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___
From the
-. *-
I4ol.ecular Biology Institute
a nd
Department of Chemistry
University of California
Los Angeles, Calif. 90d2.4
- ~
.~ .- - .
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AESTMCT
Sarcoplasmic reticulum vesj.cics prepared from rabbit skeletal nuscle
catalyze a rapid Fi 2 ::OH exchange in the presence of Mg*
and absecce of
ATP and CaH.
Tlie cclpacity for oxygen exchange is about fourteen tines the
potential capacity for .?TF cleavage in the preseiicc of Ca*,
No detectable
exchange is found vithout added bIgC12.
The exchange is unaffected by
oligomycin, 2,4-dinitropkienol or ouabain, but strong1.y inhibi-ted by low
concentrations of Ca++
in the medi:.im.
The Ca*
ion concentration giving a
half-maximum inhibition is 2.0 pX in .Che presence of 5 mPf llgC1.2,
A Iiil.1 plot
of the Ca* infiibicion gives a straighr lizle with a Hill coefficient of 1.8.
The Catt
inhibition is conipetitively overcome by additional. Ng-tf.
The
Pi $ HOH exchxge i.s ahest coxpletely inhibited by the deterger,t Triton
X-100 at low co11cei-itratiC)ns where the ATPase activity is not disturbed,
Sarcoplasr,;ic reti’ CLI~L~~E vesicles are phosphorylated by Pi in the presence of
Mg- and absence of (:a’* u:ider conditions similar to those for the Pi HOH
exchange.
The. p~ocplior~l.;i.c:ion requires Mg*
and is strongly inhibited by low
concentrations of Ca-;--t-.
similar to that: si the F’i
The response of the phosphorylation to Caft is quite
2 HOE exchange; the Caff ion concentration giving
a half-maximum inhibition is 2.0 pH in the presence of 5 mY FlgC12 and the
Hill coefficient i.s about 2.0.
The various properties of the exchange give
strong support to t h ~ .:!ro5aSility that it results from reversal of steps in
the overall process associzted with Ca*
transport driven by ATP cleavage.
.-

i
RUNNING TITLE:
Oxygen Exchanges 01 Sarcop1asmi.c Reticulum

Sarcoplasriiic reticc!.um vPsLcles isolated from skeletal musc.le actively
take up Caw from the medium in the presence of I'fg* znd ATP (1-3).
This
transport is coupled to A.TP hydrolysis catalyzed by membrane-bound Ca*,
%*--ATPase which is activated by concurrent preseme of Caf-'- and $?g* (2-6).
Considerable iiifomati.on~ have accumulated that give insight into the ATPase ,
and its coupling 13 the calcium transport.
The hydrolysis of Alf by this
enzyme occurs thrctlgh a phosphorylated ictermediate (7-11).
Fomation and
decomposj tion of tlie intermediate show vectorial requirements for Ca*
aiid
Mg-# (12,13), suggesting
intimate involvernent of the intermediate ill the
transport process.
ATP synthesis from Pi and ADP cwpled to outflov of Ca-H
from sarcoplzsric reticuiiim vesicles has recently been demonstrated (1.4,15>.
This indicates t.Le Tevei:.~iSili.ty of the entire process of calcium trai.isp~rt
in sarcoplasmic i'ei.icilli.rn1 vesicles e
In the prescrit article, it has been demonstrated that sarcc~pLasn!ic
reticulum vesicles catalyze a rapid Pi 8 IIOH exchange, which can be reasonab3.y
attributed to the rcversnl of steps in the process associated wit11 calcium
transport driven by ;?TP hydrolysis.
The characteristics of the Fi
I!OH
exc-haiige recently fcund in Na',
K+-ATPase of cell membranes (1.6) are quite
simi.lar to those of sarcoplasmic reticulum vesicles.
s im i 1 a r i t i e s b e t TJ c- c z: c 11 e t!:l o t r a n s p o 1: t s y s t ems .
This suggests niechariis tic

-5-
Materials.
Water of approximately 1.4 atom % excess l8O was obtained
from Yeda Research and DC?VelOpmE!;>.t
Company, Rehovoth, Israel.
Ic112PO~,-1sC~ vitb
about 1.7 etom % excess I8O was prepared as described by Cohn and Drjr:;3ale
(17) and appropriately dj lstcd with carrier P before use. Disodium sCi Its oT
i
ATP and ADI' were purcFd.sed Erci;n Boehringer and Mannheim. y-32P-Iabeli?d ATP
was prepared as described by Glynn and Chappell (18).
Acetylphosphate viis
obtained as the lithium salt from Sigma.
Ouabain was purchased froni Cn?biochem
and o1igomyci.n from Si.pa.
All the other chemicals were of reagent grade.
€re pa r a t i s
.of. -L a I- c cLpLgsn&.- re t i c u 1.um ye s i c I. e s . Sa r c op la sm i c r e? t i c i urn
vesicles were prepared from rabbit skeletal muscle by the method of iJeb:r
-- et - al. (3) with slight iacdific3tion.s.
About 300 g of white dorsal and tiL:id
leg ,

i
-6-
Ilcterrvinntion Of- ~ij~-~IIIi Exchclnge-. The P 2 HOH exchni;ge CataljrZcd by
i
sai-cnplasmic reticulun vesicles vas determined by two different methods. In
the first method, sarcoplasiitic reticulurr. vesicles !?ere incubated with P
i
HL8G!I with approximately 0.8 to 1.2 atom % excess l8O under conditions
in
described under "Results. " Keactiocs were queiichcd by addition of perchloric
acid, and the denatured vesicles and precipitate of potassium perchlorate
were renoved by centrifugation.
Pi Fn tlie supernatant was isolated as KE2P04,
and
inc3rporated from I',18@H
into Fi was determined as describcd belolw.
Total iirount of oxygen atonis exchanged vas calculated, as described by Boycr
and Gryan (19), from the equation,
patoms oxygen exchanged = 4 x po x In 1
1-F
where p
0
atom % excess 1.80 in
= ~IN Pi. in the reaction medium and I? = . -.- ~-
- .I
atom 7, Excess '80 in KOH
The second method vas utilized to measure the extent of Pi 2 HOH
excliarige during cleavage of ATP or acetyl phosphate a
Sarcoplasnic reticLluw
vesiclcs were incubeted with Pi-180 of approximately 0.43 atom X excess l80
urcier Loliditions described under "Results."
Reacrioiis were quenched by adding
pcrc:hloi:ic
acid at zero time and 40 minutes after the sKart of the incubation.
Subsequent: procedures for isolation of Pi and determination of lC9 i n the Pi
WC'I-C'
the same as described for the first method with the excepticn that in the
cxPcrirnenr: where ATP was present in the medium isolation of Pi was performed
at 4", while in the experiment where acetylphosphate bras present the samples
were all.owed to stand for 1 hour at rooni temperature after addition of
anti>1aiiuro molybdate in the process of Pi isolation described below to allow
decvinposit ion o€ acetylphosphnte.
The amount of oxygen exchanged during

