The Ca2+ Pump of Plasma Membranes 1st Edition

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 A C K N O W LED G M EN TS

We wish to express our gratitude to our colleagues H. Barrabin, A. J. Caride, R. B.

Kratje, J. N. Larocca, H. Mugica, D. E. Richards, and J. P. F. C. Rossi for their
experimental work on the C a2* pum p perform ed in our laboratory and to all the sci­
entists whose studies have made this book possible. The authors’ research programs
have been supported by the Consejo Nacional de Investigaciones Cientificas y Tecnicas
(CONICET) of A rgentina, the University of Buenos Aires, the Secretaria de Ciencia y
Te’cnica of A rgentina, the RLA 78/024 program from UNESCO and the Fundacion
Roemmers.
 TA B LE O F C O N TEN TS

C hapter 1
The Cellular Calcium
A. F. Rega

I. The M easurem ent of C a2* in the Cytosol...................................................................... 1

A. Total and Free-Ionic (Ca2+) C alcium ................................................................1
B. M ethods to Determine Cytosolic Ca2+ C oncentration.................................. 1
1. C a2+-Binding D yes................................................................................... 2
2. C a2+-Activated P hotoproteins................................................................3
3. C a2*-Selective Electrodes........................................................................ 3
4. Null P oint T itratio n .................................................................................5
II. The Distribution of Calcium Among the Cell C o m ponents....................................6
III. Calcium in the C y to so l..................................................................................................... 7
R eferences..................................................................................................................................... 10

C hapter 2
The C a2* Hom eostasis
A. F. Rega

I. How Does C a2* Enter and Leave the C ell?................................................................. 13

A. Excitable C ells..................................................................................................... 13
B. Nonexcitable C ells..............................................................................................14
II. T ransport of C a2* by Intracellular Organelles........................................................... 15
III. The Role of the Transport Mechanisms in the Regulation of C a2+
C oncentration in the C y to so l......................................................................................16
R eferences..................................................................................................................................... 19

C hapter 3
Calcium and Cell Function
P . J. G arrahan

I. Introduction....................................................................................................................... 21
II. The Messenger Role of C a2*...........................................................................................21
A. The M echanism of the Increase in Cytosolic C a2* inStimulated
Cells.......................................................................................................................21
1. C a2* Channels in Excitable M em branes........................................... 22
2. Cell Membrane Phospho- and Polyphosphoinositides and
Receptor-M ediated Ca2t M obilization.............................................24
3. The Release of Ca2* from Intracellular S to re s................................24
III. C a2+-Binding Proteins.................................................................................................... 25
A. General Properties..............................................................................................25
B. C alm odulin...........................................................................................................26
1. D istribution.............................................................................................27
2. Chemical and Physical P ro p erties..................................................... 27
3. Binding of Ca2*....................................................................................... 27
IV. Enzymes that Depend on C a2* and C alm odulin..................................................... 29
A. Enzymes Involved in Glycogen M etabolism ................................................ 29
1. Skeletal Muscle Phosphorylase Kinase.............................................. 29
2. Glycogen Synthetase Kinase................................................................ 30
3. P rotein-Phosphatases............................................................................30
 B. Enzymes Involved in Cyclic Nucleotide M etabolism .................................. 31
C. Enzymes Involved in Regulation of Muscle C ontraction and Other
Motile P ro cesses.................................................................................................31
1. Myosin Light-Chain K inase..................................................................31
2. Phospholam ban K in ase.........................................................................31
3. Protein Kinases of Sarcoplasmic Reticulum of Skeletal
M uscle...................................................................................................... 31
D. Nervous Tissue Protein K inases.......................................................................32
E. M em brane Protein Kinases in Other Tissues................................................ 32
F. Plasm a M embrane Ca2*-ATPases....................................................................32
G. Other E nzym es.....................................................................................................33
1. NAD Kinase..............................................................................................33
2. Platelet Phospholipase A 2 .................................................................... 33
3. 15-Hydroxyprostaglandin Dehydrogenase........................................ 33
V. C a2*-Dependent Enzymes that Do Not Require C alm odulin.................................33
A. P rotein Kinase C ..................................................................................................33
B. Calpain and C alpastatin.....................................................................................34
VI. Regulation of Cell Functions by Ca2* ....................................................................... 34
A. Endo- and Exocytosis......................................................................................... 34
B. Cell Motility and Self-Assembling Cell Com ponents.................................. 35
C. Cell D ivision.........................................................................................................36
D. M em brane C h an n els...........................................................................................37
1. The Gardos E ffect.................................................................................. 37
2. Intercellular C om m unication...............................................................38
R eferences..................................................................................................................................... 39

