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Studies on the role of Ca²⁺ in the pancreatic acinar cell Ansah, Twum-Ampofo 1980

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STUDIES ON CA2+-TRANSPORT PROCESSES IN CYSTIC FIBROSIS PATIENTS AND CONTROLS by TWUM-AMPOFO(ANSAH B.Sc, The University of Science and Technology, 1975 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Pharmacology and Toxicology of the Faculty of Pharmaceutical Sciences We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1980 (^) Twum-Ampofo Ansah, 1980 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that 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 for reference and study. I further agree that permission for extensive copying of t h i s thesis for s c h olarly purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Pharmacology and Toxicology The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date February 12, 1980.  - i i -ABSTRACT Plasma membrane-enriched preparations obtained from cultured human skin fibroblasts by differential centrifugation and sucrose density 2+ centrifugation techniques were found to contain a Ca -stimulated ATPase 2+ activity. This enzyme was Mg -dependent and was stimulated by calmodulin 2+ 2+ and thus was similar to the (Mg + Ca )-ATPase activity observed in other 2+ 2+ tissues. The specific activity of the (Mg + Ca )-ATPase present was 4-5 fold higher than that present in crude membrane preparations and 80-2+ 2+ 100 fold higher than that present in homogenates. The (Mg + Ca )-ATPase activity of both crude membrane and plasma membrane-enriched preparations of cultured fibroblasts from Cystic Fibrosis (CF.) patients was s i g n i f i -cantly reduced when compared to that activity observed in age-matched controls (p^O.Ol in the crude membrane preparations; p<.0.05 in the purified plasma membrane preparations). Reciprocal plots indicated that i t i s the maximal activation of the enzyme (vca2+) and not the a f f i n i t y of the enzyme system (K^igg) for calcium that i s altered in CF. strains. 2+ 2+ In order to determine i f this decrease in (Mg + Ca )-ATPase activity in CF. was related to a decrease in the ab i l i t y of these cells to transport calcium, inside-out (10) vesicles were prepared from red cells 2+ obtained from C F. patients and age-matched controls. Ca -uptake activity was found to be significantly reduced in the preparations derived from CF. patients. This reduction was apparent at a l l time points studied (10 seconds - 120 minutes) and at a l l free calcium concentrations used (10-150 um). Reciprocal plots of the data revealed that the K d i s s for calcium of the calcium pump, was similar in control and CF. samples but - i i i -t h a t the V £ & 2 + was s i g n i f i c a n t l y reduced (p ( 0.001) i n the C F . pre p a r a t i o n s . Calmodulin prepared from red c e l l hemolysates of c o n t r o l s was found to 2+ s t i m u l a t e Ca - t r a n s p o r t a c t i v i t y to a s i m i l a r extent i n both C F . and c o n t r o l 2+ samples; i t d i d not, though, r e t u r n Ca -uptake a c t i v i t y i n C F . prepara-t i o n s to c o n t r o l l e v e l s . An a l t e r a t i o n i n calcium t r a n s p o r t a c t i v i t y i n C F . may have a number of i m p l i c a t i o n s that may e x p l a i n some of the manif e s t a t i o n s of the disease. - i v -TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS v i i i INTRODUCTION 1 Pathophysiology of cystic fibrosis 1 Transport mechanisms 4 Factors related to cystic fibrosis 5 2+ 2+ 2+ Ca -Transport and (Mg + Ca )-ATPase activity 7 2+ 2+ RBC activator of the (Mg + Ca )-ATPase activity 12 AIMS OF THE PRESENT STUDY 15 MATERIALS AND METHODS 17 Establishment of fibroblast cultures 17 Preparation of plasma membrane-enriched fractions of human skin fibroblasts 18 Preparation of red blood c e l l inside-out vesicles 19 Enzyme assays 20 2+ 2+ A. Measurement of (Mg + Ca )-ATPase activity 20 B. 5'-Nucleotidase assay 21 C. Adenylate cyclase assay 21 D. Acetylcholinesterase assay 22 E. Glyceraldehyde 3-phosphate dehydrogenase assay 22 -v-PAGE Measurement of calcium transport activity 23 Preparation of calmodulin 23 Preparation of lymphoblast plasma membranes 24 Protein assay 24 Statistics 24 Materials 24 RESULTS 25 Partial purification of plasma membranes from cultured skin fibroblasts 25 2+ 2+ Partial characterization of the (Mg + Ca )-ATPase activity in fibroblast membrane preparations 25 2+ 2+ Comparison of (Mg + Ca )-ATPase activity in cultured fibroblasts derived from C F. patients and controls 29 Studies on inside-out vesicle preparations derived from red cells of controls and CF. patients 44 2+ Comparison of Ca -uptake activity in 10 vesicle preparation obtained from CF. patients and controls 45 2+ 2+ (Mg + Ca )-ATPase activity in purified plasma membrane preparations of human cultured lymphoblasts 52 DISCUSSION 56 BIBLIOGRAPHY 63 - v i -LIST OF TABLES TABLE 1. 5'-Nucleotidase and adenylate cyclase activities of fibroblast membrane preparations 2* K d i s s a n d v ca 2 + o f t h e ^ M 8 2 + + Ca2+)-ATPase of fibro-blast membrane preparations 2+ 3. K d i s s a n c* V C a 2 + °^ t* i e ^ a ~ u P t a ^ e system in control and C F . preparations of 10 vesicles 2+ 2+ 4. (Mg + Ca )-ATPase activity in purified plasma membrane preparations of human cultured lymphoblasts - v i i -L IST OF FIGURES PAGE FIGURE 2+ 2+ 1. The f u l l r e a c t i o n sequence o f t h e (Mg + Ca )-ATPase 11 2+ 2+ 2. Mg ^dependency o f t h e Ca - s t i m u l a t e d ATPase a c t i v i t y i n f i b r o b l a s t membrane p r e p a r a t i o n s 28 2+ 2+ 3. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane and i n p u r i f i e d plasma membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o -b l a s t s i n t h e p r e s e n c e and absence o f added c a l m o d u l i n 31 2+ 2+ 4. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane and i n p u r i f i e d plasma membrane p r e p a r a t i o n s of c u l t u r e d f i b r o -b l a s t s i n t h e p r e s e n c e and absence o f added c a l m o d u l i n 33 2+ 2+ 5. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane p r e p a r a -t i o n s o f c u l t u r e d f i b r o b l a s t s 35 6. K j i s g . a n d V Q 2+ o f t h e (Mg 2 ++ C a 2 + ) - A T P a s e a c t i v i t y or crude membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o b l a s t s 38 2+ 2+ 7. (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o b l a s t s 40 8. K d i s s and VCg2+ o f t h e (Mg 2 ++ C a 2 + ) - A T P a s e a c t i v i t y o f p u r i f i e d plasma membrane p r e p a r a t i o n s o f c u l t u r e d f i b r o -b l a s t s 442 2+ 9. Time-course o f Ca -uptake a c t i v i t y i n 10 v e s i c l e p r e p a r a t i o n s from c o n t r o l and C F . samples 47 2+ 10. I n i t i a l r a t e s o f Ca -uptake i n 10 v e s i c l e s from c o n t r o l and C F . samples 49 2+ 11. Ca -uptake a c t i v i t y i n t h e p r e s e n c e and absence o f added c a l m o d u l i n i n 10 v e s i c l e p r e p a r a t i o n s from C F . p a t i e n t s and c o n t r o l s 51 - v i i i -LIST OF ABBREVIATIONS AChE acetylcholinesterase ADP adenosine 5'-diphosphate AMP adenosine 5'-monophosphate ATP adenosine 5'-triphosphate ATPase adenosine 5'-triphosphatase Ca total calcium 2+ Ca free calcium ion cyclic AMP cyclic 3', 5'-adenosine monophosphate DEAE diethylaminoethyl DTNB 5, 5'-dithiobis-(2-nitrobenzoic acid) E enzyme EDTA ethylene diamine tetraacetate, disodium salt EGTA ethyleneglycolbis-( -aminoethyl) , N'-tetracetate E^P phosphorylated enzyme intermediate G3-PD glyceraldehyde 3-phosphate dehydrogenase GTP guanosine triphosphate 10 inside-out K^£ s s dissociation constant MBP modulator binding protein /3-NAD fb -nicotinamide adenine dinucleotide P i inorganic phosphate RBC red blood c e l l , - i x -S.E.M. standard error of the mean TCA t r i c h l o r o a c e t i c a c i d T r i s t r i s (hydroxymethyl) aminomethane V,-, 2+ maximum v e l o c i t y ACKNOWLEDGEMENTS I am deeply g r a t e f u l to Dr. S. Katz for h i s guidance and encouragement throughout t h i s study. I would l i k e to thank Drs. J.H. M c N e i l l , D.A. Applegarth and B.D. Roufogalis for t h e i r constructive c r i t i c i s m s of t h i s work. I also wish to acknowledge the excellent co-operation of Dr. A.G.F. Davidson and Dr. Lawrence Wongtand the s t a f f and patients of the Health Centre f o r Children C F . c l i n i c , Vancouver, B.C. Canada. - 1 -INTRODUCTION C y s t i c f i b r o s i s of the pancreas ( C F . ) i s now recognized as the most common genetic disease a f f e c t i n g people of Caucasian o r i g i n . I t was f i r s t r eported by Fanconi (1936) i n S w i t z e r l a n d , but i t was not recognized as a separate and d i s t i n c t d i s o r d e r u n t i l Andersen (1938) gave the f i r s t d e t a i l e d p a t h o l o g i c a l d e s c r i p t i o n of the disease. C y s t i c f i b r o s i s i s an inborn e r r o r of metabolism which i n v o l v e s exocrine glands and i s t r a n s m i t t e d as an autosomal r e c e s s i v e t r a i t (Lobeck 1972; d i Sant' Agnese and Davis 1976). I t i s estimated that approximately one i n 1600 newborns i s a f f e c t e d by C F . and that one i n 20 i n d i v i d u a l s i s a healthy c a r r i e r (Danks et a l 1965; Lobeck 1972). Pathophysiology of C y s t i c F i b r o s i s A s t r i k i n g i n c r e a s e i n the l e v e l of sodium, c h l o r i d e and, to a l e s s e r extent, potassium i s present i n the sweat of v i r t u a l l y a l l homozygote p a t i e n t s w i t h C F . and i s the most c o n s i s t e n t and e a s i l y recognizable chemical abnormality i n t h i s disease ( d i Sant' Agnese et a l 1953). The sweat e l e c t r o l y t e abnormality i s present from b i r t h and throughout l i f e and i s unrelated e i t h e r to s e v e r i t y of the u n d e r l y i n g disease or to which other exocrine organ i s i n v o l v e d ( d i Sant' Agnese and Talamo 1967). The "sweat t e s t " i s '•' - t h e r e f o r e g e n e r a l l y accepted as the simplest and most r e l i a b l e l a b o r a t o r y procedure i n the diagnosis of C F . ( d i Sant' Agnese and V i d a u r r e t a 1960 ; Shwachman'. and Antonowicz 1962). M o r p h o l o g i c a l l y , no d i f f e r e n c e has been seen between the e c c r i n e sweat glands of p a t i e n t s w i t h C F . and those of c o n t r o l subjects e i t h e r by l i g h t microscopy or by electron-microscopy techniques (Munger et a l 1961). In s k i n b i o p s i e s , Munger and co-workers (19.61) d i d not f i n d s i g n i f i c a n t d i f f e r e n c e s between the -2-two groups i n the f i n e structure of the c e l l s of the duct of sweat glands both before and a f t e r sweating. Using i n - v i t r o microperfusion, Mangos (1973) demonstrated that the duct of the sweat gland i n C F . behaved normally when perfused with normal sweat, whereas both normal and C F . sweat gland ducts be-haved abnormally when perfused with C F . sweat. I t i s highly probable that the C F . eccrine sweat abnormality i s due to an e f f e c t of a sodium reabsorption i n -h i b i t i n g f a c t o r exerted i n t r a l u m i n a l l y by the sweat of affected persons. The factor i s so l a b i l e that i t has been impossible so f a r to i s o l a t e and characterize i t . Wiesmann et a l (1972) demonstrated that i t i s s p e c i f i c a l l y the s a l i v a from the o r a l submucosal minor s a l i v a r y glands i n which sodium i s consistently i n -creased i n patients with C F . Thus, t h i s i s the only other exocrine gland system, besides the eccrine sweat glands to manifest t h i s sodium abnormality consistently i n the disease. In contrast to the serous, hypotonic secretions of patients with C F . ( v i z , sweat and sa l i v a ) which show elevated sodium and chloride l e v e l s ( d i Sant' Agnese et a l 1953; Wiesmann et a l 1972), the more p r o t e i n - r i c h or mucoid, i s o t o n i c secretions ( v i z , from the pancreas and tracheobronchial mucus glands) are found to have sodium concentrations near normal or even somewhat decreased (di Sant 1 Agnese and Talamo 1967). However these secretions have a s i g n i f i c a n t l y reduced water content (di Sant' Agnese and Talamo 1967). Physicochemically, the mucus secretions behave i n an abnormal way; they p r e c i p i t a t e , and obstruct organ passages, thus giving r i s e to chronic pulmonary disease, pancreatic i n s u f f i c i e n c y , hepatic c i r r h o s i s , i n t e s t i n a l obstruction and other complications. Elevated calcium concentrations have been noted i n submaxillary s a l i v a (Gibson et a l 1971) and parotid s a l i v a ( d i Sant' Agnese and Talamo 1967; Wotman et a l 1971) of C F . patients. Gibson et a l demonstrated experimentally that C F . -3-mucus derived from submaxillary s a l i v a was permeable to water and e l e c t r o l y t e s and also showed that normal mucus became hyperpermeable when calcium was added. They proposed that hypersecretion of calcium by the exocrine glands i s a primary defect i n the disease, rendering the mucus hyperpermeable to water and thus producing the other e l e c t r o l y t e abnormalities. Elevated calcium concentrations have also been observed i n f e c a l material (Johansen 1963), duodenal contents (Kopito 1964) and tracheobronchial secretions (Chernick and Barbero 1963; Potter et a l 1963). Boat and co-workers (1974) studied a calcium preciptable protein previously shown by Gugler (1967) to be one of two f r a c t i o n s which cause t u r b i d i t y i n submaxillary gland s a l i v a of patients with C F . This protein was shown to be a phosphoglycoprotein with a molecular weight of 12,000 daltons, with f i v e