drs
ATP cleavage was calculated as describcd previoiisly (20) by the following;
where a = initial mM ATP, p = n&.I Fi released fron ATP,
po = initial mM Pi,
k = rate constsnt (sec'l) for ATP hydrolysis governed by
= k(a-p), t =
seconds of i.ncubation, and I8O1 and = initial and final atom X excess
at
l8O in Pi=
The amount of oxygen exchanged ii-i the absence of ATP or acetylphosphat?
was calculated by the equation,
patoms oxygen exchanged = 4p,ln
1801.
The an!ouilt of oxygen exchanged in the presence of acetylphosphate was also
calcuinted using the above equation because 110
significant cleavage of acetyl-
phosphate occurred during incubation.
P. isolation and analysis. The procedure oE Eoyer and Bryan (19) was
-1
used for P. isolation and l8O analysis, xich scme inodifications.
1
After denatured
vesicles :.rere removed by centrifugation, ccn~entra ted HC1 was added to the
samp1.e to a final Concentration of 1.1 N.
The sample was washed once with
about 2 ml of isobutanol-benzene (l:l),
then 50 nM aqueous ammonium molybdate
was addcd to a final concentration of 12 nd4.
The resulting phosphomolybdate
complex tias removed by two extractions wi.tfi LsoSutanol-benzene.
The
isobutanol-benzene mixture was washed with 2 ml of 1 R HC1,
then Pi was
extracted into 1.5 tnl of 7.5 N NH40H.
After discarding the isobutanol-benzene
layer, 1.5 ml of cold magnesia mixture was addcd to the extract, and the sample
was refrigerated overnight for precipitati-on of ?1gNHL.PO4.
The precipitate was

--I_
-8-
\~n:licd wjLb 1 m l of cold 5 N NH4011 and dissolved by addirion of 0.1 ml of
6 N HC1.
Uhcn completely dissolved, 1.1 ml of 7.5 N Nk1;OFI
was added to
reprccipita te the phosphate.
Af 1 er 0verr.i ght refrigcra ti on, the preci-pita te
was washed again with 1 m l of 5 N JW4OI-I and then 1.yophilized.
5 vl of 0.1%
aqtieous bromocresol greeii was added to the dry precipitate, and the sample
was titrated with 0.1 N HC1 to a pH slightly higher than 4 (yellow end point).
After addition of water to a final volume of 0.3 inl,
the sample was passed
through a Dowex-50-Kf
column to convert the phosphate to the KH2POc+ form.
The eluent was lyophilized and '*O
determined by the guanidine-JIC1 pyrolysis
procedure (21).
~ Determination of -pi liberated and phosphoryj.ated protein formed by the
A4T12a::c -l_l_l reaction.
ATPase reactions catalyzed by sarcoplasmic reticulum were
pe1---F'omeil ~ isirig AT32?? as substrate as described under 'iXesults, '' and 32 Pi
l-iberztcd and the phosphorylated protein formed from
were measured as
--
described by Kanazawa et al. (13) using Mil-lipore filtration (1.2 p pore size),
Radioactivity of 32P was measured using Bray's solution ('22) with a Nuclear
Chicago l~icluid scintillation system, Model 725.
-_ -
Peterininat ion of phosphorylated protein fnned from P and SarcoDlasmic
-i ---
reticulum -vesicles. Sarcoplasmic reticulum vesi.c?es were incubated with 32
pi
fer 3@ sec cit 15' and pII 7.0 in 1.0 ml of a medium as described under "Results."
r.i-i.011 was quenched by adding .lo ml of 4.X psrchloric acid.
The denatured
vesicles were washed 7 times by centrifugation procedure with 10 ml of 4%
perchloric acid containing 20 mM carrier Pi and then suspended in 2 ml of
0.O:i. PI! I\JnOII containing 0.1 mM carrier Pi.
After heating at looo for 5 min,
the :;ui;p~nsi.on was centrifuged and 32P in an ali-quot of the supernatant was
i:;iii-ed
as described above.

-I)-
Ad iustrncnt of free Caw' c0nc.enCrati.oi-i &I -t& medium.
Free Ca
*
concentrations in the reac.ti.on inedium were ad justcd using a Ca-EGTA buffer,
where the concentration of CaCIZ added to the medium was fixed at 0.5 mM
and EGTA concentrations in the medium were varied,
Calculations of free Caw
concentrations were carried out assumicg that apparent stability constant of
Ca-EGTA comi>lcx c?t pH 7.0, association constant of CaHP04 from Caw and
HPOqr= and pK2 for Pi at 1-5' were, respectively, 1.3 x lo6 Ifm1 (23), 50 M-I-
(24) and 7.23 (25).
Protein concentrations were determined by Lhe method of Lowry 22. (26)
with bovine semm albumin as a standard.
hcstylphosphate was measured by the
method of Lipmann and Tuttle (27). Contamimnt Pi in acetylphosphate was
measured a.s descrihed hy Lowry and Lopez (28).
RESULTS
-- The P. 2 ROH exc?mse catalyzed by sarcoplasnic reticulum vesicle?.
-1----------
Sarcc.plasIni.c reticulum vesicles prepared f~on rabbit skeletal muscle were
incubated with 40 mM Pi at pH 7.0, 37", in the preserlce and absence of
14.5 pM free CaS3 in the medium containing Fi1-8CJI and other reagents as
indicated iil Fig. 1. As shown in Fig. 1, the vesicles were found to catalyze
a rapid incorporation of water oxygen atoms into Pj, when any free Ca*
ion
was almost cornpletel~y chelated by addition o€ 5 rnM EGTA,
a potent chelatiilg
reagent for Ca'-.
The arnouz;t of water GXygCil atoms incorporated into Pi
molecu1.e increased with time and readily approached isotopic equilibrium
when higher concentrations of sarcoplasmic reticulum vesicles and longer incubation
periods than those shown in Fig. 1 were used.
The rate of Pi 2 HOH exchange
calculated as described under "Experimental Procedure" was 520 gram atoms oxygen
bJ exchanged/sec. 1 Ob gin protein. This rate was app2ox:mately fourteen-fold higher