C hapter 4
From the Discovery of the C a2* Pum p in Plasma Membranes to the Demonstration of
Its Ubiquitousness
A. F. Rega

I. A Historical Review....................................................................................................... 45
A. The First R e p o rt..................................................................................................45
B. The Finding of the Ca2* Pump in Plasma Membranes Other than
the E rythrocyte.................................................................................................. 47
C. How to Identify a Ca2*-Transporting System with the Ca2* Pump
from Plasma M em brane................................................................................. 49
II. Some Properties of the Ca2+ Pum p from Various Cell T y p es.............................. 51
A. Circulating C e lls..................................................................................................52
B. Excitable C ells......................................................................................................52
C. Tissue Cells............................................................................................................53
D. O ther C e lls........................................................................................................... 54
E. C o nclusion............................................................................................................55
R eferences.....................................................................................................................................56

C hapter 5
Isolation and Purification of the C a2* Pump
P. J. G arrahan

I. The M ain Difficulties and the First A ttem pts.......................................................... 59

II. The Use o f Calm odulin Affinity Chrom atography to Purify the
C a2*-A TPase................................................................................................................... 60
III. Properties of the Purified C a2*-ATPase.................................................................... 62
A. S tab ility .................................................................................................................62
B. Molecular Weight and C om position..............................................................62
C. Kinetic P roperties................................................................................................63
D. Reconstitution of the Purified Enzym e..........................................................64
E. Immunological Reactivity..................................................................................64
R eferences..................................................................................................................................... 64

Chapter 6
Transport of Ca2* and A TP Hydrolysis by the Ca2* Pump
A. F. Rega

I. T ransport of Ca2*............................................................................................................ 67
A. Introduction..........................................................................................................67
B. Preparations Used for Transport S tu dies.................................................... 67
1. Intact Red Blood Cells..........................................................................67
2. Resealed G hosts...................................................................................... 68
3. Inside Out Vesicles.................................................................................69
4. Reconstituted L iposom es..................................................................... 69
5. Squid A x o n s ............................................................................................ 70
C. Dependence on Ca2* C oncentration................................................................71
1. Activation by Ca2*.................................................................................. 71
2. Inhibition by Ca2*................................................................................... 72
D. Substances and Treatments that Increase the Rate of T ran sp o rt..............74
E. The Electrical Balance During Transport of Ca2*....................................... 75
1. Electrogenic T ra n sp o rt..........................................................................75
2. Electroneutral T ransport.......................................................................76
II. A TP H ydrolysis............................................................................................................... 77
A. Dependence on C a2* C o ncentration............................................................... 77
1. Activation by Ca2*..................................................................................77
2. Inhibition by Ca2*....................................................................................78
3. The Mechanism of the Inh ibition....................................................... 79
III. Dependence on A T P ...................................................................................................... 80
A. Substrate Specificity........................................................................................... 80
B. The Substrate C u rv e .......................................................................................... 81
C. Kinetic Analysis of the Substrate C urve........................................................ 83
1. Kinetic Schemes that Give Biphasic Substrate Curves......................83
a. Two Different Enzym es........................................................... 83
b. Two Active Sites in the Same E nzym e..................................83
c. The Substrate as A c tiv a to r..................................................... 84
2. Com parison of the Kinetic Equations................................................ 85
a. The M athematical Equivalence of Rate E q u atio n s..........85
D. On the State of ATP as the Substrate for the Overall R eactio n ............... 87
R eferences..................................................................................................................................... 88