phosphate groups per molecule, which may aggregate i n the presence of calcium owing to reduction of e l e c t r o s t a t i c charge. Bettelheim (1971) using material furnished by Boat, proposed that aggregation may be associated with conformational change exposing hydrophobic s i t e s for non-covalent bonding. Calcium-precipitable protein i s i d e n t i c a l i n patients with C F . and controls i n many chemical features, including amino acid composition (Boat et a l 1964)'. Calcium-precipitable protein i s found i n comparable quantities i n s a l i v a from patients and normal controls. I t thus appears to be the calcium concentration i n s a l i v a of C F . patients that produces aggregation. Though the protein i t s e l f i s not detectable i n bronchial or duodenal f l u i d , i t has-been shown that rat i n t e s t i n a l mucins have decreased s o l u b i l i t y i n the presence of increased calcium concen-t r a t i o n (Forstner and Forstner 1976). -4-Transport Mechanisms Elevations of sweat sodium and chloride concentrations i n the face of normal concentrations of these substances i n precursor f l u i d (Schultz 1969) suggest that plasma membrane transport of e l e c t r o l y t e s by duct epithelium i s abnormal. C F . erythrocytes have been used as a simple model to delineate t h i s abnormality. Balfe et a l (1968) reported abnormal erythrocyte sodium transport i n C F . and obligate heterozygotes. These workers concluded that i f the postulated defect of sodium transport i n the sweat and s a l i v a r y ducts i n C F . represents a fundamental abnormality i n t h i s diseased state, a study of t h i s abnormality i n erythrocytes may help to determine the pathogenic defect. Lapey and Gardner (1971) observed that only the ouabain-insensitive, ethacrynic a c i d - s e n s i t i v e component of sodium e f f l u x was diminished i n erythrocytes from males and post-puberty females with C F . S i m i l a r l y , Cole and Dirks (1972) found that whereas the ouabain-sensitive component of erythrocyte ATPase a c t i v i t y was un-changed i n C F . patients, the ouabain-insensitive component was s i g n i f i -cantly decreased. This implied that an ATPase a c t i v i t y other than the one intimately r e l a t e d to Na-transport was affected by the disease state. 2+ 2+ Subsequently, Horton et a l (19 70) measured the Mg -dependent Ca -ATPase a c t i v i t y of - erythrocyte membranes from-both normals and C F . .patients and 2+ found a decreased.Ca -ATPase a c t i v i t y i n the C F . erythrocytes. The extent of enzyme a c t i v i t y depression was r e l a t e d to the s e v e r i t y of the disease. Other workers "(McEvoy et a l 1974; Feig et a l 1974; Duffy et a l 1974) did 2+ not observe any a l t e r a t i o n i n the^Ca ^ATPase a c t i v i t y i n erythrocytes from 2+ C F . patients. Recent studies (Katz 1978 a) have indicated that the (Mg + 2+ Ca )-ATPase ^ a c t i v i t y of erythrocytes obtained from C F . patients i s -5-s i g n i f i c a n t l y decreased when compared to the a c t i v i t y noted i n controls. This a l t e r a t i o n i n a c t i v i t y was found to be due to an a l t e r a t i o n i n the 2+ -2+ maximal a c t i v a t i o n of the (Mg + Ca )-ATPase and not i n the a f f i n i t y of the enzyme system f o r calcium. It was also found that t h i s decrease . 2+ 2-k xn (Mg - + Ca )-ATPase,activity i s not a part of a generalized membrane or membrane-bound enzyme a l t e r a t i o n i n C F . since studies on the Na +, K +-2+ ATPase and Mg - ATPase i n erythrocyte membranes indicated no a l t e r a t i o n i n the a c t i v i t y of these enzymes. Studies done using f i b r o b l a s t membrane 2+ 2+ preparations also indicated a decrease i n (Mg + Ca )-ATPase a c t i v i t y i n C F . s t r a i n s compared to normal s t r a i n s (Katz 1978 b) . F e i g a l and Shapiro (1979 a) reported an enlarged i n t r a c e l l u l a r calcium pool i n skin f i b r o b l a s t derived from subjects with C F . and obligate heterozygotes The observation of an a l t e r a t i o n i n f i b r o b l a s t s from obligate'heterozygotes suggested that t h i s abnormality i s r e l a t e d to the basic gene defect i n C F . (Feigal and Shapiro 1979 a). Subsequently, i t was shown that mitochondrial f r a c t i o n s i s o l a t e d from skin f i b r o b l a s t s of subjects with C F . accumulated more calcium than those from control c e l l s (Feigal and Shapiro 1979 b). Factors Related to C y s t i c F i b r o s i s Spock et a l (1967) observed that sera from C F . patients and t h e i r parents caused normally synchronous c i l i a r y a c t i v i t y i n rabbit tracheal explants to become i r r e g u l a r and dyskinetic. Oyster g i l l preparations were used by Bowman and co-workers (1969) to detect a c i l i a r y i n h i b i t o r i n sera and s a l i v a from C F . genotypes. These workers (Bowman et a l 1970) suggested that the factor which affected c i l i a t e d tissue might a f f e c t membrane transport and energy exchange. A s i m i l a r factor (or factors) has since been demonstrated - 6 -i n urine, cultured skin f i b r o b l a s t s (Beratis et a l 1973) and lymphoblasts (Conover et a l 1973) from C F . subjects, as well as i n obligate heterozygotes. The c i l i a r y i n h i b i t o r from serum i s a small molecular weight polypeptide within the range of 1,000 to 10,000 daltons (Conover et a l 1973; Bowman et a l 19 75) and i s presumed to be c a t i o n i c since i t i s eluted on chromatography i n a basic f r a c t i o n (Bowman et a l 1973). Recently, c i l i a r y dyskinesia a c t i v i t y was demonstrated i n a l l sera from C F . homozygotes and hetero-zygotes as well as i n sera from patients with bronchial asthma. However, i s o e l e c t r i c focusing showed that the presence of C F . protein corresponded with that of dyskinesia a c t i v i t y i n a l l sera tested except for the a c t i v e samples from the asthma patients (Wilson et a l 1977). These workers speculated that C F . protein i s r e l a t e d s t r u c t u r a l l y or metabolically to a C F . - s p e c i f i c c i l i a r y dyskinesia factor and that patients with bronchial asthma and other r e s p i r a t o r y and autoimmune disorders harbor a substance that can produce c i l i a r y dyskinesia but that d i f f e r s from C F . - s p e c i f i c c i l i a r y dyskinesia factor. The presence of C F . protein i n the sera from C F . and o b l i g a t e heterozygote i n d i v i d u a l s has been confirmed by other workers (Scholey et a l 1978; T u l l y et a l 1979). Bogart et a l (1977) has demonstrated that calcium ionophore A23187 produces a C F . - l i k e mucociliary disturbance i n rabbit tracheal explants when added to the culture medium. Although no s i g n i f i c a n t c o r r e l a t i o n was found between the amount of serum factor a c t i v i t y and the c l i n i c a l status of C F . patients (Nagy et a l 1979) i t i s possible that C F . c i l i a r y factor can modify passive and/or act i v e calcium transport across plasma membranes (Vincenzi; 1979). -7-O j 2~T~ O | Ca - Transport and (Mg + Ca ) - ATPase A c t i v i t y Calcium i s involved i n the regulation of a number of c e l l u l a r functions including stimulus-secretion coupling (Rubin 1974), ex c i t a t i o n - c o n t r a c t i o n coupling (Huxley 1973), control of membrane transport (Porzig 1972) and release of transmitter from motor nerve terminals (Katz and M i l e d i 1965). Many researchers have used the human red blood c e l l (RBC) as a model for studying the r o l e of the plasma membrane i n the regulation of c e l l u l a r calcium content. Harrison and Long (1968) found that the RBC contained -5 2+ 1.58 x 10 moles Ca per l i t e r of packed c e l l s . EDTA or s i m i l a r non-penetrating chelating agents could remove about 90% of RBC c e l l u l a r calcium i n i s o t o n i c medium without causing hemolysis. These workers (Harrison and Long 1968) suggested that most of the c e l l u l a r calcium was bound to the e x t r a c e l l u l a r surface of the membrane. The remaining 10% was believed to be bound mostly to proteins and small molecules leaving a cytoplasmic free Ca of about 10 M (Schatzmann 1973). The low i n t r a c e l l u l a r Ca concentration i n normal red blood c e l l s i s thought to be maintained by (i) the low calcium permeability of the plasma membrane of fresh red blood c e l l s (Porzig 1972; F e r r e i r a and Lew 1975), ( i i ) by chelation of calcium to i n t r a c e l l u l a r anions and to high a f f i n i t y membrane binding s i t e s (Weed et a l 1969; Long and Mouat 1971) or ( i i i ) by active calcium extrusion by the plasma membrane-bound calcium pump (Schatzmann 1966; Schatzmann and Vincenzi 1969; Romero and Whittam 1971). Maintenance of low i n t r a c e l l u l a r 2+ Ca has been shown to be e s s e n t i a l for normal red c e l l shape and function. 2+ An increase i n i n t r a c e l l u l a r Ca was reported to cause discoid-spheroe-chinocyte shape transformations (Weed et a l 1969; Palek et a l 1974), an + increase i n K permeability (Gardos 1958; Lew 1971), decreased deform-a b i l i t y (Weed et a l 1969), increased v i s c o s i t y (La C e l l e 1973) and i n -h i b i t i o n of Na"^  K + - transport (Dunn 1974) . In s p i t e of the fact that RBCs have r e l a t i v e l y low permeability 2+ to Ca (Porzig 1972; F e r r e i r a and Lew 1975), i t was shown that i n the 2+ 2+ presence of "AmM external Ca , the maximum passive leak of Ca into ATP - starved RBCs was about 6 umoles per l i t e r of c e l l s per hour ( F e r r e i r a and Lew 1977). This suggested that i n order to maintain low i n t r a c e l l u l a r o I _ ^ O j Ca i n the face of approximately 10 M Ca i n the e x t r a c e l l u l a r f l u i d , 2+ the RBC must have assystem for removing Ca . In 1966, Schatzmann (1966) reported transport of calcium from reversibly-hemolyzed RBC "ghosts" loaded with calcium and ATP. Later, Schatzmann and Vincenzi (1969) showed that calcium was extruded from resealed RBC "ghosts" against an e l e c t r o -chemical gradient. This transport, which was ATP and temperature dependent, 2+ 2+ required Mg and Ca i n s i d e the c e l l . The active transport of calcium from resealed RBC "ghosts" was confirmed by a number of laboratories (Lee and Shin 1969; Olson and Cazort 1969;:Qulst and Roufogalis 1975). 2+ The requirements for ATP and Mg l e d Schatzmann and Vincenzi (1969) 2+ 2+ to propose that the Mg - dependent - Ca activated adenosine triphosphatase 2+ 2+ (ATP Phosphohydrolase E C 3.6.1.3; (Mg + Ca ) - ATPase) a c t i v i t y , f i r s t reported i n RBC membranes by Dunham and Glynn (1961) and l a t e r by Wins and 2+ Schoffeniels (1966), was the biochemical expression of t h i s Ca transport process. There now e x i s t s ample evidence to support t h i s hypothesis (Schat-mann 1969; Vincenzi and Hinds 19*76) . The mechanism by which energy from the h y d r o l y s i s of ATP i s u t i l i z e d 2+ to transport Ca against an electrochemical..gradient i s not c l e a r l y under--9-stood. In recent years considerable progress has been made i n the e l u c i -2+ 2+ dation of the p a r t i a l reactions of the (Mg + Ca )-ATPase i n red 2+ 2+ c e l l membranes. The (Mg + Ca )-ATPase reaction proceeds.through a phosphorylated-protein intermediate. Formation of t h i s intermediate i s 2+ enhanced by Ca (Knauf et a l 1974; Katz and B l o s t e i n 1975; Rega and Garrahan 19 75) i n a manner s i m i l a r to the concentration dependence of 2+ Ca stimulation of the ATPase a c t i v i t y (Katz and B l o s t e i n 1975; Rega and Garrahan 1975; Szasz et a l 1978). The phosphorylated intermediate i s a 150,000 dalton protein (Knauf et a l 1974; Katz and B l o s t e i n 1975; Wolf et a l 1977) which i s chemically d i s t i n c t from the Na , K - ATPase phosphoenzyme of molecular weight around 100,000 daltons (Knauf et a l 1974; Katz and B l o s t e i n 1975). I t has been proposed that the substrate for the ATPase i s free ATP (Rega and Garrahan 1975; Schatzmann 1977; Richards et a l 1978). Substrate binding r e s u l t s i n the formation of an i n i t i a l phosphoenzyme, E-P. The K for the formation of E-P (1-6 uM) i s s i m i l a r to the K for m " m ATP hydrolysis-(2-5 uM) (Richards et a l 1978). Phosphorylation i n the 2+ 2+ presence of Ca occurs i n the absence of added Mg (Rega and Garrahan 2+ 1975). The major e f f e c t of Mg appears to be the conversion of E-P to a second state, E-P (Rega and Garrahan 1975; Garrahan and Rega 19.78). The t E-P intermediate i s more rea c t i v e than E-P and can undergo rapid hydrolysis 2+ (Rega and Garrahan 1978), consistent with the higher turnover of (Mg_ + 2+ 2+ Ca )-ATPase i n the presence of Mg (Katz and B l o s t e i n 1975; Garrahan and Rega 1978) and the higher steady-state l e v e l s of phosphoenzyme intermediate (Garrahan and Rega 1978; Schatzmann and Burgin 1978). The f i n a l step i n the sequence i s the conversion of E' to E, the regulation of which remains un-2+ known. The actual t r a n s l o c a t i o n of Ca across the plasma membrane i s -10-th ought to be through conformational changes i n the translocating protein (Schatzmann and Burgin 1978). Figure 1 describes the f u l l reaction sequence. Attempts to determine the number of calcium ions pumped per ATP hydrolyzed have y i e l d e d c o n f l i c t i n g reports. Schatzmann and Vincenzi (1969) and Schatzmann (1973) suggested a calcium to P i r a t i o of one. Quist and Roufogalis (1975) and Sarkadi et a l (1977) showed that maximum 2+ i n h i b i t i o n of Ca transport by lanthanum was associated with only a 50% 3+ i n h i b i t i o n of ATPase a c t i v i t y . They concluded that La - i n s e n s i t i v e ATPase 2+ a c t i v i t y was not associated with Ca transport and should be deducted from the t o t a l ATPase a c t i v i t y y i e l d i n g a stoichiometry estimate of 2 calcium ions pumped per P i released. Recently, Larsen et a l (1978 a) using an ion-s e l e c t i v e electrode method f o r r a p i d , continuous assessment of C a 2 + e f f l u x from resealed RBC "ghosts" reported a stoichiometry of one calcium ion 2+ pumped per ATP hydrolyzed. This may indic a t e therefore that the Ca pump has more than one mode of operation. 