- 1.0-
t-han the rote of Cau-dcpendent:
ATP hydrol.ysis, which was 37 moles Pi liberatedl
~ec.10~ gm protein, as measured i.n the preseiice of 5 ~nFf ATP, 0.5 mM CaC12
aiid 0.5 inP1 ETA without added Pi under otherwise the same conditions.
c
Without
addition of HgCl, to the medium, no dctcctab1.e Pi 2 IIOH exchange was found.
In coiltras t, the Pi 8 HOII exchange w2.s strikingly i.nhibited by the presence
of 14.5 pl\I free Ca*
in the medium.
Absence - of effects- cf ol.igornycin, 2,4-dinitrophenol, and ouabain 011 the
-l----.--- P. 2 I-IOH exchangs. As tests of the possibility that the observed Pi 2 HOli
exchange might be due to mitochondria or Na -I- ,K +- ATPase present as contaminants
in the sarcoplasmic reticulum preparation, effects of oligomycin, 2,4-dinitrophei101
and ol~abain on the Pi 2 HOH exchange vrere investigated.
Pi 2 tIOIS exchange observed in the absence of free Ca'-was
As seen in Fig. 1, the
quite unaffected by
concentrations of oligomycin, 2,4-dinitrophenol, or ouabain that sharply inhibit
cxchariges of mitochondri-a or Ma f , I(+- ATJ?asc.
Similarly, the slight Pi $ HOH
exclL3r:Le aciivit:y. remaining in the presence of 14.5 IJJM free Cau
also was not
affected by these reagents.
--- Tine -__. courbe - of -- the pi 2 HOH exchanf:c.
Data of Fig. 1 indicates that the
c
.cr'ange proceeded approximately linearly with time at 37" when the reaction
WNS i-xirriated by addition of Pi.
Additional check of Linearity with tine was
felt desirable, in particular in view of the interesting lag in onset of
t ~ i r nored ~ ~ ; ~ with the actomyosin system (29,30).
As a further assessment,
:~:~~~ri.rcc!iits reported in Fig. 2 were conducted with initiation of exchange by
addition of the sarcoplasmic reticulum vesicles, and at 15' instead of 37'
to increase the possibility of detecting a lag phase.
As seen in the figure,
c~jJo:i addition of the sarcoplasmic reticulum vesicles, the Pi 8 HOH exchange
jJ1'(.'LC!edCd 1.inearly with time without a detectable lag phase. The exchange

eciprocal pl ot-s of the rates of Pi 2 ROH exchange against conc:entration Of
MgC12 c?ddcd,
"1
~ I I P
plocs give straight lines both in the abscncc IT.^ presciice
YfCl, calculated from these plots was markedly increased by 'ihe prcscnce of
3.6 pM CaS+, being 4.9 and 77 mM, respectively, in the absence and presence of
3.6 pM Caf-b. 01: the otlier hand, the maximum rate obtained by extrapolating

-12-
/ \
observed calcium inllibilSon can be conipetitively overcome Ly additional 1&C12,
_- Caft --- jon effects zji ptiosp!io~-ylatccl- prot-cin 256 ATPasc.
In thc experiment
reported in Fig. 6, the rates of Pi liberation and rlinounts of phosphorylated
protein present during the steady state of thc ATPase reaction catalyzed by
sarcoplasmic reticuluni vesi.cl.es were measured at varying concentraticns of
free Ca"
in the rnedium.
The reactions were performed under the sac:e conditions
as those for the P. 8 310H exchange measurement shoxn in Fig. 3 except that
1
5 ~ E I A T ~ ~ P at a filial concentration was used in place of 40 m~ pi. AS seer,
in the figure, the steady' state rate of Pi 1.iberation was inc-reased with
i.ncrease in free Ca*
concentration in the medium and reached a maximum at
19 pM free CaH-, followeJ by decrease with further increase in the free CaS+
concentration. The amount of phosphorylated protein showed B quite similar
response, but was increased i.n proportion to the increase in the rate with
less increase above 8 pM CaHa
Thus, the ratio of the rate of Pi lFberation
to amount of: phosphorylated protein in the steady state remained approximately
coiistant (1.0 sec-I), particularly in the samples up to 8 la11 Ca'-+.
shows the Hill plot of the rates of Pi libei-ation givcn i.n Fig. 6.
Fig. 7
The Hill
coeff icicnt was 1.05 and the free Ca'+
concentration giving half-maximum
activation was 2.6 pM.
Effects of ATP, ADP & acetylpliosphate on the Pi 2 HOII exchange.
Table I shows the erc'fect: of ATP on the Pi 2 HOH exchangesas measured i.11 rhe
.
absence and presence of 60 PI4 free Caw in the medium. The Pi 2 HOH exchmge
in the absence of both free Cau- and ATP showed a high value of 23 to 24 gram
atoms oxygen exchanged/sec.lO 6 gm protei.n, in $ood agreement wi.th that obtained
from 180 incorporation frem J11.80H into P. under the same conditions (Figs. 2
and 3). The important poinr of ~clblc I is that the P ii 1101-1 c;xcIiari,ge in the
i
1.

absence 02 free Ca+j‘ in the mdium was proiicun.cecily inhibited by presence of
4 n$I AT]’.
‘The ATP addecl was hydrolyzed by 287, ir; the absence of free Ca*
during ~hc! 4C ininutcs incubation.
As anticipated from tlx previous experimencs, the Pi
was conipl.etcly iiiliibit-ed in. the presence of 60 I.LM free Cas’’-
HOI-I exchange
in the medium.
The inliibi.z!.o:i
of Pi 8 H09 exchange by the Ca”+ was unaffected by addition of
4 nfi! ATP: r:hile PLTP !iyd?.-olysis was activated by the ea* and 62% of the added
ATP hias lvjdrolyzed in the presence of 6G ;LM CS-~‘ diiring the 40 niinctes incubation,
Siinil.iir nieasui-einents were performed to measuLe the effect of acetyl-
phosphate on the P. 2 KJH exchange in ths abseilce and pl-.csence of 60 p1’I Ca-!+.
1
As sh0~7n i.11 ‘Ilab1.e TI, addition of 3.3 mN acetylpliospbate showed no apprcciab1.e
or a :iligli.tI.y inhihitor)7 effec.t on the Pi 2 HOH exchange in the absence of
free Caw- iil the IneJiurri, while the Pi 2 HOH exchange inhibited by 60 pV Ca”
appeared io i~c :,mewliat reactivaLed by the additj.c:i of acetyiphosphate.
No
hydi:oI.ysis o? ncetylphosphate was detected either i.n the ahse.nce of or in the
presence of La
-++.
Fig. 8 s!iov:s the effect of ADP on the Pi 2 H311 exchange catalyzed by
sarcop1as~mj.c retr.i.cuLuni vesicles in the absence of free Gaff
in the medium.
A rather high concentration (20 mM)
of QC12 was used to minimize change in
the free I.Ig.i-i- ccncentracion due to addition of A3P.
The rate of Pi 8 HOH
exchange without added ADP, equivalent to 37.6 grarr! a toms oxygen exchanged/
sec-10
6
gm protcin, was considerably higher than that: measured at 5 to 7 mFf
MgC12 under otherwise the same conditions (Fi.gs. 2,3 and 9, Tables I and 11).
As seen in the figure, the rate of Pi r! HOH exchange was markedly decreased
with incrczse i.n concentration of ADP added.
caused, rcspectivcly, 67 and 87% inhibition.
Additiun of 0.5 and 3.0 tnM ADP
HOWCVC‘~, the j-nliibition was not