C hapter 7
O ther Properties and Coupling of C a2* T ransport and A TP Hydrolysis
A. F. Rega

I. O ther P ro p e rtie s..............................................................................................................91

A. Specificity for Ca2* ............................................................................................. 91
 B. The A pparent Affinity for C a2* ...................................................................... 91
1. Modifiers of the Apparent Affinity for Ca2*................................... 93
2. The EGTA E ffect................................................................................... 93
C. The Num ber of Ca2* Sites................................................................................. 95
D. Dependence on p H ............................................................................................ 96
E. Dependence on Tem perature............................................................................97
II. The Coupling Between Ca2* Transport and A TP Hydrolysis.................................99
A. Energetics of Ca2* T ran sp ort........................................................................... 99
B. The Stoichiometry of Ca2* T ra n sp o rt............................................................ 99
C. Reversal of the Ca2* P u m p .......................................................................... 101
R eferences....................................................................................................................................102

Chapter 8
Partial Reactions of the Ca2* ATPase
P. J. G arrahan

I. The Elementary Steps of ATP H ydrolysis.............................................................105

A. Introduction..................................................................................................... 105
B. P h o sp h o ry latio n .............................................................................................105
1. Kinetics of the Phosphorylation R eactio n ...................................106
2. Reversal of Phosphorylation...........................................................107
3. Chemical Properties of the Phosphoenzym e...............................107
C. D ephosphorylation........................................................................................ 109
1. The E ^ P ^ E j ^ P Transition......................................................... I l l
D. The E2^ E, T ran sitio n ................................................................................. 113
II. Reaction Scheme for the Hyrolysis of A T P .......................................................... 114
III. Energy Changes During the Elementary S teps......................................................115
IV. The Phosphatase Activity of the C a2*-A T Pase.................................................... 116
A. General P roperties.......................................................................................... 116
B. Kinetics.............................................................................................................. 116
1 The Substrate C u rv e ......................................................................... 116
2. Dependence on Mg2*......................................................................... 117
3. Activation by Ca2*............................................................................. 118
4. Effects of M onovalent C ations......................................................... 118
C. The Interaction Between the Sites for pN PP and the Sites for
A T P ...................................................................................................................119
1. The High-Affinity Site.........................................................................119
2. The Low-Affinity S ite .........................................................................119
3. ATP Hydrolysis During Phosphatase Activity.............................. 119
D. Phosphatase Activity and Active C a2* T ra n s p o rt..................................... 122
R eferences................................................................................................................................... 123

Chapter 9
Activation by Magnesium and by Alkali Metal Ions
P. J. G arrahan

I. M agnesium .....................................................................................................................127
A. Ca2*-ATPase Activities in the Absence of Added Mg2*........................... 127
B. The Kinetics of Activation by M g2*.............................................................. 127
1. Activity vs. Mg2* Concentration........................................................128
2. Activity vs. MgATP C oncentration.................................................128
3. Activation by Mg2* under Steady-State C onditions......................129
 C. The R elation between Ca2*-ATPase Activity and the Concentration
of Mg2*.............................................................................................................129
1. Activation by Mg2* ............................................................................130
2. Inhibition by Mg2*............................................................................. 131
D. The Mechanism of the Activation by Mg2* ..............................................131
1. M gATP as the S u b strate..................................................................131
2. Direct Binding of Mg2* to the A T P ase.......................................... 132
II. Alkali Metal I o n s ........................................................................................................ 132
A. The Kinetics of Activation by Alkali Metal Io n s ....................................133
B. The Sideness of A ctiv ation .......................................................................... 134
C. The Effects of Alkali Metal Ions on the Elementary Steps of the
ATPase R e a c tio n .......................................................................................... 134
D. The Physiological Meaning of A ctivation.................................................134
R eferences.................................................................................................................................135

C hapter 10
Calm odulin and O ther Physiological Regulators of the Ca2* Pump
P . J. G arrahan

