BIOCHIMIE, I979, 61, 615-624.

Exchange ot" calcium between muscle Ca2+-binding proteins. Hans J. MOESCHLER t, Dean A. MALENCIK, Sitivad PO,CINNVONG, Olusola ALABA +, Glenn L. KERRICK ° and Edmond H. FISCHER .

From the Depariment o[ Biochemistry, University of Washington, Seattle, Washington 98195 USA. ° From the Department o[ PhyMoIogy, University o[ Washington, Seattle, Washington 98195 USA. + From the National Institutes o[ Health, Bethesda, Maryland 2001~ USA. From the D e p a r t m e n t o[ Biochemistry, University o[ Geneva, Switzerland.

Rdsum~. Le reticulum s a r c o p l a s m i q u e a 6t6 isol6 d e s m u s c l e s squeletfiques de l a p i n et de rousseffe du Pacifique (Pacific doqfish, S q u a l u s a c a n thias) et ses propri6t6s de p o m p a q e du C a 2~ ont 6t6 6tudi6es & diff6rentes t e m p 6 r a t u r e s . Les RS des deux e s p ~ c e s ont d e s vitesses de p o m p a q e du C a 2+ c o m p a r a b l e s d o n s les conditions de t e m p 6 r a t u r e b a s a l e de l ' o r g a n i s m e (5°C et 37°C, respectivement). Lorsque les v6sicules purifi6es ont 6t6 d 6 b a r r a s s 6 e s d e s p a r t i c u l e s de q l y c o q ~ n e c o n t a m i n a n t e s , elles n e pr6sentent plus q u ' u n tr~s foible p o u r c e n t a g e de phosphor y l a s e , p h o s p h o r y l a s e k i n a s e et p h o s p h a t a s e . Les flux d e C a ~÷ p e n d a n t les cycles de cont r a c t i o n - r e l a c h e m e n t m u s c u l a i r e ont 6t6 6tudi&s d o n s un s y s t ~ m e reconstitu6 e n m e s u r a n t l'inhibition de la vitesse de p o m p a q e de C a 2+ p a r le reticulum s a r c o p l a s m i q u e d ' u n e part, et de l'activit6 stimul6e p a r le C a 2÷ de la p h o s p h o r y l a s e kinase, d ' a u t r e part, a p r ~ s addition de troponine, d e troponine C, de p a r v a l b u m i n e et de p h o s p h o r y l a s e k i n a s e d6calcifi6es ; le s t a n d a r d interne est r E G T A . Les c a p a c i t 6 s de liaison du C a 2+ d e s d i v e r s e s prot6ines ont 6t6 d6termin6es s 6 p a r ~ m e n t p a r des e x p 6 r i e n c e s de filtration sur qel. Les donn6es o b t e n u e s sont en a c c o r d a v e c les v a l e u r s publi6es p o u r la troponine, la troponine C et la p a r v a l b u m i n e ; p o u r la phosphor,/This work was supported by grants from the National Institutes of Health, United States Public Health Service (AM 07902), and National Science Foundation (BMS 7516260), and the Muscular Dystrophy Association, Inc. Supported in part by a fellowship to H.J.M. [rom the Schweizrischer Nationalfonds zur Foerderung wissenscha[tlicher Forschung. 0 To whom reprint requests should be addressed.

l a s e kinase, u n e v a l e u r de n = 8 a 6t6 trouv6e quel q u e soft l'~tat d e p h o s p h o r y l a t i o n de l'enz y m e . Ces d e u x t y p e s d ' e x p 6 r i e n c e s ont montr6 q u e les affinit~s a p p a r e n t e s p o u r le C a 2÷ d6croissent d o n s l'ordre s u i v a n t : p h o s p h o r y l o s e k i n a s e _~ p a r v a l b u m i n e > EGTA > trop o n i n e ~ troponin C, s a n s q u ' o n o b s e r v e de diff6rence entre les formes p h o s p h o r y l 6 e et non p h o s p h o r y l 6 e de la p h o s p h o r y l a s e kinase. Les c o n s t a n t e s d'6quilibre a p p a r e n t e s de ces c o m p l e x e s a v e c le C a 2÷, p a r r a p p o r t & I'EGTA, sont e n a c c o r d a v e c les v a l e u r s publi6es & partit d ' 6 t u d e s c l a s s i q u e s ~ l'~quilibre. Les prot6ines isol~es de m u s c l e s de l a p i n ou d e roussette donnent d e s r6sultats s e m b l a b l e s . Dons ce s y s t ~ m e reconstitu6, on n ' o b s e r v e a u c u n indice d'interaction prot6ine-prot6ine modifiant les flux calciques, s a u f d o n s le c a s particulier de la p h o s p h o r y l a s e k i n a s e : l'inhibition de l a k i n a s e p a r la p a r v a l b u m i n e est 16q~rement plus m a r qu6e a p r ~ s p h o s p h o r y l a t i o n de r e n z y m e .

Summary. S a r c o p l a s m i c reticulum isolated from s k e l e t a l m u s c l e of the r a b b i t a n d the Pacific doqfish (Squalus a c a n t h i a s ) w a s c h a r a c t e r i z e d in t e r m s of its Ca2÷-uptake at different t e m p e r a t u r e s . SR

Abbreviations used : SR, sarcoplasmie reticulum ; TN, troponin ; TN-C, Ca~+binding subunit of troponin ; EDTA, ethylene diamine-N,N'-tetraacetate ; EGTA, ethylene glycol bis [aminoethyl ether]-N,N'-tetraacetate.

616

H. J. M o e s c h l e r

from both species s h o w e d c o m p a r a b l e C a 2+u p t a k e rates at their b a s a l b o d y t e m p e r a t u r e s (5°C a n d 37°C, respectively). O n l y v e r y low p e r c e n t a g e s of p h o s p h o r y l a s e , p h o s p h o r y l a s e k i n a s e a n d p h o s p h a t a s e w e r e found to be associated with the purified vesicles, once these were freed of c o n t a m i n a t i n g g l y c o g e n particles. Ca2~-fluxes during contraction/relaxationcycles in m u s c l e s were studied in a reconstituted system b y m e a s u r i n g the inhibition of the rate of Ca2+-uptake b y the s a r c o p l a s m i c reticulure a n d of the Ca2*-dependent activity of phosp h o r y l a s e kinase, respectively, b y metal-free troponin, troponin-C, p a r v a l b u m i n a n d phosphor y l a s e k i n a s e ; EGTA w a s u s e d a s a n internal s t a n d a r d . Ca2*-binding capacities of the v a r i o u s proteins w e r e determined s e p a r a t e l y b y gel filtration experiments. Data o b t a i n e d a g r e e d with published v a l u e s for troponin, troponin-C a n d p a r v a i b u m i n ; for p h o s p h o r y l a s e kinase, a

