X-100 concentration on 2+ total ATPase act iv i ty at 100 yM free Ca in inside-out vesicles. This experiment was performed with each inside-out vesicle preparation in order to determine the percentage of right-side out vesicles. The data represent:; a typical experiment. Total ATPase Activity nmoles Pi released/mg protein 52 l eaky . Table 2 i n d i c a t e s the e f f e c t of T r i t o n X-100 concen t ra t i ons on t o t a l ATPase a c t i v i t y i n two r ep re sen ta t i v e exper iments . The peak a c t i v i t y observed and the a c t i v i t y i n the absence of T r i t o n X-100 (where on ly i n s i d e - ou t v e s i c l e s and fragments hydro lyse ATP) were used to determine the p ropo r t i on o f r i g h t s i d e - ou t v e s i c l e s , es t imated as f o l l ows ( f o r experiment a o f Table 2 ) . Peak a c t i v i t y = a c t i v i t y due to IOV, ROV and fragments = 0.038 ymoles P^mg/hr = 100% A c t i v i t y i n the absence of T r i t o n X-100 = a c t i v i t y due to IOV and fragments = 0.024 ymoles P^/mg/hr. % of a c t i v i t y due to i n s i d e - ou t v e s i c l e s and 0 024 fragments = Q*Q33 x 100 = 63.15% . ' . % of r i g h t s i de -ou t v e s i c l e s i n the p repa ra t i on = 100 - 63.15 = 36.85% The p ropo r t i on o f IOV i n the p repa ra t i on was c a l c u l a t e d by a s i m i l a r procedure, u t i l i z i n g the ra te of a c e t y l c h o l i n e s t e r a s e a c t i v i t y i n the presence o f 0.2% T r i t o n X-100 ( t o t a l a c e t y l c h o l i n e -s te rase a c t i v i t y ) and the r a t e i n the absence o f T r i t o n X-100, where on ly r i g h t s i de -ou t v e s i c l e s and fragments r e a c t . By determin ing the p ropo r t i on o f IOV and ROV, the p ropo r t i on of fragments were es t imated as shown i n the Methods. In Table 3 , the p ropor t i ons of IOV, ROV and fragments (unsealed v e s i c l e s ) 53 TABLE 2: E f f e c t o f Inc reas ing T r i t on X-100 Concent ra t ion on Tota l ATPase a c t i v i t y (at 100 uM C a 2 + f ree.) i n two sample determinat ions (a and b ) . mg/ml T r i t o n - 100 /P ro t e i n Rat io ymoles P./mg/min TX-100 (w/w) a b a b 0 - - 0.024 0.024 0.10 1.78 1.88 0.029 0.030 0.125 - 2.34 - 0.036 0.150 2.68 2.81 0.038 0.035 0.175 - 3.28 - 0.027 0.20 3.57 3.75 0.026 0.025 0.30 5.36 5.63 0.023 .021 0.40 7.14 7.50 0.021 .019 0.80 14.29 _ 0.015 _ 54 Table 3: P ropor t i on of i n s i d e - ou t v e s i c l e s , r i g h t - s i d e out v e s i c l e s and unsealed v e s i c l e s i n v e s i c u l a r p repa ra t i on in three r ep re sen ta t i v e exper iments as determined by the procedures desc r ibed i n the Methods, Exper imental # % IOV % ROV % Unsealed V e s i c l e s 1 2 3 57 50 62 29 35 27 14 15 11 56.3±6.0 30.3±4.2 13.3+2.1 5b i s p resented . The percentage of ATPase a c t i v i t y con t r i bu t ed by fragments was c a l c u l a t e d as f o l l o w s : Mean % of ATPase a c t i v i t y con t r i bu ted by fragments = (5b\3 + 13.3) X 1 0 0 = 1 9 1 2+ 2+ This % o f a c t i v i t y was sub t rac ted from t o t a l (Ca + Mg )-ATPase a c t i v i t y to ob ta i n a c t i v i t y due to on ly sea led IOV. Cha r a c t e r i z a t i o n of l eak i nes s o f v e s i c l e s S ince we were i n t e r e s t e d in determin ing the amount of 2+ Ca taken up by the v e s i c l e s per energy u t i l i z e d , i t was important to show tha t the v e s i c l e s were not leaky to ca l c i um . 2+ F igure 8 shows the t ime course of Ca uptake i n non-EDTA t r ea t ed v e s i c l e s , and t h e . e f f e c t o f add i t i on s of 1 yM ionophore A23187 or 4.12 mM EGTA a f t e r 6 min. S i m i l a r r e s u l t s were obta ined i n EDTA - t r ea t ed IOV (F igure 9 ) , and EDTA t r ea ted IOV i n the presence of 2 yg/ml ca lmodu l in (F igure 10) . E f f e c t o f i n c r ea s i ng ca lmodu l in concen t ra t i on on. 2+ 2+ 2+ Ca t r anspo r t and (Ca + Mg )-ATPase a c t i v i t y I t was demonstrated tha t i n c r ea s i ng ca lmodu l in 2+ concen t ra t i on had a g rea te r e f f e c t on the Ca t r an spo r t 2+ 2+ parameter than on the (Ca + Mg )-ATPase a c t i v i t y a t 4 yM 2+ f r ee Ca concen t ra t i on (F igure 11) . Whereas the enzyme a c t i v i t y 56 F igure 8: Cha r a c t e r i z a t i o n of the l eak i nes s of i n s i d e - ou t v e s i c l e s not t r ea ted w i th EDTA. 2+ ( • • ) , Time course of Ca uptake at 2+ 100 yM f r ee Ca ; (o o ) , t ime course of 2+ Ca uptake in the absence of ATP; ( • • ) , e f f e c t o f a dd i t i o n of 1 yM ionophore A23187 a f t e r 6 min ( f i n a l concen-t r a t i o n ) ; (A A), e f f e c t of a dd i t i o n of EGTA ( f i n a l concen t ra t i on of 4.12 mM) a f t e r 6 min. (+ ) . Data po in t s represent the mean of a t l e a s t dup l i c a t e exper iments . 57 F igure 8 TIME (min.) 58 F igure 9: Cha r a c t e r i z a t i o n of the l eak i nes s of i n s i d e - ou t v e s i c l e s t r ea ted in EDTA (1 mM) f o r 30 min at 37°C. 2+ (A A), Time course of Ca uptake at 100 yM 2+ 2+ f ree Ca ; (A A ) , time course of Ca uptake in the absence of ATP; (0 0),effect o f 1 yM ionophore A23187 added a t 6 min.(+) ( f i n a l c on cen t r a t i o n ) ; (o o) e f f e c t of EGTA (4.12 mM f i n a l concen t ra t i on ) added a f t e r 6 min (+). Data po in t s represent the mean of at l e a s t dup l i c a t e exper iments . 60 F igure 10: Cha r a c t e r i z a t i o n of the l eak i nes s of EDTA-treated i n s i d e - ou t v e s i c l e s i n the presence of 2 yg/ml 2+ ca lmodu l i n . ( • • ) , Time course of Ca 2+ uptake i n the presence o f 100 yM f r ee Ca ; 2+ (o o ) , 'time course of Ca uptake i n the absence of ATP; (4 • ) , e f f e c t o f 1 yM ionophore A23187 ( f i n a l concen t ra t i on ) added a f t e r 6 m in . (+) ; (A A) e f f e c t of EGTA (4.12 mM f i n a l concen t ra t i on ) added a f t e r 6 min. (•!•). Data po in t s represent the mean of dup l i c a t e exper iments . 62 2+ sa tu ra ted a t about 0.8 yg/ml ca lmodu l i n , the Ca t r an spo r t inc reased w i th i n c r ea s i ng ca lmodu l in c oncen t r a t i on . However, at a f r ee ca l c ium concen t ra t i on o f 100 yM, both the enzyme a c t i v i t y 2+ and Ca t r an spo r t appeared to sa tu ra te a t 0.4 y'g/ml ca lmodu l in (F igure 12) . 2+ E f f e c t o f i n c r ea s i ng ATP concen t ra t i on on Ca t r an spo r t and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y . The e f f e c t of i n c r ea s i ng ATP concen t ra t i on was 2+ i n v e s t i g a t ed a t 100 yM f r ee Ca (F igure 13) i n the presence of 2 yg/ml ca lmodu l i n . Whereas the peak t r anspo r t a c t i v i t y was observed a t 2 mM ATP ( f i n a l c on c en t r a t i o n ) , the peak enzyme a c t i v i t y was ev iden t between 1 and 1.5 mM f i n a l ATP concen t ra t i ons . 2+ 2+ E f f e c t of i n c r ea s i ng Ca concen t ra t i on on Ca 2+ 2+ t r an spo r t and (Ca + Mg )-ATPase a c t i v i t y 2+ 2+ The Ca /ATP s t o i ch i ome t r y o f the Ca pump was i n v e s t i g a t ed i n non-EDTA t r ea t ed v e s i c l e s , i n the absence (F igure 14) and i n the presence of 2 yg/ml ca lmodu l in (F igure 15) 2+ over a wide range o f Ca concen t r a t i on s . The peak ca lmodu l in a c t i v a t i o n from these r e s u l t s was c a l c u l a t e d to be about 200%. 2+ 2+ 2+ In both Ca t r an spo r t and the (Ca + Mg )-ATPase a c t i v i t y , 2+ 2+ Ca a c t i v i t a t i o n was obta ined over a wide Ca concen t ra t i on 2+ range (0.71 to 300yM Ca ) . Table 4 shows the enzyme a c t i v i t y , 63 F igure 11: E f f e c t o f i n c r ea s i ng ca lmodu l in concen t ra t i on on C a 2 + Transpor t (0 0); and ( C a 2 + + M g 2 + ) -2+ ATPase a c t i v i t y • ) a t 4 pM f r ee Ca i n EDTA-treated i n s i d e - ou t v e s i c l e s . Resu l t s represent the means of dup l i c a t e exper iments . The dup l i c a t e va lues agreed wi th a range of l e s s than 5% C a 2 + Transport or (Ca 2 + +Mg 2 + )-ATPase Activity (pmoles mg protT • hr. ) 6b F igure 12: E f f e c t o f i n c r ea s i ng ca lmodu l in concen t ra t i on on C a 2 + Transpor t (O 0); and ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y ( • • ) at 100 yM f ree C a 2 + i n EDTA t r ea t ed i n s i d e out v e s i c l e s . Data po in t s represent the mean of dup l i c a t e exper iments , which agreed w i t h i n a range of l e s s than 5%. C a 2 + Transport or (Ca 2 % Mg 2 + )-ATPase Activity (pmoles mg protT1- hr."1) o -* to co 67 F igure 13: E f f e c t o f i n c r ea s i ng ATP concen t ra t i on on C a 2 + Transport (A A), and ( C a 2 + + Mg 2 ) -ATPase a c t i v i t y (A A) a t 100 yM f r ee Ca concen t ra t i on i n the presence of 2 yg/ml ca lmodu l i n . Each po in t represents the mean of two exper iments . C a 2 + Transport or ( C a 2 + + Mg 2 + )-ATPase Activity (umolesmg protT1- hr.~1) 69 2+ F igure 14: E f f e c t o f i n c r ea s i ng Ca concen t ra t i ons on C a 2 + t r anspo r t (4> • ) , and ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y ( • #) i n non-EDTA t rea ted i n s i d e - ou t v e s i c l e s . The data represent the mean of two exper iments . ATPase a c t i v i t i e s were terminated a f t e r 20 min, o f i ncuba t i on a t 37°C i n the presence of 2 mM ATP ( f i n a l c on cen t r a t i o n ) . C a 2 + Transport or Mg 2 + )-ATPase Activity O ro (pmoles mg protT • hr." ) o o <j) 09 P o 0 ) (Q o ro • 01 \ \ 1^ 71 F igure 15: E f f e c t of i n c r ea s i ng Ca c^ concen t ra t i on on C a 2 + t r anspo r t ( • • ) and ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y ( • • ) i n non-EDTA t rea ted i n s i d e - ou t v e s i c l e s i n the presence of 2 yg/ml ca lmodu l i n . ATPase a c t i v i t i e s were terminated a f t e r 20 min of i ncuba t i on a t 37°C i n the presence of 2 mM ATP ( f i n a l c o n c e n t r a t i o n ) . C a 2 + Transport or (Ca 2 Mvlg 2 + ) -ATPase Activity (pmoles mg protT1- hr.