I. C alm odulin.................................................................................................................... 137

A. Binding of Calmodulin to the C a2* A T P ase.............................................138
1. Role of Ca2* in Calmodulin B indin g.............................................140
2. Extent of Calmodulin D ependence............................................... 141
3. Binding of Calmodulin under Physiological C o n d ition s..........141
B. Effects of Calmodulin on the Steady-State Kinetics of the
Ca2*-A TPase...................................................................................................142
C. Effects of Calmodulin on the Elementary Steps of the
Ca2*-A TPase...................................................................................................144
II. Conditions and Treatm ents that Mimic the Effect of Calm odulin................... 144
A. The Lipid E n v iro n m en t................................................................................. 145
B. Proteolysis........................................................................................................ 146
C. The Mechanism of the Calmodulin-Like Effects of Acidic Lipids
and P roteolysis...............................................................................................147
III. O ther Physiological R egulators...................................................................................148
A. Protein A ctivators and Inhibitors................................................................. 148
B. P hosphoinositides.......................................................................................... 148
C. Regulation by P h o sp h o ry latio n ....................................................................148
R eferences................................................................................................................................... 149

C hapter 11
Inhibitors o f the C a2* Pum p
P . J. G arrahan

I. Introduction...................................................................................................................153
II. Inorganic Io n s .................................................................................................................153
A. L anthanides........................................................................................................153
B. V anadate............................................................................................................. 154
III. Calm odulin A n tag o n ists.............................................................................................. 156
IV. C om pounds th at React with P ro tein s.........................................................................159
A. N-Ethylmaleimide (N E M ).............................................................................. 159
B. Anion Channel (Band III) Inhibitors........................................................... 161
C. Fluorescein Derivatives....................................................................................161
V. O ther In h ib ito rs.......................................................................................................... 162
A. Q u e rcetin ............................................................................................................162
B. Ruthenium Red..................................................................................................162
R eferences.................................................................................................................................162

Index........................................................................................................................................... 165
 C hapter 1

T H E C E L L U L A R C A L C IU M

A. F. Rega

I. T H E M E A S U R E M E N T O F C a 2+ IN T H E C Y T O S O L

This subject has received much attention during the last few years as a consequence
of the increased awareness of the crucial role of cytosolic C a2* as a second messenger
in a num ber of processes. Ashley and Cam pbell' have edited a book on detection and
m easurem ent of free C a2* in cells, and an authoritative and up-to-date review of the
subject by R. Y. Tsien2 has been published recently.

A. Total and Free-Ionic (Ca2*) Calcium

The actual am ount o f loosely bound or free-ionic calcium in the cytosol is much
lower than the total am ount of calcium, because most of the calcium in the cells is
either sequestered by subcellular organelles or bound to cell structural components or
molecules in the cytosol. To predict chemical reactions in which calcium participates,
it is necessary to know the chemical potential of calcium which can be calculated know­
ing the concentration of free-ionic calcium under ideal conditions. The concentration
of solutes in the cytosol makes it a nonideal solution. As a consequence of this, as with
any other charged solute, free-ionic calcium will be exposed to electrical interactions
with the charged com ponents in the cytosol so that the concentration of free-ionic
calcium will be lower than that of free calcium. There are two ways to overcome this
difficulty: one is to use correction factors3 (activity coefficients) which account for the
decrease in concentration or in chemical potential due to electrical interactions, and
the other is to use C a2*-selective electrodes which allow one to measure the actual
am ount of ionized calcium, because they respond to free-ionic calcium concentrations.
The use of correction factors presents a difficulty in that they vary in value from author
to author and are not reliable for complex mixtures. Anyway, measurements with elec­
trodes have always had to be made relative to standard solutions to which numerical
values of free-ionic calcium concentration have been assigned. From a practical point
of view, this means th at (as has been clearly stated by Tsien2) “ when one says, for
example, th at the free-ionic calcium concentration in a cell was measured to be 1 y.M,
that really means that the calcium activity in the cell was the same as the calcium
activity in a certain calibrating solution in which the other m ajor ionic constituents
were considered to be similar to those of cytosol and which contained 1 p M o f calcium
ions not tightly bound to ligands.” This definition is also valid for all other techniques
used to m easure free-ionic calcium concentration. Throughout this book, C a2* will
stand for free-ionic calcium.