Introduction. The linkage between glycogen metabolism and the contractile process in muscle is provided by the absolute Ca2+-requirement of phosphorylase kinase, one of the enzymes involved in the regulation of glycogenolysis (for revie~v, see Fischer et al., 19'71 ; Fischer et al., 1976; Cohen, 1974). Release of Ca 2+ from the sarcoplasmic reticulum (SR) upon membrane depolarization causes contraction and, simultaneously, triggers glycogen breakdown needed to provide for the energy necessary to maintain contraction (for review, see Ebashi et al., 1968 ; MacLennan et al., 1975 ; Fischer et al., 1976). Although the concerted regulation of these two physiological processes is understood in its main line, many questions remain. First what could be the role of the low molecular weight, CaZ÷-binding parvalbumins abundantly present in the white skeletal muscle of lower and higher vertebrates (Pech~re et al., 1.971 ; Lei~ky et al., 1974 ; Pechbre, 1974). mould they mediate calcium exchanges between phosphorylase kinase and some of the proteins involved in the regulation of muscle contraction, or between these elements and the SR system ? Or could they be implicated in the hormonal stimulation of glycogenolysis that can be observed even in the relaxed state where free Ca e+ concentration in the sarcoplasm is extremely low (Fischer et al., 1971). For instance, one could imagine that parvalBIOCHIMIE, 1979, 61, n ° 5-6.

a n d coll.

v a l u e of n = 8 w a s found r e g a r d l e s s of the state of p h o s p h o r y l a t i o n of the e n z y m e .

Results obtained from both t y p e s of experiments established a p p a r e n t Ca2+-affinities in the following d e c r e a s i n g order : phosphorylase kinase ,,~ p a r v a i b u m i n > EGTA > troponin troponin-C, with no difference f o u n d b e t w e e n the n o n - p h o s p h o r y l a t e d a n d p h o s p h o r y l a t e d forms of p h o s p h o r y l a s e kinase. A p p a r e n t equilib r i u m constants for these Ca2+-complexes, relative to EGTA, w e r e in a g r e e m e n t with published v a l u e s o b t a i n e d from classical equilibrium studies. Similar results w e r e o b t a i n e d with proteins isolated from rabbit a n d dogfish muscle. No indication of protein-protein interactions affecting Ca2*-fluxes could be f o u n d in s u c h a reconstituted system, except for the sinqular c a s e of a slight i n c r e a s e d inhibition of p h o s p h o r y l a s e kinase b y p a r v a i b u m i n following p h o s p h o r y l a tion of the e n z y m e .

bumin could serve as a Ca 2+ sink to provide for the metal ion essential for the activation of phosphorylase kinase. If it could be shown that the phosphorylated form of phosphorylase kinase has a higher affinity for catcium than the non-activated enzyme, then a mechanism could be envisioned by which calcium could be removed from the parvalbumin pool under the influence of the cAM;Pdependent protein kinase. In order to examine some of these questions and further clarify Ca2+-fluxes during .contraction/ relaxation cycles in muscle, two procedures were selected that would show whether Ca 2+ exchanges between the various components could be affected by specific protein-protein interactions. This manuscript describes the use of kinetic approaches that measure both the uptake of calcium by purified elements of the SR and the state of activity of phosphorylated and non-phosphorylated phosphorylase kinase. A preliminary account of this work was presented (Moeschler et at., 1976).

Materials and Methods. Sarcoplasmie reticulum vesicles were prepared from white skeletal muscle of either rabbits New Zealand white or dogfish (Squalus aeanthias) according to a slight modification of the procedure of MaeLennan (1970) (carried out to the Rl-washed step), in that the extraction buffer contained KC1 instead of NaCl, and the SR suspension was washed and spun three times

M u s c l e Cae+-binding p r o t e i n s . (instead of once) at 10,000 X g for complete removal of mitochondria. The isolated vesicles were stored at 0 ° and used w i t h i n 5 days. Purified SR f r a g m e n t s f r o m the dogfish were less stable and were used w i t h i n 3 days.

Troponin (TN) and its Ca2+-binding subunit (TN-C) were isolated f r o m r a b b i t (Graeser and Gergely, 1971) and dogfish (Malencik el al., 1975) skeletal muscle. P a r v a l b u m i n was obtained f r o m dogfish and mouse skeletal musele (Alaba, 1975). Rabbit musele phosphoryIase was isolated aceording to Fischer and Krebs (1962) and phosphorylase kinase according to Krebs et al. (1959) and Cohen (1973). Purified bovine h e a r t cAMP-dependent protein kinase or its pure catalytic s u b u n i t (Peters et at., 1977) were generously provided by Dr. J. Demaille. Crystalline h u m a n salivary a-amylase, protease free, was prepared according to Fischer and Stein (1954). The Cae+-free forms of TN, TN-C and p a r v a l b u m i n were p r e p a r e d either b y dialysis against a 1 : 1 m i x t u r e of 0.1 M EDTA and EGTA or by gel filtration on a Sephadex G-25 column in the same medium. Maximum residual Ca2+ eoneentration varied f r o m 0.5 to 5 per cent (up to 10 per cent for troponin). Because i n s t a b i l i t y problems, p h o s p h o r y l a s e kinase was stored in the presence of 2 mM EDTA. It was dialyzed before use against the a p p r o p r i a t e reaction buffer. Ca~+-conlent was d e t e r m i n e d b y atomic a b s o r p t i o n on a P e r k i n - E l m e r 303 spectrometer. All buffers and solutions were passed, t h r o u g h Chelex columns w h e n necessary and eontained less t h a n 1 ~M Ca2+; they were stored exclusively in polyethylene or polypropylene containers. Protein eoneentration was d e t e r m i n e d according to Lowry el al. (1951) or by the b i u r e t procedure (Layne, 1957), using bovine s e r u m a l b u m i n as a standard, or f r o m absorbanee indiees at 280 n m (or at 260 n m for mouse p a r v a l b u m i n (Alaba, 1975)). The rate of Ca2+-uptake by SR was m e a s u r e d by the Millipore filtration teehnique of Martonosi and Feretos (1964) ; Millipore filters (0.45 iJ,m) were washed w i t h O.1 M EDTA, t h e n distilled water. Reactions were carried out at 20 ° w i t h ca. 0.1 m g / m l SR protein in 30 mM imidazole, HCI buffer, pH 7.0, containing 50 mM KC1, 5 mM MgC12, 5 mM p o t a s s i u m oxatate and 5 mM ATP. To remove c o n t a m i n a t i n g Ca2+, the SR suspension was always preineubated w i t h ATP. The total concentration of calcium (approximately 106 epm/Fxmol) was 10 lxM during the reaction. The reaction was i n t e r r u p t e d after 15 s l~y filtration and the 45Ca2+ in the filtrate was counted. Virtually all of the soluble proteins present were recovered in the filtrate.