~1) 73 2+ Table 4: E f f e c t of i n c r ea s i ng Ca concen t ra t i on on the 2+ s t o i ch i ome t r y of the Ca pump i n non-EDTA t r ea t ed IOV. (mean of dup l i c a t e exper iments) 2+ f r ee Enzyme A c t i v i t y ymoles/mg / hour No CaM* 2yg/mlCaM 2+ Ca Transport ymoles/mg /h r No CaM 2yg/m CaM Ca 2 + /ATP No CaM 2y g/nil CaM 0.117 0.234 0.315 0.423 0.506 0.634 0.686 0.772 0.888 0.867 0.194 0.586 0.736 1.017 1 .235 1.816 2.111 2.315 2.484 2.349 0.207 0.437 0.575 0.661 0.766 0.894 1.030 0.984 1.108 1.063 0.309 1 .190 1.719 2.138 2.380 2.578 2.820 2.909 2.469 2.622 1.77 1 .85 1.83 1.56 1.51 1 .41 1.50 1.28 1.25 1.23 1.60 2.03 2.33 2.10 1.93 1.42 1.34 1.26 0.99 1.12 *CaM = Calmodul in 74 2+ Ca - t r an spo r t and the c a l c u l a t e d s t o i c h i ome t r i c r a t i o s a t 2+ var ious Ca concen t r a t i on s . A s t o i ch i ome t r y of 2 was observed 2+ 2+ i n the low Ca range (0.71 to 24 yM Ca ) and a s t o i ch i ome t r y 2+ approaching 1 was observed a t high Ca concen t ra t i on 2+ 2+ (50 to 302.6 yM Ca ) . The e f f e c t of i n c r ea s i ng Ca concen t ra t i ons 2+ 2+ 2+ on Ca t r an spo r t and (Ca + Mg )-ATPase a c t i v i t y i n the absence and presence o f 0.2 yg/ml ca lmodu l in i n EDTA t r ea ted i n s i d e - ou t v e s i c l e s i s shown i n F igure 16. I t was apparent from the r e s u l t s t ha t t h i s ca lmodu l in concen t ra t i on was not enough to 2+ 2+ abo l i s h the low Ca a f f i n i t y component of e i t h e r the Ca t r an spo r t or the enzyme a c t i v i t y . When ca lmodu l in concen t ra t i on was r a i s ed 2+ to 0.5 yg/ml (F igure 17) the low Ca a f f i n i t y po r t i on of the 2+ a c t i v a t i o n appeared to be abo l i s h ed , p a r t i c u l a r l y i n the Ca 2+ t r an spo r t . In the data shown peak Ca t r anspo r t was observed 2+ a t 50 yM f r e e Ca c on cen t r a t i o n , whereas peak enzyme a c t i v i t y 2+ was observed a t 200 yM Ca concen t r a t i on . F igure 18 shows tha t whereas the t r anspo r t parameter i s maximal ly a c t i v a t ed about 400% a t 13 yM f r ee C a 2 + , the ( C a 2 + + Mg 2 + ) -ATPase a c t i v a t i o n 2+ 2+ reached maximum a t 76 yM Ca the range of Ca concen t ra t i ons where the s t o i ch i ome t r y approaches 1. Tables 5 and 6 represent the t abu la ted s t o i c h i o m e t r i c r a t i o s i n the absence and presence of 0.5 yg/ml , c a lmodu l i n , r e spec t i v e l y . ' In both ins tances the 2+ s t o i c h i ome t r y approached 2 i n the low Ca range of 0.71 yM to 24 yM, beyond which the r a t i o decreased g radua l l y towards 1. 75 F igure 16: E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on 2+ 2+ 2+ Ca t r an spo r t and (Ca + Mg )-ATPase a c t i v i t y i n EDTA t rea ted v e s i c l e s i n the absence and presence of 0.2 yg/ml ca lmodu l i n . Resu l t s are the mean of dup l i c a t e exper iments . 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y i n the absence (o o ) ; and presence (• • ) of ca lmodu l i n . 2+ Ca Transpor t i n the absence (A — A) and presence (A — A) of ca lmodu l i n . 77 F igure 17: E f f e c t of i n c r ea s i ng Ca z+ concen t ra t i ons on C a 2 + "transport and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y i n the absence and presence of 0.5 ug/ml ca lmodu l in i n EDTA t rea ted i n s i d e - ou t v e s i c l e s . 2+ 2+ (Ca + Mg )-ATPase a c t i v i t y i n the absence (o o) and presence ( • • ) o f 2+ ca lmodul in;Ca t r an spo r t i n the absence (A — A) and presence of (A — A) o f ca lmodu l i n . Data i s t y p i c a l of a t l e a s t 5 exper iments . 5 C a 2 + Transport or ( C a 2 + + Mg 2 + )-ATPase Activity (pmoles mg protT1- hr.~1) o cb rb p> 79 F igure 18: Per cent a c t i v a t i o n by ca lmodu l in of Ca 2+ 2+ t ' ransport and (Ca + Mg )-ATPase a c t i v i t y i n EDTA-treated i n s i d e - ou t v e s i c l e s ( c a l c u l a t ed from data i n F igure 17.) 2+ ( • • ) Ca Transport ( 0 0) ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y % Activation f *• Transport or ( C a 2 t + M g » ) - A T P a s e Activity oo o 81 Table 5: 2+ E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s t o i ch i ome t r y o f the 2+ Ca pump in EDTA-treated 10V i n the absence of Ca lmodu l in . The data represents mean ± S.D. of the mean o f 7 de te rm ina t ions . C a 2 + f r ee (yM) Enzyme A c t i v i t y ymoles/mg / ' hour Mean ± S.D. 2+ Ca Transpor t ymoles/mg / hour Mean ± S.D. C a 2 + / A T p Mean± S.D. 0.71 2.50 . 4.00 13.10 24.00 50.00 76.00 100.00 200.20 302.58 0.065 ± 0.020 0.110 ± 0.028 0.132 ± 0.037 0.168 ± 0.044 0.194 ± 0.054 0.275 ± 0.075 0.290 ± 0.067 0.335 ± 0.068 0.433 ± 0.071 0.483 ± 0.081 0.146 ± 0.038 0.202 ± 0.077 0.238 ± 0.062 0.300 ± 0.067 0.317 ± 0.072 0.385 ± 0.058 0.414 ± 0.046 0.436 ± 0.056 0.523 ± 0.052 0.592 0.140 2.21 ± 0.64 1.82 ± 0.44 1.81 ± 0.17 1.81 ± 0.30 1.67 ± 0.20 1.46 ± 0.27 1.41 ± 0.17 1.33 ± 0.22 1.22 ± 0.10 1.23 ± 0.27 2+ 2+ The mean s t o i c h i o m e t r i c r a t i o from 0.71 .yM Ca to 24 yM Ca was c a l c u l a t e d to be 1.86 ± 0.20 82 Table 6: 2+ E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the s t o i ch i ome t r y of the 2+ Ca pump i n EDTA t r ea ted 10V i n the presence of 0.5 yg/ml Calmodul in The data represent the mean ± S.D. o f the mean o f 5 de te rm ina t i ons . C a 2 + f r e e Enzyme A c t i v i t y ymoles/mg /hour C a 2 + Transpor t ymoles/mg /• hour 0 a 2 + / ATP 0.094 ± 0.012 0.282 ± 0.031 0.368 ± 0.024 0.582 ± 0.020 0.729 ± 0.029 1.056 ± 0.090 1.299 ± 0.176 1.470 ± 0.224 1.454 1.326 0.235 0.247 0.168 ± 0.012 0.504 ± 0.098 0.805 ± 0.077 1.173 ± 0.179 1.368 ± 0.178 1.454 ± 0.264 1.378 ± 0.286 1.416 ± 0.328 1.341 ± 0.292 1.232 ± 0.417 1.81 ± 0.23 1.80 ± 0.34 2.19 ± 0.21 2.01 ± 0.26 1.88 ± 0.22 1.38 ± 0.21 1.07 ± 0.26 1.00 ± 0 . 3 3 0.94 ± 0.27 0".95 ± 0.36 The mean s t o i c h i o m e t r i c r a t i o from 0.71 yM Ca to 24 was c a l c u l a t e d to be 1.94 ± 0.16 yM Ca 83 The r e s u l t s obta ined when ca lmodu l in concen t ra t i on was inc reased to 2 yg/ml (F igure 19) were not very d i f f e r e n t from tha t obta ined a t 0.5 yg/ml ca lmodu l i n . The percent a c t i v a t i o n by ca lmodu l in was a l so s i m i l a r to t ha t shown i n F igure 18. Table 7 shows tha t i n the three experiments performed the s to i ch i omet r y was h igher than 1 ( i e . 1 . 5 5 ± 0.12) i n the low C a 2 + range of 0.71 to 2+ 2+ 24 yM Ca and 1 i n the h igh Ca concen t ra t i on range. F igure 20 represents the Ead ie -Ho f f s tee p l o t o f the data i n F igure 19, obta ined from a non - l i n ea r computer a na l y s i s o f the da ta . In a l l the exper iments done, the observed v e l o c i t i e s were analysed and p l o t t e d from a computer program u t i l i z i n g the equat ion V , • [ C a 2 + ] - 2 + v = — L o i + K ? . [Ca^ ] ' ' [Ca ' ] 2+ where and represent the maximum v e l o c i t y and Ca d i s s o c i a t i o n constant of the high a f f i n i t y component and v 2 2+ 2 = -£=- , where V 2 and K 2 are the maximum v e l o c i t y and Ca 2+ d i s s o c i a t i o n constants of the low Ca a f f i n i t y component. 2+ E f f e c t o f Mg concen t ra t i on The e f f e c t o f dec reas ing the MgC l 2 concen t ra t i on 2+ from 6.4 mM to 3 mM and 1.0 mM on the k i n e t i c s o f Ca 84 F igure 19: E f f e c t o f i n c r ea s i ng Ca^ + concen t ra t i on 2+ 2+ 2+ on Ca t r anspo r t and (Ca + Mg )-ATPase a c t i v i t y i n the absence and presence of 2 yg/ml ca lmodu l in i n EDTA t rea ted IOV. ( C a 2 + + M g 2 + ) -ATPase a c t i v i t y i n the absence (o o) and 2+ presence ( • #} of ca lmodu l in;Ca t ranspor t i n the absence (A — A) and presence (A •) of ca lmodu l i n . Data i s t y p i c a l o f a t l e a s t 3 experiments C a 2 * Transport or (Ca2 ++ Mg 2*)-ATPase Activity (pmoles mg protT1- hr."1) 86 Table 7 : 2+ E f f e c t o f i n c r e a s i ng Ca concen t ra t i on on the s t o i ch i ome t r y of the 2+ Ca pump i n EDTA t r ea t ed 10V i n the presence 2 yg/ml Calmodul in The data represent the mean ± S.D. of the mean of 3 determinat ions C a 2 + f r ee -(y M ) Enzyme A c t i v i t y ymoles/mg /hour C a 2 + Transpor t ymoles/mg /hour C a 2 + / ATP 0.71 2.50 4.00 13.10 24.00 50.00 76.00 100.00 200.20 302.58 0.133 ± 0.021 0.418 ±: 0.190 0.580" ± 0.159 0.844 ± 0.202 0.997 ± 0.292 1.405 ± 0.293 1.490 ± 0.308 1.508 ± 0.319 1.558 ± 0.147 1.546 ± 0.164 0.223 ± 0.094 0.706 ± 0.360 0.961 ± 0.443 1.258 ± 0.388 1 .353 ± 0.216 1.641 ± 0.301 1.657 ± 0.299 1 .646 ± 0.368 1.536 ± 0.212 1.578 ± 0.375 1.64 ± 0.47 1.67 ± 0.17 1.59 ± 0.34 1.47 ± 0.10 1 .39 ± 0.20 1.17 ± 0.03 1.11 ± 0.04 1.09 ± 0.03 0.98 ± 0.05 1.01 ± 0.13 2+ 2+ The mean s t o i c h i ome t r i c r a t i o from 0.71 yM Ca to 24 yM Ca was c a l c u l a t e d to be 1.55 ± 0.12 87 F igure 20: Ead ie -Ho f f s tee p l o t o f the data i n f i g u r e 19. 2+ 2+ A. (Ca + Mg )-ATPase a c t i v i t y i n the presence (X) and absence (+) of 2 yg/ml ca lmodu l i n . 2+ B. Ca t r anspo r t i n the presence (X) and absence (+) of 2 yg/ml ca lmodu l i n . The graphs are computer p l o t s f i t t e d to the non l i nea r k i n e t i c equat ion desc r ibed i n the t e x t . 88 F igure 20 to C D V/Ca 2 + 89 t r anspo r t were examined i n F igures 21 and 22. A t 3 mMMgC^ the C a 2 + a c t i v a t i o n o f both ( C a 2 + + Mg 2 + ) -ATPase and C a 2 + -2+ t r an spo r t was of high Ca a f f i n i t y i n the presence of 2+ 2 yg/ml ca lmodu l i n , the peak Ca a c t i v a t i o n being observed a t 2+ 17 yM Ca i n both systems (F igure 21 ) , compared to 76-200 yM 2+ at 6.4 mM Mg (F igure 19) . Lowering the MgC^ concen t r a t i on 2+ to 1.0 mM f u r t h e r s h i f t e d the Ca a c t i v a t i o n curve to lower 2+ concentrat ions, peak Ca a c t i v a t i o n of both t r anspo r t and 2+ enzyme a c t i v i t y o c cu r r i ng a t 1.8 yM Ca i n the presence of 2 yg/ml ca lmodu l in (F igure 22 ) . 