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Kinetics of uric acid transport in human erythrocytes
IMICA ET BIOPHYSICA ACTA
RIC ACID T R A N S P O R T I N t ERYTHROCYTES U L R I K V. L A S S E N
)epartment A, University o/Copenhagen
(Received A p r i l
7th, 1961)
SUMMARY
I. The transport of uric acid in erythrocytes has bee been studiec ibrated ;tern b y means of labelled uric acid. 2. The necessary assumptions and calculations for f such ats are ~sented. 3. The transport is, in contrast to simple diffusion, diffusio~ concen endent. e Ks value for the transporting system is 3.2 mM. 4. From the effect of temperature on the transport, the Q10 v~ to be ___/ . . . . . . . I; the corresponding energy of activation is 158oo cal/mole. cl 5. The transport velocity is accelerated when lowering lowerir pH. This effect is presummablLy due to alterations in the fraction of total uric acid concentration being present ~resent a s the undissociated acid. Thus it is concluded that this transport is an example of o "non-ionic transfer". me 6.~. The kinetics for uric acid reveal that an "active" mechanism must be responsibh ,nsible for the transport, even though concentrative uptake or o~ extrusion has not been dede. monstrated.
INTRODUCTION
The presence of uric acid in hum~ human erythrocytes has previously been demonstrated 1-4 Until recently, the lack of a concentrative uptake was considered as indicative of uric uric acid being transported thr"ough the membrane only by diffusion. As described in a preliminary note 5, several of ~f the factors influencing uric acid transport in erythrocyte, ,tes permit the conclusion thatt diffusion cannot be the only means b y which uric acid passes the erythrocyte membrane. However, the results previously presented did not give sufficiently detailed tetailed information to permit a sugl;gestion of the kinetic behavior of the transporting system rstem. The aim of the present stud' dy is to describe in detail the method and the calculation, ttions used to determine the veloccity of uric acid transport through the human erythrocyte zte membrane and also to present ~ent results from experiments employing variations in the uric acid concentration andd the temperature of the system. These data support the concept of a "facilitated" or "carrier-linked" transport mechanism. This kind ot of kinetics has been demonstrated rated for the transport of glucose and other monosacchasaccharides through the erythroc3yte membrane 6-14. Biochim. Biophys. Acta, 53
(1961) 557-56g 369
U. V. LASSEN
xs in the p H of the suspending me the erythrocyte membrane in the a
XPERIMENTAL
te t h a t ciated)
METHODS
1 (3000 I.U. heparin/1) from health ~s used ?eriment blood from only one do: ,,d. The lasma and "buffy coat" sucked off ls were ............... ~ .......... /2. . . . . . . . . . . . . . . . J . . . . . hed two or three times by resuspension and subsequen uent centrifu ooxg 3o min. The wash fluids and the media of incubatior ttion were K] -bicarate solutions. Sufficient sodium bicarbonate was added adde to give p H at temperature of incubation with a moistened 5 % carbon carl dioxi Lure as gas phase 1~. The wash fluids were identical with the final media ~dia of incuba' ltained of glucose/l, and, unless otherwise stated, 3oo txmoles moles of uric ac experiLts with high uric acid concentrations, uric acid was dissolved taining lculated amount of lithium cabonate. In these experilI ~eriments all t~ ~ed the e lithium concentration, either as the urate or the chloride. chk Due to the instability of very dilute solutions of uric acid, a even state, 18-14Cjoactive uric acid was prepared immediately prior to t~ each e~ ! Centre Centre, Art Amersham, England) oxantine (2.4-2. 7 mC/mmole, The Radiochemical Ce oxidized b y means of milk xanthine oxidase TM in o.o 5 M Tris buffer at p H 7.5. To ~owerful substrate inhibition of [d blocking of the enzyme action due to the powel avoid thine oxidase ~*, the radioactive hypoxanthine )oxanthine was added addc repeatedly to the enzyme. xanthine 'y instance by spectrophotoThe termination of the oxidation was checked in every further addition of ry at 292 m/~ (Beckman model DU). At this wavelength wav metr, absorbancy, apart from the thine oxidase should give no additional increase in xanthine oxidase was inactivated ease caused by the enzyme. After control, the xanthine xanth increase ~oiling for 3o sec. This solution was made isotonic with sodium chloride or glucose. b y boiling The ~4C-measurements were performed by plating 50 5o ~1 of the supernatants from minium the incubates on 24 m m aluminium um discs. The total dry matter was between o.2 and tion was made for selfabsorption. The planchettes were o.3 mg/cm ~, and no correction measured for radioactivity b)y a thin-window, gas-flow, proportional counter (Frieseke "uck, Ge Germany, German model F H 4o7). Samples were counted to a and Hoepfner, Erlangen, Bruck, statistical error of less than 2 %. The p H of the incubates was measured at the end of each experiment to ascertain td occurred during the incubation. Variations between if major p H variations had individual flasks in a single experiment did not usually exceed o.o 5 p H unit. When differences were more thanL o.I p H unit, the experiment was discarded (except of here different p H values were intended), p H was measured course in the experiments where meter, Copenhagen, Denmark, model P H M 22). b y a glass electrode (Radiometer ormed in a water b a t h with provisions for heating and Incubations were performed cooling and with a shaking device ( 7 o 8 o oscillations/min). The incubating vessels were Ioo-ml conical flasks, siliconised before use. ..re determined in graduated tubes centrifuged at 25oo Hematoerit values were terminations being carried out in duplicate. rev./min for 30 rain, the determina Biochim. Biophys. Acta,
53 (I961)
557-569
ID TRANSPORT IN RED CELLS
zere carried out b y the spectrophoi ~d uricase. dculated according to standard fl determination of regression coerce
thod of ing the
nts
d cells were shaken in the incubato carbon dioxide atmosphere and tl dram. l he incubating mixture re had a hematocrit laematocnt of ot 45-6o 4.~ %. A )eriment, o.o5-o.1/zC of [8-z4C]uric acid/ml of total incubat [erent time intervals, 5-12 samples of 1.5-2.o ml were wer, taken o "'Internation~ ttrifuged in a refrigerated high speed centrifuge ("Int ~oo × g for at least 2 min. The "time" of each sampl)le was th tition of 14C to the vessel to the centrifuge rotor speed sI pa s ,out io sec after the starting of the centrifuge). The supernat for plating and, in some experiments, for uric acid aci deterrr. :line in counting rate of the supernatant, the velocit,y of uric culated as shown below.
min to of the for the Led. At 'diately R I) at om the ev./min ipetted om the ~rt was
l~C]uric acid eOlux experiments The washed erythrocytes were incubated for I h in a~i medium lll~[lUlli ~ U l | L c l i l l l l l ~ 0.I--0.2 U.I.--U.2 [8-i*CJuric acid/ml. The cells were then spun down and washed once with the th¢ Krebs-Rin ebs-Ringer-bicarbonate solution in the cold. At "zero" "z time, the z4C loaded loaded erythroc~ 'throcytes were added to the incubating medium to giive a hematocrit of lO-2O %. % Fro)m this incubate, samples were taken out and treated trea as described for influx influx th~ supernatant, the uric acid e x l )eriments. From the increase in counting rate of the cid transport was calculated.
Calculation of transport rate Influx of labelled uric acid: The erythrocytes and their thc medium of suspension arc are tS19. considered as two compartments TM ments TM. These are separated b y a membrane which exerts exert, a characteristic influence onn the velocity or uric acid transport between the two com. cornpartments. Prior to the start trt of the experiment, the system is equilibrated with nnononlabelled uric acid. At "zero" f ' time labelled uric acid is added to compartment A and starts to pass the membrane ne towards compartment B. Assuming immediate mixin~g on both sides of the memxbrane, the decrease in s u p e m a t a n t counting rate (comcornp a r t m e n t A) with time must mt depend on three factors: (a) the "permeability" of the th¢ membrane for uric acid, (b) b) the mean affective gradient of labelled uric acid, and (c) the relative volumes of the two compartments. This dependence holds true either eithei when there is no effect of the total uric acid concentration upon the transport rate, or when the molar amount of labelled uric acid is small compared to the concentration tration of non-labelled uric acid in the system. In this last situation the amount of uric acid passing the membrane from m each side per unit time must be the same. In the exexperiments reported here, the molar amount of labelled uric acid is small (about about 25/anoles/l). The following symbols are used in the calculations:
Biochim. Biophys. Acta, 53 (I96I) 557-56g 369
U. V. LASSEN
mt (compartment A). "ic acid distribution in the eryth e (same units as a and b) cent. erythrocyte volume available for ( ater phase). rtment A when extrapolated to zel rtment A at time "t". ka, Rate constant for change in counting rate in cornpartmeni
mpart-
of uric
re.