appreciably enhanced by Eurther Li-~crease in added ADP up to 4 nlM.
To estimate
the adenylate kinase activity which might obscure the experimental results,
decrease in ADP concentration in the 20 minutes incubation was measured under
the same conditions as those for Fig. 8 using the method of Reynard st al,
(31). The result showed that decreases in ADP concentration were 26, 8 and 4X
in the 20 minutes incubation when ADP was added to the medium to give, respectively,
0.5, 2 and 4 nlM. Thus, the adenylate kinase activity was not so high as to
disturb accurate measurements of Pi 8 IIOII exchange.
Effect - of --
Triton - S-100 - on --
the Ei z! HOH --
excha-.
Fig. 9 sho1-7~ the effect
of Triton X-100 on the Pi 8 HOH excliange and ATPase reactions catalyzed by
sarcoplasmic reticulum vesi cles.
In this experiment sarcoplasniic reticulum
vesicles were incubated with Triton X-100 of varioils concentrations in the
presence of 0.625 n*I
EGTA and other reagents as indicated in the figure without
added CaCIZ,
The Pi 2 if013 exchange was started by adding Pi to the preincubation
mixture, while the ATPasc reaction was initiated by addition of CaC12 and AT32P.
As seen in the figure, the rate of Pi 8 HOH exchange was progressively decreased
with increase in the Triton X-100 concent-ration; 92% of the exchange activity
was lost when Triton X-100 was added to a final concentration of 0.4 L o 0.6 pl/ml.
In contrast, the rate of Pi
liberation and the amount of phosphorylated protein
formed in the ATPase reaction remained on the same level as that of control
even when 0.6 pl/ml Triton X-100 was present in the medium.
Phosphorylation of sarcoplasmjc reticulum vesicles & pi.
The occurrence
of the Pi @ IZOH exchange suggested that the phosphorylated enzyme is formed
from P. and sarcoplasniic reticulum vesicles with elimination of water under
1
conditions where the Pi 8 llOH exchange takes place.
In the experiment reported
in Table 111, sarcoplnsuiic rcticulutn vesicles were incubated with 5 11121 32 Pi

for 30 sec under conditions similar ta [-i:myc for the P 2 HOH exchange.
i
32
measurement, and P incorporared into tlic protein was measured. As seen in
the table, a quite siuall but significant .f1:ncl:Lcn of the enzyme was
phosphorylated by P when 5 nN @CI2 was present: in the medium and any free
i
Ca* was rernoved by additio:i of EGTA, 'The phosphorylation disappeared almost
completely when 0.1 mM CaC12 vJas added witliout EGTA or when no MgCIZ was
added and free f4gtt ion contaminntFiig the lilcdiuni was chelated by EDTA.
Fig.
10 shows a double reciprocal. plot of the ainsuxit of phosphorylated protein
fcrmed from Pi again,st P. concentration in the presence of 20 mM MgC12 and
3.
absence of free Ca* ion at pH 7.0 and 15': The maximum amount of the
phosphorylation
was equivalent to 1.25 moles of phosphoryl group per
----I Effect .- of - Ca"
ion -_. c_ciLice~nt,ratj.cn on . inhibition
. .. .. ... . . of ... - the formation __--
of
~ . _
phosl>hoi^ylat:ecl Ljrotein from Pi.
In the expe;:imcnt slioim in Fig. 11, the
foi:i1:a.tiQns of phosphorylated protein f YOX P were measured at varying
i
concentrations of free Ca* ion in the c1edi:1.m. The conditions for the
measurement were the same as those for- the ?. 8 HOW exchange reaction given
in Fig. 3 except that sarcoplasmic reticiiiuiii vesic1.e~ (3 mg of protein per
1.
ml) were incubated with 5 mM 32Pi for 30 sec.
As is clear from comparison
with Fig. 3, the inhibitory effect of Cat'
ion on the formation of
phosphorylated protein from P showed quite similar features to the inhibition
i
nf the Pi 2 IIOH exc.hange. Fig. 12 shosJs the Hill plot taken from the data
of Fig. 11. A Hill coefficient obtained Erom this plct was about 2.0, and
the Ca+'- ion concentration giving a hal€-rn~ximuin inhibition was 2 .O pM.
Tllesc values were in good agreement wit11 tliose obtained from the Ca
i-t
i nlii.bi.tion of Pi 8 HDH exchange given :i.ri Pi 2s. 3 and 4,

DISCUSSION
The results clea?-ly demonstrate that the sarcoplasmic reticulum
preparation has the capacity to catalyze a rapid Pi
$ HOE1 exchange.
The
various properties of the exchange gi-ve strong support: to the probability
that it results froni rev?i-sal of steps in the overall process associated IJith
calcium transport driven by ATP cleavage,
The implications and possible
nature of the exchange i-eactioi~ inay be conveniently discussed in relation to
the scheme prescrltcd iu Fig. 13 that gives steps iil the transport process
consistent with the present and earlier findings (13,32).
The oxygen exchange could quite logically result frGiIl the dyziarnic
reversal of steps 7 and 8 of this sequence, namely the foi-mation of an
en~y1ne.P~ cornplex in presence of Mg:, and the formation rjf a covalent
;’-
en i;pi - L,yiG p;-13 t 5 ij 1; e 1 ill1 ..: u ’ * ~ ~ ui u ~ VJaier.. ! i; sErii;j.;-ig Eeatcre is tiie
:Inusual facility of this reaction. As noted in Table LIT, 0nl.y a snia1.i
fraction of the enzyme is phosphorylated by P. in presFnce of bigii-
1
and
absence of ATP and CFI-~.
Yet the capacity for oxygen cxchange is some ten
to fifteen times the pot-entia1 capacity for ATP cleavage iil the presence of
calcium.
An estimate is that the small fraction of E-? present must be turning
approximately 300 times per second at 15’ to account for the sxchange.
The data shewing 1-hc lack of effect Df inhibitors of mitochondrial
exchange processes (Fig. 1 and text), the pronounced izhibition of the
exchange by Ca’*
(Figs. 1 and 2) and the similar concentration dependencies
of the exchange (Fig. 3) and the ATP hydrolysis (Fig. 6) on Ca’+,
as well as
the sharp decl-ease caused by eaft
in the capacity for phosphoryl enzyme
formation from Pi (Table JI1 and Fig. il) all suggest that thc oxygc~n exchange
is a property of the transport AT1’as:~ sys!.ea.
The similjr characteristics of

the Ca' inhibj.tion of p1:os;~'iioi:yI enzyme formation from Ti and oi. the
i
Pi $ I-IOH exchange give nddj tjoiial evidence that the observed oxygcn cschange
is a property of tlic transport- ATPase system.
On thc othzr hand, nlthougli
the ATP hydrolysis is activated in nearly the same range of Catt
ion COIIC~II-
tration as the Pi 8 HOX exchange is inhibited, the concentration depndexies
are soaewhat different as seen from the observed difference in I!i
i.1 cocfficients
(1.05 in the ATPase reaction an3 1.8 in the Pi 8 HOH exchange).
The Caw
inhihit ion o€ the exchange, in terms of the schciile given in
Fig. 13, can be explain~?d i€ the Ca++ can combine with the free en;ryve LS a
Gf
partial reaction oE step 2 to give 8 Forin/a complex unable to cataiyzc the
exchange. Such a niecha-iim cen explain the observed cooperativi ty of Ca f-1-
assuming that in steii i CJCCU~S a conformational change which results i1-1
simultaneoas formation of two Caft-binding
sites e
The Gaff responses as well as the lack of effect of ouabain, 2 potent
inhibitor of the exchange catelyzcd by Na+,K+-ATPase
(16), effectivcly
eliminate the Na',
!