2+ The enzyme a c t i v i t y , Ca t r anspo r t and the 2+ s to i ch i ome t r y a t each Ca concen t ra t i on i n the presence'of 1 mM MgC^ concen t ra t i on and 2 yg/ml ca lmodu l in are t abu la ted i n Table 8. The r e s u l t s i n d i c a t e a s t o i ch i ome t r y approaching 2 over 2+ the e n t i r e range of Ca concen t ra t i ons s t ud i ed . Comparison o f bu f f e r used f o r study 2+ ' 2+ 2+ The Ca - t r an spo r t parameter and the (Ca + Mg ) -ATPase a c t i v i t y was compared i n T r i s -ma lea te bu f f e r and sodium -Hepes b u f f e r . I t i s apparent from F igure 23 tha t whereas on ly 2+ the h igh Ca a f f i n i t y component was observed i n the sodium -2+ Hepes b u f f e r , both the low and high Ca a f f i n i t y systems were observed i n T r i s -ma l ea te bu f f e r . 90 Figure 21: Effect of increasing C a t T concentration on C a 2 + transport and (Ca 2 ++ Mg2+)-ATPase 2+ act iv ity in the presence of 3 mM Mg and in the absence and presence of 2 ug/ml calmodulin. 2+ 2+ (Ca + Mg )-ATPase act iv i ty in the absence (A A) and presence (A A) of calmodulin. 2+ Ca Transport in the absence ( 0 — <>) a n d presence (• •) of calmodulin. Data points represent the means of two experiments. C a 2 + Transport or (Ca 2 Mvlg 2 + ) -ATPase Activity (pmolesmg protT1- hr."1) -s o 9 CD ro o to O 0> O) 01 7 t — r w > o w CO 3 92 F igure 22: E f f e c t o f i n c r ea s i ng C a ' + concen t ra t i on 2+ on Ca / t r a n s p o r t ( 0 <>) and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y ( + — • ) i n the 2+ presence of 1 mM Mg concen t ra t i on and in the presence of 2 yg/ml ca lmodu l i n . Data represent,; the mean of dup l i c a t e exper iments . C a 2 + Transport or (Ca 2 % Mg 2 + )-ATPase Activity (pmoles mg protT1- hr."1) 94 Table ° : E f f e c t o f i n c r ea s i ng Ca concen t ra t i on on the 2+ sto. ich iometry of the Ca pump i n the presence 2+ of 1 mM Mg and 2 yg/ml ca lmodu l in (as per f i g u r e 22) C a 2 + f r ee (uM).. Enzyme A c t i v i t y ymoles/mg /hour C a 2 + Transpor t ymoles/mg /hour Ca / ATP .0.53 0.442 0.709 1.58 1,84 0.791 1.095 1.38 11.33 0.486 0.625 1.29 20.22 0.370 • 0.560 1.51 52.43 0.181 0.401 2.21 100.00 0.181 0.371 2.05 180.00 0.152 0.379 2.09 Mean = 1.75±0.42 The above r e s u l t s represent the mean of dup l i c a t e exper iments 95 F igure 23: Comparison of Ca^ T Transport ( ) and ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y ( ) i n T r i s - b u f f e r and Hepes bu f f e r . In the Hepes. b u f f e r , 55 mM sodium-Hepes was s ub s t i t u t e d f o r 55 mM T r i s -ma l ea t e . A l l o ther agents and cond i t i on s were kept the same as desc r i bed i n the methods a t 6.4 mM MgC l 9 c oncen t r a t i on . 96 F igure 23 if • KM -Log [ C a 2 + ] ( M ) 97 DISCUSSION 2+ Measurement o f Mg - ATPase a c t i v i t y as a Test f o r ca l c ium contaminat ion I t was impera t i ve i n t h i s work to a s c e r t a i n t ha t the v e s i c u l a r p repa ra t i on d i d not con ta i n r e s i dua l l o o s e l y bound c a l c i u m , as dur ing t h e i r p repa ra t i on v e s i c l e s were resea led i n the presence 2+ 2+ of 500 yM Ca . Measurement of Mg - ATPase a c t i v i t y i n the 2+ absence of added Ca , and in the absence and presence of ca lmodul in ,was used f o r t h i s purpose, as ca lmodu l in a c t i v a t i o n 2+ of ATPase i s depended on Ca . The f a c t tha t the time course of 2+ the Mg - ATPase a c t i v i t y d i d not change i n the absence or presence - 2+ of 2 yg/ml" ca lmodu l in suggests the absence of Ca contaminat ion in the v e s i c u l a r p repa ra t i on and supports the e f f e c t i v ene s s of the washing procedure f o r removal of the r e s i dua l ca l c ium a f t e r r e s e a l i n g . I n t e g r i t y o f V e s i c u l a r P repara t i on a) Cha r a c t e r i z a t i o n of Leak iness : Larsen e t a l . ( 7 2 ) have suggested tha t 60 percent of ghosts prepared by sub j e c t i ng out -dated red c e l l s to repeated hemolys is do not r e s e a l . A l though on ly f r e sh red c e l l s (not more than 4 days o ld ) were used i n t h i s s tudy , i t was necessary to 98 determine tha t the v e s i c l e s prepared were not l eaky , even a f t e r r e s e a l i n g . Evidence to support the f a c t t ha t the prepared v e s i c l e s 2+ were t i g h t to Ca and smal l molecules i n c l ude the f o l l ow i ng obse rva t i ons : 2+ i . Ca uptake i n the presence of 2 mM.'ATP was l i n e a r up to 10 min. 2+ i i . No uptake of Ca was ev ident i n the absence of ATP, 2+ suggest ing a l s o tha t the Ca uptake process i s e ne r ge t i c . i i i . When EGTA concen t ra t i on was r a i s ed (from 0.1 mM to 2+ 4.12 mM f i n a l concentrat ion)^ f u r t h e r uptake of Ca 2+ was a r r e s t ed wh i l e Ca l e v e l s i n the v e s i c l e s were not decreased. i v . Add i t i o n of Ionophore A23187 r e su l t e d i n the r ap i d 2+ ' leak o f Ca from the v e s i c l e s i n t o the medium, -2+ i n d i c a t i n g tha t the Ca was taken up by the v e s i c l e s r a the r than being l oo se l y :_associ a ted . These obse rva t i ons were t rue i n both i n s i d e - ou t v e s i c l e s sub jec ted to EDTA treatment and those not t r ea ted w i th EDTA, suggest ing tha t 2+ EDTA d i d not make the v e s i c l e s l eaky to Ca and ATP, probably because the cy toske l e ton of the v e s i c l e s was s t i l l i n t a c t . The idea tha t the c y t o ske l e t on (o r spectr in)may mediate the r e s ea l i n g process de r i ves from experiments i n resea led ghosts ( 77 ) , 99 showing tha t the l o s s o f the a b i l i t y to recover impermeab i l i t y to smal l molecules i s accompanied by the s p e c i f i c l o s s o f s p e c t r i n bands 1 and 2 from the membrane ( 77 ) . I t i s suggested tha t the i n a b i l i t y to resea l ghosts i n low i o n i c s t reng th media may be due to an a l t e r a t i o n i n s p e c t r i n or i t s s p e c i f i c b ind ing 2+ 2+ s i t e (77) . The requirement o f Ca and Mg f o r r e s ea l i n g of ghosts as u t i l i z e d i n the r e s e a l i n g medium supposedly r e s u l t s i n i n t e r a c t i o n o f the d i v a l e n t ca t i ons w i th the c y t o s k e l e t on , hence b r i ng i ng about c l o su r e to both ca t i ons and smal l mo lecu les . Presumably, carboxy l groups of s p e c t r i n c r o s s - l i n k through 2+ Ca br idges w i th the carboxy l and hydroxyl groups o f phospha t i -d y l s e r i n e and phospha t i dy l e thano l am ine , r e spec t i ve l y , on the inner sur face of the membrane. b) Cha r a c t e r i z a t i o n of S idedness: Whereas a c e t y l c h o l i n e s t e r a s e a c c e s s i b i l i t y i n the presence or absence of T r i t o n X-100 (Steck and Kant (12)) was u t i l i z e d to determine the p ropo r t i on of i n s i d e - ou t v e s i c l e s i n the p r epa r a t i o n , severa l attempts to employ the g l y ce ra ldehyde-3-phosphate dehydrogenase assay ou t l i n ed by the same authors to determine the p ropo r t i on o f sea led r i g h t - s i d e out v e s i c l e s i n the p repa ra t i on f a i l e d . S ince the a c t i v i t y of the enzyme was found i n the hemolysate, i t became apparent t ha t the method of 10U v e s i c l e p repa ra t i on r e su l t e d i n a washing o f f o f the g lycera ldehyde-3-phosphate dehydrogenase from the i n t r a c e l l u l a r su r f a ce . Hence i n determin ing the p ropo r t i on of r i g h t - s i d e out v e s i c l e s , measurement of t o t a l ATPase a c t i v i t y i n the absence and presence o f a narrow range of T r i t o n X-100 concen t ra t i ons was used. The important c r i t e r i o n here was the T r i t o n X-100 to p r o t e i n r a t i o at which maximum a c t i v i t y was ob ta ined . Th is va lue was r o u t i n e l y determined and found to f a l l between 2.34 to 2.86 (Table 3 ) , which represents the c r i t i c a l concen t ra t i on of T r i t o n X-100 above which l o s s of a c t i v i t y due to denatu ra t ion of the enzyme was a t t a i n e d . The e f f e c t o f T r i t o n X-100 i s to make the sea led v e s i c l e s leaky, hence exposing the t o t a l ATPase enzyme. E x t r a c t i o n of Calmodul in from Ins ide -ou t V e s i c l e s w i th EDTA • Scha r f f (86 ) i n 1972 showed tha t t reatment of red c e l l 2+ 2+ membranes w i th EDTA reduced the (Ca + Mg )-ATPase a c t i v i t y . I t i s g ene r a l l y assumed tha t EDTA d i s s o c i a t e s ca lmodu l in from red c e l l membranes, ghosts or v e s i c l e s , presumably by c he l a t i n g 2+ Ca , hence prevent ing ca lmodu l in s t imu l a t i o n of the enzyme. 2+ I t was ev ident i n t h i s work tha t the basal l e v e l s o f Ca 2+ 2+ t r an spo r t and the (Ca + Mg )-ATPase a c t i v i t y i n i n s i d e - ou t v e s i c l e s t ha t had not been sub jec ted to EDTA treatment were much h igher than those obta ined in i n s i d e - ou t v e s i c l e s t r ea ted wi th 101 EDTA, suggest ing tha t EDTA treatment was success fu l i n s t r i p p i n g i n s i d e - ou t v e s i c l e s o f ca lmodu l i n . Furthermore, whereas v e s i c l e s t ha t had not been subjected to EDTA treatment cou ld be maximal ly s t imu la ted about 200 per cent by 2 yg/ml ca lmodu l i n , EDTA treatment r e su l t e d in about 400% maximal a c t i v a t i o n s at the same concen t ra t i on of ca lmodu l i n . I t i s not known, however, t ha t a complete d i s s o c i a t i o n of ca lmodu l in was ach ieved . A recent study i n e r y th ro cy t e ghosts (87) suggests t ha t there may be some ca lmodu l in s t i l l bound to the membrane tha t i s i n a c c e s s i b l e to EDTA t reatment . I t must be r e a l i z e d , however, t ha t t h i s supposedly t i g h t l y bound ca lmodu l in was est imated from the s t imu l a t i o n of phosphodiesterase enzyme from a b o i l e d e x t r a c t o f the membranes, but i t i s known tha t phosphodiesterase i s s t imu la ted app rec i ab l y by o ther agen t s , i n c l ud i ng proteases and phospho l i p ids (88) . Neve r the l e s s , the use of EDTA t r ea t ed i n s i d e - ou t v e s i c l e s i n t h i s work a l lowed the study 2+ of the i n f l u ence o f Ca - bound ca lmodu l in on the s t o i ch i ome t r y 2+ ' o f the Ca pump. A l though the use o f EDTA and other c h e l a t i n g .agents i n red c e l l ghost p repara t ions has been c r i t i c i z e d by severa l workers (47 ,86 ) , i t must be emphasized tha t the enzyme e n t i t y 2+ r e spons i b l e f o r the Ca pump i s s t i l l i n t a c t , desp i t e the f a c t tha t o ther p ro te i n s tha t may be f un c t i on i ng s y n e r g i s t i c a l l y w i t h , or independent ly o f , ca lmodu l in to regu la te the pump may have been removed by t h i s t reatment . Th is l a t t e r f a c t i s even more 102 important s i nce an i n h i b i t o r p ro t e i n (89) and a 63,000 da l ton a c t i v a t o r p r o t e i n (90) have been i s o l a t e d from the human e ry th rocy te and are both recogn ized to a f f e c t the enzyme a c t i v i t y . Homogeneity o f Prepared Ca lmodu l in : I t i s c l e a r from the SDS- PAGE" gel e l e c t r o p h o r e s i s -t ha t the ca lmodu l in prepared from human e ry th rocy te s and used i n t h i s work was homogeneous. The molecu lar weight of 16,430 compares favourab ly w i th t ha t obta ined by Vanaman e t a l . ( 91 ) , who a r r i v e d at a molecu lar weight of 16,723 from amino a c i d sequence ana l y s i s of ca lmodu l i n . The p r o f i l e o f enzyme a c t i v a t i o n of the ca lmodu l in prepared i n t h i s study was no d i f f e r e n t from bovine b r a i n ca lmodu l in supp l i ed to our l abo ra to r y by Dr. John T. Penniston ( r e s u l t s not shown). 