The ATP-ase activity of the SR vesicles was determ i n e d : b y measuring the a m o u n t of inorganic phosp h a t ~ liberated during the reaction according to Marsh (1959). Phosphorylase kinase was assayed by a modification of the m e t h o d of Krebs (1966), as described by Poc!nwong (1975). The incubation m i x t u r e contained 30 mM imidazole buffer, pH 7.0, 50 mM KC1, 5 mM p o t a s s i u m oxalate, 15 mM MgC12, 3 mM ATP, 10 ~tM Ca2+ and 8 m g / m l p h o s p h o r y l a s e b. After p r e i n c u b a t i o n at 30°C for 1 min, the raetion was started by the addition of p h o s p h o r y l a s e kinase. P h o s p h o r y l a s e a was assayed according to Hedrick and Fischer (1965). Phosphorylase phosphatase was assayed according to Gratecos el al. (1977). BIOCHIMIE, 1979, 61, n ° 5-6.

617

Phosphorylation of phosphorylase kinase was carried out by incubating the enzyme w i t h cAMP and bovine h e a r t cAMP-dependent protein kinase or its isolated catalytic s u b u n i t (Beavo el al., 1974; Bechtel et al., 1977 ; Peters et al., 1977). The reaction was followed by m e a s u r i n g the increase in the pH 6.8/8.2 activity ratio of p h o s p h o r y l a s e kinase. The Ca2+-eompetition curves were c o m p u t e r - d r a w n f r o m a n o n - l i n e a r least-square fitting program.

Ca~+-bindiny capacities were d e t e r m i n e d by a gel filtration procedure, using a Sephadex G-25 column in the same m e d i u m as the SR Ca2+ uptake experiments. In the control. ATP and oxalate were omitted. For t r o p o n i n C and the p a r v a l b u m i n s , the column was preequilibrated w i t h I00 IxM 45Ca2+ (only 50 ~tM in the ease of p h o s p h o r y l a s e kinase). To ensure t h a t equilib r i u m was reaehed f r o m b o t h sides, e x p e r i m e n t s were carried out w i t h Ca2+-free and Ca2+-saturated proteins.

Results. Characterization of the SR vesicles. S e v e r a l reports have indicated that various enzymes involved in the c o n t r o l of glycogen m e t a b o l i s m r e m a i n a s s o c i a t e d w i t h t h e SR d u r i n g its i s o l a t i o n ( W a n s o n a n d D r o c h m a n s , 1972 ; B a i l e y et at., 1974 ; H S r l el at., 1978 ; H S r l a n d H e i l m e y e r , 1978). It a p p e a r e d i m p o r t a n t to e s t a b l i s h w h e t h e r o r n o t t h e s e w e r e a c t u a l l y a n i n t e g r a l p a r t of t h e Ca 2+t r a n s p o r t s y s t e m or, a l t e r n a t i v e l y , a s s o c i a t e d w i t h g l y c o g e n p a r t i c l e s k n o w n to a c c o m p a n y t h e SR fraction. Therefore, phosphorylase, phosphorylase kinase and phosphatase activities were monitored d u r i n g t h e p u r i f i c a t i o n of t h e SR. T a b l e I s h o w s t h a t o n l y n e g l i g i b l e a m o u n t s of e n z y m e a c t i v i t i e s remain associated with the isolated fragments. U p o n f u r t h e r w a s h i n g by r e s u s p e n s i o n of the v e s i c l e s i n t h e i s o l a t i o n (KC1) o r s t o r a g e ( s u c r o s e ) b u f f e r s a n d r e c e n t r i f u g a t i o n at 78,000 × g, r e s i d u a l a c t i v i t i e s cou.ld b e f u r t h e r r e d u c e d w i t h m o s t o f t h e r e l e a s e d e n z y m e s f o u n d in t h e s u p e r n a t a n t ( t a b l e I). D i r e c t t r e a t m e n t of t h e SR p e l l e t w i t h a - a m y l a s e (0.:5 m g / m l f o r 3 h r ) also d r a s t i c a l l y r e d u c e d all e n z y m e a c t i v i t i e s p r e s e n t . T h e r a t e of Cae+-uptake b y t h e v e s i c l e s w a s r e d u c e d b y 35 p e r c e n t i n t h e KC1 w a s h a n d b y m o r e t h a n 90 p e r c e n t i n t h e l a t t e r t w o c a s e s . At t i m e s , r e a d d i t i o n of glyc o g e n to a f i n a l c o n c e n t r a t i o n of 2 p e r c e n t ( w / v ) led to s o m e r e s t o r a t i o n of Ca2+-uptake w h e r e a s r e a d d i t i o n of e n z y m e s s u c h as p h o s p h o r y l a s e , p h o s p h o r y l a s e k i n a s e or p h o s p h a t a s e , either w i t h or w i t h o u t glycogen, s h o w e d no significant additional effects. Similar k i n d s of results w e r e obtained when skinned rabbit muscle fibers were u s e d i n s t e a d of i s o l a t e d SR v e s i c l e s . U p t a k e of Ca "-'+

H. J. M o e s c h l e r and coll.

618

was r e d u c e d by a-amylase t r e a t m e n t (14 ttg/ml, c o r r e s p o n d i n g to 1 txg/mg muscle for 30 m i n at 20°C) and was not r e s t o r e d by r e a d d i t i o n of glycogen ( K e r r i c k et al., unpublished). At r o o m temp e r a t u r e , SR-vesicles f r e s h l y isolated f r o m the dogfish h a v e an a p p a r e n t initial rate of Ca2+-uptake about five times h i g h e r t h a n those of the rabbit,

vity f r o m 32P-labeled ATP and no change in Ca 2+uptake p r o p e r t i e s . Glucagon and insulin (15 aM) w e r e l i k e w i s e w i t h o u t effect.