2+ 2+ 2+ K i n e t i c analysis of the Ca a c t i v a t i o n of (Mg + Ca )-ATPase a c t i v i t y 2+ 2+ i n i s o l a t e d RBC membranes revealed the presence of two (Mg + Ca )-ATPase 2+ 2+ a c t i v i t i e s . These are ref e r r e d to as high and low a f f i n i t y (Mg + Ca )-2+ ATPase to r e f l e c t t h e i r d i f f e r e n t a f f i n i t i e s f o r Ca (Horton et a l 1970; 2+ Schatzmann and Rossi 1971; Scharff 1972). The Ca d i s s o c i a t i o n constant (K(j^ s s) f ° r the high a f f i n i t y enzyme was estimated to be between 1 and 4 2+ uM Ca (Schatzmann and Rossi 1971; Scharff 1972; Schatzmann 1973). The 2+ low a f f i n i t y enzyme was reported to have a K ( j i s s ' r for Ca of 46-100 uM (Schatzmann and Rossi 1971; Scharff 1976; Scharff and Foder 1977). It i s - 1 1 -E + ATP Ca 2+ Mg 7PT E ~ P + ADP E-vP Mg 2+ E'~P E-xP H 9 0 E + P i 2+ 2+ Figure 1. The f u l l r e a c t i o n sequence of (Mg + Ca )-ATPase -12-2+ 2+ s t i l l not c l e a r whether there are two d i s t i n c t (Mg + Ca )-ATPase or 2+ 2+ two states of a sing l e (Mg + Ca )-ATPase regulated by other proteins i n the membrane. 2+ It has now been demonstrated that plasma-membrane bound ac t i v e Ca 2+ 2+ transport and/or (Mg + Ca )-ATPase exists i n a number of mammalian c e l l s , i n cluding those of l i v e r (Lamb and Lindsay 1971; Garnett and Kemp 1975; Lindsay 1976), kidney (Moore et a l 1974), adrenal medulla ( L e s l i e and Borowitz 1975), brain (Robinson 1974), heart (St. Louis and Sulakhe 1976) and uterus (Janis et a l 1977; Janis and Daniel 1977; Kroeger et a l 1977). 2+ 2+ RBC Activator of the (Mg + Ca )-ATPase A c t i v i t y The RBC a c t i v a t o r or calmodulin was f i r s t reported by Bond and Clough X19 73) who noted that a non-hemoglobin protein present i n the hemolysate of 2+ 2+ human RBCs increased the (Mg + Ca )-ATPase a c t i v i t y of i s o l a t e d RBC membranes. P a r t i a l p u r i f i c a t i o n was accomplished by Luthra et a l (1976 a). The presence of calmodulin i n the RBCs of several mammalian species has been reported (Luthra et a l 1976 b). Recently, more p u r i f i e d f r a c t i o n s of calmodulin have been obtained (Luthra et a l 1977; J a r r e t t and Penniston 1978). Calmodulin i s an a c i d i c protein with an i s o e l e c t r i c point of approximately 4.0. It has a molecular weight of about 16,500 daltons and has no inherent ATPase a c t i v i t y (Jung 1978) . Calmodulin was shown to bind to the inner surface of RBC membranes (Farrance and Vincenzi 1977 a): binding 2+ was dependent on Ca (Farrance and Vincenzi 1977 b). The requirement for 2+ Ca becomes important i n the l i g h t of the fac t that calmodulin was found to be present i n the RBC at a much higher concentration than was necessary 2+ 2+ to achieve maximum a c t i v a t i o n of the (Mg + Ca )-ATPase a c t i v i t y . I t 2+ 2+ has been observed that low a f f i n i t y - l i k e (Mg + Ca )-ATPase - membranes 2+ 2+ resemble high a f f i n i t y - l i k e (Mg + Ca )-ATPase - membranes i f incubated i n the presence of added calmodulin (Farrance and Vincenzi 1977 b). Thus, i t was suggested that the presence or absence of calmodulin may account for the high and low a f f i n i t y behavior r e s p e c t i v e l y , i n i s o l a t e d RBC membrane preparations (Schatzmann and Burgin 1978). Calmodulin i s o l a t e d from RBCs shares many :physicochemical properties with a pr o t e i n p u r i f i e d from bovine b r a i n and heart shown to a c t i v a t e c y c l i c AMP phosphodiesterase (Kakiuchi et a l 1972) and adenylate cyclase (Brostrom et a l 1975) a c t i v i t i e s . The f i n d i n g that t h i s l a t t e r protein produced 2+ 2+ s e l e c t i v e a c t i v a t i o n of RBC membrane (Mg + Ca )-ATPase (Gopinath and Vincenzi 1977; J a r r e t t and Penniston 1977) and that RBC calmodulin activ a t e d cyclic-AMP phosphodiesterase a c t i v i t y ( J a r r e t t and Penniston 1977) indicated that the two proteins could be i d e n t i c a l . Recent studies have 2+ 2+ shown that the mode of stimulation of human RBC membrane (Mg + Ca )-ATPase by calmodulin i s s i m i l a r to the mode of stimulation by calmodulin of adenylate cyclase and cyclic-AMP phosphodiesterase (Lynch and Cheung 1979). In 1977, Wang and Desai (1977) described the presence of a pro t e i n from bovine brain named modulator binding protein (MBP). This protein could antagonize the a c t i v a t i o n of phosphodiesterase by binding the calmodulin -2+ Ca complex. Thus MBP and phosphodiesterase compete for the calmodulin -2+ Ca complex. Larsen et a l (1978 b) found that MBP would also antagonize 2+ 2+ the a c t i v a t i o n of RBC membrane (Mg + Ca )-ATPase a c t i v i t y by p u r i f i e d RBC calmodulin, providing further evidence for the s i m i l a r i t y of these two - 14 -proteins. Though i t s r o l e i s not known, MBP could be another c e l l u l a r regulatory protein. -15-Aims of the Present Study Although C F . a f f e c t s a number of ti s s u e s , the search continues f o r a possible common denominator that w i l l explain the cause of the disease. Many of the problems associated with the disease state involve the viscous mucoid secretion of the exocrine glands (di Sant' Agnese and Talamo 1967). An increase i n the calcium content of many of the glycoprotein-rich secretions i n C F . patients has been reported (Johansen 1963; Potter et a l 1963; Gibson et a l 1971; Wotman et a l 1971). This suggests that a major defect i n C F . might be associated with a calcium-dependent process such as secretion or transport. 2+ 2+ Previous studies have revealed a s p e c i f i c decrease i n the (Mg + Ca )-ATPase a c t i v i t y of erythrocyte membrane preparations (Katz 1978 a). Further 2+ 2+ studies revealed a decrease i n (Mg + Ca )-ATPase a c t i v i t y i n crude membrane homogenates of cultured f i b r o b l a s t s (Katz 1978.»•'- from .CF. patients compared to age-matched controls. The objective of t h i s present study was to determine 2+ 2+ i f the (Mg + Ca )-ATPase a c t i v i t y noted i n the crude membrane homogenates of cultured f i b r o b l a s t s was indiginous to the plasma membrane and functioned, as i n the red blood c e l l , to maintain low i n t r a c e l l u l a r free c a l c i lum 2+ 2+ concentrations. This required the determination of the (Mg + Ca )-ATPase a c t i v i t y present i n p u r i f i e d plasma membrane preparations and an evaluation 2+ 2+ of whether the (Mg + Ca )-ATPase a c t i v i t y was alt e r e d i n samples derived from C F . patients. 2+ 2+ The (Mg + Ca )-ATPase i s generally accepted as the biochemical basis of the calcium transport system i n a number of tissues (Katz et a l 1975; Schatzmann 1973). As a more d i r e c t approach to the evaluation of the calcium pump a c t i v i t y i n C F . , t h i s study sought to e s t a b l i s h whether calcium -16-transport i n c e l l membranes derived from C F . patients was defective. 2+ Thus, i n t h i s present work, the Ca - transport a c t i v i t y of in s i d e -out v e s i c l e preparations derived from red c e l l s of C F . patients and age-matched controls was evaluated. Parameters of the calcium pump have been shown to be affected by 2+ calmodulin (Sarkadi et a l 1978; Larsen and Vincenzi 1979). This Ca binding p r o t e i n appears to play an important r o l e i n the regulation of the calcium pump. It' remained to be determined whether calmodulin function was a l t e r e d i n C F . ti s s u e . This p o s s i b i l i t y was therefore investigated i n t h i s study. 2+ 2+ F i n a l l y , to ascertain the generalized nature of the (Mg + Ca )-ATPase a c t i v i t y i n C F . , another c e l l type derived from C F . patients was i n -vestigated; lymphoblast plasma membrane preparations were obtained and 2+ 2+ the (Mg + Ca )-ATPase a c t i v i t y of C F . samples compared to control preparations evaluated. -17-Materials and Methods Establishment of f i b r o b l a s t cultures . The c e l l s t r a i n s used i n the experiments described were derived from skin biopsies of C F . ch i l d r e n and age-matched controls obtained from the f i b r o b l a s t bank of the Children's H o s p i t a l , Vancouver, through the co-operation of Dr. D.A. Applegarth and from Dr. M. Buchwald, Department of Medical Genetics, Hospital for Sick Children, Toronto. A l l the C F . patients had high chloride l e v e l s i n t h e i r sweat and varying degrees of pulmonary and pancreatic involvement. The cont r o l strains were derived from skin biopsies of ch i l d r e n with diseases unrelated to C F . In a l l , 4 C F . and 3 control f i b r o b l a s t c e l l cultures were established by con-ventional procedures (Krooth 1969). When confluent, the c e l l s were sub-cultured 1:4 using 0.25% t r y p s i n (Flow Laboratories)' i n Hank's Balanced 2 Salt s o l u t i o n and grown i n Falcon Flasks (75 cm , Corning Co.) for 10-14 days with feeding every three days. The Flasks were maintained at 37° i n an incubation chamber (Fisher Isotemp Incubator). C e l l s t r a i n s were frozen i n culture medium with 10% dimethyl sulfoxide (DMSO) and stored i n l i q u i d nitrogen. When needed, culture s t r a i n s were reconstituted with culture medium containing 20% (v/v) F e t a l Bovine Serum. The growth medium contained Dulbecco's Modified Eagle Medium (MEM, Grand Island B i o l o g i c a l Co.) with a lower than normal amount of sodium bicarbonate to prevent a l k a l i n i t y , with heat i n a c t i v a t e d 15% (v/v) F e t a l Bovine Serum (Flow Laboratories) and an a n t i b i o t i c - a n t i m y c o t i c mixture ( p e n i c i l l i n 10,000 units/ml, fungizone 25 mcg/ml and streptomycin 10,000 mcg/ml, Grand Island Bio-l o g i c a l Co.; 10 ml per l i t e r of preparation). A l l the f i b r o b l a s t s t r a i n s -18-were subcultured several times then transferred to 1 l i t e r r o l l e r b o t t l e s continually rotated (Wheaten Co. Rotator apparatus) and fed and maintained as previous. Growing i n r o l l e r b o t t l e s increased the t i t e r of c e l l s to the l e v e l required for preparation of plasma membrane-enriched f r a c t i o n s . The experiments described were performed on f i b r o b l a s t s of passages 7-and 8, Preparation of plasma-membrane-enriched f r a c t i o n s of human skin f i b r o b l a s t s Plasma-membrane-enriched f r a c t i o n s of f i b r o b l a s t s derived from C F . and control subjects were prepared by the method of Kartner et a l (1977). T y p i c a l l y , two r o l l e r b o t t l e s containing f i b r o b l a s t s , one C F . and one control s t r a i n , were i n i t i a l l y washed 6 times with 25 ml Phosphate Balanced Salt s o l u t i o n (0,15 M NaGl, 0.01 M"NaH^P04 and 11'mM KH 2P0 4, pH.7.4), The washed c e l l s were swollen by slow rotation.of b o t t l e s containing- " V 25 ml of 1 mM sodium bicarbonate s o l u t i o n (pH 7.4)v','for 1 minute. This step was repeated twice. The swollen c e l l s could then be removed e a s i l y from the b o t t l e surface and disrupted by manual shaking of the b o t t l e containing 25 ml of 1 mM sodium bicarbonate s o l u t i o n (pH 7.4). The d i s -rupted suspension was centrifuged i n 0.5 mM EDTA'at 27,000 xg for 20' minutes. The addition of EDTA was necessary to prevent aggregation and clumping of the various types of p a r t i c u l a t e material present. The p e l l e t was resuspended i n 10 ml of 10% '(w/v) sucrose and designated, the crude membrane preparation. A part of t h i s preparation was then layered on a discontinuous density gradient of 30%, 48% and 60% sucrose and centrifuged at 76,000 xg for 2 hours. M a t e r i a l banding at the 10/30 i n t e r f a c e and the crude membrane f r a c t i o n were washed free of sucrose i n 1 mM sodium bicarbonate (pH 7.4) by c e n t r i f u g a t i o n at 33,000 xg for 20 minutes. The -19-p e l l e t s were resuspended i n 1 mM sodium bicarbonate (pH 7.4) and assayed 2+ 2+ for 5'-nucleotidase, adenylate cyclase and (Mg + Ca )-ATPase a c t i v i t i e s . Preparation of red blood c e l l inside-out v e s i c l e s C F . blood samples were obtained from patients attending the Vancouver Health Center Cyst i c F i b r o s i s C l i n i c through the co-operation of Dr. A.G.F. Davidson, Dr. Lawrence Wong and s t a f f . Approximately'8 ml blood was drawn from each patient d i r e c t l y into heparin. Control samples were obtained from student volunteers at the same time and under exactly the same conditions. The age range of the C F . donors was 12-29 years with a mean age of 20 years and that of the control subjects was 22-34 years with a mean age of 27 years. The patients chosen were attending the C F . c l i n i c f o r routine follow-up. A l l had been i d e n t i f i e d as having C F . on the basis of elevated sweat chloride. The patients had varying degrees of r e s p i r a t o r y and digestive problems. Some of the patients were re c e i v i n g pancreatic enzymes and some were re c e i v i n g a n t i b i o t i c s . Preparation of red c e l l inside-out (10) v e s i c l e s was started within 2 hours of obtaining the blood samples. C F . and control red blood c e l l s were washed 3 times i n