2+ E f f e c t o f ATP on the Ca pump and S to i ch i omet ry : In t h e i r ca l c ium t r an spo r t s tud i e s Larsen e t a l . (92) used 3 mM ATP ( f i n a l c on cen t r a t i o n ) . Whi le t h i s ATP concen t r a t i on may have been opt imal f o r t h e i r type o f i n s i d e - ou t v e s i c l e s , i t 2+ 2+ 2+ was found to i n h i b i t both Ca t r anspo r t and the (Ca + Mg ) -ATPase a c t i v i t y i n the i n s i d e - ou t v e s i c l e s used i n t h i s s tudy. Th is d i f f e r e n c e may be due to the d i f f e r e n t procedures employed i n the p repa ra t i on of i n s i d e - ou t v e s i c l e s . In t h i s s tudy , maximum 103 opera t i on of the t r anspo r t mechanism and the enzyme a c t i v i t y was observed a t 2 mM and 1 mM to 2 mM f i n a l ATP concen t r a t i on s , • r e s p e c t i v e l y . 2 mM ATP was chosen as a near optimum concen t ra t i on f o r measuring t r anspo r t and enzyme a c t i v i t i e s i n the present s t u d i e s . At 100 yM f r e e C a 2 + , Ca Z+/P. s t o i ch i omet ry of the C a 2 + pump i n v e s i c l e s t r ea t ed w i th EDTA approached 1 i n the absence or presence of 2 yg/ml ca lmodu l in over the range of ATP concen t ra t i ons s t ud i ed . At ATP concen t ra t i ons o f 1 mM and 2 mM the s t o i ch i ome t r y 2+ of the Ca pump was es t imated to be 2 when the ca l c ium ( f r ee ) concen t r a t i on was 2.5 yM. 2+ 2+ E f f e c t o f Ca and Calmodul in on the S to i ch iomet ry of the Ca pump The s i n g l e most e f f e c t i v e determinant of the s t o i ch i ome t r y o f the ca l c ium pump was found t o be the f r ee ca l c i um concen t r a t i on . 2+ The r a t i o of Ca t ranspor ted per ATP u t i l i z e d was found to 2+ approach 2 at the lower f ree Ca ;•• range ( i e . between 1 yM to 2+ 24 yM Ca ) . The s t o i ch i ome t r y was found to approach 1 i n the 2+ h igher Ca range - 50 to 300 yM. These es t imates of s t o i ch i ome t r y were the same i n both unt reated v e s i c l e s and those t r ea t ed w i th EDTA to remove endogenous ca lmodu l i n . 104 In a recent paper by Waisman e t a l . (93 ) , the s to i ch i omet r y 2+ o f the Ca pump was determined to be approx imate ly 1.5 a t 2+ 12 yM f r ee Ca concen t ra t i on and below i n the absence o f ca lmodu l i n . However, they i nd i c a t ed tha t the s t o i ch i ome t r y i n the presence of ca lmodu l in was 0 .73. The authors hence concluded tha t ca lmodu l in decreases the e f f i c i e n c y of the pump and may i n d i c a t e an a dd i t i o n a l r o l e of ca l c ium in r egu l a t i n g the p r ope r t i e s of the membrane pump. In t h i s study the presence of ca lmodu l in a t 2+ var ious concen t ra t i ons d id .not a l t e r the s t o i ch i ome t r y of the Ca pump i n e i t h e r unt reated or EDTA-treated v e s i c l e s ; i n the low 2+ f r ee Ca range (1 to 24 yM)the s t o i c h i ome t r i c r a t i o was again 2+ found to be 2,whereas i n the h igh Ca f r ee range, the r a t i o was est imated to be 1. The obvious e f f e c t o f ca lmodu l in i n t h i s study was to 2+ inc rease the maximum v e l o c i t y ( V m a x ) o f both Ca t r an spo r t and the ( C a 2 + + Mg 2 + ) -ATPase a c t i v i t y by about 3 to 4 f o l d and 2+ a!sb : .to i nc rease - the apparent a f f i n i t y o f the enzyme f o r Ca 2+ The es t imated K d o f the high Ca a f f i n i t y component of the 2+ enzyme (3.5 yM f r ee Ca ; ) i n the absence or presence of ca lmodu l in compared favourab ly w i th the r e s u l t s o f Gopinath and V incenz i (59) who obta ined a K d o f 4 yM and Schatzmann (47) who a l so found the high a f f i n i t y form to have a K d o f 2.35 yM 2+ 2+ Ca . The low a f f i n i t y component of the Ca t r an spo r t system 105 i n EDTA t r ea ted v e s i c l e s was found to have a value ranging from 200 yM to 300 y M 2+ 2+ 2+ Ana l y s i s o f both Ca t r an spo r t and (Ca + Mg ) -ATPase data?, by Ead ie -Hof f s tee plots i n the absence of ca lmodul in i n d i c a t e d a mixture of both high and low a f f i n i t y c h a r a c t e r i s t i c s 2+ o f the enzyme and the Ca pump. In the presence o f s a t u r a t i n g 2+ ca lmodul in concentrat ion. , the Ead ie -Hof f s tee p l o t o f both Ca 2 + 2 + t r an spo r t and (Ca + Mg )-ATPase a c t i v i t y i n d i c a t e d predominance of the high a f f i n i t y component, a l though the enzyme p l o t showed a smal l c on t r i b u t i o n by a low a f f i n i t y component. Table 9 presented 2+ below summarizes the k i n e t i c parameters o f the Ca t r anspo r t 2+ 2+ and (Ca + Mg )-ATPase i n human e r y th rocy te i n s i d e - ou t v e s i c l e s as determined i n t h i s s tudy. The lower K 2 va lues of 84.66 yM C a 2 + and 106.89 yM C a 2 + obta ined i n the presence o f ca lmodul in i n non-EDTA t r ea t ed and ... EDTA t r ea ted v e s i c l e s . respec t i ve l y ,may not cha r a c t e r i z e the low 2+ Ca a f f i n i t y component but a s t a t e i n the t r a n s i t i o n process 2+ 2+ from a low Ca a f f i n i t y to a predominant ly h igh Ca a f f i n i t y T 1 component i n the presence of ca lmodu l i n , s i n ce the K 2 f o r the low a f f i n i t y component approaches zero i n the presence o f ca lmodu l i n . A l t e r n a t i v e l y , the lower K 2 values cou ld be due to f a i l u r e of the computer a na l y s i s to complete ly separate out the high and low 2+ Ca a f f i n i t y components. I t i s ev i den t from t h i s study t h a t the Table 9: Summar, and Ca 2+ 2+ •y o f the K i n e t i c parameters of the (Ca + Mg )-ATPase a c t i v i t y i 2 + Transport i n human e ry th rocy te i n s i d e - o u t v e s i c l e s . ( C a 2 + + Mg 2 + ) -ATPase A c t i v i t y V-j (y moles/mg/hr) K-| (yM) (ymoles/mg/hr) K 2 (yM) Non-EDTA t rea ted v e s i c l e s  No Calmodulin 2y g/ml CaM 0.72 10.01 0.214 214 1.93 9.47 0.554 84.66 • EDTA t r ea t ed IOV No Calmodul in (n=5) 2i\x g/ml CaTmodulin (n=3) 0.29±0.07 1.32±0.17 3.81±1.36 3.70±0.91 0.267 0.31 267 106.89 2+ Ca Transport Non-EDTA t rea ted v e s i c l e s EDTA t r ea t ed IOV No Calmodulin 2ug/m1 CaM No. Calmodul in ;(n=5) 2 ,x g/.ml Calmodul in (n=3) V-j (ymoles/mg/hr) K1 (yM) V 2 (ymoles/mg/hr) KQ (M M) 0.87 3.53 0.37 218 2.89 3.95 0.36±0.07 2.75±1.20 0.31 282 1.49±0.29 2.38±0.48 Values for non-EDTA t r ea ted v e s i c l e s represent the means o f two e s t ima t i on s . In the above t a b l e , V-j and K-| were est imated by a curve f i t t i n g computer ana l y s i s . ; V 2 est imated from the d i f f e r ence between the observed V m = v and the es t imated V n va lue . max i K 2 was obtained from the equat ion K 2 = V 2 / K 2 ' where the value o f K 2 was ob ta ined from a 3 parameter ana l y s i s (see Resu l t s ) o f the da ta . was 107 t r a n s i t i o n u o r conformat iona l change from a low Ca'"' a f f i n i t y to a 2+ high Ca a f f i n i t y i n the presence of ca lmodul in i s best a t t a i n ed 2+ at lower Mg concen t ra t i ons . The s l i g h t l y h igher values o f K-j es t imated f o r the enzyme a c t i v i t y i n non-EDTA t r ea t ed v e s i c l e s may be due to s t imu l a t i o n o f o ther ATPases not r e l a t e d to the t r an spo r t mechanism,since the K-j value f o r the t r anspo r t parameter i n non-EDTA t r ea t ed v e s i c l e s seems to approximate the values ob ta ined f o r 2+ the enzyme and the Ca - t r anspo r t system i n EDTA t r ea t ed v e s i c l e s . 2+ The E f f e c t o f Mg on the K i n e t i c s and S to i ch iomet ry .-. of the C a 2 + pump. The standard assay medium employed i n t h i s study conta ined 2+ 6.4 mM Mg concen t r a t i on . Although ca lmodu l in was seen to 2+ inc rease the apparent a f f i n i t y o f the enzyme f o r Ca , the p e r s i s -tence o f a low a f f i n i t y component o f the enzyme even a t s a t u r a t i n g ca lmodul in concent ra t ions was qu i t e i n t r i g u i n g . S ince Larsen and V incenz i (92 )observedonly the high a f f i n i t y form o f the enzyme 2+ a t s a t u r a t i n g concent ra t ions o f ca lmodul in a t 3 mM Mg concen t r a t i on , i t was necessary to i n v e s t i g a t e whether the pe r s i s t ence o f the low a f f i n i t y form o f the enzyme (a t s a t u r a t i n g ca lmodu l in concent ra t i on) was the r e s u l t o f d i f f e r ence s i n 2+ membrane preparat ion: o r an e f f e c t o f Mg ; and whether lower ing 2+ Mg concen t ra t i on had an e f f e c t on the s to i ch i omet r y of the 108 2+ 2+ Ca pump. I t was conf i rmed i n t h i s study t ha t lower ing Mg concen t ra t i on i n the presence o f ca lmodul in r e s u l t e d i n the 2+ predominance o f the high Ca a f f i n i t y form o f the enzyme. The 2+ V m , „ o f both the enzyme a c t i v i t y and Ca t r an spo r t , and the max 2+ concent ra t ions o f Ca a t which both e n t i t i e s s a t u r a t e , 2+ decreased w i th decreas ing Mg c o n c e n t r a t i o n ; i n f a c t , a t 1 mM 2+ Mg concen t r a t i on , i n c r e a s i ng f r ee ca l c ium concen t ra t i on beyond 2 yM r e s u l t e d i n a r ap i d i n h i b i t i o n of a c t i v i t i e s o f both the 2+ ATPase enzyme and t r anspo r t . At 3 mM Mg concen t r a t i on , the peak 2+ 2+ a c t i v i t y was ev iden t a t 17 yM free" Ca , wh i l e a t h igher Ca concent ra t ions i n h i b i t i o n o f both enzyme a c t i v i t y and t r an spo r t o c cu r r ed . 