Inhibition of Ca~+ uptake into SR by the Ca z*binding proteins. C o m p e t i t i o n for Ca 2+ a m o n g the several muscle C a - b i n d i n g p r o t e i n s was studied

TABLE I.

Distribution of enzyme activities during purification of the SR vesicles.

Fraction

Phosphorylase kinase per cent

Phosphorylase per cent

Phosphorylase Phosphatase per cent

Cau+ Uptake rate per cent

Crude extract (1600 g sup.) 100 100 100 -Soluble fraction (44,000 g sup.) 100 97 62 -SR Vesicles (78,000 g pellet) 0.03 0.03 2.1 -SR Vesicles (a) (78,000 g pellet) 0.03 0.03 2.1 Control (100) (b) (100) (b) (100) (b) (100) (b) SR Vesicles before spin (80) (100) (100) KCI treatment 78,000 g pellet(e) (17) (d) (71) (65) SR Vesicles before spin (70) (100) (28) (100) Sucrose treatment 78,000 g pellet (c) (18) (26) (fi3) (8) SR Vesicles before spin (82) (42) (16) (100) ~-amylase treatment 78,000 g pellet (e) (12) (21) (4) (2) a. SR vesicles (1 mg/ml) are incubated for 3 hr at 20°C either in the imidazole HC1 buffer, pH 7.0, either alone (control) or in the presence of KCI (isolation buffer), sucrose (storage buffer) or a-amylase (0.5 mg/ml). b. Number in parenthesis represents percentages of the activities remaining in the SR vesicles taken as 100. c. Pellets were brought back to original volume for activity measurements. d. By further washes, the concentration of enzyme could be lowered to 2 % (0.0006 per cent of crude extract).

but the t w o rates are c o m p a r a b l e at t h e i r respective b o d y t e m p e r a t u r e s (1.0 ___ 0.1 and 2.4 ___ 0.1 lxmol Ca2÷/mg SR at 5 ° and 20 ° for the dogfish, r e s p e c t i v e l y , as c o m p a r e d to 0:5 ___ 0.1 and 1.6 -40.1 at 20 ° and 37 ° for the rabbit).

Role of EHectors. T h e initial rate of Ca 2+ uptake ( m e a s u r e d after 115 sec) and the extent of Ca 2÷ s e q u e s t e r e d ( m e a s u r e d after 15 rain) by purified SR vesicles w a s essentially u n a f f e c t e d by a n u m b e r of l o w m o l e c u l a r w e i g h t c o m p o u n d s and metabolites (tested at 1 mlV[ unless o t h e r w i s e stated). These i n c l u d e d glucose-6-P (2.!5 raM), g l u c o s e - l - P (2.5 raM), glycerol-P, pyruvate, p h o s p h o e n o l p y r u vale w i t h and w i t h o u t p y r u v a t e '.kinase (0.1 m g / m l ) o x a l o a c e t i c acid, c r e a t i n e (15 m ~ ) and c r e a t i n e p h o s p h a t e (15 raM,) w i t h and w i t h o u t c r e a t i n e p h o s p h o k i n a s e (0.1 m g / m l ) , NAD and NADH, NADP and NADPH and r e d u c e d or o x i d i z e d glutathione. Glycerol or sucrose a d d e d at 10 p e r cent w i t h or w i t h o u t 1 mM Pi w e r e equally w i t h o u t effect. A d d i t i o n of 10 ~M cAY~P or 0.1 m g / m l purified c a t a l y t i c subunit of c A ~ P - d e p e n d e n t p r o t e i n k i n a s e resulted in no i n c o r p o r a t i o n of r a d i o a c t i BIOCHIMIE, 1979, 61, n ° 5-6.

by i n c u b a t i n g SR vesicles w i t h i n c r e a s i n g a m o u n t s of the metal-free p r o t e i n s at a fixed c o n c e n t r a t i o n of 4~Ca2~ (10 ~M) ; the a p p a r e n t initial rate of Ca 2÷ u p t a k e was then m e a s u r e d . T h i s a p p r o a c h elimin a t e d p r o b l e m s of m e t a l .contamination w h i c h can o c c u r w h e n w o r k i n g at l o w Ca2+-concentrations, and of possible i n h i b i t o r y effects o b s e r v e d at high Ca2+-concentrations. In o r d e r to c h e c k the v a l i d i t y of the p r o c e d u r e , t w o a p p r o a c h e s w e r e used. First, m e t a l free protein and SR w e r e p r e i n c u b a t e d t o g e t h e r and Ca 2*uptake w a s i n i t i a t e d by a d d i t i o n of 45Ca 2÷. Second, to simulate m o r e closely p h y s i o l o g i c a l c o n d i t i o n s , m e t a l free p r o t e i n s w e r e p r e i n c u b a t e d w i t h 45Ca 2÷ and uptake i n i t i a t e d by a d d i t i o n of the SR. Since p h o s p h o r y l a s e k i n a s e is labile w h e n c o n v e r t e d to its metal-free form, it w a s used only in the f o r m e r type of e x p e r i m e n t in w h i c h Ca 2+ is r e m o v e d d u r i n g p r e i n c u b a t i o n w i t h SR. A c o n t r o l e x p e r i m e n t c a r r i e d out w i t h Ca2+-free and Ca2+-contai n i n g TN-C gave v i r t u a l l y i d e n t i c a l results. T h e overall Ca2+-exchange and uptake r e a c t i o n s consist of t w o s i m u l t a n e o u s p r o c e s s e s :first, rever-

M u s c l e Ca~÷-binding p r o t e i n s .

T h e c o n c e n t r a t i o n of e x t e r n a l f r e e c a l c i u m at the o n s e t of t h e r e a c t i o n is

sible Ca2+-binding a n d r e l e a s e f r o m a p r o t e i n w i t h w h i c h f r e e a n d b o u n d Ca :÷ are in t h e r m o d y n a m i c e q u i l i b r i u m , t h e n i r r e v e r s i b l e a c c u m u l a t i o n of Ca ~÷ by the S R - s y s t e m , a c c o r d i n g to the f o l l o w i n g set of e q u a t i o n s :

[Mq

[Me] --

[P] ~

[PMe]

(1)

k_l ks

[Me] + [ T ] ~

ks

k4

.[TM~] ~

k_2

[TM~] ~

k_3

(2i)

M e r e p r e s e n t s t h e e x t e r n a l Ca e÷ a n d Mi, t h e i n t e r n a l Ca e+ b o u n d to t h e v e s i c l e s ; P is t h e soluble C a : + - b i n d i n g p r o t e i n a d d e d to t h e system, a n d T, t h e Ca u+ t r a n s p o r t s y s t e m of t h e SR. W h e n t h e i n w a r d t r a n s p o r t of Ca 2+ is t r u l y i r r e v e r s i b l e ~k s = 0), as o b s e r v e d in the p r e s e n c e of oxalate, t h e i n i t i a l r a t e a s s u m e s t h e f o r m of t h e Michaelis-Menten equation Ve V =

(3,~ 1 + Ke/[Me] in w h i c h V e = k 3 [To], K(, = k_2/k 2 w h e n k 3 < k. 2 (Wold, 1971).