From the definition of these symbols: V
O~ = - - (IO0 - - H) IOO
p.H.V
b--
Ioo
At time "t" the counting rate in compartment B is: (x 0 - - x)
time Counting rate in compartment A will then decrease with v
-'~ --
(x
(xo-- x) a.)
or by rearrangement and integration:
((°) I
fd
--
a
x
I +~
o
--.g'o~) a
J
kadt
By solution of this integral: I
ct
a
(~)
a I 27-
b
integration. where M is the constant of inte Eqn. i describes a strailight line with a slope of --ka and an intercept on the vertical axis of M. In addition to the symbols used above, we have: E ~ u x of labelled uric acid: In mpartment B at zero time. Zo, Counting rate in corn mpartment B at time t. z, Counting rate in com ats the extracellular counting rate at zero time when the I n this case Xo represents Lve lost no radioactivity. In a similar way, the rising 14C-loaded erythrocytes have ernatant, x, represent the initial counting rate plus the counting values of the supernatan radioactivity lost from the erythrocytes (compartment B). This gives: B i o c h i m . BiopP~ys. A c t a , 53 (1961) 557-569 i69
~O.l.
I D T R A N S P O R T I N R E D CELLS I
a
(x - - xo) or z = zo - - ~ (x - - x0) influx calculation it follows:
k=(ZO--b(x--xo)--x )
=
ttion gives: a
f
a
x+~
d zo+
This integral m a y be solved to give: I
( zo
In
o Xo--x
+
=----kat
I +
+ N
(2)
~+g is the constant of integration. Before the application of Eqn. 2 to the experiment~ ~erimental data wl 5, x, and Xo values, it is necessary to calculate zo. TI This is don mula:
a+b
Z0 ~ Xeq
nprises to the
a
b
X0~
aere Xeq is the counting rate of the s u p e m a t a n t (comp~ Where )artment A) when the system totem is e(:quilibrated with respect to radioactive uric acid. Ex t )erimentally Xeq (thoughh not theoreticaU' ~oretically obtainable within a limited time) is taken as a the counting value when prolonge ~longed incubation will give no significant increase in supernatant s counting rate. From the Eqns. I and 2 it is possible to calculate the t rate constants for super)ern a t :ant a n t counting rate when using either influx or effiux efflu of [ 1 4 C ~ u r i c acid for the determination. :ermination. The aim of the determination is, however, howeve to correlate the transporl )ort of uric acid to the amount of red cells being present. The rate constant for the intracellular uric acid (kb) is simpl aply correlated to the ka value: kb = ka .b When inserting kb in the Ec ~qns. I and 2 we have: a
a+b a - -
a+b
-in
In
x
+
i +
a
--xo
=--h#
g
+M1 ÷
(3) (4)
The k~ values calculated ed f, for the same batch of cells under the same experimental Lmental conditions should be inde 1?endent of whether influx (Eqn. 3) or efltux (Eqn. 4) was wa~ used for the determination. n. From this it is possible to test the assumptions used used: i.e. the equilibrium of non-r~ L-radioactive uric acid and an insignificant molar amount maount Biochim. Biophys. Acta, 53 ( I 9 6 I ) 5 5 7 - 5 6 c9-
U. V. LASSEN
e correct, the two kb's should not
y from
sport process, half of the uric acid i :om the suspending medium. The t:
rocytes e could
c.p V =
-
c.p.kb
(5)
-
2×T½
2×0.693
ime, where C is the concentration
in the
erna~ant. RESULTS AND DISCUSSION
,rmination of distribution factor P The value found in the present work was calculated from the upernatant counting rate when labelled uric acid was wa added hed erythrocytes. Table I compares the value of p with wit figures kers.
ecrease sion of y other
TABLE I RATIO OF D I S T R I B U T I O N OF URIC ACID B E T W E E N P L A S M A OR S U S P E N D I N G
Reference
BENEDICT
2
FOLIN AND SVEDBERG 1 HELLER 8
Wu48 JORGENSEN
AND NIELSEN 4
Present work
RE~ RED MEDIUM
BLOOD
Rativ Erythrocyteuric acid concentration* Plasma uric acid concentration
71.3 22.o 54.2 53.8 55
58.7 ~- ~.o**
C
o,
,o
determination
Colorimetric Colorimetric Colorimetric Colorimetric Spectrophotometricenzymic Labelled uric acid
Referring to e r y t h r o c y t e volume, n o t corrected to w a t e r pha ,hase of the cells. S t a n d a r d deviation on a series of i o determinations.
The distribution factorr is expressed as the apparent part of the erythrocyte volume available for the distributio tribution ltion of uric acid. This is of course a fictional value as it ought to be corrected for the water phase of the cells and for the trapped volume between the erythrocytes in the hematocrit determination. The last correction is not very important in this work k as the error thus introduced (about 2-3 %) is at least partially eliminated when p is applied to the uncorrected hematocrits of the experiments. When p is corrected acted to a water phase of 65 % (see ref. 2o), the ratio of intracellular uric acid concentratior ntration tion to uric acid concentration of the medium is 0.9 o. The possible significance of this value will be discussed in relation to the experiments at different pH.
Influx of labelled uric acid THODS, the labelled uric acid was added to a red blood As described under METHODS. cell suspension which was in equilibrium with non-labelled uric acid. The results le samples are treated according to Eqn. 3- Fig. I A and ]3 from the counting rate of the B i o c h i m . B i o p h y s . Acla, 53 (I961) 557-569
2ID TRANSPORT IN RED CELLS
Du.3
as described b y this expression. T lel e x p e r i m e n t s on the same batcL nd t h e correlation coefficients wer( possible to e s t i m a t e " t h e fit of the ~ns described, t h e t r a n s p o r t has kb f t i m e s of 24.8 a n d 23.1 min. Assun ustified when inspecting the figm correlation of t h e e x p e r i m e n t a l d; )elled uric acid, does not reveal chanism u n d e r l y i n g t h e transfer. I t is only shown tt]h a t the a =ulation of the k0 values m u s t be essentially correct. Thus ~ the = acid c a n n o t seriously affect t h e s t a t e of e q u i l i b r i u m ir in t h e syst, %, as it is shown below, is influenced b y the t o t a l ur uric acid cc d e v i a t i o n fr( dlibrium h a d been distorted, one m i g h t e x p e c t dev ',ervable within the e x p e r i m e n t a l error.
O+~
Io
i< o1~ %
3.5
/ -kb=(0"028"~0"001) rain-1 6 "t
7
~
.
~NN
o"1~
0~28-* 0.002) m
4.5
i
a
I.
r =-u.~l~
=
.
s of t h e -om the As t h e experi~28 a n d e points a high xpected out the for the labelled gnitude • If t h e h t line,
,ff °1~
3.~
.kb, (0~0-* ~O0~in "1
6
10
20 minutes
30
Fig. . I. Influx of labelled uric acid into h u m a n re n a t u r a l logaerythrocytes. The ordinate are rithmic plots according to Eqn.• 3. The abscissa Ls of t h e figure, are in minutes. The two parts A and B, represent data from parallel experills. T emperature ments on the same batch of cells. Tem -ic acid concen27.6 °, pH 7.14 and 7.16. Uric tration 15o/~moles/1.
10
20
30
40
minutes
man m Fig. 2. Efflux of ( labelled uric acid from h u m cells were loaded with witl erythrocytes. The labelled uric acid prior to t h e experiment. The Th )resent "two p a r t s of t h e figure, A and B, represen d a t a from parallel e x p e r i m e n t s on t h e slame am b a t c h of cells. The ordinate are n a t u r a l logaar i t h m i c plots according to Eqn. 4. The abscissa absciss~ T.I2 are in minutes. T e m p e r a t u r e 27.6 °, p H 7.1: and 7.12. Uric acid concentration 15o #moles/l1.
Efflux of labelled uric acid from 'allel e x p e r i m e n t s w i t h t r a n s p o r t of labelled uric acid fron T h e results of two parallel are shown in Fig. 2. I n a quite similar w a y as for the th( t h e interior of t h e e r y t h r o c"ytes 3 calcu influx e x p e r i m e n t s , t h e reg~ression lines a n d t h e coefficients of correlation were calcu~ere trea t r eeaa t e d according to Eqn. 4lated. The o b s e r v e d d a t a were lid. A g a i n t h e a s s u m p t i o n ss for the calculation of the t r a n s p o r t r a t e seem to be valid ations T h e Fig. 2 also gives an im pression of t h e difference between t h e two d e t e r m i nlations. ~rlhis d u p l i c a t e analysis t h a n was found in the influx experi The g r e a t e r s p r e a d i n g of this due ,bserved u n d e r similar conditions. Most p r o b a b l y it is du m e n t s was several times obs Biochim. Biophys. Acta, 53 (1961) 557-569 557-56
U. V. LASSEN
en determining low hematocrit nd of the effective gradient at timq ce when investigating factors influ
would reason, :ransfer
7 the effiux of labelled uric acid a influx experiments. As mentioned in complete equilibrium. Thus it i~, r systems) to describe the velocity c rane by a first order process; even ---o J ; system, in response to altered concentrations of uric uri( acid, do lple kinetics.
ficantly was to s it has :ransfer nsportthese
.
.
.
.
.
-
-
d
.
.
.
.
.
.
.
.
.
.
.
,/
.
.
.
.
.
.
.
.