2+ I t can be i n f e r r e d from these r e s u l t s t ha t a Ca 2+ i n h i b i t o r y s i t e e x i s t s on the enzyme,and tha t Mg competes w i th ca l c ium f o r t h i s s i t e , s i n ce i n h i b i t i o n o f both enzyme a c t i v i t y and 2+ 2+ Ca t r an spo r t are supp res sedby i n c r ea s i ng Mg concen t r a t i on . Although t h i s i n t e r p r e t a t i o n i s i n agreement w i th t ha t o f V incenz i 2+ ( 9 4 ) , who observed s i m i l a r i n h i b i t i o n a t h igh Ca concent ra t i ons i n the presence o f ca lmodu l i n , i t must be s t r e s sed tha t the 2+ 2+ i n h i b i t i o n occurs even a t very low Ca concen t ra t i on when Mg 2+ 2+ concen t ra t i on i s a l s o very low. The s i t e o f Ca - Mg i n t e r -a c t i on may be i n f e r r e d from a cons i de r a t i on o f the e f f e c t o f 2+ 2+ 2+ Ca and Mg on the sequence o f events i n the Ca - t r an spo r t 109 ATPase, as desc r ibed by the work o f Garrahan and Rega (63-65) . The scheme, p r ev i ous l y desc r ibed i n the I n t r o d u c t i o n , i s again summarized below: ATP where E^ P and ^ a r e t n e phosphory lated forms:, o f the enzyme 2+ and E^  and E^  are the high and low Ca a f f i n i t y forms of the 2+ enzyme, r e spe c t i v e l y . Ca i s r equ i r ed f o r the phosphory la t i on o f 2+ E-j by ATP and Mg i s r equ i r ed f o r the convers ion of E^ P (of low 2+ c a t a l y t i c a c t i v i t y ) to E£P(of high c a t a l y t i c a c t i v i t y ) . (Mg 2+ can a l s o form E-jP from E-| and ATP i n the absence o f Ca 2+ (63, 65, 66 ) . A t low Mg , more o f the enzyme e x i s t s i n the low 2+ c a t a l y t i c s t a t e E-jP and lower Ca concent ra t ions are; . requ i red 2+ to compete w i th Mg and decrease the ra te o f format ion o f E^? 2+ from E-jP, hence lower ing the v e l o c i t y o f Ca t r an spo r t . At 2+ . 2+ h igher Mg concen t r a t i ons , more Ca i s necessary to compete 2+ wi th Mg f o r E^P. In the presence o f ca lmodu l i n , more of the t r an spo r t complex i s i n the form o f E-,P (66),making i t more n o su s cep t i b l e to Ca i n h i b i t i o n . 2+ At 1 mM Mg concen t r a t i on , the s to i ch i omet r y was 2+ found to be 2 a t a l l ca l c ium concen t ra t i ons . At 3 mM Mg concen t r a t i on a s to i ch i omet r y approaching 2 was found i n the low ?+ 2+ Ca range (0.71 yM to 24 uM) and 1 i n the h igher Ca range 2+ (50 - 300) , s i mil ar to the r e s u l t s obta ined a t 6.4 mM ..Mg concen t r a t i on . P h y s i o l o g i c a l Role of the High and Low A f f i n i t y Components 2+ I t i s gene ra l l y be l i e ved tha t Ca pumping i s coupled 2+ 2+ to the (Ca + Mg )-ATPase. However,there i s some cont roversy 2+ as to whether the enzyme operates i n the high or low Ca 2+ a f f i n i t y mode, as i t was suggested t ha t the low Ca a f f i n i t y s t a t e may be an a r t i f a c t o f the membrane p repa ra t i on ( 5 ) . I t i s the view o f V incenz i (94) t ha t ca lmodul in i s a subun i t o f the 2+ Ca pump ATPase and i s hence bound to the pump a t a l l t imes . This p o s t u l a t i o n consequent ly suggests tha t the enzyme operates on ly i n the high a f f i n i t y mode. Qu i s t and Roufoga l i s ( 9 ) , who used e r y th rocy te ghosts ,and Sarkadi e t a l . (10),who used i n t a c t c e l l s , s u g g e s t t ha t the low a f f i n i t y form o f the enzyme 2+ may represent the basal l e ve l o f the Ca - t r anspo r t ATPase 2+ a c t i v i t y , s i n c e i t a l s o supports Ca t r anspo r t and a l s o forms a phosphory lated i n te rmed ia te . Furthermore, Pennis ton e t a l (95 ) I l l 2+ i n d i c a t e t ha t apar t from the Ca - t r anspo r t ATPase, a l a rge number of o ther b ind ing s i t e s f o r ca lmodu l in e x i s t i n the 2+ e r y th rocy te and tha t some of these p ro t e i n s do not r equ i r e Ca f o r ca lmodu l in b i nd i ng . The observa t ions of Penniston e t a l . 2+ support the f a c t t ha t Ca - t r anspo r t ATPase may normal ly operate i n the low a f f i n i t y mode. I t i s suggested by Roufoga l i s (96) tha t ca lmodu l in b ind ing to the pump would occur dur ing t raumat i c s i t u a t i o n s , s u c h as s i c k l i n g , t o b r i ng about a maximum ra te of ca l c ium e f f l u x from the c e l l . In t h i s i n s t an ce , the enzyme w i l l operate predominant ly i n the high a f f i n i t y mode. In f luence of the Choice of Bu f fe r on the K i n e t i c s Of the 2+ Ca - t r an spo r t - ATPase I t i s apparent from t h i s study tha t the k ind o f bu f f e r 2+ used can i n f l u ence the k i n e t i c s of Ca t r an spo r t . In 2+ 2+ 2+ T r i s -ma lea te bu f f e r , both Ca - t r anspo r t and (Ca + Mg ) -2+ 2+ ATPase a c t i v i t y were s t imu la ted by Ca w i th high and low Ca a f f i n i t y components. However, the low a f f i n i t y form of the enzyme was d im in i shed i n sodium-Hepes bu f f e r . The s t o i ch i ome t r y 112 i n Hepes bu f f e r was found to be 2 over the e n t i r e range o f 2+ f r ee Ca concent ra t ions s tud i ed (0.71 yM to 300 yM) . S i m i l a r 2+ s i n g l e h igh Ca a f f i n i t y s t a te s o f the enzyme have been obta ined 2+ 2+ i n ghosts and s o l u b i l i z e d (Mg + Ca )-ATPase p repara t ions i n our l abo ra to r y by A l - Joboree e t a l . ( 97) i n Hepes b u f f e r , i n the presence o f some po ly -an ions and when EGTA concent ra t i on was inc reased to 1 mM and above. The l a t t e r f a c t i s i n t e r e s t i n g , s i n ce i t suggests tha t the enzyme probably recogn izes the 2_ an i on i c Ca-EGTA complex. 2+ A Model o f Ca T r ans l o ca t i on i n Human E r y th ro cy t e s . 2+ a) Low Ca range : S to i ch iomet ry of 2. 2+ 2+ The (Ca +. Mg )-ATPase o f human e ry th rocy te s was r e c en t l y p u r i f i e d and shown to be a p ro te i n o f mo lecu la r weight 125,000 by SDS-PAGE e l e c t r opho r e s i s (98 ) . However the s t a t e o f the enzyme i n the membrane i s unknown. In sarcop lasmic r e t i c u l um i t has been suggested tha t the a c t i v e enzyme e x i s t s as a t r ime r o r tet ramer (99 ) . Assuming t ha t the lowest a c t i v e ' 2+ form o f the Ca - t r an spo r t complex i n red c e l l s i n a dimer, i t i s f u r t h e r suggested tha t the two subun i ts must be i n c l o se 2+ 2+ p rox im i t y to support Ca t r anspo r t . When Ca concen t ra t i on i s low, i t i s po s s i b l e tha t on ly one subun i t needs to be phosphory lated by ATP f o r the t r an spo r t o f two ca l c ium ions to 113 occu r . This model assumes c o o p e r a t i v i t y between the subun i t s ; 2+ coopera t i ve i n t e r a c t i o n s between Ca s i t e s have been repor ted 2+ a t low Ca concent ra t ions QOO). The phosphory la ted enzyme, i n the form o f E-j, undergoes a conformat iona l change r e s u l t i n g i n 2+ the format ion o f E 2 P and the re l ease o f Ca to the e x t r a c e l l u l a r space. Here, the s t o i ch i ome t r y i s 2,as the h yd r o l y s i s o f one ATP 2+ 2+ by one (Ca + Mg )-ATPase u n i t can lead to the t r a n s l o c a t i o n 2+ o f two Ca ions as i l l u s t r a t e d d iagrammat i ca l l y below: Conformat ional change 2+ b) High Ca Range: S to i ch iomet ry o f ! . . 2+ At high Ca concen t r a t i ons , a t l e a s t two processes may be pos tu l a ted to account f o r the change i n s t o i ch i ome t r y to 1. i ) C r o s s - l i n k i n g o f phospho l i p i d head groups by 2+ Ca when ca l c ium concent ra t ions on the c y t o s o l i c su r face o f the c e l l i s too h igh. . I t i s po s s i b l e tha t ca l c ium may r e s u l t i n the c r o s s - l i n k i n g of negat ive head groups o f a c i d i c phospho l i p i d s , 114 r e s u l t i n g i n changes i n the membrane b i l a y e r s t r u c t u r e such as decreased f l u i d i t y o f the b i l a y e r (101)- As a consequence o f t h i s , the two subun i ts may become l e s s t i g h t l y coupled such t ha t both subun i ts must hydro lyse ATP i n order to t r an spo r t one ca l c ium each as i l l u s t r a t e d below: INSIDE OUTSIDE 2+ Support f o r t h i s mechanism may be found i n the Ca pump from 2+ sarcop lasmic r e t i c u l um , which has a Ca : s t o i c h i o m e t r y of 2 i n i n t a c t membranes but a s to i ch i omet r y o f 1 or l ess , i n r e c on s t i t u t e d systems (102). i i ) Hyd ro l y s i s o f ATP by other enzymes not a s soc i a t ed 2+ wi th Ca e f f l u x : 2+ 2+ I t i s pos tu l a ted tha t Ca s t imu la tes a Ca -dependent 2+ ATPase tha t i s not i nvo l ved i n Ca pumping,but hydro lyses ATP wi th a r e s u l t a n t re l ease o f P.-. This process can be der i ved from the work o f Qu is t and Roufoga l i s (9) and Sakard i e t a l . ( 1 0 ) , who showed tha t they cou ld i n h i b i t complete ly the t r an spo r t 115 mechanism w i th lanthanum but cou ld on ly i n h i b i t 50% of the ATPase a c t i v i t y . 