T h e r e s u l t s are i l l u s t r a t e d in figure 1 A a n d 1 B. T h e i n i t i a l r a t e of c a l c i u m u p t a k e as a f u n c t i o n of free calcium concentration shows a typical hyperb o l i c s a t u r a t i o n b e h a v i o r , as i n d i c a t e d b y t h e l i u e a r i t y of t h e d o u b l e r e c i p r o c a l p l o t s h o w n in f i g u r e 2. F r e e C a 2 + - c o n c e n t r a t i o n s w e r e c a l c u l a t e d f r o m t h e E G T A - i n h i b i t i o n c u r v e , u s i n g an

B i n d i n g of Ca 2+ to p r o t e i n s w i t h i n t h e SR w i l l affect t h e t r a n s p o r t p r o c e s s o n l y in so f a r as it w i l l m a i n t a i n a f a v o r a b l e Ca 2+ g r a d i e n t ; a d d e d

~_ ~°t-

(4)

If [Me] is l o w e n o u g h to e n s u r e a l i n e a r d e p e n d e n c e on t h e r a t e of Ca2+-uptake, t h e n t h e i n h i b i t o r y effects of t h e c h e l a t o r p r o t e i n s w i l l be a d i r e c t e x p r e s s i o n of t h e i r Ca2+-binding c h a r a c t e r i s t i c s . V a r i a t i o n of p r o t e i n c o n c e n t r a t i o n , [Po], at fixed t o t a l c a l c i u m , [M~], y i e l d s a t i t r a t i o n c u r v e w i t h a m i d - p o i n t d e p e n d e n t on Ka. If o n e w o r k s at too h i g h Ca u+ c o n c e n t r a t i o n s , t h e t i t r a t i o n c u r v e s w i l l be d i s t o r t e d as o n e starts to s a t u r a t e the Ca2+-transport s y s t e m ; n e v e r t h e l e s s , e x p e r i m e n t s c a r r i e d out in t h e p r e s e n c e of a m i x t u r e of C a - b i n d i n g p r o t e i n s w i l l still y i e l d d a t a as to t h e i r relative Ca2+-binding c a p a b i l i t i e s .

[ T ] + [M~]

k_~

1 d- Ka [el

w h e r e Mo r e p r e s e n t s t o t a l C a u+ a d d e d to t h e s y s t e m a n d K a = kl/k_l, t h e e q u i l i b r i u m c o n s t a n t for the PM e c o m p l e x .

kl

[Me] +

619

7- I Ill

8

7

,6 5 4 -Iog[Xo] (N4)

3

8

7 6 5 4 -IogiX,:]/fi (M)

Fro. 1. - - Inhibition of Ca~+-aptake by SR vesicles in the presence of increasing concentrations of calcium binding proteins or EGTA (added as a standard). In A, the data are plotted as a function of the molar concentration of the added protein : [Xo] ; in B, per Ca2+-binding site corrected to yield Kd at 50 per cent inhibition : [Xo]/n. ( I ) phosphorylated form of phosphorylase kinase, ([]) non-phosphorylated form o f p h o s p h o r y l a s e kinase, (A), parvalbumin, (O) troponin, (V/), troponin-C, (O) EGTA.

oxalate s e r v e s t h e s a m e f u n c t i o n b y p r e c i p i t a t i n g c a l c i u m i n s i d e the v e s i c l e s ( H a s s e l b a c h a n d Makinose, 19'63).

BIOCHIMIE, 1979, 61, n ° 5-6.

a p p a r e n t K a v a l u e of 1.0" 106 M -1 for the Ca 2÷E G T A c o m p l e x ( B r e m e l and W e b e r , 1975). Values of K e = 0.65 ~M a n d V e : 1.0 ~mol Ca ~÷ " r a g -1

620

H. J. Moeschler and coll.

min-1 were determined for the Ca2+-transport syst e m of SR, i n a g r e e m e n t ~vith p u b l i s h e d d a t a ( Y a m a m o t o a n d T o n o m u r a , 1967). It c a n b e s e e n

p l e x e s (PM e) is f a s t c o m p a r e d to Ca2+-uptake b y t h e SR (k 1 > k3).

the

rate

of

I n o r d e r to e x p r e s s t h e s e d a t a i n t e r m s of Ca2+ a f f i n i t i e s of t h e d i f f e r e n t p r o t e i n s P, o n e m u s t d e t e r m i n e t h e n u m b e r of C a 2 + - b i n d i n g s i t e s m T h e s e w e r e m e a s u r e d i n d e p e n d e n t l y b y gel f i l t r a tion under the same conditions. The data are l i s t e d i n t a b l e II. F o r TN-C, T N a n d p a r v a l b u m i n , v a l u e s i d e n t i c a l to t h o s e r e p o r t e d i n t h e l i t e r a t u r e w e r e o b t a i n e d ( B e n z o n a n a et al., 1972 ; P o t t e r a n d G e r g e l y , 1975). F o r t h e p h o s p h o r y l a t e d a n d n o n p h o s p h o r y l a t e d f o r m s of p h o s p h o r y l a s e k4nase, a

10

Vmax V 5

0

5

10

l / [ C a ++]

v a l u e of n : 8 g r a m a t o m Ca 2÷ b o u n d p e r e n z y m e t e t r a m e r of M W ---- 1.3 , × 10 o w a s f o u n d , i n c o n t r a s t t o n ~_ 24 o b t a i n e d b y B r o s t r o m et al. (1971) (see also K i l i m a n a n d H e i l m e y e r (1977)) i n t h e a b s e n c e of Mg ~*. C o n t r o l e x p e r i m e n t s c a r r i e d o u t without ATP and oxalate gave identical results•

15

uM -1

Fro. 2. - - Double reciprocal plot of calcium uptake by SR vesicles vs. tree calcium concentration (assuming Ks : 1.0.106 M-1 for the Ca2+-EGTA complex at pH 7) (Bremel & Weber, 1975).