E
-
-
-
periments with diBerent uric acid concentrations "Simple diffusion" might very well account for the t[ data 1~ rove. If s was the only mechanism by which uric acid could p~ )ass the m e value kb should be independent of the concentration of uric acid pre system. m a y be seen from Eqn. 5, if the transport from one side sl to the tirectly )portional to the concentration even when no net f1l u x o c c u )uld be istant to give direct proportionality between v and C. C This is •om the niliar "Fick's Law I " : J - - - - D (dC/dx) (where J is the tt numbe s transred per unit time, D is a constant, dC the change in ii concent dx the rage in distance ~1. However, when experiments with different uric acid ac concentrations were per)erformed med, the k~ value did not prove constant. Fig. 3 s] shows the variation of kb at al various :ious uric acid concentrations. This finding rules out the t possibility of diffusion ir m the,~common sense of the word. Thus the membrane see seems to show a limited transtransporting :ting capacity for uric acid. The v values (total transp( port per unit erythrocytes pel fer unitit time) were calculated from the uric acid concentratk ms and the corresponding kb ks values Lues according to Eqn. 5. The total transports and the uric acid concentrations are are
k'~1024. v-l~lo2
2-
./[,(~.o:t,x,(-:o.s)l ~ ~ g 10 m m o l e s uric acid I liter supernatant •
Fig. 3. Effect of different uri,ic acid concertt r a t i o n s on t h e influx of labelled !led uric acid in h u m a n e r y t h r o c y t e s . O r d i n a t e,~: : rate constant for e x c h a n g e of i n t r a c e l l u l a r uuric n acid × IO2. A b s c i s s a : uric acid c o n c e n t r a t iion o n in mmoles/1 s u p e r n a t a n t . T e m p e r a t u r e 25.o °, p H 7.IO-7.i5.
%12''°2
K~' ( rn m o l e s
o:5 uric
i
l:s
acid / liter supernatant)"I
Fig. 4. C o n c e n t r a t i o n d e p e n d e n c e of uric acid t r a n s p o r t in h u m a n e r y t h r o c y t e s w h e n p>lotted lotted a c c o r d i n g to LINEWEAV~-R AND BURK2~. Ordi-ih a t e : io 2 × (velocity of uric acid e x c h a n g e in ra /~moles/1 red cells/min) -1. A b s c i s s a : (mmoles/] nmoles/1 s u p e r n a t a n t ) -1. r is t h e coefficient of ccorreorrelation. O t h e r d a t a as in Fig. 3.
Biochim. Biophys. Acta, 53 (1961) 557-569 ~69
ID TRANSPORT
IN R E D CELLS
Do3
VER AND BURK22 in Fig. 4. "(v) as may be judged from inspectio~ lation. Just the same would be the when (velocity of substrate trar ntration)-l. A parallellism is thus so called MICHAELIS-MENTENeql ~rovided the comparison is justifie firm this, it is possible from the Ks, and the maximal velocity of th( (see ref. 25). From the expression for the regressic resslon line, tt ~e 3.2 mmoles uric acid/1 supernatant and the Vmax to t be 63 :hrocytes/min. Similar values were found in other exF~eriments The demonstration of a limited transporting capacity capacil of the ae for uric acid, without the creation of a consideraable conc~ mbles the findings for transport of glucose 6-12. W WILBRA l u c o s e 6-12. I L B R A N D T , FRE WILBRANDT9 have submitted the kinetics of net gluco lucose transp elation to glucose concentration, to a critical treatm( nent. The 1 t of transport seems to be (when using the same syml ~nbols as in •
v,
Ce v = Vmax
~ts and action i was
teen an nd the finding g. 4 to ~rocess, :ulated acid/1 ', memadient, NBERG 8
:ocytes or this
'
-- / ~ r
re C~ and C, are the concentrations on the two sides of the m ul d K r i1s~ dissociation constant of a postulated "substrate-canrrier complex". This type of ~tics, involving the thought of a "mobile carrier", may m~ explain the controversial kinetics. ings of a slow net transport and a fast exchange tra findin transport when C >~ Kr. Thus RE26 found that the exchange at hiigh substrate concentrations MCGINNISSAND LEFEvR velocit, city was 5O-lOO times faster than the net transport. The same kinetic behavior dc acid transport might be expected, but due to the low lo affinity of uric acid to the of uric valu and the low solubility of transsporting system (shown by a relatively high Ks value) uric acid, it is difficult to investigate this possibility. From the concentration dependence of uric acid trai transport through the erythrocyte membrane, it seems reasonabl asonable able to conclude that the uric acid enters a chenfical reaction or is coupled to a "carrier" in the membrane. The limiting factor for entry and exit of uric acid is thus presumably located in the membrane, as a tight binding es not seem to o c c u r 27. of intracellular uric acid does LII~
IILE;IIIUIO.II~
O.tlLl
15.~*
Experiments at diGerent tern1peratures If uric acid is transported ed byg aassystem with enzyme kinetics (as indicated above) temperature must heavily influence the transport rate. The variations of the rate n response to temperature changes should then be of a constants for the transfer in similar kind as for enzymic reactions. The results from an experiment with determinations of the kb values at 16 °, 23 °, 3 °° and 37 ° are shown in Fig. 5 when plotted according to the Arrhenius equation *s: + constant /~ is the apparent energy of activation of the transport process (or the enzymic rant (I.987 cal/degree) and T is the absolute temperature. reaction), R is the gas constant Biochim. Biophys. Acta, 53 ( I 9 6 I ) 5 5 7 - 5 6 9
U. V. LASSEN
~ints in Fig. 5, this mode of plott have a good degree of correlation (~ ssion line, it is possible to calculat ge 27 ° to 37 ° has a value of 2.21. T l, ~, of 15800 cal/mole. ndency supports the concept that me kind of "active" intervention 1 e diffusion process ~, and assuming Qlo of the range 27°-37 ° would b for the passively transported chloride ion, the tempe1 )erature coe LUCKNER~9 measured potentiometrically the Qto value w in t the chloride-bicarbonate shift in erythrocytes. The alteratic :entration was (in these experiments) mediated b y sudden c dde pressure. As the carbonic-anhydrase activity of the t] erythr, red a limiting role, the found value of 1.2-1. 4 must be a maxim; tture coefficient for uric acid transport is still much hi gher thar a that found for glucose transport b y LEFEVRE AND ][ DAVIES 3° ( If a considerable part of the transport at 16 ° had been be~ passiw zming a two component system, the plot in Fig. I woul would not ha~ lrve with a convexity towards zero. As the line seems seem to be s erimental error, one mechanism must be responsible for h the ma he transport.
to be From rature nds to )f uric brane. cosity ~¢ever, )t that )0-200
~onate "arbon t have e temlower ~tively m, b u t dn the lot all,
Experiments a different DH values During the investigations it was several times note( ted that uric acid was transported ted more rapidly at low than at high p H values in the t physiological range 5. I n the experiment shown in Fig. 6, the p H of the equilibrat uilibrated supernatant was varied b y the the addition of bicarbonate at constant carbon dioxid dioxide tension. In order to be able to correlate the velocity of uric acid ac transport to the percentage of undissociated uric acid, it was necessary to determine det the p K t value, as In(kb.103)
kb'q5-02~0
A •.
],love
y • (0.71tO.O?)x9) [
at
~2
[77~
r-*.t03"
3:s
Fig. 5. Influence of temperature re on r a t e const.ants for exchange of intracellular llular uric acid. T e m p e r a t u r e s 1 6 ° , 2 3 % 3 0 ° a n d [ 3 7 °. O r d i n a t e : n a t u r a l l o g a r i t h m of r a t e c o n s t a n t s × lO s. A b s c i s s a : T -x × io 3, w h e r e T is t h e a b s o l u t e t e m p e r a t u r e , r is t h e coefficien't of correlation, p H of t h e s o l u t i o n s were: 7.18,, 7.19, 7.24 a n d 7.2o. Uric acid c o n c e n t r a t i o nn 3oo #moles/1 supernatant.
-I
undluoclated uric ocid
Fig. 6. V a r i a t i o n s in r a t e c o n s t a n t s for e x c h a n g e of i n t r a c e l l u l a r uric acid × xo s in relation to, t h e c a l c u l a t e d fraction of u n d i s s o c i a t e d uric acid a t different p H v a l u e s (6.72-7.42). Abscissa in p e r cent. T e m p e r a t u r e 24.9 °. Uric acid c o n c e n t r a t i o n 5oo #moles/1 s u p e r n a t a n t , r i s t h e coefficient of correlation.