i s o t o n i c s a l i n e to remove the buffy coat and were lysed i n 35 volumes of i c e - c o l d 5 mM Na-phosphate buffer (pH 8.0). The lysed c e l l were cehtrifuged at 12,000 xg for 10 minutes i n a Beckman J2-21 centrifuge using a JA 20 rotor. The p e l l e t s were washed and centrifuged two more times i n the same buffer. The r e s u l t i n g white membrane p e l l e t s were suspended i n 35 volumes of i c e - c o l d 0.5 mM Na-phosphate buffer (pH 8.0) for 30 minutes and centrifugedaat 27,000 xg for 30 minutes. The membranes -20-were resuspended i n 17.5 volumes of 0.5 mM Na-phosphate buffer (pH 8.5) and stored overnight on i c e . The next morning, the suspensions were centrifuged at 27,000 xg f o r 30 minutes. The p e l l e t s were dispersed i n an equal .volume of the 0.5 mM Na-phosphate buffer (pH 8.0) and were passed through a 1 inch No. 27 hypodermic needle f i v e times. The membranes were then washed once i n 10 mM t r i s - g l y c y l g l y c i n e buffer (pH 7.1) containing OV025 mM MgCl2 and once i n 20 mM t r i s - g l y c y g l y c i n e buffer (pH 7,1) con-t a i n i n g 0.05 mM MgCl2 by c e n t r i f u g a t i o n at 31,000 xg f o r 35 minutes. The membrane-vesicle p e l l e t s were then stored i n 20 mM t r i s - g l y c y l g l y c i n e buffer (pH 7.1) at a p r o t e i n concentration of 4 to 5 mg/ml and used within s i x hours. Enzyme Assays 2+ 2+ A. Measurement of (Mg + Ca )-ATPase a c t i v i t y 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y was determined e s s e n t i a l l y as described by Katz and B l o s t e i n (1975). The reaction mixture contained 5 mM MgS04, 50 mM Tris-HCL (pH 7.4), 3 mM Y - 3 2 p (ATP) (20,000 cpm/sample) and 0.75 ml of membranes (0.5-0.8 mg/ml of the crude membrane preparation or 0.1-0.3 mg/ml of the preparation enriched i n plasma membranes) i n the presence or absence of calmodulin and/or the desired free calcium concentration. When calcium was present i n the incubation medium i t was added together with EGTA i n order to obtain the desired free calcium concentration. The free calcium concentration was then determined by the association 2+ constant for the i n t e r a c t i o n of EGTA with Ca at pH 7.4, using the equations of Katz et a l (1970) taking into consideration the ATP and 2+ Mg concentrations used. Ouabain, 0.1 mM, was added to the membrane preparations during pre-incubation to i n h i b i t Na +, K +-ATPase (ATP -21-phosphohydrolase EC 3.L6.3) a c t i v i t y . The assay at 37 was started by the addition of the membrane preparation and terminated a f t e r 20 minutes by the addition of 5% t r i c h l o r o a c e t i c a c i d (TCA) containing 5 mM ATP and 2 mM KR^PO^. A suspension of charcoal - TCA (1.5 g/10 ml; 0.45 ml/ sample-) was then added to each sample and the samples incubated at 0° for one hour with intermittent mixing and, following c e n t r i f u g a t i o n , an a l i q u o t of the clear supernatant counted f o r r a d i o a c t i v i t y . B. 5' - Nucleotidase assay The method of M i c h e l l and Hawthorne (1965) was followed and i n -volved the c o l o r i m e t r i c measurement of inorganic phosphate-produced by the hydrolysis of AMP. The membranes were incubated for 15 minutes at 37° i n a medium containing 100 mM KC1, 10 mM MgCl 2, 50 mM Tris-HCl buffer (pH 7.4), 5 MM AMP and 10 mM sodium potassium t a r t r a t e . The reaction was stopped with 1 ml of 25% '(w/v) TCA. An aliquot* of the supernatant was d i l u t e d to 2 ml and the color developed following the a d d i t i o n of a 1:1 mixture of 1% ammonium molybdate and 1% ascorbic a c i d and measured at 660 nm using a Beckman Model 25 spectrophotometer. C. Adenylate Cyclase Assay For the measurement of c y c l i c AMP accumulation, membrane preparations were incubated with 50 mM tris-HCI!(pH 7.4), 1 mM D i t h i o -t h r e i t o l , 1 mM EDTA, 5 mM theophylline, 10 mM KC1, 5 mM MgCl 2, 20 mM phosphoenolpyruvate and 0.35 mg/ml pyruvate kinase, 0.01 mM GTP, and 2.5 mM T r i s ATP i n the presence and absence of 10 mM NaF i n a f i n a l volume of 0.25 ml. Following a 10 minute pre-incubation period at 37°, the reaction was started by the addition of ATP and proceeded for 15 -2-2-minutes. The re a c t i o n was terminated by placing the samples i n a b o i l i n g water bath. The samples were then centrifuged at 3,000 xg and aliquots of the c l e a r supernatant assayed for the presence of c y c l i c AMP using the Gilman protein binding assay (Gilman 1970). D. Acetylcholinesterase (AChE) assay AChE-- a c t i v i t y was determined by a modification of the method of Ellman et a l (1961) as described by Steck and Kant (1974). In t h i s assay, 0.05 ml of the red c e l l v e s i c l e preparation was pre-incubated for 5 minutes with an equal volume of 20 mM t r i s - g l y c y l g l y c i n e ( p H 7.1), i n the presence or absence of 0.2% T r i t o n X-100 (v/v). H i s t i d i n e -imidazole buffer (pH 7.1; 9 mM) was added to produce a f i n a l volume of 3.0 ml.DTNB and a c e t y l t h i o c h o l i n e chloride 0.1 ml each were then added and the re a c t i o n at room temperature was followed using a Beckman model 25 recording spectrophotometer at a wavelength of 412 nm. E. Glyceraldehyde 3-phosphate Dehydrogenase (G3-PD) assay E s s e n t i a l l y , the assay (Cori et a l 1948) involved pre-incubation of 0.05 ml of the red c e l l v e s i c l e preparation with an equal volume of 20 mM t r i s - g l y c y l g l y c i n e buffer (pH. 7.4) for 5 minutes, i n the presence of or absence of 0.2% T r i t o n X-100 (v/v). Sodium pyrophosphate (30 mM; 0.3 ml) and cystine-IICl (4 mM; 1.75 ml) were then added followed by sodium arsenate (0.4 M; 0.09 ml) and 20 mM >^-NAD (20 mM; 0.16 ml). To s t a r t the reaction, 0.1 mM glyceraldehyde 3-Phosphate (0.03 ml). was added. •' The reaction at room temperature was then followed using a Beckman Model 25 recording spectrophotometer, at 340 nm. -23-Measuremetit of calcium transport a c t i v i t y Ca 2 +-uptake experiments were performed at 30°. The 10 v e s i c l e preparations (90 ug/ml f i n a l incubation volume) were pre-incubated for 30 minutes i n a medium containing 9 mM h i s t i d i n e - i m i d a z o l e buffer (pH 7.1), 0.6 mM t r i s - g l y c y l g l y c i n e (pH 7.1), 40 mM NaCl, 7.5 mM KC1, 3 mM MgCl 2 and CaCl2 (containing 5 x 10~* cpm/samples of ^ 5 c a C l 2 ) • The desired free calcium concentration was maintained by the addition of EGTA and the free calcium concentrations present determined by the equations of Katz et a l (1970) 2+ taking into consideration the Mg and ATP concentrations used. When calmodulin was added, i t was present i n a f i n a l concentration of 0.8 ug/ml. The reaction was started by the a d d i t i o n of 3 mM ATP. At s p e c i f i c time i n t e r v a l s aliquots of 100 u l were then taken and quenched i n 1.0 ml of cold 40 mM t r i s - g l y c y l g l y c i n e b u f f e r (pH 7.1) containing 0.1 mM MgCl 2. Ves i c l e s were trapped on a 0.45 um f i l t e r ( M i l l i p o r e Co.), the f i l t e r s washed once with the same buffer and counted for r a d i o a c t i v i t y i n Aquasol (New England Nuclear). Preparation of calmodulin Calmodulin was prepared from red c e l l s of outdated human blood e s s e n t i a l l y according to the method of Jung (1978) by hemolysis i n hypotonic medium and the i s o l a t i o n of the calmodulin protein by DEAE-Sephadex column chromatography. The active f r a c t i o n s were eluted by a buffered NaCl gradient s o l u t i o n and concentrated by a M i l l i p o r e separator technique. Desalting of the a c t i v e f r a c t i o n s was done on Sephadex G-15 and G-25 columns. A concentration curve of the a c t i v i t y of the calmodulin preparations was obtained by the assay of 2+ 2+ the stimulation of (Mg + Ca )-ATPase a c t i v i t y of an EDTA-washed membrane -24-preparation from control erythrocytes. The maximal ac t i v a t o r concentration (by volume) obtained by t h i s procedure was then used i n subsequent experi-ments. The calmodulin preparations were then stored at -80° and used within 3 weeks. Preparation of lymphoblast plasma membranes Preparation was done by Dr. Riordan's group, Research I n s t i t u t e , Sick Children's H o s p i t a l , Toronto. The preparation was stored at -80° and used within 3 weeks. Protein assay Protein concentrations were measured by the method of Lowry et a l (1951) using bovine serum albumin as a standard.. S t a t i s t i c s Student's " t " test f o r unpaired, common variance data was used as a measure of s i g n i f i c a n c e . Standard error of the mean (S.E.M.) was used as a measure of v a r i a t i o n . Materials A l l chemicals used were a n a l y t i c a l grade. ATP, DEAE-Sephadex, EGTA, \/ 3 2 EDTA and Trizma base were purchased from Sigma Chemical Company. • -P 45 (ATP) (10-20 Ci/mmole and C a C l 2 (lOCCi/mmole) were purchased from Amersham Company.Charcoal (Norit'A) was purchased from Fisher S c i e n t i f i c and Aquasol s c i n t i l l a t i o n f l u i d from New England Nuclear Company. -25-Results P a r t i a l p u r i f i c a t i o n of plasma membranes from cultured skin f i b r o b l a s t s Table 1 i l l u s t r a t e s the re s u l t s of assays done to determine the presence of 5'-nucleotidase and adenylate cyclase a c t i v i t y i n the crude plasma mem-brane f r a c t i o n and i n the "10/30" f r a c t i o n obtained following sucrose density gradient ce n t r i f u g a t i o n . When the crude plasma membrane f r a c t i o n , obtained by d i f f e r e n t i a l c e n t r i f u g a t i o n , was separated on a sucrose den-s i t y gradient there was a 5-fold increase i n the a c t i v i t y of 5'-nucleotidase and adenylate cyclase i n the f r a c t i o n present at the "10/30" i n t e r f a c e . These enzymes are known to be present i n the plasma membrane of f i b r o b l a s t s (Solymon and Trams 1972; Kartner et a l 1977). The enhancement i n a c t i v i t y of these enzymes i n the "10/30" f r a c t i o n therefore suggests that t h i s f r a c t i o n i s enriched i n plasma membranes. This f r a c t i o n of f i b r o b l a s t membranes was therefore u t i l i z e d as the plasma membrane-enriched preparation i n further experiments. 2+ 2+ P a r t i a l c h a r a c t e r i z a t i o n of the (Mg + Ca )-ATPase a c t i v i t y i n f i b r o b l a s t membrane preparations  A high degree of the ATP h y d r o l y t i c a c t i v i t y noted i n both the crude 2+ membrane and plasma membrane-enriched preparations of f i b r o b l a s t s was Ca 2+ 2+ dependent. Figure 2 i l l u s t r a t e s the Mg -dependency of the Ca stimulated component of the ATPase a c t i v i t y i n the crude membrane and plasma membrane 32 -enriched f r a c t i o n . The res u l t s are expressed as nmoles P i released per 2+ 2+ mg pr o t e i n per minute. (Mg + Ca )-ATPase a c t i v i t y r e f e r s to that a c t i v i t y present following subtraction of the a c t i v i t y measured i n the absence of 2+ added calcium and i n the presence of 0.1 mM EGTA. The Ca stimulated component of t h i s a c t i v i t y was markedly increased i n the presence of f r om 2+ 0.5-5.0 mM Mg with maximal stimulation i n both preparations obtained at 20.0 mM MgS04. -26-Table 1. 5'-Nucleotidase and adenylate cyclase a c t i v i t i e s of f i b r o b l a s t membrane preparations . Adenylate-Cyclase  Fraction 5'-Nucleotidase Basal NaF  Crude 5.11+1.20 3.35+0.75 6.75+1.20 10/30 26.36 + 2.80 14.00 + 2.50 76.00 + 5.20 Assays were performed as i n Methods. The a c t i v i t y of 5'-nucleotidase i s expressed as pinoles. P i released per mg protein per hour. Adenylate cyclase a c t i v i t y i s expressed as pmoles c y c l i c AMP per mg protein per minute. -27-Figure 2. Mg -dependency of the Ca -stimulated ATPase a c t i v i t y i n f i b r o b l a s t membrane preparations. 2+ ATPase a c t i v i t y i n the presence of 200 uM Ca free was determined as described i n Methods i n the absence 2+ (0.1 mM EGTA) and presence of varying Mg concentra-tions (0.5-50 mM) i n crude plasma membrane ( O O ) and plasma membrane-enriched preparations © ) of cultured f i b r o b l a s t s . The r e s u l t shown i s a t y p i c a l experiment of duplicate determinations. -28--29-2+ 2+ In order to further characterize the (Mg + Ca )-ATPase a c t i v i t y present i n the f i b r o b l a s t membrane preparations, the e f f e c t of calmodulin was investigated. This protein has previously been shown to s p e c i f i c a l l y 2+ 2+ stimulate red c e l l membrane (Mg + Ca )-ATPase a c t i v i t y (Bond and Clough 1973; Luthra et a l 1976 a&b). Figure 3 and 4 i l l u s t r a t e that calmodulin 2+ obtained from red c e l l hemolysates s i g n i f i c a n t l y stimulated the (Mg + 2+ Ca )-ATPase a c t i v i t y present i n these f r a c t i o n s . Two d i f f e r e n t calmodulin preparations were used i n t h i s study: One of the calmodulin preparations 2+ 2+ utili'zed produced a much higher stimulation of the (Mg + Ca )-ATPase a c t i v i t y i n the membrane fr a c t i o n s than the other; the extent of a c t i v a t i o n over that a c t i v i t y noted i n the absence of added calmodulin was 150% and 296% i n the crude and the plasma membrane-enriched f r a c t i o n s , respectively (Figure 4) as compared to 83% and 114% stimulation when the less active calmodulin preparation was used (Figure 3). The amount of calmodulin preparation (Figure 4) necessary to produce half-maximal a c t i v a t i o n was 0.63 ug i n the crude membrane and 1.52 ug i n the plasma membrane-enriched f r a c t i o n . 2+ 2+ Comparison of (Mg + Ca )-ATPase a c t i v i t y i n cultured f i b r o b l a s t s derived from C F . patients and controls  In a l l , 4 C F . and 3 control s t r a i n s were used i n t h i s study. 