U s i n g t h e v a l u e s r e p o r t e d i n t a b l e H, t h e e x t e n t of i n h . i b i t i o n r e p o r t e d i n F i g u r e 1 A c a n b e r e c a l -

TABLE II.

Ca2+-binding

characteristics

o[ m u s c l e

proteins.

Equilibrium binding studies

This study Ka (.uM-1) (c)

Effectors

~"

EGTA

1

1.8 2.1 4.1 2.1

Troponin-C Troponin Parvalbumin Phosphorylase kinase dephospho form phospbo form

Ka(~M -1}

--Mg e+ -+Mg ~+ --Mg ~+ --~Mg~+

~! -t~-

1.0

0.1 0.1 0.5 0.3

24 -t- 1 6.8 Jr- 1 24 _____.1 --

2.8 0.1 5.4 2.2

-+-t-4q-

33 3 20

Ref 1

1.6 0.06 0.9 0.5

2 2 2 3,4 5 6 5

--

n'(a)

N (b)

SR

D ephospho Phospho Ph-K Ph-K

(1)

(1.0)

(1.0) •

(1.0)

3 . 7 -4- 0 . 3

4

0.35

0.45

o.32

3 . 8 +___0 . 5 1.7 -+- 0.2

4 2

0.63 3.2

1.0 3.2

08 6.3

7.8 Jr- 0.5

8

2.7

7.9 "t- 0 . 6

8

3.2

~. N u m b e r of Ca2+-binding sites. • I n t e g r a l n u m b e r of Ca2+-binding sites chosen to calculate the a p p a r e n t e q u i l i b r i u m constants. c. Association c o n s t a n t for Ca2÷ d e t e r m i n e d e i t h e r in the SR i n h i b i t i o n e x p e r i m e n t (SR) or i n h i b i t i o n of p h o s p h o r y l a s e k i n a s e activity u s i n g the d e p h o s p h o r y l a t e d a n d p h o s p h o r y l a t e d f o r m s of the enzyme. 1. R. D. Bremel ~ A. W e b e r (1975) Biochim. Biophys. Acta, 376, 366. 2. J. D. P o t t e r ~ J. Gergely (1975) J. Biol. Chem., 250, 4628. 3. Benzonana, G., Capony, J. P. & Pech6re, J. F. (1972) Biochim. Biophys. Acta, 278, 110. 4. J. O. A l a b a ( u n p u b l i s h e d results). 5. C. O. B r o s t r o m et al. (1971) J. Biol. Chem., 246, 1961. 6. M. K i l i m a n n & L. M. G. Heilmeyer, Jr. (1977) Eur. J. Biochem., 73, 191.

t h a t t h e i n h i b i t i o n of Ca 2÷ u p t 'alke d e c r e a s e s i n t h e following order : phosphorylase ldnase > parvalbumin > TN >TN-C > EGTA. No difference was found between non-phosphorylated and phosphorylated kinase. Identical results were obtained w h e t h e r t h e p r o t e i n s w e r e p r e i n c u b a t e d "with SR o r w i t h 45Ca2+ ( s e e M a t e r i a l s a n d M e t h o d s ) , i n d i c a t i n g t h a t t h e f o r m a t i o n of t h e p r o t e i n - m e t a l c o m -

BIOCHIMIE, 1979, 61, n ° 5-6.

culated per Ca2÷-binding site for each protein. T h e p l o t s o b t a i n e d (figure 1 B) s h o w t h a t t h e a p p a r e n t Ca2+-affinities r e l a t i v e to E G T A ( g i v e n b y t h e c u r v e m i d - p o i n t s ) d e c r e a s e i n t h e o r d e r of p h o s p h o r y l a s e k i n a s e ~ p a r v a l b u m i n > E G T A _~ T N > TN-C. T h e d a t a a r e i n g o o d a g r e e m e n t w i t h values obtained by classical equilibrium techn i q u e s ( t a b l e II), i n d i c a t i n g t h e a b s e n c e of a n y

621

Muscle Ca~*-binding proteins.

Inhibition of phosphorylase ,kinase activity by SR and muscle Ca2÷-binding proteins. As s h o w n

significant i n t e r a c t i o n b e t w e e n the i n d i v i d u a l proteins and the SR system. T h e same can be said for possible i n t e r a c t i o n s i n v o l v i n g p a r v a l b u m i n since mixtures of this p r o t e i n w i t h TN=C or p h o s p h o r y lase k i n a s e s h o w e d p u r e l y a d d i t i v e effects.

p r e v i o u s l y , r a b b i t p h o s p h o r y l a s e kinase is inactiv a t e d by isolated SR vesicles (Ozawa et at., 19,67 ; B r o s t r o m el at., 197.1 ; F i s c h e r et at., 1976). T h a t

A

[]

~ 50~ , ~ ~ ~

o

7

6 5 -IoglX]/5

4 (M)

3 7

6 5 4 -Iog[Xo}/5 (M)

FIG. 3. - - Inhibition of phosphorglase kinase activity by increasing concentration of calcium binding proteins and EGTA (added as a standard). In A, it is plotted as

the dephosphorylated form or in B as phosphorylated form. (A) parvalbumin, ( . ) troponin, (V) troponin-C, (0) EGTA.

6 Vmax

V

4

2

0

I

5

I

I

I

10 15 20 1/[Ca +,] (UM-1)

25

Fie. 4. - - Double reciprocal plot of phosphorglasc kinase actipity (non-phosphorylated) vs. free Ca2+-concentration (calculated as in figure 2).

V i r t u a l l y i d e n t i c a l results w e r e o b t a i n e d w h e n e x p e r i m e n t s w e r e c a r r i e d out w i t h dogfish SR, TN-C and p a r v a l b u m i n , or w h e n r a b b i t TN-C and p a r v a l b u m i n w e r e e x p o s e d to dogfish SR or v i c e versa.