Biochim. Biophys. Acta, 53 (I96I) 557-56~)
ID TRANSPORT
IN
RED
CELLS
7 between the values found in earl ophotometrically as described b b method, was 5.79 =~ 0.04 (SD) as 9 o to 3o4 mt~. This figure was used uric acid at different p H values (t
Ou/
38. The t al. 34.
~termialation r.NDER-
ested that cell membranes might le only dous compounds. This has been sh umber lenon of "non-ionic diffusion" ha: rather ~ly accepted. I t has been extensively reviewed recently recent b y MI 1 effect If the acceleration of uric acid transport, when lowering loweri pH, :he ratio between undissociated and dissociated uric acid, the direct ( in the )ortionality between the rate constants and the percentage per regresissociated form. Fig. 6 shows this kind of plot. From the tl two va points line and the coefficient of correlation were calculated, tAs it m a y mantly yell to the line, and the intercept of the line on the Y axis i t proof ;rent from zero. The finding of such proportionality is b y no m ~ver, if uric acid is only able to permeate the membrane in the acid iations port (as for Lher p H dependent factor was influencing the transpoI etween that direct propo substrate-carrier dissociation), it is not likely dir lat the t above variables would be found. So far it seems reasonable rea,. _! " ~, sport of uric acid, though not passive in nature, is an example ot "'non-ionic sport. found in this work to be o.587 The distribution ratio for uric acid in red cells was fol to the water phase he total erythrocyte volume. When this figure was corrected c( of the he cells, the concentration in intracellular water was wa 9 ° % of the supernatant in the "entration. This is considerably higher than the relative relati, chloride concentration of concentrat 3o % which presumably is distributed according to the tl Donnan potential *°, 38,39. 78-80 of the intracellular m using the formula of VANSLYKE et al. 4° for the calculation cal~ When wh( m a n y of the experiments pH, and when using a supernatant p H of 7.00 (a value where his work were performed), the intracellular p H was found to be 6.94. JACOBS36 in this suggested a simple formulat for the determination of intracellular concentration of ording to non-ionic diffusion: weak acids, distributed accordin I "4- I o P H f - p K
C~ =
I - - I o P H o - pK
Co
"epresent inner and outer respectively. This equation m a y where the symbols i and o re be applied to the above p H values of 6.94 and 7.oo. The inner (total) concentration is :he outer concentration. However, in this p H range, the then found to be 88 % of the :ide will also approach unity. Variations in p H from 6.90 distribution factor for chloride Lhe distribution factor in this work, did not significantly to 7.2o when determining the alter the value found, and these figures are part of the calculated mean presented Lions it can be said, that the magnitude of intraceUular above. From these calculatk ther supports nor contradicts the theory of transport of uric acid concentration neither investigation with more exact methods for the estimation the undissociated acid. An investi of the distribution factor (and involving a wider p H range) might elucidate this problem. Biochim. Biophys. Acta,
53 (1961) 557-569
U. V. LASSEN C O N CLUSION
netic behavior of uric acid transp¢ xpectations for simple diffusion. I strong inhibitory effect on uric aci ibitory action is not confined only being equally or more potent as iI cid has kinetics in common with m ['he conclusive proof or active tran tt of particles against a concentration or electrical gradient t for the transport of sodium ions in m a n y systemse, 4~. Such an not been demonstrated for uric acid transport in erycthrocyte~ uric acid presumably belong to the group of substan ances bein~ ailibrating carrier mechanism ''*. If the transporting system, s~ wl Lmon to those purines which are able to penetrate the red cell c these substance, then the formation of nucleosides or nucleotid ht possibly occur. This formation might be expected to be el would account for uric acid transport being concentration concentr~ depl ' as is found for the turnover of substrate in an enzymi rmlc reactior exact nature of the transport system has not been elucidated. el
ocytes r been Work lthine, where movesically nsport "eason, abya ably is pecific ebrane lature, similar wever,
ACKNOWLEDGEMENTS
E KLENOW for stimulating The author wishes to thank Drs. T. ROSENBERG and H. discussion ussion, and Dr. K. OVERGAARD-HANSENfor generous assistance with some of the experiments. eriments. Dr. A. CODDINGTON'Scorrection of the manuscript manu is gratefully acknowledgled. This investigation was aided b y a grant (L38/6o ) from Statens Almindelige Videnskabsfond. REFERENCES 1 0 . . FOLIN AND A. SVEDBERG, J. Biol. Chem., 88 (193o) 715 . * S . R. BENEDICT AND J. A. EEHRR, J. J . Biol. Chem., 92 (1931) 16 i6i. 3 j , HELLER, Biochem. Z., 279 (1935) I49. 4 S. JORGENSEN AND AA. T. NIELSEN mLSRN, Scan& J. Glin. Lab. Invest., 8 (1956) Io8. 5 K. OVERGAARD-HANSI~NAND U. V. LASSEN, Nature, 184 (1959) 5538 T. ROSENBERG, Symposia Soc.:. Exptl. Biol., 8 (1954) 27. T. ROSENBERG AND W. WILBR, mANDT, J. Gen. Physiol., 41 (I957) 289. 8 W . WILBRANDT, S. F R E I AND T. ROSENBRRG, Exptl. Cell Research, I I (1956) 599 W. WILBRANDT in A. KLEINZELLER NZELLER AND A. KOTYK, Membrane Transport and MetabolismSymposium, Prague, I96O, p. 205. 18 p. G. LEFEvRE, J. Gen. Physiol., ~iol., 31 (1948) 505 . lX p. G. LEFRVRE AND G. F. McGZNNISS cGINNISS, J. Gen. Physiol., 44 (196o) 87. 12 W. F. WIDDAS, J. Physiol. (London), London), 118 (1952 ) 23 . 13 R. EGE, Biohem. Z., 114 (1921) 88. 14 O . BANG AND S. L. ORSKOV, J. Clin. Invest., 16 (1937) 279. 15 W. W. UMBREIT, R. H. BURRIS RRIS AND J . F . STAUFFRR, Manometric Techniques, Minneapolis, 1957, p. 149. 18 H. KLRNOW AND R. EMBERLAND AND, Arch. Biochem. Biophys., 58 (1955) 276. 17 B. MACKLRR, H. R. MAHLER AND D. E. GREEN, J. Biol. Chem., 21o (1954) 149. 18 E . PRARTORIUS AND H . E . P oOULSRN, l Scand. J. Clin. Lab. Invest., 5 (1953) 273. 18 C. W . SHRPPARD AND W . R . MARTIN, J. Gen. Physiol., 33 (1949) 703 • 20 B. VESTERGAARD-BOGIND AND 'D T. HESSELBO, Biochim. Biophys. Acta, 44 (196o) 117. t t A. MAURO in Symposium on Salt and Water Metabolism, N e w Y o r k H e a r t Association, 1959, p. 845.
Biochim. Biophys. Acta, 53 (1961) 557-569
TRANSPORT IN RED CELLS
Du~
• Chem. Soc., 56 (1934) 658. 9chem. Z., 49 (I913) 333. Federation Proc., 18 (196o) IOI. n. Lab. Invest., 9 (I957) 194. istry, New York, I948. ~er's, 241 (1939) 753. Gen. Physiol., 34 (1951) 515 . Hoppe-Seylers, 31 (19oo) i. N, Helv. Chim. dcta, 23 (194 o) 245. 133. BEAVEN, E. R. HOLYDAY AND E. A. JOHNSON in E. CCHAR, HARGAFF
AND
BON,
•ucleic Acids. Vol. I, Academic Press, Inc., New York, 1955, p. ] 496. BER, J. Cellular Comp. Physiol., 7 (1936) 367. JACOBS, Cold Spri~*g Harbor Symposia Quant. Biol., 8 (194o) 3 ° • MILNE, B. H. SCRIBNER AND M. A. CRAWFORD, Am. J. Med. Med., 24 (1958) .~ALD'~VELL, Intern. Rev. Cytol., 5 (1956) 229. jLYNN, Progress in Biophys., 8 (1957) 242. VANSLYKE, H. ~VIT AND F. C. MCLEAN, J. Biol. Chem., 56 (I, (1923) 765 • LASSEN AND K. OVERGAARD-HANSEN, unpublished experimel ~eriments. USSING, in H. H. USSING, P. I~RUHOFFER, J. HESS THAYSEN AND A N. A. "]
kali
Ions in Biology, Heidelberg, 196o. J, J. Biol. Chem., 51 (1922) 21. Biochim. Biop)~Izys. Acta,
ABNORMAL
-569
HUMAN HAEMOGLOIBINS
VII. VII. THE COMPARIS~JN OF NORMAL HUMAN HAEMOGLOBIN F. AND H A E M O G L O B I N DCmCAGO B A R B A R A BOWMAN AND V. M. INGRA ;RAM Division ~ision oo] Biochemistry, Department o[ Biology, Massachusetts Institute o] Technology, 3ambridge, Mass. (U.S.A.) Cambrid
(Received april 7th, 1961)
SUMMARY
peptide from haemoglobin Dchleago carries a chemical I t has been shown that a tryptic peg d i f f e r e n c e w h e n c o m p a r e d t o t:he h e corresponding peptide in normal haemoglobin A. T h e a l t e r e d p e p t i d e , c a l l e d n u mnber b e r 5, occurs in the fl-chain of the molecule.