2+ Ca - dependent breakdown o f a k inase-phosphod ies terase system may a l so con t r i bu t e to P.. fo rmat ion . Recent l y , Qu i s t and Reece ( 48 ) i nd i c a t ed tha t Mg-ATP induces shape t rans fo rmat ion o f e r y th rocy te ghosts by s t imu l a t i n g the format ion o f membrane d i -and t r i - p h o s p h a t i d y l i n o s i t o l through the phosphory la t ion o f a membrane 2+ subs t ra te by a Mg - dependent k inase . S i m i l a r l y , i t has been shown by o ther wo rke r s , t ha t high concent ra t ions of ca l c ium can s t imu l a t e dephosphory la t ion o f membrane d iphosphat idy l i n o s i t o l and t r i p h o s p h a t i d y l i n o s i t o l (37,46).The f o l l ow i ng r eac t i ons can hence be env isaged: 2+ 2+ Mg , ATP Ca^ PI > D_.PI > PI + P. 2+ 2+ -Mg -K inase Ca - dependent monoesterase 2+ Mg Kinase D..PI » T r i PI > D.PI + P. M g 2 + , ATP C a 2 + dependent Phosphodiesterase I f these processes a l so occur i n i n s i d e - o u t v e s i c l e s , i t may 2+ r e s u l t i n ove r - e s t ima t i on of ATP h yd r o l y s i s coupled to Ca t r an spo r t when i n t r a c e l l u l a r ca l c ium concen t ra t i on i s h i gh . Thus the ac tua l s t o i ch i ome t r y may be h igher than 1, po s s i b l y 2 116 2+ 2+ even a t h igh Ca concen t r a t i on . The requirement f o r Mg by the k inase enzyme may e xp l a i n why the s t o i ch i ome t r y was 2 a t a l l 2+ 2+ Ca concent ra t i ons when Mg concen t ra t i on was lowered to 1 mM, s i n ce the above r eac t i ons may now not s i g n i f i c a n t l y c on t r i bu t e 2+ to P.j r e l e a s e . To e xp l a i n the s t o i ch i ome t r y o f 2 a t a l l Ca concen t ra t i ons i n the presence of HEPES, i t may be pos tu l a ted tha t Hepes b u f f e r , which i s a n i o n i c , i n t e r f e r e s w i th the above r e a c t i o n s , hence g i v i n g r i s e to the s t o i c h i ome t r i c r a t i o of 2. A l though ca lmodu l in has 4 b ind ing s i t e s f o r c a l c i um , i t i s a n t i c i p a t e d tha t the b ind ing o f ca lmodu l in doe.s not 2+ con t r i bu t e i t s Ca i n the t r anspo r t sequence, but on ly inc reases 2+ 2+ the apparent a f f i n i t y o f the enzyme f o r Ca at i t s own Ca b ind ing s i t e , as suggested by Penniston e t a l . (95) . The 2+ f a i l u r e o f ca lmodu l in to a l t e r the s t o i ch i ome t r y o f Ca t r an spo r t i s c on s i s t en t w i th the view tha t ca lmodu l in does not c on t r i bu t e i t s ca l c ium to t r an spo r t . Aga in , when ca l c ium concen t ra t i on i s low i t i s a n t i c i p a t e d tha t on ly one subun i t hydro lyses ATP even in the presence of ca lmodu l i n . S ince the s t o i ch i ome t r y 2+ decreases to 1 a t h igher Ca concen t r a t i on s , i t i s argued tha t 2+ the uncoupl ing o f the subun i ts or the Ca - dependent s t imu l a t i o n 2+ 2+ 2+ of o ther Ca - dependent ATPase or a Mg - k inase/Ca phosphodiesterase at h igher ca l c ium concent ra t i ons a l s o occurs i n the presence of ca lmodu l i n . 117 SUMMARY AND CONCLUSIONS I n t r a c e l l u l a r ca l c ium concen t ra t i on was determined 2+ to be the major determinant o f the s t o i ch i ome t r y o f the Ca pump i n human e r y th rocy te i n s i d e - o u t v e s i c l e s . Whereas the s to i ch i omet r y was found to be two a t low ca l c ium concen t r a t i on s , high ca l c ium concent ra t ions r e su l t e d i n s to i ch i omet r y o f 1. The reason f o r t h i s apparent decrease i n the e f f i c i e n c y o f the pump i s unknown a t present but may be due to s p l i t t i n g o f ATP 2+ by o ther Ca dependent processes unre la ted to the t r an spo r t o f ca l c ium. The change i n s t o i c h i o m e t r i c r a t i o s observed a t low 2+ 2+ and high Ca concent ra t ions was observed a t Mg concent ra t ions o f 3 mM and above. However, a s t o i ch i ome t r y o f 2 was observed 2+ 2+ a t 1 mM Mg concen t ra t i on even a t high Ca concen t r a t i on ; t h i s 2+ may suggest d ea c t i v a t i o n o f a non--pump-Ca - -ATPase o r k i nase -2+ phosphodiesterase tha t a l so s p l i t s ATP. Mg was a l so observed 2+ to compete wi th ca l c ium f o r a Ca i n h i b i t o r y s i t e , po s s i b l y the 2+ Mg s i t e normal ly r e spons i b l e f o r convers ion o f E-|P to the c a t a l y t i c a l l y more a c t i v e form E£P. Calmodul in was found not to a f f e c t the s t o i ch i ome t r y 2+ of the Ca pump. The predominant e f f e c t o f ca lmodul in was to inc rease both the V m a x and the a f f i n i t y o f the enzyme fo r 118 ca l c ium. Lowering M g t T concen t ra t i on i n the presence o f s a t u r a t i n g concent ra t i ons o f ca lmodul in r e s u l t e d i n the predominance o f the 2+ high a f f i n i t y form o f the Ca t r an spo r t complex. I t must be emphasized t ha t the i n s i d e - ou t v e s i c l e s used in t h i s study were sea led and hence represent a good model 2+ 2+ f o r the study o f the k i n e t i c parameters o f (Ca + Mg )-ATPase 2+ and the s t o i ch i ome t r y o f the Ca pump. L a s t l y , i t must a l s o be emphasized tha t the bu f f e r used can a f f e c t the k i n e t i c s o f 2+ 2+ 2+ Ca t r an spo r t and (Ca + Mg )-ATPase, and hence the i n t e r p r e t a t i o n of data ob ta ined . 119 REFERENCES 1. Baker, P.F. 1972. Transpor t and Metabol ism o f Calc ium ions i n Nerve. P rogr . B iophys. Mol . B i o l . 24, 177-223 2. Ebash i , S. 1976. E x c i t a t i o n - Con t rac t i on Coupl ing Ann. Rev. P h y s i o l . 38, 293-314. 3. Rasmussen, H. and Goodman, D.B.P. (1977). Re l a t i onsh i p s between Calc ium and C y c l i c Nuc leo t ides i n Ce l l A c t i v a t i o n . P h y s i o l . Rev. 57_, 421-509. 2+ 4. Schatzmann, H . J . 1966. ATP dependent Ca e x t r u s i on from Human red c e l l s . E xpe r i e n t i a (Base l ) 22: 364. 5. Schatzmann, H . J . 1975. Ac t i ve ca l c ium t r an spo r t and C a 2 + a c t i v a t e d ATPase in human red c e l l s . I n Current t op i c s i n membrane Transpor t Volume 6 ed i t e d by F. Bronner and A. K l e i n z e l l e r pp.125-168. Academic P r e s s , New York. 6. S a ka r d i , B., and Tosteson, D.C. 1979. A c t i v e ca l c ium t r an spo r t i n human red c e l l s . In Membrane t r anspo r t i n B i o l ogy . Ed i t ed by G. G i e b i s c h , D.C. Tosteson and H.H. Uss ing , Sp r i nge r - V e r l a g , B e r l i n pp.117-160. 7. O l son , E. J . , and Cazor t , R . J . 1969. A c t i v e ca l c ium and s t ron t i um t r an spo r t i n human e r y th rocy te ghosts . J . Gen. P h y s i o l . 53^ 311-322. 8. Young N. Cha; Bak C. Sh i n ; and Kwang S. Lee. 1971. A c t i v e uptake o f C a 2 + and C a 2 + - A c t i v a t ed Mg2+ ATPase i n Red Ce l l membrane fragments. Journa l o f General Phys io logy 57, #2 pp.202-215. 9. Qu i s t , E .E . , and Rou foga l i s , B.D. 1975b. Determinat ion o f the S to i ch iomet ry o f the Ca-pump i n human e ry th rocy tes us ing Lanthanum as a s e l e c t i v e i n h i b i t o r . FEBS L e t t . 50, 135-139. 10. S a r k ad i , B., Szasz , I . , Ge r l o c zy , A . , and Gardos, G. 1977 Transpor t parameters and s to i ch i omet r y o f a c t i v e ca l c ium ion e x t r u s i on i n i n t a c t human red c e l l s . B ioch im. B iophys. Ac ta , 464, 93-107. 120 11. Mol lman, J . E . , and P l ea su re , D.E. 1980. Calc ium t r an spo r t i n Human Ins ide -ou t E ry th rocy te V e s i c l e s . J . B i o l . Chem. 255, #2, 569-574. 12. S teck , T . L . , and Kant, J . A . 1974. P repa ra t i on o f impermeable i n s i d e - ou t and r i g h t - s i d e out v e s i c l e s from e ry th rocy te membranes. In "Methods i n Enzymology" Vol XXXI Par t 4, (S. F l e i s c h e r and L. Packer eds. 172-180. 13. Schatzmann, H . J . 1969. Transmembrane ca l c ium movements i n resea led human red c e l l s . In Calc ium and c e l l u l a r f u n c t i o n . Ed i t ed by A.W. Cuthbert MacMi l lan Company, London, pp.85-95. 14. P o r z i g , H. 1972. ATP independent ca l c ium net movements i n human red c e l l ghosts . J . Memb. B i o l . 8, 237-258. 15. F e r r e i r a , H .6 . , and Lew, V . L . 1975. Ca t r an spo r t and Ca pump reve r sa l i n human red b lood c e l l s . J . P h y s i o l . (London), 252, 86-87P. 16. Long, C. , Mouat, B. 1971. The b ind ing of ca l c ium ions by e r y th rocy te s and ' g h o s t ' - c e l l membranes. Biochem. J . 123, 829-836. 17. Weed, R . I . , L aCe l l e , P . L . , M e r r i l l , E.W., C r a i g , G . , Gregory, A . , Karch, F. and P i c kens , E. 1969. Metabo l i c dependence o f a c i d c e l l d e f o rmab i l i t y . J . C l i n . Inves t . 48 795-809. 18. Chau-Wong, M., and Seeman, P. 1971. The con t ro l o f membrane bound C a 2 + by ATP. B ioch im. B iophys. A c t a , 241, 473-482. 19. Edmondson,J.W. and L i , T-K. 1976. The e f f e c t s o f ionophore A23187 on e r y t h r o c y t e s . Re l a t i on sh i p o f ATP and 2 ,3 -d iphosphog lycerate to ca l c ium b ind ing capac i t y . B ioch im. B iophys. Ac ta . 443, 106-113. 20. La C e l l e , P .L . -1969. A l t e r a t i o n of d e f o rmab i l i t y o f the e r y th rocy te membrane i n s to red b lood t r a n s f u s i o n , 9: 238-245. 21 . L aCe l l e , P . L . , F.H. K i r k p a t r i c k , and M. Udkow. Re la t i ons o f a l t e r e d d e f o rmab i l i t y , ATP, DPG and Ca2+ concent ra t i ons i n senescent e r y t h r o c y t e s . In : E r y t h ro cy t e s , thrombocytes, Leukocytes , e d i t e d by E. G a l a r d i , K. Moser, E. Deutsch and W. Wilmanns. S t u t t g a r t : Thieme 1973 pp.49-52. 121 22. Dunn, M.J. 1974. Re: Blood Ce l l ca l c ium and magnesium: e f f e c t s upon sodium and potassium t r an spo r t and c e l l u l a r morphology. B ioch im ica B ipphys i ca Acta 352, 97-116. 23. Marches i , V . T . , and S t ee r s , E. , J r . 1968. S e l e c t i v e s o l u b i l i z a t i o n o f a P ro t e i n component o f the Red Ce l l Membrane. Sc ience 159, 203-204. 24. C l a r ke , M. 1971. I s o l a t i o n and Cha r a c t e r i z a t i o n o f a Water -so lub le P ro te i n from Bovine E ry th rocy te Membranes. Biochem. B iophys. Res. Comm. 45, 1063-1070. 25. T i l n e y , L .G . , Detmers, P. 1975. A c t i n i n E ry th rocy te Ghosts and i t s A s s o c i a t i o n w i th S p e c t r i n . J . Ce l l B io logy 65_, 508-520. 26. K i r k p a t r i c k , F .H . , 1976. S p e c t r i n : cu r ren t understanding o f i t s p h y s i c a l , b iochemica l and f un c t i ona l p r o pe r t i e s . L i f e Sc ience 1_9, 1-18 27. Pa lek , J . , W.A. Curby and F . J . L i o n e t t i . 