B. M ethods to Determ ine Cytosolic C a2* Concentration

These m ethods were not available until a few years ago because of the difficulties
em anating from the fact that: (1) cytosolic C a2* is always in the 10~6 to 10~9 M range
and has to be m easured in the presence of levels of Mg2*, Na*, and K* which are orders
of m agnitude higher; (2) the m ethod to determine cytosolic C a2* concentration must
allow the insertion of the C a2* probe into the cell without causing any damage to the
structure and function of the cell; and (3) the C a2* probe must not bind significant
am ounts of C a2* from the cytosol.
Four m ethods are now available to measure the concentration of intracellular C a2*:
2The Ca2+ Pump o f Plasma Membranes

yM Ca2+
00m
/^ V O O

nH Mg2+
B

AA-0.017
T

425 450 475 500 525 550 575 600 625 650 675 700
X (nm)
X

FIG U R E 1. D ifferential spectrum o f arsenazo III vs. arsenazo III and var­
ious concentrations o f C a2* (A) and M g2* (B). (From Scarpa, A ., D etection
an d M easu rem en t o f Free C a 2* in Cells, A shley, C. C. and Cam pbell, A . K .,
E d s., E lsevier/N orth -H ollan d , A m sterdam , 1979, 96. W ith perm ission.)

(1) C a2*-binding dyes; (2) C a2*-activated photoproteins; (3) C a2*-selective electrodes;

and (4) null point titration.

1. Ca2*-Binding Dyes
There are two main classes of substances that undergo changes in their optical spec­
tra upon C a2* binding: metallochrom ic indicators and tetracarboxylate dyes. Arsenazo
III and antipyrylazo III are the m ost com m on m etallochromic indicators.4 Both are
derivatives of 2,7-bisazo-l,8-dihydroxy-3,6-naphthalenedisulfonic acid. Ca2* binds to
arsenazo III with a Kj near 60 \jlM and increases arsenazo absorbance at 595 and 658
nm (Figure 1). The change can be detected measuring the differential spectrum of free
arsenazo III vs. arsenazo III plus Ca2*, and it is not a linear function of C a2*. Mg2*
produces a single broader change in absorbance with a maximum at 608 nm (Figure 1)
so that, although arsenazo III is not specific for C a2*, it can be made selective through
the use of an appropriate pair of wavelengths. Com bination of C a2* with antipyrylazo
III increases the absorbance of the dye in the red region of the visible spectrum, while
M g2* produces no change in this region. This property of antipyrylazo III makes it
suitable for m easuring C a2* concentrations w ithout the interference of Mg2* (but not
Sr2*) by differential absorbance at 720 to 790 nm. The Kd of antipyrylazo III for C a2*
is higher than th at of arsenazo III and lies between 60 and 500 ^M. A t C a2* concentra­
tions near to or greater than the Kd, the changes in absorbance are nonlinear with C a2*
concentration.
Because of the relatively low differential extinction coefficient between the free-indi-
cator and the calcium-indicator complexes, considerable am ounts of the indicators are
needed for the assay. This may cause undesirable changes in the concentration of C a2*
in the presence of the indicators. A nother problem is that the stoichiometry of the dye-
calcium complex changes form 1:1 to 2:1 at concentrations of either dye greater than
1 nM . M etallochromic indicators do not permeate and have to be injected into the cells.
Their use for measuring cytosolic C a2*, therefore, is limited to large cells. One m ajor
advantage of these C a2* indicators is their fast response time which becomes im portant
when changes in C a2* concentration are to be measured.
 coodoc- ( 'OOC—\
N XN^COO‘ 'N O O C v^N ^-
-ooc
X I.
EGTA X X

X
BAPTA or benzl: X =H
benz2: X =Me quinl: X =H
broml: X =Br quin2: X =MeO
FIG U R E 2. Structure o f E G T A and som e carboxylate d yes.(F rom Tsien, R. Y ., A n n . Rev. B ioph ys.
B ioen g., 12, 91, 1983. W ith perm ission.)