2+ 2+ Figure 5 i l l u s t r a t e s a t y p i c a l experiment of the measurement of (Mg + Ca )• ATPase a c t i v i t y i n crude membrane preparations of C F . and control f i b r o -2+ 2+ bl a s t s t r a i n s . (Mg + Ca )-ATPase a c t i v i t y was increased with increasing free calcium concentration i n both C F . and control s t r a i n s to a maximum 2+ 2+ 2+ at 200 uM Ca free. The (Mg + Ca )-ATPase a c t i v i t y noted i n the control -30-Figure 3. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane and i n p u r i f i e d plasma membrane preparations of cultured f i b r o -b l asts i n the presence and absence of added calmodulin. 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n the presence of 200 uM 2+ Ca free was determined as described i n Methods i n the absence and presence of varying calmodulin concentrations (3.25-9. 75 ug) i n the crude plasma membrane ( O O ) and plasma membrane-enriched preparations (@ ® ) of cultured f i b r o b l a s t s . The res u l t shown i s a t y p i c a l experiment of duplicate determinations. - 3 1 --32-Figure 4. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane and i n p u r i f i e d plasma membrane preparations of cultured f i b r o -b l a sts i n the presence and absence of added calmodulin. 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n the presence of 200 pM 2+ Ca free was determined as described i n Methods i n the absence and presence of varying calmodulin concentrations (0.85-^7-6.4 ug) i n the crude plasma membrane (O — - O ) and plasma membrane-enriched preparations (<@ @) of cultured f i b r o b l a s t s . The r e s u l t shown i s a t y p i c a l experiment of duplicate determinations. - 3 3 --34-Figure 5. (Mg + Ca )-ATPase a c t i v i t y i n crude membrane preparations of cultured f i b r o b l a s t s . 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y was measured as described i n Methods i n the absence (0.1 mM EGTA) and presence (50-600 2+ uM Ca free) of added calcium i n crude membrane prepara-tions derived from control ( O O ) a n d C F . (© @) f i b r o b l a s t s t r a i n s . The re s u l t s shown are a t y p i c a l experiment of duplicate determinations. -35-nmoles 3 2Pi released mg - 1 min--36-s t r a i n s was consistently higher than that noted i n the C F . s t r a i n s at a l l free calcium concentrations tested. Figure 6 i s a r e c i p r o c a l p l o t of t h i s 2+ 2+ data i n d i c a t i n g that the ^ Qa2+ a n d not the K^iss of the (Mg + Ca )-ATPase for calcium i s alte r e d i n the C F . s t r a i n s . 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y i n the plasma membrane-enriched prepara-tions was greatly increased over that observed i n the crude membrane preparations. Figure 7 i l l u s t r a t e s a t y p i c a l experiment of the determination 2+ 2+ of the (Mg + Ca )-ATPase a c t i v i t y i n plasma membrane-enriched preparations 2+ 2+ of both C F . and control s t r a i n s . Again, the (Mg + Ca )-ATPase a c t i v i t y of t h i s preparation increased with increasing free calcium concentration 2+ 2+ 2+ to a maximum at 200 uM Ca free.(Mg + Ca )-ATPase a c t i v i t y i n the control str a i n s was consistently higher than that i n the C F . s t r a i n s . Figure 8 i s a r e c i p r o c a l p l o t of t h i s data i n d i c a t i n g again, that i t i s the V^ a2+ and 2+ 2+ not the K^gg of the (Mg + Ca )-ATPase for calcium that i s altered i n the C F . s t r a i n s . Table 2 shows a comparison of the crude membrane and plasma membrane-enriched preparations used-in- t h i s study. P u r i f i c a t i o n of the crude membrane 2+ 2+ preparation increased the s p e c i f i c a c t i v i t y of the (Mg + Ca )-ATPase approximately 4-5 f o l d . This i s an 80-100 f o l d increase over the a c t i v i t y noted previously i n f i b r o b l a s t membrane homogenate preparations (Katz 1978 b; not shown). The v^ a2+ was s i g n i f i c a n t l y decreased i n both the crude membrane (p<0.01) and the plasma membrane-enriched preparations (p^ 0.05) obtained from C F . f i b r o b l a s t s t r a i n s compared to preparations obtained from c o n t r o l s t r a i n s . The K^gg for calcium was increased - 3 7 -Figure 6. K,. and V r 2+ D f the (Mg^+ Ca^)-ATPase a c t i v i t y of crude membrane preparations of cultured f i b r o b l a s t s . 2+ Reciprocal plots of a t y p i c a l determination of (Mg + 2+ Ca )-ATPase a c t i v i t y of crude membrane preparations derived from control ( O — - O ) and C F . (® «®) f i b r o b l a s t s t r a i n s . The K^gg i s expressed i n pM and V^ &2+ i n nmoles mg-"*" min-"^". -38--39-Figure 7. (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of cultured f i b r o b l a s t s . 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y was measured as described i n Methods i n the absence (0.1 mM EGTA) and presence 2+ (50-600 uM Ca free) of added calcium i n p u r i f i e d plasma membrane preparations derived from control ( O - O ) and C F . («@ @) f i b r o b l a s t s t r a i n s . The r e s u l t s shown are a t y p i c a l experiment of-duplicate determinations. - 4 1 -Figure 8. K d l s g and ^Qg2+ of the (Mg + Ca )-ATPase a c t i v i t y of p u r i f i e d plasma membrane preparations of cultured f i b r o b l a s t s . 2 + Reciprocal p l o t s of a t y p i c a l determination of (Mg + 2 + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations derived from control (O O ) and C F . (<g) @) f i b r o b l a s t s t r a i n s . The K d^ s g i s expressed i n uM and the V F 2 + in~ nmoles mg-"'" min \ - 4 2 --43-Table 2. K,. and V p 2+ of the (Mg + Ca )-ATPase of f i b r o b l a s t Q l S S L<cL membrane preparations. K d i s s XpM) V c &2+ (NMOLES MG 1 MIN -1) CRUDE PLASMA MEMBRANES: CONTROL STRAINS C F . STRAINS 39.1 + 5.3 41.5 + 2.2 48.9 + 27.8 + 3.8 2.1 a PURIFIED PLASMA MEMBRANES: CONTROL STRAINS C F . STRAINS 113.0 + 6.1 103.4 + 6.6 207.0 + 102.7 + 39.4 10. 8 b (Mg 2 ++ Ca 2 +)-ATPase a c t i v i t y of f i b r o b l a s t s membrane preparations was determined as described i n Methods i n the presence of varying Calcium' concentrations (50-600 uM Ca2+) f r e e ) . Reciprocal p l o t s of duplicates of i n d i v i d u a l experiments were then drawn and the i n t e r -cepts determined by regression analysis. The r e s u l t s are the mean + S.E.M. of 3 control s t r a i n s and 4 C F . s t r a i n s . a s t a t i s t i c a l l y s i g n i f i c a n t compared to the controls (P < 0.01) b s t a t i s t i c a l l y s i g n i f i c a n t compared to the controls (P< 0.05) approximately 2 1/2 f o l d i n the plasma membrane-enriched f r a c t i o n compared to the crude membrane f r a c t i o n . No difference was observed, though, i n the K^iss f ° r calcium i n the C F . strains compared to the control s t r a i n s . Studies on inside-out v e s i c l e preparations derived from red c e l l s of controls and C F . patients  In t h i s present work, subsequent studies were directed at determining 2+ 2+ whether the a l t e r a t i o n i n (Mg + Ca )-ATPase a c t i v i t y observed i n C F . samples could s i g n i f i c a n t l y a f f e c t calcium transport i n C F . c e l l s . To accomplish t h i s , red c e l l v e s i c l e preparations obtained from C F . patients and controls were used. The r e l a t i v e proportion of inside-out, right-side-out and unsealed membranes i n the red c e l l v e s i c l e preparations was estimated by determining the a c t i v i t y of the enzymes located on the outer surface (AChE) and the inner surface (G3-PD) of the red c e l l membrane. The r a t i o of the enzyme a c t i v i t y measured i n the absence and i n the presence of 0.1% T r i t o n X-100 was used as a measure of the percentage a c c e s s i b i l i t y of the marker enzymes .{Cohen and Solomari 1976). Only 20-25% of the t o t a l AChE was accessible whilst 55-60% of the t o t a l G3-PD was accessible. The remaining a c t i v i t y , 15-25% unaccounted f o r , could be due to the presence of membrane frag -ments. Thus, the preparations were found to consistently contain 55-60% 10, v e s i c l e s . 10 v e s i c l e s were not separated from right-side-out v e s i c l e s and broken membranes due to the s i g n i f i c a n t loss i n Ca -uptake a c t i v i t y produced by the separation procedures a v a i l a b l e . - 4 5 -2+ Comparison of Ca -uptake a c t i v i t y i n 10 v e s i c l e preparation obtained from C F . patients and controls  2+ Figure 9 compares the Ca -uptake a c t i v i t y of f i v e d i f f e r e n t c o n t r o l and s i x d i f f e r e n t C F . 10 v e s i c l e preparations, each obtained from a 2+ d i f f e r e n t subject. The r e s u l t s are expressed as Ca -uptake i n nmoles per mg protein following subtraction of that a c t i v i t y noted i n the absence of ATP. It can be observed that calcium accumulation was time-dependent and varied l i t t l e from preparation to preparation. The 10 red c e l l v e s i c l e s prepared from control blood samples i n t h i s present study were found to 2+ . have a s i m i l a r Ca -transport a c t i v i t y to those prepared by other workers (Sarkadi et a l 1978; Larsen and Vincenzi 1979). I t can be seen that the 2+ amount of Ca -uptake observed i n the c o n t r o l preparations was considerably higher than that observed i n the C F . preparations at a l l time points studied. Experiments were then c a r r i e d out to determine whether t h i s difference was apparent at the i n i t i a l times of incubation. Figure 10 2+ i l l u s t r a t e s the Ca -uptake a c t i v i t y of both C F . and control preparations at sample times between 10 seconds and 10 minutes. In both C F . and control 2+ preparations, there was a l i n e a r r e l a t i o n s h i p between the Ca -uptake 2+ a c t i v i t y and the time of incubation. Ca -uptake a c t i v i t y was found to be considerably reduced i n the C F . preparations at the e a r l i e s t time of assay and at a l l subsequent assay times. 2+ Ca -uptake a c t i v i t y i n 10 v e s i c l e preparations derived from C F . and control red c e l l s was then determined at various free calcium concentrations 2+ 2+ (10-150 uM Ca f r e e ) . Figure 11a i l l u s t r a t e s that Ca -uptake a c t i v i t y 2+ increased with increasing free Ca concentration to a maximum at 100 uM -46-Figure 9. Time-Course of Ca -Uptake A c t i v i t y i n 10 v e s i c l e preparations from Control and C F . samples. 2+ Ca -uptake was determined as described i n Methods 2+ i n the presence of 150 pM Ca free i n control (O -O) and C F . preparations (® ® ) . Results represent the mean + S.E.M. of f i v e control and s i x C F . preparations each from a d i f f e r e n t donor. -47-ujajojd Bui/«»|ouiu :»])04dn ++ 03 - 4 8 -Figure 10. I n i t i a l rates of Ca -uptake i n 10 v e s i c l e s from Control and C F . samples. 2+ Ca -uptake was determined as described i n Methods i n the 2+ presence of 150 pM Ca free i n con t r o l ( O O ) and C F . preparations (@ ® ) . Results represent the mean + S.E.M. of f i v e control and si x C F . preparations each from a d i f f e r e n t donor. - 4 9 -C 'tt) Time (min) -50-Figure 11. Ca -uptake a c t i v i t y i n the presence and absence of added calmodulin i n 10 v e s i c l e preparations from C F . patients and controls. 2+ a) Ca -uptake was measured as described i n Methods 2+ i n the presence of various free Ca concentrations (10-150 uM) i n f i v e control ( O O ) and s i x C F . preparations ( © @) each from a d i f f e r e n t donor and i n the presence of added calmodulin (0.8 pg/ml f i n a l incubation volume) i n these control (A A) and C F . (^ A ) preparations. b) ' Reciprocal plots of data shown i n Figure 11a. -52-2+ 2+ Ca free in both C F . and control preparation; Ca -uptake activity was consistently reduced in the CF. preparations at a l l free calcium concen-trations studied. Calmodulin has been shown to be present i n a number of c e l l types and to regulate the activity of the calcium pump system in red c e l l preparations (Luthra et al a&b; Larsen and Vincenzi 1979), Its role or possible alteration in disease states i s not yet known. Figure 11a illustrates that when calmo-dulin from red c e l l hemolysates was added to the incubation medium i t 2+ stimulated Ca -uptake activity in both C F . and control preparations to approximately the same extent (35-39%). Calmodulin, though, did not 2+ restore the Ca -uptake activity of the CF. samples to levels observed in the control preparations. 2+ Figure l i b and Table 3 il l u s t r a t e the K d^ g g for Ca and the V^a2+ 2+ of the Ca -transport system in the 10 vesicle preparations of the control and CF. preparations in the presence and absence of added calmodulin. 2+ It can be noted that there was l i t t l e alteration in the K^gg for Ca in the CF. samples compared to the controls. Calmodulin had l i t t l e effect 2+ on the K^gg for Ca in either preparation. It can v_>- also be noted that the V^a2+ was significantly decreased in CF. samples (p ^  0.001) compared to the controls. Calmodulin significantly increased the V^&2+ in both groups (p<0.01 in the CF. group and p ^ 0.001 in the control group) but did not restore the V C a2+ of the CF. preparations to control values. 2+ 2+ (Mg + Ca )-ATPase activity in purified plasma membrane preparations of human cultured lymphoblasts  Lymphoblasts obtained from CF. patients have been reported to be -53-Table 3. K c j i s s and V C a2+-uptake system i n control and C F . preparations of 10 v e s i c l e s . Control (5) a CF. (6 ) a - calmodulin + calmodulin - calmodulin + calmodulin K d i s s ^ ) V C a 2 + (nmoles/mg) 22.17 + 1.54t 24.30 + 1.83 20.75 + 1.54 18.56 + 2.19 61.23 + 1.63 85.24 + 1.82c 28.47 + 1.48 c 38.50 + 2.25°' d Ca"'-uptake was assayed as described i n Methods. When calmodulin was added i t was present i n a concentration of 0.8 pg/ml f i n a l incubation volume. a Number of preparations i n each group, each from a d i f f e r e n t subject, b Mean + S.E.M. i n each case. c S i g n i f i c a n t (P K 0.001) when compared to control i n the absence of added calmodulin. d S i g n i f i c a n t (P< 0.01) when compared to C F . i n the absence of added calmodulin. -54-abnormal with respect to the a c t i v i t y of oC.