INTRODUCTION T h r e e v a r i a n t s of h u m a n h a e m oLoglobin D have been investigated by BENZER et al. 1. E a c h v a r i a n t c a r r i e d a c h e m i c a fl l change in a different part of the haemoglobin molecule. I n h a e m o g l o b i n D~ ( f r o m La Turkish Cypriot) the a-chain tryptic peptide 23 D a (from a Gujerati Indian) the fl-chain (ref. 2) h a d d i s a p p e a r e d ; i n hLaemoglobin aemo
Biochim. Biopt~ys. Acta, 53 (1961) 569-573
RIC ACID T R A N S P O R T I N t ERYTHROCYTES U L R I K V. L A S S E N
)epartment A, University o/Copenhagen
(Received A p r i l
7th, 1961)
SUMMARY
I. The transport of uric acid in erythrocytes has bee been studiec ibrated ;tern b y means of labelled uric acid. 2. The necessary assumptions and calculations for f such ats are ~sented. 3. The transport is, in contrast to simple diffusion, diffusio~ concen endent. e Ks value for the transporting system is 3.2 mM. 4. From the effect of temperature on the transport, the Q10 v~ to be ___/ . . . . . . . I; the corresponding energy of activation is 158oo cal/mole. cl 5. The transport velocity is accelerated when lowering lowerir pH. This effect is presummablLy due to alterations in the fraction of total uric acid concentration being present ~resent a s the undissociated acid. Thus it is concluded that this transport is an example of o "non-ionic transfer". me 6.~. The kinetics for uric acid reveal that an "active" mechanism must be responsibh ,nsible for the transport, even though concentrative uptake or o~ extrusion has not been dede. monstrated.
INTRODUCTION
The presence of uric acid in hum~ human erythrocytes has previously been demonstrated 1-4 Until recently, the lack of a concentrative uptake was considered as indicative of uric uric acid being transported thr"ough the membrane only by diffusion. As described in a preliminary note 5, several of ~f the factors influencing uric acid transport in erythrocyte, ,tes permit the conclusion thatt diffusion cannot be the only means b y which uric acid passes the erythrocyte membrane. However, the results previously presented did not give sufficiently detailed tetailed information to permit a sugl;gestion of the kinetic behavior of the transporting system rstem. The aim of the present stud' dy is to describe in detail the method and the calculation, ttions used to determine the veloccity of uric acid transport through the human erythrocyte zte membrane and also to present ~ent results from experiments employing variations in the uric acid concentration andd the temperature of the system. These data support the concept of a "facilitated" or "carrier-linked" transport mechanism. This kind ot of kinetics has been demonstrated rated for the transport of glucose and other monosacchasaccharides through the erythroc3yte membrane 6-14. Biochim. Biophys. Acta, 53
(1961) 557-56g 369
U. V. LASSEN
xs in the p H of the suspending me the erythrocyte membrane in the a
XPERIMENTAL
te t h a t ciated)
METHODS
1 (3000 I.U. heparin/1) from health ~s used ?eriment blood from only one do: ,,d. The lasma and "buffy coat" sucked off ls were ............... ~ .......... /2. . . . . . . . . . . . . . . . J . . . . . hed two or three times by resuspension and subsequen uent centrifu ooxg 3o min. The wash fluids and the media of incubatior ttion were K] -bicarate solutions. Sufficient sodium bicarbonate was added adde to give p H at temperature of incubation with a moistened 5 % carbon carl dioxi Lure as gas phase 1~. The wash fluids were identical with the final media ~dia of incuba' ltained of glucose/l, and, unless otherwise stated, 3oo txmoles moles of uric ac experiLts with high uric acid concentrations, uric acid was dissolved taining lculated amount of lithium cabonate. In these experilI ~eriments all t~ ~ed the e lithium concentration, either as the urate or the chloride. chk Due to the instability of very dilute solutions of uric acid, a even state, 18-14Cjoactive uric acid was prepared immediately prior to t~ each e~ ! Centre Centre, Art Amersham, England) oxantine (2.4-2. 7 mC/mmole, The Radiochemical Ce oxidized b y means of milk xanthine oxidase TM in o.o 5 M Tris buffer at p H 7.5. To ~owerful substrate inhibition of [d blocking of the enzyme action due to the powel avoid thine oxidase ~*, the radioactive hypoxanthine )oxanthine was added addc repeatedly to the enzyme. xanthine 'y instance by spectrophotoThe termination of the oxidation was checked in every further addition of ry at 292 m/~ (Beckman model DU). At this wavelength wav metr, absorbancy, apart from the thine oxidase should give no additional increase in xanthine oxidase was inactivated ease caused by the enzyme. After control, the xanthine xanth increase ~oiling for 3o sec. This solution was made isotonic with sodium chloride or glucose. b y boiling The ~4C-measurements were performed by plating 50 5o ~1 of the supernatants from minium the incubates on 24 m m aluminium um discs. The total dry matter was between o.2 and tion was made for selfabsorption. The planchettes were o.3 mg/cm ~, and no correction measured for radioactivity b)y a thin-window, gas-flow, proportional counter (Frieseke "uck, Ge Germany, German model F H 4o7). Samples were counted to a and Hoepfner, Erlangen, Bruck, statistical error of less than 2 %. The p H of the incubates was measured at the end of each experiment to ascertain td occurred during the incubation. Variations between if major p H variations had individual flasks in a single experiment did not usually exceed o.o 5 p H unit. When differences were more thanL o.I p H unit, the experiment was discarded (except of here different p H values were intended), p H was measured course in the experiments where meter, Copenhagen, Denmark, model P H M 22). b y a glass electrode (Radiometer ormed in a water b a t h with provisions for heating and Incubations were performed cooling and with a shaking device ( 7 o 8 o oscillations/min). The incubating vessels were Ioo-ml conical flasks, siliconised before use. ..re determined in graduated tubes centrifuged at 25oo Hematoerit values were terminations being carried out in duplicate. rev./min for 30 rain, the determina Biochim. Biophys. Acta,
53 (I961)
557-569
ID TRANSPORT IN RED CELLS
zere carried out b y the spectrophoi ~d uricase. dculated according to standard fl determination of regression coerce
thod of ing the
nts
d cells were shaken in the incubato carbon dioxide atmosphere and tl dram. l he incubating mixture re had a hematocrit laematocnt of ot 45-6o 4.~ %. A )eriment, o.o5-o.1/zC of [8-z4C]uric acid/ml of total incubat [erent time intervals, 5-12 samples of 1.5-2.o ml were wer, taken o "'Internation~ ttrifuged in a refrigerated high speed centrifuge ("Int ~oo × g for at least 2 min. The "time" of each sampl)le was th tition of 14C to the vessel to the centrifuge rotor speed sI pa s ,out io sec after the starting of the centrifuge). The supernat for plating and, in some experiments, for uric acid aci deterrr. :line in counting rate of the supernatant, the velocit,y of uric culated as shown below.
min to of the for the Led. At 'diately R I) at om the ev./min ipetted om the ~rt was
l~C]uric acid eOlux experiments The washed erythrocytes were incubated for I h in a~i medium lll~[lUlli ~ U l | L c l i l l l l l ~ 0.I--0.2 U.I.--U.2 [8-i*CJuric acid/ml. The cells were then spun down and washed once with the th¢ Krebs-Rin ebs-Ringer-bicarbonate solution in the cold. At "zero" "z time, the z4C loaded loaded erythroc~ 'throcytes were added to the incubating medium to giive a hematocrit of lO-2O %. % Fro)m this incubate, samples were taken out and treated trea as described for influx influx th~ supernatant, the uric acid e x l )eriments. From the increase in counting rate of the cid transport was calculated.
Calculation of transport rate Influx of labelled uric acid: The erythrocytes and their thc medium of suspension arc are tS19. considered as two compartments TM ments TM. These are separated b y a membrane which exerts exert, a characteristic influence onn the velocity or uric acid transport between the two com. cornpartments. Prior to the start trt of the experiment, the system is equilibrated with nnononlabelled uric acid. At "zero" f ' time labelled uric acid is added to compartment A and starts to pass the membrane ne towards compartment B. Assuming immediate mixin~g on both sides of the memxbrane, the decrease in s u p e m a t a n t counting rate (comcornp a r t m e n t A) with time must mt depend on three factors: (a) the "permeability" of the th¢ membrane for uric acid, (b) b) the mean affective gradient of labelled uric acid, and (c) the relative volumes of the two compartments. This dependence holds true either eithei when there is no effect of the total uric acid concentration upon the transport rate, or when the molar amount of labelled uric acid is small compared to the concentration tration of non-labelled uric acid in the system. In this last situation the amount of uric acid passing the membrane from m each side per unit time must be the same. In the exexperiments reported here, the molar amount of labelled uric acid is small (about about 25/anoles/l). The following symbols are used in the calculations:
Biochim. Biophys. Acta, 53 (I96I) 557-56g 369
U. V. LASSEN
mt (compartment A). "ic acid distribution in the eryth e (same units as a and b) cent. erythrocyte volume available for ( ater phase). rtment A when extrapolated to zel rtment A at time "t". ka, Rate constant for change in counting rate in cornpartmeni
mpart-
of uric
re.