Recently a new series of tetracarboxylate dyes (Figure 2) derived from l,2-bis(o-

am inophenoxyJethane-N .N .N '.N -tetracetic acid (BAPTA), a molecule very close
to EGTA , or from 2,2-bis(ethoxycarboxyl)methylamino-5-methylphenoxymethyl-6-
methoxy-8-bis-(ethoxycarbonyl)m ethylam inoquinoline (quin2), have been introduced
as C a2* indicators.5-6 These chelators show high affinity (Kd 0.11 and 0.08 for
BAPTA and quin2, respectively), 1:1 stoichiometry for C a2*, and high selectivity for
C a2* over M g2*. Their absorbance and fluorescence increase largely upon Ca2*-binding
in a way which is not directly proportional to C a2* concentration (Figure 3). These
tetracarboxylic acids are hydrophilic and do not permeate the membrane except when
masked with esterifying groups. The resulting esters are hydrophobic and readily dif­
fuse into all cells of a given population. Cytoplasmic esterases then hydrolyze the ester
groups and restore the parent tetracarboxylic dye. This m ethod of entrapping the tetra­
carboxylate chelators is particularly applicable to small cells in suspension.2 6 This,
together with their negligible binding to biological material, makes either BAPTA or
quin2 a highly satisfactory means for nondestructively measuring C a2* concentrations
in the cytosol.

2. Ca2*-Activated Photoproteins
A equorin is a protein of M r near 31,000 (isolated from the jellyfish Aequorea fos-
kalea) which, in the excited state, reacts with C a2* to emit one photon of light of about
460 nm wavelength. It combines with two C a2* with an apparent K* of the same order
as those of the m etallochrom ic indicators. However, in an ionic environment similar
to th at within the cell, the apparent Kd increases to 10~3 to 10~4 M. This is fortunate,
because at C a2* concentrations of about 0.1 pM , the percentage of aequorin bound to
Ca2* will be low, it will be consumed at a low rate, and no disturbances in cytoplasmic
C a2* concentration will occur. Aequorin is highly selective for C a2*. Mg2* retards the
rate of light emission and at physiological concentrations lowers the sensitivity of ae­
quorin to pH changes. A problem with aequorin is that its light output is an exponen­
tial function of C a2* concentration (Figure 4). A relation of this type complicates at­
tem pts at quantification of transient changes in light output, because the same
increm ent in total C a2* will give different increments in light if it is confined to a small
volume or distributed evenly throughout a cell. A nother drawback to the use of ae­
quorin is that it has to be introduced into cells by microinjection or reversible lysis.

3. Ca2*-Selective Electrodes
The simplest m ethod for the measurem ent of C a2* concentration is the C a2*-selective
electrode (Figure 5). The m ain advantage of selective electrodes is that they measure
4 The Ca~+ Pump o f Plasma Membranes

TOO — E x c ita tio n s p e c tra . I

\1 /IOOjjM E m is s io n s p e c tra .
r\oo\\
E m is sio n 4 9 2 n m / / \l E x c ita tio n 3 3 9 n m
/ lU
/ M\ \

i/i I
fanool
Ml 5 0 0 \
nM \
z
D
>-
cr 200 \
<
cr
' 200\ nM \
5a: / nM\ 1
<
U 50 —
I /ioo\\ 1
/ \lI oo « m \
UJ
u
l/l
UJ
a:
OD /5o\\v iO nM \
nM\ l

/
/S<\ V
nM\\ 0 nM \
// n M \
D nM ^

O-Co 3- Ca

o' -----------------------»-----------------------1-------------- _ ^ s l ----------------------- I_______________ 1_______________ |_______________ I

-^ o 300 350 400 450 500 nm 550 600

W A V E L E N G T H / nm

FIG U R E 3. Excitation and em ission spectra o f 20 ^ M q u in 2 with varying C a2+ concentration in a solution
containing 120 to 135 m M K \ 20 m M N a \ 1 m M M g2+, pH 7.05.(From Tsien, R. Y ., P ozzan, T ., and
R ink, T. J., J. Cell. B io l.,9 4 , 325, 1982. W ith perm ission.)