- L - fucosidase (Maler and Riordan 1979). Other membrane-bound enzyme a c t i v i t i e s may also be al t e r e d 2+ 2+ in t h i s c e l l - t y p e . I t was therefore investigated whether (Mg + Ca )-ATPase a c t i v i t y was present i n lymphoblast membrane preparations and i f t h i s a c t i v i t y 2+ 2+ was al t e r e d i n C F . samples. Table 4 i s a comparison of the (Mg + Ca )-ATPase a c t i v i t y of p u r i f i e d plasma membrane preparations of cultured lympho-2+ 2+ bla s t s derived from C F . patients and controls. (Mg + Ca )-ATPase a c t i v i t y observed i n the lymphoblast membrane preparations was much lower than that noted i n the p u r i f i e d plasma membrane preparations of cultured f i b r o b l a s t s (see Table 2). No s i g n i f i c a n t difference was detected i n the 2+ 2+ (Mg + Ca )-ATPase a c t i v i t y present i n the C F . plasma membrane samples compared to that a c t i v i t y present i n the controls. -55-Table 4. (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of human cultured lymphoblasts. nmoles P-; released mg min' CONTROL PREPARATIONS PM 23 36.0 PM 17 33.7 PM 19 38.4 + S.E.M. 36.0 + 1.3 Cystic F i b r o s i s PREPARATIONS PM 22 46.0 PM 18 39.8 PM 16 30.5 + S.E.M. 38.8 + 4.5 a P u r i f i e d plasma membranes from cultured lymphoblasts were prepared by Tom Maler and Dr. J. Riordan, Hospital for Sick Children, Toronto. These preparations were frozen and thawed twice p r i o r to use and i n -cubated at 37° for 30 minutes i n the presence of 5 mM MgCl2,50 mM Tris - H C l , pH 7.4, 3 mM Y = 3 2 P(ATP) and 0.10 mM free Ca . The reaction was terminated with 5% t r i c h l o r o a c e t i c acid as described previously. The re s u l t s shown are duplicates following the subtraction of the ATPase 2+ a c t i v i t y measured i n the absence of added free Ca a not s t a t i s t i c a l l y s i g n i f i c a n t when compared to the controls. -56-Discussion 2+ 2+ In the present study, the presence of a (Mg + Ca )-ATPase a c t i v i t y i n p u r i f i e d plasma membrane preparations of cultured f i b r o b l a s t s was demon-2+ 2+ strated. The s i g n i f i c a n t decrease i n (Mg + Ca )=ATPase a c t i v i t y observed i n plasma membrane preparations of f i b r o b l a s t s and red c e l l s (Katz 1978 a) 2 + derived from C F . st r a i n s could be related to the altered Ca -uptake a c t i v i t y observed i n 10 v e s i c l e preparations from C F . red blood c e l l s i n thi s present study. The preparation of plasma membrane-enriched fractions from cultured f i b r o b l a s t s involved mechanical disruption of the harvested c e l l s under hypotonic conditions. This has advantages over many of the e x i s t i n g methods (Warren et a l 1966; Brunette and T i l l 1971),in that i t avoids the use of proteases for harvesting of c e l l s and the use of denaturing and c r o s s l i n k i n g agents, l i k e heavy metals or aldehydes, which influence c e l l function. It appears evident from these studies that the plasma membrane-enriched 2+ 2+ preparations of cultured f i b r o b l a s t s contain a high degree of (Mg + Ca )-ATPase a c t i v i t y . P u r i f i c a t i o n of the membrane preparations by sucrose 2+ 2+ density gradient c e n t r i f u g a t i o n revealed a 4-5 f o l d increase i n (Mg + Ca )-ATPase a c t i v i t y which i s 80-100 f o l d higher than that previously observed i n homogenates (Katz 1978 b). Also a 4-5 f o l d increase i n 5'-nucleotidase and adenylate cyclase a c t i v i t i e s was observed i n the "10/30" f r a c t i o n . The fact that these two enzymes, believed to be indiginous to the plasma 2+ 2+ membrane, "co-purify" with t h i s f r a c t i o n indicates that the (Mg + Ca )-ATPase a c t i v i t y i s indiginous to the plasma membrane and possibly functions to regulate the i n t r a c e l l u l a r free calcium concentration, s i m i l a r to i t s 2+ 2+ r o l e i n the red blood c e l l . The (Mg + Ca )-ATPase of f i b r o b l a s t plasma membranes was found to share many of the same c h a r a c t e r i s t i c s as the 2+ 2+ (Mg + Ca )-ATPase from other tissues (Vincenzi and Hinds 1976). This 2+ includes a c t i v a t i o n by micromolar concentrations of calcium and a Mg -dependency. This source of the enzyme i s also stimulated by calmodulin prepared from red c e l l hemolysates. Recently, E l d i k and Watterson (1979) p u r i f i e d and characterized calmodulin from transformed chicken embryo f i b r o b l a s t s . Their preparation was i n d i s t i n g u i s h a b l e from calmodulin prepared from chicken gizzard and brain and bovine brain i n terms of p h y s i c a l , chemical and f u n c t i o n a l properties. 2+ It was observed that whilst the degree of stimulation of the (Mg + 2+ Ca )-ATPase a c t i v i t y by calmodulin increased markedly i n p u r i f i e d plasma membranes compared to crude preparations, the apparent K^iss of calmodulin for stimulation.'increased. S i m i l a r l y , i t was observed that although the 2+ 2+ VQa2+ of the (Mg + Ca )-ATPase increased s i g n i f i c a n t l y as the f i b r o b l a s t plasma membranes were p u r i f i e d , the calcium a f f i n i t y of the enzyme system present i n t h i s preparation was lower. This implies that there i s a higher 2+ 2+ a f f i n i t y - calmodulin-sensitive (Mg + Ca )-ATPase.system i n .another f i b r o -b l a s t organelle. Moore and Paston (1977) have shown that microsomal prep-arations from f i b r o b l a s t s a c t i v e l y accumulate calcium. They also observed that i n growing cultures the s p e c i f i c a c t i v i t y of the microsomal calcium transport system increased up to 8 f o l d as a function of c e l l density. It thus appears that at l e a s t two energy-dependent calcium transport systems may e x i s t i n cultured f i b r o b l a s t s ; one associated with the microsomal f r a c t i o n and the other associated with the plasma membrane. The former -58-may be involved i n the modulation of c e l l u l a r movement and p r o l i f e r a t i o n (Moore and Paston 1977) at the l e v e l of the cytoplasm and the l a t t e r i n the control of the i n t r a c e l l u l a r free calcium concentration (Kartner et a l 1977). 2+ 2+ It was demonstrated that the (Mg + Ca )-ATPase a c t i v i t y present i n plasma membrane-enriched preparations of f i b r o b l a s t s was s i g n i f i c a n t l y re-duced i n C F . s t r a i n s . K i n e t i c analysis indicated that the K^gg for calcium was s i m i l a r i n the C F . and c o n t r o l s t r a i n s but that there was a s i g n i f i c a n t 2+ 2+ decrease i n the maximal a c t i v a t i o n of the (Mg + Ca )-ATPase i n the C F . f i b r o b l a s t s t r a i n s . Fibroblasts represent a c e l l - t y p e that many studies have indicated might be abnormal i n C.F.; abnormally high rates of glycosaminoglycan synthesis and release (Matalon and Dorfman 1968) have been observed i n C F . f i b r o b l a s t s as well as an increased occurrence of cytoplasmic metachromasia with t o l u i d i n e blue (Danes and Beam 1969) and a decreased incorporation of thymidine into DNA (Barranco et a l 19 76). There are few reports i n the l i t e r a t u r e regarding p o s s i b l e a l t e r a t i o n s i n ion-transport i n C F . f i b r o b l a s t s t r a i n s ; Fletcher and L i n (1972) i n a study of two C F . f i b r o b l a s t s t r a i n s found no a l t e r a t i o n i n Na +, K +-ATPase a c t i v i t y and Q u i s s e l and P i t o t (1974) observed no di f f e r e n c e i n 3 H -ouabain binding i n C F . f i b r o b l a s t s . F e i g a l and Shapiro (1979a) have recently reported an enlarged i n t r a -2+ c e l l u l a r Ca pool i n skin f i b r o b l a s t s from subjects with C F . and obligate 2+ heterozygotes and indicated that the a l t e r e d Ca pool represented a change 2+ inomitochondfia'l Ca content. These workers (Feigal and Shapiro 1979b) 2+ have also shown that mitochondria from C F . c e l l s accumulate more Ca than those from c o n t r o l c e l l s . These studies are i n agreement with our findings since a l e s s e f f e c t i v e plasma membrane calcium pump would lead to the accumulation of i n t e r n a l c e l l calcium and suggests that the i n t r a c e l l u l a r pool of calcium that i s enhanced exists i n the mitochondria. 2+ Ca -transport studies were done i n 10 v e s i c l e preparations from red blood c e l l s because i n such an inverted system the conditions at the ATP, 2+ 2+ Mg and Ca -binding s i t e s could be easily, c o n t r o l l e d . Although the 10 v e s i c l e preparations were not p u r i f i e d by elimination of right-side-out v e s i c l e s and broken fragments they were found to y i e l d extremely reproducible r e s u l t s . The s p e c i f i c a c t i v i t y of these preparations was also found to be equal to or higher than that of s i m i l a r preparations used by other workers (Larsen and Vincenzi 1979; Sarkadi et a l 1978) when the proportion of 10 v e s i c l e present to t o t a l p r o t e i n i s taken into consideration. 2+ This study indicated that Ca -uptake a c t i v i t y i n ;IQ v e s i c l e prep-arations obtained from C F . patients was s i g n i f i c a n t l y reduced when compared to controls; t h i s reduction i s observed at a l l time-points and at a l l 2+ free calcium concentrations tested. That the Ca -uptake a c t i v i t y i n the C F . preparations i s reduced at the i n i t i a l times studied indicates the p h y s i o l o g i c a l relevance of t h i s f i n d i n g . The fact that t h i s difference i n 2+ Ca -uptake a c t i v i t y i s noted at r e l a t i v e l y low free calcium concentrations i s also s i g n i f i c a n t . Calmodulin prepared from red c e l l hemolysates of normal volunteers was 2+ found to stimulate Ca -transport i n both control and C F . preparations; the degree of calmodulin stimulation appeared to be s i m i l a r i n both groups. -60-The low degree of calmodulin stimulation observed compared to the that found by other workers i n similar;preparations could e i t h e r be due to the presence of indiginous i n h i b i t o r s i n the calmodulin preparation or to the p o s s i b i l i t y that some calmodulin was associated with the v e s i c l e s and could not be removed during the preparation. I t i s important to note, though, that addition of calmodulin (0.8 ug/ml f i n a l incubation volume) did not 2+ return the Ca -uptake a c t i v i t y i n the C F . preparations to those l e v e l s present i n the c o n t r o l preparations. This suggests that regulation of the 2+ Ca -transport activity by,calmodulin i n C F . red cells i s unaffected by the disease state. The p o s s i b i l i t y e x i s t s though, that e i t h e r C F . calmodulin i s a l t e r e d i n some way compared to calmodulin from control preparations or that red c e l l membranes from C F . patients respond d i f f e r e n t l y to C F . calmodulin. Studies to date i n d i c a t e that no s i g n i f i c a n t differences e x i s t i n plasma membrane structure, with respect to t o t a l protein, carbohydrate and l i p i d content, i n C F . red c e l l s (McEvoy et a l 1974), f i b r o b l a s t s (Fletcher and L i n 1973) or lymphoblasts (Maler and Riordan 1978). A 2+ 2+ s i g n i f i c a n t decrease i n the V C a2+ of the (Mg + Ca )-ATPase a c t i v i t y of 2+ f i b r o b l a s t membrane preparations and Ca -uptake a c t i v i t y of 10 v e s i c l e preparations derived from C F . patients without an a l t e r a t i o n i n the K d i s s ^ o r c a l c i u m m a y therefore be due to an e f f e c t on the active s i t e of the enzyme. There may be l e s s enzyme per mg protein, a defect i n the mechanisms that regulate calcium transport or the enzyme may be a l t e r e d at a s i t e other than the c a t a l y t i c s i t e (an a l l o s t e r i c s i t e ) which ultimately influences the c a t a l y t i c a c t i v i t y of the enzyme. - 6 1 -2+ 2+ A decreased (Mg + Ca )-ATPase a c t i v i t y i n C F . c e l l s may represent a basic a l t e r a t i o n i n the a b i l i t y of these c e l l s to transport calcium. This i s indicated by the experiments conducted using the 10 v e s i c l e preparations, and may be a generalized phenomenon i n C F . No a l t e r a t i o n 2+ 2+ in (Mg + Ca )-ATPase a c t i v i t y was observed, though, i n lymphoblast membrane preparations derived from C F . patients. Lymphoblasts are trans-2+ formed c e l l s and th i s c h a r a c t e r i s t i c of low Ca -transport a c t i v i t y may be l o s t during transformation. It i s noted that i n non-diseased samples, differences e x i s t i n Na , K -ATPase a c t i v i t y between transformed and non-transformed cultured f i b r o b l a s t s (Kimelberg and Mayhew 1976). In these studies no attempt was made to corr e l a t e the r e s u l t s obtained with the seve r i t y of the disease state due to the r e l a t i v e l y small sample size of each group studied. Mangos (1978) using i s o l a t e d rat parotid acinar c e l l s observed that C F . s a l i v a produced a s i g n i f i c a n t increase i n i n t r a c e l l u l a r calcium i n comparison with c e l l s incubated i n s a l i v a from control subjects. I t i s possible that the alte r e d calcium pump a c t i v i t y noted i n C F . ce l l - t y p e s may be a response to a c i r c u l a t i n g factor which acts as a modulator of calcium pump a c t i v i t y . An a l t e r a t i o n i n calcium pump a c t i v i t y i n C F . may have a number of possible implications that may explain some of the 2+ manifestations of the disease; Ca i s involved i n many secretory processes inc l u d i n g macromolecular secretion (Douglas 1968). An a l t e r a t i o n i n calcium pump a c t i v i t y would thus a f f e c t the secretion of glycoproteins. A number of workers have also shown that a l t e r e d e x t r a c e l l u l a r calcium l e v e l s w i l l a f f e c t glycoprotein f l u i d i t y ( v i s c o s i t y ) and thus mucus clearance (Bettelheim 1971; Boat et a l 1974; Forstner and Forstner 1976), an important and ce n t r a l problem i n C F . (Boat et a l 1974). Calcium l e v e l a l t e r a t i o n s may also + + 2 + greatly a f f e c t Na reabsorption and Na -Ca exchange systems (Russell and Blaustein 1974). 