From the definition of these symbols: V
O~ = - - (IO0 - - H) IOO
p.H.V
b--
Ioo
At time "t" the counting rate in compartment B is: (x 0 - - x)
time Counting rate in compartment A will then decrease with v
-'~ --
(x
(xo-- x) a.)
or by rearrangement and integration:
((°) I
fd
--
a
x
I +~
o
--.g'o~) a
J
kadt
By solution of this integral: I
ct
a
(~)
a I 27-
b
integration. where M is the constant of inte Eqn. i describes a strailight line with a slope of --ka and an intercept on the vertical axis of M. In addition to the symbols used above, we have: E ~ u x of labelled uric acid: In mpartment B at zero time. Zo, Counting rate in corn mpartment B at time t. z, Counting rate in com ats the extracellular counting rate at zero time when the I n this case Xo represents Lve lost no radioactivity. In a similar way, the rising 14C-loaded erythrocytes have ernatant, x, represent the initial counting rate plus the counting values of the supernatan radioactivity lost from the erythrocytes (compartment B). This gives: B i o c h i m . BiopP~ys. A c t a , 53 (1961) 557-569 i69
~O.l.
I D T R A N S P O R T I N R E D CELLS I
a
(x - - xo) or z = zo - - ~ (x - - x0) influx calculation it follows:
k=(ZO--b(x--xo)--x )
=
ttion gives: a
f
a
x+~
d zo+
This integral m a y be solved to give: I
( zo
In
o Xo--x
+
=----kat
I +
+ N
(2)
~+g is the constant of integration. Before the application of Eqn. 2 to the experiment~ ~erimental data wl 5, x, and Xo values, it is necessary to calculate zo. TI This is don mula:
a+b
Z0 ~ Xeq
nprises to the
a
b
X0~
aere Xeq is the counting rate of the s u p e m a t a n t (comp~ Where )artment A) when the system totem is e(:quilibrated with respect to radioactive uric acid. Ex t )erimentally Xeq (thoughh not theoreticaU' ~oretically obtainable within a limited time) is taken as a the counting value when prolonge ~longed incubation will give no significant increase in supernatant s counting rate. From the Eqns. I and 2 it is possible to calculate the t rate constants for super)ern a t :ant a n t counting rate when using either influx or effiux efflu of [ 1 4 C ~ u r i c acid for the determination. :ermination. The aim of the determination is, however, howeve to correlate the transporl )ort of uric acid to the amount of red cells being present. The rate constant for the intracellular uric acid (kb) is simpl aply correlated to the ka value: kb = ka .b When inserting kb in the Ec ~qns. I and 2 we have: a
a+b a - -
a+b
-in
In
x
+
i +
a
--xo
=--h#
g
+M1 ÷
(3) (4)
The k~ values calculated ed f, for the same batch of cells under the same experimental Lmental conditions should be inde 1?endent of whether influx (Eqn. 3) or efltux (Eqn. 4) was wa~ used for the determination. n. From this it is possible to test the assumptions used used: i.e. the equilibrium of non-r~ L-radioactive uric acid and an insignificant molar amount maount Biochim. Biophys. Acta, 53 ( I 9 6 I ) 5 5 7 - 5 6 c9-
U. V. LASSEN
e correct, the two kb's should not
y from
sport process, half of the uric acid i :om the suspending medium. The t:
rocytes e could
c.p V =
-
c.p.kb
(5)
-
2×T½
2×0.693
ime, where C is the concentration
in the
erna~ant. RESULTS AND DISCUSSION
,rmination of distribution factor P The value found in the present work was calculated from the upernatant counting rate when labelled uric acid was wa added hed erythrocytes. Table I compares the value of p with wit figures kers.
ecrease sion of y other
TABLE I RATIO OF D I S T R I B U T I O N OF URIC ACID B E T W E E N P L A S M A OR S U S P E N D I N G
Reference
BENEDICT
2
FOLIN AND SVEDBERG 1 HELLER 8
Wu48 JORGENSEN
AND NIELSEN 4
Present work
RE~ RED MEDIUM
BLOOD
Rativ Erythrocyteuric acid concentration* Plasma uric acid concentration
71.3 22.o 54.2 53.8 55
58.7 ~- ~.o**
C
o,
,o
determination
Colorimetric Colorimetric Colorimetric Colorimetric Spectrophotometricenzymic Labelled uric acid
Referring to e r y t h r o c y t e volume, n o t corrected to w a t e r pha ,hase of the cells. S t a n d a r d deviation on a series of i o determinations.
The distribution factorr is expressed as the apparent part of the erythrocyte volume available for the distributio tribution ltion of uric acid. This is of course a fictional value as it ought to be corrected for the water phase of the cells and for the trapped volume between the erythrocytes in the hematocrit determination. The last correction is not very important in this work k as the error thus introduced (about 2-3 %) is at least partially eliminated when p is applied to the uncorrected hematocrits of the experiments. When p is corrected acted to a water phase of 65 % (see ref. 2o), the ratio of intracellular uric acid concentratior ntration tion to uric acid concentration of the medium is 0.9 o. The possible significance of this value will be discussed in relation to the experiments at different pH.
Influx of labelled uric acid THODS, the labelled uric acid was added to a red blood As described under METHODS. cell suspension which was in equilibrium with non-labelled uric acid. The results le samples are treated according to Eqn. 3- Fig. I A and ]3 from the counting rate of the B i o c h i m . B i o p h y s . Acla, 53 (I961) 557-569
2ID TRANSPORT IN RED CELLS
Du.3
as described b y this expression. T lel e x p e r i m e n t s on the same batcL nd t h e correlation coefficients wer( possible to e s t i m a t e " t h e fit of the ~ns described, t h e t r a n s p o r t has kb f t i m e s of 24.8 a n d 23.1 min. Assun ustified when inspecting the figm correlation of t h e e x p e r i m e n t a l d; )elled uric acid, does not reveal chanism u n d e r l y i n g t h e transfer. I t is only shown tt]h a t the a =ulation of the k0 values m u s t be essentially correct. Thus ~ the = acid c a n n o t seriously affect t h e s t a t e of e q u i l i b r i u m ir in t h e syst, %, as it is shown below, is influenced b y the t o t a l ur uric acid cc d e v i a t i o n fr( dlibrium h a d been distorted, one m i g h t e x p e c t dev ',ervable within the e x p e r i m e n t a l error.
O+~
Io
i< o1~ %
3.5
/ -kb=(0"028"~0"001) rain-1 6 "t
7
~
.
~NN
o"1~
0~28-* 0.002) m
4.5
i
a
I.
r =-u.~l~
=
.
s of t h e -om the As t h e experi~28 a n d e points a high xpected out the for the labelled gnitude • If t h e h t line,
,ff °1~
3.~
.kb, (0~0-* ~O0~in "1
6
10
20 minutes
30
Fig. . I. Influx of labelled uric acid into h u m a n re n a t u r a l logaerythrocytes. The ordinate are rithmic plots according to Eqn.• 3. The abscissa Ls of t h e figure, are in minutes. The two parts A and B, represent data from parallel experills. T emperature ments on the same batch of cells. Tem -ic acid concen27.6 °, pH 7.14 and 7.16. Uric tration 15o/~moles/1.
10
20
30
40
minutes
man m Fig. 2. Efflux of ( labelled uric acid from h u m cells were loaded with witl erythrocytes. The labelled uric acid prior to t h e experiment. The Th )resent "two p a r t s of t h e figure, A and B, represen d a t a from parallel e x p e r i m e n t s on t h e slame am b a t c h of cells. The ordinate are n a t u r a l logaar i t h m i c plots according to Eqn. 4. The abscissa absciss~ T.I2 are in minutes. T e m p e r a t u r e 27.6 °, p H 7.1: and 7.12. Uric acid concentration 15o #moles/l1.
Efflux of labelled uric acid from 'allel e x p e r i m e n t s w i t h t r a n s p o r t of labelled uric acid fron T h e results of two parallel are shown in Fig. 2. I n a quite similar w a y as for the th( t h e interior of t h e e r y t h r o c"ytes 3 calcu influx e x p e r i m e n t s , t h e reg~ression lines a n d t h e coefficients of correlation were calcu~ere trea t r eeaa t e d according to Eqn. 4lated. The o b s e r v e d d a t a were lid. A g a i n t h e a s s u m p t i o n ss for the calculation of the t r a n s p o r t r a t e seem to be valid ations T h e Fig. 2 also gives an im pression of t h e difference between t h e two d e t e r m i nlations. ~rlhis d u p l i c a t e analysis t h a n was found in the influx experi The g r e a t e r s p r e a d i n g of this due ,bserved u n d e r similar conditions. Most p r o b a b l y it is du m e n t s was several times obs Biochim. Biophys. Acta, 53 (1961) 557-569 557-56
U. V. LASSEN
en determining low hematocrit nd of the effective gradient at timq ce when investigating factors influ
would reason, :ransfer
7 the effiux of labelled uric acid a influx experiments. As mentioned in complete equilibrium. Thus it i~, r systems) to describe the velocity c rane by a first order process; even ---o J ; system, in response to altered concentrations of uric uri( acid, do lple kinetics.
ficantly was to s it has :ransfer nsportthese
.
.
.
.
.
-
-
d
.
.
.
.
.
.
.
.
.