1971. Regu la t ion o f Ca2+ a c t i v a t e d ATPase to C a 2 + - l i n k ed shr inkage o f human red c e l l ghosts . American Journa l o f Phys io logy 220; 1028-1032. 28. Weed, R . I . L aCe l l e , R.L. and M e r r i l l , E.W.. 1969. Metabo l i c dependence o f red c e l l d e f o rmab i l i t y . Journa l o f C l i n i c a l I n v e s t i g a t i o n 48: 795-809. 29. L aCe l l e , P . L . , K i r k p a t r i c k F .H . , Udkow, M.P. and A r k i n , B. 1972. Membrane f ragmentat ion and C a 2 + membrane i n t e r a c t i o n : p o t e n t i a l mechanisms o f shape changes i n senescent red c e l l s . In Red Ce l l Shape, Be s s i e s , W., Weed R . I . and LeBlond, P.F. ( e d s . j - pp.789-798. 30. A l l a n , D., and R.H. M i c h e l l . 1975. Accumulat ion of acy l 1, 2-di acy l g l y ce ro l i n the plasma membrane may l ead to ech inocyte t rans fo rmat ion o f e r y t h r o c y t e . Nature 258; 348-349. 31. A l l a n , D., and R.H. M i c h e l l . Calc ium ion dependent d i a c y l g l y c e r o l accumulat ion i n e r y th rocy te i s a s soc i a t ed w i th m i c r o v e s i c u l a t i o n but not w i th e f f l u x of potassium ions Biochem J . 166 ( 3 ) : 495-9, 15 Sep.77. 122 32. Gardos G. The f unc t i on o f ca l c ium i n the potass ium pe rmeab i l i t y o f human red c e l l s . 1958. B ioch im ica B iophys i ca Acta 30 653-654. 33. Blum R.M., and J . F . Hoffmann Ca- induced K t r an spo r t i n Human red c e l l s : L o c a l i z a t i o n of the s e n s i t i v e s i t e to the i n s i d e of the membrane. 1972. B ioch im. B iophys i ca Research Communications 4£; 1146-1152. 34. Szasz , I . , and Gardos, G. Mechanism o f var ious drug e f f e c t s on the C a 2 + -dependent K+ e f f l u x from human red b lood c e l l s . FEBS:..Xett. 44, 213-223. 35. Gardos, G. 1958. E f f e c t o f Ethylenediamine - t e t r a a ce t a t e on the pe rmeab i l i t y o f human e r y t h r o cy t e s . Acta Phy s i o l og i c a Aca. Sc i . (Hungary) 14: 1-5. 36. Dunham, E.T. , and I .M. G lynn. 1961. ATPase a c t i v i t y and the a c t i v e movements o f a l k a l i metal i on s . Journa l o f Phys io logy (London), 156: 274-293. 37. A l l a n , D., and M i c h e l l , R .H. . 1978. A ca l c ium a c t i v a t e d po l yphospho inos i t i de phosphodiesterase i n the plasma membrane o f human and r a bb i t e r y t h r o c y t e s . BBA 508: 277-286. 38. S e i f r i n g , G .E . , A p o s t o l , A . B . , Ve lasco , P.T. and Lorand, L.: Enzymatic bas i s f o r the C a 2 + induced cross l i n k i n g of membrane p ro te ins i n i n t a c t human e r y th rocy te B iochemis t ry 17: 2598-2604. (1978). 39. Dunham, E.T. , and Glynn, I .M. .1961. Adenosine t r i phosphatase a c t i v i t y and the a c t i v e movements o f a l k a l i metal i ons . Journa l o f Phys io logy (London) 156; 274-293. 40. Schatzmann H . J . , and V i n c en z i , F.F. (1969). Calc ium movements across the membrane o f human red c e l l s . J . Phys io l (London). 201, 369-395. 41. Lee, K.W., and Sh i n , B.C. 1969. S tud ies on the a c t i v e t r an spo r t o f ca l c ium i n human red c e l l s . J . Gen. P h y s i o l . 54, 713-729. 42. V i n c e n z i , F.F. and H inds , T.R. (1976). Plasma membrane ca l c ium t r an spo r t and membrane - bound enzymes. In the enzymes o f b i o l o g i c a l membranes, Vo l . 3, e d i t e d by A. Mar tonoz i , New York, pp. 261-281. 123 43. Chav Y . N . , Sh i n , B .C. , and Lee, K .S . . 1971. A c t i v e uptake of Ca2+ and Ca2+ a c t i v a t e d Mg2+ATPase i n red c e l l membrane fragments. J . Gen. P h y s i o l . 57, 202-215. 44. Wins, P., and S c h o f f e n i e l s . 1966b. S tud ies on r e d - c e l l ghosts ATPase. systems; P rope r t i e s o f a (Mg2+ + Ca2+)-dependent ATPase. B ioch im. B iophys. A c t a , 120, 341-350. 45. P f l e g e r , H. , and Wolf , H.U. 1975. A c t i v a t i o n of a membrane bound h igh a f f i n i t y ca l c ium i o n - s e n s i t i v e adenosine t r i phosphatase of human e ry th rocy te by d i v a l e n t metal i o n s . Biochem J . 147, 359-361. 46. Lang, V . , P r y h i t k a , G. , and Buck ley, J . T . 1977. E f f e c t o f neomycin and ionophore A23187 on ATP l e v e l s and turnover of po l yphospho inos i t i des i n human e r y t h r o c y t e s . Can J . Biochem. 55_ 1007-1012. 47. Schatzmann, H .J . 1973. Dependence on ca l c ium concen t ra t i on and s to i ch i ome t r y o f the ca l c ium pump i n human red c e l l s . J . P h y s i o l . (London), 235, 551-569. 48. Qu i s t , E . E . , and Reece, K.L. 1980. The Role o f D iphosphat idy-1 i n o s i t o l i n e r y t h ro cy t e membrane shape r e g u l a t i o n . Biochem. B iophys. Res. Comm. 95_, #3, 1023-1030. 49. Lu th ra , M.G., H i l denbrand t , G.R., and Hanahan, D.J . 1976. S tud ies on an a c t i v a t o r o f the (Ca 2+ + Mg 2+)-ATPase o f human e r y th rocy te membranes. B ioch im. B iophys. A c t a . 419, 164-179. 50. Bond, G .H . , and Clough, D.L. 1973. A s o l ub l e p r o t e i n a c t i v a t o r o f (Mg2+ + Ca2+)-dependent ATPase o f red c e l l membranes. B ioch im. B iophys. Ac ta .323 , 592-599. 51. Cheung, W.Y., Lynch, T . J . , and Wa l l ace , R.W. 1978. An endogenous Ca2+ dependent a c t i v a t o r p r o t e i n o f b r a i n adeny late cyc l a se and c y c l i c nuc l eo t i de phosphod ies terase. I n . Advances i n C y c l i c Nuc leo t i de Research (George, W.J . and Igna r ro , L . J . eds . ) 9, 233-251. 52. Stevens, F .C . , Walsh, M., Ho, H . , Teo, T . S . , and Wang, J . H . 1976. Comparison o f ca l c ium b ind ing p r o t e i n s : bovine hear t and b ra i n p r o t e i n a c t i v a t o r s o f c y c l i c nuc l eo t i de phosphodiesterase and r a bb i t s k e l e t a l muscle t ropon in C. J . B i o l . Chem. 251, 4495-4500. 124 53. Cheung, W.Y. 1980. Calmodul in P lays a P i v o t a l Role i n c e l l u l a r Regu l a t i on . Sc ience 207, 19-27. 54. Wins, P. (1969) The i n t e r a c t i o n o f red c e l l membrane ATPase w i th ca l c ium. Arch . I n t . P h y s i o l . B ioch im. 77:245-250. 55. Wolf , H.U. (1970): P u r i f i c a t i o n o f the C a 2 + -dependent ATPase of human e r y th rocy te membranes. B ioch im. B iophys. A c t a . 21_9: 521-524. 56. Schatzmann, H . J . , and R o s s i , G.L. (1971): ( C a 2 + + M g 2 + ) -a c t i v a t e d membrane ATPase i n human red c e l l s and t h e i r po s s i b l e r e l a t i o n s to ca t i on t r an spo r t . BBA 241: 379-392. 57. Qu i s t , E.E. and Rou foga l i s , B.D. (1975): Calciurn t r an spo r t i n human e r y t h r o c y t e s : sepa ra t i on and r e c o n s t i t u t i o n o f h igh and low Ca a f f i n i t y (Mg + Ca)-ATPase a c t i v i t i e s i n membranes prepared a t low i o n i c s t r eng t h . A r ch . Biochem. B iophys . 168: 240-251. 58. Scha r f , 0 . , and Foder, B. (1977) : Low Ca^ T Concent ra t ion c o n t r o l l i n g two k i n e t i c s t a t e s o f Ca 2 + -ATPase from human e r y t h r o c y t e s . BBA 483: 416-424. 59. Gop inath, R.M., and V i n c e n z i , F .F . , 1977. Phosphodiesterase p r o t e i n a c t i v a t o r mimics red blood c e l l cy top lasmic a c t i v a t o r o f ( C a 2 + + Mg 2 + ) -ATPase. B ioch im. B iophys. Res. Commun. 77, 1203-1209. 60. Hanahan, D . J . , and ..Ekholm, J . E . 1978. The express ion of optimum ATPase a c t i v i t i e s i n human e r y t h r o c y t e s . A . compar is ion o f d i f f e r e n t l y t i c procedures. A r ch . Biochem. B iophys. 187, 170-179. 61. Mac ln ty re , J . , and Green, J .W. .1978. S t imu l a t i o n of ca l c ium t r an spo r t i n i n s i d e - o u t v e s i c l e s o f human e r y t h ro cy t e membranes by a s o l ub l e cy top lasmic a c t i v a t o r B ioch im. B iophys. Ac ta . 510, 373-377. 62. S a ka r d i , B. A c t i v e ca l c ium t r an spo r t i n human red c e l l s . 1980. B ioch im. B iophys. Ac t a . 604, 159-190. 63. Rega, A . F . , and Garrahan, P . J . 1975. Calc ium ion dependent phosphory la t ion o f human e r y th rocy te membranes. J.Memb. B i o l . 22, 313-327. 125 64. Rega, A . F . , and Garrahan, P . J . 1978. Calc ium ion-dependent dephosphory la t ion o f the Ca2+-ATPase o f human red c e l l s by ADP. B ioch im. B iophys. Acta 507, 182-184. 65. Schatzmann, H . J . , and Bu rg i n , H. 1978. Ca lc ium i n human red b lood c e l l s . Ann. N.Y. Acad. S c i . 307, 125-146. 66. Mual lem, S . , and K a r l i s h , S . J . D . 1980. Regulatory i n t e r a c t i o n between ca lmodu l in and ATP on the red c e l l Ca2+ pump. B ioch im. B iophys. A c t a , 597, 631-636. 2+ 67. Ka t z , S . , and B l o s t e i n , R. 1975. Ca - s t imu l a t ed membrane phosphory la t i on and ATPase a c t i v i t y o f the human e r y t h r o c y t e . B ioch im. B iophys. A c t a , 389, 314-324. 68. Enyed i , A . , S a ka r d i , B. , Szasz , I . , Bot , G . , and Gardos, G. 1980. Mo lecu la r p r ope r t i e s o f the Red Ce l l Ca lc ium Pump. C e l l Ca lc ium 1_, 299-310. 69. Rega, A . F . , and Garrahan, P . J . 1980. E f f e c t s o f ca lmodu l in on the phosphoenzyme o f the Ca2+-ATPase o f Human Red Ce l l Membranes. B ioch im. B iophs . A c t a . 596, 487-489. 70. . J e f f e r y , D.A., Rou foga l i s , B.D. , and Katz , S. 1981. The e f f e c t o f ca lmodul in on the phosphoprote in in te rmed ia te of M g 2 + - dependent Ca2+ s t imu l a t ed adenosine t r i phosphatase in human e r y th rocy te membranes. The Biochem. J . (London), 198, #194, 481-486. 71. Hasse lbach, W., and Makinose, M. (1963). Uber Mechanismus des Ca l c i umt ranspor te r s durch d i e membranen des sarkop lasmat ischen Ret i cu lums. BioChem. Z.339, 94. 72. La rsen , F .L . , H inds, T .R . , V i n c e n z i , F .F . 1978. On the Red Blood Ce l l Ca2+ pump: An Est imate o f S to i ch i ome t r y . J . Membrane B i o l . 41_, 361-376. 73. J . P . F . C . Ro s s i , P . J . Garrahan and A . F . Rega. (1978). Reversal o f the Calc ium pump i n Human Red C e l l s . J . Membrane B i o l . 44, 37-46. 