Ca'+,PIg*-RTPase,
of sarcopinsinic L-C!I:~.CL!].III~~ that the sys tern is activated by
very low Ca*
concentration.
The pi:~:j~~?t: results showed thn t a half-maxiinuin
inhibiti.on of the Pi $ HOH exchange was caused by 2 pb5 Ca*
which was found
to be nearly equi-valent to that givii:g a li~I~.f-inaxinium activation of the.
Ca*,
Mg*-ATPase
measured under sirni1k~- coilclitiolls to those for th.e P,
1' HCH
exchange measurement.
Such results acid to the probability that the observed
Pi ;F! HCII exchange is catalyzed by tlle L1:ijf-, hig'+-ATPase
of sarcoylasmtc
ret icu1u:n.
Implications of the tiilie course of Clie exchange warrant comment.
\\lheri
sarcoplasmic reticulum vesicles vere added to the reaction medium containi.ng
Pi and excess EGTA,
the Pi $ HOH excbc-?nge occurred immediately without showi,ng
any appreciable lag phase and pr-oc~.e:lccl 1-inenriy with t5me (Fig, 2), while
the exchange was prevented from the st.art of the i.nci.ihai:i on when s2r~npj.?s~:! P
retlculum vesic1.e~ were added t:o tkie mcldiuiii containing P
i end free Ca-++.
These observations indicate that the ink~ibition of the Pi 2 HON exchange is
caused by external Cat3 located outzide mcinbranes, but not by internal LaCi--.
Vesicles usually contain about 2G m,c~.cs or nore of endogenous Ca*
per mg
prctein (34,35) and this CaSf
does not cliffuse out in a few minutes at
neutral pEI when medium Ca* is chelated by excess EGTA (15,36).
EGTA added
to the inedium cannot diffuse intc the vesicles (3).
As mentioned earlier, the fracti:in ijf the enzyme that is phosphorylated
in the presence of Pi and is small. It amounts to only about 1/50 of
that present during the steady-state hydrolysis of ATP in presence of Ca
st-
and ~g*,
and suggests a particularly rapid turnover for participation of
this phosphoryl enzyme in the oxygen cxcliange.
Whether the same phosphoryl
enzyme participates in the synL1iesi.s ('i A'I'P
lrom ADP and Pi upon outward fli:.

-13-
of Ca’i- reported by Flalcirtose and tIasseI.bach (1.4) is uncertain.
In our
experiments, addition of external Ca*
inh:Lbi.ter? fornia’iioi.? of tkc phosphoryl
enzyme, but it cou1.d form froin Pi in presence of .LEterr>al Ca* when ATP synthesis
accompanies Ca* efflux, Indeed Hasselbach gt s. (37) have qui.te recently
reported that 32P.was incorporated into the protein of sai-coplasnic reticu1.um
vesicles preloaded with Ca*-.
The amount of 321’ incorporated (2 moles/106 gm
protein) was much larger than that obtain.ed by us (0.125 rnoles/lC6 gm protein)
using Ca*-unloaded
b
sai-coplaemic re+,iculuin vesicles.
thc
behavior can Le explained by shifts of steps 4 and 5 toward/right due to binding
of preloaded Ca+’with free E-Pat step 4.
Such apparent contrast in
The cooperaLivity observed in the Ca.’+. j.nkibition of the Pi 2 HOE exchange
suggests that an allosteric transition cou?d be induced by Caw- in the Ca*-
trznsport system of sarcoplasmic reticulum.
A possi.billty previously suggested
(13), based on the dependence of the breakclown of the phosphory1.ate.d intermediate
on internal i?:gfs’,
is that NgH- is transported by sarcoplasmic reticulum as
counter ion to Galt..
The cooperative inhibition of the Pi C!
lI0H exchange may
ref~ect a Ca”+-i.nduced transition of the transport system from one foim
(Mg*-carrier), which has a binding site with high affinity for Zig* and
catalyzes trsnslocation of Ng*
across membranes coupled with Ng*-dependent
P 2 HCUI exchange, to’ the other form (Caff-carri.er) which has two binding sites
i
with high affi.nity for Ca*
and catalyzes translocation of CaS+.
This explanation
is consistent with the fact that the breakdown of the phosphorylated intermediate
was unaffected by external Ca* as shown in Fig. b in spite of almost complete
-1-1-
inhibition of the Pi 2 IiOH exchange by extcriial . Cd
The in?ii biLim of the Pi 2 IIOH exchange lty n i lcaotjdes ~ ~ such as ATP and
ADP could bc c;:piaiiled by assuming that Mg-H--c::>) i(’r form of the enzyme is

-20-
co11\71:rt:ed to Caif'-carrier f01.m by these nucliiot:i.cllis even in the absence of
Cau.
i+
This assumption is compatible wi.th the present observation that the Ca
acti\TntioiI of the ATPase measured in the preseilcil of ATP shows no cooperativity.
IC i s also compaCi.ble with de Ileis and Hssselhach Is recent observation (38,39)
that- t!ie Ca*
uptake by sarcop1.asmic reticulum vesi cles shows a hyperbolic
c a -E+
ion concentration dependence with high CaS-'
i m affinity when ATP is used
3s a11 cmergy SOlirce, but a sigmoid dependence wi!:l-i Lov Ca* ion affinity when
acetyI.pf:nspb;-ite is used in place of ATP.
Ho~ever~ this assumptioil seems not
to be in liarmony with the facr that Catt acti.miti.:m of the ATPase occurred in
the concentrarion range si.milar to that for Ca'li
irlhi.bition of the Pi 8 HOIi
(Pigs 3 arid Gj and with the cooperat:ive activation of phosphorylated
i-xl.tr?!:!fi~d:i-1.t~ fr>nnpti.nn frnn ,A.TP hy (!aff- (1-3) A].i-lin~~gh a!: pre.Fnf: +h~sp
diEf Lcul ties cannot
be readily resolved, it shc~~:id be nclt-ed that the activation
o 1 13 12 L 2 s p?iLc ry 1 a t e d in t e rme d i a t e f o rina ti on ha s b c> c :I
in c a s u I: cd L: t 1 ow t emp e r a t u r e
(0 " ) igli i CFI might be unfavorable to nuc ie o t id c - i L-icI i i c t i. on of the t i-a ns it i on.
A ziiied for memh-ane integrity for the Pi ;? Z(X r:xc!!nnge
i.s implied by
the almost: coinplete inhibition by low concentral-ion of Triton X-100.
Such low
detergent concentrations did not disturb the ATPiise activity.
Transport capacity
is li k.?vise destroyed by detergents, again i.nd:ica!:-i.ng that the exchange is
inti Iy relatcd to the transport mechanism, i'i s ji~ti.i.L~I: inhibition by a
detergent, deoxycholate, has been found in thc Pi :.!
HVd exchange catalyzed by
Na-', I:+-/i?'l~asc (16)
1:t has recently been found by lhhins and Iioycr (4.1.) that Na 4-
,K-I.-
ATPase
also c:?::nl.yzc.s Pi 2 HOB exchange which is activaterl by I:i ~::tf~iis transported across