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periments with diBerent uric acid concentrations "Simple diffusion" might very well account for the t[ data 1~ rove. If s was the only mechanism by which uric acid could p~ )ass the m e value kb should be independent of the concentration of uric acid pre system. m a y be seen from Eqn. 5, if the transport from one side sl to the tirectly )portional to the concentration even when no net f1l u x o c c u )uld be istant to give direct proportionality between v and C. C This is •om the niliar "Fick's Law I " : J - - - - D (dC/dx) (where J is the tt numbe s transred per unit time, D is a constant, dC the change in ii concent dx the rage in distance ~1. However, when experiments with different uric acid ac concentrations were per)erformed med, the k~ value did not prove constant. Fig. 3 s] shows the variation of kb at al various :ious uric acid concentrations. This finding rules out the t possibility of diffusion ir m the,~common sense of the word. Thus the membrane see seems to show a limited transtransporting :ting capacity for uric acid. The v values (total transp( port per unit erythrocytes pel fer unitit time) were calculated from the uric acid concentratk ms and the corresponding kb ks values Lues according to Eqn. 5. The total transports and the uric acid concentrations are are
k'~1024. v-l~lo2
2-
./[,(~.o:t,x,(-:o.s)l ~ ~ g 10 m m o l e s uric acid I liter supernatant •
Fig. 3. Effect of different uri,ic acid concertt r a t i o n s on t h e influx of labelled !led uric acid in h u m a n e r y t h r o c y t e s . O r d i n a t e,~: : rate constant for e x c h a n g e of i n t r a c e l l u l a r uuric n acid × IO2. A b s c i s s a : uric acid c o n c e n t r a t iion o n in mmoles/1 s u p e r n a t a n t . T e m p e r a t u r e 25.o °, p H 7.IO-7.i5.
%12''°2
K~' ( rn m o l e s
o:5 uric
i
l:s
acid / liter supernatant)"I
Fig. 4. C o n c e n t r a t i o n d e p e n d e n c e of uric acid t r a n s p o r t in h u m a n e r y t h r o c y t e s w h e n p>lotted lotted a c c o r d i n g to LINEWEAV~-R AND BURK2~. Ordi-ih a t e : io 2 × (velocity of uric acid e x c h a n g e in ra /~moles/1 red cells/min) -1. A b s c i s s a : (mmoles/] nmoles/1 s u p e r n a t a n t ) -1. r is t h e coefficient of ccorreorrelation. O t h e r d a t a as in Fig. 3.
Biochim. Biophys. Acta, 53 (1961) 557-569 ~69
ID TRANSPORT
IN R E D CELLS
Do3
VER AND BURK22 in Fig. 4. "(v) as may be judged from inspectio~ lation. Just the same would be the when (velocity of substrate trar ntration)-l. A parallellism is thus so called MICHAELIS-MENTENeql ~rovided the comparison is justifie firm this, it is possible from the Ks, and the maximal velocity of th( (see ref. 25). From the expression for the regressic resslon line, tt ~e 3.2 mmoles uric acid/1 supernatant and the Vmax to t be 63 :hrocytes/min. Similar values were found in other exF~eriments The demonstration of a limited transporting capacity capacil of the ae for uric acid, without the creation of a consideraable conc~ mbles the findings for transport of glucose 6-12. W WILBRA l u c o s e 6-12. I L B R A N D T , FRE WILBRANDT9 have submitted the kinetics of net gluco lucose transp elation to glucose concentration, to a critical treatm( nent. The 1 t of transport seems to be (when using the same syml ~nbols as in •
v,
Ce v = Vmax
~ts and action i was
teen an nd the finding g. 4 to ~rocess, :ulated acid/1 ', memadient, NBERG 8
:ocytes or this
'
-- / ~ r
re C~ and C, are the concentrations on the two sides of the m ul d K r i1s~ dissociation constant of a postulated "substrate-canrrier complex". This type of ~tics, involving the thought of a "mobile carrier", may m~ explain the controversial kinetics. ings of a slow net transport and a fast exchange tra findin transport when C >~ Kr. Thus RE26 found that the exchange at hiigh substrate concentrations MCGINNISSAND LEFEvR velocit, city was 5O-lOO times faster than the net transport. The same kinetic behavior dc acid transport might be expected, but due to the low lo affinity of uric acid to the of uric valu and the low solubility of transsporting system (shown by a relatively high Ks value) uric acid, it is difficult to investigate this possibility. From the concentration dependence of uric acid trai transport through the erythrocyte membrane, it seems reasonabl asonable able to conclude that the uric acid enters a chenfical reaction or is coupled to a "carrier" in the membrane. The limiting factor for entry and exit of uric acid is thus presumably located in the membrane, as a tight binding es not seem to o c c u r 27. of intracellular uric acid does LII~
IILE;IIIUIO.II~
O.tlLl
15.~*
Experiments at diGerent tern1peratures If uric acid is transported ed byg aassystem with enzyme kinetics (as indicated above) temperature must heavily influence the transport rate. The variations of the rate n response to temperature changes should then be of a constants for the transfer in similar kind as for enzymic reactions. The results from an experiment with determinations of the kb values at 16 °, 23 °, 3 °° and 37 ° are shown in Fig. 5 when plotted according to the Arrhenius equation *s: + constant /~ is the apparent energy of activation of the transport process (or the enzymic rant (I.987 cal/degree) and T is the absolute temperature. reaction), R is the gas constant Biochim. Biophys. Acta, 53 ( I 9 6 I ) 5 5 7 - 5 6 9
U. V. LASSEN
~ints in Fig. 5, this mode of plott have a good degree of correlation (~ ssion line, it is possible to calculat ge 27 ° to 37 ° has a value of 2.21. T l, ~, of 15800 cal/mole. ndency supports the concept that me kind of "active" intervention 1 e diffusion process ~, and assuming Qlo of the range 27°-37 ° would b for the passively transported chloride ion, the tempe1 )erature coe LUCKNER~9 measured potentiometrically the Qto value w in t the chloride-bicarbonate shift in erythrocytes. The alteratic :entration was (in these experiments) mediated b y sudden c dde pressure. As the carbonic-anhydrase activity of the t] erythr, red a limiting role, the found value of 1.2-1. 4 must be a maxim; tture coefficient for uric acid transport is still much hi gher thar a that found for glucose transport b y LEFEVRE AND ][ DAVIES 3° ( If a considerable part of the transport at 16 ° had been be~ passiw zming a two component system, the plot in Fig. I woul would not ha~ lrve with a convexity towards zero. As the line seems seem to be s erimental error, one mechanism must be responsible for h the ma he transport.
to be From rature nds to )f uric brane. cosity ~¢ever, )t that )0-200
~onate "arbon t have e temlower ~tively m, b u t dn the lot all,
Experiments a different DH values During the investigations it was several times note( ted that uric acid was transported ted more rapidly at low than at high p H values in the t physiological range 5. I n the experiment shown in Fig. 6, the p H of the equilibrat uilibrated supernatant was varied b y the the addition of bicarbonate at constant carbon dioxid dioxide tension. In order to be able to correlate the velocity of uric acid ac transport to the percentage of undissociated uric acid, it was necessary to determine det the p K t value, as In(kb.103)
kb'q5-02~0
A •.
],love
y • (0.71tO.O?)x9) [
at
~2
[77~
r-*.t03"
3:s
Fig. 5. Influence of temperature re on r a t e const.ants for exchange of intracellular llular uric acid. T e m p e r a t u r e s 1 6 ° , 2 3 % 3 0 ° a n d [ 3 7 °. O r d i n a t e : n a t u r a l l o g a r i t h m of r a t e c o n s t a n t s × lO s. A b s c i s s a : T -x × io 3, w h e r e T is t h e a b s o l u t e t e m p e r a t u r e , r is t h e coefficien't of correlation, p H of t h e s o l u t i o n s were: 7.18,, 7.19, 7.24 a n d 7.2o. Uric acid c o n c e n t r a t i o nn 3oo #moles/1 supernatant.
-I
undluoclated uric ocid
Fig. 6. V a r i a t i o n s in r a t e c o n s t a n t s for e x c h a n g e of i n t r a c e l l u l a r uric acid × xo s in relation to, t h e c a l c u l a t e d fraction of u n d i s s o c i a t e d uric acid a t different p H v a l u e s (6.72-7.42). Abscissa in p e r cent. T e m p e r a t u r e 24.9 °. Uric acid c o n c e n t r a t i o n 5oo #moles/1 s u p e r n a t a n t , r i s t h e coefficient of correlation.
