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Calcium related properties of plasma membranes from guinea pig placenta Shami, Yehezkel 1974

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CALCIUM RELATED PROPERTIES OF PLASMA MEMBRANES FROM GUINEA PIG PLACENTA by YEHEZKEL SHAMI B.Sc, The Hebrew University of Jerusalem, 1969 M.Sc, The Hebrew University of Jerusalem, 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of PHYSIOLOGY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1974  In p r e s e n t i n g t h i s  thesis  an advanced degree at  fulfilment  the U n i v e r s i t y of  the L i b r a r y s h a l l make it I further  in p a r t i a l  freely  of  the  requirements  B r i t i s h Columbia, I agree  available  for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  of  this  representatives. thesis for  It  financial  that  reference and study. this  thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  or  i s understood that copying or p u b l i c a t i o n gain s h a l l not  written permission.  Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada  be allowed without my  ABSTRACT  Calcium transport across the placenta i s asymmetrical  and i s  believed to be an active transport. An essential step in such a transport i s translocation of the ion across a single plasma membrane. The 2+ objective of this thesis was to study the Ca -related properties of the placental plasma membranes and to gain some knowledge of their role 2+ in Ca  -transport. 2+ Three Ca -related properties were studied: 2+ 1. Ca -binding to the placental plasma membranes; 2+ 2. The membrane bound enzyme Ca -ATPase; and 2+ 3. Ca -uptake by the placental plasma membrane vesicles. 2+ Ca -binding properties of the membrane preparation were studied  by the use of a new method, the flow dialysis system.  Two types of sites  2+ for Ca were found: 1) high a f f i n i t y , low capacity s i t e s , and 2) low a f f i n i t y , high capacity sites. The high a f f i n i t y sites had 10-fold 2+ 2+ higher a f f i n i t y for Ca than for Mg . 2+ A calcium-stimulated, membrane-bound enzyme, namely Ca  -ATPase,  was located in the placental plasma membranes. This enzyme i s d i s t i n c t from the Na , K -ATPase and alkaline phosphatase. +  +  activated by Mg  but with lower efficiency.  the enzyme at the same s i t e .  The enzyme can be  Both Ca  and Mg  activate  A formula was derived, enabling one to  predict very precisely the velocity of the enzyme incubated under any 2+ combination of Ca dimensional model.  2+ and Mg  ; this relationship i s presented in a three  The formula can be used for other enzymes or other i i substrates, as was demonstrated with ATP and ADP.  Ill  The placental plasma membrane vesicles are capable of accumulating Ca 2+ . Ca 2+ -uptake was defined as the amount of Ca 2+ which i s not available for rapid exchange and cannot be displaced by a high concentration of competitor in the presence of ATP.  This definition i s  different from and more accurate than the one which i s widely used and 2+ cited in the l i t e r a t u r e .  concentration of 190 mM 2+ was recorded, which was 24-fold higher than the external Ca concen2+ tration (8 mM). Ca -uptake was dependent on ATP hydrolysis by the 2+ 2+ placental Ca -ATPase. This process was independent of Mg . 2+ It i s suggested that while the substrate for Ca -ATPase i s Ca2+ 2+ ATP, the substrate for Ca -uptake i s Ca . 2+ The overall Ca -related properties of the placental plasma 2+ membranes are independent of Mg and the entire process from binding 2+ to membrane through activation of the enzyme and f i n a l l y Ca -uptake 2+ is dependent on Ca  An intravesicular Ca  alone. This situation i s unique to the placental  plasma membranes. It i s tempting to speculate that the link between the maternal and the fetal circulation i s achieved by forming vesicles loaded with 2+ Ca on the maternal side and unloading them through fusion with the basal plasma membrane on the fetal side. 2+ The Ca  -related properties of placental plasma membranes des-  cribed in this thesis, provide many answers regarding the f i r s t step in 2+ the asymmetrical transplacental Ca  -transport.  Further investigation  is required before a f u l l understanding of the entire process i s achieved.  TABLE OF CONTENTS  Page ABSTRACT  i i  LIST OF TABLES  vi  LIST OF FIGURES  vii  LIST OF PLATES  x  ACKNOWLEDGMENT  xi  GENERAL INTRODUCTION  .  1  DIVISION I CHAPTER I - CALCIUM BINDING TO THE PLACENTAL PLASMA MEMBRANES . . . INTRODUCTION  *  10 10  MATERIALS AND METHODS  13  RESULTS  25  DISCUSSION  36  SUMMARY  45 DIVISION II  CHAPTER II - CHARACTERIZATION OF CALCIUM-STIMULATED ATPase  47  INTRODUCTION  47  MATERIALS AND METHODS  48  RESULTS  50  DISCUSSION  69  SUMMARY  76 iv  V  Page CHAPTER III - THE EFFECT OF Ca /Mg 2+  2+  CONCENTRATION RATIO ON  PLACENTAL (Ca2+-Mg2+)-ATPase ACTIVITY  78  INTRODUCTION  78  MATERIALS AND METHODS  83  RESULTS  84  DISCUSSION  89  SUMMARY  91  CHAPTER IV - COMPARISON BETWEEN ALKALINE PHOSPHATASE AND C a 2+  ATPase  92  INTRODUCTION  92  MATERIALS AND METHODS  ..  93  RESULTS  ..  95  DISCUSSION  99  SUMMARY  104 DIVISION III  CHAPTER V - Ca -UPTAKE BY PLACENTAL PLASMA MEMBRANE VESICLES 2+  ...  107  INTRODUCTION  107  MATERIALS AND METHODS  109  RESULTS  110  DISCUSSION  119  SUMMARY  124  GENERAL CONCLUSIONS  125  REFERENCES  130  LIST OF TABLES  Table I  Page Specific a c t i v i t i e s of marker enzymes in the different fractions  II III  A f f i n i t y and capacity of placental plasma membranes and Bovine Albumin 45 2+ 40 2+ Apparent Km's for Ca displacement by Ca , Mg , S r 2+  IV  18  2 +  32 39  Comparison between alkaline phosphatase a c t i v i t y and ATPase a c t i v i t y i n the membrane preparation and the purified alkaline phosphatase fraction  V  The effect of phosphate on Ca  -uptake  2+  VI  The effect of 5.4 mM Mg  H  7  2+  on Ca  (diffusion rate) by 3 x 10 M _1  uptake  101  4 0  Ca  displacement 2 +  and Ca 2+  118  vi  LIST OF FIGURES  Figure  Page  1  Diagram of the apparatus used for measuring 22  calcium binding by rate of dialysis 45 2+ Measurement of Ca diffusion rate at various 45 2+ Ca concentrations •45 2+ Measurement of Ca diffusion rate as a function of cpm concentration i n the upper chamber Flow dialysis profiles of calcium binding at various calcium concentrations . . . • 2+ 2+ Ca concentration effect on Ca -binding levels by placental plasma membranes and Bovine Albumin 2+  2  3 4  5  6  26 27 29  30  Scatchard plot of Ca -binding by placental plasma 31  membranes and Bovine Albumin 2+ 7 Ca -binding as a function of placental plasma membrane protein concentration , 2+ 8 pH effect on Ca -binding 9A ^ C a displacement by divalent cations 45 2+ 9B,C,D Double reciprocal plots for Ca displacement 4  2+  by C a , Mg 4 0  2 +  2+  and S r  34  . . .  35 37  38  2 +  10  Stimulation of ATP hydrolysis by divalent cations  11  Variations in Lineweaver-Burk plots of ATPase  . . . .  52  2+ a c t i v i t y at various concentrations of ATP, Ca vi i and Mg  53  2+ 2+ Enzyme activation by Ca and Mg  55  2+  12  viii Fi gure 13 14  Page The effect of incubation  time on Pi release and pH  of the incubation medium  56  Protein concentration effect on Pi release  58  +  2+  15  Effect of Na on enzyme activation by Ca  59  16  Effect of pH on enzyme a c t i v i t y  60  17  Effect of inhibitors  62  18  2+ Effect of mersalyl on Ca activation of the enzyme  ...  63  2+  19  Effect of EDTA on enzyme activation by Ca 2+  64  2+  20  EGTA effect on Ca  and Mg  21  The effect of blocking NH  22  Substrate s p e c i f i c i t y :  + 3  activation of the enzyme. . .  65  groups with maleic anhydride .  67  Hydrolysis of ATP compared to  that of other high energy t r i - and diphosphate nucleotides  68  23  Temperature effect on enzyme a c t i v i t y  70  24  Postulated scheme for ATP hydrolysis i n the presence of C a  2+  and Mg  80  2+  25  The effects of C a  26A  The effect of changing the concentration ratio of Mg  2+  to C a  2+  2+  and Mg  2+  on ATP hydrolysis  on ATP hydrolysis  86  26B  The change in -K^ as a function of [Mg ]/[Ca ]  27  A three dimensional model describing the relationship 2+ 2+  2+  between [Mg  85  86  2+  ]/[Ca ] and the a c t i v i t y of the placental  Ca -ATPase The elution p r o f i l e of alkaline phosphatase and protein on sephadex G-200 gel f i l t r a t i o n The effect of pH on ATP hydrolysis by the purified alkaline phosphatase  88  2+  28 29  96 .  97  ix Figure 30  Page The effect of pH on p-nitrophenyl phosphate hydrolysis by the purified alkaline phosphatase  31 32 33  34A  34B 35A 35B 36  The effect of storage at 4°C on enzyme a c t i v i t y  98 100  2+ Ca -ATPase s p e c i f i c a c t i v i t y as function of embryo age. . 102 The effect of the major constituents of the incubation 45 2+ -1 40 2+ medium on Ca displacement by 3 x 10 M Ca .... I l l 2+ The effect of incubation time on Ca -uptake and ATP hydrolysis 112 2+ The relationship between Pi release and Ca -uptake by the placental plasma membrane vesicles 114 2+ 2+ The effect of Ca concentration on Ca -uptake 115 2+ The effect of Ca concentration on Pi/Ca ratio 115 2+ 2+ The effect of Ca concentration on Ca -binding and 2+ on the velocities of ATP hydrolysis and Ca uptake . . . 126  LIST OF PLATES  Plate 1 2  Page Electron microscopic picture of the trophoblastic layer of guinea pig placenta Detail of placental membrane between the maternal blood space and the fetal capillary  3 4 5  ^ 7  Electron microscopic picture of the f i n a l membrane preparation (x 24,282)  20  Electron microscopic picture of the f i n a l membrane preparation (x 61,560)  21  Histochemical localization of ATPase a c t i v i t y i n the guinea pig placenta  51  x  ACKNOWLEDGMENT  The assistance of a number of people contributed to the accomplishment of this thesis.  F i r s t , I would l i k e to thank my research super-  visor, Dr. D. H. Copp, for the interest he showed in the subject and for the continuous encouragement which made the conclusion of this study possible.  The help of Dr. Harold Messer in editing this thesis, and  the helpful advice he provided me throughout the entire period, i s greatly appreciated.  I thank Dr. C. F. Cramer for his responsiveness while acting  as the chairman of my research committee. The just c r i t i c i s m of Dr. V. Palaty, which was done in the t r a d i tional manner of a good teacher, contributed to reaching a higher scient i f i c 1evel.  I am particularly indebted to Dr. I. C. Radde for introducing me to the subject and guiding me in the early stages. The good and encouraging atmosphere which was provided by a l l the people in the laboratory was greatly appreciated. Special thanks are due to Kurt Henze and Ralph Assina for the preparation of the thesis i l l u s t r a t i o n s . F i n a l l y , I wish to thank my wife, Chava, for her help, understanding and love, for without these I could not have made i t .  xi  GENERAL INTRODUCTION  Calcium ion has important roles associated with c e l l u l a r function.  These include: 1. Coupling between excitation - secretion at nerve endings (Eccles, 1964). 2. Coupling in excitation - contraction in muscle (Ebashi and Endo, 1968; Bianchi, 1969). 3. Maintenance of membrane integrity (Poste and A l l i s o n , 1973). 4. Membrane fusion. Thus a l l c e l l u l a r events which include a stage of fusion of membrane are calcium dependent (Poste and A l l i s o n , 1973).  These include cell fusion, endocytosis, exocrine (Hagen,  1959) and endocrine secretion (Hales and Milner, 1968; Curry et al_., 1968) (exocytosis). 2+ 5. Regulation of enzyme a c t i v i t y .  Ca  acts as an inhibitor of a  large number of i n t r a c e l l u l a r enzymes while i t serves as an activator for only a few of them (Bianchi, 1968). 6. Control of hormone secretion (Copp, 1970). Calcium ion concentration in the cytosol i s estimated to be in the range of 10~ M-10" M (Hodgkin and Keynes, 1957; Nanninga, 1961), _3 in contrast to 10 M in extracellular f l u i d . The necessity for low 5  8  2+ Ca  concentration in the cytosol was explained some years ago as follows:  • high Ca  in the cytosol may react1 with the high (as then believed)  2 inorganic phosphate i n the cytosol to form a calcium phosphate precipitate, which w i l l impair c e l l function (Manery, 1969).  This argument  l o s t much of it's basis with the finding that inorganic phosphate concentration in the cytosol i s very low (Seraydarian et al_., 1961). Some calcium phosphate precipitates can be found i n cell organelles which 2+ are involved in maintaining low Ca concentration i n the cytosol (Martin 2+ and Matthews, 1970; Borle, 1973).  By examining the roles for Ca  in  c e l l u l a r function, one can j u s t i f i a b l y conclude that calcium ion i s a powerful regulator of c e l l function, and for this reason i t s i n t r a c e l l u l a r concentration i s controlled very closely at a low l e v e l .  The extracellu-  lar f l u i d i s the most obvious "sink" for disposal of the excess i n t r a c e l l u l a r calcium.  The best available description of such a regulatory  mechanism i s that of the red blood c e l l (Vincenzi, 1971). 2+ 2+ this model, a membrane bound enzyme, namely a Ca -Mg  According to  -ATPase, i s respon-  sible for the active extrusion of calcium from the red blood c e l l .  Since  red blood c e l l s i n circulation contain no organelles, the plasma membrane "calcium pumps" i s the only effective mechanism regulating i n t r a c e l l u l a r Ca 2+ concentration. Though Ca2+ -ATPase i s found i n plasma membranes of several other tissues (Martin et al_., 1969; Parkinson and Radde, 1971; 2+ Ma e_t al_., 1974), i t s involvement i n Ca  extrusion i n those tissues  remains speculative at this stage. Unlike the red blood c e l l , a l l other c e l l s are equipped with 2+ organelles which can respond effectively to changes in cytosol Ca 2+ concentration, accumulating or releasing Ca as needed. The most e f f i 2+ cient c e l l organelle i n controlling cytosol Ca i s the sarcoplasmic  reticulum (Martonosi and Feretos, 1964). implicated.  2+ Once again Ca -ATPase i s  However, sarcoplasmic reticulum i s unique to muscle tissue.  The less specialized endoplasmic reticulum (microsomal fraction) i s 2+ 2+ also capable of accumulating Ca (Alonso and Walser, 1968). This Ca accumulation involves ATP hydrolysis. The mitochondrion i s another 2+ c e l l u l a r organelle which accumulates Ca (Reynafarje and Lehninger, 1969) 2+ The fate of the accumulated Ca within the c e l l u l a r organelles and the way i t i s f i n a l l y extruded from the c e l l i s not clear. One can speculate that at least the microsomes can fuse with the plasma membrane and secrete their contents into the extracellular f l u i d s .  Alternatively  2+ Ca  uptake by the c e l l u l a r organelles may become important only when  the plasma membrane calcium pump i s unable temporarily to handle a large 2+ influx of Ca . Thus the c e l l u l a r organelles act as a "buffer" mechanism. Borle (1973) proposed a model of c e l l u l a r calcium regulation in which 2+ Ca  influx i s passive and the mitochondria act as the main regulator  of cytoplasmic calcium a c t i v i t y and of calcium transport. He estimated that the efficiency of the plasma membrane calcium same of that of the mitochondria.  pump i s about the  However, since the surface area of  the mitochondria is so much larger than that of the plasma membrane, 2+ the contribution of the mitochondria to c e l l u l a r Ca regulation is 97% where the plasma membrane contributes only 3%. Thus i n this model there 2+ are three major parameters: passive Ca i n f l u x , active uptake by the 2+ mitochondria which acts as an ion buffer, and active extrusion of Ca by the plasma membrane calcium pump. Hormones affecting calcium metabolism  4 (PTH, Calcitonin and Vitamin D Metabolite 1-25 DHCC) can act by modifying the three parameters that regulate the exchange between the cytos o l , the mitochondria and the extracellular f l u i d . 2+ The importance of maintaining i n t r a c e l l u l a r Ca at a low level 2+ creates special problems in transport of Ca from one body compartment to another across a c e l l u l a r barrier, since this must occur without 2+ increasing s i g n i f i c a n t l y the Ca concentration in the cytosol of the 2+ c e l l s involved.  Such transport, which involves a net transfer of Ca  in one direction (asymmetric), occurs in the gut, bone, kidney, and placenta. The placenta provides a suitable system for studying calcium transport from one body compartment to another.  I t was chosen as the  subject of this study for the following reasons: 1) By the 60th day of gestation the placental barrier consists of fetal endothelial and trophoblastic layers.  I t may be assumed that the  l a t t e r i s involved in active transport of calcium. 2) It is easy to isolate significant quantities of plasma membranes primarily of the trophoblast. 2+ 3) In many mammalian species transfer of Ca is asymmetrical  across the placenta  (net transfer from dam to fetus) and occurs against a  concentration difference (Papadopoulos e_t al_., 1967; Macdonald et a l . , 1965; Twardock and Austin, 1970). 4) This concentration difference can be easily measured. 2+ 5) Because of the large amounts of Ca  required by the fetus (Comar,  1956), i t is possible that the transport system i s operating at high velocity.  5 6) In vivo studies (Papadopoulos et al_., 1967; Macdonald et a l . , 1965; Twardock and Austin, 1970) have suggested the presence of an active 2+ Ca transport across the placenta. Comar (1956) estimated the net 2+ hourly transfer of Ca  from dam to fetus to be 7% of the total maternal  plasma calcium i n the human, 50% i n the cow, 100% i n the rabbit and 400% i n the guinea pig. In these four species the stress placed on calcium homeostatic mechanisms by pregnancy i s far the greatest i n guinea pig and the least in man.  