-21-
mer lit. ra ne s a re s ini 1 a r be tw e c:i 1:12 e ~a +I-,
+-I-- P~T PI? s e 05 sa r c op 1 ii sin i. r-
re t i. c u I urn
and the Na', KS-ATPase of cell me!nbrr!nes Th.e Pi &? KOH exchanges catalyzed
by these ti40 transport-ATPases may ref Icct mechanistic similarities between
the two systen~s '
Similarly, the o~curren~e of the exchanges suggcsts possible
me chani s t ic s i;ni 1 a r I t i e s to 1: he inemb :a ne a c 1.5. v a I; i. on accompanying ox i d a t iv e
phosphorylation (42) and ac.tive fi.-~nsporf of sugars ar.d amino acids (43)

1.
2.
3.
4.
Ebashi, S, and Lilmanii, FC, .J. Ccl.1 Biol., 14, 389 (1962).
Hasselbach, W, and KI-kj.r,ose, $le , Biocheifi. Z. , 339, '34 (1963)
Weber, A., Ikrz,, R. a13 Reiss, I,, Riochem. Z., 345, 329 (1966).
Hasselbach, I$., Prog, Biopliys Eiophys. Chem., 14, 169 (1.964).
5.
Weber, A.,
Current Topics i n fiioenergetics, 1, 203 (19GG).
6.
7.
8.
9.
10.
11.
7.7
LL .
13.
Yamada, S., Yam;lIliOt:c), T. and Tonomura, Y., J. Bioc.hem., 67, 789 (1970).
Yamamoto, T, and TO~OITILI~C~, Yo, .J. Biochem., 62, 558 (1967).
Nartonosi, A.. , Biochem. Biophys. Res. Commun., 29, 753 (1967).
Yamamoto, Te 3.n.d %Qnomra, Y., J. Eicchein,, 64, 137 (1968).
Kakinose, M., Eur, J. Bioc'hem., l(j, 74 (1969).
Eartonosi, A,, 2, Ciol, Chem,, %, 613 (1969).
r-
m -.
E;aIliii:iiwa, L. ? .~aii!iicia, S, aiid Tuttuiiwra, Y. , .To Zioc'nem. 68, 593 (i57Gj.
Kanazawa, T., Yamadz, S., Yamamotc, T. and Tonomura, Y., J. Biochem., 77,
95 (1971).
14. Makinose, Pie a~td lhsselbach !a,, FEES Lett., 12,271 (1971).
15. Panet, R. am? SelLng:er, Z., Riocbim. Eiophys. Acta, 255, 34 (1972).
16. Dahms,A.S. and Ijoy~dr, P. D., J. Biol. Chem., submitted as conipanion paper.
17. Colin, M. and brysclz!c, G. R,, J. Biol, Chem., 23, 831 (1955).
18. Glynn, I, M. aw! Chal:pe!l, J. E., Biochem, J., 22,147 (1964).
19. Boyer, P. 1). aid ~rydn, 1). $I., Fn K. W. Estabrook and M. E. Pullman, Xd->,,
"Methods in Enzymology, Vol.. X," Academic Press, New York, 1967, p. 60,
20. Dempsey, $1. E., Boycr, P. D. and Benson, E. S., J. Biol. Chem., 238, 2708 (1963).
21. Boyer, P. I)., Craves, D. J., Suelter, C. H. and Dempsey, M. E., Anal. Chclrl.,
- 33, 1906 (1961).
22. Bray, G. A., hiLil. i;iochwi., 2,279 (1960).

23. Og

42. Roycr, P. D., Cross, 1%. L., Chudc., 0.. Iklins3 A, S. an3 Iianazawa, T,,
in Proceedings of "lnterna'ij ona1 Symposium 01. Diochemistry and Biophysics
of Mitochondrial Nenbrclnrs, 'I
Brecsanone-Pcidova, Italy, June 1971, in press.
43. Boyer, P. D,, Klein, W. L. and Dalms, A. S., in Proceedings of "Inter-
national Colioquirn on Bioenergetics, '' Pugnochiuso, Italy, September 1970,
In press.

-2.S-
FOOTNOYCS
* Supported in part by Contract AT(04-3)-39, Project 102 of thc U. S.
Atomic Energy Conuri-ssicn aild by Grant G1T-13094 of the Institute of General
Medical Sciences of the E, So Public Iiealth Setvice.
T
On leave from the Ucpartrnent of Biology, Faculty of Science, Osaka
Lh iv e r s i t y , Ja pa n .

- . . . . - - . - . . . . . - . . . . . . . . . . . . . . . . .
- 26-
LEGEND FOR FIGLTtE 1
SONE CILIRACTERISTICS OF T!'E
Pi, 8 HOII EXCHANGE
CATAIS Z ED BY SARC 0 P JA SMI C RET IC ULUM VE S ICLE S
Sarcopl.asrnic reticulum vesicles, equivalent to 0.5 mg of protein, \rere
incubated for 3 min at 37' and pi1 7.0 without potential. inhibitors or with
5.56 pg/ml oligomycin, 0.222 mi.I 2,4.-dinitrophenol or 1.11 mM ouabain en C.9 nl
18
of H OH (0.88 atom % excess containiilg 5.S6 mK EGTA, 5.56 mM MgC12, 15.1
ml\l KC1 and 111 m$f
Tris-HC1. A sec.ond incubation szries had the same cornposi.tion
as alJOVe except for 0.556 mPi EGTA and 0.556 nlM CaCI2.
Pi 2 HOE1 exchange
reactioiis were initiated by sdding 0.1 ml of 400 nN potassium phosphate at pII
7.0 to give final concentrations of 40 rd4 Pi and 5 niki EGTA or 40 n-3 Pi, 0.5 d\I
+c:, 97-a
2 '.'II"-
t-t free Ca
EGTA left
indicated
incorporated into Pi were made as described under "Experinental Procedu;-e "
Ir! a control the procedure vas the same as above except that the preincubation
mixture contained none of oligomycin, 2,4-dinitrophenol and ouabain. 9 -- 3,
no further cdditions; 0 - 8, 5 yg/ml oligomycin; -- A, 0.2 d4 2,4-
dinitrophenol; u - 0, 1 rilM ouzbain;

lu'

LF,CEl\lD FOR FLGLXE 2

' -
-
--I--
-I-----
I
-
IO 20
MINUTES
30

LEGEND FOR FIGURE 3
Pi, 2 HOH exchange reactions were carried out for 30 min a:; described ix
Fig. 2 except that free CaSf
concentrations in the reaction riiedium were varied
using a Ca-ECTh buffer, where final concentration of CaC12 in the reaction
medium was fixed at 0.5 1~29 and final concentration of EGTA was changed in the
range from 0,3 to 5.0 inM.
Removal of nearly a1.1 free Ca*
ion from the medtuiz
was achieved bg7
addjiig RGTA t-n gi.~!e 8 finel c~ncentretim cf 5 yM, v:ithe::t:
added CaC12
rieteriiiiriations of l8O incorporated into Pi
and calculations GE
the Pi if. HOH ~~cba~igtz? rates and free Ca'H concentrations in the medium were
made as describe6 under "Experimental Procedure. ''

I.
-----/f-TI-- ---I------.--
--l-----1
2c
15
io
5

-32 -
LEGENU FOR PIGLqIE 4
The rates 01 I'i 2 HO1I exchailgc (V) are tahcn from those given in Fig. 3,
Vo represents t-he rdte of Pi i? HOH exchange in the absence of free
i.e.
in the prcsencc of 3 ]+I
EGTA without added CaC12.