BIOCHIMIE, 1979, 61, n ° 5-6.

this effect is due to c a l c i u m r e m o v a l is i n d i c a t e d by the fact that aged vesicles w h i c h h a v e lost t h e i r ability to pump c a l c i u m w e r e ~ i t h o u t effect ; also, i n h i b i t i o n by SR is r e v e r s e d by excess calcium. The i n h i b i t o r y effect of TN-C, TN, p a r v a l -

622

H . J. M o e s c h l e r a n d coil.

b u m i n a n d EGTA on the c a t a l y t i c a c t i v i t y of phosp h o r y l a s e k i n a s e w a s e x a m i n e d u s i n g essentially the same a p p r o a c h as for the SR e x p e r i m e n t s . The d a t a o b t a i n e d f o r the p h o s p h o r y l a t e d a n d nonp h o s p h o r y l a t e d forms of the enzyme, e x p r e s s e d as a f u n c t i o n p e r c a l c i u m b i n d i n g site, are s h o w n in figure 3'A a n d B, r e s p e c t i v e l y . T h e a p p a r e n t c a l c i u m affinities d e c r e a s e in the same o r d e r as f o u n d w i t h the SR e x p e r i m e n t s , i.e., p a r v a l b u m i n > E G T ~ ~ TN > TN-C. T h e s e values, calcul a t e d f r o m the c u r v e m i d p o i n t s , are also l i s t e d in table II. W h e r e a s curve m i d p o i n t s s h o w e d good r e p r o d u c i b i l i t y , v a r i a t i o n s in the c o m p u t e r d e r i ved Hill coefficients w e r e o b t a i n e d in the k i n a s e i n h i b i t i o n e x p e r i m e n t s . As this w a s o b s e r v e d even in the case of EGTA, it c a n n o t be a t t r i b u t e d to c o o p e r a t i v i t y in Ca 2+ b i n d i n g to the different p r o teins, The o n l y slight b u t c o n s i s t e n t d e v i a t i o n f r o m the g e n e r a l p a t t e r n is an i n c r e a s e d i n h i b i t i o n of the p h o s p h o r y l a t e d f o r m of p h o s p h o r y l a s e k i n a s e b y p a r v a l b u m i n . T h e fact that in all o t h e r e x p e r i ments, the enzyme s h o w s the same Ca2+-binding c h a r a c t e r i s t i c s r e g a r d l e s s of its state of p h o s p h o r y lation, suggests t h a t some specific i n t e r a c t i o n m i g h t o c c u r b e t w e e n the two proteins. The observ e d effects are not t i m e - d e p e n d e n t a n d t h e r e f o r e c a n n o t be a t t r i b u t e d to an e n z y m a t i c r e a c t i o n in w h i c h p a r v a l b m n i n w o u l d affect p h o s p h o r y l a s e k i n a s e activity. A double r e c i p r o c a l plot of p h o s p h o r y l a s e k i n a s e a c t i v i t y vs. free Ca2+-concentration, analogous to t h a t d e p i c t e d in figure ~, s h o w s a d o w n w a r d c u r v a t u r e (figure 4') that can be a t t r i b u t e d to heterogeneous Ca 2+ b i n d i n g sites. This p r e c l u d e s a r e a d y e s t i m a t i o n of the k i n e t i c p a r a m e t e r s for the Ca 2÷ d e p e n d e n c e of the enzyme (see also Brost r o m et al. (1971). H o w e v e r , since the p o s i t i o n s of the curve m i d p o i n t s are c o m p a r a b l e for all chelators used in b o t h the SR and p h o s p h o r y l a s e k i n a s e i n h i b i t i o n studies, this suggests the b i n d i n g constant of the e n z y m e for Ca 2+ m u s t be s i m i l a r to t h a t of the SR t r a n s p o r t system (i.e. ca. 0.6'5 ~M).

Discussion.

SR vesicles i s o l a t e d from b o t h dogfish a n d rabbit skeletal muscle d i s p l a y e d n o r m a l Ca2+-uptake p r o p e r t i e s and ATP-ase activities ; these i n c r e a s e d w i t h t e m p e r a t u r e as e x p e c t e d f r o m t h e r m o d y n a m i c c o n s i d e r a t i o n s . Rates of Ca2+ uptake w e r e c o m p a r a b l e w h e n m e a s u r e d at t h e i r r e s p e c t i v e

BIOCHIM1E, 1979,

61, n ° 5-6.

b o d y t e m p e r a t u r e s (7°C a n d 37°C). T h i s suggests that b o t h have s i m i l a r t r a n s p o r t systems w i t h an a c t i v i t y d e p e n d e n t u p o n the s u r r o u n d i n g l i p i d phase. In this c o n t e x t the r e c e n t findings b y Sumida and T o n o m u r a (1'974) and b y I k e m o t o (1975) are of interest. T h e y s h o w e d that the ratio of Ca 2÷u p t a k e p e r ATP split b y r a b b i t SR is r e d u c e d from 2.0 at 22°C to 1.0 at 0°C. T h i s ~vas a t t r i b u t e d to the b l o c k i n g of one or two high affinity Ca 2÷t r a n s p o r t sites b y a c h a n g e in the m o b i l i t y of adjacent p h o s p h o l i p i d molecules. Only v e r y l o w p e r c e n t a g e s of g l y c o g e n o l y t i c enzymes are a s s o c i a t e d w i t h isolated SR-vesicles, in a g r e e m e n t w i t h the r e c e n t results of H6rl et al. (1976). R e s i d u a l p h o s p h o r y l a s e , p h o s p h o r y l a s e k i n a s e a n d p h o s p h o r y l a s e p h o s p h a t a s e activities c o u l d be f u r t h e r r e d u c e d b y a - a m y l a s e t r e a t m e n t of the vesicles or b y s i m p l e r e p e t i t i v e w a s h i n g w i t h KCI or sucrose solutions. The Ca2÷-binding c h a r a c t e r i s t i c s found for the v a r i o u s muscle p r o t e i n s b y the i n d i r e c t competitive S.R a p p r o a c h are g e n e r a l l y in good a g r e e m e n t w i t h p u b l i s h e d values o b t a i n e d from classical e q u i l i b r i u m studies (table II). The s t o i c h i o m e t r y of Ca2÷-binding f o u n d for TN-C and TN (n = 4) is consistent w i t h the values r e p o r t e d by P o t t e r and Gergely (1975). The value n - - 8 o b t a i n e d in the p r e s e n c e of Mg2* for the t e t r a m e r i c fo.rm of phosp h o r y l a s e k i n a s e of m o l e c u l a r w e i g h t ca. 1.3 X 106 is s i m i l a r to those of K i l i m a n and He~lmeyer (1977), i n d i c a t i n g that two e q u i v a l e n t s of c a l c i u m are b o u n d p e r e n z y m e m o n o m e r (of s u b u n i t s t r u c t u r e a~7 or a,~vS). I n d e e d , a r e p o r t that has a p p e a r e d s i n c e this w o r k w a s com]~leted (Cohen et al., 1978) i n d i c a t e s that the e n z y m e m i g h t also c o n t a i n s t o i c h i o m e t r i c amounts of CDR, the calciumd e p e n d e n t r e g u l a t o r of cAMP p h o s p h o d i e s t e r a s e . T h e r e is no i n d i c a t i o n as yet as to w h i c h k i n a s e s u b u n i t or subunits s p e c i f i c a l l y b i n d c a l c i u m . F r e e CDR is s a i d to b i n d 2 to 4 Ca 2÷ a t o m s / t o o l w i t h an affinity in the o r d e r of K~ ~ 10 -5 M (Yazawa et at., 1978 ; Wolff et at., 1977 ; Cox, u n p u b l i s h e d results ; D e d m a n et at., 1977). These c o r r e s p o n d to the low affinity sites of TN-C (Potter a n d Gergely, 1975), a n d a r e at least one o r d e r of m a g n i tude l o w e r than o b s e r v e d for p h o s p h o r y l a s e k i n a s e (table II). T h e d a t a w o u l d i m p l y that if CDR is the sole c a l c i u m - b i n d i n g s u b u n i t of p h o s p h o r y l a s e kinase, its c a l c i u m - b i n d i n g p r o p e r t i e s w o u l d have to be g r e a t l y affected b y i n t e r a c t i o n w i t h the other s u b u n i t s of the enzyme. A value of n = 24 was o b t a i n e d by B r o s t r o m et al. (197'1) for p h o s p h o r y lase k i n a s e in the absence of Mg2*, p r o b a b l y due to speci~fic a n d non-specific Ca 2+ b i n d i n g .