This fact and the s i m i l a r i t y i n the structure of the guinea  pig placenta to that of man (both are c l a s s i f i e d as haemochorial), made the guinea pig placenta very attractive for use as a model. The structure of the guinea pig placenta as shown by electron microscopy (Bjorkman, 1970) i s presented in Plates 1 and 2. They show that the maternal and the fetal circulation are separated by a continuous layer of syncytiotrophoblast. This would appear to be the logical s i t e 2+ for active Ca transport across the placenta. However, the obvious limitations of i n vivo studies preclude answers concerning the transport 2+ of Ca at the c e l l u l a r l e v e l . 2+ Since Ca transport occurs across the trophoblastic layer, i t is assumed that the plasma membranes should demonstrate some calcium2+ related properties which could assist i n Ca transport. 2+ The subject of this thesis i s to characterize the Ca -related properties of the placental plasma membranes and to try to u t i l i z e them 2+ 2+ in understanding Ca transport, i n the same way that Ca -related properties of the sarcoplasmic reticulum (Martonosi and Feretos, 1964),  Plate 1.  Survey picture showing the great variation i n thickness of the trophoblastic layer (Tr) and i t s relation to the foetal c a p i l l a r i e s (FCp). Note the m i c r o v i l l i (Mv) projecting into the maternal blood spaces (MS). Em. x 3600.  7  Plate 2. Detail of placental membrane between a maternal blood space ( M S ) and a foetal capillary (FCp). The intervening layers comprise a simple layer of trophoblast (Tr), a basal lamina (BL), and foetal endothelium (FE). The trophoblast appears to be highly differentiated with a well developed rough surfaced endoplasmic reticulum and infoldings of the basal plasma membrane (BI). Em. x 23,300.  8 mitochondria  (Reynafarje and Lehninger, 1969), and red blood cell  (Schatz-  mann and Rossi, 1971) membranes contributed to understanding of i n t r a 2+ c e l l u l a r Ca regulation. Since the sarcoplasmic reticulum has been 2+ most intensively studied with respect to i t s Ca -related properties, i t often serves as a reference i n this thesis. However, one should keep 2+ 2+ in mind that unlike the placental Ca transport, the sarcoplasmic Ca transport i s symmetric and i s exposed to large changes i n velocity i n a very short time as part of i t s role i n contraction-relaxation of muscle. 2+ Three Ca -related properties of the placental plasma membranes 2+ were chosen for study: Ca binding to the membranes, membrane bound Ca 2+ activated ATPase, and Ca2+ uptake by the placental plasma membrane vesicles. This thesis i s divided into three divisions, and the studies 2+ on each of the three Ca -related properties are described and discussed in separate divisions. The reasons for choosing these properties are given in the introduction to each division. 2+ To date there has been no f u l l description of these three Ca related properties of any plasma membranes involved i n asymmetrical 2+ Ca gap.  transport.  Therefore the experiments were designed to f i l l this 2+  An attempt to relate these properties to Ca -transport i s made  in the general conclusion of the thesis.  DIVISION I CALCIUM BINDING TO THE PLACENTAL PLASMA MEMBRANES  9  CHAPTER I CALCIUM BINDING TO THE PLACENTAL PLASMA MEMBRANES  INTRODUCTION The present concepts of the mechanism of active transport require that a preliminary and essential step for calcium transport i s the binding of the ion to the membrane involved. This binding i s assumed to be passive.  A knowledge of the number of calcium-binding s i t e s , their  s p e c i f i c i t y , relative a f f i n i t i e s , and capacity i s essential to an understanding of this process (binding to the membrane) and i t s relationship to active transport. Perhaps the most important property i s the s p e c i f i c i t y of the site for the ion to be transported.  The a b i l i t y of the membrane to  transport ions selectively i s dependent on the s p e c i f i c i t y of the s i t e . The best described s e l e c t i v i t y i n biological membranes, i s that between Na  +  and K . +  The "Sodium Pump" system has specific sites for Na on the +  inner surface of the c e l l membrane and specific sites for K on the +  outer surface of the c e l l membrane. The apparent a f f i n i t i e s of each site are at least 50 times higher for the one ion than for the other (Garay and Garrahan, 1973).  If we exclude the monovalent cations as  2+ competitors for the Ca s i t e , the only divalent cation whose pnysiological 2+  concentration enables i t to act as a competitor for Ca 10  2+  i s Mg .  11  The driving force underlying s p e c i f i c i t y , i s the difference between the free energy of cation-site electrostatic interaction and 2+ 2+ the free energy of hydration of the cation. When Ca and Mg are present in the medium in equimolar concentration, the distribution of 2+ 2+ the occupancy of the sites by Ca and Mg i s dependent on the ratio between the -AG (free energy) for the corresponding reactions. The free energies of the reactions can be estimated from the following equations.  2+ For Ca : AG  where  A  ^jca  =  Ca t  '  =  i e  AG  I C a " HCa AG  c n a n  9  e  ^  i n  9 Y of the system after the 2+  r e e e n e r  interaction of Ca Ca  2+  + X  with the s i t e (x)  '  Ca-X 2+  and  AG  HCa  =  t h e  c n a n  9  e  "  1  n t n e  f r e e  is hydrated Ca + n H 0 " 2+  2  e n e r  9 y °f the system when Ca Ca  2+  (HgO) n  2+ Since Ca  i n aqueous solution i s mainly hydrated, i t must f i r s t be  dehydrated (+AG^) before i t can interact with the s i t e .  I f the AG of  the overall reaction is negative, i t w i l l proceed spontaneously. (AG Mg2+ can be calculated in the same way as for Ca2+ .) In the presence 2+ 2+ of both Mg  and Ca  in equimolar concentrations, the f i n a l ratio of  calcium-occupied sites (Ca - X) to magnesium-occupied sites (Mg - X) w i l l be as follows: Ca - X  Mg - X  - Ca A G  2 +  -AG 2+ Mg  12 Since the diameter of C a  2+  (0.99 A°) i s larger than that of Mg  i t s dehydration energy wi11 be smaller.  2+  (0.65 A ) 0  Since the dehydration energies  are constant under physiological conditions, the only way for the c e l l to increase i t s s e l e c t i v i t y i s by construction of a specific s i t e . Since 2+ 2+ the charge density of Ca i s smaller than that of the Mg , i t w i l l react selectively with sites with low f i e l d . Strong f i e l d s i t e (weak 2+ acid) w i l l react favorably with Mg (Diamond and Wright, 1969). 2+ In this chapter, the Ca -binding properties of the placental 2+ plasma membrane are described.  Their possible relationship to Ca  transport i s dealt with i n the general conclusion. 2+ The most popular technique for measurement of Ca -binding i s ultrafiltration.  However, in the present study some uncontrolled non-  specific binding to the f i l t e r was found. For example, i t was observed 2+ that the binding of Ca to the f i l t e r was time dependent. Thus with increasing membrane concentration of the f i l t e r e d sample, the f i l t r a t i o n 2+ time increased as did the uncontrolled non-specific Ca -binding to the filter.  Because of these d i f f i c u l t i e s , a method based on the measurement  of the rate of diffusion in a flow d i a l y s i s system was used (Colowick and Womack, 1969).  This method has not previously been used for measuring  2+ Ca  -binding, so i t had to be modified as described i n the methods section  of this chapter.  The advantages of the flow dialysis method are given  in the discussion.  13 MATERIALS AND METHODS Isolation of Placental Plasma Membranes The method of Post and Sen (1967) for the isolation of renal plasma membranes was adapted for this study of placental plasma membranes. The purity of the final preparation was checked by electron microscopy and by enzyme markers to ensure that the method was suitable for isolation of the placental plasma membranes. Pregnant guinea pigs around the 60th day of gestation were anaesthetized with Na pentobarbital (100 mg/kg). The abdomen was opened and the amniotic sacs with the fetuses were removed.  Each placenta was  carefully freed from membranes, gross blood vessels and uterine tissue and placed in ice-cold 0.9% NaCl solution.  Using a Thomas tissue grinder,  size C with Teflon pestle, for 10 strokes at 1500 rpm,the placentas were homogenized i n 25 ml of a solution containing 87 g sucrose, g NaCl, 1.860 litre.  1.169  g Na^EDTA, 0.2 g MgCl 6 H 0 and 0.68 g imidazole per 2  2  s  The tissues were then processed i n a manner similar to that  described by Post and Sen (1967) for the isolation of renal plasma membranes, omitting the urea stage.  The procedure consists of a series  of centrifugations at 35,000 xg for 30 min in different solutions, and is given in detail in the accompanying flow chart. were f i n a l l y suspended in 5 x 10~ M 4  The isolated membranes  imidazole-histidine, 5 mM Tris-HCl  buffer (pH 7.6) and stored at +4°C until assay.  This procedure for  isolating the placental plasma membranes was used throughout the study except in certain cases, when i t was modified to serve different purposes.  14 Isolation of Placental Plasma Membranes  HOMOGENATE -centrifuged at 300 x g for 10 min c e l l u l a r and nuclear pellet (discarded)  supernatant centrifuged at "35,000 x g for 30 min  Particle free supernatant (discarded) •Microsomal fraction. The upper layer was scraped free and transferred to 25 ml solution (1), homogenized and centrifuged at 35,000 x g for 30 min. This procedure was repeated 3 times, in solutions (2), (3), and (4), to reduce progressively contamination by mitochondria.  I  Mitochondrial pellet (discarded)  The final microsomal fraction was suspended in 5 x 10" M imidazole-histidine, 5 mM Tris-HCL pH 8.0 and stored at +4°C. Composition of Solutions Used in Isolation of the Plasma Membranes: (1)  (2)  0.25 M Sucrose 0.02 M NaCl 5 mM Na2EDTA 1 mM MgCl 10 mM Imidazole 2  0.25 M Sucrose 2 mM Na?EDTA 0.1 mM MgCl2 4 mM Imidazole 0.02% (W/V) Na-Heparin  (3)  (4)  15 mM NaCl 1 mM Na EDTA 3 mM Imidazole 2  10 mM Imidazole 0.1 mM Na2EDTA  15 Determination of Membrane Concentration The protein concentration of the final preparation was taken as an indicator of membrane content in the medium. The total protein was measured by the procedure of Lowry ejt al_. (1951) using bovine albumin standards.  A l l the results are expressed per mg of membrane protein.  Enzyme Marker Assays The supernatants from the 35,000 x g centrifugations were pooled, as were the pellets.  These fractions and the f i n a l preparations were  assayed for alkaline phosphatase, glucose-6-phosphatase and succinate dehydrogenase. Alkaline Phosphatase (EC 3.1.3.1) Alkaline phosphatase as a marker for plasma membranes (Dixon and Webb, 1964a) was assayed by the method of Bessey et al_. (1946) as modified by Kelly and Hamilton (1970). Aliquots (50 y l ) from the different fractions were incubated for 30 min at 30°C i n 2-amino-2-methyl-l-propanol buffer (Eastman Organic Chemical) 0.75 M pH 10.0 using 5 mM disodium p-nitrophenyl phosphate (Sigma) as substrate. ml.  The final volume was  0.55  The reaction was terminated by adding 5 ml of 0.05 N NaOH, and p-  nitrophenol release was determined spectrophotometrically by reading absorbence at 410 nm, compared to a standard p-nitrophenol curve. Blank samples (in the absence of enzyme) were incubated under the same conditions.  16 Glucose-6-Phosphatase (EC 3.1.3.9) Glucose-6-phosphatase i s a predominantly microsomal enzyme (Ginsburg and Hers, 1970) and was used in this study as a marker for the assessment of the purity of the subcellular fractions. tested as described by Hiibscher and West (1965). ture contained:  The enzyme was  The incubation mix-  43 mM maleate buffer (pH 6.0), 2 mM KF, 4 mM EDTA, 28.5  mM glucose-6-phosphate as substrate and approximately 1 mg protein from the different fractions.  The final volume was 0.7 ml and i t was incu-  bated for 15 min at 37°C. The reaction was terminated with 1.3 ml 10% (W/V) TCA and the amount of inorganic phosphate liberated was measured by the G0M0RI method (Gomori, 1942) using the auto analyzer (Technicon). The Pi released in the absence of protein was subtracted before calculating the specific a c t i v i t y . Succinate Dehydrogenase (EC 1.3.99.1). Succinate dehydrogenase i s suitable as a marker for mitochondria (Dixon and Webb, 1964a). The a c t i v i t y was measured as described by Pennington (1961) using sodium succinate (50 mM) as substrate, INT  0.1%  [2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Eastman) as hydrogen acceptor i n 50 mM potassium phosphate buffer pH 7.4 in the presence or absence of aliquots (0.1 ml) from the different fractions.  The samples (final volume 1 ml) were incubated for 15 min  at 37°C and the reaction terminated with 1 ml 10% (W/V) TCA.  The for-  mazan was extracted with 4 ml ethyl acetate and i t s absorbence measured at 490 nm.  Non-enzymatic reduction of INT was measured and subtracted  from the total a c t i v i t y .  17 Electron Microscopic Examination of the Final Preparation An aliquot from the final preparation was centrifuged at 90,000 x g for 10 min.  The pellet was fixed in phosphate-buffered  osmium tetra-  oxide (1%), dehydrated, and embedded in Epon. The sections were stained with uranyl acetate and lead c i t r a t e .  Evaluation of the Method Marker Fnzymes Table I summarizes the distribution of marker enzymes. specific a c t i v i t y of a l l the enzymes in the supernatant was low.  The Glucose-  6-phosphatase and alkaline phosphatase were distributed i n the same ratio of 1.1 between the "final preparation" and the p e l l e t , indicating that the final preparation is truly a microsomal fraction.  The high specific  a c t i v i t y of the two phosphatases in the pellet is due to incomplete recovery of the heavier plasma membranes fragments and microsomes. However, the distribution of succinate dehydrogenase showed a ratio of 0.1, indicating that the "final preparation" contains very few mitochondria. No attempt was made to separate the plasma membranes from the microsomes. The electron microscopic picture of the trophoblastic layer (plates 1, 2) showed a continuous network of endoplasmic reticulum with the plasma membranes which project into the maternal circulation as micro-vil1i. Thus, following homogenization, both form fragments with the same density and s i z e , which together comprise the microsomal fraction. 2+ was used for studying the Ca components.  This fraction  -related properties of these placental  18  TABLE I. Specific a c t i v i t i e s of marker enzymes in different fractions, and ratio between the specific a c t i v i t y found in the final preparation (F.P.) over the specific a c t i v i t y found in the pellet.  Alkaline * Phosphatase  Glucose-6- * Phosphatase  Succinate Dehydrogenase**  Supernatant  0.22  0.015  0  F.P.  2.35  0.688  0.398  Pellet  2.05  0.602  3.76  Ratio F.P./Pellet  1.14  1.14  0.105  * Activity of Phosphatases (ymole Pi/mg protein per 30 min)  **  O.D./mg protein/ 15 min.  19 Electron Microscopic Examination The electron microscopic examination of the final preparation revealed no contamination with mitochondria confirming the results with the enzyme markers.  The preparation consisted mostly of plasma membranes  in the form of vesicles and fragments (Plates 3 and 4).  Calcium binding assay The calcium-binding properties were investigated i n a flow dialysis system based on that described by Colowick and Womack (1969). The method i s based on the principle that the rate of diffusion i s proportional to the concentration of the free d i f f u s i b l e molecule; this rate w i l l be constant when equilibrium i s achieved with the macromolecule in the upper chamber of the d i a l y s i s c e l l .  Equilibrium w i l l be reached  within seconds, and a constant diffusion rate w i l l occur within 1.5-2 minutes when the effluent volume pumped through the lower chamber i s about 5 times i t s volume (Colowick and Womack, 1969). The dialysis c e l l (Figure 1) was prepared as described by Colowick and Womack (1969) using a standard cellophane dialysis membrane 45 (Fisher S c i e n t i f i c Company).  The placental plasma membranes and  Ca  (New England Nuclear) were premixed, and the pH adjusted to 8.0 i f necessary, before addition to the upper chamber. A l l experiments were con45 2+ ducted at room temperature (24°C).  Initial  Ca  concentration was  1.5 x 10" M (approximately 0.04mCi/ml i n f i n a l reaction mixture) unless 5  indicated d i f f e r e n t l y .  l_  Plate 3.  —  The final membrane preparation (x 24,282).  21  Plate 4.  The final membrane preparation (x 61,560).  22  •  •  •  •  • —  4 5  Ca  2 +  •  •  • • • . T T T  •  —  Ca-Mem  *Mem  DIALYSIS MEMBRANE  ^  —'  ff  S  r  | A A A A A A A A A A A A A A A A A A A A A A |  f UUU FRACTION COLLECTOR  Purrfp  r  BUFFER  Figure 1. Diagram of the apparatus used for measuring calcium binding by rate of d i a l y s i s .  23 The dialysis buffer contained 20 mM Tris-HCl (pH 8.0) and 100 mM NaCl. The NaCl concentration was raised to 100 mM (as suggested by Reed (1973)) in order to prevent an excessive non-specific binding to the d i a l y s i s membrane. The flow rate was maintained at 8 ml/min and the effluent was collected in 2 ml aliquots with a fraction collector. One ml samples were added to 10 ml s c i n t i l l a t i o n f l u i d (aquasol-New England Nuclear) and were counted i n a Beckman model LS-233 l i q u i d scint i l l a t i o n counter. Protein was determined by the procedure of Lowry et aV. (1951). Calculation of calcium binding The difference between the control diffusion rate without plasma membranes and the diffusion rate observed in the presence of plasma 2+ branes represents the fraction of bound Ca  mem-  i n the medium. However,  the control diffusion rate i s not constant because d i l u t i o n occurs with each addition to the upper chamber, and because of cpm ( C a ) loss 45  in the effluent during the experiment.  