0 r 160 mMKCI 21°C

-1 - 2mM Mg**

y ' 3 '

-7 - --------------
------------- / / -------1--------- 1--------- 1______ i______i______ i______i
EQTA -8 -7 -6 -5 -4 -3-2
log fca*j(M)

FIG U R E 4. The relation betw een the fractional light em ission and
Ca2+ concentration for aequorin. Filled circles, C a2* concentration es­
tablished by dilution o f 1 M C aC l2; blank circles, C a2+ concentration
established by C aEG T A b uffer m ixtures. (From A llen, D . G. and
Blinks, J. R ., D etectio n a n d M easu rem ent o f Free C a 2* in Cells, A sh ­
ley, C. C. and Cam pbell, A . K ., E d s., E lsevier/N orth -H ollan d , A m ­
sterdam , 1979, 96. W ith perm ission.)
 n 40

- 0

- -40

- mV

- -60

- -120

i r
»_______i_______ i----------- 1----------- 1----------- 1----------- 1--------- -*-ieo
9 8 7 6 6 4 3 2 1

pc«2+

F IG U R E 5. C alibration curve o f a C aJ*-selective electrode. Circles

d enote solu tion containing 200 m M K* and squares denote solutions
containing 200 m M K* and 1 m M M g2*.(From O wen, J. D . and Mack
B row n, H ., D etectio n a n d M easu rem ent o f Free C a 2*in Cells, A shley,
C. C. and Cam pbell, A . K ., E d s., E lsevier/N orth -H ollan d , Am ster­
dam , 1979, 96. W ith perm ission.)

the actual C a2* concentration w ithout disturbing the equilibrium between bound and
free calcium th at the indicators that react with C a2* could alter.
The perform ance of ion-selective electrodes is highly dependent on the composition
o f the m em brane in the electrode.7 The use of electrically neutral ionophores for the
m em brane has m ade available C a2*-selective electrodes which have a detection limit of
0.1 fiM or less and a selectivity for C a2* so high that it makes them almost immune
from Mg2* and other ionic species within cells.7 There are, however, some difficulties
in the use of electrodes for measuring the concentration of C a2* in the cytosol. The cell
has to be punctured to allow the tip of the electrode to enter the cell, so that the method
could dam age the plasm a m em brane of the cell and cause leakage. On the other hand,
because of the tip size, electrodes cannot be used in small cells. A nother difficulty with
electrodes is their low response speed which makes them unsuitable for measuring tran ­
sients in C a2* concentration. M icroelectrodes measure the local C a2* concentration in
the surroundings of their tip. This is advantageous when point sampling within the
cytoplasm of a cell is desired, but it represents a m ajor drawback to the use of elec­
trodes for measuring the mean C a2* concentration in the cytoplasm.

4. N u ll P oint Titration
This m ethod consists of measuring the change in C a2* concentration of the suspend­
ing medium due to penetration of C a2* after the plasma membrane of the cells has been
m ade permeable to small solutes.8 Digitonin is generally used to render the membrane
perm eable to Ca2*, because it is effective on cholesterol-rich membranes, but has little
effect on m itochondrial membranes. The C a2* of the suspending medium can be meas­
ured with a C a2*-selective electrode. A series of measurements at various initial extra­
cellular C a2* concentrations are made, and the change in extracellular C a2* concentra­
tion is plotted as a function of the initial extracellular C a2* to obtain a straight line.
The intercept of the line with the abscissa gives a null point when there is no net move­
m ent of C a2* into or out of the cells after the plasma membrane has been made perme­
able. If there is no m em brane potential, the null point should correspond to the C a2*
in the cytosol. Null point titration cannot be used for determining transient changes in
Ca2* concentration.
 II. T H E D IS T R IB U T IO N O F C A L C IU M A M O N G T H E C E L L
COM PONENTS