Muscle

T h e data in this m a n u s c r i p t i n d i c a t e no significant p r o t e i n - p r o t e i n i n t e r a c t i o n a m o n g the comp o n e n t s p r e s e n t in the r e c o n s t i t u t e d system emp l o y e d here. No e v i d e n c e w a s found, for instance, that p a r v a l b u m i n could facilitate c a l c i u m exchange b e t w e e n p h o s p h o r y l a s e k i n a s e a n d the C a ~ - t r a n s p o r t system of the SR. It should be noted, h o w e v e r , that these studies w e r e c a r r i e d out w i t h a purified p r e p a r a t i o n of soluble p h o s p h o r y l a s e kinase. Using fluorescent antibodies, H6rl et al. (1976) h a v e s h o w n that the e n z y m e is p r e d o m i n a n tly localiz.ed along the s a r c o l e m m a . F u r t h e r m o r e , Sulakhe and D r u m m o n d (19743 d e m o n s t r a t e d that p h o s p h o r y l a t i o n of a s a r c o l e m m a l p r e p a r a t i o n by c A M P - d e p e n d e n t p r o t e i n kinase results in an i n c r e a s e d a c c u m u l a t i o n of calcium. T h e p o s s i b i l i t y t h e r e f o r e r e m a i n s that p a r v a l b u m i n .could affect c a l c i u m e x c h a n g e s w i t h such p r e p a r a t i o n s of b o u n d e n z y m e s or a n y o t h e r m e m b r a n e - a s s o c i a t e d , Ca2+-dependent p r o t e i n l~inase. A d i r e c t i n v o l v e m e n t of p a r v a l b u m i n in m o d u lating muscle c o n t r a c t i o n or glycogenolysis is u n l i k e l y because of its u n e v e n tissue d i s t r i b u t i o n (very l o w c o n c e n t r a t i o n , if any, in red or c a r d i a c muscle or other organs such as the liver). Its a b u n d a n c e in w h i l e (fast-t~vit.ch glycolytic) skeletal muscle still raises the p o s s i b i l i t y of an involv e m e n t in the c o n c e r t e d r e g u l a t i o n of glycogen m e t a b o l i s m and muscle c o n t r a c t i o n . These t w o processes are coupled p r i m a r i l y t h r o u g h Ca 2÷ reIease f r o m the SR to p r o v i d e the energy necessary to m a i n t a i n c o n t r a c t i o n . T h e fact that one can initiate glycogenolysis u p o n h o r m o n a l stimulation in a r e l a x e d muscle w i t h o u t t r i g g e r i n g cont r a c t i o n (Stull and Mayer, 1971) m i g h t be explained on the basis of the h i g h e r affinity of p h o s p h o rylase k i n a s e for Ca 2÷, as c o m p a r e d to TN and TN-C (fig. 2. and table II). P a r v a l b u m i n has an a p p a r e n t Ca2+-affinity s i m i l a r to that of p h o s p h o rylase kinvse, but it is p r e s e n t in m u c h h i g h e r c o n c e n t r a t i o n s in the sarcoplasm. T h e r e f o r e , parv a l b u m i n could f u n c t i o n in a Ca2+-buffering capac i t y (Pech~re et al., 1~975 ; P e c h b r e e t at., 1977), m a i n t a i n i n g Ca 2÷ c o n c e n t r a t i o n s at a m i n i m a l basal level, h i g h enough to allow for some a c t i v a t i o n of p h o s p h o r y l a s e k i n a s e but too l o w to initiate contraction. E v e n lo~v lev~ls of p h o s p h o r y l a s e kinase a c t i v a t i o n w o u l d result in a c o n s i d e r a b l e activation of p h o s p h o r y l a s e and, thereby, glycogen b r e a k d o w n , because of the e n z y m a t i c nature of the r e a c t i o n and the amplification afforded by enzyme cascade systems ( F i s c h e r et at., 1971 ; S t a d t m a n and Chock, 1977 ; Chock and Stadtman: 1977). A hyloothesis that p h o s p h o r y l a t i o n of p h o s p h o rylase kinase m i g h t i n c r e a s e its affinity for Ca 2+ and thus, i n c r e a s e its ability to r e m o v e C,a:+ f r o m BIOCHIMIE, 1979, 61, n ° 5-6.

623

Ca~*-binding proteins.

a p a r v a l b u m i n pool d u r i n g r e l a x a t i o n could not he c o n f i r m e d : both f o r m s of the e n z y m e seem to have i d e n t i c a l Ca2+-binding c h a r a c t e r i s t i c s ; if anything, p h o s p h o r y t a t i o n a p p e a r e d to facilitate r e m o v a l of c a l c i u m f r o m the enzyme by Ca2+-free parvalbumin.

Acknowledgements. The authors t h a n k Mr. Dorr Tippens and Bill Buck f o r their technical assistance and Mrs. R o b i n Colby and Dr. Steoe Robertson f o r their assistance w i t h the c o m p u t e r program.

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