2+  For these reasons the i n i t i a l  control diffusion rate must be corrected for dilution and cpm loss for each step.  The d i l u t i o n effect can be duplicated i n the control simply  by adding the same volumes to the upper chamber without plasma membranes present.  The cpm loss cannot be duplicated because differences i n the  diffusion rates i n the presence and absence of plasma membranes produce different rates of loss from the upper chamber. For each experiment, the loss of cpm for each step was calculated from the cpm found in the effluent collected i n each step.  These  figures were then subtracted from the i n i t i a l cpm in the upper chamber  24 to give actual cpm concentration for each step.  The total loss of cpm  during experiments of 25-30 min, with a flow rate of 8 ml/min was up to 10%.  The control diffusion rate for each step was calculated by  multiplying the experimental i n i t i a l control diffusion rate by the d i l u tion factor and by the cpm loss factor. C.D.R. = I.C.D.R. x D.F. x Cpm.L.F. Where:  C.D.R. = Control Diffusion Rate, I.C.D.R. = I n i t i a l Control Diffusion Rate at time zero in the absence of plasma membranes, D.F.  = Dilution Factor (due to volume increase with each addition to the upper chamber),  and  Cpm L.F.  = Cpm Loss Factor (due to loss of Cpm from the upper chamber to the effluent).  Thus:  % Ca  2+  bound  =  C  -  D ,  ^ " °- ' D  D  R  R  x 100  Where O.D.R. = Observed Diffusion Rate i n the presence of plasma membranes.  Calcium Binding by Bovine Albumin 2+ For comparison, Ca -binding by Bovine Albumin (Fraction V, Armour Pharm. Comp.) was also studied, under the same conditions as described for the placental plasma membranes.  I t i s assumed that Bovine 2+  Albumin i s representative of a class of proteins which do not bind Ca specifically.  25 RESULTS Evaluation of the Method 2+ The s u i t a b i l i t y of the flow dialysis method for Ca -binding 45 2+ studies i s demonstrated i n Figure 2. The addition of  Ca  to the  upper chamber i n the absence of plasma membranes (Figure 2, upper curve) caused a linear increase i n diffusion rate (Figure 3), suggesting that 45 2+ only a small fraction of  Ca  was bound. The lower curve i n Figure 2  represents the results obtained under the same conditions but i n the presence of plasma membranes i n the upper chamber. The difference i n the diffusion rates i n the absence and the presence of plasma membranes 45  2+ is due to Ca binding by the placental plasma membranes. 40 2+ -2 The addition of a large excess of Ca (10 M) i n the absence of plasma membranes produced a sharp spike i n the diffusion rate as a 45 2+ 40 2+ result of Ca displacement from the dialysis membrane by Ca . However the amount bound was negligible (0.1% of the total C a present) 4 5  2 +  and after a few samples the diffusion rate returned to the same level 40 2+ as before the addition of  Ca  . In the presence of plasma membranes,  40 2+ -2 Ca (10 M) increased the diffusion rate to the level of the 45 2+ control, indicating complete displacement of Ca from the plasma membranes. The absence of a spike i n the presence of plasma membranes 45 2+ is due to a reduced binding of Ca to the d i a l y s i s membrane because 45 2+ of the higher a f f i n i t y of the plasma membranes for the available Ca . excess  26  cpm  x10~3  5 .  44  3J  C O N T R O L without membranes anes I ^  [  *  v  40c  | 4.7x10- M Ca 7  3.8"10" M  45  2<  J  I  7  27-10 7»10  /'  <'i  ri  /  M  >' with membranes  •  t  10  1 20  7 4^ ?• 1.5x10"'M Ca^ 4 5  1  1  30  40  SAMPLE  r50  1  60  r— 70  -i— 80  NO.  Figure 2. Measurement of Ca diffusion rate at various Ca concentrations • • without plasma membranes • • with plasma membranes. The medium and dialysis buffer contained 20 mM Tris-HCl (pH 8.0), 100 mM NaCl. Protein concentration was 2.52 mg/ml, the effluent was collected i n 2 ml fractions of which 1 ml was counted for 45ca. Each step was allowed 15 samples before i n creasing 45rj 2+ concentration. The results are expressed in cpm/ml effluent. a  27  Cpm/ml  0  Effluent  0.5  1.0  1.5  C p m x l O ~ / m l . o f upper c h a m b e r 6  Figure 3.  "1  2.0  fluid  Measurement of Ca diffusion rate as a function of cpm concentration in the upper chamber. The data presented here were obtained from Figure 2 (upper curve).  28 A typical 4.  45  Ca  2+  diffusion rate p r o f i l e i s presented i n Figure  The control diffusion rate after the f i r s t step (samples 1-15) was  45 2+ corrected for dilution and loss of Ca , as described i n the Methods section. For a l l experiments, the data were f i r s t plotted in this manner, 2+ to allow calculation of the extent of Ca  -binding for each step.  The  2+ Ca  -binding was then replotted against the different parameters used  in subsequent experiments. Calcium Binding by Placental Plasma Membranes 2+ -7 -7 At low Ca concentrations (10 -5 x 10 M) calcium binding per mg protein was linear (Figure 5A: data derived from Figure 2), i n d i cating a constant percentage bound i n this concentration range.  In a  double reciprocal plot the l i n e passed through the o r i g i n , suggesting non-saturable  kinetics. O  At higher Ca  i  C  0_|_  O  concentration (10" -10~ M) the amount of Ca  bound per mg protein increased sigmoidally (Figure 5B). A Scatchard 2+ plot (Figure 6) of Ca  -binding revealed two types of s i t e s :  high af-  f i n i t y sites accommodating 2 5 + 2 (mean + S.E.) nmoles per mg protein -5 and dissociation constant 3.1 +_ 0.4 x 10  M; and low a f f i n i t y sites  accommodating 266 +_ 27 nmoles per mg protein and dissociation constant 1.1 + 0.1 x 10" M (Table I I ) . 3  Figure 4:  Flow dialysis profiles of calcium binding at various calcium concentrations: The medium and d i a l y s i s buffer contained 20 mM Tris-HCl (pH 8.0), 100 mM NaCl. At time zero 1.6 x 10"5M 45r, 2+ was added to the medium i n the presence or absence of plasma membranes. The lower curve represents the diffusion rate p r o f i l e of 45ca2+ i n the presence of plasma membranes (protein concentration 2.4 mg/ml) at various 40Ca2 concentrations, as indicated under the arrows. The upper curve i s a control curve in the absence of plasma membranes. Up to sample No. 16 the control curve represents experimental values. The remainder i s a corrected control curve compensated for dilution and cpm loss for each step. The flow rate was 8 ml/min, each step was allowed 8 samples and results are expressed as cpm per ml effluent. a  +  30  Log  [Ca  2 +  ]  Figure 5. Ca^ concentration effect on Ca^-binding levels by placental plasma membranes and Bovine Albumin. (A)Ca -binding at low C a concentration (1 • 10" -5 • 10 M). This curve was derived from Figure 1. The results are expressed i n nmoles C a bound per mg protein. (B)Ca2+ binding at higher Ca2 concentrations (1 -10-6-1 . 10" M). This curve was derived from four different experiments carried out under the same conditions as described i n Figure 2. The Ca2+ concentration was varied s l i g h t l y from one experiment to another to cover the wide range. The bound calcium i s expressed i n nmoles Ca2+ per mg protein. T  2+  2+  7  _7  2+  +  2  31  30CH  2 *t" Bound Ca Free  Figure 6.  (nmoles/mg Protein) Ca  2 +  (io- M) 5  Scatchard plot of Ca -binding by placental plasma membranes and Bovine Albumin (derived from the steady values which were used to construct Figure 5B).  32  Table II. Affinity and capacity of placental membranes and Bovine Albumin for Ca2+  K  Sl  *  n **  H  n ** 2  K  *s2*  Placental Plasma Membranes  c 3.1+0.4x10"^  26+2  o 1 .l+O.lxlO^M  266+27  Albumin  1.4+0.06x10" M  28+1  9.1+0.6xlO~ M  99+5  4  All the results are expressed as mean +_ S.E. * K = dissociation constant * 2+ nmoles Ca per mg protein s  4  33 Calcium Binding by Bovine Albumin The results are plotted in Figures 5-6 and summarized i n Table 2+ II.  Two types of sites for Ca  were revealed.  The high a f f i n i t y sites  have the same capacity as the high a f f i n i t y sites of the plasma membranes but the a f f i n i t y i s almost five-fold lower.  The low a f f i n i t y sites have  the same a f f i n i t y with only 40% of the capacity of the low a f f i n i t y sites of the plasma membranes. Time, Temperature, Protein Concentration and pH Effects The maximal diffusion rate was achieved after 1.5 min, and when no further additions or treatments were performed, the diffusion rate was constant (after compensating for cpm loss) for at least 30 min. Thus calcium binding by placental plasma membranes reached a maximum within 1.5 min, and thereafter was independent of incubation time. Reducing the temperature from 24°C to 4°C did not change s i g n i 2+ ficantly the extent of Ca -binding. 2+ The binding of Ca increased linearly with protein concentration 2+ -5 over the range 0.25-2.7 mg protein/ml when [Ca ] was 1.5 x 10 M (Figure 7). Calcium binding was dependent on pH, and practically no binding was detected at pH 4.0. The binding increased with increasing pH up to pH 11.0, and leveled o f f (Figure 8).  Since the membranes were sub-  jected to each pH for no more than 2.5 min, denaturation effects of extreme pH's were minimized; the pH effect was reversible over the entire pH range (3.0-11.8).  /  / 1  0  1  1  1 3  2  mg  1 4  r  5  Protein/ml  Figure 7. Ca binding as a function of placental plasma membrane protein concentration. The medium contained 20 mM TrisHC1 (pH 8.0), 100 mM NaCl, and 1.5 • 10" M 45ca . The plasma membranes were added to the upper chamber to give the indicated concentration. The results are expressed as total nmoles Ca2 bound. 5  +  2  35  40 J  Figure 8. pH effect on Ca*" binding. The pH i n the upper chamber was measured with a pH electrode, and controlled by addition of HC1 or NaOH (0.1 M). The plasma membranes were exposed to each pH for not more than 2.5 min. The medium contained 20 mM Tris-HCl, 100 mM NaCl 2.2 • 10"3M 45 2+ d protein concentration was 2.5 mg/ml. The results are expressed as total nmoles Ca2 bound. T  Ca  +  a n  36 2+  Ca  Displacement from Its Sites by Mg Figure 9A shows the  Mg  2+  2+  , and Sr  . The percent  4c  2+  and Sr  2+  Ca displacement from i t s sites by  45  Ca  2+  40  Ca  2+  ,  bound at the end of the f i r s t step 2+  2+  was regarded as zero percent displaced. The a b i l i t y of Mg and Sr 45 2+ 40 2+ to displace Ca from i t s sites was much lower than that of Ca (Figure 9A). 45 The double reciprocal plots for displacement of 9J-  AC)  OX.  2+ Ca  from the  94-  plasma membranes by Ca , Mg , and Sr (Figure 9B, C, D) revealed two apparent Km's for each ion. These values appear in Table I I I . The a f f i n i t y of Mg  2+  2+  2+  and Sr  for the high a f f i n i t y Ca sites was approxi40 2+ 2+ mately 10-fold lower than that of Ca . The a f f i n i t y of Mg for the 2+  high a f f i n i t y Ca  sites was higher than that of Sr  2+  . The wide gap of  a f f i n i t i e s was narrower for the low a f f i n i t y sites (4-fold lower for Mg 2+ and 8-fold lower for Sr 2+ ). These results indicate high s p e c i f i c i t y of these sites for calcium, with greater s p e c i f i c i t y i n the high a f f i n i t y sites.  DISCUSSION The flow dialysis method was found in the present study to be a 2+  very useful tool for studying Ca  -binding. The process of binding has  met the requirements for the use of this method as defined by Colowick and Womack (1969): seconds; predicted;  1) chemical equilibrium was achieved within a few  2) a constant diffusion rate was established after 1.5 min as 3) the dissociation constants of this binding reaction were  37  Figure 9A.  Ca displacement by divalent cations. (A) The experiments were carried out as described for Figure 2. 40ca2 , Mg2+, and Sr^+ were added i n increasing concentrations to the upper chamber as indicated. The results are expressed as a percentage of ^5r; 2+ displaced from the plasma membranes. Zero displacement i s defined as the amount of 45ca2+ bound at the end of the f i r s t step. +  a  38  Figure 9B, C and D. Double reciprocal plots for Ca displacement by C a , Mg , and S r , respectively. D i s expressed as percent 45ca2+ displaced and [S] as millimolar divalent cation. These plots were derived from data used in Figure 4 0  9A.  2 +  2+  2 +  39 A^ ?+ 40 2+ 2+ TABLE I I I . Apparent Kin's for Ca displacement by ™Ca , Mg . S r . The Km's were calculated from the double reciprocal plots presented i n Figure 9B-D. 1  2 +  ION 4 0  Ca  Mg Sr  2+  2 +  2 +  Krm (M+S.E.)  Km2 (M+S.E.  (2.2 + 0.3) x 10~ M  (2 + 0.2) x 10" M  (1.4 + 0.15) x 10~ M  (9 + 1) x 10~ M  (2.2 + 0.25) x 10" M  (1..5 +0.1) x 10" M  4  3  3  3  3  2  40 -3  -6  within the effective range of the method (10" -10" M). The use of 45 2+ Ca as the d i f f u s i b l e molecule, which has a relatively high diffusion rate through the dialysis membrane, made this system sensitive to changes in free  45 2+ Ca i n the upper chamber. 2+ The flow dialysis system offers several advantages for Ca -  binding measurements over equilibrium dialysis and u l t r a f i l t r a t i o n . It i s faster than the equilibrium dialysis method, since each step can be completed within 2 minutes.  Washing the membrane, which i s necessary  in the u l t r a f i l t r a t i o n method, i s avoided here; this eliminates a possible 45 displacement of  Ca, particularly from the low a f f i n i t y s i t e s . 45 2+  continuous monitoring of the  Ca  The  diffusion rate gives a clearer picture  of the kinetic behavior of the investigated reaction. In studying pH effects, the membranes are exposed to each pH for not more than 2.5 min. Thus denaturation effects by extreme pH's can be minimized.  Using the  other methods, the exposure to each pH i s much longer and denaturation 2+ effects cannot be excluded.  However i n calculating Ca -binding using  the flow dialysis method two effects must be taken into account: dilution effect and cpm loss during the experiments.  the  These two factors  should be calculated for each step to give the actual control diffusion 2+ rate for each step.  An excessive nonspecific Ca -binding to the d i a l y s i s  membrane was successfully prevented by raising NaCl concentration up to TOO mM, as suggested by Reed (1973).  In contrast to the u l t r a f i l -  tration method the flow dialysis method (without modfication) i s not sensitive enough to measure calcium uptake.  Because uptake i s a rela-  tively slow process, the effect on the diffusion rate w i l l be small,  41 and d i f f i c u l t to detect.  Another limitation of this method i s that i t  requires high protein concentrations (2-3 mg/ml). 2+ Two types of Ca -binding sites can be distinguished on the placental plasma membranes: high a f f i n i t y sites with a capacity of 26 -5 nmoles per mg protein and K = 3.1 x 10 M,and low a f f i n i t y sites with -3 a capacity of 266 nmoles per mg protein and K = 1.1 x 10 M. Bovine Albumin which was used as a reference in this study, was also shown 2+ s  to possess two types of Ca  -binding s i t e s .  While the capacity of the  high a f f i n i t y sites for albumin was equal to that of the membranes, the a f f i n i t y was f i v e - f o l d lower (K  = 1.4 x 10~ M). It i s tempting to 2+ speculate that the higher a f f i n i t y for Ca of the placental plasma 2+ 4  sl  membranes may be related to a function in Ca  transport.  Membranes from several sources show more than one class of calciumbinding s i t e s .  The sarcoplasmic reticulum has been most intensively 2+  studied with respect to i t s Ca  -binding properties, but even with this  one type of membrane, different methods have yielded conflicting results. Cohen and Selinger (1969) reported two classes of sites with the same -5 dissociation constant (K = 4 x 10 g  M), while a more recent study by  Chevallier and Butow (1971) revealed three classes of sites with the following dissociation constants:  1.3 x 10~ M; 3.2 x 10~ M; 3.2 x 10" M. 2+ 6  5  Despite the d i f f i c u l t i e s one faces trying to compare Ca  4  -binding  properties of preparations obtained by the various methods, the existence of high a f f i n i t y sites i s typical of membranes that are involved in the active regulation of i n t r a c e l l u l a r calcium.  In addition to the sarco-  plasmic reticulum, high a f f i n i t y sites for calcium are found in the  42 mitochondria (Reynafarje and Lehninger, 1969) and in cardiac microsomes (Repke and Katz, 1972).  Lower a f f i n i t y sites are found in rat l i v e r  plasma membranes (Schlatz and Marinetti, 1972)  (K = 2.5 x 10~ M 4  g  and  3.1 x 10  M), and in red blood cell membranes (Gent e_t al_., 1964) (K = 2.8 x 10 -4 M). The placental plasma membranes, in respect to Ca 2+ s  -5 binding a f f i n i t y (K  g  = 3.1 x 10  M), ranks with the sarcoplasmic  culum, mitochondria and cardiac microsomes.  reti-  I t i s interesting to note  that l i v e r plasma membranes (Schlatz and Marinetti, 1972) have binding sites with a 10-fold higher dissociation constant and a  correspondingly  lower a f f i n i t y for calcium than those sites in the placental plasma membranes. It i s tempting to attribute this difference in a f f i n i t y for 2+ Ca to the fact that the l i v e r plasma membranes, unlike the placental 2+ plasma membranes, are not involved in active Ca  -transport between body  compartments. 2+ The pH-dependent Ca  binding profile by the placental plasma  membranes shows a pH optimum of 11.0, and at pH 4.0 there i s practically no binding.  Up to pH 7.0 the pH profile agrees with that already des-  cribed for l i v e r plasma membranes (Schlatz and Marinetti, 1972), sarcoplasmic reticulum (Cohen and Selinger, 1969), and muscle microsomes (Carvalho, 1966).  Above pH 7-8 a decrease was reported (Cohen and  Selinger, 1969; Gent et al_, 1964). was observed even above pH 11.0.  