F;
I .t,
I .c
>
I -2
0
c.9
a d
0.5
8.
-0.5
- 1.0
6.0

-34 -
EFFECT OF Ca* ION ON THE Mgi4-DEP1?P!DEMC1: OF TIE P, 8 HOH E:CCHifiGE:
I-'. 2 KOlI exchange reactioas were j:.nir:iated by adding 0.05 ml of sarc.oplzsrnic
i
reticulum vesicles (20 mg of protein per ml) to 0.95 in1 of 11i801i containing all
J.
other reagents at pH 7.0 and 15'.
The reaction iil.ixti1L-e had 8 final coxposition
of H~~OH (1.19 atom x excess
18
01, sarcoplasmic reticul-urn vesicles (1.0 mg
protein per ml), 40 TEN potassium pfiospliatc, 190 d f KCl, LOO m3"L Tris-11C1, l.:gC12
as indicated and 1 inFl EGTA,
or had the s~?il:e composition as above exr;ept for
0.5 niX CaCl and 0.6 nix EGTA. 1 riH EGTA uithout added CaCl almost compietel-y
re::,5.2ed
2 2
r,++ i3nc v7i-;i .:- thn -----~-,.,--F /.n -1K T, --a n c - % ' r8-C-
A i aiiu u a -> ,LICL .L,a~~ .I ,-
, V
d'L4-L.- .:I., b L ' L ,JI.CI;>CILLc: UL -/u ILUI
/ >
0.6 mIv1 EGTA left 3.6 pM free Caw ions.
The rate of the Pi, 2 HOIJ exc:hzligr: ('Jj
If
WGS detemined as described under Experimerltal Procedure. " 0 - d, '-' 1 inM
EGTA. without added CaCl-2, 30 min reacticm; 3 -'- a,
0.5 CaC12 -+ 0,6 !??>!
EGTA,
60 min reaction.

. i ',
(9
b

-36-
LEGEND FOR I'LC'LTRE
G
DEPENDENCE OF THE RATE OF IJi Td TC1:'IUTION
AXD AMOUNT OF PHOSPHORYLATED
PROTETN FOWD IN THE STE,\EY S'ZA'LE OB THE Cau
ION CONCENTlLlT1ON
ATPase reactions were initi:-lt-.ed by adding 0.1 1111 of 50 plI AT32P to 0.9 ml
of reaction mix-tures containing sarcop1asmj.c reticulum vesicles (O.Ol1 ing protein),
5.56 inI4 bIgC12, 111 mN KCI, 111 n!X Tris-EC1, 0.556 r&f CaC12 atid 0.3 to 5.0 nil4
EGTA at pH 7,O or to G,9 ml of a reaction mixture with the same ccmposition. as
above except for 5.56 :d+ EGTA witliuut added CaC12, at 15'. Reactions were
qucnclied by adding 1 ml of a inixtcre of 8X perchloric acid contairiinZ 1 ii$l
carrier ATP and 0.04 mM carric~: Pi 10 and 60 sec after the start of ccactions.
17
-'Li> 1.i berated
i
and amounts of pf:osy:hc\ryl.atcd piroteic formed were ineasiired as
descrilJed under "Experimental Procedure. The amounts of phosplioryis tcd pi:otein
WCI--C xenszred only with the sa:upI.es of 60 sec reactions, while thE rates of Pi
1lber.ati.o:i were calcul.atec! froin tlc difference in the amount of Pi Liberated
between 10 and 60 sec after the start of reacttons. These are pre.c:entcd in
thc figure after subtracting the r-ltc of I)i liberation and anout af
incorporatcd into protein in the presence of 5 d f EGTA without added CaC12,
3L
P
respecrivcly, froin those with added CaCl?.
-
Free Cau
ion concentrations were
calcvlatcd as described under "Cxperimental Procedure. I'

-35-
I,EGEI

t .c
0.5
- 0.5
- 1.0
(
6.0 5.5 5.0

-&)-
LEGEND FOR FIGURE 8
INHIEITION OF THE Pi 2 HOB EXCllANGE EY ADDITION OF ADP
Pi 2 HI)H exchailge reactions were initizted as described in Fig. 2 by
adding sarcoplasmic reticulum vesicles to give a final. composition of I€18011
(1.19 atom % excess l8O), sarcoplasmic reticulum vesicl.cs (0.5 rtig prorein per
ml),
40 my potassiu.m phosphzte, 5 nii EGTA, 20 ixM IfgC12, 1.00 mM IC1, 100 rdI
Tris-HC1 and AD1 as Lridica'ieci at pli 7.0, 15'.
Keactiorrs were quexiched by
adding 1 nil of 8% perchl-cric acid 20 min after additioa of the vesi.cles, and
determi.iiations of "0 incorporated into Pi and caicul.atioris of the Pi $ HOH
exchange rates were made as described under "Experimental Procedure. "

EFFECT OF TKITON X-100 ON THE Pi ?r HOII EXCXiNGE AI'JI! ATPase REAC'LIOZ\IS
For the Pi 8 I-IOi-I exchacge measurements, sarcopl.asmLc reticulum vesicles
(0.5 mg of protein) were incubi2tsd in 0.8 ml of H 1.13 OII (0.96 atom % excess l8O)
containins Triton X-103 at 17aryin.g concentrations, 0.625 1111.1 EGTA, 6.25 mY
I\lgC12, 125 mM. KCl and 125 it24 Tris-ZC? at pH 7.0, 15', for 20 to 30 min, and
then Pi 2 HOH cxchnn.ge reactions were initiated by adding C.2 ml of 200 mM
potassium phosphate zit plI 7.0 to the incubation mixture at 15'.
Reactions were
quenched by adding 1 ml of 8% perchloric acid 40 min after the addition of pi,
and determinations of isO : incorporated into P. and cxlculaticns of the Pi ? HOE
exchange rates were made as described under "ExperimririIla1 Procedure, " For. the
1
ATPase measurmentis
sazcopl-asc1i.c i:eticulum vesicics (3.5 mg of protein) r~ere
2- -..L -L- 3
AuLuuaLcu ii-i 3. 775 u ~ i .I .J. iiiediuni ci.iiiaiiiiiig Trj-kc.)il i

P -
i / Q
i
Q
in
V
M 0 i, E s P 1.4 os P H 0 RY L AT E D F R i! 7 E I N FO R M E D/ I 0 6C-JM P R 07- E i N
.- v-