2+ Studies to determine the mechanisms underlying the a l t e r e d Ca pump a c t i v i t y i n C F . are presently underway. - 6 3 -BIBLIOGRAPHY Andersen, D.H.: C y s t i c f i b r o s i s of pancreas and i t s r e l a t i o n to c e l i a c disease: c l i n i c a l and pathological study Am. J. Dis. C h i l d . 56: 344-399, 1968. Balfe, J.W., Cole, C. and Welt, L.G.: Red-cell transport defect i n patients with c y s t i c f i b r o s i s and i n t h e i r parents. Science 162: 689-690, 1968. Barranco, S.C, Bolton, W.E. , Haenelt, B.R. and A b e l l , C.W. : Differences i n the incorporation of thymidine into DNA of normal and c y s t i c f i b r o s i s f i b r o b l a s t s i n v i t r o . J. C e l l . P h y s i o l . 88: 33-42, 1976. Bettlehim, F.A.: On the aggregation of calcium p r e c i p i t a b l e glycoprotein from human submaxillary s a l i v a . Biochim. Biophys. Acta. 236: 702-705, 1971. B e r a t i s , N.G., Conover, J.H. , Conod, E.J., Bonforte, R.J. and Hirschorn, K. : Studies on c i l i a r y dyskinesia factor i n c y s t i c fibrosis.1111. Skin f i b r o b l a s t s and cultured amniotic f l u i d c e l l s . Pediat. Res. 7_: 958-964, 1973. Boat, T.F., Wiesmann, U.N., P a l l a v i c i n i , J . C : P u r i f i c a t i o n and properties of the calcium-precipitable protein i n submaxillary s a l i v a of normal and c y s t i c f i b r o s i s subjects. Pediat. Res. _8: 531-539, 1974. Bogart, B.L., Conod, E.J. and Conover, J.H.: The b i o l o g i c a c t i v i t y of c y s t i c f i b r o s i s serum I. The e f f e c t of c y s t i c f i b r o s i s sera and calcium ionophore A23187 on rabbit tracheal explants. Pediat. Res. 11: 131-134, 1977. Bond, G.H. and Clough, D.L.: A soluble protein activator of (Mg +CCa )-dependent ATPase i n human red c e l l membranes. Biochim. Biophys. Acta. 323: 592-599, 1973. Bowman, B.H., Barnett, D.R., Matalon, R.: C y s t i c f i b r o s i s : F r a c t i o n a t i o n of f i b r o b l a s t media demonstrating c i l i a r y i n h i b i t i o n . Proc. Nat'l. Aca. S c i . _70: 548-551, 1973. - 6 4 -Bowman, B.H., Lankford, B.J., F u l l e r , G.M.: Cystic f i b r o s i s : The c i l i a r y i n h i b i t o r i s a small polypeptide associated with immuno-globin G. Biochem. Biophys. Res. Commun. 6_4: 1310-1315, 1975. Bowman, B.H., Lockhart, L.H. and McCombs, M.L.: Oyster c i l i a r y i n -h i b i t i o n by c y s t i c f i b r o s i s f actor. Science 164: 325-326, 1969. Bowman, B.H. , McCombs, N.L. and Lockhart, L.H.: Cystic f i b r o s i s : Characterization of the i n h i b i t o r to c i l i a r y a c tion i n oyster g i l l s . Science 167: 871-873, 1970. Brostrom, CO., Huang, Y., Breckenridge, B.M. and Wolff, D.J.: I d e n t i f i c a t i o n of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase. Proc. Nat. Aca. S c i . 72: 64-68, 1975. Brunette, D.M. and T i l l , J.E.: A rapid method for the i s o l a t i o n of L - c e l l surface membranes using an aqueous two-phase polymer system. J. Membrane B i o l . _5: 215-224, 1971. Chernick, W.S. and Barbero, G.J.: Studies on human tracheobronchial and submaxillary secretions i n normal and pathophysiological conditions. Ann. New York Acad. Sc. 106: 698-708, 1963. Cohen, CM. and Solomon, A.K. : Ca binding to the human red c e l l membrane: Characterization of membrane preparations and binding s i t e s . J. Membrane B i o l . _29: 345-372, 1976. Cole, CH. and Dirks, J.H.: Changes i n erythrocyte membrane ATPase In patients with c y s t i c f i b r o s i s of the pancreas. Pediat. Res. 6_: 6.16-621, 1972. Conover, J.H. , B e r a t i s , N.C, Conod, E.J., Ainbender, E. , and Hirschhorn K.: Studies on c i l i a r y dyskinesia f a c t o r i n c y s t i c f i b r o s i s . II Short term leukocyte cultures and long term lymphoid l i n e s . Pediat. Res. li 224-228, 1973. Conover, J.H., Conod, E.J. and Hirschhorn, K.: Studies on c i l i a r y dyskinesia factor i n c y s t i c f i b r o s i s . I V . I t s possible i d e n t i f i c a t i o n as anaphylatoxin (C3a)-Ig G. Complex. L i f e S c i . 14: 253-266, 1974. -65-Cori, G.T., Stein, M.W. and C o r i , C.F.: C r y s t a l l i n e D-glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle. J . B i o l . Chem. 173: 605-618, 1948. Danes, B.S. and Beam, A.G.: Cystic f i b r o s i s of the pancreas: a study i n c e l l culture. J. Exp. Med. 129: 775-793, 1969. Danks, D.M. , A l l a n , J. and Anderson, CM.: A genetic study of f i b r o -c y s t i c disease of the pancreas. Ann. hum. Genet. 2J3: 323-56, 1965. di Sant' Agnese, P.A., Darling, R.C, Perera, G.A. and Shea, E. : Abnormal e l e c t r o l y t e composition of sweat i n c y s t i c f i b r o s i s of pancreas: c l i n i c a l s i g n i f i c a n c e and r e l a t i o n s h i p to disease. P e d i a t r i c s 12: 549-563, 1953. di Sant' Agnese, P.A. and Davis, P.B.: Research i n c y s t i c f i b r o s i s . N. Engl. J. Med. 295: 481-485, 534-541, 597-602, 1976. di Sant' Agnese, P.A. , Talamo, R.C: Pathogenesis and physiopathology of c y s t i c f i b r o s i s of the pancreas: f i b r o c y s t i c disease of the pancreas (mucoviscidosis). N. Engl. J. Med. 277: 1287-1294, 1344-1352, 1399-1408, 1967. di Sant' Agnese, P.A. and Vidaurreta, A.M.: Cystic f i b r o s i s of pancreas. J.A.M.A. 172: 2065-2072, 1960. Douglas, W.W.: Stimulus-secretion coupling: the concept and clues from chromaffin and other c e l l s . B r i t J . Pharmacol. 34: 451-474, 1968. Dunham, E.T. and Glynn, I.M.: Adenosinetriphosphatase a c t i v i t y and the acti v e movement of a l k a l i metal ions. J . Physiol (London) 156: 274-293, 1961. Dunn, M.J.: Red blood c e l l calcium and magnesium: E f f e c t s on sodium and potassium transport and c e l l u l a r morphology. Biochim. Biophys. Acta. 352: 97-116, 1974. - 6 6 -Duffy, M.J., Cohn, E.V. and Schwarz V.: Ca -uptake and binding by i s o l a t e d erythrocyte membranes from c y s t i c f i b r o s i s and control subjects. C l i n . Chim. Acta. 50: 97-101, 1974. E l d i k , L.J.V. and Watterson, M.D.: Characterization of a calcium-modulated protein from transformed chicken f i b r o b l a s t s . J . B i o l . Chem. 254: 10250-10255, 1979. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M.: Anew and rapid c o l o r i m e t r i c determination of ac e t y l c h o l i n e s t r a s e a c t i v i t y . Biochem., Pharmacol. 7_: 88-95, 1961. Fanconi, G., Uehlinger, E., and Knauer, C : Das coeliokiesyndrom b e i angeborener zy s t i s c h e r pankreasfibromatose und bronchiektasien. Wien. med. Wchnschr. 86: 753-756, 1936. 2+ 2+ Farrance, M.L. and Vincenzi, F.F.: Enhancement of (Ca + Mg )-ATPase a c t i v i t y of human erythrocyte membranes by hemolysis i n isosmotic imidazole buffer. I. General properties of v a r i o u s l y prepared membranes and the mechanism of the isomotic imidazole e f f e c t . Biochim. Biophys. Acta. 471: 49-58, 1977a. 2+ 2+ Farrance, M.L. and Vincenzi, F.F.: Enhancement of (Ca + Mg )-ATPase a c t i v i t y of human erythrocyte membranes by hemolysis i n isosmotic imidazole buffer. II Dependence on calcium and a cytoplasmic a c t i v a t o r . Biochim. Biophys. Acta. 471: 59-66, 1977b. Feig, S.A., Segel, G.B., Kern, K.A., Osher, A.B. and Schwartz, R.H.: Erythrocyte transport function i n c y s t i c f i b r o s i s . Pediat. Res. 8: 594-597, 1974. F e i g a l , R.J. and Shapiro, B.L.: Altered i n t r a c e l l u l a r calcium i n f i b r o -b l a s t s from patients with c y s t i c f i b r o s i s and heterozygotes. Pediat. Res. 13:" 764-768, 1979a. F e i g a l , R.J. and Shapiro, B.L.: Mitochondrial calcium uptake and oxygen consumption i n c y s t i c f i b r o s i s . Nature 278: 276-277, 1979b. F e r r e i r a , H.G. and Lew, V.L.: Ca transport and Ca pump revers a l i n human red blood c e l l s . J . Physiol (London) 252: 86-87, 1975. - 6 7 -F e r r e i r a , H.G. and Lew, V.L.: Passive Ca transport and cytoplasmic Ca b u f f e r i n g i n i n t a c t red c e l l s . In: Membrane Transport i n  Red C e l l s , J.C. E l l o r y and V.L. Lew (eds) pp 5 3 - 9 1 , Academic Press, London, 1977. Fletcher, D.S. and L i n , T.Y.: Incorporation of 1-Leucin and d-Glucosamine into skin f i b r o b l a s t s derived from c y s t i c f i b r o s i s and normal i n -di v i d u a l s . C l i n . Chim. Acta. 44 : 5 - 19 , 1973 . Forstner, J.F. and Forstner, G.G.: E f f e c t s of calcium on i n t e s t i n a l mucin: implications f o r c y s t i c f i b r o s i s . Pediat. Res. 10 : 6 0 9 - 6 1 3 , 1976. Gardos, G.: The function of calcium i n the potassium permeability of human red c e l l s . Biochim. Biophys. Acta. J30_: 653 - 654 , 1958. Garnett, H.M. and Kemp, R.B. (Ca +MMg )- A c t i v i a t e d ATPase i n the plasma membrane of mouse l i v e r c e l l s . Biochim. Biophys. Acta. 382: 5 2 6 - 5 3 3 , 1975. Garrahan^ P.J. and Rega, A.F.: A c t i v a t i o n of p a r t i a l reactions of the Ca -ATPase from human red c e l l s by Mg 2 + and ATP. Biochim. Biophys. Acta. 5 13 : 5 9 - 6 5 , 1978. Gibson, L.E., Matthews, W.J.Jr., Minihan, P.T.: Relating mucus calcium, and sweat i n a new concept of c y s t i c f i b r o s i s . P e d i a t r i c s 4 8 : 6 95 - 710 , 1971 . Gilman, A.G.: A protein binding assay f o r adenosine 3 ' : 5 ' - c y c l i c mono-phosphate. Proc. N a t l . Acad. S c i . U.S.A. 6 7 : 305 -312 , 1970. Gopinath, R.M. and Vincenzi, F.F.: Phosphodiesterase p r o t e i n a c t i v a t o r mimics red blood c e l l cytoplasmic a c t i v a t o r of (Ca2++ Mg 2 +)-ATPase Biochem. Biophys. Res. Commun. J7_: 1 203 -1209 , 1977. Gugler, E. , P a l l a v i c i n i , J . C , Swerdlow, H. , d i Sant' Agnese, P.A.: Role of calcium i n submaxillary s a l i v a of patients with c y s t i c f i b r o s i s . J . Pediat. 71 : 5 85 - 588 , 1967. -68-Harrison, D.G. and Long, C.: The calcium content of human erythrocytes. J . Physiol (London) 199: 367-381, 1968. Horton, CR. , Cole, W.Q. , and Bader, H. : Depressed (Ca )-transport ATPase in cystic fibrosis erythrocytes. Biochem. Biophys. Res. Commun. 40: 505-509, 1970. Huxley, H.E.: Muscular contraction and c e l l motility. Nature 243: 445-449, 1973. Janis, R.A., Crankshaw, D.J. and Daniel, E.E. : Control of intracellular Ca2+ activity in rat myometrium. Amer. J. Physiol. 232: C50-C58, 1977. Janis, R.A. and Daniel E.E. In: The Biochemistry of smooth muscle, N.L. Stephens ed., University Park press, Baltimore, M.D. pp 653-671, 1977. Jarrett, H.W. and Penniston, J.T.: Partial purification of the (Ca 2 ++ Mg2+)-ATPase activator from human erythrocytes: Its slmilarrty. to- the a c t i -vator of 3':5'-cyclic mucleotide phosphodiesterase. BiochemT Biophys. Res. Commun. 77: 1210-1216, 1977. Jarrett, H.W. and Penniston, J.T.: Purification of the Ca 2 +-sEImulated ATPase activator from human erythrocytes. Its membership in the class of Ca 2 +-binding modulator proteins. J. Biol. Chem. 253: 4676-4682, 1978. Johansen, P.G.: Physieochemical investigations on two unusual s i a l i c acid-rich mucoids from intestinal tract of patients with fibrocystic disease of pancreas. Biochem. J. 87: 63-70, 1963. Jung, N.S.G.T.: Purification and characterization of the cytoplasmic (Ca 2 ++ Mg2+)-ATPase activator protein of human red blood cells. Master's thesis, University of Washington, Seattle, Washington, 1978. Kakiuchi, S., Yamazaki, R. and Teshima, Y.: Regulation of brain phosphodie-sterase activity: C a + + plus Mg"*-*"-dependent phosphodiesterase and i t s activating factor from rat brain. Adv. Cyclic. Nucleotide Res. 455-477, 1972. -69-Kartner, N., Alon, N., S w i f t , M., Buchwald, M. and Riordan, J.R. : I s o l a t i o n of plasma membranes from human s k i n f i b r o b l a s t s J . Membrane B i o l . 36: 191-211, 1977. Ka t z , A.M., Repke, 0.1., Upshaw, J.E. and P o l a s c i k , M.A.: C h a r a c t e r i z a t i o n of dog c a r d i a c microsomes. Use of zonal cenferifugation to f r a c t i o n a t e fragmented sarcoplasmic r e t i c u l u m , (Na + + K + ) - a c t i v a t e d ATPase and mito-c h o n d r i a l fragments. Biochim. Biophys. Acta. 205: 473-490, 1970. Katz , A.M., Tada, M. and K i r c h b e r g e r , M.A.: C o n t r o l of calcium t r a n s p o r t i n the myocardium by the c y c l i c AMP-protein kinase system. Advan. C y c l i c N u c l . Res. _5: 453-472, 1975. Katz , B. and M i l e d i , R.: The e f f e c t of calcium on a c e t y l c h o l i n e r e l e a s e from motor nerve t e r m i n a l s . Proc. Roy. Soc. Lond. B. 161: 496-503, 1965. K a t z , S.: Calcium and sodium t r a n s p o r t processes i n p a t i e n t s w i t h c y s t i c f i b r o s i s . I . A s p e c i f i c decrease i n Mg 2 +-dependent, Ca 2 +-adenosine triphosphatase, a c t i v i t y i n e r y t h r o c y t e membranes from c y s t i c f i b r o s i s p a t i e n t s . P e d i a t . Res. 12: 1033-1038, 1978a. K a t z , S.: Calcium and^odium t r a n s p o r ^ p r o c e s s e s i n p a t i e n t s w i t h c y s t i c f i b r o s i s . 