In the present study no such decrease These differences can be at least  partly accounted for by the different methods used.  The time of exposure  to each pH varied from 40 min (10 min incubation and 30 min centrigugation) (Cohen and Selinger, 1969) up to 42 hr (Shlatz and Marinetti, 1972) using the equilibrium dialysis method. The use of flow dialysis  43 reduced the exposure time to 2.5 min, minimizing long term pH effects. In addition, no attempt to reverse the pH effect was mentioned i n these studies (Cohen and Selinger, 1969; Shlatz .and Marinetti, 1972) so that long term effects cannot be excluded. The a f f i n i t y of Mg 2+ and Sr2+ for the Ca2+ high a f f i n i t y sites 2+ 2+ was found to be 10-fold lower than that of Ca . The binding of Mg 2+ and Sr was not studied, so that the p o s s i b i l i t y of specific sites f o r + 2+ these ions cannot be excluded. The effect of Na on Ca binding could not be studied i n detail since i t was necessary to maintain a high Na 2+ +  concentration to prevent nonspecific Ca -binding to the dialysis membrane.  However, i n several short experiments when NaCl (100 mM) was  added after the f i r s t step (before measurable amounts of ^ C a 4  2 +  were  bound to the d i a l y s i s membrane, which i s time dependent) (Reed, 1973) 45 2+ no significant changes i n Ca diffusion rate could be detected. The 45 2+ 2+ 2+ more e f f i c i e n t displacement of Ca by Mg and Sr from the low 2+ a f f i n i t y sites indicates a reduced s p e c i f i c i t y for Ca . 2+ The s p e c i f i c i t y of Ca -binding sites of different membranes shows wide variations. The sites on the l i v e r mitochondria (Reynafarje 2+ 2+ 2-1" and Lehninger, 1969) are insensitive to Mg , but Sr displaces Ca quite effectively.  The sarcoplasmic reticulum (Cohen and Selinger, 1969)  sites are unaffected by Mg  up to 10~ M, while Sr displaces at a level 2+ close to that found i n this study. The Ca -binding sites of another plasma membrane (derived from l i v e r ; Sclatz and Marinetti, 1972) are •f*  "4~  insensitive to K and Na while Mg a f f i n i t y sites only.  2*^~  24~  reduced Ca -binding to the low  The degree of s p e c i f i c i t y observed i n the present  44 study enables us to conclude that at physiological concentrations of Mg2+ , Ca2+ and normal levels of Sr2+ , practically no binding other than 2+ Ca  to these sites w i l l be detected. 2+ The concept behind this study i s that a passive binding of Ca  to the membrane i s an essential f i r s t step i n the process of an active 2+ Ca  transport.  The placental plasma membranes were found to contain  2+ sites for Ca with capacity, s p e c i f i c i t y and a f f i n i t y within the range 2+ reported for other membranes involved i n active transport of Ca . Unlike the sarcoplasmic reticulum and mitochondria, which are exposed 2+ to only two different Ca concentrations, the placental plasma membranes 2+ are exposed to three different Ca concentrations. The outer plasma 2+ membranes on the maternal side are exposed to a Ca concentration of 2.9 mequiv/L, and on the fetal side to 3.8 mequiv/L (Papadopoulos et a l , 2+ 1967). The inner surface of the membranes i s exposed to a low Ca -5 -6 concentration, estimated to be around 10 -10 M.  I t i s premature to  assign the distribution of the two classes of sites to the different surfaces of the placental plasma membranes. However, i t should be noticed that the dissociation constants obtained are close to the physio2+ logical concentrations of Ca to which the membranes are exposed. 2+ Although high a f f i n i t y specific Ca sites are present i n placental plasma membranes, the direct involvement of these sites i n active trans2+ port of Ca remains speculative at this stage.  45 SUMMARY The Ca-binding properties of placental plasma membranes were studied using a flow dialysis system. Ca-binding was not detectable at pH 4.0, but increased at higher pH's to a maximum binding at pH 11.0. Two types of Ca-binding sites were i d e n t i f i e d : high a f f i n i t y -5 sites with dissociation constant K = 3.1 x 10 M and a capacity of _3 26 nmoles per mg protein; low a f f i n i t y sites with K = 1.1 x 10 M and a capacity of 266 nmoles per mg protein. 2+ 2+ The a f f i n i t i e s of Mg and Sr for the high a f f i n i t y sites 2+ g  were 10-fold lower than that of Ca  , and for the low a f f i n i t y sites  were 4- and 8-fold lower respectively. 2+ The placental plasma membranes contain sites for Ca with capacity, s p e c i f i c i t y and a f f i n i t y within the range reported for other 2+ membranes involved i n an active transport of Ca plasmic reticulum, cardiac microsomes).  (mitochondria, sarco-  DIVISION II -STIMULATED ATPase OF THE GUINEA PIG PLACENTAL PLASMA MEMBRANES  46  CHAPTER II CHARACTERIZATION OF CALCIUMSTIMULATED ATPase  INTRODUCTION It has been proposed that calcium transport across the placenta is an active transport (Papadopoulos et al_., 1967).  Most energy-utili-  zing processes are driven by the breakdown of ATP (Dixon and Webb, 1964b). To be effective the hydrolysis of ATP must be enzymatic and coupled with the energy-utilizing process, i n this case, calcium transport. The coupling between the two events must take place within the membrane, so that the enzyme hydrolyzing the ATP i s fixed i n space; this makes possible the coupling between the chemical reaction (ATP hydrolysis) and translocation of the molecule (Curie-Prigogine principle) (Katchalski and Curran, 1967). Skou (1965) has established the relationship between Na and K + + 2+ fluxes and ATP hydrolysis by the membrane-bound (Na , K )-activated Mg dependent ATPase (EC 3.6.1.3).. Other membrane-bound ATPases activated +  +  by Ca2+ and Mg 2+ , and involved i n Ca2+ -transport, have been described in the red blood cell (Schatzmann and Rossi, 1971) and the sarcoplasmic reticulum (Martonosi and Feretos, 1964). 47  48 2+  2+  In this chapter the properties of such a (Ca , Mg )-activated ATPase located in the placental plasma membranes are described, and 2+ 2+ compared with the properties of other (Ca , Mg ) ATPases.  MATERIALS AND METHODS 2+ Histochemical Localization of Ca  -ATPase in the Placenta  The placenta, after removal, was frozen (to conserve enzymatic a c t i v i t y ) and sectioned (20p).  The sections were incubated for an hour  in 20 mM Tris-HCl buffer pH 8.5, 30 mM CaCl and 12 mM ATP.  The sections  2  were stained as described by McManus et_ al_ (I960).  The staining method  is based on precipitating the released Pi as calcium phosphate, replacing the calcium with cobalt by washing with 2% cobalt acetate and replacing the Pi with sulfide by placing the sections for one minute i n 2% aqueous yellow ammonium sulfide.  The result i s that the ATPase sites are stained  brown-black by the precipitate of cobalt-sulfide. Though this method 2+ 2+ is not specific for Ca -ATPase, the presence of high Ca concentration (30 mM) and 12 mM ATP in the incubation medium make the contributions of other phosphatases to ATP hydrolysis negligible.  ATP Hydrolysis by the Placental Plasma Membranes To test the various enzymatic properties, 0.1 ml of membrane suspension containing 10-20 ug of protein, was incubated with 1.0 ml of solutions containing calcium or magnesium i n appropriate amounts (0.1 to 10 mM); 20 mM Tris-HCl buffer (pH 8.2) and 70 mM Na Na ATP (Sigma) was added to make 5 mM. ?  +  (as NaCl);  Blank specimens did not contain  bivalent cations.  The samples were incubated for 30 min in a Dubnoff  shaking water bath at 37°C. The reaction was terminated by plunging the tubes into an ice-water bath and adding 1.0 ml 10% (W/V) trichloroacetic acid.  The rate of ATP hydrolysis was determined by measuring  the amount of Pi released from the samples. Using the AutoAnalyzer inorganic phosphate was measured by G0M0RI (1942) method, total protein by the Lowry et al_. (1951) procedure. Results are expressed as umole Pi released per mg of protein in 30 min.  Every value of the various  experiments was the mean of t r i p l i c a t e samples and every whole experiment was repeated at least three times. The following compounds were obtained from the sources indicated 1. Na ATP (Sigma No. A-3127); £  2. Tris-ATP [Di-Tris (hydroxymethyl)-amino-metane s a l t ] ; 3. ITP-Inosine 5'-triphosphate sodium s a l t (Sigma No. 1-5000); 4. GTP-Guanosine 5'-triphosphate sodium s a l t (Sigma No. G-8752); 5. ADP-Adenosine 5' diphosphate disodium s a l t (Sigma No. A-0127); 6. AMP-Adenosine 5'-monophosphoric acid sodium s a l t (Sigma No. A1877); 7. EDTA-disodium ethylenediamine-tetraacetate (Fisher S-311); 8. EGTA - ethyleneglycol-bis-(-aminoethyl ether)n, n'-tetraacetic acid (Sigma No. E-3251); 9. Mersalyl acid (Sigma No. M-8125); 10. Oligomycin (Sigma No. 0-1295); 11. Ruthenium Red (K and K Laboratories No. 2603-A); and 12. Maleic anhydride (Fisher A-168).  50 RESULTS Histochemical Localization of ATPase Activity in the Placenta Plate 5 shows clearly that the ATPase a c t i v i t y (black-brown staining) i s localized i n the trophoblastic layer between the maternal and the fetal circulations. 2+ Distribution of Ca -ATPase Activity 2+ The Ca -ATPase a c t i v i t y of the different fractions (see Chapter I for d e f i n i t i o n of the fractions) was assayed i n the presence of 5 mM 2+ Ca  under standard conditions as described i n the Methods section.  The distribution was as follows: supernatant, 1.3; final preparation, 16.8; and p e l l e t , 15.0 (nmoles Pi/mg protein per 30 min).  This follows  the distribution of alkaline phosphatase and glucose-6-phosphatase (Table I ) , with the same ratio (1:1) between the a c t i v i t y i n the final preparation and the pellet.  These results indicate that the three phos-  phatases are located in the membranes of the microsomal fraction. Activation of the Enzyme by Divalent Cations Activation of the enzyme by calcium ions was assessed by varying the calcium concentration in the incubation medium from 0.02 to 10 mM. Figure 10 depicts the rate of Pi production at the various calcium con2+ centrations.  The apparent  for Ca  of placentas from 5 different  guinea pigs was 0.26 +0.01 mM (m^SErn) (Figure 11). The rate of Pi release at peak a c t i v i t y ranged from 15.0 to 22.0 pmole Pi per mg protein in 30 min.  51  Plate 5. Histochemical localization of ATPase a c t i v i t y in the guinea pig placenta. F = fetal c i r c u l a t i o n , M = maternal circulation, and TR = trophoblast. The brown staining indicates ATPase activity.  52  mM divalent cation  Figure 10. Stimulation of ATP hydrolysis by divalent cations. Unless indicated incubation fluids contained 70 mM Na (as NaCl), 20 mM Tris-HCl (pH 8.2) and 5 mM Na2ATP. • ©activation by Ca2+; • • Ca2 + Mg2+ (in equimolar concentrations); X X Mg2+; • • Mn2+; 0 0 Sr2+. +  +  53  "i  0 1  2  1  1——i  1  3 4 ,5 1/tS] (mM)"  6  7  1  8  1  9  1  1-  10  1  Figure 11. Variations in Lineweaver-Burk plots of ATPase a c t i v i t y at various concentrations of ATP, C a and Mg2+. V i n ymole Pi formed per mg protein in 30 min [S] in mM. The incubation mixture contained 20 mM Tris HCl (pH 8.2), 70 mM Na . . mM ATP + Mg^ X .2+ mM ATP + Ca' 0 2+  +  9  mM Mg  2+  + ATP  mM C a  2+  + ATP  54 In the absence of calcium, magnesium ion also activated the enzyme but always less effectively than calcium (Figure 10).  The apparent  for magnesium was 0.56 +_ 0.03 mM (m+SEm) (Figure 11). When instead 2+ 2+ of the single cation a 1:1 combination of Ca + Mg was used, the resultant curve lay between the two curves obtained from incubation with Ca2+ or Mg2+ alone (Figure 10). 2+  2+  The effect of other divalent cations (Mn , Sr ) on enzyme a c t i vation was also tested.  Figure 10 shows that manganese activated the  enzyme but strontium did not.  Maximal activation of the enzyme with  2+ Mn occurred at 2 mM, higher concentrations producing i n h i b i t i o n . 2+ 2+ Activation of enzyme by Ca  and Mg  was further tested by  2+ using a constant Ca concentration (5 mM) and adding increasing amounts of Mg2+, as well as by using a constant Mg2+ concentration (5 mM) and 2+ 2+ adding increasing amounts of Ca (up to 8 mM). The addition of Ca 2+ 2+ to 5 mM Mg led to increased stimulation, whereas the addition of Mg 2+ to 5 mM Ca led to inhibition of the enzyme. Figure 12 gives the curve so obtained. The point of intersection of the two curves was at 5 mM 2+ 2+ Ca^ + 5 mM Mg .  The Effect of Incubation Time on the Concentration of H and Pi i n the Medium" +  Frequent recording of pH and Pi release (Figure 13), revealed that during 4 hrs of incubation the pH dropped only 0.1 pH unit, and Pi release was linear.  During the f i r s t 30 min the drop in pH was very  small indicating that the selection of Tris as buffer was appropriate.  55  5  5+2  Divalent  5+4 cation  5+6  5+8  mM  Figure 12. Enzyme activation by Ca and Mg . 2+ 2+ o oConstant Ca (5 mM) + varying Mg concentrations. 2+ 2+ • • Constant Mg (5 mM) + varying Ca concentrations. 2+ • "Ca alone • • Mg alone Incubation fluids contained 20 mM Tris HCl (pH 8.2) 70 mM Na+ (as NaCl) and 5 mM NaJ\TP. 2+  lOO-i I  c  90-  o  80-  CO  »_ CL  E  7060 50  _0)  o  6  4 OH  30-1 20 IOH r 0  1 0.5  1 I  1 2  Incubation Time  1 3  1 4  (Hours)  Figure 13. The effect of incubation time on Pi release and pH of the incubation medium. The incubation f l u i d contained 5 mM Ca2+, 70 mM NaCl, 5 mM Na2ATP and 20 mM Tris-HCl (pH 8.2).  57 Protein Concentration Effect on Pi Release Inorganic phosphate release was linear within the protein concentration range tested 5-60pg/ml incubation f l u i d (Figure 14).  Sodium Independence of the Enzyme The requirement of sodium ions for enzyme activation was tested by incubating the enzyme in 0 or 70 mM Na and adding Tris-ATP instead of Na ATP. 2  Specimens incubated without sodium showed s l i g h t l y higher  activation than those incubated with sodium (Figure 15).  The Effect of pH on ATP hydrolysis The calcium and magnesium activation curves of the enzyme were 2+ obtained by incubating the enzyme preparation with 0 or 5 mM Ca  or  2+ Mg at a pH ranging from 6.5 to 10.7. In each instance the pH was adjusted both i n the incubation f l u i d and Na,>ATP solutions and determined before and after incubation.  Figure 16 shows the curves obtained.  The  2+ pH optimum of Ca ATPase was between 8.2 and 8.5. At pH 7.1 and 9.6 the enzyme was stimulated by calcium ions to only 50% of peak a c t i v i t y . 2+ The pH optimum of Mg ATPase was between 8.2 and 9.3 with approximately 60% of the activation by C a . Below pH 7.0 and above pH 9.7, Mg 2+  2+  2+ produced higher activation than Ca . Inhibitors The effects of different inhibitors on calcium activation of the enzyme were tested.  Ouabain (1 mM) was added to the solutions  58  Figure 14. Protein concentration effect on Pi release. The incubation medium contained 5 mM ATP, 20 mM TrisHC1 (pH 8.2) and membrane protein at the concentrations as indicated.  59  o CO  c Q) O  a E aT  JD  o E 3.  3.0 4.0 8.0 mM Ca 2+  Figure 15. Effect of Na on enzyme activation by Ca e • without Na , Owith 70 mM Na . Incubation medium contained 20 mM Tris-HCl (pH 8.2), and 5 mM Tris-ATP. +  +  60  I c E  o co  c o  i_ D_  E •  <D  o E  3.  pH  Figure 16. Effect of pH on enzyme a c t i v i t y . Incubation solutions contained 5 mM Ca2+, 20 mM Tris HC1, 70 mM Na and 5 mM Na ATP. 2+ •e pH effect with 5 mM Ca 2+ - B pH effect with 5 mM Mg +  2  61 containing 0 or 5 mM calcium, 20 mM Tris HCl buffer (pH 8.2), 70 mM Na  +  and 5 mM Na ATP. 2  Figure 17 shows that ouabain did not i n h i b i t the  enzyme. Ethacrynic acid, added to the samples to produce concentrations 2+ between 0.15 and 5.0 mM, inhibited enzyme a c t i v i t y by Ca  (Figure 17);  5.0 mM ethacrynic acid producing 55% inhibition. When the enzyme was preincubated at 37° with 1 mM mersalyl, time-dependent inhibition was shown (Fig. 18A-a).  The inhibitory effect -5 -2 of mersalyl was then tested with a concentration range of 10 M to 10 M. The resulting inhibition curve (Figure 18A-b) shows three stages of i n -3 -2 hibition with a f a l l - o f f in a c t i v i t y between 10 M and 10 M. The effect 2+ of 1 mM mersalyl on Ca the V  m a x  activation of the enzyme (Figure 18B) showed  to be 80% of the control and  remaining unchanged (Figure 18C).  When Na H EDTA was added i n increasing amounts to the incubation 2+ 2+ 2  2  solutions containing 5 mM Ca creased as shown i n Figure 19.  , the Ca -sensitive ATPase a c t i v i t y deThe increase in degree of chelation of  calcium ions led to decrease in enzyme a c t i v i t y ; the enzyme activation 2+ by Ca could be inhibited completely by the addition of 5 mM Na H EDTA. However, the inhibition obtained with EDTA was greater than expected 2+ from the non-chelated Ca activation curve. 2+ EGTA, a specific chelator of Ca , was used to i n h i b i t the enzyme. 2+ The inhibition curve was a symmetrical mirror image of the Ca activation curve of the enzyme (Figure 20A). When the incubation medium contained 2+ 2+ equimolar concentrations of Ca , Mg , and EGTA, EGTA led to a decrease 2+ in the a c t i v i t y to a level similar to that obtained by Mg alone (Figure 20B). Increasing the EGTA concentration to 10 mM did not cause further 2+ inhibition and the Mg activation of the enzyme was not affected. 2  2  62  • Ouabain  Eth acrynic Acid 5 H O  E  00  Figure 17.  1.0  ~i  r  r~  2.0 3.0 4.0 mM inhibitor  5.0  Effect of inhibitors. Incubation contained 5 mM Ca 70 mM Na+, 20 mM Tris HCl (pH 8.2) and 5 mM Na2ATP. • • Ouabain, • • Ethacrynic acid.  ,  63  Figure 18. Effect of mersalyl on Ca*" Incubation media contained 8.2), 5 mM Na2ATP and Ca2+ A c t i v i t y i s expressed as % r  activation of the enzyme. 70 mM NaCl, 20 mM Tris-HCL (pH and mersalyl as indicated. of Pi production with 5 mM Ca ". 24  A-a: • • effect of preincubating the enzyme with 1 mM mersalyl on the V obtained with 5 mM C a . The Na ATP was added at the time indicated; ATP hydrolysis was determined after 30 min of further incubation. 