Biochim. Biophys. Acta, 53 (I96I) 557-56~)
ID TRANSPORT
IN
RED
CELLS
7 between the values found in earl ophotometrically as described b b method, was 5.79 =~ 0.04 (SD) as 9 o to 3o4 mt~. This figure was used uric acid at different p H values (t
Ou/
38. The t al. 34.
~termialation r.NDER-
ested that cell membranes might le only dous compounds. This has been sh umber lenon of "non-ionic diffusion" ha: rather ~ly accepted. I t has been extensively reviewed recently recent b y MI 1 effect If the acceleration of uric acid transport, when lowering loweri pH, :he ratio between undissociated and dissociated uric acid, the direct ( in the )ortionality between the rate constants and the percentage per regresissociated form. Fig. 6 shows this kind of plot. From the tl two va points line and the coefficient of correlation were calculated, tAs it m a y mantly yell to the line, and the intercept of the line on the Y axis i t proof ;rent from zero. The finding of such proportionality is b y no m ~ver, if uric acid is only able to permeate the membrane in the acid iations port (as for Lher p H dependent factor was influencing the transpoI etween that direct propo substrate-carrier dissociation), it is not likely dir lat the t above variables would be found. So far it seems reasonable rea,. _! " ~, sport of uric acid, though not passive in nature, is an example ot "'non-ionic sport. found in this work to be o.587 The distribution ratio for uric acid in red cells was fol to the water phase he total erythrocyte volume. When this figure was corrected c( of the he cells, the concentration in intracellular water was wa 9 ° % of the supernatant in the "entration. This is considerably higher than the relative relati, chloride concentration of concentrat 3o % which presumably is distributed according to the tl Donnan potential *°, 38,39. 78-80 of the intracellular m using the formula of VANSLYKE et al. 4° for the calculation cal~ When wh( m a n y of the experiments pH, and when using a supernatant p H of 7.00 (a value where his work were performed), the intracellular p H was found to be 6.94. JACOBS36 in this suggested a simple formulat for the determination of intracellular concentration of ording to non-ionic diffusion: weak acids, distributed accordin I "4- I o P H f - p K
C~ =
I - - I o P H o - pK
Co
"epresent inner and outer respectively. This equation m a y where the symbols i and o re be applied to the above p H values of 6.94 and 7.oo. The inner (total) concentration is :he outer concentration. However, in this p H range, the then found to be 88 % of the :ide will also approach unity. Variations in p H from 6.90 distribution factor for chloride Lhe distribution factor in this work, did not significantly to 7.2o when determining the alter the value found, and these figures are part of the calculated mean presented Lions it can be said, that the magnitude of intraceUular above. From these calculatk ther supports nor contradicts the theory of transport of uric acid concentration neither investigation with more exact methods for the estimation the undissociated acid. An investi of the distribution factor (and involving a wider p H range) might elucidate this problem. Biochim. Biophys. Acta,
53 (1961) 557-569
U. V. LASSEN C O N CLUSION
netic behavior of uric acid transp¢ xpectations for simple diffusion. I strong inhibitory effect on uric aci ibitory action is not confined only being equally or more potent as iI cid has kinetics in common with m ['he conclusive proof or active tran tt of particles against a concentration or electrical gradient t for the transport of sodium ions in m a n y systemse, 4~. Such an not been demonstrated for uric acid transport in erycthrocyte~ uric acid presumably belong to the group of substan ances bein~ ailibrating carrier mechanism ''*. If the transporting system, s~ wl Lmon to those purines which are able to penetrate the red cell c these substance, then the formation of nucleosides or nucleotid ht possibly occur. This formation might be expected to be el would account for uric acid transport being concentration concentr~ depl ' as is found for the turnover of substrate in an enzymi rmlc reactior exact nature of the transport system has not been elucidated. el
ocytes r been Work lthine, where movesically nsport "eason, abya ably is pecific ebrane lature, similar wever,
ACKNOWLEDGEMENTS
E KLENOW for stimulating The author wishes to thank Drs. T. ROSENBERG and H. discussion ussion, and Dr. K. OVERGAARD-HANSENfor generous assistance with some of the experiments. eriments. Dr. A. CODDINGTON'Scorrection of the manuscript manu is gratefully acknowledgled. This investigation was aided b y a grant (L38/6o ) from Statens Almindelige Videnskabsfond. REFERENCES 1 0 . . FOLIN AND A. SVEDBERG, J. Biol. Chem., 88 (193o) 715 . * S . R. BENEDICT AND J. A. EEHRR, J. J . Biol. Chem., 92 (1931) 16 i6i. 3 j , HELLER, Biochem. Z., 279 (1935) I49. 4 S. JORGENSEN AND AA. T. NIELSEN mLSRN, Scan& J. Glin. Lab. Invest., 8 (1956) Io8. 5 K. OVERGAARD-HANSI~NAND U. V. LASSEN, Nature, 184 (1959) 5538 T. ROSENBERG, Symposia Soc.:. Exptl. Biol., 8 (1954) 27. T. ROSENBERG AND W. WILBR, mANDT, J. Gen. Physiol., 41 (I957) 289. 8 W . WILBRANDT, S. F R E I AND T. ROSENBRRG, Exptl. Cell Research, I I (1956) 599 W. WILBRANDT in A. KLEINZELLER NZELLER AND A. KOTYK, Membrane Transport and MetabolismSymposium, Prague, I96O, p. 205. 18 p. G. LEFEvRE, J. Gen. Physiol., ~iol., 31 (1948) 505 . lX p. G. LEFRVRE AND G. F. McGZNNISS cGINNISS, J. Gen. Physiol., 44 (196o) 87. 12 W. F. WIDDAS, J. Physiol. (London), London), 118 (1952 ) 23 . 13 R. EGE, Biohem. Z., 114 (1921) 88. 14 O . BANG AND S. L. ORSKOV, J. Clin. Invest., 16 (1937) 279. 15 W. W. UMBREIT, R. H. BURRIS RRIS AND J . F . STAUFFRR, Manometric Techniques, Minneapolis, 1957, p. 149. 18 H. KLRNOW AND R. EMBERLAND AND, Arch. Biochem. Biophys., 58 (1955) 276. 17 B. MACKLRR, H. R. MAHLER AND D. E. GREEN, J. Biol. Chem., 21o (1954) 149. 18 E . PRARTORIUS AND H . E . P oOULSRN, l Scand. J. Clin. Lab. Invest., 5 (1953) 273. 18 C. W . SHRPPARD AND W . R . MARTIN, J. Gen. Physiol., 33 (1949) 703 • 20 B. VESTERGAARD-BOGIND AND 'D T. HESSELBO, Biochim. Biophys. Acta, 44 (196o) 117. t t A. MAURO in Symposium on Salt and Water Metabolism, N e w Y o r k H e a r t Association, 1959, p. 845.
Biochim. Biophys. Acta, 53 (1961) 557-569
TRANSPORT IN RED CELLS
Du~
• Chem. Soc., 56 (1934) 658. 9chem. Z., 49 (I913) 333. Federation Proc., 18 (196o) IOI. n. Lab. Invest., 9 (I957) 194. istry, New York, I948. ~er's, 241 (1939) 753. Gen. Physiol., 34 (1951) 515 . Hoppe-Seylers, 31 (19oo) i. N, Helv. Chim. dcta, 23 (194 o) 245. 133. BEAVEN, E. R. HOLYDAY AND E. A. JOHNSON in E. CCHAR, HARGAFF
AND
BON,
•ucleic Acids. Vol. I, Academic Press, Inc., New York, 1955, p. ] 496. BER, J. Cellular Comp. Physiol., 7 (1936) 367. JACOBS, Cold Spri~*g Harbor Symposia Quant. Biol., 8 (194o) 3 ° • MILNE, B. H. SCRIBNER AND M. A. CRAWFORD, Am. J. Med. Med., 24 (1958) .~ALD'~VELL, Intern. Rev. Cytol., 5 (1956) 229. jLYNN, Progress in Biophys., 8 (1957) 242. VANSLYKE, H. ~VIT AND F. C. MCLEAN, J. Biol. Chem., 56 (I, (1923) 765 • LASSEN AND K. OVERGAARD-HANSEN, unpublished experimel ~eriments. USSING, in H. H. USSING, P. I~RUHOFFER, J. HESS THAYSEN AND A N. A. "]
kali
Ions in Biology, Heidelberg, 196o. J, J. Biol. Chem., 51 (1922) 21. Biochim. Biop)~Izys. Acta,
ABNORMAL
-569
HUMAN HAEMOGLOIBINS
VII. VII. THE COMPARIS~JN OF NORMAL HUMAN HAEMOGLOBIN F. AND H A E M O G L O B I N DCmCAGO B A R B A R A BOWMAN AND V. M. INGRA ;RAM Division ~ision oo] Biochemistry, Department o[ Biology, Massachusetts Institute o] Technology, 3ambridge, Mass. (U.S.A.) Cambrid
(Received april 7th, 1961)
SUMMARY
peptide from haemoglobin Dchleago carries a chemical I t has been shown that a tryptic peg d i f f e r e n c e w h e n c o m p a r e d t o t:he h e corresponding peptide in normal haemoglobin A. T h e a l t e r e d p e p t i d e , c a l l e d n u mnber b e r 5, occurs in the fl-chain of the molecule.
INTRODUCTION T h r e e v a r i a n t s of h u m a n h a e m oLoglobin D have been investigated by BENZER et al. 1. E a c h v a r i a n t c a r r i e d a c h e m i c a fl l change in a different part of the haemoglobin molecule. I n h a e m o g l o b i n D~ ( f r o m La Turkish Cypriot) the a-chain tryptic peptide 23 D a (from a Gujerati Indian) the fl-chain (ref. 2) h a d d i s a p p e a r e d ; i n hLaemoglobin aemo
Biochim. Biopt~ys. Acta, 53 (1961) 569-573