2. Mg -dependent, Ca -ATPase a c t i v i t y in-" f i b r o b l a s t membrane preparations from c y s t i c f i b r o s i s p a t i e n t s and c o n t r o l s . Res. Commun. Chem. Pathol., and Pharmacol. 19: 491-503, 1978b. K a t z , S. and B l o s t e i n , R.: Ca - s t i m u l a t e d membrane phosphorylation and ATPase a c t i v i t y of the human er y t h r o c y t e . Biochim. Biophys. Acta. 389: 314-324, 1975. Kimelberg, H.K. and Mayhew, E.: C e l l growth and ou a b a i n - s e n s i t i v e °"Rb' uptake and (Na ++ K +)-ATPase a c t i v i t y i n 3T3 and SV 40 transformed 3T3 f i b r o b l a s t s . Biochim. Biophys. Acta. 455: 865-875, 1976. Knauf, P.A., P r o v e r b i o , F., and Hoffman, J.F.: E l e c t r o p h o r e t i c separation of d i f f e r e n t phosphoproteins a s s o c i a t e d w i t h Ca-ATPase and Na, K-ATPase i n human red c e l l ghosts. J . Gen. P h y s i o l . 63_: 324-336, 1974. -70-Kopito, L. and Shwachman, H.: Spectroscopic analysis of tissues from patients with cystic fibrosis and controls. Nature (London) 202: 501-502, 1964. Krooth, R.S.: Genetics of cultured somatic cel l s . Medical cli n i c s of North America 53: 795-811, 1969. La Celle, P.L., Kirkpatrick, F.H., Udkow, M.P and Arkin, B.: Membrane fragmentation and ea2+-membrane interaction: Potential mechanisms of shape change in the senescent red c e l l . In: Red Cell Shape. W. Bessis, R.I. Weed, and R.F. Le Blond (eds) pp 789-798, 1973. Lamb, J.L. and Lindsay, R.: Effect of Na, metabolic inhibitors and ATP on Ca movements in L cel l s . J. Physiol (London) 218: 691-708, 1971. Lapey, A and Gardner, J.D.: Abnormal erythrocyte sodium transport in cystic fibrosis of the pancreas. Pediat. Res. 5_: 446-451, 1971. 2+ Larsen, F.L., Hinds, T.R. and Vincenzi, F.F.: On the red blood c e l l Ca pump: An estimate of stoichiometry. J. Membrane. Biol. 4J.: 361-376, 1978a. Larsen, F.L., Raess, B.U., Hinds, T.R. and Vincenzi, F ' F A L Modulator binding protein antagonizes activation of (Ca + Mg )-ATPase and C a 2 + transport of red blood c e l l membranes. J. Supramolec. Struct. 9_: 269-274, 1978b. Larsen, F.L. and Vincenzi, F.F.: Calcium transport across the plasma membrane: Stimulation by calmodulin. Science 204: 306-309, 1979. Lee, K.S. and Shin, B.C.: Studies on the active transport of calcium in human red c e l l s . J. Gen. Physiol. 54: 713-729, 1969. Leslie, S.W. and Borowtiz, J.L.: Evidence for a plasma membrane calcium pump in bovine adrenal medulla but not adrenal cortex. Biochim. Biophys. Acta. 39_4: 227-238, 1975. 2+ + Lew, Y.L.: Effect of ouabain on the Ca -dependent increase in K permeability in depleted guinea pig red c e l l s . Biochim. Biophys. Acta. 249: 236-239, 1971. -71-Lindsay, R. : P r o p e r t i e s sof the calcium-activ.'ated adenosine triphospha-tase from L - c e l l membranes. Quartely J . Exp. P h y s i o l . 61: 95-104, 1976. Lobeck, C.C. i n : The Metabolic B a s i s of I n h e r i t e d Disease. J.B. Stanbury, J.B. Wyngaarden and D.S. Fre d e r i c k s o n (eds). McGraw-Hill Co., New York, pp 1605, 1972. Long, C. and Mouat, B.: The b i n d i n g of calcium ions by er y t h r o c y t e s and ghos"t>'ce±l membranes. Biochem. J . 123:.. 829-836, 19 71 ; Lowry, O.H., Rosebrough, N.J., F a r r , A..L. and R a n d a l l , R.J.: P r o t e i n measurement w i t h the f o l i n phenol reagent. J . B i o l . Chem. 193: 265-275, 1951. L u t h r a , M.G. , Au, K.S. and Hannahan^D. J . £ + P u r i f i c a t i o n of a c t i v a t o r of human ery t h r o c y t e membrane (Ca + Hg )-ATPase. Biochem. Biophys. Res. Commun. _77: 678-687, 1977. Lu t h r a , M.G., Hildenbrandt, G.R. and Hannahan, D.J.: Studies on an a c t i v a t o r of the (Ca2++ Mg 2 +)-ATPase of human er y t h r o c y t e membranes. Biochem. Biophys. Acta. 419: 164-179, 1976a. Luthra, M.G., Hildenbrandt, G.R., Kim, O.H. and Hannahan D.J.: Observations on the (Ca 2 ++ Mg 2 +)-ATPase a c t i v a t o r found i n v a r i o u s mamalian e r y t h r o c y t e s . Biochem. Biophys. Acta. 419: 180-196, 1976b. 2+ 2+ Lynch, T.J. and Cheung, W.Y. : Human erythrocyte i(;Ca +Mg )-ATPase: Mechanism of s t i m u l a t i o n by C a ? + Arch. Biochem. Biophys. 194: 165-170, 1979. Maler, T. and Riordan, J.R.: I s o l a t i o n and c h a r a c t e r i z a t i o n of plasma membranes of c u l t u r e d lymphoblasts from p a t i e n t s w i t h c y s t i c f i b r o s i s . Can. Fed. B i o l . Soc. 21: 117, 1978. Maler, T. and Riordan, J.R.: C u l t u r e d lymphoblasts from p a t i e n t s w i t h c y s t i c f i b r o s i s c o n t a i n reduced l e v e l s of o^-L-fucosidase. T h i r d Canadian C y s t i c F i b r o s i s P r o f e s s i o n a l Conference H a r r i s o n , B.C. 1979. Mangos, J.A.: Microperfusion study of the sweat gland abnormality in cystic fibrosis. Tex. Rep. Biol. Med. 31: 651-663, 1973. Mangos, J.A.: Cystic Fibrosis in: Physiology of Membrane Disorders. T.E. Adreoli, J.F. Hoffman, D.D. Fanestil (eds) Plenum Medical, New York, N.Y. pp 941-953, 1978. Matalon, R. and Dorfman A.: Acid mucopolysaccharides in cultured fibroblasts of cystic fibrosis of the pancreas. Biochem. Biophys. Res. Commun. 33_: 954-958, 1968. McEvoy, F.A. , Davies, R.J. , Goodchild, M.C. and Anderson, CM: Erythrocyte membrane properties in cystic fibrosis. Clin. Chem. Acta. 54: 195-204, 1974. Michell, R. and Hawthorne, J.N.: The site of phosphoinositide synthesis in rat l i v e r . Biochem. Biophys. Res. Commun. 21: 333-338, 1965. Moore, L., Fitzpatrick, D.F., Chen, T.S. and Landon, E.J.: Calcium pump activity of the renal plasma membrane and renal microsomes. Biochem. Biophys. Acta. 345: 405-418, 1974. Moore, L. and Paston, I.: Energy-dependent calcium uptake activity in cultured mouse fibroblast microsomes. Regulation of the uptake system by c e l l density. J. Biol. Chem. 252: 6304-6309, 1977. Munger, B.L., Brusilow, S.W., Cooke, R.E.: Electron microscopic study of eccrine sweat glands in patients with cystic fibrosis of pan-crease. J. Pediat. 59: 497-511, 1961. Nagy, E.C, Khan, S. and Sturgess, J.M.: Serum factor in cystic fibrosis: Correlation with c l i n i c a l parameters. Pediat. Res. _13: 729-732, 1979. Olson, E.J. and Cazort,. R.J.: Active calcium and strontium transport in human erythrocyte ghosts. J. Gen. Physiol. _53: 311-322, 1969. Palek, J., Stewart, G. and Lionetti, F.J.: The dependence of shape of human erythrocyte ghosts on calcium, magnesium and adenosine triphosphate. Blood 44: 583-597, 1974. -73-Porzig, H.: ATP-independent calcium net movements in human red c e l l ghosts. J. Membrane Biol. _8: 237-258, 1972. Potter, J.L., Matthews, L.W., Lemm, J. and Spector, S.: Human pulmonary secretions in health and disease. Ann. New York,Acad. Sc. 106: 692-697, 1963. Quissel, D.O. and Pitot, H.C.: Number of ouabain-binding sites in fibro-blasts from normal subjects and patients with cystic fibrosis. Nature 247: 115-116, 1974. Quist, E.E. and Roufogalis, B.D.: Determination of the stoichiometry of the calcium pump in human erythrocytes using lanthanum as a selective inhibitor. FEBS Lett. _50_: 135-139, 1975. Rega, A.F. and Garrahan, P.J. : Calcium ion-dependent phosphorylation of human erythrocyte membranes. J. Membrane Biol. _2_2: 313-327, 1975. Rega, A.F. and Garrahan, P.J.: Calcium ion-dependent dephosphorylation of the Ca2+-ATPase of human red-cells by ADP. Biochem. Biophys. Acta. 507: 182-184, 1978. Richards, D.E. , Rega, A.F. and Garrahan, P.J.: Two classes of site for ATP in the Ca2+-ATPase from human red c e l l membranes. Biochem. Biophys. Acta. 511: 194-201, 1978. Robinson, J.D.: (Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation. Arch. Biochem. Biophys. 176: 366-374, 1976. Romero, P.J. and Whittman.: The control of internal calcium of membrane permeability to sodium and potassium. J. Physiol. (London), 214: 481-507, 1971. Rubin, R.P. In: Calcium and the Secretory Process. Plenum Press, New York, 1974. Russell, J.M. and Blaustein, M.P.: Calcium efflux from barnacle muscle fibres. Dependence on external cations. J. Gen. Physiol. 63: 144-167, 1974. -74-Sarkadi, B. , Szasz, I., Gerloczy, A. and Gardos, G.: Transport parameters and stoichiometry of active calcium ion extrusion in intact human red c e l l s . Biochem. Biophys. Acta. 464: 93-107, 1977. Scharff, 0.: The influence of calcium ions on the preparation of the (Ca 2 ++ Mg2+) -activated membrane ATPase in human red ce l l s . Scand. J. Clin. Lab. Invest. 30_: 313-320, 1972. 2~H 21 21 Scharff, 0.: Ca activation of membrane-bound (Ca + Mg )-dependent ATPase from human erythrocytes prepared in the presence or absence of C a 2 + Biochem. Biophys. aActa. 443: 206-218, 1976. 2+ Scharff, 0. and Foder, B.: Low Ca concentrations controlling two kinetic states of Ca2+-ATPase from human erythrocytes. Biochem. Biophys. Acta. 483: 416-424, 1977. Schatzmann, H.J.: ATP-dependent Ca -extrusion from human red cell s . Experientia, 22: 364-365, 1966. Schatzmann, H.J.: Dependence on calcium concentration and stoichiometry of the Ca pump in human red c e l l . J. Physiol (London) 235: 551-569 1973. 2+ 2+ Schatzmann, H.J.: Role of magnesium in the (Ca + Mg )-stimulated membrane ATPase of human red blood cel l s . J. Membrane Biol. 35: 149-158, 1977. Schatzmann, H.J. and Burgin, H.: Calcium in human red blood cel l s . Ann. New York Acad. Sci. 307: 125-146, 1978. 2+ 2+ Schatzmann, H.J. and Rossi, G.L.: (Ca + Mg )-activated membrane ATPases in human red cells and their possible relation to cation transport. Biochem. Biophys. Acta. 241: 379-392, 1971. Schatzmann, H.J. and Vincenzi, F.F.: Calcium movements across the membrane of human red ce l l s . J. Physiol. (London) 201: 369-395, 1969. Scholey, J., Applegarth, D.A., Davidson, A.G.F. and Wong, L.T.K.: Detection of cystic fibrosis protein by electrofocusing. Pediat. Res. 12: 800, 1978. -75-Schultz, I . J . : Micropuncture studies of the sweat formation i n c y s t i c f i b r o s i s patients. J . C l i n . Invest. _48: 1470-1477, 1969. Shwachman, H. and Antonowicz, I.: Sweat test i n c y s t i c f i b r o s i s Ann. New York Acad. Sc. 93_: 600-620, 1962. Solymon, A., Trams, E.G.: Enzyme markers i n c h a r a c t e r i z a t i o n of i s o l a t e d plasma membranes. Enzyme 13_: 329-372, 1972. Spock, A., Heick, H.M.C., Cress, H. and Logan, W.S.: Abnormal serum factor i n patients with c y s t i c f i b r o s i s of the pancreas. Pediat. Res. 1: 173-177, 1967. St. Louis, P.J. and Sulakhe, P.V.: Adenosine triphosphate-dependent calcium binding and accumulation by guinea p i g cardiac sarcolemma. Can. J . Biochem. 54: 946-956, 1976. Steck, T.L. and Kant, J.A.: Preparation of impermeable inside-out and rig h t - s i d e - o u t v e s i c l e s from erythrocyte membranes,In: Methods - i n Enzymology Vol. XXXI, Part A., S. F l e i s c h e r and L. Packer, (eds)., Academic Press, New York, N.Y. pp 172-180, 1974. Szasz, I., Sarkadi, B., Schubert, A., Gardos, G.: E f f e c t s of lanthanum on calcium-dependent phenomena i n human red c e l l s . Biochem. Biophys. Acta. 512: 331-340, 1978. T u l l y , G.W., Nevin, G.B., Young, I.R. and Nevin, N.C.: Detection of c y s t i c f i b r o s i s p r o t e i n by i s o e l e c t r i c focusing of serum. Pediat. Res. 13: 1078, 1979. Vincenzi, F.F.: A calcium ;pump i n red c e l l membranes.In: C e l l u l a r mechanisms for calcium t r a n s f e r and homeostasis. G. Nichols and R.H. Wasserman (eds). Academic Press, New York pp 135-147, 1971. Vincenzi, F.F.: Calcium transport and c y s t i c f i b r o s i s . Third Canadian Cysti c F i b r o s i s P r o f e s s i o n a l Conference, Harrison, B.C. 1979. Vincenzi, F.F. and Hinds, T.R.: Plasma membrane calcium transport and membrane-bound enzymes Iri: The enzymes of b i o l o g i c a l membranes, Vol. 3, A. Martonosi (eds), New York, pp 261-281, 1976. - 7 6 -Wang, J.H. and Desai, R.: Modulator binding protein. Bovine brain protein e x h i b i t i n g the Ca 2 +-dependent ass o c i a t i o n with the protein modulator of c y c l i c nucleotide phosphodiesterase. J. B i o l . Chem. 2 5 2 : 4 1 7 5 - 4 1 8 4 , 1 9 7 7 . Warren, L., Glock, M.C., Nass, M.K.: Membranes of animal c e l l s . I Methods of i s o l a t i o n , of the surface membrane. J . C e l l . P h y s i o l . £ 8 : 2 6 9 - 2 8 7 , 1 9 6 6 . Weed, R.I., La C e l l e , P.L., M e r r i l l , E.W., Craib, G., Gregory, A.; Kareh, F. and Pickens, E.: Metabolic dependence of red c e l l deformability. J . C l i n Invest. 4J5: 7 9 5 - 8 0 9 , 1 9 6 9 . Wiesmann, U.N., Boat, T.F., d i Sant' Agnese, P.A.: Flow rates and e l e c t r o -l y t e s i n minor s a l i v a r y - g l a n d - s a l i v a i n normal subjects and patients with c y s t i c f i b r o s i s . Lancet 2: 5 1 0 - 5 1 2 , 1 9 7 2 . Wilson, G.B., Monsher, M.T. and Fudenberg, H.H.: Studies on c y s t i c f i b r o s i s using i s o e l e c t r i c focusing I I I . C o r r e l a t i o n between c y s t i c f i b r o s i s p r o t e i n and c i l i a r y dyskinesia a c t i v i t y i n serum shown by a modified rabbit tracheal bioassay. Pediat. Res. 1 1 : 1 4 3 - 1 4 6 , 1 9 7 7 . Wins, P. and Schof f e n i e l s , E.: Studies on r e d - c e l l ghost ATPase systems: properties of a (Mg 2 ++ Ca 2 +)-dependent ATPase Biochem. Biophys. Acta. 1 2 0 : 3 4 1 - 3 5 0 , 1 9 6 6 . Wolf, H.H., Diechross, G. and Lichtner, R.: P u r i f i c a t i o n and properties of h i g h - a f f i n i t y Ca 2 +-ATPase of human erythrocyte membranes. Acta. B i o l . Med. Germ. 3 6 : 8 4 7 - 8 5 8 , 1 9 7 7 . Wotman S., Mandel, I.D., and'Mercadante J . : P a r o t i d and submaxillary calcium i n human c y s t i c f i b r o s i s . Arch. Oral B i o l . J-6_: 6 6 3 - 6 6 5 , 1 9 7 1 . 

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