2+  m a x  2  A-b: • • effect of increasing mersalyl concentrations on the V of the enzyme incubated with 5 mM C a . 2+  m a x  B: Effect of 1 mM mersalyl on Ca2+ activation of the enzyme, • H control, • • + mersalyl.. C: Lineweaver-Burk plot of the effect of 1 mM mersalyl on Ca2+ activation of the enzyme. E a control, • • with 1 mM mersalyl. V i s expressed as percent Pi production with 5 mM Ca2+.  64  + CN  0  1.0  2.0 3.0 4.0 mM EDTA  5.0  2+ Figure 19. Effect of EDTA on enzyme activation by Ca , 0 0 "Observed" curve obtained from incubation with 5 mM Ca2+, 20 mM Tris-HCL (pH 8.2), 70 mM Na and 5 mM Na ATP. © • "Theoretical" curve obtained with Ca2+ concentrations i n the incubation f l u i d corresponding to the non-chelated Ca2+ of the "observed" curve. +  2  65  X ACTIVITY  Figure 20. EGTA effect on C a  activation of the enzyme. 2+ A: e • activation of the enzyme by Ca , • • a c t i v i t y obtained when the incubation medium contained 5 mM Ca2+ plus increasing amounts of EGTA. 2+ B: • • activation by Ca ; • • activation by Mg2+ alone; a • activation by 4.2 mM Ca2+ + 4.2 mM Mg2+; * * activation by (4.2 mM Ca2+ + 4.2 mM Mg2+) + EGTA. CT  and Mg  tT  66 Complete modification of amino groups on the enzyme with 25 mg maleic anhydride per mg of protein was carried out as described by Freedman et al_. (1968).  This modification led to complete inactivation of  the enzyme (Figure 21 A).  When the enzyme was treated with 1 mg maleic  anhydride per mg protein the  was 70% of the control (Figure 21A)  but the K was unchanged (Figure 21B). ffl  Ruthenium red (prepared by the method of Luft (1971)) in a concentration range of 10" M to 10" M did not i n h i b i t the Ca  or the Mg  activation of the enzyme. Oligomycin in a final concentration of 1 yg/ml also did not i n h i b i t the enzyme.  Substrate  Specificity  The substrate s p e c i f i c i t y of the enzyme was tested by adding increasing amounts (up to 5 mM) of Na GTP, Na ITP, Na ADP or AMP to the 2  2  2  incubation f l u i d plus enzyme and comparing the Pi release to that produced from Na ATP. 2  When the Na ATP concentration was varied between 0.1 and 5 2  mM, maximal calcium activation occurred at 5 mM (Figure 22) with an apparent  of 0.08 mM-0.10 mM.  The two other triphosphates and ADP  also served as substrates, but GTP and ADP gave a greater K (0.38 and ffl  0.15, respectively), whereas the apparent same as that for ATP.  No change in the  of ITP (0.08 mM) was the for Na ATP (0.08-0.1 mM) was 2  2+ 2+ obtained when 5 mM Ca was replaced by 5 mM Mg (Figure 11) but the V  m a w  with ATP was higher.  AMP was not hydrolyzed at a l l .  67  % AC TIVI TY  Figure 21. The effect of blocking NhS^ groups with maleic anhydride. Incubation media contained 70 mM NaCl, 20 mM Tris-HCl (pH 8.2), 5 mM Na ATP, 5 mM CaCl and maleic anhydride as indicated. 2+ A: B B activation by Ca ; • 6 effect of 1 mg maleic anhydride per mg protein on C a activation of the enzyme; • • 25 mg maleic anhydride per mg protein. B: Lineweaver-Burk plot of the effect of 1 mg maleic anhydride per mg protein on Ca2+ activation of the enzyme. o a control, « e with maleic anhydride; 1 ug/mg protein. 2+ V i s expressed as percent of Pi production with 5 mM Ca ; and S in mM Ca . 2  2  2+  2+  68  0  Figure 22.  1.0  2.0  3.0 4.0 mM substrate  5.0  Substrate s p e c i f i c i t y : Hydrolysis of ATP compared to that of other high energy t r i - and diphosphate nucleotides. O  • ATP:  n  B  ITP:  • 0  • 0  GTP: ADP:  X 2+  Incubation f l u i d contained 5 mM Ca Tris-HCl (pH 8.2).  X AMP. +  , 70 mM Na , 20 mM  69 Temperature Effect on ATP Hydrolysis Samples were incubated at temperatures in the range 1°C to 80°C in 5 mM C a , 20 mM Tris-HCl (pH 8.2), and 5 mM Na ATP. ATP hydrolysis 2+  2  was stimulated at temperatures up to 50°C, levelled between 50°C and 70°C, and dropped sharply between 70°C and 80°C (Figure 21A).  The Q-JQ values  (calculated according to the method of Giese (1968) decreased from 1.9 for the range 1 C-11°C to 1.3 for the 27-37°C range and became 0.15 0  above 70°C (Figure 23B).  The arrhenius plot (Figure 23B) revealed that  the activation energy of ATP hydrolysis was 6.31 KCal/mole between 1°C45°C.  S t a b i l i t y of the Enzyme A decrease of only 10% i n enzyme a c t i v i t y was noted after two months of storage at 4°C. Freezing the samples resulted i n rapid loss of a c t i v i t y .  DISCUSSION 2+ A Ca -stimulated ATPase i s found i n several tissues i n which active transport of calcium i s thought to occur, such as the intestinal mucosa (Martin et al_., 1969) and renal tubules (Parkinson and Radde, 1971).  Calcium-sensitive ATPases have also been described i n other  tissues i n which calcium ions are needed for specific functions, such as sarcoplasmic reticulum (MacLennan, 1970), brain and nerve tissue 2+ (Berl and Puszkin, 1970; Nakamaru e_t al_., 1967).  Another Ca -sensitive  ATPase has been characterized i n the red cell (Schatzmann and Vincenzi, .1969; Cha et al_., 1971).  70  Figure 23.  Temperature effect on enzyme a c t i v i t y . A - Arrhenius plot for ATPase a c t i v i t y B - Q,  n  at different temperatures.  71 This chapter describes such an enzyme isolated from the guinea 2+ pig placenta.  Histochemically the placental Ca  -ATPase was  localized  in the trophoblastic layer, and more s p e c i f i c a l l y in the microsomal fraction, as concluded from the distribution of enzyme markers.  The  enzyme appears to be membrane bound as indicated by the low a c t i v i t y in the 35,000 x g supernatant. 2+ The common properties of the various Ca they do not require Na  +  or K  +  2+ Mg  ATPases are that  for activation (MacLennan, 1970;  Berl and Puszkin, 1970), and that ouabain does not i n h i b i t their a c t i v i t y . 2+ This i s also true for the placental Ca -ATPase. However, the cation requirements d i f f e r for each ATPase. In sarcoplasmic reticulum, whereas Mn 2+ can substitute 2+for both Mg 2+ and Ca 2+ , Sr 2+ can substitute for 2+ Ca but not for Mg (MacLennan, 1970). In the erythrocyte (Schatzmann and Vincenzi, 1969; Rosenthal e_t al_., 1970) the enzyme i s stimulated by Ca 2+ and inhibited by Mg 2+ . In the kidney (Parkinson and Radde, 1971) 2+ and intestinal mucosa (Martin e_t al_, 1969), Mg always stimulates the 2+ enzyme more than Ca but either ion could be substituted for the other. Brain Ca ATPase (Berl and Puszkin, 1970; Nakamaru et al_., 1967) can 2+ be stimulated equally well by Ca  2+ and Mg  .  Calcium ions are the preferential cations for stimulation of 2+ the placental Ca -ATPase. Only 60% of maximal a c t i v i t y produced by 2+ 2+ 2+ Ca ions can be achieved by substituting 5 mM Mg for 5 mM Ca . However, there i s no essential requirement by the enzyme for either, and both produce stimulation.  Maximal activation produced by either ion  can be modified by the other ion.  Thus, an inhibition was produded by 2+ 2+ adding increasing concentrations of Mg to 5 mM Ca , and stimulation  72 2+ 2+ by adding increasing concentrations of Ca to 5 mM Mg . I t seems 2+ 2+ therefore, that there is competition between Ca and Mg for the active 2+ s i t e s , and since the a f f i n i t y and the V of Ca is higher than that max 2+ of Mg , any combination of these two ions w i l l give lower a c t i v i t y 2+ 2+ than Ca alone, but higher than with Mg alone. J  3  The role of the divalent cations i n this enzyme system i s believed to be the production of a divalent ion - ATP complex (Melancon and Deluca, 1970; Hyde and Rimai, 1971) which serves as a substrate for the enzyme. Although the same 5 mM C a  for Na ATP was obtained by replacing 2  with 5 mM Mg , the V _ for Mg ATPase was only 60% of the max 2+ 2+ V for Ca ATPase. This finding indicates that the Ca ATP-complex is the preferential substrate. The kinetics of the competition between 2+  2+  2+  m a x  2+ Ca  2+ and Mg w i l l be described in the next chapter. 2+ The optimal pH for the Ca -activated ATPase of the placenta  also differs from that for similar enzymes in other tissues. For example, i t ranges from 7.5 in sarcoplasmic reticulum (Martonosi and Feretos, 1964) to pH 9.0 in brain (Nakamaru e_t al_, 1967).  Although the enzyme  was not isolated, the narrow pH curve suggests that only a few or perhaps a single enzyme i s active. Since the optimal pH of this enzyme i s 8.2, i t i s unlikely that an alkaline phosphatase is activated, whose optimum is at 10.3 in guinea pig piacenta(Manning  e_t al_, 1970).  A more detailed  comparison with alkaline phosphatase w i l l be given later in the thesis. There are contradictory reports in the literature about substrate s p e c i f i c i t y for (Na , K ) ATPase. +  +  Some claim that ATP is the only sub-  strate, while others showed hydrolysis of GTP and ITP as well.  These  73 results have been reviewed by Whittam and Wheeler (1970).  A more recent  study by Watson et al_. (1971a) on the red blood c e l l ( C a  + Mg )-  2+  2+  ATPase suggests that other nucleotides cannot serve as substrate. In this study ATP was the preferential substrate, but GTP, ITP, and ADP were also hydrolyzed to a great extent.  Similar results were  2+ obtained for other Ca  -ATPases from plasma membranes of bacteria (Davies  and Bragg, 1972; Mirsky and Barlow, 1971), kidney (Parkinson and Radde, 1971), g i l l s (Ma et al_., 1974), and to a certain extent in the sarcoplasmic reticulum (MacLennan, 1970). The absence of substrate s p e c i f i c i t y makes i t l i k e l y that this enzyme is a general triphosphatase with ATP as the preferential substrate, 2+ and this is the main j u s t i f i c a t i o n for c a l l i n g the enzyme a Ca -ATPase. 2+ The search for a specific inhibitor of Ca  -ATPase has thus far  been unsuccessful. Ouabain (Berl and Puszkin, 1970) and oligomycin (Dunham and Glynn, 1961), specific inhibitors of (Na + K )-ATPase, 2+ have no effect on Ca -ATPases, and so i t is in this study. Ruthenium 2+ red, an inorganic dye, has been shown to i n h i b i t selectively Ca -ATPase +  +  of the red blood c e l l (Watson e_t a}_., 1971b); i t had no effect on the 2+ placental Ca -ATPase. Some non-specific inhibitors of enzyme a c t i v i t y such as ethacrynic acid (Vincenzi, 1968) and mersalyl (Schatzmann and 2+  Vincenzi, 1969) also i n h i b i t Ca  -ATPase.  The inhibition obtained with ethacrynic acid suggests that there is an SH group in or near the active center of the enzyme, since ethacrynic acid is believed to block SH groups (Davis, 1970).  This i s similar  + +  to i t s action in the kidney where i t inhibits Na K -ATPase to cause  74 diuresis (Davis, 1970; Duggan and N o l l , 1965).  The essentiality of  SH-groups for Ca 2+ -ATPase and Ca 2+ -transport has been demonstrated in sarcoplasmic reticulum (Hasselbach, 1966a) by using other SH-blockers. 2+ 2+ In the red blood cell both Ca -ATPase and the Ca pump are inhibited by ethacrynic acid (Vincenzi, 1968). an SH-blocker (Slater, 1967).  Mersalyl is also believed to be 2+  Therefore inhibition of Ca  -ATPase  a c t i v i t y caused by this compound i s an additional indication that free SH-groups are essential for f u l l expression of the enzyme activation. The time-dependent as well as the three-stage inhibition of the enzyme by mersalyl suggest that there are several types of SH-groups, presumably located in different layers of the plasma membrane. I f the i n h i b i t o r has to penetrate the membrane to different depths to interact with SHgroups, this w i l l produce time-dependency. -5 mersalyl (10  Thus low concentrations of  -5 M to 5 x 10  M) would saturate only external SH-groups, -5 -3 while higher concentration (5 x 10 M to 10 M) may block deeper groups _3 and concentrations above 10  appear to give the greatest i n h i b i t i o n .  The innermost SH-groups are presumably the most essential ones because the greatest degree of inhibition i s obtained by saturating them.  The  existence of three different types of -SH groups was demonstrated in sarcoplasmic membranes (Hasselbach, 1966b), although not a l l of them 2+ are involved in ATPase a c t i v i t y .  Since the IC^ for Ca  was not affected  by mersalyl, the inhibition i s non-competitive. The positive charge on the free amino groups probably plays a major role in the binding of the metal-ATP complex to the enzyme. Treating the enzyme with maleic anhydride replaced the positive charge  donated by the free amino group with a negative charge (Freedman et a l . , 1968), and led to a complete loss of a c t i v i t y . 2+  Wolf (1972) in his model  2+  of the Ca Mg -ATPase in the erythrocyte described the role of the + 2+ NHg in the s p l i t t i n g site of the active center. Since the for Ca was unchanged with the lower concentration of maleic anhydride (1.0 yg/mg protein), while the V was reduced to 70%, i t seems that treat' max ment of the enzyme with maleic anhydride reduced the number of sites J  J  r  available for ATP hydrolysis. 2+ The placental Ca  -ATPase was found to be r e l a t i v e l y heat resis-  tant and a marked f a l l in velocity was noticed only between 70°C-80°C. 2+ The activation energy for the hydrolysis of ATP by the placental Ca  -  ATPase was 6.31 Kcal/mole. A higher activation energy for hydrolysis of ATP was reported for the acto-heavy meromyosin ATPase (25-30 Kcal/ mole) (Barouch and Moos, 1971).  A discontinuity in the Arrhenius plot  for the hydrolysis of ATP by (Ma , K ) ATPase was found in a few cases. +  +  Gruner and Avi-Dor (1966) obtained 29.5 Kcal/mole activation energy for the 0°C-6°C range and 7.8 Kcal/mole for the 20°C-37°C range.  Simi-  lar results were reported by Bowler and Duncan (1968). A possible discontinuity in Arrhenius plot around 23°C can be detected in this study (Figure 23). However, even i f two lines are drawn instead of one, the difference in the activation energies i s not impressive (8.7 Kcal/mole for the 1°C-23°C range and 3.5 Kcal/mole for the 23°C-45°C range). The transition temperature of brain phospholipid dispersed i n water i s 23°C (Luzzati and Husson, 1962).  Thus the p o s s i b i l i t y that 2+ the activation energy of ATP hydrolysis by the placental CA -ATPase  is affected to a certain degree by the state of the phospholipidis in the membrane i s not excluded.  SUMMARY 2+ Guinea pig placental homeogenate was found to contain a Ca ATPase.  Further p u r i f i c a t i o n and tests for marker enzyme showed that  2+ the Ca  -ATPase i s located i n the plasma membranes. This enzyme i s preferentially activated by calcium ions, i n  the presence of 5 mM ATP, maximal enzyme a c t i v i t y being obtained at 2+ 3 to 5 mM Ca . The maximal rate of ATP hydrolysis varies between 15 and 22 ymole Pi/mg protein i n 30 min. 2+ Mg also activates the enzyme, but always to a lesser degree than Ca . Mn , but not Sr , activates the enzyme. At optimal c a l 2+ cium concentration, addition of magnesium i s always inhibitory. Ca 2+ and Mg  seem to act on the same s i t e . + The enzyme does not require Na or 2+ The optimal pH for Ca activation 8.2 and 8.5; at pH 7.1 and 9.5 only 50% of  + 2+ K for activation by Ca . of the enzyme l i e s between maximal activation occurs. -6  Ruthenium red, in the concentration range of 10  -4  M to 10  M, Ouabain  _o  (10  ), and oligomycin (1.0 yg/ml) did not i n h i b i t the enzyme. Other triphosphates may serve as substrate; but the Vmax „ for r  J  ATP i s highest. Addition of increasing amounts of Na2H2EDTA leads to a progressive decrease in a c t i v i t y , complete inhibition occurring at 5 mM when 2+ the incubation f l u i d contains 5 mM Ca .  77 2+  EGTA, a specific chelator of Ca 2+ Ca activation of the enzyme.  , symmetrically reversed the 2+ ' The activation of the enzyme by Mg  was not affected. The inhibition of the enzyme by mersalyl, an SH-blocker, was such that i t suggested the presence of three types of SH-groups involved i n the activation of the enzyme.  Mersalyl acted as a non-competitive i n -  hibitor of the enzyme. Free amino groups are essential for ATP-hydrolysis.  Complete  modification of the amino groups with maleic anhydride led to total inactivation of the enzyme, whereas partial blocking of NHg -groups 2+ reduced the V without affecting the 1^ of Ca . 2+ In conclusion, the-properties of the placental Ca -ATPase are 2+ +  m a x  s u f f i c i e n t l y similar to the properties of other Ca -ATPases involved 2+ in Ca -transport, to suggest that i t may possibly have a similar role 2+ in translocation of Ca i n the placenta.  CHAPTER III THE EFFECT OF Ca /Mg 2+  2+  CONCENTRATION RATIO ON  PLACENTAL (Ca -Mg )-ATPase ACTIVITY 2+  2+  INTRODUCTION 2+  2+  The antagonism between Ca and Mg on enzyme a c t i v i t y i s well documented. Dixon and Webb (1964c) and Williams (1959) give l i s t s of 2+ antagonistic effects on different enzymes. Since Ca i s the major 2+ divalent cation in the extracellular f l u i d and Mg i s the major d i 2+ valent cation found i n t r a c e l l u l a r l y , i t has been suggested that Ca  /  2+ Mg  concentration ratio acts as regulator of enzyme a c t i v i t y (Bianchi,  1968). 2+ In the previous chapter i t was suggested that Ca 2+ 2+ compete for the same s i t e on the placental (Ca  -Mg  2+ and Mg  )-ATPase.  This  suggestion was based on the results that are presented in Figures 10 and 12.  To prove this postulate, an equation expressing ATPase a c t i v i t y 2+  as a function of Ca  2+  /Mg  concentration ratio  has been derived.  The  following assumptions were made: 1. Calcium ions and magnesium ions compete for the available ATP 78 to form divalent cation-ATP complexes, which serve as alternate substrates for the enzyme on the same s i t e .  79 2+  2. The association of Ca  2+  , Mg  , and ATP directly with the enzyme  is low compared with Ca-ATP and Mg-ATP association constants. The postulated pathways are presented i n Figure 24. 