Total calcium in cells is represented by bound and free calcium, a large portion of
the latter being Ca2+. Bound calcium is combined mainly to anionic sites of sugars,
proteins, phospholipids, phosphate, and phosphate esters. The concentration of cal­
cium goes from 0.02 m m ol/ I cell in cells containing no subcellular organelles up to
about 2 m m o l/i cell in those cells that contain organelles. Measuring cellular calcium
and fractionating it am ong the different cell com ponents is a difficult task, because
cellular com ponents can be damaged during fractionation, changing the concentration
of calcium they had originally in the intact cell. Few studies on the m atter have been
reported. Electron m icroprobe X-ray analysis, for instance, represents a nondestruc­
tive m ethod of high specificity for calcium which, provided the biological sample is not
altered by the preparation procedure, may be suitable to measure calcium distribution.
The m ethod, however, is complex and expensive.
M urphy et a l.8 applied a rapid cell fractionation technique to fractionate the calcium
content of isolated hepatocytes from rat liver among the cellular subfractions. They
found th at the cells contained a total of 5.01 /umol calcium /g cell protein. A fter frac­
tionation, 60 to 80% was isolated with the m itochondria and of it, only about 0.6%
was Ca2+. M ost of the rem ainder was found associated to a microsomal fraction which
m ay represent the endoplasmic reticulum . M urphy et al.8 also found that the distribu­
tion of calcium m entioned above was independent of total cellular calcium between 3.2
to 31 ptmol/g cell protein. Essentially similar results have been reported by Foden and
R andle.9 These results show that in hepatocytes, intracellular organelles are the main
reservoir of calcium.
Blaustein et a l.10 have reported that total calcium content of presynaptic nerve ter­
minals at rest isolated from rat brain is 5 to 10 fjmol/g protein. A bout 70% of it
remains associated to intracellular organelles after lysis, and 10% may represent cal­
cium in the axoplasm; 5 to 10% of calcium in the axoplasm may be bound to proteins.
O f the calcium in the organelles, about 50 to 75% is associated with m itochondria and
can be released with uncouplers of oxidative phosphorylation like FCCP, and 20 to
25% is probably associated with components of the sm ooth endoplasmic reticulum and
can be released with the calcium ionophore A23187.
Results of electron probe analysis in cryosections of skeletal and smooth m uscle"
suggest that much of intracellular calcium is accum ulated within the sarcoplasmic re­
ticulum rather than the m itochondria. This assertion is supported by the finding that:
(1) electron probe analysis in skeletal muscle cells reveals that the terminal cisternae of
sarcoplasmic reticulum contains 66 mmol total calcium /kg dry weight, whereas total
calcium in the cytosol is 1 m m ol/kg dry weight, and (2) m itochondria from smooth
muscle cells do not appear loaded with calcium even after the cytoplasmic C a2+ concen­
tration is m aintained at 1 for 30 min. It seems reasonable to think that the abun­
dance of sarcoplasmic reticulum and its high affinity for calcium will lower the concen­
tration of calcium in the cytosol of muscle cells below the level where effective Ca2*
uptake by m itochondria, whose affinity for C a2+ uptake is orders of m agnitude lower
than that of the sarcoplasmic reticulum, is feasible. In light of the results mentioned
above, it seems that in some specialized cells, the role of m itochondria in calcium
storage might not be im portant.
The red blood cell can be taken as an example of a cell without intracellular organ­
elles. The total content and the distribution of calcium in red blood cells have been
studied by H arrison and L o n g .12 They washed hum an and red blood cells with isotonic
sodium chloride and after dry-ashing, m easured calcium by atomic absorption spec­
trom etry. The mean value of total calcium they found for norm al subjects was 0.015