1,EGEND FOR FIGURE EO
DCIUBLE EECTPROCBT,
PLOT OF THE AFfOXlNT GI;’ PKOSPl~?OilYLATED
PROTEIN FOHNED YKOM Pi AGAINST 1’.
1.
CONCENTiGTION
Sarcop1asygi.c reti.cu1.um vesicles, equivaler-t to 3 ng of protei-n, were
inccibated for 30 sec. at pll 7 .O and 1.5” in l.C 1111 of a icediuin coiitair,ing
1.67-10 mM 32P.
13
1.’ -
30 ITPI I.IgCL2, 106 iiiY KCI, 1.00 1d1 TrLs-liC1 and 5 n+l EGTA. The
amount of -“-F inccrporated i:ito t!~e proLei.n was measured as desej:il:cd u.ndcr
“Experj.mcnta1 ?ri!cedure “ The amomt of: 32P- incorpora tion ind epeixlerit of >ig sf-
wzs measured at each 32P. concentrrc1:ion under the same conditions as above
1
except for 5 mM ECTA without added I.IgC12 slid was subtracted from the above
value obtnincc~ at corresporiciing ‘ 2 concentration ~ ~
before plotting.

0. i 0.2 0.3 0.4
[Pi]-'
(mM1-l
0.5 0.6

LEGEND FOR F%GijRE 1.1
Sarcoplasmic retj.c.ilLum vesicles, eq;ivalent to 3 ng of prctein, were
incubated for 30 sec at pH 7.0 and 1.5" in 1.0 in1 of a medium containifig 5 mbl
32Pi, 5 idf NgClz, 100 inN IXI, 100 nit4 Tris-HC1, 0 or 0.5 ndl c~C.1~) and 0.3-5.0
mM EGTA. Free Caft concentxations in the is!c:ubation iiiedii.im vere varied using
a Ca-EGTA buffer, where CaC12 ConcenCratioLi ir? the medium V ~ S i'ixeci ai: 0.5 mbi
and EGTA concentration was changed i:i the range froin 0.3 ho 2 .O iiil.1.
Rcmoval
of n.ear1.y all free CaH ions froin the n;ediuni xt7as achieved by addir);; 11GTA to
give a final concentration of 5 n2.i without add.c?d CaC12.
The mount oF 32P
incorporated into the protein was measured 3s described under "Experim~iital
Procedure" and was plotted after subtracting the amount of 32P incorporated
under the same conditions as above except for S nd3 EDTA without added P!gC12.
Free CaH- ion concencrations in the medium were ca1c:~latecl as described under
11 Experimental Procedure" except that formation of P&X?O4
wi.th association constant of 71) ~ - (24) 1 was taken account of for this
calculation.
FIE*
-
2nd lIPO4-

-48-

I .c
0
- 0.5
5.8 5.6 5.4
- LOG CCa+"3 (M)
5.2

-59-
E and El-' rcpreserit, respectively, nonphospho~ylated and phcsphorylated
enzynies which have two bindizig sites with high affinity for CaH- but not for
~x-~.+i.
K L&
cG zzcb, , ~ , ~ l ~ ~ Zfid ~ : CGtdlyitI: c : ~ ~ a ~ : ~ ui ~ f:a++
i ~ at:ruys ~ ~ j rrLciilbralles
, o ~ ~
(Ca*-carrj.E:rs) E?.: and E"P indicate, respectively, nonphospkorylated and
phosphory1,ated cnzymcs which have one bindin::
site with an affinity for Mg -I+
but not for CaSi
on each rnol.ecule and catalyze transl.ocation of Mg#- across
meinbra ne s (Mg '-$- cii r i: i e rs ) ,

__-_I
Reactions were ini.ti.a';ed
by adding 0.05 in1 of sarcoplnsniic reticclun
vesicles to 0.45 ml. nf z reaction medium containing all. other constituents.
Reaction mixtures contained
at the start of reactions, sarcop1.nsmi.c reticuluni
vesicles (0.5 mg proteiil per ml.), 40 rrJ4 potassium phosphnte-1LSD iqj.tl-, 0,432
atom Z, excess 1'0, 7 d l X?CI~, 1-00 mi'i KCI
and 100 inM ~ris-.~~. al: p~ 7.9, 150,
as well as 0 or 0.5 mPi CaCJ.2, 0,4 or 5,0 mI$ EGTA and 0 or 4.9 n!M
as
indicated.
0.5 mN CaCI,, 0.b. nLN EGTA and 40 nM Pi gave 60,4 pJ1 free Ca'-t. ta
i
the reaction medikm. 1..5 nil of 5.37, perchloric acid w ~ ediied s to the reaction
mixture before (as zero Ciii-e) OT 40 min after additicn of sarcopiasmic reticulum
vesicles.
Loss of '-'O €zox Pi-''(; and hydrolysis of A$']? in the 40 minute
reactions were measured and the rates or' Pi $ HGH exchange \:ere calculated as
described under "Experi.n~nta1. Procedure, 'I
CaC1 added EGYA added ~
2
--___.
mM mM mM
ATP concentration Rat€: :>f Pj i! HOIi
Initial Fina 1 exchz I?gE
- ---_-
0 5.0 0 0 22.5
0 5.0 0 0 24 . 3
0 5.0 4.00 2.90 1,1
0 5.0 4.00 2.89 0 . 1
0.5 0 .4 0 0 0
0.5 0.4 0 0 0
0.5 0 e 4 4.00 1.52 0

Pi $ HOH exchange re3ct-';ons \.rere perfomed at pH 7.0, 15', Cor 40 min as
described in Table i excepl: that: acetylphosphate was added in place of AT3?P
to the medium to give 3..3 u!bI at the start of reacti.ons.
Tile addition of
acetylphosphate resulted :i.ii ;-;n ~ J I C ~ P in ~ Pi S ~ concentration by G. 65 mM. at the
start of reactions since ?ccc:ty1.p~iosp!?ate used was contaminated wj-l:h Pi.
: 8- the
Determination of loss of '- 0 froin I'i-].'O i.n/40 minute
reactions and calcula-
tion of the rate of Pi T! I-iOTi e>;c:hGnjie VCJ:~ made as described under "Experimental
Procedure. "
0 5 * 0 0 23.3
0 5.0 0 28.8
0 5.0 3.3 20.6
0 5.0
0.5 0.4
0.5 0 , !:
3.3
0
0
22.5
0
0
0.5 0.4 3.3 7.0
0.5 0.4 3.3 3.6

i c
- .5 rt -
Sarcoplasinl c reticulvm vcsicli-fs, equivalent to 3 nig of protein, were
iucubatcd for 30 scc at pi-1 7,,0 and 15'
in 1.0 nil of a medium containing 5 r,ll\I
32Pi;
100 inl.f IXlj 100 IY~M 'i'ri.s-EC1 and other reagents as indicated.
The reaction
was quenched by adding 10 1111 of 4X p~rchloric acid and 3GP incorporat:.xl into
the pxotein was measure4 as ciescri bed under "Experimental Proc.edure."
3
5 0 0 5 0,94
5 0-1
.o 0
0 0
1. 5
0,05
0,03