2+ In accordance with the preceding assumptions Ca reacts in the following way: k  Ca  l  + ATP  2+  - Ca ATP  (1)  The s t a b i l i t y constant of the Ca ATP complex w i l l be:  K  ^ [CaAJl] 2 [Ca^ ] [ATP]  =  =  1  k  k  3  Ca ATP + E - — • E Ca ATP K  4 2 k k^ =  Ca ATP] [E] E Ca ATPJ k  (3) (4)  5  E Ca ATP  - E + Products  V = k [E Ca ATP] 1  ( 2 )  +  5  (5)  (6)  2+  Similarly for Mg  the following holds: k  Mg  2+  6  + ATP ,  Mg ATP k  7  (7)  2+ Figure 24. Postulated scheme for ATP hydrolysis i n the presence of Ca  2+ and Mg .  Where: K = Association constant E = Enzyme, P = Products and broken lines indicate minor interactions.  ^  o  81 The s t a b i l i t y constant of Mg ATP Complex w i l l be: 6__  [Mq ATP]  7  [Mg ] [ATP]  k =  3  k  k  8  E + Mg ATP „  E Mg ATP k  1Q  g  ( 1 0 )  E Mg ATP]  E Mg ATP  2  (9)  "9 ATP] [E]  9_  V = k  ( 8 )  2+  —  -  E + Products  (11)  [E Mg ATP]  (12)  2+ Since there i s competition between Ca  2+ and Mg  for ATP i n  the f i r s t stage and again between Ca ATP and Mg ATP for the enzyme (E), the ratio [E Ca ATP] to [E Mg ATP] after introducing the terms obtained from equations 2, 4, 8, 10, w i l l be:  E Ca ATP]  TWJW] ~  l K  K  3  K  4 [Ca ] IC, 2 2+  [ M g  (13)  + ]  Saturating the enzyme by using at least one of the ions at .the optimal concentration, the total [E] w i l l form complexes with both substrates i n the following way:  82  [E-Mn-ATP]  =  ^  TOO  ^  [  K K 3  C  a  2  +  ^ ^  ]  (  1  4  )  [Mg ] 2+  2  This i s the fraction of the enzyme which i s complexed with Mg ATP. Since the enzyme i s saturated, the rest of the enzyme which i s not complexed with Mg ATP, w i l l complex with Ca ATP. Thus E Ca ATP complex in percent of total enzyme w i l l be:  E  EC a  100  fJ P  fE  1 0 0  K [Ca ]  (15)  2+  K ]  4  K K 3  [Mg ] 2+  2  Since the total a c t i v i t y i s A  =  V + V = k [E Ca ATP] + K 1  2  5  1Q  [E. Mg ATP],  (16)  we can write: 100 K. [ C a ]  \ )  'K K [Mg ]  '  A = K  2+  2+  3  2  '100  1 0 0  K K [Ca ]  (17)  2+  +  1 0  X  K K [Mg ] 2+  3  5  2  Introducing the constants into equation 17 and substituting Vmax for k , Vmax for k , we get the total a c t i v i t y i n percent by 1  5  2  1Q  2+  referring to the Vmax-j for Ca  as 1.0.  83 100 \ A = ( K, K,[Ca ] ) 5 — + l / Vmax K K [Mg ] ' 2+  / +  v  0  2+  3  1 0 0  100 K, K. [ C a ] (18) — o r - + 1 / Vmax, K K [Mg^ ] 2+  V  2  +  2  1  3  Since we assume that the last stages of the pathway for equations 5 and 11 are irreversible we can use Krn^ instead of IC,, and Km instead 2  of K^.  (K and 2  are the dissociation constants.)  and K are the 3  s t a b i l i t y constants of Ca ATP and Mg ATP. Thus the only variable i s 2+ 2+ either [Ca ] or [Mg ] while the other ion i s at optimal concentration. Finally the formula w i l l be: 100  2+-] A = ^ K-j Km [Ca*"  (19)  Km [ C a ] 2+  2  +  K Krr^ [Mg ]  100  100 1  2  Vrnax,  +  2+  K  3  1  Vmax,  Km [Mg ] 2+  3  1  MATERIALS AND METHODS Guinea pig placental plasma membranes were isolated according to the method of Post and Sen (1967).  The-experiment was carried out  as described i n the previous chapter.  The amount of inorganic phosphate  released was measured by the Gomori (1942) method and the protein i n the sample by the Lowry et_ al_. (1951) procedure.  84 RESULTS 2+ The activation curves obtained by either of the two ions, Ca 2+ and Mg , are rectangular hyperbolas of typical substrate concentration curves (Figure 25). 2+ Adding increasing amounts of Mg  2+ to 5 mM Ca  resulted i n i n -  hibition of ATP hydrolysis; stimulation was produced by adding increasing amounts of Ca  to 5 mM Mg  shown i n Figure 25.  or 10 mM Mg  . The results obtained are  The apparent K (calculated from a Lineweaver-Burk m  plot) was 0.24 mM for C a  2+  and 0.52 mM for Mg . 2+  For C a , the Vmax = 2+  1  2+ 1, and for Mg  , Vmax = 0.6. The s t a b i l i t y constants for Ca ATP and 2  Mg ATP (Hyde and Rimai, 1971) (^ and K ) are log ^ =3.9 and log K = 3  4.5.  3  The curves I, II and III i n Figure 25 are the theoretical curves  obtained by using equation 19.  Each experimental value was the mean of  samples done i n t r i p l i c a t e and the whole experiment was repeated four times.  The goodness of f i t between the theoretical curves and the ex-  perimental points was tested by using x2 test, and the values were for each of four experiments:  0.99 > P > 0.97; 0.995 > P > 0.99; P = 0.97; 1  2  3  0.9 > P > 0.75. 4  Since the concentration ratio i s the only variable and a l l other 2+ values are constant, i t i s possible to plot A (activity) against [Mg ]/ 2+ 2+ 2+ [Ca ] while either Mg or Ca i s at optimal concentration (5 mM). The calculated theoretical curve so obtained by using the above equation and the experimental results are shown i n Figure 26A.  I t i s also possible  to calculate the change in the apparent Km for the total divalent cations  85  Divalent cation mM  Figure 25. The effects of Ca^ and Mg^ on ATP hydrolysis. The incubation medium contained 70 mM NaCl, 20 mM Tris-HCl (pH 8.2), 5 mM Na ATP and C a or Mg2+ or C a + Mg2+ i n concentration as indicated. Results expressed as percent of a c t i v i t y at 5 mM C a which ranged between 15 and 22 ymole/Pi per mg protein i n 30 min at 37°. o o activation by C a , • ° activation by Mg ; I ,H,m, theoretical curves obtained by using the equation. , • , experimental values for 5 mM Ca2+ and increasing Mg concentrations; , • , experimental values for 5 mM Mg2+ and increasing Ca2+ concentrations, *, experimental values for 10 mM Mg2+ and increasing Ca2+ concentration. T  2+  2+  2  2+  2+  2+  2+  86  Figure 26. A - The effect of changing the concentration ratio of Mg^' to C a , while at least one cation i s at the optimal concentration of 5 mM. The s o l i d line represents the theoretical, calculated curve and individual dots (• •) are experimental values obtained when the Vmax2 f o r Mg was 60% of Vmax] for Ca . 2+ 2+ B - The change in Km as a function of Mg /Ca . V values for the various combinations obtained from Figure 26A. 2+  2+  2+  87 2+ 2+ as a function of Ca /Mg concentration 0.54 mM for Mg  to 0.25 mM for C a  2+  2+  ratio.  The "Km" drops from  (Figure 26B).  Finally a three dimensional model describing the relationship 2+ 2+ 2+ between [Mg ]/[Ca ] and the a c t i v i t y of the placental Ca -ATPase was built.  In this model the Y and the X axes are the concentration  of Ca  2+  2+ and Mg  respectively, and the Z axis i s the velocity of the reaction. 2+ 2+ The surface formed by the activation curves at different [Mg ]/[Ca ] represent the velocity of the reaction under any possible combination 2+ 2+ up to the 10 mM Ca  +10 mM Mg  . The model i s shown in Figure 27.  To test further the v a l i d i t y of equation (19) for competition between substrates, the formula was used for predicting the velocity of the reaction as a function of ATP/ADP concentration  ratio.  ADP i s a  production of ATP hydrolysis by ATPase and can i t s e l f be hydrolyzed further by the enzyme.  The hypothesis was that i f ADP and ATP compete for the  same s i t e , Pi release w i l l be inhibited. The following pathway was assumed: K  ADP  as  ATP  Kas ADP  Ca-ATP-  Ca-ADP'  3C  Km ATP  Km ADP  E-Ca-ATP  E + P  E-Ca-ADP  E + P  The formation of the Ca-ADP and Ca-ATP complexes i s dependent on the association constants.  Log K for Ca-ATP = 3.6, and log K for Ca-ADP =  2.78 as quoted by Williams (1959).  The Km for ATP i s 0.1 mM (Figure 11)  88  Figure 27. A three dimensional model describing the relationship between [Mg2+]/[Ca2+] and the a c t i v i t y of the placental Ca2 -ATPase. The Y and X axes are the concentration of Ca2+ and Mg2+ respectively, and the Z axis i s the velocity of the reaction. +  89 and for ADP Km = 0.15 mM (calculated from Figure 22). Samples were incubated with 5 mM ATP ADP and 2.5 mM ATP + 2.5 mM ADP.  V for ATP was ]  considered as 1.0, and V with 5 mM ADP was 0.69. Using a l l the constants 2  mentioned, equation (19) w i l l be as follows:  A =[ K ATP(K^ATP)LATPj  ' VgADP + MOO - K ATP(K^ATP)|ATPj  as  ^K ADP(K ADP)LADPJ as  as  +  m  A = ( 7782 (0?15) (2.5) \ 892 (0.10) (2.5)  V  \  , )°'/  69  +( \  1 0 0  K ADP(y\DP) LADPJ  ) 1 V  A T P  + 1  gs  ~ 7782 ( 0 J 5 ) (2.5T , ) 892 (0.10) (2.5) '/  1 , 0  A = 4.89 + 92.9 = 97.8%  Thus the calculated velocity i n the presence of 2.5 mM ATP + 2.5 mM ADP i s 97.8% of that with 5 mM ATP alone.  The actual experimental value  was 96% for 1:1 [ATP]/[ADP] concentration r a t i o , which i s i n good agreement with the predicted value.  Thus ATP and ADP appear to be hydrolyzed  by the same s i t e , and the inhibitory effect of ADP i s very small.  Even  when [ATP]/[ADP] = 0.1, Pi release w i l l decrease only 13% (calculated value).  DISCUSSION The use of the formula presented i n this chapter was very success2+ 2+ ful i n predicting the velocity of the placental (Ca -Mg )-ATPase incu2+ 2+ bated with numerous combinations of [Ca ]/[Mg ]. I t i s concluded  90 2+  2+  that Ca and Mg compete for the same s i t e , or more precisely the corresponding complexes with ATP compete for the same s i t e . I t has been shown very clearly that the placental ATPase a c t i v i t y can be regu2+ lated by the Ca  2+ to Mg  concentration ratio.  The role of these two  ions in regulating c e l l u l a r enzymes has been the subject of several studies.  Shikama (1971) calculated the standard free energy for ATP 2+ 2+  hydrolysis as a function of pMg and pCa.  He found that the [Ca ]/[Mg ]  ratio plays an important role as a sensitive modifier of the thermodynamic potential of the ATP molecule.  Thus the energy that can be derived from  ATP hydrolysis by ATPase is regulated by at least two steps, both dependent on [ C a ] / [ M g ] . 2+  2+  Step I, the rate of ATP hydrolysis; Step I I ,  the change of free energy (-AG°) for ATP hydrolysis. 2+ 2+ The role of Ca  and Mg  in the ATPase of sarcoplasmic  was studied by Panet et al_. (1971).  reticulum  Their results suggest that phosphory-  lation of the enzyme (an intermediate step in ATP hydrolysis) i s dependent 2+ 2+ on Ca , while dephosphorylation i s increased by Mg and decreased by 2+ 2+ 2+ Ca . In the placental (Ca -Mg )-ATPase i t i s unlikely that this i s true, since the hydrolysis of ATP proceeds with either ion alone.  Though  the p o s s i b i l i t y of formation of a phosphorylated intermediate exists for the placental enzyme as w e l l , no attempt was made to study this aspect. The a b i l i t y to predict accurately the velocity of Pi release in the presence of ATP and ADP indicates the wider use that can be made of this formula.  It also suggests that ATP and ADP are hydrolyzed by  the same site and that product inhibition due to an increase in ADP concentration i s negligible.  91 In addition, i f one is interested in the contribution of each substrate to the total a c t i v i t y , i t can be derived easily since each term in the formula represents the respective contributions. Though i t is possible to adopt this equation to predict the a c t i v i t y of an unsaturated enzyme, the prediction w i l l be less accurate than for the saturated enzyme.  SUMMARY 2+ The velocity of the reaction of a Ca  2+ Mg  -stimulated ATPase  in placental plasma membranes of the guinea pig was found to depend on 2+ the [Mg  2+ ]/[Ca  ] concentration ratio.  The observed activation curve  of the enzyme while changing the concentration ratio agrees with the theoretical equation, derived for the case in which two ions activate the same s i t e while the enzyme i s saturated.  Because of the good agree2+ 2+ ment, i t i s concluded that the two divalent cations, Ca and Mg , activate this placental ATPase at the same s i t e . 2+ of [Mg  The regulatory effect  2+ ]/[Ca  ] on the placental ATPase a c t i v i t y , can be visualized  by a three dimensional model.  The equation may be used to determine  whether or not two substrates compete for the same s i t e , and what i s the relative contribution to the total a c t i v i t y by each substrate.  This  was demonstrated with ATP and ADP, which are both hydrolyzed at the same site. The equation i s not specific for this enzyme and i t can be used for other enzymes as well, provided the necessary constants are available, or can be calculated.  CHAPTER IV COMPARISON BETWEEN ALKALINE PHOSPHATASE AND Ca -ATPase 2+  INTRODUCTION Alkaline phosphatase i s one of the most studied placental enzymes, and i t s specific a c t i v i t y level serves as a general index of placental function (Curzen and Morris, 1968).  However, i t s physiolo-  gical role and even i t s naturally occurring substrate have not been 2+ defined.  I t has been suggested that i n the intestine Ca -ATPase a c t i -  vity i s just another manifestation of alkaline phosphatase (Haussler ejtal_., 1970; Russel et al_., 1972). 2+  Since Ca -ATPase i s implicated 2+  in Ca -transport, alkaline phosphatase can be matched loosely with 2+ Ca  -transport.  While this study does not oppose this suggestion re-  garding the physiological role of alkaline phosphatase, i t does argue that the two enzymes are not identical.  As described i n previous chapters,  i t was found that the two enzymes are located i n the microsomal fraction. Since no specific inhibitor i s available for either enzyme, a different approach was taken to separate the two enzymes by selective purification. The dissociation of these two phosphatase a c t i v i t i e s (ATPase and monophosphatase), i s made by comparing key properties of the two throughout 92 the process of purification of alkaline phosphatase. If the two enzymes  93 are not i d e n t i c a l , the purified alkaline phosphatase fraction should 2+ not demonstrate Ca  -ATPase a c t i v i t y .  An additional subject described in this chapter i s the specific 2+ a c t i v i t y p r o f i l e of Ca  -ATPase in developing guinea pig placenta through-  out the gestation period.  Its relation with the reported transplacental  calcium flux and fetal calcium deposition (Twardock and Austin, 1970; Twardock, 1967) is considered.  The logic behind this study i s that  since the placenta is a dynamic organic which responds to the changing requirements of the fetus, i t may be expected that the placental enzymatic picture w i l l be modified accordingly to meet the changing requirements of the fetus for calcium. MATERIALS AND METHODS Purification of Guinea Pig Placental Alkaline Phosphatase Alkaline phosphatase was purified by the method of Ghosh and Fishman (1968), in a procedure involving three major steps:  1) Butanol  extraction to disrupt the lipoprotein association; 2) Ammonium sulphate precipitation;  3) Gel f i l t r a t i o n using sephadex G-200.  Lipids were extracted from the membrane preparation by addition of butanol (20 ml butanol to 50 ml membrane preparation).  The enzyme  passes to the aqueous phase with slow s t i r r i n g for 1 hr at 4°C and then for 10 min at 37°C. The extract was centrifuged (Sorvall RC2-B) for 30 min at 14,600 x g and the aqueous phase was centrifuged again for 30 min at 35,000 x g.  The supernatant (which contains the enzyme) was  94 dialyzed overnight against 50 mM Tris-HCl pH 8.6 (10 volumes) at 5°C6°C.  The proteins were then precipitated with 90% (NH^)^ SO^, extracted  with 40% (NH ) S0 and reprecipitated with 80% (NH ) S0 - The resul4  2  4  4  2  4  tant precipitate was suspended i n 50 mM Tris-HCl pH 8.6 and dialyzed overnight under the same conditions as the previous d i a l y s i s .  The column  was prepared as described by Ghosh and Fishman (1968) using a 60 x 2.5 cm column and sephadex G-200 gel. The enzyme solution was layered on the top of the column, eluted with 50 mM Tris-HCl pH 8.6 and collected i n 10 ml fractions. The protein concentration of the fractions was determined by reading abosrbence at 280 nm (Perkin-Elmer spectrophotometer, Coleman124). Alkaline phosphatase a c t i v i t y was measured as described i n the Methods section of Chapter I, using carbonate-bicarbonate  buffer (100 mM)  2+ except when Ca  was added to the incubation medium and glycine buffer  (50 mM) was used. Estimation of the Gestational Age of the Guinea Pigs Pregnant guinea pigs at different stages of gestation were handled as described previously (Chapter I ) .  The fetuses were weighed  and the average weight per l i t t e r was used to estimate the placental age by using growth tables given by Draper (1920).  Each placenta was  processed separately and the membrane preparation of each placenta was 2+ assayed for Ca -ATPase (see Chapters I and III for d e t a i l s ) . 2+  The mean  and the standard error of the specific a c t i v i t y of Ca -ATPase for each l i t t e r (3-5 fetuses) were calculated.  95 RESULTS Purification of Alkaline Phosphatase The elution p r o f i l e of alkaline phosphatase a c t i v i t y and protein i s presented in Figure 28. The alkaline phosphatase a c t i v i t y peak appeared between fractions No. 11-16; the highest total a c t i v i t y and specific a c t i v i t y (500 pmole/mg protein) was found in fraction No. 14. 2+ Comparison Between Alkaline Phosphatase A c t i v i t y and Ca -ATPase Activity 2+ The eluate was tested for Ca -ATPase a c t i v i t y under the standard 2+ + conditions for this enzyme (5 mM Ca ,5 mM ATP, 70 mM Na , and 20 mM Tris-HCl pH 8.2); no measurable a c t i v i t y could be detected in any fraction. 2+ Studying the Ca -ATPase a c t i v i t y in the previous steps revealed that 2+ the butanol extraction abolished completely the Ca -ATPase a c t i v i t y . 2+ However, though Ca  -ATPase a c t i v i t y could not be detected, ATP was hydro-  lyzed to a certain extent by the purified alkaline phosphatase fraction. the pH profile of ATP hydrolysis (Figure 29), shows that the pH optimum for ATP hydrolysis shifted from pH 8.2-8.5 (in the membrane preparation) 2+ (Figure 16) to pH 9.0-9.5; and only slight activation by 10 mM Ca 2+ was observed at the peak (pH 9.0-9.5). Ca was completely ineffective 2+ as a stimulator at a lower pH range.  