74. Benz inger , T . , K i t z i n g e r , T . , Hems, R. and Bu r t on , K. (1959). Free energy changes o f the glutaminase r e a c t i on and the h yd r o l y s i s o f the te rmina l pyrophosphate bond o f adenosine t r i phospha te . Biochem. J . 71, 400-407. ^Z6 75. Hoffman, J . F . 1962. The a c t i v e t r an spo r t o f sodium by ghosts of human red b lood c e l l s . J . Gen. P h y s i o l . 45, 837. 76. Bodemann, H. , and Passow, H. (1972). Factors c o n t r o l l i n g the Resea l ing o f the Membrane of Human E ry th rocy te Ghosts a f t e r Hypotonic Hemolysis J . Membrane B i o l . 8, 1-26. 77. Robert M. Johnson and Douglas H. Kirkwood (1978). Loss o f Resea l ing A b i l i t y i n E ry th rocy te membranes. E f f e c t o f D i va l en t Cat ions and Spec t r i n Release. B. 509, 58-66. 78. Ka tz , S . , Rou foga l i s , B .D. , Landman, AUD., and Ho, L. 1979. P r ope r t i e s o f ( M g 2 + + Ca 2 + ) -ATPase o f E ry th rocy te membranes prepared by d i f f e r e n t procedures: In f luence o f M g 2 + , C a 2 + , ATP and P ro t e i n a c t i v a t o r . J . Supramolecular S t ru c tu re J_0, 215-225. 79. S h r i e r , S .L . 1967. ATP syn thes i s i n human e r y th ro cy t e membranes. B ioch im. B iophys. A c t a , 135, 591-598. 80. F i s k e , C .H . , and Subba Row, Y . J . 1925. The c o l o r i m e t r i c de te rminat ion o f phosphorus. J . B i o l . Chem. 66^ , 375-400. 81. Ka tz , A . M . , Repke, D.L . , Upshaw, J . E . , and P o l a s c i k , M.A. 1970 Cha r a c t e r i z a t i o n of Dog ca rd i a c microsomes Use of zonal c e n t r i -f uga t i on to f r a c t i o n a t e fragmented sarcop lasmic r e t i cu l um (Na + + K + ) - a c t i v a t e d ATPase and m i tochondr i a l f ragments. Biochim B iophys. Acta 205, 473-490. 82. Sparrow, M.P., and Johnstone, B.M. 1964. A r ap i d micromethod f o r e x t r a c t i o n o f ca l c ium and magnesium from t i s s u e B ioch im. B iophys. A c t a . , 90_, 425-426. 83. Raess, B .U . , and V i n c e n z i , F.F. 1980. A semi-automated method f o r the determinat ion o f mu l t i p l e membrane ATPase a c t i v i t i e s . J . Pharmacol. Meth. 4, 273-283. 84. Lowry, I . H . , Rosenbrough, N . J . , Fa r r , A . L . , and Randa l l , R . J . 1951. P r o t e i n measurement w i th f o l i n phenol reagent . J . B i o l . Chem. 193, 265-275. 85. Bensadoun, A . , and We ins te i n , D. 1976 Assay of p ro te i n s i n the presence o f i n t e r f e r i n g ma t e r i a l s . Ana l . Biochem. 70, 241-250. 127 86. S c h a r f f , 0 . 1972. The i n f l u en ce o f ca l c ium ions on the p repa ra t i on o f ( C a 2 + + Mg2 + ) - a c t i v a t ed membrane ATPase i n human red c e l l s . Scand. J . C l i n . Lab. Inves t . 30, 313-320. 87. K l i n g e r , R., Wetzker, R., F l e i s c h e r , I . , and Frunder , H. 1980. E f f e c t o f Ca lmodu l in , Ca2+ and Mg2+ on the ( C a 2 + + Mg 2 + ) -ATPase o f E ry th rocy te membranes. Ce l l Ca lc ium, 1, 229-240. 88. Wol f , D . J . , and Brostrom, C O . 1976. Calc ium dependent c y c l i c nuc l eo t i de phosphodiesterase from b r a i n : I d e n t i f i c a t i o n o f phospho l i p ids as ca l c ium independent a c t i v a t o r s . A rch . Biochem. B iophys. 173, 720-731. 89. Au, K.S. 1978. An Endogenous I n h i b i t o r o f E ry th rocy te Membrane (Oa 2 * + Mg 2 + ) -ATPase In t . J . Biochem. 9_, 477-480. 90. Maul d i n , D., Rou foga l i s , B.D. 1980. A p r o t e i n o f M g 2 + Dependent Ca2 + s t imu l a t ed ATPase i n human E ry th rocy te membranes .d i s t inc t from ca lmodu l in Biochem J . 187, 507-513. 91. Vanaman, T . C . , S h a r i e f , F., and Watte'rson, D.M. 1977. S t r u c t u r a l homology between b ra i n modulator P r o t e i n and muscle Tn C's In : Calc ium B ind ing P ro te in s and Calc ium Func t i on , ed . by R.H. Wasserman e t a l . , 107-117. 92. Larsen , F . L . , and V i n c e n z i , F .F . 1979. Ca lc ium Transpor t Across the Plasma Membrane: S t imu l a t i on by ca lmodu l in . Sc ience 201, 306-308. 93. Waisman, D.M., Gimble, J . M . , Goodman, B .P . , and Rasmussen, H. 1981. S tud ies o f the C a 2 + Transpor t Mechanism i n Human E ry th rocy te Ins i de -ou t Membrane V e s i c l e s . 1. Regu la t ion of the Ca2+ Pump by ca lmodu l i n . J . B i o l . Chem. 256, #1, 409-414. 94. V i n c en z i , F . F . , H inds , T .R . , and Raess, B.U. 1980. Calmodul in and the Plasma membrane ca l c ium pump. Anna ls . N.Y. Academy o f Sc i ence . 356_, 232-244. 95. Penn i s ton , J . T . , Gra f , E. , and I tano , T. 1980. Calmodul in Regu la t ion of the C a 2 + Pump o f E ry th rocy te Membranes. Anna ls . N.Y. Academy o f S c i . 356, 245-257 128 96. Rou foga l i s , B.D. 1979. Regu la t ion o f calc iurn t r a n s l o c a t i o n across the red b lood c e l l membrane. Canad. J . Phys i o l Pharmacol. 57, 1331-1349. 97. A i - J obo r e , A . , Arda Minocherhomjee, and Rou foga l i s , B.D. 1980. E f f e c t o f anions on (Ca2+ + Mg2+)-ATPase i n human E ry th rocy te membranes. 1980 Banf f Conference on Biomembranes. 98. N i g g l i , V . , Penn i s ton , J . T . , and C a r a f o l i , E. 1979. P u r i f i c a t i o n of the (Ca2+ + Mg2+)-AIPase from Human E ry th rocy te membranes us ing a ca lmodul in A f f i n i t y column. The J . B i o l . Chem. 254, #2, 9955-9958. 99. Le Mai r e , M., L i n d , E .K. , J o rgensen ,K .E . , Ro igaard , H. , and Mo l l e r , J . V . 1978. Enzymat i ca l l y A c t i v e Ca2+ ATPase from Sarcop lasmic Ret icu lum membranes, s o l u b i l i z e d by Non- ion i c Detergents . Role o f l i p i d f o r aggrega t ion . J . B i o l . Chem. 253, 19, 7051-7060. 100. S cha r f f , 0 . , and Foder, B. 1978. Reve rs ib l e s h i f t between two s t a t e s of Ca2 +-ATPase i n Human E ry th rocy tes mediated by Ca2+ and a membrane bound a c t i v a t o r . B ioch im. B iophys. A c t a , 509, 67-77. 101. L i v i n g s t one , C . J . , and Schachter , D. 1980. Calc ium modulates the L i p i d Dynamics o f Rat Hepatocyte Plasma Membranes by d i r e c t and i n d i r e c t mechanisms. Biochem. ]9_, 4823-4827. 102. Wang, C. , S a i t o , A . , and F l e i s c h e r , S. 1979. Co r r e l a t i o n o f U l t r a s t r u c t u r e o f r e c on s t i t u t e d sarcop lasmic r e t i c u l um membrane v e s i c l e s w i t h v a r i a t i o n i n phospho l i p i d to p r o t e i n r a t i o . J . B i o l . Chem., 254, #18, 9209-9219. 129 APPENDIX ?+ Assumptions of the Energe t i c s of the Ca Pump The f o l l ow i ng represent the va r ious assumptions made i n 2+ a r r i v m g at the c a l c u l a t i o n s regard ing the ene rge t i c s o f the Ca pump i n f r e sh human red c e l l s : [ATP]. = 1.5 x 1 0 " 3 M (47, 73) [ADP] i = 0.32 x 10"3 M (47, 73) [ P ] i = 0.36 x 1 0 " 3 M (47, 73) [ C a 2 + ] i = 5 x 1 0 " 7 M (47) [ C a 2 + ] Q = 1.5 x 1 0 " 3 (47) Energy A v a i l a b l e ATP ADP + P i chemical energy a v a i l a b l e (AG) i s represented by: n . [ADP][P.] AG = G° + RT In — [ATP] where AG 0 ' = standard f r ee energy = -7 .3 Kcal/mole at 37°C, T = 310°A. R = gas constant = 1.98 c a l o r i e s . 130 . . . /\G = -73 3 1 0 x In (0.32 x 0.36 x l O - 6 ) ) I W J 1.5 x 10~ 3 = (-7.3 + ( -5 .82)) Kcal/mole chemical energy a v a i l a b l e = -13.12 Kca l /mo le . 2+ The energy r equ i r ed to t r an s l o ca t e n Ca (n C a 2 + > n C a 2 + ) i s represented by the equat ion n R T In [ C \ , ] o • + n ZFE [Ca 2 .] 2+ where Z = i o n i c charge of Ca = 2 F = Faradays Constant = 96,492.. coulombs E = membrane po t en t i a l = 0.01 Vo l t s (ex te rna l minus i n t e r n a l ) For n = 1, - 1.98 x 310 • , • 1.5 x 1 0 " 3 . ,-, v 96493 x .01 energy r equ i r ed = ' - 3 x In — : = - r — + I x — . 2 1 Q Q Q 1000 5 x 1 0 " 7 X = (4.91 + 0.46) ..Kcal/mole = 5.37 K ca l /mole f o r n = 2, Energy r equ i r ed = (2 x 5'. 37) K ca l s /mo le = 10.75 K ca ls /mole 131 Determinat ion of the f ree Ca^' concen t ra t i on 2+ The f r ee Ca concent ra t ions were determined by means o f a computer program based on the f o l l ow i ng equat ions and cons tan ts : [ATP]" 4 =[ATP] 4 " + [H-ATP] 3 " +[Ca-ATP] 2 " + [Mg-ATP] 2" [ M g ] 2 + ] - = . [Mg-ATP] 2" + m g 2 + [ C a ] 2 + = [Ca-ATP] 2 " + C a 2 + e e + [Ca-EGTA] 2 ' [EGTA] 4 - = EGTA 4" + [Ca-EGTA] 2~ C a 2 + + ATP > [Ca-ATP] 2 " rCa-ATP] = K = 88000 [ C a 2 + ATP] a S S M g 2 + + ATP > .[Mg-ATP] 2" f M 9 " A T P ^ - K = 31,500 [Mg] [ATP] H + + ATP — > [H-ATP] 3" [H-ATP] 3 " = K = 1 Q 6 . 9 7 [H+] [ATP] a S S 132 The a s s o c i a t i o n constant f o r Ca-EGTA complex was dependent on PH. C a 2 + + EGTA 4" > [Ca-EGTA] 2~ [Ca-EGTA] 2~ = = 0.13611 x TO 7 [Ca* ][EGTA] a s s at PH 6 . 1 , K = 34501 ass These a s s o c i a t i o n constants f o r Ca-EGTA were c a l c u l a t e d by means o f a computer program based on four other constants l i s t e d below from Por t zhe l e t a l . (1964). C 1 = 2.89 x 1 0 " 9 C 2 = 7.09 x 1 0 " 8 C 3 = 4.78 x 1 0 " 2 133 Phosphate Determinat ion P.j determinat ions were performed us ing automated mod i f i ed F iske and SubbaRow procedure. A Technicon Autoana lyser pump w i th a 16 channel man i fo ld and a Technicon Sampler f i t t e d w i th a 40-2/1 cam tha t p rov ies 40 samples i n c l u d i n g washes was used. The se t -up employs two l a rge mix ing c o i l s w i t h i n which samples and reagents r e a c t . The output o f the autoana lyser was connected to a f low through c e l l l o ca ted i n a Technicon spectrophotometer . Absorbance was monitored a t 660 nm v i a the same Technicon c a l o r ime t e r , and recorded on a Technicon Recording System. Reagents used i nc l ude 1. A c i d molybdate s o l u t i o n (139 ml o f concent ra ted HgSO^, 25 g o f ammonium molybdate and enough de ion i zed water to make 2 l i t r e s ) . 2. 6% SDS ( e l e c t r o pho r e t i c grade); 3. 2% SDS ( e l e c t r o p h o r e t i c grade) 4. 9% A s co rb i c a c i d (w/v) . A phosphate s t anda rd i z a t i on curve rang ing from 0-400 nm/ml K^PO^ was run w i th each exper iment and was found to be l i n e a r over the range of concent ra t ions used. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095303/manifest

Comment

Related Items