Mg  showed basically the same  p r o f i l e with somewhat better efficiency as a stimulator of enzyme a c t i v i t y . The pH profile of p-nitrophenyi-phosphate hydrolysis by fractions No. 13-14 was typical of alkaline phosphatase with a pH optimum of 10.02+ 10.2, and only Mg  stimulated hydrolysis.  (Figure 30). The specific  96  UJ  < -J  I  Fraction na  Figure 28. The elution p r o f i l e of alkaline phosphatase and protein on Sephadex G-200 gel f i l t r a t i o n carried out by the method of Ghosh and Fishman (1968). X X, protein; © ©, enzyme a c t i v i t y . The protein was eluted with 50 mM TrisHCl pH 8.6 and collected i n 10 ml fractions.  97  100% = 2 7 / j m o l e s / m g Protein-hr-  o  Figure 29.  The effect of pH on ATP hydrolysis by the purified alkaline phosphatase (fraction no. 13-14). o o, without divalent cations; X X, with 10 mM Ca2+; e ©, with 10 mM Mg . ATP concentration was 5 mM. 2+  98  1 0 0 % = 500 ju:moles/mg Protein • hr —  ^ o  I  2  o  J  i  1  1  1  1  1  7  8  9  10  II  12  PH  Figure 30.  The effect of pH on p-nitrophenyl phosphate hydrolysis by the purified alkaline phosphatase (fractions no. 13-14). o o, without divalent cations; X X, 10 mM C a ; • with 10 mM Mg2+. The samples were incubated in the presence of 5 mM p-m'trophenyl phosphate. 2+  99  a c t i v i t y at the peak was 500 ymole/mg protein compared with only 27 ymole/mg protein (5.4% efficiency) when ATP served as a substrate under 2+ the same conditions (pH 10.0, 10 mM Mg  ).  The s t a b i l i t y on storage  of the two enzyme a c t i v i t i e s in the membrane preparation was compared. Alkaline phosphatase was very unstable when stored at 4°C, and less than 2+ 10% of the i n i t i a l a c t i v i t y was l e f t after 7 days (Figure 31). Ca ATPase, on the other hand, lost only 10% of i t s i n i t i a l a c t i v i t y during 2+ the same period (Figure 31) and no further decrease in a c t i v i t y of Ca ATPase was observed up to 2 months at 4°C. The results of the comparison 2+ study between alkaline phosphatase and Ca  -ATPase a c t i v i t i e s in the  membrane preparation and the purified alkaline phosphatase fraction, are summarized in Table'IV. 2+ Ca  -ATPase Specific A c t i v i t y as a Function of Placental Age The results obtained from 21 guinea pigs with 3-5 fetuses per 2+  l i t t e r revealed that Ca  -ATPase a c t i v i t y increased up to the 50th day,  leveled between the 50th and the 60th day, and dropped after the 60th day (Figure 32). DISCUSSION It has been demonstrated clearly that the purified alkaline 2+ ' phosphatase does not possess any Ca -ATPase a c t i v i t y , and nor do any of the other eluted fractions. The only ATPase a c t i v i t y present is 2+ not dependent on Ca  for hydrolyzing ATP.  It seems very l i k e l y from  100  Figure 31.  The effect of storage at 4 C on enzyme a c t i v i t y . •  e , Ca  2+  -ATPase; A  • , alkaline phosphatase.  Table IV. Comparison between alkaline phosphatase a c t i v i t y and ATPase a c t i v i t y in the membrane preparation and the purified alkaline phosphatase fraction.  Substrate  Preparation  pH optimum  5 mM p-nitrophenyl phosphate  Membrane preparation  10.0-10.2  Purified [Alkaline phosalkaline phatase a c t i v i t y ) phosphatase fraction  10.0-10.2  5 mM Na ATP  Membrane preparation  8.2- 8.5  (ATPase activity)  Purified alkaline phosphatase fraction  9.0- 9.5  2  Tris-HCl buffer (20 mM) Glycine buffer (50 mM) Carbonate-bicarbonate buffer (100 mM)  ymoles Pi release per mg protein per hour at 37°C Net activation by (1) and (2) (1) (2) No divalent 10 mM Mg 10 mM C a cation 2+  ** 5  *** 200 * 0.75  * 20  ** 12  300  ***  * 30  * 7  ** 0  2+  Stability at 4°C Very unstable  ** 0 50  *  * 3  Very stable  102  O  25-\  a. E .1  20  CL  o E  I5H  S  o <  10-J  CD CO D  CL  5H  i -•-  CM O  o  -T 30  1  1  1  1  40  50  60  70  Embryo Age in Days  2+  Figure 32. Ca -ATPase specific a c t i v i t y as a function of embryo age. Each experimental value represents the mean + S.E. for each litter.  103 the pH p r o f i l e that ATP is being hydrolyzed by alkaline phosphatase and not by ATPase.  Felix and Fleisch (1974) investigated the p o s s i b i l i t y  that purified alkaline phosphatase from calf bone might demonstrate 2+ 2+ (Ca -Mg )-ATPase a c t i v i t y . They found that the only activation of 2+ 2+ ATP hydrolysis by Ca was obtained in the absence of Mg and always 2+ was substantially lower than the activation by Mg . Their conclusion 2+ 2+ was that calf bone alkaline phosphatase i s not a "true" (Ca -Mg )ATPase, as i t has been suggested for the intestinal alkaline phosphatase (Curzen and Morris, 1968; Haussler et_ al_., 1970). It has been shown that the extraction of the l i p i d s with butanol 2+ from the placental plasma membranes abolished completely the Ca -ATPase a c t i v i t y of the preparation.  Emmelot and Bos (1968) found that  (Na , +  K )-ATPase a c t i v i t y depended on the l i p i d component of the preparation. 2+ Similar results were reported by MacLennan (1970) for the Ca -ATPase of the sarcoplasmic reticulum. He was able to restore 50% of the original 2+ +  a c t i v i t y by adding phospholipid to Ca digested with phospholipase C.  -ATPase which was previously  The exact role of the l i p i d i s not known,  however, i t might be that the lipoprotein association keeps the protein in the conformation required for enzymic a c t i v i t y .  The lack of depen-  dence of alkaline phosphatase on the l i p i d , and i t s i n s t a b i l i t y on storage 2+ at 4°C compared with the very high s t a b i l i t y of the Ca -ATPase, are 2+ additional indications that Ca alkaline phosphatase.  -ATPase i s not just a manifestation of  Another argument against this hypothesis can be  drawn from the work of Manning et al_. (1970), who found that alkaline phosphatase from the guinea pig placenta i s very heat-sensitive (16 min  104 2+ at 55°C resulted i n 95% inactivation). On the other hand, the Ca ATPase was inactivated only at temperatures above 70°C (Figure 23). 2+ The peak of Ca -ATPase a c t i v i t y in the developing placenta appears to occur between the 50th and 60th day of pregnancy, with an increase i n a c t i v i t y up to the 50th day and a decrease after the 60th day.  A similar p r o f i l e was reported for alkaline phosphatase i n the guinea  pig (Hard, 1946).  Twardock (1967) reported that trans-placental calcium  transport i n the guinea pig (in vivo study), peaks around the 55th day of pregnancy, and drops thereafter.  Thus this profile i s i n agreement  2+ with the Ca -ATPase a c t i v i t y p r o f i l e i n the developing placenta.  However,  these profiles cannot account for the continuous increase of calcium deposition in the fetus throughout pregnancy (Twardock, 1967). SUMMARY The purified alkaline phosphatase fraction does not possess any Ca  2+ -ATPase a c t i v i t y .  Disruption of the lipoprotein association of 2+ the membranes by butanol destroyed completely the Ca -ATPase a c t i v i t y ,  while increasing the alkaline phosphatase a c t i v i t y .  In the membrane  2+ preparation Ca -ATPase i s very stable on storage at 4°C, while alkaline phosphatase a c t i v i t y decayed very rapidly.  The specific a c t i v i t y profile  2+ of Ca -ATPase throughout the gestation period revealed a peak between the 50th and the 60th day of pregnancy, similar to the alkaline phosphatase p r o f i l e . The peak of enzyme a c t i v i t y corresponds with the re2+ 2+ ported trans-placental Ca flux,^ but not with Ca -deposition i n the fetus.  105 2+  In conclusion, i t was demonstrated very clearly that Ca  -ATPase  and alkaline phosphatase from the guinea pig placenta are two separate enzymes.  DIVISION I I I  -UPTAKE BY PLACENTAL PLASMA MEMBRANE VESICLES  106  CHAPTER V Ca -UPTAKE BY PLACENTAL PLASMA 2+  MEMBRANE VISICLES  INTRODUCTION Transplacental calcium transport i s asymmetrical.  Presumably  the ion must be transferred across at least the two plasma membranes which comprise the trophoblastic layer that separates the maternal and 2+ the fetal circulations. Studying Ca -transport in such a system as one unit is extremely d i f f i c u l t i f not impossible. However, active 2+ Ca  -transport can be studied with less d i f f i c u l t y i f i t occurs across  a single plasma membrane, which i s clearly a necessary step for any asymmetrical active transport. To be able to measure such transport 2+ one must have a system i n which Ca  w i l l be concentrated.  This require-  ment i s met by the placental membrane preparation which consists predominantly of vesicles (Plates 3, 4) of plasma membrane origin. In previous chapters i t was demonstrated that the placental 2+ plasma membrane vesicles contain Ca -ATPase. This enzyme i s s u f f i 2+ 2+ ciently similar to other Ca -ATPases involved in active Ca -transport, to suggest that these vesicles may also be involved. 2+ Ca  -uptake by sarcoplasmic reticulum vesicles has been studied 107 by many investigators for almost two decades, but numerous questions  108 are s t i l l unresolved, and to date there is no widely accepted model 2+ for Ca  -transport. Part of the literature on this subject i s reviewed  by Inesi (1972).  The relevant data w i l l be compared with the findings  of this study in the discussion. This chapter was designed to answer very simple questions: 1) Can the placental plasma membrane vesicles accumulate calcium; i f so, how e f f i c i e n t l y ? 2) Is the uptake dependent on ATP hydrolysis? 2+ 2+ 3) What i s the effect of the external Ca concentration on Ca uptake? It i s realized that many more questions must be answered before a firm 2+ conclusion can be made concerning the relationship of the Ca -related properties of the placental plasma membranes to the active transport of 2+ Ca  . However, positive answers to the basic questions may  suggest  that the system i s operating generally in the same manner as the sarcoplasmic reticulum (Inesi, 1972) and cardiac microsomes (Repke and Katz, 1972). Before any measurement of Ca  2+  -uptake can be made, the  term  uptake must be defined and distinguished from binding. The most widely 2+ used working definitions of Ca  -uptake and binding are given by Entman  et al_. (1973) and Repke and Katz (1972).  Binding i s defined as C a  2+  accumulation in the presence of ATP but in the absence of a calciumprecipitating anion (oxalate or phosphate). reaches a maximum within 1 min.  This process i s rapid and 2+  Uptake i s Ca  presence of ATP and calcium-precipitating anion.  accumulation in the This is a slower process.  109 When calculating uptake the binding should be subtracted.  This i s done  by extrapolating the uptake to time zero. In this study binding and uptake are defined differently.  Bound  calcium i s accessible for an immediate exchange and can be displaced by a competitor. 4 5  Ca  2 +  Bound calcium i s measured by labeling the membrane with  (in the absence of ATP) and displacing i t with "cold" calcium. 45  Uptake i s represented by the fraction of 40 2+ displaced by a large excess of Ca .  2+ Ca  which cannot be rapidly  The flow dialysis system described in Chapter I was adapted for 2+ measuring Ca  -uptake.  MATERIALS AND METHODS 2+ Measurement of Ca  -uptake  Placental plasma membrane vesicles were incubated at 26°C in 20 mM Tris-HCl buffer pH 8.0, 100 mM NaCl, 10" M 5  4 5  Ca  2 +  and  indicated, with or without 5 mM ATP (final volume 1.5 ml).  4 0  Ca  2 +  as  At the end  of the incubation period 3 x 0.1 ml aliquots were taken for the determination of Pi release.  One ml of the incubation medium was introduced 40 2+  to the upper chamber of the flow dialysis c e l l , 0.5 ml of 1 M was added, and the diffusion rate was measured.  Ca  The diffusion rate of  the control without ATP was also measured. The difference in the d i f fusion rates represents the fraction of calcium which i s not available 2+ for displacement; thus Ca  -uptake can be calculated. After each measure-  ment the upper chamber was emptied and washed three times with the  effluent buffer (20 mM Tris-HCl pH 8.0, 100 mM NaCl) before the next 45 sample was introduced.  Ca counting was carried out as described i n  Chapter I, protein was determined by the Lowry method (1951), and Pi released by the Gomori (1942) method. RESULTS 2+ Evaluation of the Flow Dialysis Method for Measuring Ca -uptake The usefulness of the method for measuring calcium uptake was tested by incubating vesicles for 2 hours i n the presence of ATP. Displacement of C a 4 5  cell.  2 +  by 3 x 10 M -1  4 0  Ca  2 +  was measured i n the flow dialysis  The results are represented i n Figure 33. I t is evident that i n 2+  the presence of 5 mM ATP, 5 mM Ca  and the placental plasma membrane  vesicles (Figure 33, column B), part of the C a 4 5  2 +  (25%) i s not available  for displacement after 2 hrs of incubation at 24°C. From the results of the various controls i t i s concluded that this fraction of non2+ displacable Ca represents uptake by the vesicles and i s not the result of  nonspecific  unexchangable  incubation f l u i d .  binding  t o any o f  the  constituents  of  the  Thus the s u i t a b i l i t y of the modified flow dialysis  2+ method for Ca -uptake studies i s demonstrated. 2+ The Effect of Incubation Time on Ca -uptake and ATP Hydrolysis Calcium uptake and ATP hydrolysis by the placental plasma mem2+ brane vesicles showed similar kinetic behaviour (Figure 34A).  No Ca -  uptake was detected in the absence of ATP. The ratio between ATP  m  2.5  D  E  F  w 2.0 CD  LU IO  O  g  0  1.5  E °CL 3  CL O  E rr E o>  .2<o  o  v)  0.5 h  Q Q0.0  L  Membrane protein mg /ml 4 0 . 2+ . Ca ATP  Figure 33.  in mM  1.6  1.6  5  5  5  5  5  in mM  The effect of the major constituents of the incubation medium on 45ca displacement by 3 x ItHM 40ca . The samples were incubated for two hours i n 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10-5M 45 2+, nd different combinations of ATP, 4 0 c a and protein as indicated. 4 0 c a (3 x 10~1M) was added after two hours of incubation at 24°C, and the diffusion rate obtained was divided by the cpm concentration i n the upper chamber to give cpm/ml effluent per 10 cpm/ml upper chamber f l u i d . 2+  2+  Ca  a  2+  6  112  0  60  120  180  Time in Minutes  Figure 34A. The effect of incubation time on Ca "^-uptake and ATP hydrolysis. The samples) were incubated under standard conditions. Protein concentration was 1.78 mg/ml. © o x  2+ Ca -uptake i n the presence of 5 mM ATP and 5 mM Ca o, Pi release under the same conditions 2+ x, Ca -uptake in the absence of ATP  113 2+ hydrolysis and Ca  -uptake was 7.7 (Figure 34B). Thus for every 7.7 2+ ymole ATP hydrolyzed 1 ymole Ca was taken up. This ratio was constant 2+ throughout the incubation time.  These results demonstrate Ca  -uptake  in the absence of calcium-precipitating ion (except for the Pi released). However, the process was slow (even after 2 hrs of incubation steadystate conditions were not reached) and required high protein concentration (1.5-2.0 mg protein/ml). 2+ An attempt to improve the efficiency of Ca -uptake by somcation (sonic dismembrator, Artek N.Y.) for up to 30 min at 40 watts, 20 Kh, did 2+ not affect the efficiency of Ca  -uptake.  However, i t was observed that  2+ Ca  -uptake by freshly prepared vesicles was very low.  "Aging" the  vesicles by storing for 2 weeks at 4°C improved substantially the e f f i 2+ ciency of Ca  uptake, and the Pi/Ca ratio dropped to 4 (in the presence  of mM ATP ofandExternal 5 mM C aCa )2+. Concentration on Ca 2+ -uptake and Pi/Ca Ratio The 5 Effect 2+  At Ca  2+  concentrations below 10  -3  M, Ca  2+  -uptake was very low  (Figure 35A) (less than 0.05 ymoles/mg protein per 2 hrs) and Pi/Ca ratio values were very high, indicating extremely low efficiency (Figure 35B). Ca 2+ -uptake showed a sharp increase at Ca 2+ concentrations between 10 -3 M-10 -2 M (Figure 35A), with a similar increase in Ca 2+ -uptake e f f i -2 2+ ciency. The Pi/Ca ratio dropped to 2 at 10" M Ca (Figure 35B). Estimation of Calcium Concentration in the Vesicles The estimation of the intravesicular calcium concentration requires the knowledge of vesicular volume.  An attempt to measure this  114  0.6  -I  0  1.0  2.0  3.0  4.0  5.0  /jmoles Pi released mg Protein  Figure 34B.  The relationship between Pi release and Ca -uptake. The data to construct this figure were obtained from Figure 34A.  115  Figure 35A. The effect of Ca^ concentration on Ca^-uptake. The samples were incubated for two hours under the standard conditions with different concentrations of Ca2+. The results are expressed i n ymoles C a accumulated per mg protein. Protein concentration was 1.72 mg/ml. T  2+  2+ B. The effect of Ca concentration on Pi/Ca ratio. Pi release i s expressed i n ymoles/mg protein (was measured in the same samples which were used for constructing Figure 35A), and Ca -uptake i n ymoles/mg protein. 2+  116 volume with  labelled sorbitol (which does not penetrate membranes)  was not successful. It was found that after centrifugation of 5 ml of a suspension of vesicles for 30 min at 35,000 x g, the sorbitol concentration was lower in the supernatant (top 4 ml) than in bottom 1 ml which contained the vesicles.  Thus some binding of sorbitol to the  vesicles made this method for measuring vesicular volume ineffective. The vesicular volume of a sarcoplasmic reticulum preparation, using ultracentrifugation, was estimated to be 10 yl per mg membrane protein (Weber ejt al_., 1966).  Using this approximate figure, the intravesicular  2+ Ca -concentration was calculated to be 190 mM (1.9 ymole/10 yl = mg 2+ protein) after 2 hrs of incubation. The i n i t i a l Ca concentration was 10 mM and after two hours 20% was taken up by the vesicles, so that 2+ the final external Ca  concentration dropped to 8 mM.  Thus the intra-  vesicular calcium was concentrated approximately 24-fold (190/8 = 23.8). The data was obtained from Figure 35A. 2+ in the presence of 5 mM Ca  When the vesicles were incubated  and 5 mM P i , the vesicles concentrated  calcium 39-fold (calculated from Table V). . 2+ Phosphate Effect on Ca -uptake 2+ To exclude the p o s s i b i l i t y that Ca accumulation in the presence of ATP i s due to the release Pi which acts as a calcium-precipitating ion, samples were incubated with 0.3 and 5 mM ^HPO^. 7^0 in the 2+ absence of ATP under the standard conditions (5 mM Ca 2+ No Ca -uptake was found.  for two hours).  When the samples were incubated with Pi (5 mM) 2+  and ATP (5 mM) a marked increase in Ca -uptake was observed and the Pi/Ca ratio dropped from 4.0 to 1.7. The results so obtained are presented i n Table V.  117  Table V. The effect of phosphate on Ca -uptake. The samples were incubated for 2 hours i n 20 mM Tris-HCl pH 8.0 100 mM NaCl, 5 mM Ca2+, ATP and Pi as indicated. Protein concentration was 2.1 mg/ml.  Additions 5 mM ATP  Ca^-uptake ymoles/mg protein  Pi release ymoles/mg protein  Pi/Ca ymoles Pi ymoles Ca"  0.52  2.2  4.0  + 0.3 mM Pi  0.52  2.2  4.0  5 mM ATP + 5 mM Pi  1.2  2.2  1.70  5 mM ATP  118  Table VI.  The effect of 5.4 mM Mg*"''" on ^Ca displacement (diffusion rate) by 3 x 1(HM 40ca2+ and Ca2+-uptake. The samples were incubated for two hours at 24° C in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10~ M 45ca, and 2.2 mg membrane protein, i n final volume of 1.85 ml. The concentrations of Ca2+, Mg2+, and ATP were varied as indicated. 5  Additions .2+ mM Ca' 2+ mM Mg  5.4  5.4  Diffusion rate _, cpm/ml effluent" per 106 cpm/ml upper chamber fluid 2+ Ca -uptake nmoles C a mg protein 2+  2,540  -  0.02  0.02 5.4  5.4  5.4  mM ATP  5.4  5.0  5.0  5.0  5.0  2,020  1,990  2,520  2,330  934  986  -  1.4  119 2+ 2+ Mg Effect on Ca -uptake 2+ 2+ The effect of Mg (5.4 mM) on Ca -uptake was studied when the 2+ vesicles were incubated in the presence of high (5.4 mM) Ca , and low ( 2 x 10" M) C a . Mg (5.4 mM) had no effect on Ca -uptake when 2+ the external medium contained 5.4 mM Ca . However, when the external -5 2+ medium contained only 2 x 10 M Ca , the diffusion rate dropped from 2,520 for the control (without Mg ) to 2,330 (cpm/ml effluent per 10 2+ cpm/ml upper chamber f l u i d ) when Mg (5.4 mM) was present, indicating 2+ 5  2+  2+  2+  2+  6  that the vesicles accumulated about 1.4 nmoles Ca  per mg protein.  The results are presented i n Table VI. DISCUSSION The flow d i a l y s i s method, which was used successfully for studying ?+ 2+ Ca -binding (Chapter I ) , was also found suitable for measuring Ca uptake.  Taking advantage of the flow dialysis system, a different and 2+ 2+ more simple definition of uptake was possible; Ca taken up i s the Ca which cannot be rapidly displaced by a high concentration of competitor, due to accumulation i n the vesicles (in the presence of ATP).  This  2+ definition enables one to distinguish between Ca bound to the external 2+ surface of the vesicles and the Ca stages of the incubation period.  accumulated in the vesicles at a l l  This definition contrasts with other  definitions of binding and uptake (Entman e_t al_., 1973; Repke and Katz, 1972) i n which the distinction between binding and uptake i s possible only at time zero.  Thus any event during the incubation period that  120 alters the binding of calcium to the external surface of the vesicles from the time zero value may distort the measurement of uptake. For example, the hydrolysis products of ATP (which are not present at time 2+ zero) can form a complex with Ca (e.g. Ca-ADP). The concentration of such complexes w i l l be time dependent and so w i l l their binding to the external surface of the vesicles.  Thus taking measurements later than  time zero without being able to distinguish between binding and uptake, 2+ can overestimate Ca -uptake. Since the definition presented here enables one to distinguish between binding and uptake throughout the incubation 2+ period, such overestimation of Ca -uptake i s avoided. The f i r s t question which this study was designed to answer related to the a b i l i t y of the vesicles of the placental plasma membrane to accumulate calcium. Clearly these vesicles are capable of accumulating calcium, even without a calcium precipitating ion (except for the Pi released due to ATP hydrolysis).  After two hours of incubation the  concentration of calcium i n the vesicles was calculated to be 190 mM and in the external medium 8 mM.  Thus calcium was concentrated approximately 2+  24-fold.  A 40-fold increase i n Ca  was observed when the vesicles were 2+  incubated in the presence of 5 mM Ca  and 5 mM P i .  In this study the  use of oxalate as calcium-precipitating ion was avoided, since i t would result i n a major deviation from physiological conditions. Calcium accumulation by the sarcoplasmic reticulum was calculated 2+ to reach a concentration of 50 mM (500-fold over the external Ca tration) in the absence of oxalate (Hasselbach, 1964).  concen-  Meisner (1973)  calculated the total calcium concentration the vesicles of sarcoplasmic  121 reticulum to be 30-35 mM, and the free calcium concentration 13-18  mM  (Inesi, 1972).  In less specialized calcium accumulating systems such 2+ as platelet membranes, the intravesicular Ca concentration can reach 2+ only 1 mM, a 10-fold increase over the external Ca  concentration,  in the absence of oxalate and in the presence of ATP (Robblee et a l . , 1973).  Evidently the calcium concentration within the placental plasma  membrane vesicles (as measured in the absence of oxalate) exceeds that of the sarcoplasmic reticulum and platelet membrane vesicles.  However,  the concentrating capability f a l l s far behind that of the sarcoplasmic reticulum, although i t is higher than that of the platelet membrane vesicles. 2+ The stoichiometry of Ca -uptake was somewhat different from one preparation to another, and fluctuated between 4-8 ATP molecules 2+ hydrolyzed for each Ca molecule taken up (measured i n the presence 2+ 2+ of 5 mM Ca ). A constant ratio of 2 molecules of Ca taken up for each ATP molecule hydrolyzed, has been repeatedly reported for sarcoplasmic reticulum (Martonosi and Feretos, 1964; Weber e_t al_., 1966; Hasselbach and Makinose, 1972).  and  However, when the vesicles are leaky,  higher Pi/Ca ratios (4-8) were reported for the sarcoplasmic reticulum (Racker and Eytan, 1973; Huxtable and Bressler, 1973). membranes the Pi/Ca ratio i s between 10-20  For platelet  (Robblee, 1973).  The lowest  Pi/Ca ratio observed in this study was 1.8 (when the incubation medium 2+ contained 10 mM Ca  ).  The high Pi/Ca ratio can be explained as follows:  1) The percentage of closed vesicles as seen by electron microscopy i s approximately 50%.  2) It i s quite possible that some of the closed  122 2+ vesicles are inside-out; thus no accumulation of Ca takes place. 3) The calcium which i s complexed with ATP i s not taken up by the vesi2+ 2+ cles. Only free Ca i s available for uptake; when the Ca concentration _3 is 10 M very l i t t l e calcium is available. On the other hand, Ca-ATP PH-  I'S the substrate for the Ca -ATPase, with Km = 0.25 mM.  Thus, with  2+ increasing Ca concentration, ATP hydrolysis w i l l approach a maximum 2+ before significant Ca -uptake occurs. This might explain the logarithmic 2+ decrease i n Pi/Ca ratio as the external Ca  concentration rises. A l l  of the above factors and others w i l l tend to increase the apparent Pi/Ca ratio so that the actual Pi/Ca may be much lower and might even be as low as was reported for the sarcoplasmic reticulum (0.5) (Weber et a l . 1966). It was noted that i n the presence of 5 mM P i , but i n the absence 2+ of ATP, no Ca bation.  was accumulated i n the vesicles after two hours of incu-  This indicates that the vesicles are f a i r l y impermeable to  calcium. The exact role of ATP was not revealed i n this study. However, the p r o f i l e of ATP hydrolysis as a function of incubation time was the 2+ 2+ same as the p r o f i l e of Ca -uptake, and no uptake of Ca was detected in the absence of ATP. As has been mentioned already, the most obvious role of ATP i s to serve as the source of energy for the uptake process. Nakamura and Konishi (1974), observed that for brain microsomes, though 2+ 2+ no Ca -uptake occurred without ATP, Ca uptake did not follow the p r o f i l e of Pi release.  Their conclusion was that the brain microsomes 2+ 2+ exhibit ATP-dependent Ca -uptake without the participation of Ca ,  123 Mg 2+ -dependent ATPase. They suggested that the role of ATP is to provide the phosphate for the formation of the phospholipid triphosphoinositide through the action of the enzyme diphosphoinositide kinase (EC 1.68).  2.7.  The s t a b i l i t y constant of the complex between this phospholipid 2+  and Ca  i s higher (10-fold) in the presence of ATP than in i t s absence  (Hendrickson and Reinertsen, 1969), and this might explain the role of 2+ ATP in Ca  -uptake by brain microsomes.  The profiles of Pi release and  2+ Ca  -uptake by placental plasma membrane vesicles show the same kinetic  behaviour, unlike the case of brain microsomes.  Thus i t i s unlikely  that the role of ATP in the uptake process i s other than acting as a source of energy. 2+ The increase in Ca -uptake in the presence of 5 mM Pi and 5 mM ATP, i s most l i k e l y due to precipitation of calcium-phosphate i n the 2+ vesicles, thereby decreasing the leakage of Ca . 2+ 2+ A positive effect of Mg on Ca -uptake was noted only at low Ca concentration, and i s interpreted as follows. Mg frees Ca 2+ from the complex with ATP. Since Ca i s the substrate of the uptake 2+ system, there i s more Ca available. The enzymes can s t i l l be a c t i 2+ vated by Mg-ATP so that the overall result w i l l be an increase in Ca 2+ uptake. However, this i s true only when the Ca concentration i s very 2+ 2+ 2+ low compared with that of Mg . At 5 mM Ca and 5 mM Mg the increase 2+ in Ca -uptake was not significant. 2+ 2+ Ca -uptake by the sarcoplasmic reticulum i s dependent on Mg , as i s the ATPase a c t i v i t y (Martonosi and Feretos, 1964; Weber et a l . , 1966; Hasselbach and Makinose, 1972).  No such dependency was noted in _3 this study, provided that calcium concentration was above 10 M.  124 In conclusion, the present study demonstrated that the placental 2+ plasma membrane vesicles are capable of accumulating Ca , and this 2+ appears to be dependent on ATP hydrolysis by the placental Ca -ATPase.  SUMMARY The placental plasma membrane vesicles are capable of accumu2+ lating up to 190 mM Ca . This i s 24-fold higher than the external 2+ Ca  concentration. This process i s dependent on ATP hydrolysis by the placental  Ca -ATPase. 2+  2+ The Pi/Ca ratio i s dependent on the external Ca concentration, 2+ and reaches the value of 2 at 10 mM Ca . 2+ Phosphate (5 mM) can double Ca -uptake when measured i n the 2+ presence of 5 mM Ca . 2"t~ 2*4* 2"f* Mg increased Ca -uptake only at a low Ca concentrations, 2+ and had no significant effect at 5 mM Ca .  GENERAL CONCLUSIONS 2+ The objective of this thesis was to investigate the Ca -related properties of the placental plasma membranes, and to f i l l a gap created by the fact that there i s no f u l l description of these properties for any plasma membranes involved in asymmetrical  calcium transport.  This  section w i l l attempt to correlate these properties with each other and to speculate on their possible role in calcium transport across the placenta. 2+ 2+ The effect of Ca concentration on Ca -binding, and the veloc 2+ 2+ c i t i e s of Ca -ATPase and Ca -uptake, was the key parameter which was investigated. Figure 36 shows this effect. It i s evident that at a 2+ -4 2+ 2+ Ca concentration of 10 M only Ca -binding and Ca -ATPase reached significant level.  Ikemoto (1974) found that, in the sarcoplasmic reticu-  lum, the high a f f i n i t y sites for Ca  2+  have a Ks = 4 x 10  -6  M.  This figure  2+  was the same as the Km of the Ca  -ATPase. He concluded that high af-  f i n i t y sites are involved in activation of the enzyme. 2+ the Km for Ca  In this study  -4 (2.5 x 10  M) with respect to ATPase a c t i v i t y does not 2+ correspond with either of the dissociation constants for the Ca -binding -5 -3 sites (Ks-j = 3 x 10 M, KSr, = 10 M). There i s no evidence that these 2+ sites are involved in the activation of Ca  -ATPase. However, theore-  t i c a l l y only the high a f f i n i t y sites could take part, since ATP forms a 2+ -4 2+ complex with Ca (Ks ^ 10~ M) which w i l l dissociate most of the Ca from the low a f f i n i t y sites. 125  126  + CM  Figure 36. The effect of Ca concentration on Ca -binding and on the velocities of ATP hydrolysis and on Ca -uptake. 2+ 2+ 100% Ca -binding = 250 nmoles Ca /mg protein 2+  100% ATP hydrolysis = 4 ymoles Pi/mg protein 2+ 2+ 100% Ca -uptake =1.9 pmoles Ca /mg protein Protein concentration i n the incubation medium was 2 mg/ml.  127 2+ The presence of high a f f i n i t y sites for Ca was reported for 2+ membranes which are involved i n active transport of Ca (see Chapter 2+ I). The suggested role for these sites i s binding Ca passively as 2+ the f i r s t step i n Ca transport. However, because no specific inhibitor 2+ is available, either for the sites or for Ca -ATPase, the role of these sites remains speculative. The a b i l i t y of the placental plasma membrane vesicles to accumu2+ late Ca has been demonstrated. This accumulation i s dependent on ATP 2+ 2+ hydrolysis by the placental Ca -ATPase. The involvement of Ca -ATPase 2+ in Ca -uptake by the sarcoplasmic reticulum i s well documented (see Chapter V). In sarcoplasmic reticulum a single protein acts as both 2+ ATPase and ionophore in promoting Ca -uptake a c t i v i t y (Warren et a l . , 2+ 1974). The present study suggests that the substrate of Ca -ATPase 2+ is Ca-ATP complex, while only free Ca can be transported. Thus i n 2+ -3 the presence of ATP at Ca concentration below 10 M there was very 2+ 2+ l i t t l e free Ca and Ca -uptake was i n s i g n i f i c a n t . 2+ The s p e c i f i c i t y of the Ca -related properties was studied by 2+ introducing Mg  into the incubation medium and analysing i t s effect 2+ on the measured values. The a f f i n i t y of Ca for the high a f f i n i t y 2+ sites was 10-fold higher than for Mg . Thus i f these sites face the 2+ 2+ extracellular f l u i d where the Ca concentration i s higher than the Mg 2+ concentration these sites w i l l be occupied by Ca . However, i f the 2+ high a f f i n i t y sites face the cytosol whei;e the Ca concentration i s 2+ much lower than the Mg concentration, these sites w i l l be occupied 2+ to a large extent by Mg .  128  The effect of [Ca ]/[Mg ] concentration ratio on Ca -ATPase a c t i v i t y was pronounced. 2+  From the kinetic behaviour of the enzyme as a 2+  function of the [Ca ]/[Mg ] r a t i o , i t i s concluded that the two ions activate tiie enzyme at the same s i t e .  The derived formula enables one  to predict very precisely the velocity of the reaction under any com2+ bination of Ca  2+ and Mg  concentrations.  There was no requirement for  2+ Mg , and the reaction could proceed with either of the two ions, although Ca 2+ was more e f f i c i e n t . The Ca2+ -ATPase of sarcoplasmic reticulum has 2+ an absolute requirement for Mg (MacLennan, 1970) and no activation by 2+ 2+ 2+ Ca was obtained in the absence of Mg . The same i s true for Ca uptake by the sarcoplasmic reticulum (Martonosi and Feretos, 1964; Weber e_t al_., 1966; Hasselbach and Makinose, 1972). 2+ Magnesium did not affect Ca -uptake by the vesicles of placental 2+ -5 plasma membranes when the Ca concentration was 5 mM. At 2 x 10 M Ca 2+ the effect of Mg 2+ on Ca2+ -uptake was pronounced compared with Ca 2+-uptake in the absence of Mg2+ . However, the actual value was very 2+ -2 2+ low relative to Ca uptake at 10 M Ca '(1.4 nmoles/mg compared with 1.9 ymoles/mg).  One can speculate that under physiological conditions 2+ 2+ two systems are active: 1) a low Ca concentration, Mg dependent 2+ 2+ system with low efficiency, and 2) a high Ca concentration, Mg i n 2+ dependent system with higher efficiency. The overall Ca -related 2+ properties of the placental plasma membranes are independent of Mg and the entire process from binding to the membrane through activation 2+  of the enzyme and f i n a l l y to Ca -uptake, appears to be dependent on Ca  alone.  This situation appears unique to the placental plasma  129 membranes. The other systems described i n the literature deal with the Ca  2+ 2+ -related properties of membranes involved i n i n t r a c e l l u l a r Ca -  regulation. 2+ The physiological role of this system i n Ca -transport across the placenta cannot be defined clearly. The objective of this thesis 2+ was to study the relation between Ca Ca  and the membrane involved i n  2+ -transport.  However, one can speculate that the vesicles which 2+ are loaded with Ca , may be an in vivo part of the continuous tubular network which has been seen with electron microscope i n the trophoblast 2+  (Plates 1 and 2).  Croley (1973) localized Ca  network within the human trophoblast.  histochemically i n this  Since the human placenta and  guinea pig placenta have the same basic structure (haemochorial) one 2+ can expect similar localization of Ca i n the guinea pig trophoblast. Thus fusion of segments of this network with the basal plasma membrane w i l l provide the l i n k between the maternal and the fetal c i r c u l a t i o n . 2+ The Ca -related properties of placental plasma membranes described i n this thesis, provides possible explanations of the f i r s t steps 2+ in the asymmetrical transplacental Ca -transport.  Further investigation  is required before the entire process i s f u l l y understood.  REFERENCES  Alonso, G. and Walser, M. (1968). ATP s p l i t t i n g and calcium binding by brain microsomes measured with a rapid perfusion method. J. Gen. Physiol. 52:111. Barouch, W. W. and Moos, C. (1971). The effect of temperature on actin of heavy meromyosin ATPase. Biochim. Biophys. 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